RMCP Vol. 12 (2021): Supl 3 [english version] by Revista Mexicana de Ciencias Pecuarias

Edición Bilingüe Bilingual Edition

Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 12 Suplemento 3, pp. 1-307, NOVIEMBRE-2021

ISSN: 2448-6698

Rev. Mex. Cienc. Pecu. Vol. 12 Suplemento 3, pp. 1-307, NOVIEMBRE-2021

REVISTA MEXICANA DE CIENCIAS PECUARIAS Volumen 12 Suplemento 3, Noviembre 2021. Es una publicación trimestral de acceso abierto, revisada por pares y arbitrada, editada por el Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Avenida Progreso No. 5, Barrio de Santa Catarina, Delegación Coyoacán, C.P. 04010, Cuidad de México, www.inifap.gob.mx Distribuida por el Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, Km 15.5 Carretera México-Toluca, Colonia Palo Alto, Cuidad de México, C.P. 05110. Editor responsable: Arturo García Fraustro. Reservas de Derechos al Uso Exclusivo número 042021-051209561700-203. ISSN: 2428-6698, otorgados por el Instituto Nacional del Derecho de Autor (INDAUTOR). Responsable de la última actualización de este número: Arturo García Fraustro, Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, Km. 15.5 Carretera México-Toluca, Colonia Palo Alto, Ciudad de México, C.P. 015110. http://cienciaspecuarias. inifap.gob.mx, la presente publicación tuvo su última actualización en noviembre de 2021. Concurso Nacional de Fotografía INIFAP en tu Vida 2020 1er Lugar, Categoría Pecuaria Autor: Guillermo Martínez Velázquez Título: Mujer cuidando vacas en pastoreo

DIRECTORIO EDITOR EN JEFE Arturo García Fraustro

FUNDADOR John A. Pino EDITORES ADJUNTOS Oscar L. Rodríguez Rivera Alfonso Arias Medina

EDITORES POR DISCIPLINA Dra. Yolanda Beatriz Moguel Ordóñez, INIFAP, México Dr. Ramón Molina Barrios, Instituto Tecnológico de Sonora, Dr. Alfonso Juventino Chay Canul, Universidad Autónoma de Tabasco, México Dra. Maria Cristina Schneider, Universidad de Georgetown, Estados Unidos Dr. Feliciano Milian Suazo, Universidad Autónoma de Querétaro, México Dr. Javier F. Enríquez Quiroz, INIFAP, México Dra. Martha Hortencia Martín Rivera, Universidad de Sonora URN, México Dr. Fernando Arturo Ibarra Flores, Universidad de Sonora URN, México Dr. James A. Pfister, USDA, Estados Unidos Dr. Eduardo Daniel Bolaños Aguilar, INIFAP, México Dr. Sergio Iván Román-Ponce, INIFAP, México Dr. Jesús Fernández Martín, INIA, España Dr. Maurcio A. Elzo, Universidad de Florida Dr. Sergio D. Rodríguez Camarillo, INIFAP, México Dra. Nydia Edith Reyes Rodríguez, Universidad Autónoma del Estado de Hidalgo, México Dra. Maria Salud Rubio Lozano, Facultad de Medicina Veterinaria y Zootecnia, UNAM, México Dra. Elizabeth Loza-Rubio, INIFAP, México Dr. Juan Carlos Saiz Calahorra, Instituto Nacional de Investigaciones Agrícolas, España Dr. José Armando Partida de la Peña, INIFAP, México Dr. José Luis Romano Muñoz, INIFAP, México

Dr. Alejandro Plascencia Jorquera, Universidad Autónoma de Baja California, México Dr. Juan Ku Vera, Universidad Autónoma de Yucatán, México Dr. Ricardo Basurto Gutiérrez, INIFAP, México Dr. Luis Corona Gochi, Facultad de Medicina Veterinaria y Zootecnia, UNAM, México Dr. Juan Manuel Pinos Rodríguez, Facultad de Medicina Veterinaria y Zootecnia, Universidad Veracruzana, México Dr. Carlos López Coello, Facultad de Medicina Veterinaria y Zootecnia, UNAM, México Dr. Arturo Francisco Castellanos Ruelas, Facultad de Química. UADY Dra. Guillermina Ávila Ramírez, UNAM, México Dr. Emmanuel Camuus, CIRAD, Francia. Dr. Héctor Jiménez Severiano, INIFAP., México Dr. Juan Hebert Hernández Medrano, UNAM, México Dr. Adrian Guzmán Sánchez, Universidad Autónoma Metropolitana-Xochimilco, México Dr. Eugenio Villagómez Amezcua Manjarrez, INIFAP, CENID Salud Animal e Inocuidad, México Dr. José Juan Hernández Ledezma, Consultor privado Dr. Fernando Cervantes Escoto, Universidad Autónoma Chapingo, México Dr. Adolfo Guadalupe Álvarez Macías, Universidad Autónoma Metropolitana Xochimilco, México Dr. Alfredo Cesín Vargas, UNAM, México Dra. Marisela Leal Hernández, INIFAP, México Dr. Efrén Ramírez Bribiesca, Colegio de Postgraduados, México

TIPOGRAFÍA Y FORMATO: Oscar L. Rodríguez Rivera

Indizada en el “Journal Citation Report” Science Edition del ISI . Inscrita en el Sistema de Clasificación de Revistas Científicas y Tecnológicas de CONACyT; en EBSCO Host y la Red de Revistas Científicas de América Latina y el Caribe, España y Portugal (RedALyC) (www.redalyc.org); en la Red Iberoamericana de Revistas Científicas de Veterinaria de Libre Acceso (www.veterinaria.org/revistas/ revivec); en los Índices SCOPUS y EMBASE de Elsevier (www.elsevier. com).

REVISTA MEXICANA DE CIENCIAS PECUARIAS La Revista Mexicana de Ciencias Pecuarias es un órgano de difusión científica y técnica de acceso abierto, revisada por pares y arbitrada. Su objetivo es dar a conocer los resultados de las investigaciones realizadas por cualquier institución científica, relacionadas particularmente con las distintas disciplinas de la Medicina Veterinaria y la Zootecnia. Además de trabajos de las disciplinas indicadas en su Comité Editorial, se aceptan también para su evaluación y posible publicación, trabajos de otras disciplinas, siempre y cuando estén relacionados con la investigación pecuaria. Se publican en la revista tres categorías de trabajos: Artículos Científicos, Notas de Investigación y Revisiones Bibliográficas (consultar las Notas al autor); la responsabilidad de cada trabajo recae exclusivamente en los autores, los cuales, por la naturaleza misma de los experimentos pueden verse obligados a referirse en algunos casos a los nombres comerciales de ciertos productos, ello sin embargo, no implica preferencia por los productos citados o ignorancia respecto a los omitidos, ni tampoco significa en modo alguno respaldo publicitario hacia los productos mencionados. Todas las contribuciones serán cuidadosamente evaluadas por árbitros, considerando su calidad y relevancia académica. Queda entendido que el someter un manuscrito implica que la investigación descrita es única e inédita. La publicación de Rev. Mex. Cienc. Pecu. es trimestral en formato bilingüe Español e Inglés. El costo

total por publicar es de $ 7,280.00 más IVA por manuscrito ya editado. Se publica en formato digital en acceso abierto, por lo que se autoriza la reproducción total o parcial del contenido de los artículos si se cita la fuente. El envío de los trabajos de debe realizar directamente en el sitio oficial de la revista. Correspondencia adicional deberá dirigirse al Editor Adjunto a la siguiente dirección: Calle 36 No. 215 x 67 y 69 Colonia Montes de Amé, C.P. 97115 Mérida, Yucatán, México. Tel/Fax +52 (999) 941-5030. Correo electrónico (C-ele): rodriguez_oscar@prodigy.net.mx. La correspondencia relativa a suscripciones, asuntos de intercambio o distribución de números impresos anteriores, deberá dirigirse al Editor en Jefe de la Revista Mexicana de Ciencias Pecuarias, CENID Salud Animal e Inocuidad, Km 15.5 Carretera México-Toluca, Col. Palo Alto, D.F. C.P. 05110, México; Tel: +52(55) 3871-8700 ext. 80316; garcia.arturo@inifap.gob.mx o arias.alfonso@inifap.gob.mx. Inscrita en la base de datos de EBSCO Host y la Red de Revistas Científicas de América Latina y el Caribe, España y Portugal (RedALyC) (www.redalyc.org), en la Red Iberoamericana de Revistas Científicas de Veterinaria de Libre Acceso (www.veterinaria.org/revistas/ revivec), indizada en el “Journal Citation Report” Science Edition del ISI (http://thomsonreuters. com/) y en los Índices SCOPUS y EMBASE de Elsevier (www.elsevier.com)

VISITE NUESTRA PÁGINA EN INTERNET Artículos completos desde 1963 a la fecha y Notas al autor en: http://cienciaspecuarias.inifap.gob.mx Revista Mexicana de Ciencias Pecuarias is an open access peer-reviewed and refereed scientific and technical journal, which publishes results of research carried out in any scientific or academic institution, especially related to different areas of veterinary medicine and animal production. Papers on disciplines different from those shown in Editorial Committee can be accepted, if related to livestock research. The journal publishes three types of papers: Research Articles, Technical Notes and Review Articles (please consult Instructions for authors). Authors are responsible for the content of each manuscript, which, owing to the nature of the experiments described, may contain references, in some cases, to commercial names of certain products, which however, does not denote preference for those products in particular or of a lack of knowledge of any other which are not mentioned, nor does it signify in any way an advertisement or an endorsement of the referred products. All contributions will be carefully refereed for academic relevance and quality. Submission of an article is understood to imply that the research described is unique and unpublished. Rev. Mex. Cien. Pecu. is published quarterly in original lenguage Spanish or English. Total fee charges are US $ 425.00 per article in both printed languages.

Part of, or whole articles published in this Journal may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying or otherwise, provided the source is properly acknowledged. Manuscripts should be submitted directly in the official web site. Additional information may be mailed to Associate Editor, Revista Mexicana de Ciencias Pecuarias, Calle 36 No. 215 x 67 y 69 Colonia Montes de Amé, C.P. 97115 Mérida, Yucatán, México. Tel/Fax +52 (999) 941-5030. E-mail: rodriguez_oscar@prodigy.net.mx. For subscriptions, exchange or distribution of previous printed issues, please contact: Editor-in-Chief of Revista Mexicana de Ciencias Pecuarias, CENID Salud Animal e Inocuidad, Km 15.5 Carretera México-Toluca, Col. Palo Alto, D.F. C.P. 05110, México; Tel: +52(55) 3871-8700 ext. 80316; garcia.arturo@inifap.gob.mx or arias.alfonso@inifap.gob.mx. Registered in the EBSCO Host database. The Latin American and the Caribbean Spain and Portugal Scientific Journals Network (RedALyC) (www.redalyc.org). The Iberoamerican Network of free access Veterinary Scientific Journals (www.veterinaria.org/ revistas/ revivec). Thomson Reuter´s “Journal Citation Report” Science Edition (http://thomsonreuters.com/). Elsevier´s SCOPUS and EMBASE (www.elsevier.com) and the Essential Electronic Agricultural Library (www.teeal.org).

VISIT OUR SITE IN THE INTERNET Full articles from year 1963 to date and Instructions for authors can be accessed via the site http://cienciaspecuarias.inifap.gob.mx

REVISTA MEXICANA DE CIENCIAS PECUARIAS

REV. MEX. CIENC. PECU

VOL 12 (Suplemento 3)

NOVIEMBRE-2021

CONTENIDO Contents REVISIONES Reviews

Pág.

Logros, retos y perspectivas de la investigación en mejoramiento genético de bovinos productores de carne en el INIFAP Beef cattle genetic improvement research at the INIFAP: accomplishments, challenges and perspective Ángel Ríos Utrera, Guillermo Martínez Velázquez, René Calderón Chagoya, Moisés Montaño Bermúdez, Vicente Eliezer Vega Murillo ......................................................................................1 El ganado bovino Criollo Coreño del occidente de México en la producción de carne: caracterización, retos y perspectivas Criollo Coreño cattle in western Mexico: characterization, challenges and outlook Guillermo Martínez-Velázquez, Ángel Ríos-Utrera, José Antonio Palacios-Fránquez, Vicente Eliezer Vega-Murillo, Moisés Montaño-Bermúdez ……………………………………………………………………………….23 Biotecnologías reproductivas en el ganado bovino: cinco décadas de investigación en México Reproductive biotechnologies in beef cattle: five decades of research in Mexico Jorge Víctor Rosete Fernández, Horacio Álvarez Gallardo, David Urbán Duarte, Abraham Fragoso Islas, Marco Antonio Asprón Pelayo, Ángel Ríos Utrera, Sandra Pérez Reynozo, José Fernando De La Torre Sánchez ..............................................................................................................39 Principales aportes de la investigación del INIFAP a la nutrición porcina en México: retos y perspectivas Main contributions of INIFAP research to swine nutrition in Mexico: challenges and perspectives José Antonio Rentería Flores, Sergio Gómez Rosales, Luis Humberto López Hernández, Gerardo Ordaz Ochoa, Ana María Anaya Escalera, César Augusto Mejía Guadarrama, Gerardo Mariscal Landín ...................................................................................................................................79 Antecedentes y perspectivas de algunas enfermedades prioritarias que afectan a la ganadería bovina en México Background and perspectives of certain priority diseases affecting cattle farming in Mexico Carmen Rojas Martínez, Elizabeth Loza Rubio, Sergio Darío Rodríguez Camarillo, Julio Vicente Figueroa Millán, Francisco Aguilar Romero, Rodolfo Esteban Lagunes Quintanilla, José Francisco Morales Álvarez, Marco Antonio Santillán Flores, Guadalupe Asunción Socci Escatell, Jesús Antonio Álvarez Martínez ……………………………………………………………………………………………………111

III

Salud porcina: historia, retos y perspectivas Swine health: history, challenges and prospects José Francisco Rivera-Benítez, Jazmín De la Luz-Armendáriz, Luis Gómez-Núñez, Fernando Diosdado Vargas, Guadalupe Socci Escatell, Elizabeth Ramírez-Medina, Lauro Velázquez-Salinas, Humberto Ramírez-Mendoza, Maria Antonia Coba Ayala, Catalina Tufiño-Loza, Marta Macías García, Víctor Carrera-Aguirre, Rebeca Martínez-Bautista, María José Martínez-Mercado, Gerardo Santos-López, Irma Herrera-Camacho, Ignacio Siañez-Estrada, Manuel Zapata Moreno ……………149 Control y prevención de nematodosis en pequeños rumiantes: antecedentes, retos y perspectivas en México Control and prevention of nematodiasis in small ruminants: background, challenges and outlook in Mexico David Emanuel Reyes-Guerrero, Agustín Olmedo-Juárez, Pedro Mendoza-de Gives ……………….…186 Enfermedades infecciosas de relevancia en la producción caprina, historia, retos y perspectivas Important infectious diseases in goat production in Mexico: history, challenges and outlook Gabriela Palomares Resendiz, Francisco Aguilar Romero, Carlos Flores Pérez, Luis Gómez Núñez, José Gutiérrez Hernández, Enrique Herrera López, Magdalena Limón González, Francisco Morales Álvarez, Francisco Pastor López, Efrén Díaz Aparicio ……………………………………………………………..205 Resultados e impacto de la investigación en genética y mejoramiento genético de las abejas melíferas desarrollada por el INIFAP en México Results and impact of research on honeybee genetics and breeding conducted by INIFAP in Mexico Miguel Enrique Arechavaleta-Velasco, Claudia García-Figueroa, Laura Yavarik Alvarado-Avila, Francisco Javier Ramírez-Ramírez, Karla Itzel Alcalá-Escamilla ..................................................224 Rehabilitación de praderas degradadas en el trópico de México Rehabilitation of degraded pastures in the tropics of Mexico Javier Francisco Enríquez Quiroz, Valentín Alberto Esqueda Esquivel, Daniel Martínez Méndez ….243 Los pastizales y matorrales de zonas áridas y semiáridas de México: Estatus actual, retos y perspectivas The grasslands and scrublands of arid and semi-arid zones of Mexico: Current status, challenges and perspectives Pedro Jurado-Guerra, Mauricio Velázquez-Martínez, Ricardo Alonso Sánchez-Gutiérrez, Alan Álvarez-Holguín, Pablo Alfredo Domínguez-Martínez, Ramón Gutiérrez-Luna, Rubén Darío GarzaCedillo, Miguel Luna-Luna, Manuel Gustavo Chávez-Ruiz .........................................................261

Historia y perspectivas del modelo GGAVATT (Grupos Ganaderos de Validación y Transferencia de Tecnología) History and perspectives of the GGAVATT model (Groups for Livestock Technological Validation and Transfer) Heriberto Román Ponce, Miguel Arcangel Rodríguez Chessani, José Antonio Espinosa García, Tomás Arturo González Orozco, Alejandra Vélez Izquierdo, Juan Prisciliano Zárate Martínez, Martha Eugenia Valdovinos Terán, Rubén Cristino Aguilera Sosa, Rafael Guarneros Altamirano, Rubén Santos Echeverría, Héctor Macario Bueno Díaz, Ubaldo Aguilar Barradas ……………………………..…286

Actualización: marzo, 2020 NOTAS AL AUTOR La Revista Mexicana de Ciencias Pecuarias se edita completa en dos idiomas (español e inglés) y publica tres categorías de trabajos: Artículos científicos, Notas de investigación y Revisiones bibliográficas.

Los autores interesados en publicar en esta revista deberán ajustarse a los lineamientos que más adelante se indican, los cuales en términos generales, están de acuerdo con los elaborados por el Comité Internacional de Editores de Revistas Médicas (CIERM) Bol Oficina Sanit Panam 1989;107:422-437. 1.

Página del título Resumen en español Resumen en inglés Texto Agradecimientos y conflicto de interés Literatura citada

Sólo se aceptarán trabajos inéditos. No se admitirán si están basados en pruebas de rutina, ni datos experimentales sin estudio estadístico cuando éste sea indispensable. Tampoco se aceptarán trabajos que previamente hayan sido publicados condensados o in extenso en Memorias o Simposio de Reuniones o Congresos (a excepción de Resúmenes). Todos los trabajos estarán sujetos a revisión de un Comité Científico Editorial, conformado por Pares de la Disciplina en cuestión, quienes desconocerán el nombre e Institución de los autores proponentes. El Editor notificará al autor la fecha de recepción de su trabajo. El manuscrito deberá someterse a través del portal de la Revista en la dirección electrónica: http://cienciaspecuarias.inifap.gob.mx, consultando el “Instructivo para envío de artículos en la página de la Revista Mexicana de Ciencias Pecuarias”. Para su elaboración se utilizará el procesador de Microsoft Word, con letra Times New Roman a 12 puntos, a doble espacio. Asimismo se deberán llenar los formatos de postulación, carta de originalidad y no duplicidad y disponibles en el propio sitio oficial de la revista.

Por ser una revista con arbitraje, y para facilitar el trabajo de los revisores, todos los renglones de cada página deben estar numerados; asimismo cada página debe estar numerada, inclusive cuadros, ilustraciones y gráficas.

Los artículos tendrán una extensión máxima de 20 cuartillas a doble espacio, sin incluir páginas de Título, y cuadros o figuras (los cuales no deberán exceder de ocho y ser incluidos en el texto). Las Notas de investigación tendrán una extensión máxima de 15 cuartillas y 6 cuadros o figuras. Las Revisiones bibliográficas una extensión máxima de 30 cuartillas y 5 cuadros.

Los manuscritos de las tres categorías de trabajos que se publican en la Rev. Mex. Cienc. Pecu. deberán contener los componentes que a continuación se indican, empezando cada uno de ellos en página aparte.

Página del Título. Solamente debe contener el título del trabajo, que debe ser conciso pero informativo; así como el título traducido al idioma inglés. En el manuscrito no es necesaria información como nombres de autores, departamentos, instituciones, direcciones de correspondencia, etc., ya que estos datos tendrán que ser registrados durante el proceso de captura de la solicitud en la plataforma del OJS (http://ciencias pecuarias.inifap.gob.mx).

Resumen en español. En la segunda página se debe incluir un resumen que no pase de 250 palabras. En él se indicarán los propósitos del estudio o investigación; los procedimientos básicos y la metodología empleada; los resultados más importantes encontrados, y de ser posible, su significación estadística y las conclusiones principales. A continuación del resumen, en punto y aparte, agregue debidamente rotuladas, de 3 a 8 palabras o frases cortas clave que ayuden a los indizadores a clasificar el trabajo, las cuales se publicarán junto con el resumen.

Resumen en inglés. Anotar el título del trabajo en inglés y a continuación redactar el “abstract” con las mismas instrucciones que se señalaron para el resumen en español. Al final en punto y aparte, se deberán escribir las correspondientes palabras clave (“key words”).

10. Texto. Las tres categorías de trabajos que se publican en la Rev. Mex. Cienc. Pecu. consisten en lo siguiente: a) Artículos científicos. Deben ser informes de trabajos originales derivados de resultados parciales o finales de investigaciones. El texto del Artículo científico se divide en secciones que llevan estos encabezamientos:

Introducción Materiales y Métodos Resultados Discusión Conclusiones e implicaciones Literatura citada

referencias, aunque pueden insertarse en el texto (entre paréntesis).

Reglas básicas para la Literatura citada Nombre de los autores, con mayúsculas sólo las iniciales, empezando por el apellido paterno, luego iniciales del materno y nombre(s). En caso de apellidos compuestos se debe poner un guión entre ambos, ejemplo: Elías-Calles E. Entre las iniciales de un autor no se debe poner ningún signo de puntuación, ni separación; después de cada autor sólo se debe poner una coma, incluso después del penúltimo; después del último autor se debe poner un punto.

En los artículos largos puede ser necesario agregar subtítulos dentro de estas divisiones a fin de hacer más claro el contenido, sobre todo en las secciones de Resultados y de Discusión, las cuales también pueden presentarse como una sola sección. b) Notas de investigación. Consisten en modificaciones a técnicas, informes de casos clínicos de interés especial, preliminares de trabajos o investigaciones limitadas, descripción de nuevas variedades de pastos; así como resultados de investigación que a juicio de los editores deban así ser publicados. El texto contendrá la misma información del método experimental señalado en el inciso a), pero su redacción será corrida del principio al final del trabajo; esto no quiere decir que sólo se supriman los subtítulos, sino que se redacte en forma continua y coherente.

El título del trabajo se debe escribir completo (en su idioma original) luego el título abreviado de la revista donde se publicó, sin ningún signo de puntuación; inmediatamente después el año de la publicación, luego el número del volumen, seguido del número (entre paréntesis) de la revista y finalmente el número de páginas (esto en caso de artículo ordinario de revista). Puede incluir en la lista de referencias, los artículos aceptados aunque todavía no se publiquen; indique la revista y agregue “en prensa” (entre corchetes).

c) Revisiones bibliográficas. Consisten en el tratamiento y exposición de un tema o tópico de relevante actualidad e importancia; su finalidad es la de resumir, analizar y discutir, así como poner a disposición del lector información ya publicada sobre un tema específico. El texto se divide en: Introducción, y las secciones que correspondan al desarrollo del tema en cuestión.

En el caso de libros de un solo autor (o más de uno, pero todos responsables del contenido total del libro), después del o los nombres, se debe indicar el título del libro, el número de la edición, el país, la casa editorial y el año. Cuando se trate del capítulo de un libro de varios autores, se debe poner el nombre del autor del capítulo, luego el título del capítulo, después el nombre de los editores y el título del libro, seguido del país, la casa editorial, año y las páginas que abarca el capítulo.

11. Agradecimientos y conflicto de interés. Siempre que corresponda, se deben especificar las colaboraciones que necesitan ser reconocidas, tales como a) la ayuda técnica recibida; b) el agradecimiento por el apoyo financiero y material, especificando la índole del mismo; c) las relaciones financieras que pudieran suscitar un conflicto de intereses. Las personas que colaboraron pueden ser citadas por su nombre, añadiendo su función o tipo de colaboración; por ejemplo: “asesor científico”, “revisión crítica de la propuesta para el estudio”, “recolección de datos”, etc. Siempre que corresponda, los autores deberán mencionar si existe algún conflicto de interés. 12. Literatura citada. Numere las referencias consecutivamente en el orden en que se mencionan por primera vez en el texto. Las referencias en el texto, en los cuadros y en las ilustraciones se deben identificar mediante números arábigos entre paréntesis, sin señalar el año de la referencia. Evite hasta donde sea posible, el tener que mencionar en el texto el nombre de los autores de las referencias. Procure abstenerse de utilizar los resúmenes como referencias; las “observaciones inéditas” y las “comunicaciones personales” no deben usarse como

En el caso de tesis, se debe indicar el nombre del autor, el título del trabajo, luego entre corchetes el grado (licenciatura, maestría, doctorado), luego el nombre de la ciudad, estado y en su caso país, seguidamente el nombre de la Universidad (no el de la escuela), y finalmente el año. Emplee el estilo de los ejemplos que aparecen a continuación, los cuales están parcialmente basados en el formato que la Biblioteca Nacional de Medicina de los Estados Unidos usa en el Index Medicus. Revistas

Artículo ordinario, con volumen y número. (Incluya el nombre de todos los autores cuando sean seis o menos; si son siete o más, anote sólo el nombre de los seis primeros y agregue “et al.”).

VII

Basurto GR, Garza FJD. Efecto de la inclusión de grasa o proteína de escape ruminal en el comportamiento de toretes Brahman en engorda. Téc Pecu Méx 1998;36(1):35-48.

XI)

Sólo número sin indicar volumen. II) Stephano HA, Gay GM, Ramírez TC. Encephalomielitis, reproductive failure and corneal opacity (blue eye) in pigs associated with a paramyxovirus infection. Vet Rec 1988;(122):6-10.

XII) Cunningham EP. Genetic diversity in domestic animals: strategies for conservation and development. In: Miller RH et al. editors. Proc XX Beltsville Symposium: Biotechnology’s role in genetic improvement of farm animals. USDA. 1996:13.

III) Chupin D, Schuh H. Survey of present status ofthe use of artificial insemination in developing countries. World Anim Rev 1993;(74-75):26-35.

Tesis.

No se indica el autor.

XIII) Alvarez MJA. Inmunidad humoral en la anaplasmosis y babesiosis bovinas en becerros mantenidos en una zona endémica [tesis maestría]. México, DF: Universidad Nacional Autónoma de México; 1989.

IV) Cancer in South Africa [editorial]. S Afr Med J 1994;84:15.

Suplemento de revista.

XIV) Cairns RB. Infrared spectroscopic studies of solid oxigen [doctoral thesis]. Berkeley, California, USA: University of California; 1965.

V) Hall JB, Staigmiller RB, Short RE, Bellows RA, Bartlett SE. Body composition at puberty in beef heifers as influenced by nutrition and breed [abstract]. J Anim Sci 1998;71(Suppl 1):205.

Organización como autor. XV) NRC. National Research Council. The nutrient requirements of beef cattle. 6th ed. Washington, DC, USA: National Academy Press; 1984.

Organización, como autor. VI) The Cardiac Society of Australia and New Zealand. Clinical exercise stress testing. Safety and performance guidelines. Med J Aust 1996;(164):282-284.

XVI) SAGAR. Secretaría de Agricultura, Ganadería y Desarrollo Rural. Curso de actualización técnica para la aprobación de médicos veterinarios zootecnistas responsables de establecimientos destinados al sacrificio de animales. México. 1996.

En proceso de publicación. VII) Scifres CJ, Kothmann MM. Differential grazing use of herbicide treated area by cattle. J Range Manage [in press] 2000.

XVII) AOAC. Oficial methods of analysis. 15th ed. Arlington, VA, USA: Association of Official Analytical Chemists. 1990.

Libros y otras monografías

XVIII) SAS. SAS/STAT User’s Guide (Release 6.03). Cary NC, USA: SAS Inst. Inc. 1988.

Autor total. VIII) Steel RGD, Torrie JH. Principles and procedures of statistics: A biometrical approach. 2nd ed. New York, USA: McGraw-Hill Book Co.; 1980.

XIX) SAS. SAS User´s Guide: Statistics (version 5 ed.). Cary NC, USA: SAS Inst. Inc. 1985.

Publicaciones electrónicas

Autor de capítulo. IX)

XX) Jun Y, Ellis M. Effect of group size and feeder type on growth performance and feeding patterns in growing pigs. J Anim Sci 2001;79:803-813. http://jas.fass.org/cgi/reprint/79/4/803.pdf. Accessed Jul 30, 2003.

Roberts SJ. Equine abortion. In: Faulkner LLC editor. Abortion diseases of cattle. 1rst ed. Springfield, Illinois, USA: Thomas Books; 1968:158-179.

Memorias de reuniones. X)

Olea PR, Cuarón IJA, Ruiz LFJ, Villagómez AE. Concentración de insulina plasmática en cerdas alimentadas con melaza en la dieta durante la inducción de estro lactacional [resumen]. Reunión nacional de investigación pecuaria. Querétaro, Qro. 1998:13.

XXI) Villalobos GC, González VE, Ortega SJA. Técnicas para estimar la degradación de proteína y materia orgánica en el rumen y su importancia en rumiantes en pastoreo. Téc Pecu Méx 2000;38(2): 119-134. http://www.tecnicapecuaria.org/trabajos/20021217 5725.pdf. Consultado 30 Ago, 2003.

Loeza LR, Angeles MAA, Cisneros GF. Alimentación de cerdos. En: Zúñiga GJL, Cruz BJA editores. Tercera reunión anual del centro de investigaciones forestales y agropecuarias del estado de Veracruz. Veracruz. 1990:51-56.

VIII

XXII) Sanh MV, Wiktorsson H, Ly LV. Effect of feeding level on milk production, body weight change, feed conversion and postpartum oestrus of crossbred lactating cows in tropical conditions. Livest Prod Sci 2002;27(2-3):331-338. http://www.sciencedirect. com/science/journal/03016226. Accessed Sep 12, 2003.

ha hectárea (s) h hora (s) i.m. intramuscular (mente) i.v. intravenosa (mente) J joule (s) kg kilogramo (s) km kilómetro (s) L litro (s) log logaritmo decimal Mcal megacaloría (s) MJ megajoule (s) m metro (s) msnm metros sobre el nivel del mar µg microgramo (s) µl microlitro (s) µm micrómetro (s)(micra(s)) mg miligramo (s) ml mililitro (s) mm milímetro (s) min minuto (s) ng nanogramo (s)Pprobabilidad (estadística) p página PC proteína cruda PCR reacción en cadena de la polimerasa pp páginas ppm partes por millón % por ciento (con número) rpm revoluciones por minuto seg segundo (s) t tonelada (s) TND total de nutrientes digestibles UA unidad animal UI unidades internacionales

13. Cuadros, Gráficas e Ilustraciones. Es preferible que sean pocos, concisos, contando con los datos necesarios para que sean autosuficientes, que se entiendan por sí mismos sin necesidad de leer el texto. Para las notas al pie se deberán utilizar los símbolos convencionales. 14 Versión final. Es el documento en el cual los autores ya integraron las correcciones y modificaciones indicadas por el Comité Revisor. Los trabajos deberán ser elaborados con Microsoft Word. Las fotografías e imágenes deberán estar en formato jpg (o compatible) con al menos 300 dpi de resolución. Tanto las fotografías, imágenes, gráficas, cuadros o tablas deberán incluirse en el mismo archivo del texto. Los cuadros no deberán contener ninguna línea vertical, y las horizontales solamente las que delimitan los encabezados de columna, y la línea al final del cuadro. 15. Una vez recibida la versión final, ésta se mandará para su traducción al idioma inglés o español, según corresponda. Si los autores lo consideran conveniente podrán enviar su manuscrito final en ambos idiomas. 16. Tesis. Se publicarán como Artículo o Nota de Investigación, siempre y cuando se ajusten a las normas de esta revista. 17. Los trabajos no aceptados para su publicación se regresarán al autor, con un anexo en el que se explicarán los motivos por los que se rechaza o las modificaciones que deberán hacerse para ser reevaluados.

versus

gravedades

Cualquier otra abreviatura se pondrá entre paréntesis inmediatamente después de la(s) palabra(s) completa(s).

18. Abreviaturas de uso frecuente: cal cm °C DL50 g

caloría (s) centímetro (s) grado centígrado (s) dosis letal 50% gramo (s)

19. Los nombres científicos y otras locuciones latinas se deben escribir en cursivas.

Updated: March, 2020 INSTRUCTIONS FOR AUTHORS Revista Mexicana de Ciencias Pecuarias is a scientific journal published in a bilingual format (Spanish and English) which carries three types of papers: Research Articles, Technical Notes, and Reviews. Authors interested in publishing in this journal, should follow the belowmentioned directives which are based on those set down by the International Committee of Medical Journal Editors (ICMJE) Bol Oficina Sanit Panam 1989;107:422-437. 1.

Title page Abstract Text Acknowledgments and conflict of interest Literature cited

Only original unpublished works will be accepted. Manuscripts based on routine tests, will not be accepted. All experimental data must be subjected to statistical analysis. Papers previously published condensed or in extenso in a Congress or any other type of Meeting will not be accepted (except for Abstracts). All contributions will be peer reviewed by a scientific editorial committee, composed of experts who ignore the name of the authors. The Editor will notify the author the date of manuscript receipt. Papers will be submitted in the Web site http://cienciaspecuarias.inifap.gob.mx, according the “Guide for submit articles in the Web site of the Revista Mexicana de Ciencias Pecuarias”. Manuscripts should be prepared, typed in a 12 points font at double space (including the abstract and tables), At the time of submission a signed agreement co-author letter should enclosed as complementary file; coauthors at different institutions can mail this form independently. The corresponding author should be indicated together with his address (a post office box will not be accepted), telephone and Email.

Title page. It should only contain the title of the work, which should be concise but informative; as well as the title translated into English language. In the manuscript is not necessary information as names of authors, departments, institutions and correspondence addresses, etc.; as these data will have to be registered during the capture of the application process on the OJS platform (http://cienciaspecuarias.inifap.gob.mx).

Abstract. On the second page a summary of no more than 250 words should be included. This abstract should start with a clear statement of the objectives and must include basic procedures and methodology. The more significant results and their statistical value and the main conclusions should be elaborated briefly. At the end of the abstract, and on a separate line, a list of up to 10 key words or short phrases that best describe the nature of the research should be stated.

Text. The three categories of articles which are published in Revista Mexicana de Ciencias Pecuarias are the following:

a) Research Articles. They should originate in primary

works and may show partial or final results of research. The text of the article must include the following parts:

To facilitate peer review all pages should be numbered consecutively, including tables, illustrations and graphics, and the lines of each page should be numbered as well.

Introduction Materials and Methods Results Discussion Conclusions and implications Literature cited

Research articles will not exceed 20 double spaced pages, without including Title page and Tables and Figures (8 maximum and be included in the text). Technical notes will have a maximum extension of 15 pages and 6 Tables and Figures. Reviews should not exceed 30 pages and 5 Tables and Figures.

In lengthy articles, it may be necessary to add other sections to make the content clearer. Results and Discussion can be shown as a single section if considered appropriate.

Manuscripts of all three type of articles published in Revista Mexicana de Ciencias Pecuarias should contain the following sections, and each one should begin on a separate page.

b) Technical Notes. They should be brief and be evidence for technical changes, reports of clinical cases of special interest, complete description of a limited investigation, or research results which

should be published as a note in the opinion of the editors. The text will contain the same information presented in the sections of the research article but without section titles.

names(s), the number of the edition, the country, the printing house and the year. e. When a reference is made of a chapter of book written by several authors; the name of the author(s) of the chapter should be quoted, followed by the title of the chapter, the editors and the title of the book, the country, the printing house, the year, and the initial and final pages.

c) Reviews. The purpose of these papers is to

summarize, analyze and discuss an outstanding topic. The text of these articles should include the following sections: Introduction, and as many sections as needed that relate to the description of the topic in question.

f. In the case of a thesis, references should be made of the author’s name, the title of the research, the degree obtained, followed by the name of the City, State, and Country, the University (not the school), and finally the year.

10. Acknowledgements. Whenever appropriate, collaborations that need recognition should be specified: a) Acknowledgement of technical support; b) Financial and material support, specifying its nature; and c) Financial relationships that could be the source of a conflict of interest.

Examples The style of the following examples, which are partly based on the format the National Library of Medicine of the United States employs in its Index Medicus, should be taken as a model.

People which collaborated in the article may be named, adding their function or contribution; for example: “scientific advisor”, “critical review”, “data collection”, etc. 11. Literature cited. All references should be quoted in their original language. They should be numbered consecutively in the order in which they are first mentioned in the text. Text, tables and figure references should be identified by means of Arabic numbers. Avoid, whenever possible, mentioning in the text the name of the authors. Abstain from using abstracts as references. Also, “unpublished observations” and “personal communications” should not be used as references, although they can be inserted in the text (inside brackets).

Journals

Standard journal article (List the first six authors followed by et al.) I)

Basurto GR, Garza FJD. Efecto de la inclusión de grasa o proteína de escape ruminal en el comportamiento de toretes Brahman en engorda. Téc Pecu Méx 1998;36(1):35-48.

Issue with no volume

Key rules for references

II) Stephano HA, Gay GM, Ramírez TC. Encephalomielitis, reproductive failure and corneal opacity (blue eye) in pigs associated with a paramyxovirus infection. Vet Rec 1988;(122):6-10.

a. The names of the authors should be quoted beginning with the last name spelt with initial capitals, followed by the initials of the first and middle name(s). In the presence of compound last names, add a dash between both, i.e. Elias-Calles E. Do not use any punctuation sign, nor separation between the initials of an author; separate each author with a comma, even after the last but one.

III) Chupin D, Schuh H. Survey of present status of the use of artificial insemination in developing countries. World Anim Rev 1993;(74-75):26-35.

No author given

b. The title of the paper should be written in full, followed by the abbreviated title of the journal without any punctuation sign; then the year of the publication, after that the number of the volume, followed by the number (in brackets) of the journal and finally the number of pages (this in the event of ordinary article).

IV) Cancer in South Africa [editorial]. S Afr Med J 1994;84:15.

Journal supplement V) Hall JB, Staigmiller RB, Short RE, Bellows RA, Bartlett SE. Body composition at puberty in beef heifers as influenced by nutrition and breed [abstract]. J Anim Sci 1998;71(Suppl 1):205.

c. Accepted articles, even if still not published, can be included in the list of references, as long as the journal is specified and followed by “in press” (in brackets). d. In the case of a single author’s book (or more than one, but all responsible for the book’s contents), the title of the book should be indicated after the

Organization, as author VI) The Cardiac Society of Australia and New Zealand. Clinical exercise stress testing. Safety and performance guidelines. Med J Aust 1996;(164):282284.

In press VII) Scifres CJ, Kothmann MM. Differential grazing use of herbicide-treated area by cattle. J Range Manage [in press] 2000. Books and other monographs

Author(s) VIII) Steel RGD, Torrie JH. Principles and procedures of statistics: A biometrical approach. 2nd ed. New York, USA: McGraw-Hill Book Co.; 1980.

Organization as author XV) NRC. National Research Council. The nutrient requirements of beef cattle. 6th ed. Washington, DC, USA: National Academy Press; 1984. XVI) SAGAR. Secretaría de Agricultura, Ganadería y Desarrollo Rural. Curso de actualización técnica para la aprobación de médicos veterinarios zootecnistas responsables de establecimientos destinados al sacrificio de animales. México. 1996. XVII) AOAC. Official methods of analysis. 15th ed. Arlington, VA, USA: Association of Official Analytical Chemists. 1990. XVIII) SAS. SAS/STAT User’s Guide (Release 6.03). Cary NC, USA: SAS Inst. Inc. 1988. XIX) SAS. SAS User´s Guide: Statistics (version 5 ed.). Cary NC, USA: SAS Inst. Inc. 1985.

Chapter in a book IX)

Roberts SJ. Equine abortion. In: Faulkner LLC editor. Abortion diseases of cattle. 1rst ed. Springfield, Illinois, USA: Thomas Books; 1968:158-179.

Conference paper X)

XI)

Thesis XIII) Alvarez MJA. Inmunidad humoral en la anaplasmosis y babesiosis bovinas en becerros mantenidos en una zona endémica [tesis maestría]. México, DF: Universidad Nacional Autónoma de México; 1989.

Electronic publications XX) Jun Y, Ellis M. Effect of group size and feeder type on growth performance and feeding patterns in growing pigs. J Anim Sci 2001;79:803-813. http://jas.fass.org/cgi/reprint/79/4/803.pdf. Accesed Jul 30, 2003. XXI) Villalobos GC, González VE, Ortega SJA. Técnicas para estimar la degradación de proteína y materia orgánica en el rumen y su importancia en rumiantes en pastoreo. Téc Pecu Méx 2000;38(2): 119-134. http://www.tecnicapecuaria.org/trabajos/20021217 5725.pdf. Consultado 30 Jul, 2003. XXII) Sanh MV, Wiktorsson H, Ly LV. Effect of feeding level on milk production, body weight change, feed conversion and postpartum oestrus of crossbred lactating cows in tropical conditions. Livest Prod Sci 2002;27(2-3):331-338. http://www.sciencedirect.com/science/journal/030 16226. Accesed Sep 12, 2003. 12. Tables, Graphics and Illustrations. It is preferable that they should be few, brief and having the necessary data so they could be understood without reading the text. Explanatory material should be placed in footnotes, using conventional symbols.

13. Final version. This is the document in which the authors have already integrated the corrections and modifications indicated by the Review Committee. The works will have to be elaborated with Microsoft Word. Photographs and images must be in jpg (or compatible) format with at least 300 dpi resolution. Photographs, images, graphs, charts or tables must be included in the same text file. The boxes should not contain any vertical lines, and the horizontal ones only those that delimit the column headings, and the line at the end of the box.

XIV) Cairns RB. Infrared spectroscopic studies of solid oxigen [doctoral thesis]. Berkeley, California, USA: University of California; 1965.

XII

14. Once accepted, the final version will be translated into Spanish or English, although authors should feel free to send the final version in both languages. No charges will be made for style or translation services.

MJ m µl µm mg ml mm min ng

mega joule (s) meter (s) micro liter (s) micro meter (s) milligram (s) milliliter (s) millimeter (s) minute (s) nanogram (s) P probability (statistic) p page CP crude protein PCR polymerase chain reaction pp pages ppm parts per million % percent (with number) rpm revolutions per minute sec second (s) t metric ton (s) TDN total digestible nutrients AU animal unit IU international units

15. Thesis will be published as a Research Article or as a Technical Note, according to these guidelines. 16. Manuscripts not accepted for publication will be returned to the author together with a note explaining the cause for rejection, or suggesting changes which should be made for re-assessment.

17. List of abbreviations: cal cm °C DL50 g ha h i.m. i.v. J kg km L log Mcal

calorie (s) centimeter (s) degree Celsius lethal dose 50% gram (s) hectare (s) hour (s) intramuscular (..ly) intravenous (..ly) joule (s) kilogram (s) kilometer (s) liter (s) decimal logarithm mega calorie (s)

versus

gravidity

The full term for which an abbreviation stands should precede its first use in the text. 18. Scientific names and other Latin terms should be written in italics.

XIII

https://doi.org/10.22319/rmcp.v12s3.6084

Editorial Livestock

INIFAP National Photography Constest 2020 2nd Place, Livestock Category Author: Jerónimo Sepúlveda Vázquez Title: Curious sheep

production in Mexico is currently facing the pressures of globalization and growth of national and global demand for animal-source foods. Despite the 8.5% drop in the national economy caused by the Sars-CoV-2 pandemic during 2020, the primary GDP associated with livestock grew 2.8% during this period and has maintained an uninterrupted growth streak in the last five years, with a rate above the national economy.

According to the data reported by SIAP (January 2021), during 2020, the production of bovine milk increased 2.3%, table egg 2.0%, chicken meat 3.6%, pork 3.1%, bovine meat 2.6%, sheep meat 1.1%, goat meat unchanged and only honey production decreased 12.7%, compared to 2019. These positive indicators, however, contrast with the alarming figures of natural resource degradation, vulnerability to climate change and poverty levels in rural areas of high and very high marginalization. For this reason and within the framework of the 35th anniversary of our Institution, the General Directorate of INIFAP wants to share in this Supplement 3, a series of reviews on the advances and perspectives of the research developed in INIFAP on livestock issues of great interest to the national livestock, such as animal production and health. In most cases, the research works were carried out in cooperation with other research and educational institutions at the national and international levels, in addition to the active participation of federal, state authorities and producers. It is expected that, by sharing this Supplement 3, the development of new comprehensive livestock systems based on scientific research will be stimulated,

XIV

which will continue to contribute to the increase of productivity, sustainable management and protection of natural resources, the improvement of the quality of life of livestock producers and, at the same time, reduce their vulnerability to climate change. To achieve the latter, the participation of decision-makers and producer associations is essential in the search for instruments of agricultural and technological support policy that facilitate the incorporation of sustainable systems that are generated in livestock development programs at the national level. Finally, I would like to express my sincere appreciation to all the livestock researchers who, throughout these 35 years, have contributed with the generation of knowledge and technologies to the development of livestock farms in the country and to the positioning of our Institution.

SINCERELY,

THE PERSON IN CHARGE OF THE OFFICE OF THE AFFAIRS OF THE GENERAL DIRECTORATE OF INIFAP

LUIS ÁNGEL RODRÍGUEZ DEL BOSQUE PhD

INIFAP INIFAPNational NationalPhotography PhotographyConstest Constest. 2020 3th 3thPlace, Place,Livestock LivestockCategory Category Author: Author:María Maríadel delCarmen CarmenZavaleta ZavaletaCórdova Córdova Title: Title:Grass Grassweighing weighing

https://doi.org/10.22319/rmcp.v12s3.5883 Review

Beef cattle genetic improvement research at the INIFAP: accomplishments, challenges and perspective

Ángel Ríos Utrera a Guillermo Martínez Velázquez b René Calderón Chagoya c Moisés Montaño Bermúdez c Vicente Eliezer Vega Murillo d*

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Campo Experimental La Posta, km 22.5 carretera federal Veracruz-Córdoba, Paso del Toro, Municipio de Medellín, 94277. Veracruz, México. b

INIFAP. Sitio Experimental El Verdineño. Nayarit, México.

INIFAP. Centro Nacional de Investigación Disciplinaria en Fisiología y Mejoramiento Animal. Querétaro, México. d

Universidad Veracruzana. Facultad de Medicina Veterinaria y Zootecnia. Veracruz, México. *Corresponding author: vvega@uv.mx

Abstract: The National Institute of Forestry, Agricultural and Livestock Research in Mexico has been active for decades in researching genetic improvement in beef cattle. This review uses master theses, congress papers and scientific articles published from 1987 to 2020 to summarize much of the relevant research, and addresses research challenges and outlook over the short-, medium- and long-term in this area. Research done over the last 34 yr has evaluated the productive and reproductive performance of Bos indicus and Bos taurus x Bos indicus beef cattle raised under tropical conditions. Multibreed genetic evaluations have been done for Simmental-Simbrah and Charolais-Charbray populations in Mexico. Analyses have 1

Rev Mex Cienc Pecu 2021;12(Supl 3):1-22

quantified the importance of maternal effects on growth traits, and estimated heritability and genetic correlations for growth and reproductive traits in male and female Bos taurus and Bos indicus animals. The genotype-environment interaction has been confirmed to influence expression of weaning weight in Simmental cattle. Age adjustment factors have been developed for maternal age for weights at birth and weaning, and prototypes of national genetic evaluation were developed for stayability and heifer fertility. Genetic diversity has been quantified for Simmental, Charolais and Simbrah populations, and SNPs identified that are associated with growth traits in Simmental and Simbrah populations. Short-term goals include development of selection indices and prediction of the genetic merit of carcass traits. Over the medium-term, emphasis is needed on genomic evaluations for tolerance to heat stress, residual feed intake and health traits, while in the long-term the goal is to make interbreed genomic predictions. Key words: Genomic association, Beef cattle, Genetic correlations, Crossbreeding, Genetic diversity, Heritability, Genetic improvement.

Received: 27/11/2020 Accepted: 29/03/2021

Introduction In beef cattle production, crossbreeding is intended to utilize breed differences and heterosis effects to improve production outcomes. Heterosis in a crossbreeding program can increase cattle herd productivity by an estimated average of 26 % compared to a similar program using purebred animals. The greatest benefit occurs with the use of crossbred cows(1,2,3). How much productivity increases from crossbreeding depends on the breeds involved. In the early 1980s, cattlemen in Mexico did not use crossbred cows to produce calves at weaning and the availability of specialized breed bulls was limited, especially in tropical regions. In 1978, the National Institute of Artificial Insemination and Animal Reproduction, of what was then the Ministry of Agriculture and Hydraulic Resources, a federal institution, made semen available from Bos taurus breeds specialized in beef production (Angus, Chianina, Charolais, Hereford, Limousin, Simmental and European Brown Swiss)(4). Genetic improvement of livestock involves selection of above-average animals to function as parents to subsequent generations. Identification of genetically superior animals as parents of the next generation requires breed associations to integrate their respective databases such that national-level genetic evaluations can be done. The first national genetic evaluation of 2

Rev Mex Cienc Pecu 2021;12(Supl 3):1-22

beef cattle was done in 2001 by a group of researchers from the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP; National Institute of Forestry, Agricultural and Livestock Research), using the database of the Asociación Mexicana Simmental-Simbrah (AMSS). The INIFAP has since entered into agreements with other associations to allow use of their databases for research purposes. Evaluation of growth traits can usually be done using conventional genetic evaluations. However, measuring economically important traits such as reproductive performance, feed efficiency and carcass traits can be complex, difficult and/or expensive. Sequencing of the bovine genome has made evaluation of these traits more accessible. Arrays have been developed containing thousands of single nucleotide polymorphisms (SNPs), which allow research into prediction of genetic merit(5). The present review summarizes research done at the INIFAP on genetic improvement of beef cattle, as well as the challenges and research perspectives over the short-, medium- and longterm.

Results of Bos taurus x Bos indicus crosses at Las Margaritas Experimental Station, in a humid hot subtropical climate Af(c)

One study evaluated the reproductive performance to weaning of Zebu (Z) cows mated to sires from four Bos taurus (Bt) breeds [Angus (A), Charolais (Ch), Hereford (H) and Brown Swiss (S)], and two Bos indicus (Bi) breeds [Brahman (B) and Indubrasil (I)]. When mating was done in spring, weaning rate of cows mated to H (64.3%), B (60.7 %) and S sires (59.2 %) was highest, that of cows mated to A (56.1 %) and Ch sires (52.7 %) was intermediate, and that of cows mated to I sires (39.5 %) was lowest. When mating occurred in autumn, however, weaning rate of cows mated to H sires (72.4 %) was highest, that of cows mated to S sires (56.0%) was intermediate, and that of cows mated to Ch (55.9 %), I (49.8 %), A (44.9 %) and B sires (43.0 %) was lowest. In general, weaning rate of cows mated to B and I sires was 16.5 and 23.7 percentage units lower than that of cows mated to H sires. These results indicate that bull breed and mating season are important considerations when designing crossbreeding schemes for commercial production of weaned calves(6). Another study analyzed breeding data that included I and B cows, as well as F1 A x Z, H x Z, Ch x Z and S x Z cows. The F1 cows (except the Ch x Z) exhibited reproductive behavior from gestation to weaning that surpassed that of the I and B cows (Table 1). The weaning rate of F1 cows was 26 percentage units higher than that of Z cows, and calf weaning weight (WW) of F1 cows was 5.9 % higher than that of Z cows; in terms of kilograms of calf weaned 3

Rev Mex Cienc Pecu 2021;12(Supl 3):1-22

per cow exposed the overall advantage was 30.6 %. The F1 heifers also first calved at a younger age (17 mo on average) than the Bi heifers(7).

Table 1: Least square means for productivity parameters in F1 Bos taurus x Bos indicus cows crossed with Bos taurus bulls, and Zebu cows crossed with Bos indicus bulls Cow genetic PR¥ CR¥ WR¥ LP* WW210₰ TMP₰ ST2₪ ST5₪ group (%) (%) (%) (kg) (kg) (kg) (%) (%) Hereford x Zebu 82.0a Brown Swiss x 79.0a Zebu Charolais x Zebu 65.0b Angus x Zebu 85.0a Brahman 67.0b Indubrasil 66.0b

81.0a 76.0ab

73.0a 73.0a

738.8ab 667.9bc

164.0bc 185.0ab

1008b 1256a

92.0b 75.0ac

47.0b 42.0b

67.0abc 80.0a 65.0bc 56.0c

61.0ab 74.0a 56.0b 48.0b

777.3a 871.8a 521.1c ---

178.0c 191.0a 113.0d ---

1012b 1454a 902b ---

92.0b 84.0ab 66.0a ---

55.0bc 62.0c 32.0a ---

PR= Pregnancy rate; CR= Calving rate; WR= Weaning rate; LP= Lifetime productivity; WW210= Weaning weight adjusted to 210 d of age; TMP= Total milk production; ST2= Stayability at second parturition; ST5= Stayability at fifth parturition. ¥ Cows were crossed with bulls of the same breed as cow’s father (Ríos et al(7)). *Vega et al(9). ₰ Quiroz-Valiente et al(11). ₪ Vega et al(10). a,b,c,d

Different letter superscripts in the same column indicate difference (P<0.05).

A study of the causes of culling and productive life found that Bi cows were culled at an average of 66.2 % due to infertility, which is much more than the 17.9 % in F1 cows. Clearly, F1 cows had longer productive life than Bi cows; the higher stayability of the F1 cows allowed them greater reproductive capacity since the differences in mortality and maternal ability were not notable. Overall, F1 cows lived 2.8 yr longer than purebred Bi cows(8). Other studies have evaluated lifetime productivity (LP) and stayability (ST) at different ages in B and F1 A x Z, S x Z, Ch x Z and H x Z cows(9,10). Lifetime productivity was defined as cumulative weaning weight of calves weaned up to 9 yr of age per cow exposed, while ST was defined as the probability that a cow would have 2 (ST2), 3 (ST3), 4 (ST4) or 5 (ST5) calves, given that she had first calved before 3 yr of age. As part of these studies, B cows were mated to B bulls, while F1 cows were mated to Bi bulls from 1986 to 1989 and to Bt bulls from 1990 to 1994. In terms of LP, the F1 cows surpassed the B cows by 101.2 kg at 3 yr of age and by 242.9 kg at 9 yr of age; indeed, at 9 yr of age both the A x Z and Ch x Z cows had accumulated significantly more kilograms of weaned calf than the S x Z and B cows (Table 1)(9). In relation with ST, important differences were found in ST2; average ST2 for Ch x Z and H x Z cows was 92 %, while that for B and S x Z cows was 65.5 %. Similarly, significant differences were also found in ST5; A x Z cows had greater ST5 than B cows (62 4

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vs 32 %), with intermediate values for the Ch x Z, H x Z and S x Z cows (Table 1). The study showed that the F1 Bt x Bi cows were more likely to produce more calves than the B cows(10). Both studies confirm F1 cows as an option for increased productivity to weaning in cow-calf systems in the tropics of Mexico. Another study evaluated milk production in B cows and four F1 Bt x Bi breed group cows and how it correlated to calf WW. Milk yield was measured using the calf weighing technique before and after suckling. A correlation coefficient of 0.61 was observed between total milk yield (TMY) and WW adjusted to 210 d of age (WW210), and A x Z and S x Z cows were found to have higher TMY and calves with higher WW210 than Ch x Z, H x Z and B cows (Table 1)(11).

Results of Bos taurus x Bos indicus crosses at the El Macho Experimental Station in a subhumid hot tropical climate Aw1

One study evaluated the differences in productive performance of F1 Bt x Bi and Bi calves in terms of birth weight (BW), pre-weaning average daily weight gain (DWG) and WW adjusted to 232 d of age (WW232). The calves were the product of crosses between Z cows and Ch, Chianina (Ci), Limousin (L), Simmental (Sm), S and I bulls. Birth weight was generally 7 % higher in the F1 Bt x Bi calves than in the Bi calves (Table 2). The highest BW was observed in the Ch x Z calves, which was higher than that observed in the Ci x Z, Sm x Z, L x Z and S x Z calves, although these four crosses did not differ (Table 2). The lowest BW was in the I calves; BW was 13 % higher in the Ch x Z, 8 % in the Sm x Z, 6 % in the S x Z, 5 % in the L x Z and 4% in the Ci x Z. Among the six crosses, WW232 was highest in the Sm x Z and Ch x Z calves, which did not differ. These were followed by the remaining three crosses (S x Z, L x Z, Ci x Z) and I, which also did not differ. The Sm x Z and Ch x Z calves generally performed better in both traits compared to the I calves. Indeed they exhibited similar trends; in terms of WW232 the Sm x Z was 13 % heavier and the Ch x Z 12 % heavier than the I calves, while for BW the Sm x Z calves were 8% heavier and the Ch x Z 13 % heavier than the I calves(12).

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Table 2: Least square means for birth weight (BW), average daily weight gain (DWG) and weaning weight at 205 days (WW205) and 232 days (WW232) of age in Bos indicus and F1 Bos taurus x Bos indicus calves BW DWG WW232 BW DWG WW205 Genetic group ¥ ¥ ¥ ₭ ₭ (kg) (kg) (kg) (kg) (kg) (kg)₭ Simmental x 31.70b 0.713b 197.3b 30.80bc 0.620bc 157.80c Zebu Charolais x 33.08c 0.693b 193.7b 31.58b 0.634b 159.45c Zebu Brown Swiss x 31.24b 0.644a 180.5a 30.24c 0.602bc 154.44bc Zebu Limousin x 30.90b 0.617a 174.1a 30.17c 0.594cd 151.15bc Zebu Chianina x Zebu 30.61b 0.605a 171.0a 31.17b 0.602bc 154.56bc Indubrasil 30.34a 0.610a 171.3a 30.08c 0.564d 148.91b a,b,c,d

Different letter superscripts in the same column indicate difference (P<0.05). ¥ Reynoso et al(12). ₭Martínez(13).

Using a larger data set than in the previous study(12), an analysis was done for BW, DWG and WW adjusted to 205 d (WW205)(13). Birth weight was highest in the Ch x Z and Ci x Z calves, followed by the Sm x Z calves and finally the L x Z, S x Z and I calves (Table 2). Daily weight gain was highest in the Ch x Z calves and lowest in the L x Z and I calves, although the Sm x Z, Ci x Z and S x Z calves did not differ from the Ch x Z calves. Weaning weight adjusted to 205 d of age was highest in the Ch x Z and Sm x Z calves and lowest in the I calves, but the Ci x Z, S x Z and L x Z did not differ from the I calves. As occurred with BW, among the F1 crosses the Ch x Z exhibited the highest WW205 and the Ci x Z the lowest (Table 2). With these averages the direct additive genetic effects could be calculated for BW, DWG and WW205 (Table 3). Table 3: Direct additive genetic effectsa for birth weight (BW), average pre-weaning daily weight gain (DWG) and weaning weight adjusted to 205 days of age (WW205) for four Bos taurus breeds and one Bos indicus breed Breed BW (kg) DWG (kg) WW205 (kg) Indubrasil Charolais Simmental Chianina Brown Swiss

1.65 2.73 1.22 1.93 0.14 a

51 73 47 15 15

Expressed as deviation from the Limousin breed. Martínez(13).

17.9 15.4 12.3 6.3 6.1

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Overall, the results of the above studies suggest that the performance of Zebu calves is significantly surpassed by that of calves from terminal crosses of Z dams with Ch or Sm sires.

Genetic evaluations of Bos taurus and Bos indicus cattle The Asociación Mexicana Simmental-Simbrah was the first breed association to carry out a national genetic evaluation (2001). Supported by the INIFAP, the AMSS currently publishes expected progeny differences for BW, WW, yearling weight (YW), scrotal circumference (SC), frame score (FS), ST and heifer fertility (HF) for the Simmental and Simbrah breeds. In genetic evaluations, and other research on these and other breeds (e.g. Charolais, Charbray, Red Brangus), HF is defined as the probability that a heifer calved before 1,281 d (42 mo) of age. Stayability, in turn, is defined as the probability that a cow has a second calf or more before six years of age given she had a calf at three years of age. Expected progeny differences are the product of a multibreed genetic evaluation in which, generally, the animal model includes the contemporary group (herd-year-season-calf sex), dam’s age at calving (covariate), and the fixed effects of proportion of Simmental genes, heterozygosity, and recombination loss. Simmental-Simbrah population. One multibreed genetic evaluation which compared different variants of the animal model found that the most appropriate model for estimating variance components for BW, WW and YW was one which included direct and maternal genetic effects as well as the maternal permanent environmental effect. Exclusion of maternal effects (genetic and permanent environment) or inclusion of the covariance between direct and maternal genetic effects caused an overestimation of additive genetic variance and, consequently, heritability. The estimated values of direct and maternal heritability generated with the most appropriate model were 0.17 (direct) and 0.01 (maternal) for BW, 0.14 (direct) and 0.02 (maternal) for WW, and 0.15 (direct) and 0.01 (maternal) for YW (Table 4)(14). A separate multivariate analysis calculated genetic correlations of 0.26 for BW-WW, 0.26 for BW-YW and 0.62 for WW-YW, which suggest the presence of pleiotropic effects. However, given the magnitude of the estimated values, selection for higher WW can be expected to result in a higher correlated response in YW, compared to direct selection for lower BW, which is favorable to breeders(15).

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Table 4: Heritability (on the diagonal within each population) and genetic correlation estimates Population SimmentalSimbrah

CharolaisCharbray

Red Brangus

Limousin

Gelbvieh

Santa Gertrudis

Indubrasil

BWd BWd BWm WWd WWm YWd YWm SC FS ST HF BWd BWm WWd WWm YWd YWm SC FS ST HF BWd BWm WWd WWm YWd YWm SC FS ST HF BWd BWm WWd WWm YWd BWd BWm WWd WWm YWd BWd BWm WWd WWm YWd BWd BWm WWd WWm YWd

BWm

0.17

WWd

WWm

0.26

YWd

YWm

0.36

0.47

0.35

0.59 0.42

-0.24

-0.65

0.47 0.07

0.26

0.01 0.14

0 0.02

0.62 0.15 0.01

0.36 0.13 0.27

-0,81 0.15 0.30

0.37

0.42

0.21

0.15 0.25

0.12

0.40

0.41

-0.03

0.39

0.51

0.68

0.88

0.50

0.88

0.81

0.18

0.97 0.25

0.76

-0.08

0.26

0.57 0.06

0.22 0.30

-0,15 0.06

0.30 -

0.06 0.13

0.36

0.58

0.15 0.21

-0.69 0.32

0.42 0.20

0.30 0.21 0.45 0.06 0.03 0.32

-0.63 0.07 0.41

0.27 0.10 0.11

-0.27 0.09 0.13

BWd= direct birth weight; BWm= maternal birth weight; WWd= direct weaning weight; WWm= maternal weaning weight; YWd= direct yearling weight; YWm= maternal yearling weight; SC= scrotal circumference; FS= frame score; ST= stayability; HF= heifer fertility.

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A subsequent multibreed genetic analysis of young Simmental and Simbrah bulls found estimated heritability values of 0.33 for YW, 0.35 for SC and 0.42 for FS, while those for the genetic correlations were 0.36 for YW-SC, 0.47 for YW-FS and 0.59 for SC-FS (Table 4). These results suggest the feasibility of employing YW, SC and FS in direct selection, although, direct selection to improve one of these three traits would cause a correlated response in the other two(16). Breeders would therefore have to consider the wisdom of producing animals with higher YW or SC since FS of replacement females might not match with the resources available in the production system. Multibreed genetic analysis of HF showed that, when comparing heritability in the logistic sire model with the linear models, this variable did not improve prediction of breeding values, and estimation of breeding values was unaffected by distribution of the adjusted variable. However, data quality and connectedness did impact prediction and animal ranking based on their breeding values(17). When considering all the criteria used to compare the models, it was found that, for two basic reasons, genetic evaluation of HF is best done with a linear animal model. The first reason is that the database was large enough (n= 37,390) since the number of animals with expected progeny differences was greater than in the sire models. Second, interpreting sire logistic model results is far more complicated. Heritability of HF was found to be low (0.07) in this study(17), however, the observed ranges of expected progeny differences (-5.79 to 8.72 for Simmental; -9.56 to 8.84 for Simbrah) indicate that genetic change in HF may occur in response to selection(18). Two other studies addressed the association between reproductive traits of females and males. In one, estimated genetic correlation between SC and HF (-0.65; Table 4) suggests that sire selection based on SC breeding values can lead to improvement in HF. Moreover, the genetic correlation between ST and HF (0.47) suggests that cows’ ST can be increased when they are selected as replacement heifers based on HF breeding values(19). In the other study, age at first calving (AFC), calving interval (CI) and cumulative WW at second calving (CWW) were found to significantly correlate: 0.42 for AFC-CI, 0.63 for AFC-CWW, and 0.97 for CI-CWW. Direct response to selection for CI and CWW was predicted to be greater than the expected correlated response with selection for AFC, independent of calf number per sire. Indirect selection was superior to direct selection by 42 % in the AFC-CI and by 63 % in the AFC-CWW(20). A more recent study analyzed WW as a different trait in three different regions of Mexico(21). Differences between regions were observed in the estimated values for direct heritability (0.10 to 0.54), maternal heritability (0.44 to 0.71), the correlation between direct effects (0.35 to 0.69) and the correlation between maternal effects (-0.76 to 0.16). These differences suggest the presence of genotype-environment interaction since a genetic correlation of less than 0.80 is reported to indicate the existence of this interaction(22). Consequently, genetic evaluations of the Simmental breed need to consider this factor. These results also highlight 9

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the importance of knowing a selected sire’s environmental conditions, and emphasize the need to prioritize superior genotypes in production systems similar to those of the country or region where a sire was bred. Another study compared additive dam age adjustment factors for BW and WW generated using AMSS data to those recommended by the U.S. Beef Improvement Federation (BIF) and those generated by the American Simmental Association (ASA). For unadjusted BW, the sum of squares (SOS) associated with dam’s age was 8,812 kg², but when applying the respective correction factors it was 4,595 kg² with the BIF factor, 1,151 kg² with the ASA and 184 kg² with the AMSS. For unadjusted WW, the SOS associated with dam’s age was 110.138 kg², but when the correction factors were applied it was 32.0733 kg² with the BIF, 241.840 kg² with the ASA and 11.245 kg² with the AMSS. The error mean square for unadjusted BW was 881 kg² and that for WW was 11,014 kg², although after applying the estimated correction factors they were 18 and 1,124 kg², respectively. These results show that use of the adjustment factors estimated for Simmental cattle under Mexican environmental conditions is recommended. Use of the adjustment factors for dam’s age recommended by the BIF and ASA could bias estimation of breeding values because they are based on the environmental conditions of the United States of America, which differ markedly from those of Mexico. The adjustment factors for Mexican Simmental cattle are: BW, daughters of cows 2, 3, 4, 5-11 and 12 yr of age= 1.95, 1.12, 0.41, 0.00 and 0.48 kg, respectively; BW, sons of cows 2, 3, 4, 5-11 and 12 yr of age= 2.30, 1.21, 0.36, 0.00 and 0.61 kg, respectively; WW, daughters of cows 2, 3, 4, 5-7 and 8-12 yr of age= 9.30, 5.86, 1.08, 0.00 and 3.04 kg, respectively; WW, sons of cows of 2, 3, 4-8 and 9-12 yr of age= 10.20, 5.55, 0.00 and 3.56 kg, respectively(23). In one study, expression of the HSP60 gene was evaluated in Simbrah cattle in four tropical locations in Mexico (Coahuayana, Compostela, Tamazula, Puerto Vallarta) at two times: in the early morning (AM), when environmental temperature was not high; and in the afternoon (PM), when temperature was high. Mean 2-ΔΔCt values differed between times at Compostela (3.12 in AM and 5.16 in PM) and Tamazula (1.94 in AM and 2.93 in PM), but not at Coahuayana (2.02 in AM and 1.91 in PM) or Puerto Vallarta (0.21 in AM and 0.47 in PM). The results highlight the possible identification of heat-tolerant animals in the Simbrah breed which could be incorporated into genetic improvement programs(24). Charolais-Charbray population. The Charolais-Charbray Herd Book de México currently publishes expected progeny differences for the same traits assessed by the AMSS, which are also estimated using a multibreed animal model. The first study done in the CharolaisCharbray population found that the most appropriate animal model for estimating genetic parameters for BW, WW and YW included the direct additive genetic effect, the maternal additive genetic effect, the covariance between the direct and maternal genetic effects, and the maternal permanent environmental effect. Compared to five other models, this model 10

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substantially reduced the -2[logarithm of the likelihood], providing the best fit for all three traits. Using the most appropriate model, the estimated values of direct heritability, maternal heritability and variance of the permanent maternal environment as a proportion of the phenotypic variance were 0.36, 0.13 and 0.042 for BW, respectively; 0.27, 0.15 and 0.060 for WW, respectively; and 0.30, 0.12 and 0.045 for YW, respectively (Table 4). In the analysis of each trait, and in comparison to the more complex model, the simplest animal model, composed only of the direct additive genetic effect, was found to underestimate direct additive genetic variance but overestimate residual variance(25). In a later study analyzing data from young Charolais and Charbray bulls, SC, FS and YW showed to be moderately heritable with values of 0.21, 0.25 y 0.30, respectively (Table 4)(26). Also, the genetic correlations of YW-SC (0.37) and YW-FS (0.42) were found to be moderately strong, which means that some of the genes that control YW also control SC and FS (pleiotropic effect). Young bulls that excel in terms of YW can therefore also be expected to excel in terms of SC and FS. In contrast, these latter two traits were only weakly correlated (0.15). Estimated direct response to selection based on five half-sib offspring per bull was 0.38 cm for SC, 0.18 units for FS and 8.30 kg for YW. If selection is focused on higher YW, the expected correlated response in the next generation is 0.16 cm for SC and 0.08 units for FS. It follows, therefore, that indirect selection of SC and FS based on YW would not be as effective as direct selection to improve SC and FS. Finally, the direct and correlated responses to selection based on 500 half-sib offspring per bull were almost two times higher than those based on only five offspring. In an effort to genetically improve female reproduction, a national genetic evaluation prototype was developed for HF in Charolais and Charbray, which is equivalent to that developed for HF in Simmental and Simbrah. Using this prototype, estimated HF heritability was 0.06 (Table 4). The range for the expected progeny differences was -7.94 to 8.22 for Charolais and -7.29 to 6.14 for Charbray, each indicating the feasibility of identifying bulls outstanding in HF. The genetic trends estimated with this prototype indicated that HF had exhibited a favorable genetic change in both breeds during the 2007-2011 period(18). One of the most recent studies of these two breeds evaluated the association between male and female reproductive traits. A strong genetic correlation was identified between SC and ST (0.76; Table 4), suggesting that selection of bulls based on expected progeny differences for SC could induce a favorable genetic change in ST. An important genetic relationship was also found between ST and HF (0.57), highlighting the possibility of increasing a cow’s probability of remaining in a herd if, at an early age, she is selected as a replacement heifer because she has a high expected progeny difference for HF. Estimated heritability values were 0.18 cm for SC, 0.26 % for ST and 0.11% for HF, while those for direct response to selection, assuming five half-sib offspring per bull, were 0.15 cm for SC, 0.04 percentage units for ST and 0.15 percentage units for HF. If bulls are selected based on their expected 11

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progeny differences for SC, and assuming the same number of offspring, a correlated response to selection of 0.20 percentage units in ST can be expected, but no correlated response is expected in HF. However, when the estimates of the direct response to selection are based on ten offspring, the values are higher for SC (42.3 %), HF (50.0 %) and ST (40.0 %)(27). Red Brangus population. As has been done with the Simmental, Simbrah, Charolais and Charbray breeds, a large number of traits have been incorporated into genetic evaluations of the Red Brangus breed, although ST is not among them. A recent multivariate analysis found a moderately heritable direct additive genetic effect for BW (40 %), WW (30 %), YW (30 %) and FS (25 %) (Table 4), but poor heritability for SC (18 %). The maternal additive genetic effect for BW was moderately heritable (22 %), but that of WW was lowly heritable (6 %). When analyzing the relationship between traits it was found that selection for higher WW can result in a considerable correlated response in BW, YW, SC and FS. This was due to the high values of the genetic correlation estimates of WW with BW (0.41), YW (0.68), SC (0.88) and FS (0.50). There were also strong correlations between YW and SC (0.88), YW and FS (0.81), and SC and FS (0.97) (Table 4)(28). A univariate analysis with a linear animal model showed that in Red Brangus HF heritability is low (0.06)(29), as reported for the Simmental, Simbrah, Charolais and Charbray populations. Limousin population. Genetic evaluations of the Limousin breed have included BW, WW, YW and SC. One study found that the most suitable animal model for BW and WW included the direct and maternal genetic effects plus their covariance, whereas for YW it only included the direct genetic effect. When the BW and WW models did not consider the maternal effects they overestimated genetic variance and heritability. The direct and maternal genetic effects of WW (0.21 and 0.32, respectively) were found to be more heritable than those of BW (0.13 and 0.15, respectively) (Table 4), while the direct genetic effects of WW (0.21) and YW (0.20) exhibited similar heritability (Table 4)(30). In another study, a trivariate analysis found BW, WW and YW to be moderately correlated: 0.36 (BW-WW), 0.58 (BW-YW) and 0.42 (WW-YW) (Table 4). When selecting any of these traits a correlated response can therefore be expected in the other two(31). Gelbvieh and Santa Gertrudis populations. Genetic evaluations implemented by the Asociación Mexicana de Criadores de Ganado Santa Gertrudis (Mexican Association of Santa Gertrudis Cattle Breeders) and the Asociación de Criadores de Ganado Gelbvieh de la República Mexicana (Gelbvieh Cattle Breeders Association of the Mexican Republic) have calculated expected progeny differences for BW, WW and YW. One study using BW, WW and YW data to compare models for genetic evaluation of the Gelbvieh breed considered six random effects in the models: direct genetic (D), maternal genetic (M), the covariance (C) between D and M, maternal permanent environment (P) and the residual. No significant differences between the models (i.e. D, DP, DM, DMP, DMC and DMCP) were identified 12

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for any of the variables. Inclusion of C in the model underestimated direct heritability for BW and overestimated it for WW. Considering this and the importance of maternal genetic effects on both traits, the DMP model appeared to be the most suitable model for genetic evaluation of BW and WW. However, for YW the D model was more suitable given the low importance of M and P for this trait. When using the respective most suitable model the estimated values for direct heritability were 0.30 for BW, 0.21 for WW and 0.45 for YW (Table 4)(32). In a later study using data only for the Santa Gertrudis breed, it was observed that the estimates of direct additive genetic variance, as a proportion of phenotypic variance, indicated little direct additive genetic variability for BW (direct heritability= 0.06), but clear variability estimated for WW (0.32) and YW (0.41). The estimated values for maternal heritability suggest that very little of the variability in BW and WW was due to maternal genetic effects(33). Indubrasil experimental population. A study done using data from an experimental Indubrasil herd at INIFAP kept in a humid tropical climate found that for BW and WW the direct genetic effect was more important than the maternal genetic effect. In addition, the maternal permanent environmental effect did not influence expression of WW. Estimated values of direct heritability were 0.27 for BW, 0.11 for WW and 0.13 for YW, while those of maternal heritability were 0.10 for BW and 0.09 for WW (Table 4)(34). A different study which analyzed the female reproductive traits of CI, age at first service and AFC found considerable genetic variation and moderate to high heritability values (0.13 for CI, 0.31 for age at first service and 0.39 for AFC). In contrast, gestation length, days open and services per conception exhibited little genetic variation and therefore low heritability (0.08 for gestation length, 0.03 for days open and 0.03 for number of services per conception)(35).

Estimation of genetic diversity in Bos taurus and Bos taurus x Bos indicus cattle using genealogical data A population’s genetic diversity can be studied by analyzing pedigree data. These genealogical data, together with statistics based on probabilities of gene origin, provide valuable information for the study of populations that have been under selection for several years(36). An analysis of registered Simmental cattle in Mexico included animals born between 1985 and 2014; 1985 is when the first Simmental animals born in Mexico were registered. The population has had a low inbreeding coefficient, varying from 0.68 % for animals born in 2014 to 1.65 % for those born in 1997. When considering five-year subpopulations beginning in 1985, the effective population size increased from 134.7 in 1985-1989 to 186.6 in 2010-2014. The effective numbers of founders, ancestors, and founder genomes increased from 1985 to 2004, but decreased from 2005 to 2014. For animals born 13

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in the periods 2005-2009 and 2010-2014, the ratio of the effective number of ancestors to the effective number of founders suggests loss of diversity due to bottlenecks in both periods. For the same animals, the ratio of the effective number of founder genomes relative to the effective number of ancestors suggests genetic drift in both periods. Finally, for animals born in 2010-2014 one ancestor explained 3.4% of total genetic variability and 15 ancestors explained 20 % of said variability; the marginal genetic contribution of each of these 15 ancestors was similar(37). An analysis of the genetic diversity of registered Simbrah cattle in Mexico used animals born between 1990 and 2014. The inbreeding coefficient was low, with a clear tendency to decrease over time; it varied from 0.14 % for animals born in 1990 to 0.03 % for those born in 2014. When analyzing five-year subpopulations beginning in 1990, effective population size decreased from 79.3 in 1990-1994 to 36.4 in 2010-2014. The effective numbers of founders, ancestors, and founder genomes increased from 1990 to 2009, but decreased dramatically from 2005 to 2014. For animals born in the period 2010-2014, the ratio of the effective number of ancestors to the effective number of founders suggests loss of diversity due to bottlenecks. For the same animals, the ratio of the effective number of founder genomes relative to the effective number of ancestors suggests genetic drift. For animals born from 2010-2014, one ancestor explained 0.21 % of total genetic variability and 10 ancestors explained 1.4 % of said variability(38). For registered Charolais cattle in Mexico, analysis of the evolution of its genetic diversity was done using data for animals born between 1984 and 2018. The inbreeding coefficient remained between 2.1 and 1.3 % throughout the 35-yr study period. Effective population size gradually increased from 105.0 in 1984 to 237.1 in 2013, then decreased slightly to 233.2 in 2018. An increase in the effective number of ancestors was observed from 1984 to 2008, followed by a decrease in the following decade. The effective number of founder genomes increased from 1984 (130.1) to 2003 (143.7), but decreased over the next 15 yr (127.7), resulting in allele loss from 2004 to 2018. The ratio of the effective number of ancestors to the effective number of founders suggests that the loss of genetic diversity in the period 1999 to 2018 was due, in part, to formation of pedigree bottlenecks. The ratio of the effective number of founder genomes to the effective number of ancestors suggests loss of founder alleles due to genetic drift(39).

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Genome-wide association studies for growth traits in Bos taurus cattle Analyses of association have been done using the whole genome (Gn) and by chromosome (Chr) in registered Simmental(40) and Simbrah(41) cattle to identify SNPs associated with growth traits. The genotypes and phenotypes of 967 animals (473 Simmental and 494 Simbrah) were used. The phenotypes used were the deregressed breeding values of BW, direct WW (DWW), maternal WW (MWW) and YW. Genotyping was done with highdensity panels. After quality control, 105,129 autosomal SNPs were obtained. In the Simmental animals, 22 SNPs were found to be associated with BW, 25 with DWW, 28 with MWW and 42 with YW. For BW, 18 SNPs were identified in the Gn and 8 in the Chr, while for DWW, 15 SNPs were isolated in the Gn and 15 in the Chr. For MWW, 21 SNPs were found in the Gn and 8 in the Chr, while for YW, 18 SNPs were found in the Gn and 34 in the Chr. Overlap between Gn and Chr analyses was 4 SNPs for BW, 5 for DWW, 1 for MWW and 10 for YW(40). In the Simbrah animals, a total of 50 SNPs were associated with BW, 29 with DWW, 18 with MWW, and 19 with YW. For BW, 24 SNPs were significant in the Gn analysis and 38 in the Chr analysis; for DWW, 25 SNPs were found in the Gn and 9 in the Chr analysis. For MWW there were 16 SNPs in the Gn and 5 in the Chr, while for YW there were 14 in the Gn and 12 in the Chr. Overlap between the Gn and Chr analyses was 12 SNPs for BW, 5 for DWW, 3 for MWW and 7 for YW(41).

Research challenges and perspective The objectives of modern genetic improvement have evolved towards development of better evaluation methods. These have made it possible to increase the accuracy of breeding value estimates, shorten generational interval and produce more efficient crossbred animals(42). For the past 20 yr, genetic evaluations have been done for different beef cattle breeds in Mexico, but genetic improvement still faces new challenges in terms of pace and direction. Little or no progress has been made for some traits because they are expensive to measure (residual feed intake, methane production, tolerance to heat stress), their genetic variation is low (fertility), and/or genetic correlation is unfavorable between some of these traits(43). Selection in beef cattle is currently done using a large number of traits for which genetic predictions can be made and which are available to farmers. In the short-term, selection needs to be done based on traits of interest by applying selection indices, such as maternal or terminal indices(44). Greater emphasis needs to be placed on carcass traits. Some breed associations in Mexico began measuring phenotypes for these traits in 2016 and predictions 15

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of genetic merit for these traits should be available shortly. Using the traits measured currently, estimates are needed of the genetic correlations between them and their correlated response to selection. The structure of phenotypic data (i.e. greater number of progeny per sire) can be improved in a way that allows an increase in or establishment of reference populations, which will support genomic selection over the medium-term(45). In this same time frame it is also important to consider new phenotypes (although they can be expensive and difficult to measure) that contribute to production efficiency; these can include methane production, tolerance to heat stress, residual feed intake, and health traits that improve animal welfare. Periodic genomic evaluations of these traits are then needed to track them. There is great interest in developing methods that allow comparison between individuals of different breeds because it provides potential commercial opportunities. Research is needed over the medium- and long-term on crossbreed genomic prediction. This will be much more challenging because different breeds can exhibit different QTLs, dominance or epistasis may occur and allele frequencies can vary between populations(5). In beef production, genetic improvement is focused on identifying individuals that can efficiently provide high quality protein while considering environmental equilibrium, greenhouse gas emissions and growing consumer consciousness of animal welfare and food safety. The beef production strategies currently under development in Mexico are inefficient. The genetic improvement tools now available for selection and crossbreeding need to be focused on identifying animals with greater productive and biological efficiency and a reduced environmental footprint, while guaranteeing the highest standards of animal welfare and food safety.

Conclusions By using increasingly larger databases for the various beef cattle breeds used in Mexico (Bt, Bi and Bt x Bi), genetic improvement practices and research have attained many milestones. For Bi and Bt x Bi animals under tropical conditions, characterization was done for productive (growth, milk production, longevity, lifetime productivity) and reproductive traits (age at first calving, pregnancy rate, calving rate, weaning rate, kilograms calf weaned per cow exposed). A multibreed genetic evaluation model has been developed for the registered Simmental-Simbrah and Charolais-Charbray populations. The importance of maternal effects (genetic and permanent environment) for growth traits has been quantified, which has allowed identification of the most suitable animal model for genetic evaluation of BW, WW and YW. The magnitude of heritability and genetic correlation was estimated for growth and reproductive traits (including AFC, CI and CWW) in males and females, which allows 16

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prediction of the direct and correlated responses to selection based on different numbers of offspring per bull in these traits. The genotype-environment interaction has been proven to be an important factor in WW expression in the Simmental breed, suggesting that it needs to be incorporated into national-level genetic evaluations. Dam age adjustment factors for BW and WW have been developed, thus avoiding use of adjustment factors generated in other countries, which can result in biased breeding value estimates. National-level genetic evaluation prototypes have been developed for ST and HF based on the environmental and management conditions of Mexico. Expression of the HSP60 gene in Simbrah cattle exposed to heat stress in the tropics of Mexico has been evaluated. Estimates of genetic diversity in the registered Simmental, Charolais and Simbrah populations have been obtained, and, finally, SNPs associated with growth traits have been identified in the Simmental and Simbrah breeds.

Impacts In the early 1980s, when most cattlemen in the Mexican tropics used Zebu cows in cow-calf systems, a project was begun (described herein) that proved the advantages of using Bt x Bi cows to produce calves. Almost forty years later, an estimated 50 % of beef cattle production units in Mexico use Bt x Bi cows. Once the first national genetic evaluation of beef cattle was done by INIFAP researchers in 2001 using AMSS data, other beef breed associations began their own national genetic evaluations. The INIFAP currently does genetic evaluations for breeders of Simmental-Simbrah-Simangus, Charolais-Charbray, Red Brangus, Santa Gertrudis, Braford and Italian cattle. These evaluations provide breeders the tools to genetically improve beef cattle in Mexico through the approximately 14,000 bulls sold annually to commercial and registered producers. Literature cited: 1. Cundiff LV, Gregory KE, Schwulst FJ, Koch RM. Effects of heterosis on maternal performance and milk production in Hereford, Angus and Shorthorn cattle. J Anim Sci 1974;38:728-745. 2. Koger M, Peacock FM, Kirk WG, Crockett JR. Heterosis effects on weaning performance of Brahman-Shorthorn calves. J Anim Sci 1975;40:826-822. 3. Gregory KE, Cundiff LV. Crossbreeding in beef cattle: Evaluation of systems. J Anim Sci 1980;51:1224-1242.

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4. SAGARPA. Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación. Informe sobre la situación de los recursos genéticos pecuarios (RGP). México. 2002. 5. Garrick DJ. The nature, scope and impact of genomic prediction in beef cattle in the United States. Genet Sel Evol 2011;43(17):1-11. 6. Vega MVE, Ríos UA, Montaño BM. Comportamiento productivo hasta el destete de vacas Cebú apareadas con sementales Bos taurus y Bos indicus. Téc Pecu Méx 1996;34:1219. 7. Ríos UA, Vega MVE, Montaño BM, Lagunes LJ, Rosete FJV. Comportamiento reproductivo de vacas Brahman, Indobrasil y cruzas F1 Angus, Charoláis, Hereford y Suizo Pardo x Cebú y peso al destete de sus crías. Téc Pecu Méx 1996;34:20-28. 8. Ríos UA, Vega MVE, Montaño BM. Causas de desecho y vida productiva de vacas Bos indicus y cruzas Fl Angus, Charolais, Hereford y Suizo Pardo x Cebú. Téc Pecu Méx 1998;36:203-211. 9. Vega-Murillo VE, Ríos-Utrera A, Montaño-Bermúdez M. Herd life and lifetime productivity of Brahman and F1 crossbred Angus, Hereford, Charolais and Brown Swiss x Zebu cows. 7th World Congr Genet Appl Livest Prod 2002; Commun. No. 02-63. 10. Vega-Murillo VE, Ríos-Utrera A, Montaño-Bermúdez M. Stayability of Brahman and F1 crossbred Angus, Hereford, Charolais and Brown Swiss x Zebu cows. 8th World Congr Genet Appl Livest Prod; 2006;8:32-34. 11. Quiroz-Valiente J, Vega-Murillo VE, Ríos-Utrera A, Montaño-Bermúdez M. Milk yield and composition of Brahman and F1 crossbred Angus, Hereford, Charolais and Brown Swiss x Zebu cows. 6th World Congr Genet Appl Livest Prod 1994;17:379-382. 12. Reynoso CO, Villarreal PM, Vazquez PCG. Análisis del crecimiento hasta el destete de animales Bos taurus x Bos indicus criados bajo condiciones tropicales de México. Téc Pecu Méx 1987;25:271-280. 13. Martínez VG. Efectos genéticos aditivos individuales de razas bovinas para características de crecimiento en el trópico [tesis maestría]. Texcoco, Estado de México: Universidad Autónoma Chapingo; 1989. 14. Vega-Murillo VE, Ríos-Utrera A, Montaño-Bermúdez M, Martínez-Velázquez G. Multiple-breed genetic evaluation of growth traits in Simmental and Simbrah cattle. Trop Subtrop Agroecosyst 2012;15(2):403-414.

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15. Vega MVE, Torres VA, Manzanilla PC, Baeza RJJ, Borrayo ZA, Martínez VG, Ríos UA, Montaño BM. Correlaciones genéticas entre peso al nacimiento, al destete y al año de edad en ganado Simmental-Simbrah [resumen]. Reunión Nacional de Investigación Pecuaria. Campeche, Camp. 2010:210. 16. Torres-Vázquez JA, Manzanilla-Pech CIV, Borrayo-Zepeda A, Ríos-Utrera A, VegaMurillo VE, Martínez-Velázquez G, Baeza-Rodríguez JJ, Montaño-Bermúdez M. Parámetros genéticos y fenotípicos para peso al año, circunferencia escrotal y talla en ganado Simmental y Simbrah en México. Rev Mex Cienc Pecu 2012;3(3):291-298. 17. Baeza-Rodríguez JJ, Montaño-Bermúdez M, Vega-Murillo VE, Arechavaleta-Velasco ME. Linear and logistic models for multiple-breed genetic analysis of heifer fertility in Mexican Simmental-Simbrah beef cattle. J Appl Anim Res 2018;46(1):534-540. 18. Baeza-Rodríguez JJ, Vega-Murillo VE, Ríos-Utrera A, Martínez-Velázquez G, Arechavaleta-Velasco ME, Montaño-Bermúdez M. Prototipo de evaluación genética nacional para fertilidad de vaquillas Simmental-Simbrah y Charolais-Charbray. Rev Mex Cienc Pecu 2017;8(3):249-258. 19. Martínez VG, Vega MVE, Román PSI, Ríos UA, Baeza RJJ, Arechavaleta VME, Montaño BM. Correlaciones genéticas entre circunferencia escrotal, fertilidad de vaquillas y permanencia productiva en la población Simmental-Simbrah de México [resumen]. Reunión Nacional de Investigación Pecuaria. Nuevo Vallarta, Nayarit. 2018:268-270. 20. Bejarano-Cabrera DY, Vega-Murillo VE, Montaño-Bermúdez M, Ríos-Utrera Á, Martínez-Velázquez G, Román-Ponce SI, Baeza-Rodríguez JJ, Arechavaleta-Velazco ME. Respuesta directa y correlacionada a la selección para características productivas y reproductivas en ganado Simmental y Simbrah en México [resumen]. Reunión Nacional de Investigación Pecuaria. Nuevo Vallarta, Nay. 2018:283-285. 21. Rosas-García ME, Vega-Murillo VE, Montaño-Bermúdez M, Pescador-Salas N, RománPonce SI, Ríos-Utrera A, Martínez-Velázquez G, Baeza-Rodríguez JJ. Interacción genotipo-ambiente sobre el peso al destete en ganado Simmental [resumen]. Reunión Nacional de Investigación Pecuaria. Tuxtla Gutiérrez, Chiapas. 2019:338-341. 22. Robertson A. The sampling variance of the genetic correlation coefficient. Biometrics 1959;15:469-485. 23. Torres VJA, Montaño BM, Ríos UA, Martínez VG, Vega MVE. Factores de ajuste de edad de la madre para pesos al nacer y al destete en bovinos Simmental [resumen]. Reunión Nacional de Investigación Pecuaria. Querétaro, Qro. 2012:171.

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24. Guzmán LF, Martínez-Velázquez G, Villaseñor-González F, Vega-Murillo V, RománPonce S, Montaño-Bermúdez M. Expresión del gen Hsp60 en ganado bovino raza Simbrah expuesto a estrés calórico en el trópico de México [resumen]. XII SIRGEAC Uruguay. Rocha, Uruguay. 2019:149. 25. Ríos-Utrera A, Martínez-Velázquez G, Vega-Murillo VE, Montaño-Bermúdez M. Genetic effects for growth traits of Mexican Charolais and Charbray cattle estimated with alternative models. Rev Mex Cienc Pecu 2012;3(3):275-290. 26. Ríos Utrera A, Montaño Bermúdez M, Vega Murillo VE, Martínez Velázquez G, Baeza Rodríguez JJ. Genetic parameters of scrotal circumference, frame score and yearling weight of Charolais and Charbray young bulls. Rev Colomb Cienc Pecu 2018;31(3):204-212. 27. Martínez Velázquez G, Ríos Utrera A, Román Ponce SI, Baeza Rodríguez JJ, Arechavaleta Velasco ME, Montaño Bermúdez M, Vega Murillo VE. Genetic correlations between scrotal circumference, heifer fertility and stayability in CharolaisCharbray cattle. Livest Sci 2020;232:103914. 28. Resendiz-Hernández CA, Vega-Murillo VE, Montaño-Bermúdez M, García-Mateos VX Calderón-Chagoya R, Román-Ponce SI, et al. Correlaciones genéticas de características de crecimiento, circunferencia escrotal y talla en bovinos Brangus Rojo en México [resumen]. Reunión Nacional de Investigación Pecuaria. Tuxtla Gutiérrez, Chiapas. 2019:312-315. 29. Baeza RJJ, Vega MVE, Montaño BM, Ríos UA, Román PSI, Martínez VG. Comparación de modelos para el análisis genético de la fertilidad de vaquillas Brangus Rojo [resumen]. Reunión Nacional de Investigación Pecuaria. Tuxtla Gutiérrez, Chiapas. 2019:361-363. 30. Ríos-Utrera A, Vega-Murillo VE, Martínez-Velázquez G, Montaño-Bermúdez M. Comparison of models for the estimation of variance components for growth traits of registered Limousin cattle. Trop Subtrop Agroecosyst 2011;14(2):667-674. 31. Martínez-Velázquez G, Vega-Murillo VE, Ríos-Utrera A, Montaño-Bermúdez M. Parámetros genéticos de análisis univariados y multivariados para peso al nacer, al destete y al año de edad en bovinos Limousin [resumen]. Reunión Nacional de Investigación Pecuaria. Mérida, Yuc. 2008:85. 32. Martínez VG, Vega MVE, Ríos UA, Montaño BM, Borrayo ZA, Baeza RJJ, Manzanilla PC, Torres VJA. Modelos para la evaluación genética de características de crecimiento en bovinos Gelbvieh mexicanos [resumen]. Reunión Nacional de Investigación Pecuaria. León, Gto. 2011:136. 20

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33. Vega MVE, Martínez VG, Montaño BM, Ríos UA. Componentes de varianza y parámetros genéticos para pesos al nacer, al destete y al año de edad de bovinos Santa Gertrudis [resumen]. Congreso de la Asociación de Médicos Veterinarios Zootecnistas Especialistas en Bovinos del Estado de Veracruz. Boca del Río, Ver. 2016:102-104. 34. Ríos-Utrera Á, Hernández-Hernández VD, Villagómez Amezcua-Manjarréz E, ZárateMartínez JP, Villagómez-Cortés A. Direct and maternal genetic effects for growth traits of Indubrazil cattle. Rev Científ FCV-LUZ 2013;23(5):440-447. 35. Ríos-Utrera Á, Hernández-Hernández VD, Villagómez Amezcua-Manjarréz E, ZárateMartínez JP. Heredabilidad de características reproductivas de vacas Indubrasil. Agron Mesoam 2013;24(2):293-300. 36. Boichard D, Maignel L, Verrier É. The value of using probabilities of gene origin to measure genetic variability in a population. Genet Sel Evol 1997;29:5-23. 37. Ríos-Utrera A, Vega-Murillo VE, Montaño-Bermúdez M, Martínez-Velázquez G, Román-Ponce SI. Genetic diversity assessment of the Mexican Simmental population through pedigree analysis. R Bras Zootec 2018;47:e20160088. 38. Vega MVE, Ríos UA, Montaño BM, Román PSI, Martínez VG. Estudio de la diversidad genética de la población Simbrah mexicana mediante análisis de pedigrí [resumen]. Reunión Nacional de Investigación Pecuaria. Toluca, Edo. de Méx. 2015:210-212. 39. Ríos-Utrera A, Montaño-Bermúdez M, Vega-Murillo VE, Martínez-Velázquez G, BaezaRodríguez JJ, Román-Ponce SI. Genetic diversity evolution in the Mexican Charolais cattle population. Anim Biosci 2021;34(7):1116-1122. 40. Calderón-Chagoya R, Román-Ponce SI, Vega-Murillo VE, García-Ruiz A, MontañoBermúdez M, Ríos-Utrera A, Martínez-Velázquez G. Estudio de asociación de genoma completo para características de crecimiento en ganado Simmental [resumen]. Reunión Nacional de Investigación Pecuaria. Tuxtla Gutiérrez, Chiapas. 2019:342-345. 41. Calderón-Chagoya R, Román-Ponce SI, Vega-Murillo VE, García-Ruiz A, MontañoBermúdez M, Ríos-Utrera A, Martínez-Velázquez G. Estudio de asociación del genoma completo para características de crecimiento en ganado Simbrah [resumen]. Reunión Nacional de Investigación Pecuaria. Tuxtla Gutiérrez, Chiapas. 2019:346-349. 42. Hayes BJ, Lewin HA, Goddard ME. The future of livestock breeding: genomic selection for efficiency, reduced emissions intensity, and adaptation. Trends Genet 2013;29(4):206-214.

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43. Ferreira Júnior RJ, Bonilha SFM, Monteiro FM, Cyrillo JNSG, Branco RH, Silva JAIV, Mercadante MEZ. Evidence of negative relationship between female fertility and feed efficiency in Nellore cattle. J Anim Sci 2018;96(10):4035-4044. 44. Golden BL, Garrick DJ, Benyshek LL. Milestone in beef cattle genetic evaluation. J Anim Sci 2009;87(E. Suppl.):E3-E10. 45. Montaldo HH, Casas E, Sterman Ferraz JB, Vega-Murillo VE, Roman-Ponce SI. Opportunities and challenges from the use of genomic selection for beef cattle breeding in Latin America. Anim Front 2012;2(1):23-29.

https://doi.org/10.22319/rmcp.v12s3.5884 Review

Criollo Coreño cattle in western Mexico: characterization, challenges and outlook

Guillermo Martínez-Velázquez a Ángel Ríos-Utrera b José Antonio Palacios-Fránquez a Vicente Eliezer Vega-Murillo c Moisés Montaño-Bermúdez d*

Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias (INIFAP). Sitio Experimental “El Verdineño”, km 7.5 carretera Navarrete-Sauta, Municipio de Santiago Ixcuintla Nayarit, México. b

INIFAP, Campo Experimental La Posta, Veracruz, México.

Universidad Veracruzana. Facultad de Medicina Veterinaria y Zootecnia, Veracruz, México. d

INIFAP. Centro Nacional de Investigación Disciplinaria en Fisiología y Mejoramiento Animal, Querétaro, México.

Corresponding author: montano.moises@inifap.gob.mx

Abstract: The National Institute of Forestry, Agricultural and Livestock Research (INIFAP) in Mexico has been studying the Criollo Coreño (C) cattle of the Sierra Madre Occidental for over 20 yr. This review covers productive, genetic and molecular characterization, as well as short-, medium- long-term research challenges and outlooks. Evaluations have been done of 35 growth, carcass quality, fertility and milk production traits in C, Guzerat (G), CG and GC cattle generated through diallelic crossing. Individual heterosis was found to affect 23

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reproduction and milk production, while maternal heterosis influenced kilograms of calf weaned per cow exposed. Direct and maternal genetic effects had no relevant influence on the analyzed variables. In other studies, C bulls fed high energy diets were found to produce meat with favorable fatty acid profiles and good quality carcasses, while low doses of FSH can be used for superovulation in C heifers, without affecting embryo production. Overall, the results indicate that GC cows are the best option for calf production, and that the C population maintains significant levels of genetic diversity. Further genetic diversity research is needed on Mexican Criollo cattle populations using molecular genetics; Criollo herds must be included in the systematic recording of phenotypes of productivity and adaptability to be able to identify genes of interest unique to Criollo cattle. Key words: Beef cattle, Productive characterization, Criollo Coreño, Diallelic cross, Genetic diversity, Heterosis.

Received: 27/11/2020 Accepted: 09/03/2021

Introduction There are several populations of Criollo cattle in Mexico, descendants of Bos taurus cattle introduced by the Spanish during the Colonial Period. Among them is the Criollo Coreño in the Sierra Madre Occidental. Isolated for over 500 yr in different regions of the country, these populations have been subject to natural selection under adverse conditions, such as low feed availability and lack of health care(1). They have apparently developed the genetic capacity to adapt to difficult conditions and can contribute to the sustainability of cow-calf systems since they do not require radical changes in their rangelands. These populations’ hardiness originates in natural selection and is reflected in traits that allow them to overcome random and adverse environmental variations without significant reductions in productive capacity. The genetic diversity of Mexican Criollo cattle is an important genetic resource, which can contribute to design of livestock production systems with low input requirements. Crosses between imported cattle breeds and Criollo populations in Mexico have led to varying degrees of decline or genetic erosion in the latter, although each Criollo population is in a different risk situation. Successful conservation of Criollo cattle populations depends on the knowledge available about them. The main goal of conservation should be to preserve as much of a breed’s genetic

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diversity as possible. Three types of data are needed to define a strategy for conservation of Criollo cattle populations. First, effective population size depends on the number of males and females used as reproducers in each generation and helps to understand levels of consanguinity and possible loss of genetic diversity. Second, a population’s genetic diversity is quantified based on a breed’s history and/or molecular genetic data. It can be used to quantify the potential for future evolution and identify genes associated with present or future traits of interest. Third, phenotypic behavior in production and adaptation traits can be used to estimate a population’s genetic variability(2). The Criollo Coreño (C) is found in the Sierra Madre Occidental in indigenous communities in the states of Durango, Jalisco, Nayarit and Zacatecas, in western Mexico. Traditional use of C cattle is for weaned calf production in agroecological regions with limited year round feed availability and difficult environmental conditions. These challenging conditions manifest as declines in productive efficiency. There are approximately 16,000 head of C cattle in this region, which falls within the area of influence of the El Verdineño Experimental Station of the National Institute of Forestry, Agricultural and Livestock Research (Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias - INIFAP)(1,3). El Verdineño is located in the municipality of Santiago Ixcuintla, Nayarit, Mexico (21º42’ N; 105º07’ W). Regional climate is tropical warm subhumid (Aw2), altitude is 60 m asl, average annual rainfall is 1,200 mm and average annual temperature is 24 ºC. Vegetation is semi-evergreen tropical forest in secondary succession, and introduced grasses (Andropogon, Megathyrsus and Cynodon). A herd of C cattle is maintained in El Verdineño for experimental purposes. It was founded in 1982 through acquisition of 50 cows and 10 bulls from rural communities in the Sierra Madre Occidental; no data is available on the origins of these animals. By 1990, the herd included 70 C cows which had been born on site, with known genealogical and production information, as well as management conditions. Diallelic crosses between C and Guzerat (G) animals were begun to evaluate productive capacity. Estimates were made of the direct and maternal genetic effects of the C (Bos taurus) and G (Bos indicus) breeds, and the effects of individual and maternal heterosis, on economically important traits(4). The first stage of diallelic crossing (1990-1994) involved mating 14 C bulls and 12 G bulls with 70 C cows and 70 G cows to produce pure C and G animals and reciprocal F1 crosses (CG and GC). Beginning in 1993, the second stage of diallelic crossing involved mating of the females of the four breed groups (C, G, CG and GC) produced in the first stage with Red Angus bulls. In both stages, the calves remained with the cows until weaning, which occurred at 7 mo of age on average.

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This review summarizes INIFAP research on the productive, genetic and molecular characterization of C cattle from the Sierra Madre Occidental in Mexico, through analysis of the breed groups of the cows produced during diallelic crossing. Research challenges and short-, medium-, and long-term outlooks for Mexico’s Criollo cattle populations are addressed.

Productive characterization of Criollo Coreño cattle Research done at the INIFAP has shown that C cattle can contribute to improving growth calf production in the warm subhumid tropical region of western Mexico(5,6,7). Offspring of C bulls with G or Red Angus cows which are grown and finished in feedlot in the tropical region of Nayarit produce good quality carcasses with good yields(8,9). The following section reviews the productive, genetic and molecular characterization of C cattle from 1990-2019.

Breed, additive and non-additive genetic effects

In a study evaluating reproductive characteristics, C, CG and CG cows exhibited higher (P<0.10) rates of estrus (ER) and gestation (GR) than G females. For ER, the G cows were 20 % lower than C, 26 % lower than CG and 33 % lower than GC, while for GR they were 14 % lower than C, 18 % lower than CG and 26 % lower than GC (Table 1). The C, CG and GC cows were reproductively more efficient than the G females. No differences in calving rate (CR) were observed between G and CG cows (Table 1), possibly due to higher embryo or fetus loss in CG cows. This suggests that G cows provide a less favorable uterine environment than do C cows. The CR for GC cows suggests they also have a relatively unfavorable uterine environment for prenatal growth than do GC cows. Compared to G cows, weaning rate (WR) was also higher (P<0.10) in C (14%), CG (16 %) and GC (25 %) cows (Table 1). This could be due to lower maternal ability in the G cows during the birth-weaning period, lower calf survival during this period, or a combination of both. Estimated individual heterosis (hi) had a favorable influence (P<0.10) on all reproductive rates, which was manifested in a higher WR (P<0.10) in the GC and CG cows than in the C and G cows. In contrast, no differences in direct genetic effects (gi) were observed between C and G cows for any of the reproductive variables (Table 2). Differences between the four breeds of cows were minimal for maternal genetic effects (gm) for ER, GR and WR, but clearly favored C cows (15 %, P<0.10) for CR (Table 2)(5).

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Table 1: Least squared means for reproductive traits, pre-weaning growth, milk production and quality, growth in feedlot and carcass traits for Guzerat (G), Guzerat x Criollo Coreño (GC), Criollo Coreño x Guzerat (CG) and Criollo Coreño (C) cattle from sources published between 2006 and 2012 Breed Group GC CG C 1 Estrus rate, % 49a 82b 75b 69b 1 a b b Gestation rate, % 46 72 64 60b 1 a b ac Calving rate, % 42 71 56 59bc 1 a b b Weaning rate, % 38 63 54 52b 2 a ab a Birth weight, kg 31.72 30.44 31.11 28.86b 2 a a a Weaning weight, kg 177.42 187.45 183.34 153.50b 2 a a a Weight of calf at birth per cow exposed, kg 17.69 19.73 18.21 16.38a 2 Weight weaned per cow exposed, kg 98.44ac 143.50b 116.15ab 83.97c 2 Gestation length, days 283.25ab 283.87ab 280.98a 284.10b 3 Total milk production, kg 949a 1,059b 990ab 805c 3 a b ab Daily milk production, kg 4.50 5.00 4.70 3.90c 3 a b ab Peak production, kg 6.50 7.30 6.70 5.80c 3 a a a Day peak production, days 76 74 77 70a 3 a a a Lactation persistance, days 147 147 149 143a 4 Milk fat, % 2.80 2.98 3.09 2.81 4 Milk fat, kg 25.90 29.50 28.80 22.30 4 Milk protein, % 3.56a 3.83b 3.72b 3.88b 4 Milk protein, kg 33.90 38.20 37.20 31.60 4 Lactose, % 4.79 4.82 4.75 4.71 4 Lactose, kg 47.10a 52.10a 48.60a 39.50b 4 Milk non-fat solids, % 9.09 9.36 9.19 9.29 4 Milk non-fat solids, kg 88.20a 98.90a 93.20a 76.90b 5 Initial weight in the feedlot, kg 226.00a 206.70ab 208.90b 164.60c 5 Yearling weight, kg 359.50a 338.50ab 329.50b 279.40c 5 Final weight in the feedlot, kg 405.60a 388.60ab 377.10b 335.20c 5 a a Daily weight gain in the feedlot, kg 1.15 1.13 1.07a 1.09a 5 a b a Feed efficiency in the feedlot, kg 0.105 0.114 0.103 0.109ab 5 a a b Rib-eye area, inches 10.73 11.83 9.66 8.40c 5 a a a Fat thickness, inches 0.37 0.48 0.38 0.40a 5 a a b Hot carcass weight, kg 241.70 249.60 210.60 187.50b 5 a ab b Dressing percentage, % 60.20 58.60 58.20 54.20c 5 Kidney and pelvis fat, kg 8.06a 2.91b 5.34b 7.95a 5 Yield grade, units 2.61a 2.92ab 2.62a 3.21b 5 Cutability, % 50.79a 50.06ab 50.80a 49.48b 5 a ab a Retail yield, % 76.58 75.12 76.61 73.97b a,b,c 1,2 3,4,5 Different letter superscripts in the same row indicate difference: (P<0.10); (P<0.05). Sources: 1Martinez et al.(5); 2Martinez et al.(6); 3Martinez et al.(11); 4Martinez et al.(12); 5Martinez et al.(8). Variables

Reproductive performance in cows and pre-weaning growth in calves

Aspects of superovulation in C cows were evaluated in a study using reduced doses of FSH applied in three treatments (T1= 280 mg, T2= 200 mg and T3= 140 mg) to C cows (Exp 1) and C heifers (Exp 2). Treatment 1 produced higher (P<0.05) values for recovered corpuscles

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(RC) and unfertilized ovules (UFO). However, no differences were observed between treatments for transferable embryos (TE), non-transferable embryos (NTE), corpora lutea (CL), ovary volume (OV), serum progesterone concentration (P4), fertilization percentage (F%) and recovery percentage (R%). Considering the average values for NTE, CL, UFO, OV and R%, T1 was deemed the best treatment for promoting superovulation in C cows. As FSH dose decreased so did RC, most notably in T1 (10.3 RC) which differed (P<0.05) from T3 (4.1 RC). The number of UFO was also higher (P<0.05) in T1 than in T3, suggesting that T3 promoted a lower superovulatory response in the cows than T1. However, T1 did not result in higher embryo production, due to the high UFO in this treatment. In contrast to the C cows, no differences were detected between treatments for any of the variables in the C heifers. This highlights the feasibility of using reduced FSH doses in heifers without affecting their response to superovulation. A positive correlation (P<0.05) between CL and P4 was observed in all the C females, reflecting the fact that females with the highest CL also had the highest P4. Another positive correlation (P<0.05) was also detected between OV and P4, again indicating that those with the highest OV had the highest P4. Considering the results of the three treatments in both experiments, it was observed that the cows responded better to superovulation by producing higher CL (10.4, 9.1 and 8.8 for T1, T2 and T3, respectively) than the heifers (5.8, 5.5 and 4.5 for T1, T2 and T3, respectively); in contrast, cows had lower F% (44.6, 52.5 and 36.9 % for T1, T2 and T3, respectively) than heifers (96.5, 95.7 and 97.3 % for T1, T2 and T3, respectively). Overall, the results of the three treatments in both experiments showed that the cows had higher OV, CL and P4 than the heifers. Ovulatory response in terms of RC was also lower in the heifers (3.8 in T1, 2.3 in T2 and 2.0 in T3) than in the cows (10.3 in T1, 7.2 in T2 and 4.0 in T3). However, no differences were present for TE (2.3 in T1, 1.8 in T2 and 1.7 in T3) and NTE (1.3 in T1, 0.3 in T2 and 0.2 in T3) in heifers, and TE (1.2 in T1, 2.9 in T2 and 1.1 in T3) and NTE (2.0 in T1, 1.8 in T2 and 0.8 in T3) in cows. Average TE and NTE values in the cows and heifers indicated that both had similar embryo production levels, probably due to the higher fertility of heifers. No differences between treatments were detected for TE in either experiment. The results show that low FSH doses can be used to induce superovulation in C heifers, thus reducing the costs of superovulation and embryo production. The C cows responded better to superovulation than the C heifers, although the heifers had a higher F%(10).

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Table 2: Estimators of direct (gi), maternal (gm) and grandam (gn) genetic effects, and individual (hi) and maternal (hm) heterosis for reproductive traits, pre-weaning growth, milk production and quality, growth in feedlot, and carcass traits estimated from a diallel generated from Criollo Coreño (Bos taurus) and Guzerat (Bos indicus) cattle, and published between 2006 and 2012 Variables

hi 20.00β 15.00β 13.00β 13.00β

Estrus rate, % 1 Gestation rate, % 1 Calving rate, % 1 Weaning rate, % 2,4 Birth weight, kg 2,4 Weaning weight, kg 2 Weight of calves at birth per cow exposed, kg 2 Weight weaned per cow exposed, kg 2 Gestation lenght, d 3 Total milk production, kg 3 Daily milk production, kg 3 Peak production, kg 3 Day of peak production, d 3 Lactation persistance, d 4 Milk fat, % 4 Milk fat, kg 4 Milk protein, % 4 Milk protein, kg 4 Lactose, % 4 Lactose, kg 4 Milk non-fat solids, % 4 Milk non-fat solids, kg 5 Initial weight in the feedot, kg 5 Yearling weight, kg 5 Final weight in the feedot, kg 5 Daily weight gain in the feedot, kg 5 Feed efficiency in the feedot, kg 5 Rib-eye area, inches 5 Fat thickness, inches 5 Hot carcass weight, kg

Genetic effect hm gi gm -14.00 -6.00 -6.00 -7.00 -2.00 -15.0β -5.00 -9.00 0.48 3.80* 0.74 8.45 19.93* 37.60* 1.93 38.62*

-1.25 147.00* 0.60* 0.80* 2.50 3.00 0.23 5.07* 0.05 4.97* 0.03 7.04* 0.09 13.48*

-0.12 4.34 -0.21* 3.38 0.15 11.08* -0.03 16.98*

-0.11 0.68 0.11 1.06 0.07 3.48 0.17 6.64

12.51 14.60 12.40 -0.02 0.02 1.18β 0.04 15.5

Dressing percentage, % Kidney and pelvis fat, kg 5 Yield grade, units 5 Cutability, % 5 Retail yield, %

1.16 -3.88β -0.14 0.30 0.59

Direct genetic effect = Guzerat-Criollo. Maternal genetic effect = Criollo-Guzerat. Bold type indicates significant genetic effects: *(P<0.05); β(P<0.10). 1 Sources: Martínez et al(5); 2Martínez et al(6); 3Martinez et al(11); 4Martínez et al(12); 5Martínez et al(8).

2.26 -9.05 -11.47 -0.06 -0.01β -2.20β 39.02β

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In a study of pre-weaning productive traits, birth weight (BW) in the calves of G and CG cows was 2.9 kg heavier (P<0.05) than those of C cows, while BW for the calves of GC cows was intermediate (Table 1). Compared to the calves of C cows, weaning weight (WW) was heavier (P<0.05) in those of G (24 kg), CG (30 kg) and GC (34 kg) cows. In contrast, no differences (P>0.05) were observed in weight of calf at birth per cow exposed (CWB) between these breed groups. The GC cows produced more (P<0.05) weight weaned per cow exposed (CWW) than the G and C cows, while the CG cows only produced more (P<0.05) than the C cows. Gestation in the C cows was 3.1 d longer (P<0.05) than in the CG cows, while the CG and G cows had intermediate gestation length (GL) (Table 1). Individual heterosis (hi) had no effect (P>0.05) on GL, and maternal heterosis (hm) had no effect (P>0.05) on BW or CWB. However, hm did affect (P<0.05) WW and CWW (Table 2). The effect of hm on WW was reflected in an increase of 19.93 kg (P<0.05). For CWW, the hm estimator indicates that, on average, GC and CG cows produced 38.6 kilograms more calf at weaning (P<0.05) than G and C cows (Table 2). More specifically, GC cows produced between 45 and 60 kg more CWW than G or C cows (P<0.05), highlighting the advantage of using GC cows to increase productivity at weaning in G or C herds in calf-cow systems(6).

Milk production and quality

Another study compared milk production and WW in C, G, CG and GC cows. Compared to C and G cows, the GC cows had higher (P<0.10) total milk production (TMP; 1,059 vs 805 and 949 kg, respectively), daily milk production (DMP; 5.0 vs. 3.9 and 4.5 kg, respectively) and peak production (PEAK; 7.3 vs. 5.8 and 6.5 kg, respectively); the CG cows had intermediate values. Guzarat (G) cows had higher (P<0.10) values for TMP (144 kg) and DMP (0.6 kg) than C cows. No differences were observed between the genetic groups for day of peak production (DPEAK) and lactation persistence (LP) (Table 1). Individual heterosis (hi) influenced (P<0.05) TMP, DMP and PEAK, but not DPEAK or LP. The gi estimator identified higher values (P<0.05) for G cows than C cows in TMP, DMP and PEAK. In contrast, gm did not identify any differences between genetic groups for any of the milk production variables (Table 2). The TMP-WW correlation was significant (P<0.05) with all the estimators and for all genetic groups; however, the correlation was stronger for GC (r= 0.49) than CG (r= 0.25). Based on the estimator values, the authors observe that use of GC or CG cows for weaner calf production should incorporate different strategies for feeding management during lactation for each genetic group. The estimator of the overall TMP-WW correlation for all four genetic groups was 0.44 (P<0.05). The highest regression coefficients of WW over TMP corresponded to C and GC (P<0.05), which suggests that the offspring of C mothers or grandams had a lower nutritional dependence on mother’s milk and were largely dependent on lower nutritional value food sources. When considering the

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kilograms of milk per lactation required by each genetic group to produce 1 kg WW, it appears that the calves of C and GC cows made more efficient use of available milk (average= 22.5 and 27.3 kg milk/kg WW, respectively) than did the calves of G and CG cows (average= 30.9 and 37.3 kg, respectively). The 10 kg gap between the calves of GC and CG cows in this parameter is important to consider when developing recommendations for the use of these genetic groups in cow-calf systems(11). As a complement to the above study of milk production, an analysis was done of milk composition and its relationship to WW. When compared between the four genetic groups, no differences were observed in the percentages of milk fat (MF), lactose (ML) and non-fat solids (NFS) contents, nor in the weight of milk fat (MFK) and protein (MPK). In contrast, milk from C, GC and CG cows had a higher (P<0.05) protein content (MP) than that of G cows. Milk from G, GC and CG cows contained more (P<0.05) kilograms of lactose (MLK) and non-fat solids (NFSK) than that of C cows (Table 1). Guzaret (G) cows gave birth to heavier calves (P<0.05) than C, GC and CG cows, while G, GC and CG cows weaned heavier calves (P<0.05) than did C cows (Table 1). The fact that calves from crossbred cows weighed less at birth than those from G cows, and that G, GC and CG cows produced calves with similar WW was attributed to better productive performance during the birth-weaning period of crossbred cows and their calves. Of the non-additive genetic effects, hi did not influence milk component percentages, but did influence (P<0.05) milk components in terms of weight. Maternal heterosis (hm) was found to influence (P<0.05) expression of BW and WW. Its effects were significant (P<0.05) and favorable when BW decreased (hm= -1.3 kg) and WW increased (hm= 12.7 kg) (Table 2). In terms of additive genetic effects, no differences between gm were significant for any milk composition variable. Differences (P<0.05) between the gi values for ST were favorable to C by 0.20 percentage points. For the variables of milk components by weight, the differences (P<0.05) between the gi values favored G for lactose (11.1 kg) and non-fat solids (17.0 kg); G was also favored by the differences (P<0.05) between gi values for BW (3.8 kg) and WW (37.6 kg) (Table 2). The correlations between WW and milk component percentages were not significant, except for ST (r= -0.18; P<0.05), which suggests that milk quality had little influence on WW. However, for milk components in kilograms, correlations (P<0.05) were observed of WW with MFK (r= 0.16), MLK (r= 0.21) and NFSK (r= 0.19), with no correlation of WW with MPK (r= 0.13)(12).

Feedlot performance and carcass characteristics

In a study evaluating feedlot performance and carcass characteristics, initial weight, yearling weight and final weight were lower (P<0.10) in C cows than in G, GC and CG cows; indeed, the calves of G cows were up to 80 kg heavier. However, daily weight gain and feed

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efficiency (FE) of C cow progeny did not differ (P>0.05) from those of G, GC and CG cow progeny (Table 1). The progeny of G and GC cows had a larger (P<0.10) rib-eye area (REA) and hot carcass weight (HCW) than CG and C cow progeny. The progeny of C cows also had the lowest dressing percentage (P<0.10) of the four genetic groups, but fat thickness did not differ among them (Table 1). When compared to the calves of C cows, the calves of G and CG cows had higher (P<0.10) values for yield grade, cutability and retail yield, while the calves of GC cows had intermediate values. Kidney and pelvis fat (KPF) did not differ between the calves of G and C cows (Table 1). Daughters of C cows produced calves with higher (P<0.10) FE (0.01 kg) than daughters of G cows. In a similar way, the grandchildren of C grandmas had higher (P<0.10) REA (2.2 inches2) and HCW (39.0 kg) than the grandchildren of G grandmas. Overall, most differences tended to favor the grandchildren of C grandmas, although the only significant differences (P<0.10) were for FE, REA and HCW. Finally, the hm estimator was not significant (P>0.10) for any of the feedlot traits, but it was (P<0.10) for REA (1.18 inches2) and KPF (-3.88 kg) (Table 2)(8). Another study evaluated the fatty acid profile of meat from young C bulls fed different dietary energy levels (EL) (EL1 = 2.2; EL2 = 2.4; EL3 = 2.2 + 2.4; EL4 = 2.4 + 2.6 Mcal ME/kg DM). The EL4 diet had the highest (P<0.05) polyunsaturated fatty acids (PUFA) content and the lowest (P<0.05) saturated fatty acids (SFA) content, but a higher (P<0.05) content of unidentified fatty acids (Table 3)(13). Palmitic acid C16.0 and stearic acid C18.0 contents declined (P<0.05) as dietary energy level increased. Monounsaturated fatty acid (MUFA) content increased (P<0.05) beginning in EL2, although palmitoleic acid C16:1cis-9 decreased and oleic acid C18:1cis-9 content did not vary (Table 3). Linoleic acid C18:2cis9, 12 (C18:2ω-6) and linolenic acid C18:3cis-9, 12, 15 (C18: 3ω-3) contents also varied (P< 0.05) with dietary energy level, but inconsistently. The PUFA/SFA, (MUFA+PUFA)/SFA and C18:2ω-6/C18:3ω-3 ratios improved (P<0.05) as diet energy level increased (Table 3). Meat fatty acid profile is important in human health since the omega-6:omega-3 ratio can reach up to 17:1 in some Western diets(14).

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Table 3: Fatty acids profile (fatty acids g/100 g fat) in the meat of young Criollo Coreño bulls fed different energy levels Variable EL1 EL2 EL3 EL4 a c a Palmitic acid C16.0 27.0 24.2 26.6 20.5b a a a Palmitoleic acid C16:1cis-9 3.2 3.3 3.4 2.7b Stearic acid C18.0 20.7a 17.6b 16.9b 17.0b a a a Oleic acid C18:1cis-9 37.0 38.9 39.5 37.5a Linoleic acid C18:2 cis-9, 12 3.0a 4.0bc 3.6ac 3.8ac a a a Linolenic acid C18:3 cis-9, 12, 15 1.07 0.71 0.68 1.90b Saturated fatty acids 45.0a 42.8a 45.0a 35.0b ac bc b Monounsaturated fatty acids 39.7 42.4 42.6 41.2bc Polyunsaturated fatty acids 4.1b 4.6b 4.6b 5.5a a b b PUFA/SFA 0.08 0.12 0.11 0.15c b b b Unidentified fatty acids 8.7 6.6 6.4 17.6a (PUFA+MUFA)/SFA 0.98a 1.13b 1.09b 1.33c b b b Linoleic/Linolenic 0.37 0.26 0.10 0.82a EL1= 2.2 Mcal ME/kg DM throughout assay; EL2= 2.4 Mcal ME/kg DM throughout assay; EL3= 2.2 Mcal ME/kg DM start to 300 kg body weight, then 2.4 Mcal ME/kg DM to end of assay; EL4= 2.4 Mcal ME/kg DM start to 300 kg body weight, then 2.6 Mcal ME/kg DM to end of assay. PUFA= Polyunsaturated fatty acids; MUFA= monunsaturated fatty acids; SFA= saturated fatty acids. a,b,c Different letter superscripts in the same row indicate significant difference (P<0.05). Source: Bustamante et al(13).

A study evaluating carcass characteristics in young bulls fed the same diets as in the above study(9), found that dressing percentage was higher (P<0.05) in EL4 (53.1 %) than in EL2 (51.2 %), but that EL1 (52.0 %) and EL3 (51.9 %) did not differ (P>0.05) from either of these two treatments. For the variables of REA, kidney, pelvic and heart fat, and lean cut yield, EL4 (12.3 in2, 3.05 % and 50.1 %, respectively) was higher (P<0.05) than EL1 (11.2 in2, 3.95 % and 49.2 %, respectively), but did not differ (P>0.05) from EL2 (11.6 in2, 3.37 % and 48.1 %, respectively) and EL3 (11.4 in2, 3.45 % and 48.9 %, respectively) (Table 4). In terms of fat thickness, EL3 had higher (P<0.05) values (0.31 in) than EL4 (0.22 in), although EL1 and 2 did not differ (P>0.05) from either. In contrast, yield grade was clearly higher (P<0.05) in EL2 (2.4 units) than in EL4 (1.6 units), but these did not differ (P>0.05) from EL1 and 3 (Table 4). In EL4 primary cuts (53.1 %) and total cuts (81.2 %) yields were higher than in EL2 (51.2 % and 77.4 %, respectively), but these did not differ (P>0.05) from EL1 and 3 (Table 4). In summary, the study showed that young C bulls in the feedlot produced carcasses with remarkable yield and quality characteristics in response to the highenergy diets commonly used in commercial finishing of beef cattle.

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Table 4: Carcass characteristics in young Criollo Coreño bulls fed different energy levels Characteristics EL 1 EL 2 EL 3 EL 4 ab b ab Dressing percentage, % 52.0 51.2 51.9 53.1a Rib-eye area, inches2 11.2b 11.6ab 11.4ab 12.3a ab ab a Fat thickness, inches 0.26 0.25 0.31 0.22b Kidney, pelvic and heart fat, % 3.95b 3.37ab 3.45ab 3.05a ab a ab Yield grade, units 2.0 2.4 2.1 1.6b Primary cuts, % 52.0ab 51.2a 51.9ab 53.1b ab b ab Total cuts, % 79.4 77.4 78.7 81.2a Lean cuts, % 49.2b 48.1ab 48.9ab 50.1a EL1= 2.2 Mcal ME/kg DM throughout assay; EL2= 2.4 Mcal ME/kg DM throughout assay; EL3= 2.2 Mcal ME/kg DM start to 300 kg body weight, then 2.4 Mcal ME/kg DM to end of assay; EL4= 2.4 Mcal ME/kg DM start to 300 kg body weight, then 2.6 Mcal ME/kg DM to end of assay. a,b,c Different letter superscripts in the same row indicate significant difference (P<0.05). Source: Bustamante et al(9).

Molecular characterization A study using nine microsatellites to evaluate autosomal diversity among seven cattle genetic groups (Criollos from the states of Chihuahua, Durango, Nayarit [Criollo Coreño] and Guerrero; Lidia; Central American Dairy Criollo; and Guzerat) identified considerable genetic differentiation among the Criollo cattle populations. No evidence of Bos indicus influence was found in the Criollo cattle of Nayarit, Chihuahua and Durango, but it was present in those from Guerrero(15). High-density platform SNP markers were employed in an analysis of genetic diversity among C cattle from three locations in the state of Nayarit, Mexico: El Nayar (N), La Yesca (Y) and Santiago Ixcuintla (S). These populations were found to maintain moderate levels of observed average heterozygosity (from 0.29 to 0.34), and their estimated molecular coancestry values indicate that the N population differed from the S and Y populations, which can be considered portions of the same population(16). Copy number variation (CNV) analysis identified 2,170 CNV in 40 animals, located in 733 regions, with a coverage of 32.1 Mb of the autosomal genome. The functional analysis associated these CNVs with 131 genes mainly involved in inflammation and immune response, as well as 923 overlapping QTLs, classified into six different QTL-term categories: reproductive, productive, conformation, milk production, carcass, meat, and health. The group and principal component analyses showed that the animals were grouped by location of origin, although, the fact that they shared 75 of the 302 CNVs identified in more than two animals indicates they have a common genetic origin. The Y population was found to share 34 CNV with the N population and 30 CNV with the S. Between them, the N and S populations shared 56 CNV, suggesting that they are

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genetically closer, perhaps because the S population originated in part from animals brought from N 25 years ago. Total CNVs per population were 36 for N, 37 for Y and 34 for S, values which suggest the presence of genetic diversity among these populations(17).

Research challenges and outlook Conserving diversity in animal genetic resources is vital to making livestock production systems sustainable. Genetic variation in livestock populations around the world is declining, both within and between breeds. But it is necessary for improving productivity and adapting to changing environmental conditions, such as climate and production or market conditions. Research done at the INIFAP on Criollo Coreño cattle suggests that these cattle may play a prominent role as a maternal breed in commercial beef production. Short-term goals include continued systematic recording of economically interesting productive data in the experimental herd at EVSE, and identification of the most productive genotypes using highdensity arrays. This will support the medium-term goals of using quantitative approaches to identify the genomic regions subject to natural selection and find evidence of artificial selection, and using homozygous runs to estimate genomic relationships between individuals and levels of consanguinity(18). No accurate data on risk is available for Criollo Coreño cattle, so another medium-term goal is to evaluate effective size and structure of the population every five years to effectively monitor trends in population size(19). In the long-term, research is needed to identify the genes associated with characteristics of present and future interest. In other Mexican Criollo cattle populations, short-term genetic diversity studies are needed within and between groups using molecular genetic data. Over the medium-term, controlled herds should be integrated to allow systematic recording of productive and adaptive characteristics in different phenotypes. Long-term studies need to identify unique polymorphisms that distinguish these populations. Mexican Criollo cattle populations are small in size, suggesting that only small long-term financial gains can be expected from the application of genomic data(20). However, they can benefit from technological developments attained in other cattle breeds with larger populations, such as methods for optimizing genetic response and maintaining diversity, which are more easily applied in small populations(21).

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Conclusions Research done at the INIFAP over the last twenty years has found that hi influences reproduction and milk production, and that hm influences growth during offspring birthweaning, which was reflected in higher productivity at weaning per cow exposed. Direct and maternal genetic effects did not affect the analyzed traits. Promising results include that young C bulls in feedlot fed high-energy diets produce meat with favorable fatty acid profiles and good carcass quality, and that reduced doses of FSH can be used for superovulation in C heifers without affecting their response to embryo production. Guzerat-Coreño (GC) cows are a better option than CG cows for growth calf production, and both are better producers than G or C cows. Finally, genetic diversity remains high in C populations. Future research in Mexican Criollo cattle populations needs to focus on genetic diversity studies using molecular genetic data, and integration of controlled herds to allow systematic recording of phenotypes, with their respective productivities and adaptabilities, to identify genes exclusive to these populations. Literature cited: 1. SAGARPA. Informe sobre la situación de los Recursos Genéticos Pecuarios (RGP) de México. Coordinación General de Ganadería. 2002. http://www.sagarpa.gob.mx/ganaderia/Publicaciones/Paginas/InfoRGPecuariosM.aspx 2. FAO. The management of global animal genetic resources. Proc Expert Consultation, Anim Prod Health. Hodges J, editor. FAO, Rome. Paper No. 104, 1992:1-263. 3. Martínez VG. El ganado bovino Criollo en Nayarit: Ubicación y población estimada. Sitio Experimental “El Verdineño”. CIRPAC- INIFAP. Folleto Técnico Número 1. 2005. Nayarit, México. 4. Dickerson GE. Inbreeding and Heterosis in Animals. Proc Anim Breed Symp, Honor of Jay Lush. Am Soc Anim Sci & Am Dairy Sci Assoc. Illinois. 1973:54-77. 5. Martínez VG, Montaño BM, Palacios FJA. Efectos genéticos directos, maternos y heterosis individual para tasas de estro, gestación, parición y destete de vacas Criollo, Guzerat y sus cruzas F1. Téc Pecu Méx 2006;44(2):143-154. 6. Martínez VG, Montaño BM, Palacios FJA. Productividad hasta el destete de vacas Criollo, Guzerat y sus cruzas recíprocas F1. Téc Pecu Méx 2008;46(1):1-12.

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7. Martínez VG, Montaño BM. El bovino Criollo del Occidente de México. Perezgrovas GRA y De la Torre SF editores. Los Bovinos Criollos de México: Historia, Caracterización y Perspectivas. Primera edición. Tuxtla Gutiérrez, Chiapas, México: Universidad Autónoma de Chiapas; 2015: 147-174. https://www.textosdeinvestigacion.unach.mx/ebooksbd/20151023_121503/#p=174 8. Martínez VG, Bustamante GJJ, Palacios FJA, Montaño BM. Efectos raciales y heterosis materna Criollo-Guzerat para crecimiento posdestete y características de la canal Criollo Coreño. Téc Pecu Méx 2006;44(1):107-118. 9. Bustamante GJJ, Martínez VG, Basurto GR, Palacios FJA, Moreno FLA, Montaño BM. Rendimiento y calidad de la canal de toretes criollos finalizados en corral [resumen]. V Congreso Internacional de Manejo de Pastizales. I Congreso en Ciencias Veterinarias y Zootécnicas “Amado Nervo”. Reunión Red Latina en Ciencia Animal. Nuevo Vallarta, Nayarit. 2014:1-5. 10. Villaseñor GF, de la Torre SJF, Martínez VG, Álvarez GH, Pérez RS, Palacios FJA, Polanco SR, Montaño BM. Caracterización de la respuesta ovárica a la superovulación en bovino Criollo Coreño utilizando dosis reducidas de FSH. Rev Mex Cienc Pecu 2017;8(3):225-232. 11. Martínez VG, Borrayo ZA, Montaño BM, Bustamante GJJ, Palacios FJA, Vega MVE, Ríos UA. Producción de leche de vacas Criollo, Guzerat y sus cruzas recíprocas F1 y su relación con el peso al destete de las crías. Rev Mex Cienc Pecu 2012;3(4):501-514. 12. Martínez VG, Palacios FJA, Bustamante GJJ, Ríos UA, Vega MVE, Montaño BM. Composición de leche de vacas Criollo, Guzerat y sus cruzas F1 y su relación con el peso al destete de las crías. Rev Mex Cienc Pecu 2010;1(4):311-324. 13 Bustamante GJJ, Martínez VG, Palacios FJA, Basurto GR. Perfil de ácidos grasos en carne de toretes Criollo alimentados con distintos niveles de energía en la dieta [resumen]. V Congreso Internacional de Manejo de Pastizales. I Congreso en Ciencias Veterinarias y Zootécnicas “Amado Nervo”. I Reunión Red Latina en Ciencia Animal. Nuevo Vallarta, Nay. 2014;1-6. 14. Simopoulos AP. Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic variation: nutritional implications for chronic diseases. Biomed Pharmacother 2006;60:502-507. 15. Ulloa-Arvizu R, Gayosso-Vázquez A, Ramos-Kuri M, Estrada FJ, Montaño M, Alonso RA. Genetic analysis of Mexican Criollo cattle populations. J Anim Breed Genet 2008;125(5):351-359.

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16. Martínez RCP, Martínez VG, Román PSI, Cortes CM, Guzmán RLF, De la Torre SJF, et al. Estimación de la diversidad genética mediante marcadores SNP en bovino Criollo Coreño (Bos taurus) [resumen]. Reunión Nacional de Investigación Pecuaria. Toluca, Edo. De México. 2015:189-191. 17. Cozzi MC, Martinez-Ruiz CP, Roman-Ponce SI, Vega-Murillo VE, Ríos-Utrera A, Montaño-Bermúdez M, et al. Copy number variants reveal genomic diversity in a Mexican Creole cattle population. Livest Sci 2019;229:194-202. doi.org/10.1016/j.livsci.2019.09.030. 18. Puriﬁeld DC, Berry DP, McParland S, Bradley DG. Runs of homozygosity and population history in cattle. BMC Genet 2012;13:70. doi:10.1186/1471-2156-13-70. 19. Boettcher PJ, Tixier-Boichard M, Toro MA, Simianer H, Eding H, Gandini G, et al, GLOBALDIV Consortium. Objectives, criteria and methods for using molecular genetic data in priority setting for conservation of animal genetic resources. Anim Genet 2010; 41 (Suppl. 1):64–77. 20. Pryce JE, Daetwyler HD. Designing dairy cattle breeding schemes under genomic selection: a review of international research. Anim Prod Sci 2012;52(3):107–114. 21. Gandini G, Del Corvo M, Biscarini F, Stella A. Genetic improvement of small ruminant local breeds with nucleus and inbreeding control: a simulation study. Small Ruminant Res 2014; 120:196–203. doi:10.1016/j.smallrumres. 2014.06.004.

https://doi.org/10.22319/rmcp.v12s3.5918 Review

Reproductive biotechnologies in beef cattle: five decades of research in Mexico

Jorge Víctor Rosete Fernández a Horacio Álvarez Gallardo b David Urbán Duarte b Abraham Fragoso Islas a Marco Antonio Asprón Pelayo c Ángel Ríos Utrera d Sandra Pérez Reynozo b José Fernando De La Torre Sánchez b*

Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias (INIFAP). Sitio Experimental Las Margaritas. Km 9.5 carretera federal Hueytamalco-Tenampulco, Hueytamalco, Puebla. México. b

INIFAP. Centro Nacional de Recursos Genéticos. Blvd. De la Biodiversidad Nº 400, Tepatitlán de Morelos, Jalisco. México. c

Universidad Autónoma de Querétaro. Facultad de Ciencias Naturales. Av. de las Ciencias S/N, Juriquilla, Querétaro. México. d

INIFAP. Campo Experimental Paso del Toro. Km 22.5 carretera federal Veracruz-Córdoba, Medellín, Veracruz. México.

* Corresponding author: delatorre.fernando@inifap.gob.mx

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Abstract: The main bovine reproductive biotechnologies are recapitulated herein in five sections, and their historical development and current status are analyzed, including the results generated in Mexico. In the 1970s, estrus synchronization and ovulation induction began; thus, the reproductive cycle started to be controlled with the resources available at that time, based on the knowledge of bovine reproductive physiology. Over the years, hormone therapy evolved as new compounds were discovered, refining methods to standardize the effect and generating new methods for the release of hormones. The most widely used biotechnology in the world, artificial insemination, owes its expansion to advances in semen processing, among which the development of diluents, cryopreservation, semen sexing, and computerassisted sperm analysis stand out. The embryonic era began with the development of multiovulation and methods for collecting, evaluating, transferring, and cryopreserving embryos. The second embryonic era came with the fully in vitro production of embryos from immature eggs and frozen sperm, known as in vitro embryo production. Great research and material resources have been invested in this procedure, rendering it a pillar of genetic improvement and productivity, in combination with two other tools: sexed semen and genomic evaluations. A golden age of in vitro embryo production is on the horizon, with the possibility to produce accurate modifications in the embryo genome, thanks to gene editing technology. Key words: Synchronization, Sexed semen, Embryos, Multi-ovulation, Embryo transfer, In vitro production, Bovines.

Received: 04/01/2021 Accepted: 30/04/2021

Introduction The text of the FAO Convention on Biological Diversity (CBD)(1), in force since 1993, states that the term "biotechnology" refers to any technological application that uses biological systems, living organisms (or derivatives thereof) to make or modify products or processes for a specific use. In this sense, and for the purposes of the topics to be covered in this review, reproductive biotechnology will be understood as the technological applications that affect the physiological processes of animal reproduction, their gametes and embryos, for the purpose of achieving productive improvements.

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This document will cover only the bovine species and will place particular emphasis on the research carried out by institutions and universities in Mexico, particularly the National Institute for Research in Forestry, Agriculture and Livestock (INIFAP), in its 35 years of existence, and its predecessor: the National Institute of Livestock Research (INIP). While the authors do not claim this to be an exhaustive review of all the reproductive biotechnologies that have been applied to cattle, it is address those that have had the greatest impact on productivity and briefly mention those that have had minimal or no application in Mexico, such as transgenesis and cloning. The topics of estrus synchronization and ovulation induction are discussed first, as they are the topics on which research has been conducted for the longest time (50 yr) and for which INIP-INIFAP has generated the largest number of technologies. Biotechnologies developed for semen collection, dilution, and cryopreservation have underpinned the massive use of bulls of high genetic merit through artificial insemination (AI). This topic will be discussed, with emphasis on recent developments such as semen sexing and computer-assisted sperm analysis. Issues related to embryo manipulation, including multi-ovulation and embryo transfer (MOET), in vitro embryo production (IVP), and transvaginal oocyte aspiration (TVA) have seen important developments in recent years, which will be addressed in this paper. In addition to including research results on these topics, in some cases, mention will be made of government or producer organization programs that marked a milestone in the dissemination and adoption of these reproductive biotechnologies.

Estrus synchronization and ovulation induction In cattle, a common problem is prolonged postpartum anestrus, a condition characterized by a delay in the return to estrous cyclicity after parturition due to various factors(2-6). Many efforts have been made to resolve this condition: hormonal treatments have been studied to induce estrus and ovulation(7,8) by controlling breastfeeding(9,10), the frequency and quality of its stimuli(11), feeding(12) and the different mating seasons(13). This section reviews results of ovulation induction in anestrus females and synchronization of estrus and ovulation in cycling females, carried out by INIP (1971-1985) and the current INIFAP (1986-2021), as well as by other institutions.

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Estrus synchronization and ovulation induction studies

The first studies date back to 1948(14), when progesterone (50 mg/d, i.m.) was used to block estrous cyclicity for a period of up to 13 d. It was observed that, at the end of its effect, a good proportion of females presented estrus at 4 and 5 d. Later, with the addition of estrogens to the treatment, estrus presentation increased, and the progesterone blocking time was reduced(15). The use of hormonal products such as 19 alpha acetoxy-11 beta-methyl-19 norg 4-ene-3, 2-dione (a very potent progestogen known as SC21009), natural progesterone, estradiol valerate (EV), and prostaglandin F2α (PGF2α) began in the 1970s; at this time, work focused on estrus synchronization(16), the resolution of prolonged postpartum anestrus in cows, and the attempt to advance puberty in heifers(17-21). Thus, heifers of European breeds, Creole breeds, and pre-pubertal zebu crossbreeds, treated with 5 mg of EV (i.m.) + 3 mg of SC21009 (i.m.) + 6 mg of SC21009 (auricular implant removed on d 9), had 79 % of estrus presentation in the first 48 h of implant removal, compared to control heifers, of which only 6 % exhibited estrus(21). In another study(22) with anestrus cows and fattened zebu heifers, it was observed that, in females treated with progesterone (25 mg i.m. for 5 d) + estradiol cypionate (EC; 2 mg i.m. on the first day) or with SC21009 (3 mg for 5 d in subcutaneous implant) + EC (2 mg i.m. on the first day), the estrus was synchronized at 72 h, with an estrus rate (ER) of 61.1 and 73.7 %, and a conception rate (CR) of 44.4 and 31.3 %, respectively. On the contrary, in females that received the control and individual treatments (progesterone, SC21009 or EC) with the same doses, estrus was not synchronized at 72 h, and they had a low TC, from 10.5 to 21.1 %, during the 30 d of the study. In another experiment(23) with zebu cows with calf and at 60 d postpartum, the effectiveness of SC21009 + EV was evaluated. In the first 48 d, the ER in treated cows was 24 %, and 0 % in untreated cows, while the CR was 12 and 0 %, respectively. However, at the end of the AI period (d-48), 20 % of the treated cows were pregnant, and 12 % of the untreated cows were pregnant; at the end of mating (d-68; AI + natural mating), 28 % of the treated cows were pregnant, but only 12 % of the untreated cows were pregnant. However, in non-breeding zebu cows, after applying 6 mg of EV + 3 mg of SC21009 via i.m. + 6 mg of SC21009 in subcutaneous implant, it was observed that 100 % of the treated cows showed estrus in the first 48 h after the implant was removed, in contrast to the untreated cows, of which only 29.4 % exhibited estrus. The CR was 42.8 % in treated cows, and 14.7 % in untreated cows. This indicates that synchronization groups the estruses in order to facilitate AI and overall herd management(24), but provides evidence that it is more difficult to reduce the anestrus period in cows with calves, a circumstance that reveals the importance of reducing or eliminating the effect of lactation on the return to estrous cyclicity.

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Estrus synchronization with melengestrol acetate

Progestogen melengestrol acetate (0.5 mg/d for 9 d) in feed, combined with EV (single dose of 6 mg i.m. on d 1) and progesterone (single dose of 50 mg on d 1), was tested in Brown Swiss x Zebu cows and heifers. It was observed that the percentage of cows inseminated at 48 days after mating was 38.9, 51.5, and 75.8 %, respectively, in control cows, in cows conventionally inseminated at detected estrus, and in cows inseminated at any sign of estrus. The percentage of cows inseminated was significantly higher when insemination was performed before any sign of estrus. CRs were statistically similar between treatments (20.9, 28.8 and 37.1 %, respectively), despite the fact that the estruses were clustered among the cows in each treatment(25). Estrus synchronization with melengestrol acetate is not a widespread practice in Mexico, despite its proven efficacy, because it is not practical to perform it in small groups of animals, and small herds are predominant in our country.

Estrus synchronization with fixed-time artificial insemination (FTAI)

Because some females did not exhibit estrus even when treated hormonally, the decision was made to research the convenience of inseminating at pre-established schedules. Thus, in Brangus and Creole heifers, AI was performed at preset times in anestrus females because the percentage of animals in estrus in the first 5 days after removal of the implant was very low (53.1 %). Under this premise, heifers were inseminated at 48, 54 and 60 h after implant removal, resulting in CRs of 54.5, 60.6 and 47.0 %, with higher CRs at 48 and 54 h after implant removal(26). These results set the tone for the initiation of many studies on estrus synchronization with FTAI. On the other hand, studies were carried out with estrus synchronization in grazing beef cattle during 85-d mating seasons (matings), when AI is difficult to perform. Thus, in humid subtropical climate with non-breeding zebu cows, the ER in the first 60 h of mating was higher in cows treated with SC21009 (9 or 6 mg for 9 d) + EV (6 mg on d 1 of the 9-d treatment) than in untreated cows (84 vs 0 %). Similarly, CR at 26 ds after mating favored treated cows over untreated cows (59 vs 40 %), although at the end of the mating period there were no differences in CR between treatments(27).

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Inclusion of PGF2α in estrus synchronization

The availability of PGF2α in the early 1970s made it possible to start work on estrus synchronization in cycling females, a status that was confirmed through the detection of a corpus luteum(28,29). A CR of 34.8 % was obtained in cycling Brangus, Gyr and Charolais heifers treated with PGF2α and artificially inseminated 80 h after treatment; this percentage was similar to that of heifers synchronized and inseminated 12 h after estrus was observed (CR= 26.1 %). At the end of mating (90 d), in heifers inseminated at 80 h, a TC of 69.6 % was obtained, similar to the TC (56.5 %) of heifers artificially inseminated 12 h after estrus(30). Studies on estrus synchronization facilitated the reproductive management of grazing cattle, since it allows females to be served in the first days of mating(13,23-26).

Use of the progestogen norgestomet in estrus synchronization

Norgestomet (CRESTAR®), (a more potent progestogen than natural progesterone), applied subcutaneously on the dorsum of the ear and removed 9 d after implantation, associated with EV and PGF2α (only in cycling cows), also proved to be useful in estrus synchronization and ovulation(31-34). Thus, in a study(35) with zebu cows and their crosses with European bulls, without offspring, in a 63-d mating (42 d of AI and 21 d of natural mating), TES of 86.1 and 95.0 % were achieved 5 d after implant removal in anestrus and cycling cows, respectively. Subsequently, 42 ds after implant removal, the TES was 100 % in both groups of cows. When pregnancy diagnosis was performed, it was determined that 49.5 % of the anestrus cows and 54.0 % of the cyclic cows conceived during the first 5 d of mating, with the following percentages: 90.7 and 98.3 % at 42 d; and at the end of mating (63 d), with the presence of the bull from d 43 to the end of mating, 96.2 % and 98.3 %, respectively. This study demonstrated the usefulness of estrus induction and synchronization in mating cows (mainly anestrus cows) that would otherwise be delayed up to 21 d to conceive. The results with the SC21009 auricular implant combined with EV for the performance of FTAI at 56-60 h after removal of the implant (d 9) were also attractive, since a TES of 95 and a CR of 85 were achieved in 45-d matings(36).

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Use of the progesterone-releasing intravaginal device (PIDR) in estrus synchronization The introduction of the PIDR into the market revolutionized estrus synchronization(37), and, therefore, researchers from INIFAP(38,39) started using this device in cows in anestrus and cycling cows with good body condition (BC), obtaining a CR of 44.8, 77.1 and 100.0 % at 3, 30 and 60 d after PIDR withdrawal, respectively(38). In Bos taurus x Bos indicus(39) heifers, the CR with PIDR and FTAI at 84 h after the removal of the device was higher than with conventional AI at 12 h after the detection of estrus (36.4 vs 18.2 %, respectively). Similarly, the CR with CRESTAR and FTAI at 84 h after device removal was higher than with conventional AI (27.3 vs 18.2 %, respectively). In this work, the advantage of FTAI at 84 h after the removal of the device was demonstrated, since pregnancy was achieved in some heifers that did not exhibit estrus. A frequent practice in estrus and ovulation synchronization protocols with FTAI in which PIDR and PGF2α are used in association with estradiol benzoate (EB) (which is applied when inserting the PIDR on d 0 and the day after its removal) is the substitution of GnRH for EB. This is because, like EB, GnRH synchronizes the emergence of a new wave of follicular development, which culminates in the ovulation of the mature follicle, giving good results. However, if a significant proportion of the cows are suspected to be in anestrus, it is recommended to additionally apply 400 IU of equine chorionic gonadotropin eCG on d 7 of the protocol. Furthermore, EB can be replaced with GnRH (100 µg i.m.), on the first application (d 1 of PIDR), but the second application must be carried out at the time of FTAI. An example for cycling cows is shown in Figure 1(40).

Importance of body condition in estrus induction and synchronization

It has been shown that body condition (BC) is an indicator of the cow's nutritional status and that, if the cow has a good BC before and after calving, her fertility improves soon after giving birth(41). In addition, this has been shown to be associated with high concentrations of insulin-like growth factor-1 (IGF-1), leptin and insulin, allowing early resumption of postpartum estrous activity(42). Therefore, other research focused on studying changes in the blood concentration of these three hormones as metabolic indicators of the nutritional status of the animals, observing that a low BC in females (<6.0 units; scale 1 to 9) is associated with a reduction in the blood concentration of insulin and IGF-1, without changes in leptin, which diminishes the response to postpartum estrus induction in beef cows(43,44). Therefore, females that are synchronized and enter mating should have good BC (of no less than 3.0), in order to favor the occurrence of estrus and achieve gestation(45).

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Kisspeptin in estrus synchronization and ovulation induction

Kisspeptins are peptides that are named for their number of amino acids: kisspeptin-54, kisspeptin-14, kisspeptin-13 and kisspeptin-10(46). Synthesizing neurons are considered to be integrators of signals that modulate the functionality of the somatotropic and gonadal axis(47). Studies with intravenous kisspeptin-10 (kiss-10) (5 µg/kg bw) produced increases in LH, FSH, and growth hormone secretion in prepubertal male and female calves(48,49), LH increased with age in all calves, with mean values of 6.1, 7.2 and 11.6 ng/ml at 4, 7 and 11 mo of age, respectively(50). The highest LH concentration was found in 11-month-old calves. In an attempt to understand the sensitivity of the gonadotropic axis to kiss-10, other studies tested intravenous doses of 0.1, 1.0, 2.0, 3.0, 4.0 and 5.0 µg/kg bw(51) and 50.0 µg/kg(52) in prepubertal calves, showing that effective doses for inducing LH release ranged from 1.0 to 5.0 µg/kg. Therefore, the application of low doses, less than 5 µg/kg body weight, could considerably reduce the cost of treatment with this peptide(51). In a subsequent experiment with prepubertal calves using kisspeptin-10 at a dose of 5 µg/kg-bw applied every 2 h for 84 h (3.5 d), ovulation and corpus luteum formation were induced in 28.5 % of the calves; however, the corpus luteum disappeared and the calves returned to their prepubertal state(53). Later, it was found that, in (European x zebu) beef cows, at 78 d postpartum, with anestrus, and nursing their calf, kisspeptin-10 at a dose of 1 μg/kg-bw every 2 h for 24 h also augmented the serum LH concentration, an increase that emulated a brief LH pulse(54). This result prompted an ovulation synchronization study, such that kisspeptin-10 was tested at a total dose of 500 µg at the time of FTAI, compared to GnRH (100 µg at the time of FTAI) and eCG (400 IU on withdrawal of PIDR on d 7), in a protocol in which each hormone was combined with PIDR, BE (2 mg at PIDR application + 1 mg the day after PIDR withdrawal), and cloprostenol (total dose of 500 µg at PIDR withdrawal), in non-calving, 180-d postpartum beef cows inseminated between 54 and 56 h after PIDR withdrawal. The ovulation rate for the eCG, GnRH and kisspeptin-10 treatments was 89.2, 96.5 and 93.8 %, while the conception rate was 43.6, 73.8 and 54.3 % respectively, with no significant statistical differences between treatments in either case(55). This result could be of interest to the pharmaceutical industry as an alternative for ovulation synchronization and induction.

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Figure 1: General outline of estrous synchronization in cows and heifers with FTAI(40) PIDR insertion, 1.9 g + BE, 2 mg i.m.

Withdrawal of the PIDR BE, 1 mg i.m. + PGF2α, 25 mg i.m.

FTAI

Day 0

Day 7

Day 9

Day 8

In conclusion, estrus synchronization and ovulation induction in cattle in tropical climates revolutionized reproductive management, since it allowed the concentration of estrus between 52 and 56 h after the withdrawal of the PIDR in a mating season with FTAI in a way that facilitates the use of AI, with the possibility of re-inseminating females that did not conceive at the first service at manifest estrus, increasing the number of pregnant cows with AI, which no doubt favors the herd genetically.

Semen processing and artificial insemination AI was the first reproductive biotechnology applied to improve production through the more efficient use of bulls of high genetic merit. The widespread use of this technique and the achievement of its full potential required the cryopreservation of semen for long periods of time. The first report of semen cryopreservation was made in 1776(56), where it was observed that when spermatozoa from human, guinea pig and frog were cooled in snow for up to 30 min, they became inactive, but could be reactivated. In 1940(57), egg yolk began to be used to protect bull sperm cells from thermal shock upon cooling. In 1941 (58), the sperm extender was improved by using egg yolk with sodium citrate, which allowed semen preservation at 5 °C for up to 3 d. In 1949, bull spermatozoa were frozen for the first time by using glycerol in the diluent. The great discovery of the cryoprotective action of glycerol opened a successful era in the cryopreservation not only of gametes of various species, but also of other cells and tissues(59). The so-called Cornell diluent, created by Foote and Bratton in 1950(60), contained a mixture of the antibiotics penicillin, streptomycin, and polymyxin B, and was used for many years as standard. In 1952, in Cambridge, the first calf was born from frozen-thawed bovine sperm(61).

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Reports of fertility with frozen spermatozoa from bulls led to an intense development of cryopreservation methods to try to improve the results obtained, so other disciplines (such as molecular biology) associated with the cryopreservation process began to be used. Such is the case of proteomics(62) and genetic engineering(63). On these issues, a study was carried out in Mexico with INIFAP researchers, where the addition of recombinant FAA (fertilityassociated antigen) and recombinant TIMP-2 (tissue inhibitor of metalloproteinases 2) to frozen bovine semen was evaluated and proved to significantly increase its fertility(64).

Sperm sexing

Advances in AI have generated interest in using AI for sex selection in dairy cattle. For this reason, over time, many researchers have tried to separate spermatozoa with "X" and "Y" chromosomes using various techniques(65). In the 1980s, flow cytometry began to be applied, making it possible to separate spermatozoa according to their sex chromosomes and amount of DNA. It took about 20 yr for this technology to be commercialized for use in AI in cattle. This technique is based on the fact that spermatozoa with an "X" chromosome in cattle contain 3.8 % more DNA than "Y" spermatozoa(66). This technology had an efficiency of 85 to 95 % (in terms of separation of sperm with X or Y chromosome); however, it had not been fully perfected(67). The first commercial production of sexed semen took place at the Cogent company in the United Kingdom(65). Although it had a relatively slow start, bovine sexed semen production increased exponentially with an estimated 4 million doses in 2008(68). The sexed semen was marketed in 0.25 ml straws at a concentration of 2.1 million spermatozoa(69). This concentration was due to the fact that, at the time of semen sexing, approximately 80 % of the ejaculate was lost between the sperm of the unwanted sex and the sperm that could not be differentiated, in addition to the long time that the separation process took(70). Despite the limitations of sexed semen, it was clearly a welcomed development(68). Acceptable gestation percentages were achieved with the reduced dose (2.1 x 106 sperm) of sexed semen in heifers, but little work was done with lactating cows(66). Nowadays, sperm sexing technology has evolved, modifying the techniques, increasing the speed of sexing, decreasing stress on spermatozoa, increasing concentration and, therefore, improving sperm viability parameters.

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AI with low dose of sexed semen

In 1997, research was conducted with two objectives: 1) to evaluate conception rates of heifers inseminated (in the uterine horn, ipsilateral to the ovary with the largest follicle) with reduced doses of semen (1 x 105; 2.5 x 105; 2.5 x 106 sperm/0.21 ml) chilled at 5 °C under ideal conditions at the field level; 2) to evaluate conception rates of heifers inseminated (in the uterine horn, ipsilateral to the ovary with the largest follicle) with reduced doses of sexed semen (1-2 x105 sperm/0.1 ml) refrigerated at 5 °C. In experiment 1, gestation percentages at 40 d were 41, 50 and 61 % for 1 x 105; 2.5 x 105; 2.5 x 106 spermatozoa/IA respectively. In experiment 2, out of 67 heifers inseminated, 22 % were pregnant, and 82 % of the offspring were of the selected sex(71). Research indicates that AI with lower than conventional sperm doses is possible when using sexed sperm, 2.1 x 106 spermatozoa being most commonly used in the insemination dose, against at least 10x106, which is used with unsexed sperm.

Successful cryopreservation of sexed semen

Subsequently, in 1999, another research was carried out with the objective of evaluating the freezing process of the sexed semen; this was achieved because the semen was processed in a MoFlow SX™ flow cytometer, that allowed to have sufficient quantity of spermatozoa, unlike when working with the EPICS V flow cytometer. In this work it was determined that the use of the laser at a power of 100 mW had a lower impact on the progressive motility of post-thawed semen than when it was used at 150 mW. It was also observed that post-thawing progressive motility was higher when using a TRIS-based diluent than when using citrateegg yolk or TEST. Regarding the equilibration time (adaptation to the cryoprotectant) at 5 °C prior to freezing, it was concluded that progressive motility was better after thawing for 3 to 6 h than when this took 18 h. On the other hand, it was determined that it was better to keep fresh semen for 4 to 7 h at 22 °C than to dilute it with TALP medium added with Hoechst 33342 fluorochrome. These new sperm sexing procedures yielded slightly lower results in terms of motility and acrosomal integrity than conventional semen. With these results, it was considered that the use of sexed semen for commercial AI would be available in approximately 2 yr(72).

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The beginnings of sexed semen commercialization

The Monsanto Company, located in St. Louis, Mo., USA, developed a unique sperm sorting system that used 16 sorting nozzles instead of just one as in the case of the MoFlow SX™ cytometer. This equipment was intended to be commercialized, but, apparently, due to problems with low conception percentages detected in its first tests, the company decided not to commercialize it. In 2003, Genetic Resources International/Sexing Technologies in Navazota TX, USA purchased the intellectual property and the sperm sexing equipment developed by Monsanto, as well as the entire infrastructure of XY Inc. (the first company to own the intellectual property of sperm sexing and the creator of the MoFlowTM cytometers)(69). The company has since changed its name to STgenetics®(72).

SexedULTRA™ sexed semen

The difference in fertility between conventional semen and sexed semen did not improve with increasing sperm concentration by AI. The causes of lower fertility of sexed semen have been attributed to the various biochemical changes that sperm undergo during sexing. The XY technology described in previous publications(73,74) has been modified and is now a totally new sexing system called SexedULTRA™ (Navazota, TX, USA). The SexedULTRA™ technology has been designed to be more sperm-friendly during the most critical points of the process, particularly improving pH changes (buffer system) and oxidative stress. Modifications to the SexedULTRA™ sexing technique Although there is currently very little data on this new technology, it has been reported that it facilitates the entry of the Hoechst 33342 fluorochrome and retains it inside the cell, allowing for greater fluorescence and thus better discrimination between "X" and "Y" populations. The protocol was modified, with a pretreatment prior to the staining process, in addition to the use of a new staining medium that maintains the pH stable for a longer period of time. The freezing medium was also modified, taking into account the dose of sexed semen per straw(72). The success of the ultrasexing process was mainly influenced by two factors: modifications in the means and equipment used to perform sexing. The MoFlo SX™ cytometers (Cytomation Inc., Fort Collins, CO, USA) were very expensive, bulky, had low throughput and required highly trained personnel to operate them. Modern Genesis cytometers

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developed by Cytonome ST™ (Boston, MA, USA) have advanced and automated electronic features with multiple heads on one machine for parallel separation. The Genesis III™ cytometer uses a solid state laser, two orthogonal detectors (0° and 90° to the laser), an orientation nozzle and a subpopulation separation of ~8000 spermatozoa/second with ~90% purity, reaching a maximum separation rate of 500 million sperm per hour(75).

Laboratory testing of SexedULTRA™ technology

The aforementioned modifications brought about sperm motility and acrosome integrity were increased with respect to the XY Legacy technology (conventional sexing) considering the same sperm concentrations(76). On the other hand, in 2018(77), sperm quality was evaluated considering plasma membrane integrity, percentage of intact acrosomes and DNA fragmentation index of SexedULTRA™ semen compared to conventional (non-sexed) semen. In SexedULTRA™ semen at 3 h postthawing, the percentage of intact acrosomes was significantly higher than in conventional semen. In addition, SexedULTRA™ semen had a significantly lower DNA fragmentation index at all evaluation points compared to conventional semen. The authors conclude that SexedULTRA™ technology maintains semen quality and, in many cases, has greater in vitro longevity compared to conventional semen.

Field testing of SexedULTRA™ technology In the first field evaluation using SexedULTRA™ technology for AI(78,79), there was a 7.4 % increase in heifer conception rates over XY Legacy technology (conventional sexing). The second test was conducted in collaboration with the commercial company Select Sires, using eight bulls from which semen was collected and processed using both SexedULTRA™ technology and XY Legacy technology, inseminating 6,930 heifers. The results showed that SexedULTRA™ semen increased the conception rate 4.5 % over XY Legacy semen, 46.1 and 41.6 % respectively. With these tests it was observed that the deleterious effects of the XY Legacy technology were partially ameliorated with the new SexedULTRA™ technology, so the next logical step was to increase the sperm concentration per dose, although in the past increasing sperm concentration did not improve fertility. The following test was performed in collaboration with German Genetics International: semen was collected from five bulls; each ejaculate was

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divided into 4 parts and processed with XY Legacy technology of 2.1 million sperm, SexedULTRA™ of 2.1, 3 and 4 million sperm per dose. In addition, semen from these same bulls was used from contemporary conventionally frozen ejaculates, with a concentration of 15 million spermatozoa per dose. Non-return to estrus rates (NRR) at 65 d were calculated from 7,855 AI with sexed semen and 62,398 AI with conventional semen. Overall, the XY Legacy semen of 2.1 million sperm per dose resulted in lower NRRs (55.9 %) compared to all SexedULTRA™ treatments (2.1 million 59.9; 3.0 million 60.0 %; 4.0 million 66.7 %) and conventional semen (65.7 %). SexedULTRA™ treatments of 2.1 and 3 million sperm per dose were similar (59.9 and 60.0 % respectively), but lower than conventional semen (65.66 %); however, the SexedULTRA™ treatment of 4 million sperm per dose had NRRs similar to conventional semen of 15 million sperm per dose(79). The data obtained demonstrated for the first time the effect of the response to the dose when using sexed semen.

Field tests with SexedULTRA-4M™ technology The use of SexedULTRA-4M™ semen was evaluated in FTAI(80) using beef cows and heifers. The results show that there was no significant difference in the percentage of pregnancies between conventional semen (61.9 %) and SexedULTRA-4M™ semen (63.8 %) when the females were in heat prior to FTAI. Another experiment(81) compared the use of conventional semen and SexedULTRA-4M™ semen in AI using three different bulls and beef cows. In this study, fertility was found to be influenced by the bull, as only one of three bulls had no difference in the percentage of gestations when comparing conventional semen and SexedULTRA-4M™, which shows that there is a difference between bulls, as well as with the sexed Legacy. There is little research on the effectiveness of the use of sexed semen in Mexico, although it is already available from several semen processing companies and is routinely used, especially in dairy production units. Experiments have used Legacy sexed semen and found it to work for both Holstein cows and heifers, with a pregnancy rate between 80 to 90 % of that recorded for females inseminated with conventional semen and with values of 85 to 93.6 % of offspring born with the predicted sex (82-85). It was also found that the use of sexed semen does not influence the occurrence of miscarriages or dystocic births(86).

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Computer-assisted sperm evaluation Fertility is a multiparametric event related to semen quality and quantity, the exact timing and method of AI, the intrinsic fertility of the bull and proper herd management(87). Morphology, motility, viability, acrosome, and DNA concentration and integrity have been used as parameters to evaluate the sperm quality of bovine semen(87-90). Some of these parameters (such as morphology and motility) can be assessed manually by conventional methods using a visible light microscope. However, these evaluations are subject to subjective criteria and technical errors that diminish their accuracy and repeatability. In the mid-1980s, computer-assisted sperm analysis (CASA) systems were introduced commercially to maximize the accuracy and repeatability of semen evaluations(91). The main function of these CASA systems is the objective evaluation of semen quality. The basic components of this technology consist of a microscope to visualize the sample, a digital camera to capture images and a computer with specialized software to analyze the images. Motility is one of the most important sperm characteristics associated with the ability to fertilize(92). With the use of the CASA system, various motility parameters describing specific sperm movements are obtained. Total and progressive motility percentages are the most important parameters in the evaluation of sperm kinetics(87). Total motility refers to the fraction of spermatozoa that show any movement, whereas spermatozoa with progressive motility have a forward movement, essentially in a straight line. Other specific kinetic parameters determined by the CASA system are useful to evaluate several sperm characteristics simultaneously and objectively. These kinetic parameters consist mainly of three values of the speed of movement, three speed indices, and three parameters that reflect the oscillation characteristics of the spermatozoa(93). The three values of the velocity of motion are the curvilinear velocity (VCL), the rectilinear velocity (VSL) and the mean trajectory velocity (VAP). From these three values, three indices are calculated, linearity (LIN=VSL/VCL), straightness (STR=VSL/VAP) and trajectory oscillation (VAP/VCL), thus characterizing the quality of sperm movement. The parameters that show the oscillation characteristics of spermatozoa are the lateral displacement of the head, the frequency of tail beating, and the mean angular displacement. CASA system movement parameters have been used for the identification of sperm subpopulations and their subsequent correlation with freezing resistance(94,95). In addition, the effects of different media during in vitro processing on sperm function have evaluated(96).

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On the other hand, with the use of the CASA system, several groups have reported a significant correlation between the total (r=0.26-0.61) and progressive (r=0.26-0.33) motility of bull semen and its field-associated fertility(97-101). The CASA system collects a wide range of sperm kinetics parameters. Some research groups showed a positive correlation between VSL (r=0.17-0.67), LIN (r=0.28-0.46) and STR (r=0.33), and field fertility(98,102,103). Correlations between motility parameters and fertility tend to be low to medium, and their use in isolation to predict the fertility of a semen sample is not recommended(101-103). However, the combination of several motility parameters provides a better tool for predicting fertility, as the multi-parameter regression of the CASA system explained up to 0.98 (r2 value) of the variation in fertility, compared to 0.34 explained by total motility alone(103). On the other hand, recent analyses of sperm movement in the third dimension and the study of flagellar movement are new functional parameters that could be related to fertility(104). Sperm morphology is one of the most important tests in semen quality control, as it reflects the physiological or pathological state of the functionality of the testicles, epididymis and accessory glands of the reproductive tract(105); is also considered a better test to evaluate sperm DNA and genetic characteristics, compared to sperm motility(106,107). Several studies have shown a significant correlation between sperm morphology (r=0.22-0.76) and field fertility(94,98,108). However, most of the morphological analyses are performed through conventional methods, which remains a problem due to the subjectivity in the evaluation, as well as the inconsistency observed within and between technicians(109,110). For this reason, morphological analysis has not been considered reliable in predicting field fertility(94, 98). The development of specific modules for the morphological analysis of spermatozoa within the CASA system has allowed the individual evaluation of morphometric characteristics of the sperm head in terms of size (area, perimeter, length and width) and shape (ellipticity, elongation, regularity and roughness)(111). Some systems even provide information about the mid piece (area and width) and data concerning the insertion of the mid piece into the head, such as distance and angle of insertion(112). Within these parameters, the width of the sperm head showed a significant correlation (r=0.53) with field fertility(113). On the other hand, sperm subpopulations have been reported based on their morphometric structure(114). The evaluation of new parameters, as well as the identification of sperm subpopulations, could provide information on an optimal fertility-enhancing population(90). There are other parameters in the study of sperm function, such as vitality, acrosome integrity, and DNA fragmentation. Viability classifies spermatozoa as alive or dead and shows the existence of damage to the sperm plasma membrane(115). Acrosome integrity is one of the most important sperm function tests, since only a spermatozoon with an intact acrosome can penetrate the oocyte(116). In the case of sperm DNA integrity, its importance in fertilization and in the early stages of embryonic development has been demonstrated, having been recognized as a parameter indicative of sperm fertilization potential(117,118). Several studies have shown a significant correlation between vitality (r=0.19-0.40), acrosome 54

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integrity (r=0.52) and DNA fragmentation (r=-0.49), and the field fertility of bulls(94,95,97,99). In addition, when several parameters (sperm kinetics, vitality, DNA fragmentation and morphology) were combined in regression models, the correlation with fertility increased to more than 90%(12). Although most of these tests are performed through the use of flow cytometry, new generations of CASA system modules have been developed for the automatic assessment of vitality, acrosome integrity, and DNA fragmentation(92). However, few studies have been conducted in cattle using these new modules. Therefore, motility and morphology are shown to be the most important modules of the CASA system in terms of use. These studies show the great potential of CASA systems for estimating semen quality, studying sperm function, and predicting fertility. However, the parameters provided by the CASA system have also been shown to have limitations and cannot be used in isolation as reliable predictors of the fertilization ability of the sperm. In addition, spermatozoa are complex cells that require a large number of criteria in order to be considered to achieve fertilization(119). Therefore, the use of various parameters provided by the CASA system within a regression model is presented as the best option to attempt to predict the fertility of a semen sample. However, there is no consensus as to which parameters of sperm functionality to use in the spermatozoa(99), perhaps due to the differences found between working groups with respect to the parameters correlated with fertility. Currently, sperm analysis through a CASA system is widely used within the quality control protocols of semen processing centers, mainly the motility module. These protocols establish thresholds for certain variables such as total and progressive motility; ejaculates below these thresholds are usually discarded before or after freezing(99). In Mexico, several associations, institutions and companies related to the livestock sector utilize CASA systems on a regular basis mainly in the quality control of commercial doses of bovine, ovine, and porcine semen. However, studies of post-cryopreservation sperm kinetics of Pelibuey and Blackbelly rams(120), Mexican Pelon pigs(121) and Merino sheep from Socorro Island(122), and evaluations of cryopreserved semen samples from Chiapas sheep(123) and Tamaulipas Creole cattle(124) stored at the National Center for Genetic Resources of INIFAP (CNRG-INIFAP) have also been carried out. On the other hand, CASA systems have become more attractive as part of the practical evaluation of the sperm quality in semen samples of many domestic species. However, there are multiple reasons why a semen evaluation using a CASA system may vary, including system and equipment maintenance, sample handling, and technician experience(89). For this reason, it is vitally important to corroborate the effectiveness of the analysis. To ensure that the CASA system works properly, validation of technicians, protocols and equipment is crucial. In this sense, the CNRG-INIFAP has a test validated before the Mexican accreditation entity called “Evaluation of semen from domestic animals”, which is carried out with a CASA system. This allows semen evaluations to be performed with high quality standards(125). 55

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CASA systems have evolved in recent years to become powerful tools for rapid and objective evaluation of sperm quality and function of bovine semen samples. CASA systems will continue to be developed to perform new, repeatable and increasingly accurate tests in order to contribute to fertility improvement in the field.

Multi-ovulation and embryo transfer (MOET) This concept groups together a series of reproductive biotechnologies whose purpose is to increase the reproductive capacity on the maternal side and which are used as tools for genetic improvement. These biotechnologies include: selection of donors (both female and male) of high genetic merit, superovulation of donors, embryo collection and evaluation, embryo transfer to recipient cows, or embryo cryopreservation. This latter technique has had its greatest development in cattle; however, it is also widely applied in such species as equines, sheep, goats, and deer, among others(126). In addition to the aforementioned purpose (genetic improvement), the MOET has also been used for the conservation of animal genetic resources, as, according to FAO(127), it is an excellent option to conserve genetic diversity and is the fastest way to restore a population at risk. For this reason, INIFAP has the National Center for Genetic Resources, where work has been done to generate germplasm banks of bovine breeds with different risk statuses(128). The first birth from embryo transfer in mammals was achieved in 1890 with rabbit embryos(129), and the first successful surgical transfer in cattle was achieved in 1951(130). The set of biotechnologies involving MOET developed between 1940 and 1960, laying the foundation for the embryo transfer industry, which began in 1970 as a tool for the mass introduction of continental European breeds to North America(131). The modern activity of the MOET is the result of the efforts of two groups: a) scientists, who initially developed the procedures and techniques of embryo transfer, and b) field veterinarians, who applied this technology commercially, making it practical and available for the cattle industry, and later for other productive species as well(131). In the early 1970s, the first embryo transfer centers were established in North America, including Alberta Livestock Transfer (Alberta, Canada), Modern Ova Trends (Ontario, Canada), Colorado State University (Colorado, United States), Carnation Genetics (California, United States), and Codding Embryological Science, Inc. (Oklahoma, United States)(132). In our country, the first embryo transfers (ET) in cattle were performed in 1978 by North American technicians, but the results are unknown. In February 1979, the Embryo Transfer Clinic was inaugurated in Ajuchitlán, Querétaro, as part of the National Center for Animal Reproduction, under the National Institute for Artificial Insemination and Animal

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Reproduction (INIARA), of the then Ministry of Agriculture and Hydraulic Resources (Secretaría de Agricultura y Recursos Hidráulicos, SARH). In that same year, MOET's first Holstein calf was born there(132). The first TE zebu calf was born in 1981 at Carnation in Mexico(133). The Center for Genetic Improvement and Embryo Transplantation (CEMEGEN), which is attached to the state-owned dairy company Leche Industrializada Conasupo (LICONSA), began its activities in November 1986, producing embryos of the Holstein Friesian, Brown Swiss, Jersey, Simmental, Simbrah, and F1 Holstein x Gyr dairy (Gyrholando) and Holstein x Guzerat breeds. It was estimated that, by 1993, 20,000 embryos per year would be produced from 2,200 donors. The center ceased operations in 1994; between 1987 and 1993 it produced almost 42,000 embryos. In addition, 18 training courses in the technique were given to approximately 300 professionals, and work was carried out for 23 Bachelor's and 4 Master's degree theses. Most of the embryos produced were frozen and subsequently transferred to different states of Mexico(134). In 1990, the National Center for Animal Reproduction became part of the National Commission for Genetic Improvement and Animal Reproduction (CONAMEGRA), established through an agreement between the Ministry and the National Livestock Confederation (CNG). Several TE works were carried out for farmers in different parts of the country, and an agreement was established with LICONSA for the commercialization, between 1993 and 1994, of 1,343 embryos produced at CEMEGEN, at a price of $100.00 per embryo ―an amount well below the cost of production. CONAMEGRA sold this genetic material to 33 producers from 11 Mexican states, its own technicians thawed 447 embryos, transferred them, and obtained 51 % of pregnancies(135). In 1993, INIFAP received a donation of 1,558 embryos from CEMEGEN; these were F1 and ¾ Holstein x Zebu embryos, destined for the genetic programs of its experimental stations in the tropics. That same year, 800 embryos and 300 head of cattle were donated to the College of Posgraduates (Colegio de Posgraduados). In 1994, LICONSA terminated the loan agreement with UNAM, auctioned the livestock, dismissed and paid off its staff, and returned its facilities to UNAM, thus putting an end to the operation of CEMEGEN(135). The collection and transfer of embryos was initially a very complex process, since both the collection of embryos from the donor and their subsequent transfer to recipient females was done by surgical methods and using general anesthesia; this involved an enormous logistical effort, as the donors and the recipients had to be prepared for surgery at the same time(132). In the first instance, both donor and recipient surgeries were performed under local anesthesia; subsequently, procedures for the non-surgical collection of embryos and their transfer to recipients were developed, which facilitated the more widespread use of the technique(136). The results obtained using non-surgical methods gradually approached those obtained with surgical methods, which is why the latter fell into disuse(131). 57

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Regarding ovarian stimulation to induce multiovulation, there has always been a high variability in the superovulatory response, despite efforts to control the sources of variation, which is one of the main problems affecting the efficiency and profitability of MOET at the commercial level(137). A study carried out with successive superovulations in zebu cows in the "El Macho" experimental station, INIFAP, where the variables of time, FSH dose and age were considered, concluded that the initial response of a zebu cow to superovulation cannot be said to be a good predictor of subsequent responses by the same cow(138). To date, there have been considerable advances in the study of ovarian physiology, as well as factors inherent to the donor; however, there remain some aspects to be understood in order to generate more efficient ovarian stimulation protocols(139). In its beginnings, superovulation was induced with eCG; however, a few years later it was discovered that crude pituitary extract (FSH with 20% LH) generated a better superovulatory response than eCG(140). Pituitary extracts are now widely used; there is a high variability in the amount of LH present in crude extracts, while purified extracts have less variation in the amount of LH. Multiple studies have been conducted to evaluate in vivo embryo production using crude and purified extracts(131). In a 1995 study conducted at CEMEGEN in Mexico, the crude extract was found to produce more embryos than the purified extract in dairy cattle(141). Conversely, in a study conducted in 2014 at INIFAP with beef cattle, the purified extract generated more embryos than the crude extract(142); this may be due to the fact that the hepatic metabolism of dairy cattle is more accelerated compared to that of beef cattle(143). Despite many efforts to increase embryo production per cow per year by increasing ovarian response to superovulatory treatment, little progress has been made(131). The use of PIDR in combination with BE and prostaglandins has allowed the development of a protocol called Rapid Donor Recycling. This protocol reduces the interval between superovulations by almost half (33 to 35 d) with no decline in response, either in the number of embryos produced or in their quality, in successive superovulations for up to one and a half years(131,142). Thus, the production of transferable embryos per donor per year is nearly doubled. The first successful cryopreservation of embryos was reported in murines in 1972(144). One year later, the first calf was born from a previously cryopreserved blastocyst using a 2M dimethyl sulfoxide solution with a freezing and thawing rate of 0.2 °C/min and 36 °C/min, respectively(145). The first calves born from cryopreserved embryos in Mexico came from embryos frozen in Colorado, USA, and transferred to Nayarit, Mexico, into an experimental station of INIFAP(146). For more than 10 yr, glycerol was the cryoprotectant of choice for bovine embryo cryopreservation. However, in 1992, a direct transfer cryopreservation system using ethylene

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glycol as cryoprotectant was announced. This had a rapid and positive effect worldwide on the embryo transfer industry to the present day(131). According to data from the American Embryo Transfer Association, in 2009, approximately 99 % of the beef bovine embryos and 94 % of the dairy bovine embryos were frozen in ethylene glycol for direct transfer(131).

In vitro production of bovine embryos (IVP) IVP biotechnology has a variety of applications in basic and applied science; in the first instance, it has supported the production of embryos used for a wide variety of research, including the treatment of infertility in humans. It is certainly a tool to increase the productivity of farm animals by increasing the reproductive potential of animals of higher genetic merit; in addition, it plays a relevant role in the conservation of the genetic resources of animals, especially endangered ones(147). Generically known as IVF (in vitro fertilization) or IVP (in vitro production), it is a reproductive biotechnology, which, like MOET, is composed of several biotechnologies such as: in vitro maturation of eggs, sperm capacitation, in vitro fertilization, and the culture of zygotes and embryos up to pre-eclosion stages (7 to 8 d post-fertilization)(148). IVP consists of retrieving eggs or oocytes from ovarian follicles to be matured and fertilized under laboratory conditions; the resulting zygotes are cultured until post-compaction stages (morula or blastocyst), at which time they are transferred to a recipient cow or cryopreserved for subsequent transfer. Oocytes can be drawn from trace ovaries or live animals, by means of ultrasound-guided transvaginal aspiration (TVA)(148). This biotechnology dates back to the 1970s, thanks to which research and achievements in the areas of culture media development, oocyte maturation, sperm capacitation and fertilization (which occurred in that decade and the following one(126)) led to the birth, in 1987, of the first calf produced entirely in vitro(149). Although this biotechnology was initially oriented primarily to research and was based on tests carried out on ovaries obtained at the slaughterhouse, with the incorporation of TVA (making it possible to obtain immature oocytes from living donors), the commercial application was seen as a more promising tool than MOET for the mass production of offspring from progenitors of high genetic merit. The above is confirmed by observing that, worldwide, while the number of embryos collected in vivo and transferred has remained stable in recent years, the number of transferred IVF embryos has had an average annual growth rate of 12 % and, for the first time, in 2017, the number of viable embryos produced in vitro, exceeded the number of transferable embryos

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collected in vivo(150); that trend has continued as of the 2019 International Embryo Technology Society (IETS) report, with data from 2018(151). In this same report, it is noted that the vast majority of viable IVF embryos were obtained from oocytes collected through TVA (98.9 %), in contrast to the few embryos obtained from trace ovaries(151). The reason for this is the uncertainty of obtaining germplasm suitable for commercial production from the genetic and sanitary point of view. It is known that there are a significant number of research laboratories where viable IVP embryos are produced and discarded once they have fulfilled their function; however, this datum is not recorded by the IETS statistics committee(152). Globally, there are two events that have been driving forces in IVP: sexed semen biotechnology and genomic evaluations. The first is that thanks to IVP it is possible to maximize the use of sexed semen by fertilizing a large number of oocytes (100+) with a single dose of semen, and the second is that it makes it possible to intensify the power of selection by shortening generation intervals (TVA in calves, enabling the use of their germplasm before the age of service) and increasing the reproductive capacity on the maternal side. Thus, these three biotechnologies (IVP, sexed semen, genomic evaluations) play a relevant role in genetic improvement and the cattle industry in general(152). IVP had a period of great growth at the beginning of this century, especially in Brazil, where, in 2009, 85 % of the available embryos came from in vitro production; this amount was equivalent to 50 % of the worldwide production. The success of the Brazilian companies encouraged their expansion to other Latin American countries, including Mexico, where they settled, working independently or in partnership with Mexican companies or producer organizations. It has not been possible to replicate what has been achieved in Brazil in Mexico because this country does not have the competitive advantages that Brazil has (breeds, demand, size of production units, availability of receivers)(148). Nevertheless, there continues to be moderate activity by these and other domestic companies; thus, in 2018, the transfer of almost 28,000 embryos was reported in Mexico, almost all from IVP, and, in contrast, only over 4,000 embryos obtained in vivo were transferred(151). Although a considerable amount of research has been generated regarding the main components of this biotechnology (development of sequential culture media, control of potentially toxic agents, exclusion of serum components, inclusion of amino acids, vitamins, chelating agents and hormones, among others)(153-156), and so has research in oocyte maturation and in vitro fertilization processes(152-157), the fact is that it has not been possible to exceed the limit of 40 to 50 % of blastocysts obtained from fertilized oocytes, a value not very different from the 30 to 40 % that existed 20 yr ago. This has drawn attention to the lack of homogeneity in the oocyte source as the most likely cause of limited IVP success rates(155,157,158). It is therefore imperative to continue to promote IVP-IVT (in combination with other biotechnologies such as the use of sexed semen and genomic evaluations) as a 60

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valuable productive tool, and also to continue to conduct research for IVP in larger quantities with respect to the eggs left to mature, which achieve a better post-transfer development and have greater tolerance to cryopreservation. Due to the complexity and high requirements of infrastructure and equipment, as well as personnel trained in this biotechnology, IVP research in Mexico has been incipient. Only a handful of universities ―namely, Universidad Nacional Autónoma de México, Universidad Autónoma Metropolitana, Universidad Veracruzana, Universidad Autónoma de Nuevo León, Universidad Autónoma de Tamaulipas, Colegio de Posgraduados, and Universidad Autónoma de Chihuahua―, some producer organizations and livestock companies, and INIFAP have worked on certain IVP research topics. The following are some of the results obtained in research conducted in Mexico on topics related to bovine IVP: in relation to agents and culture conditions to reduce the production of reactive oxygen species (ROS) in the culture medium, in a study conducted at INIFAP, it was found that by reducing the O2 tension to 2 %, the number of cells in the blastocysts increased and the production of ROS was reduced(159). In another study also carried out at INIFAP, pterostilbene, a phytoalexin, was used as an antioxidant agent at a concentration of 0.33 μM, and it was found to reduce the production of ROS and the occurrence of intracytoplasmic lipids in 7-d-old embryos(160). The latter is presented as an alternative for improving IVP embryo cryopreservation. In a collaborative work between INIFAP and UANL, the effect of the addition of a heat shock protein (HSP70) on the development of bovine embryos produced in vitro was evaluated, and the addition of HSP70 to the culture media was found to have favorable effects on the percentage of blastocysts and cell number(161). A system for individual embryo culture called "WOW" was also evaluated at INIFAP, and, although it produces a similar number of embryos compared to group culture in microdroplets, the WOW system was found to produce a higher percentage of embryos with a better morphological quality(162). In another study carried out at UNAM, the effectiveness of two vitrification devices for cryopreserving bovine embryos was compared. Cryotop® was found to be a more effective vitrification support than Open Pulled Straw®, resulting in higher post-vitrification viability(163). Another study carried out between the Colegio de Posgraduados and the Universidad Veracruzana tested the alternative of utilizing the culture media used in the human IVF system to cultivate bovine embryos and found that it is possible to produce blastocysts with similar results(164). It should be noted that the culture media used in humans are often more readily available on the domestic market. Yet another study, carried out at the Autonomous University of Chihuahua, proved that the addition of IGF-I at different times during the IVP process did not produce a beneficial effect on the percentage of blastocysts(165). The future of IVP in cattle faces important challenges that must be resolved in order to ensure its usefulness as a productive tool, as well as support for research in multiple areas of knowledge. The evolution of culture media to provide near-physiological conditions for gametes and embryos has been one of the areas with the greatest progress(152), despite which, 61

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difficulties are still encountered today in producing similar embryos to those obtained in vivo, resulting in low pregnancy rates at transfer, low tolerance to cryopreservation and alterations in fetal and placental development(157). Research efforts have focused on the development of chemically defined culture media(166), use of oxidation level regulating compounds(167), use of delipidating compounds or lipid metabolism regulators(160,168), and mechanisms such as the use of sequential media to remove the presence of molecules that are toxic to the embryo(169), as well as on the development of cryopreservation methods that are friendlier to the embryo produced in vitro, such as vitrification(170). On the other hand, with the development of biotechnologies such as embryonic nuclear transplantation (16-cell stage blastomers) and somatic transplantation (cumulus cells, fibroblasts, etc.)(171), an important future was foreseen for the genetic modification of embryos for productive, medical and research purposes (gene function studies, xenotransplantation, recombinant protein production, genetic improvement, and food production)(172) and their eventual cloning. However, despite some encouraging results, the random insertion of the transgenes generated highly variable and unpredictable results, rendering the use of this technology unviable(173). Today, with the advent of gene editing technologies (CRISPR - Cas - 9), it is already possible to perform precise gene editing, including epigenetic reprogramming, which augurs a golden age in the genetic modification of farm animals for the aforementioned purposes, with the strong support of IVP biotechnologies(174). There will still be ethical impediments and legal pitfalls to be resolved.

Conclusions As a final reflection, it can be said that, although research institutions in Mexico, and especially INIP-INIFAP, have accompanied the development of reproductive biotechnologies in the world in the last five decades. With the passage of the years, these have evolved meteorically, and it has been increasingly difficult to maintain a solid research base that allows us to be aligned with the technological developments that are taking place. Although the information generated by Mexican institutions on current topics such as in vitro embryo production is modest, the national livestock industry is demanding immersion in these technologies. This should encourage research institutions to generate technological components that will allow the efficient use of this and other technologies under local conditions.

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125. Álvarez GH, Urban DD, Pérez RS, De La Torre SJF. Validación del ensayo de evaluación de semen bovino mediante un sistema “CASA” (Computer Assited Sperm Analyzer). VII Congreso Virtual Iberoamericano sobre la Gestión de Calidad en Laboratorios IBEROLAB. Madrid, España. 2014:259-260. 126. Moore SG, Hasler JF. A 100-Year Review: Reproductive technologies in dairy science. J Dairy Sci 2017;100(12):10314-10331. 127. FAO. Cryoconservation of animal genetic resources. FAO: Rome. Animal Production and Health Guidelines No. 12. 2012.. 128. Villaseñor GF, De La Torre SJF, Martínez VG, Álvarez GH, Pérez RS, Palacios FJA, et al. Caracterización de la respuesta ovárica a la superovulación en bovino criollo coreño utilizando dosis reducidas de FSH. Rev Mex Cien Pecu 2017;8(3):225-232. 129. Heape W. Preliminary note on the transplantation and growth of mammalian ova within a uterine foster-mother. Proc Roy Soc London B 1890;48:457-9. 130. Willett EL, Black WG, Casida LE, Stone WH, Buckner PG. Successful transplantation of a fertilized bovine ovum. Science 1951;113:247. 131. Hasler JF. Forty years of embryo transfer in cattle: a review focusing on the Journal Theriogenology, the growth of the industry in North America, and personal reminiscences. Theriogenology 2014;81(1):152-69. 132. Cerda AA. Crecen saludables en México los primeros becerros de probeta. El sol del campo 22 de abril 1980:1-4. 133. Asprón PMA. Evolución histórica de la transferencia de embriones. Revista Cebú 1989;15(3):47-54. 134. Sánchez AA, Ramírez CJ. Apoyos de LICONSA a los ganaderos lecheros en diferentes regiones de México. En: Martínez BE, et al, coord. Dinámica del sistema lechero mexicano en el marco regional y global. México: Ed. Plaza y Valdés; 1999:270-291. 135. México Holstein. CONAMEGRA tutela el mejoramiento genético de la ganadería. 1994;25:29-31. 136. Elsden RP, Hasler JF, Seidel GE Jr. Non-surgical recovery of bovine eggs. Theriogenology 1976;6(5):523-32.

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137. Córdoba SLA. Superovulación inducida en ganado bovino. Téc Pecu Méx 1988;26(1):109-119. 138. De La Torre SJF, Castro LMA, González PE, Reynoso CO. Respuesta de vacas cebú a superovulaciones sucesivas con FSH. Téc Pecu Méx 1992;30(3):223-231. 139. Mikkola M, Hasler JF, Taponen J. Factors affecting embryo production in superovulated Bos taurus cattle. Reprod Fertil Dev 2019;32(2):104-124. 140. Bó GA, Mapletoft RJ. Historical perspectives and recent research on superovulation in cattle. Theriogenology 2014;81(1):38-48. 141. Martínez BS, Sánchez AA, Anta JE, Berruecos VJM, Valencia MJ. Valoración de dos hormonas folículo estimulantes comerciales usadas en la superovulación de vacas en lactación y vaquillas en ganado lechero. Téc Pecu Méx 1995;33(1):34-38. 142. Polanco SR. Efecto de dos preparaciones de FSH-LH al inducir multiovulación usando reciclado rápido y tradicional en donadoras Brangus rojo [tesis maestría]. Xalisco, Nayarit, México: Universidad Autónoma de Nayarit; 2014 143. Hart CG, Voelz BE, Brockus KE, Lemley CO. Hepatic steroid inactivating enzymes, hepatic portal blood flow and corpus luteum blood perfusion in cattle. Reprod Domest Anim 2018;53(3):751-758. 144. Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos, frozen to -196°C and -289°C. Science 1972;178:411–414. 145. Wilmut I, Rowson LEA. Experiments on the low-temperature preservation of cow embryos. Vet Rec 1973;92:686–690. 146. de los Santos-Valadez S, Tervit HR, Elsden RP, Seidel GE. Transport of frozen cattle embryos from U.S.A. to Mexico. Theriogenology 1981;15(1):123. 147. Bavister BD. Early history of in vitro fertilization. Reproduction 2002;124:181-196. 148. Vera AHR, Santos ER, Hernández MJH, Gutiérrez ACG, de la Torre SJF, Álvarez GH, et al. Eficiencia reproductiva de los bovinos en el trópico. En: Estado del arte sobre investigación e innovación tecnológica en ganadería bovina tropical. 2ª ed. Ciudad de México, México: Red de investigación e innovación tecnológica para la ganadería bovina tropical. CONACYT; 2018. 149. Lu KH, Gordon I, Chen HB, McGovern H. In vitro culture of early bovine embryos derived from in vitro fertilization of follicular oocytes matured in vitro. Proc. Third Meet. Eur. Embryo Transf. Assoc. Lyon, France; 1987.

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150. Viana JHM. 2017 Statistics of embryo production and transfer in domestic farm animals: Is it a turning point? In 2017 more in vitro-produced than in vivo-derived embryos were transferred worldwide. Embryo Transfer Newsletter 2018;36(3):8-25. 151. Viana JHM. 2018 Statistics of embryo production and transfer in domestic farm animals, embryo industry on a new level: over one million embryos produced in vitro IETS Data Retrieval Committee. Embryo Technology Newsletter 2019;36(4). 152. Ferré LB, Kjelland, ME, Strøbech LB, Hyttel P, Mermillod P, Ross PJ. Review: Recent advances in bovine in vitro embryo production: reproductive biotechnology history and methods. Animal 2020;14:991-1004. 153. De La Torre SJF, Gardner D, Preis K, Gibbons, J, Seidel, GE. Metabolic regulation of in-vitro-produced bovine embryos. II. Effects of phenazine ethosulfate, sodium azide and 2,4-dinitrophenol during post-compaction development on glucose metabolism and lipid accumulation. Reprod Fert Develop 2006;8:597-607. 154. Lane M, Gardner D. Embryo culture medium: which is the best?. Best Pract Res Clin Obstet Gynaecol 2007;21(1):83-100. 155. Sanches BV, Zangirolamo AF, Seneda MM. Intensive use of IVF by large-scale dairy programs. Anim Reprod 2019;16(3):394-401. 156. Ferré LB, Kjelland ME, Taiyeb AM, Campos CF, Ross PJ. Recent progress in bovine in vitro‐derived embryo cryotolerance: Impact of in vitro culture systems, advances in cryopreservation and future considerations. Reprod Dom Anim 2020;55:659-676. 157. Patel O, Bettegowda A, Ireland J, Coussens P, Lonergan P, Smith G. Functional genomics studies of oocyte competence: evidence that reduced transcript abundance for follistatin is associated with poor developmental competence of bovine oocytes. Reproduction 2007;133(1):95-148. 158. Sirard MA. 40 years of bovine IVF in the new genomic selection context. Reproduction 2018;156:R1–R7. 159. Delgado TGA. Efecto de diferentes niveles de oxígeno en la atmósfera de cultivo y la adición de un antioxidante comercial en el desarrollo de embriones bovinos producidos in vitro [tesis maestría]. Nayarit, México: Universidad Autónoma de Nayarit; 2013. 160. Sosa F, Romo S, Kjelland M, Álvarez GH, Pérez RS, Urbán DD, et al. Effect of pterostilbene on development, equatorial lipid accumulation and reactive oxygen species production of in vitro-produced bovine embryos. Reprod Dom Anim 2020;55:1–11.

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161. Garza AAJ, Pérez RS, Urbán DD, Cervantes VR, De La Torre SJF. Adición de la proteína de choque térmico HSC70 en medios de cultivo sobre la producción in vitro de embriones bovinos. Reunión Nacional de Investigación Pecuaria, Chiapas, México: 2019:120-122. 162. Virrueta MLM. Evaluación de dos sistemas de cultivo in vitro de embriones bovinos [tesis licenciatura]. Saltillo, Coahuila, México: Universidad Autónoma Agraria Antonio Narro; 2018. 163. Morales PF. Viabilidad post-criopreservación de embriones bovinos producidos in vitro y vitrificados mediante las técnicas de “open pulled straw” y cryotop [tesis maestría]. Ciudad de México, México; Universidad Nacional Autónoma de México; 2013. 164. Ahuja AC, Montiel PF, Pérez HP, Gallegos SJ. Medio alternativo para la producción in vitro de embriones bovinos. Zootecnia Trop 2009;27(3):277-284. 165. Martínez GO, Antillón RJ, Rodríguez AFA. Tasa de fertilización, desarrollo y calidad de embriones bovinos Holstein producidos in vitro con semen sexado y adición de IGFI. Tecnociencia Chihuahua 2020;9(3):140-147. 166. Van der Valk J, Brunner D, De Smet K, Svenningsen ÅF, Honegger P, Knudsen LE, et al. Optimization of chemically defined cell culture media–replacing fetal bovine serum in mammalian in vitro methods. Toxicol In vitro 2020;24(4):1053-1063. 167. Sovernigo TC, Adona PR, Monzani PS, Guemra S, Barros FDA, Lopes FG, et al. Effects of supplementation of medium with different antioxidants during in vitro maturation of bovine oocytes on subsequent embryo production. Reprod Dom Anim 2017;52(4):561569. 168. Torres V, Hamdi M, Millán de la Blanca MG, Urrego R, Echeverri J, López‐Herrera A, et al. Resveratrol–cyclodextrin complex affects the expression of genes associated with lipid metabolism in bovine in vitro produced embryos. Reprod Dom Anim 2018;53(4):850-858. 169. Moore K, Rodriguez-Sallaberry CJ, Kramer JM., Johnson S, Wroclawska E, Goicoa S, et al. In vitro production of bovine embryos in medium supplemented with a serum replacer: effects on blastocyst development, cryotolerance and survival to term. Theriogenology 2007;68(9):1316-1325. 170. Do VH, Catt S, Kinder JE, Walton S, Taylor-Robinson AW. Vitrification of in vitro derived bovine embryos: targeting enhancement of quality by refining technology and standardising procedures. Reprod Fert Dev 2019;31(5):837-846.

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https://doi.org/10.22319/rmcp.v12s3.5866 Review

Main contributions of INIFAP research to swine nutrition in Mexico: challenges and perspectives

José Antonio Rentería Flores a † Sergio Gómez Rosales a Luis Humberto López Hernández a Gerardo Ordaz Ochoa a Ana María Anaya Escalera a César Augusto Mejía Guadarrama a Gerardo Mariscal Landín a*

Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias. Centro Nacional de Investigación en Fisiología y Mejoramiento Animal. km 1 Carretera a Colón, 76280 Ajuchitlán, Querétaro, México.

*Corresponding author: mariscal.gerardo@inifap.gob.mx

Abstract: This review is a retrospective of the research activities carried out in swine nutrition by INIFAP researchers during the 35 years of the Institute's existence. The main product of this activity was to lay the foundations for a better feeding of the breeding herd and pigs for slaughter, focusing on solving the particular problems of Mexican swine farming, with respect to the raw materials that are utilized in pig feeding, as well as the evaluation and improvement of the carcass quality, and the enrichment of meat with metabolites that increase its shelf life and improve its organoleptic properties. It also reflects on the challenges that swine nutrition will face in this century, proposing the areas that will have to be researched in order to guarantee the sustainability of the sector, and the actions that the

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program's researchers and INIFAP will have to take in order to adequately respond to the challenges faced by Mexican swine production. Key words: Swine nutrition, Challenges, Contributions, INIFAP.

Received: 16/11/2020 Accepted: 22/03/2021

Introduction Research in swine nutrition is important, since this species is characterized by its high prolificacy, short production cycle and high feed efficiency ―characteristics that have made it the most consumed meat in the world, representing 36 % of the market(1), and the second at the national level, representing 26 % of the country's meat consumption(2). In Mexico, livestock research began in 1947, with the creation of the Livestock Institute (Instituto Pecuario) under the Ministry of Agriculture and Livestock (Secretaría de Agricultura y Ganadería); in 1962, it became the National Livestock Research Center (Centro Nacional de Investigaciones Pecuarias, CNIP). In 1968, this became the National Institute of Livestock Research (Instituto Nacional de Investigaciones Pecuarias, INIP), which, in 1985, merged with the Institutes of Agricultural and Forestry Research, giving rise to the National Institute for Research on Forestry, Agriculture, and Livestock (Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, INIFAP). The first four articles on swine nutrition appeared in 1966; were published in issue No. 7 of Técnica Pecuaria en México (Livestock Technology in Mexico), a journal created in 1963 by CNIP, which in 2010 was transformed into Revista Mexicana de Ciencias Pecuarias (Mexican Journal of Livestock Sciences). From that time to date, the Institute's staff has conducted research that responds to the needs of the national swine industry, either by generating the necessary technology for the use of raw materials in the different production phases or by studying the impact of the feed on the quality of the product, as well as the impact of feeding on reproductive efficiency and productivity of the breeding herd. Throughout this period, INIFAP's research has contributed to the development of the national swine industry and its value chain. This manuscript summarizes INIFAP's main contributions to swine nutrition and the medium and long-term challenges faced by research across the world and particularly in Mexico. For the selection of the material, it is considered what was generated from 1985 to date by INIFAP researchers, in both printed and digital material, giving preference to published scientific articles and, in the second place, to publications in theses and conferences. The information was organized

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into breeding herd and slaughter pig nutrition, from weaning to finishing, as well as carcass yield and meat quality.

Breeding herd Litter size, the number of farrowings per sow per year and the number of pigs produced during the productive life of the sow are the parameters that establish the basis for the productivity and profitability of a pig farm. Scientific and technological advances in the disciplines of genetics, nutrition, reproduction, and animal health have made it possible to increase these variables over the last 35 yr.

Current situation

In most of the technified farms, 30 pigs per sow are expected to be weaned every year, and each sow is expected to produce more than 70 pigs for slaughter throughout her productive life. In the case of Denmark in particular, the total number of piglets weaned per litter increased from 9.9 in 1996 to 12.2 in 2009(3), while a more recent report mentions that the number of piglets weaned per litter for this country in 2017, was 14.6, which is equivalent to 33.3 piglets weaned per sow per year(4). The statistical evaluation of swine production in Mexico is oriented toward the inventory of pigs, production by state, imports and exports, costs of inputs and raw materials, price of live pigs and primal cuts. The few herd productivity analysis data available in Mexico are isolated reports from farms where detailed records of herd performance are available. When analyzing data for first and second farrowing sows of four commercial farms, Ek-Mex et al(5) found variation in the number of piglets born alive per sow per year, ranging between 17.4 and 27.2 piglets. On the other hand, Pérez Casillas(6) presented data from four commercial farms where the average number of total piglets born was 14.96; piglets born alive 13.59, and piglets weaned 11.58 ―values similar to those reported by PigChamp in 2019, for U.S. farms.

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Issues

Although the increase in reproductive efficiency has meant greater profitability for swine farms, it also represents new challenges, among which are the development of precision feed and feeding schemes that allow expressing the genetic potential in the different physiological stages, improving their permanence in the herd, and maintaining the sustainability of swine farms. Inadequate nutrient intake can influence reproductive response in several ways; it may disrupt the ovulation process and delay the onset of puberty in gilts. Overfeeding during gestation causes excessive weight gain, interferes with normal mammary gland development, and has a negative impact on milk production and voluntary feed intake during lactation. The increase in litter size and, consequently, in the demand for milk production, results in a nutritional deficit for the lactating sow, which can lengthen the duration of the weaning-estrus interval, reduce the ovulatory rate and the quality of the oocytes released, as well as the development of the corpora luteae, during the first post-weaning estrus. This is likely to result in a reduction of the ability of the embryos to develop and survive, negatively impacting litter size at the subsequent parturition. These issues are a major cause of culling, particularly in gilts, because they make it difficult to maintain the integrity of farrowing groups in the farm and induce a reduction in the number of piglets produced annually per sow(7). Pubertal sow feeding The feeding of replacement sows during their growth influences the age at which they start their reproductive life (puberty) and the number of eggs released (ovulation rate) during the first estrous cycles, as well as the conformation of their bone, muscle and fat structure. According to research by INIFAP, the feeding of replacement sows should start at 75 kg/120 days of age, limiting daily weight gain to 700 g/d, for which energy intake should be restricted to 8 Mcal of ME/d, using a feed with 0.78 % of digestible lysine and a protein level of no more than 17 %(8). Cyclic sow feeding Nutrition influences the reproductive function of sows, particularly affecting the ovulation rate, but there is little information on the specific effect of an energy or protein deficit on embryo survival. Mejía-Guadarrama et al(9) reported that protein restriction in cycling nulliparous sows decreases plasma urea concentrations without affecting ovulation rate. Increased maternal muscle mass at delivery cushions the negative impact of moderate protein

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restriction on milk production(10); however, when this restriction is severe, there are repercussions on reproduction(11-13). Feeding of the pregnant sow Globally, the competition between animal production and human beings for basic grains is worrisome; therefore, one of the lines of research that INIFAP has developed is the use of alternative ingredients and nutritional strategies that guarantee the provision of adequate nutrients to the pregnant sow, taking advantage of industry by-products and local inputs, both conventional and non-conventional. Dehydrated alfalfa and corn, sorghum or grass silage can be used as the complete ration for the pregnant sow; likewise, the substitution of 33.3 % of sorghum with cassava plant meal can be used as a complete ration(14) or the addition of 3 kg/d of cassava silage(15) improve sow weight gain during the last third of gestation, provided that they are fortified with vitamins, minerals and protein supplements(16-18), to ensure that, in the last third of gestation, the energy requirement will be met. Regarding the use of molasses as an energy source, Ángeles and Cuarón(19) report longer sow lifespan and improved productivity due to the use of ketogenic substrates. By evaluating the addition of different sources and levels of soluble and insoluble fiber to the diet of pregnant sows, the fiber composition was shown to differentially affect energy digestibility and the dietary nitrogen levels(20); therefore, it is important to know what type of fiber is being used. The 78 % of the variation in the digestibility of energy in a diet could be explained by the intake of soluble and insoluble fiber: AED= 88.74+0.083(SF)- 0.02(ISF); (P<0.01; R2=0.78), where: AED is the apparent energy digestibility, SF is the soluble fiber intake in grams, and ISF is the insoluble fiber intake in grams. Rentería et al(20) proved that the inclusion of insoluble fiber decreased apparent energy digestibility by 0.2 % and apparent nitrogen digestibility by 0.1 % at an insoluble fiber intake of 250 g/d equivalent to 13.5 % insoluble fiber in the diet(21). It was also shown that soluble fiber intake did not affect nitrogen digestibility, while energy digestibility was positively related to soluble fiber intake. This means that it is possible to include high levels of fiber in the diet of pregnant sows without compromising their productivity, provided that the influence of fiber on nutrient digestibility is considered in the formulation of the diet and in the calculation of the daily ration(22). Besides, the inclusion of fiber in the diet of the pregnant sow helps to mitigate the impact of the dietary restriction to which she is subjected and improves feed intake in lactation. Feeding of the lactating sow In general, a decrease in energy intake during lactation does not affect milk production, which is maintained thanks to the mobilization of maternal body reserves(23). However, if energy restriction is particularly severe (6.5 vs 16.5 Mcal ME/day), litter growth is reduced(24).

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For the lactating sow, the amino acid intake is calculated according to the lysine intake, which is the first limiting amino acid. Protein intakes are expressed as the amount of crude protein or lysine provided in the feed. Litters of sows subjected to protein restriction during lactation (300-400 g of protein vs. 700-900 g/d), showed a reduction in their growth rate(25), especially from the third week of lactation onwards(26). The decrease in litter growth rate is due to a reduction in the amount of milk produced, as well as to a low export of protein and lipids in the milk of rationed sows(27). However, other authors(10) observed no significant effects of severe protein restriction (350 to 410 g protein vs 830 g/d) on litter growth and milk composition. These contrasting results could be explained by differences in the state of the body reserves of the sows at the beginning of lactation, since a greater maternal muscle mass at farrowing cushions the negative impact of protein restriction on milk production(10-12). Milk production depends on the quantity and quality of feed consumed during lactation, as well as on the sows' ability to mobilize their body reserves. The level of lysine intake and metabolizable energy interact to influence milk production(28). Research conducted by INIFAP on lactating sows has been aimed at improving the consumption of energy, amino acids, vitamins and minerals, which are essential in this phase, as well as developing feeding programs and techniques that promote daily feed consumption to minimize excessive loss of weight, protein and body fat, in order to improve reproductive parameters and reduce the high rate of culling(8). Research conducted at INIFAP shows that the addition to the diet of sucrose, molasses(19,29,30), molasses-cassava plant(15), or chromium picolinate(31,32) can contribute to partially diminish the problem of low voluntary feed intake and reduced litter size at the next parturition, provided that the lysine level of the diet is taken into account(33-35); however, the best strategy to avoid this problem is to avoid overfeeding the sow during gestation. The digestible lysine demand for the highest productivity is 49 to 56 g/d(21). When the dietary lysine concentration (0.85 to 1.05 %) was evaluated in sows during their first lactation, litter weight gains in excess of 2.5 kg/day (21-d weaning litter weight ≥67 kg) were reported when the digestible lysine intake was 45 g/d, and the response increased to a digestible lysine intake of 66.2 g/d(21). With respect to mineral intake in lactating sows, selenium has been researched as an important mineral (0.2 to 0.3 ppm) because of its relation to production, the use of "organic" selenium is recommended due to its better distribution in peripheral tissues(36). With respect to vitamin intake, it was observed that the inclusion of β-carotene (250 mg/kg) before the first service and during lactation increases embryonic survival(8), and the use of 4 mg/kg of 25-OH-cholecalciferol contributes to structural robustness and cell differentiation in reproductive activity(8).

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Restarting sexual activity at weaning

Nutritional restriction during lactation can influence the post-weaning reproductive performance of the sow, inducing, firstly, an increase in the weaning-estrus interval, and in the case that the sow becomes pregnant, an increase in embryonic mortality and a decrease in litter size at the subsequent farrowing(28). Several authors report a 39 % decrease in the number of preovulatory follicles (≥ 7 mm in diameter) during the 3 to 4 d after weaning in gilts subjected exclusively to protein restriction (approximately 460 g/d), suggesting that the ovulatory rate of these sows may be negatively affected(37). For their part, MejiaGuadarrama(10,11) and Quesnel(12) established that a protein deficit during lactation does not affect embryonic survival, but it does decrease the ovulatory rate at the first post-weaning estrus in gilts, in contrast to the findings of other authors(38). This discordance is probably explained, at least partially, by the difference in the ovulatory capacity of the sows used in these studies, 20 eggs or more vs 12-15 eggs, which could accentuate the negative impact of the protein deficit on the ovulatory rate in sows with a high ovulatory potential.

Nutrition of pigs for slaughtering Piglet feeding

Current situation Weaning is one of the most stressful events in the life of the pig, predisposing it to digestive disorders in the short and medium term and negatively affecting its productivity and survival; at this stage, mortality levels of 6 to 20 % are reported(39). This mortality is linked to different stress factors associated with weaning, such as change of feed or environment, separation from the mother, interaction with piglets from other litters, and the presence of pathogens. The change of feed is relevant because, by replacing milk with a solid feed from different sources and with different proportions of nutrients derived from cereal grains and oilseed pastes, which contain starches and complex proteins, the piglet does not digest it fully because its digestive capacity has not yet reached sufficient maturity to assimilate the nutrients. In addition, sometimes these ingredients may contain antigens or anti-nutritional factors, which cause lower digestibility of the solid feed, resulting in mechanical or infectious diarrhea(40,41).

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At weaning, piglets are exposed to different types of pathogens, some of which are part of the normal microbiota of the digestive tract. Likewise, the immune system associated with the intestinal mucosa (innate and adaptive) is immature in piglets weaned at 21 to 28 d of age, due to the changes undergone by the surface of the intestinal mucosa as a result of weaning. At this stage, cortisol concentrations in the blood are also elevated, causing additional immunosuppression(42,43). This is why young pigs are susceptible to a number of bacterial diseases, including colibacillosis, caused by enterotoxigenic Escherichia (E). coli serotypes, and salmonellosis, caused by Salmonella spp(44). On the other hand, piglets exhibit a low and erratic feed intake, which causes delayed or total suspension of stomach motility, congestion of the intestinal blood vessels affecting their lining with hemorrhages and ulcerations, edema and inflammation with the presence of immature epithelial cells on the absorption surface. This leads to the deterioration of the protective function of the intestinal mucosa, because the protein matrix found in the intercellular spaces is weakened by the inflammation of the intestine, increasing the permeability of the epithelium(40,41). After a few hours of fasting and starvation, piglets may consume feed in quantities above their digestion capacity, which together with changes in mucosal permeability may lead to incomplete digestion of feed, causing osmotic and pH changes in the intestine which induce increased secretions into the intestinal lumen and result in increased motility and the presence of diarrhea. Also, undigested feed components can serve as a substrate for the growth of pathogenic microorganisms, increasing the incidence and severity of diarrhea, which can be reflected in growth retardation in the piglets(45). The duration and magnitude of growth depression caused by the above factors can vary from 7 to 14 d, depending on the age and physical condition of the piglet at weaning, the quality of the feed, the feeding program, and the environmental and sanitary management. Although most piglets manage to overcome the recession and continue to grow, there is evidence that pigs that are stunted at this stage have larger amounts of subcutaneous fat deposits and lower amounts of protein in the carcass at later stages than pigs that were not affected in their growth. It follows that post-weaning productive capacity is affected by the magnitude of the reduction in post-weaning growth rate. From the nutritional point of view, in order to shorten post-weaning growth retardation, it is necessary to formulate diets with highly digestible ingredients, and with the appropriate nutrients for the weight and age of the animals, to design a feeding program that considers the digestive development of the piglet, as well as the use of feed additives that favor the integrity of the mucosa. This results in better feed utilization and consequently better piglet productive performance.

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Intestinal health In pigs, intestinal health and the criteria defining it have not been fully elucidated(45,46). The usual focus on intestinal health has been on the prevention of infectious diseases, as well as on improving animal productivity, which includes nutrient utilization and productive performance(47). Studies on intestinal health have evaluated different components of the intestinal barrier, especially the mucosal epithelium, the components of the immune system and the microbiota, which are interdependent and, together with dietary factors, have a determining influence on the morphological and functional development of the digestive tract. The low feed intake due to weaning causes the morphological and functional changes in the intestine described above, as well as the shortening of the villi, hyperplasia of the crypts, increased mitosis, and recycling of epithelial cells, reducing brush border enzymatic activity and absorptive capacity. The aforementioned histological damage causes increased paracellular permeability, with increased transport of antigens to the lamina propria, leading to inflammation. Adequate feed intake after weaning prevents the loss of the barrier function of the tight junctions located in the intercellular spaces, indicating the importance of the presence of nutrients inside the intestine to maintain the protective function of the epithelium(44,47). The loss of intestinal integrity also reduces the development of innate immune activity by limiting its antigen-presenting capacity, as well as the release of cytokines and chemokines that regulate the local immune response(48,49). Although the enterocyte monolayer represents approximately 80 % of the epithelial cells, the remaining 20 % of the intestinal epithelium performs other important tasks and consists of: goblet cells that secrete mucins, Panneth cells that produce defensins, M cells that are part of the enteric immune system, and endocrine cells that release hormones and neuropeptides. In studies of intestinal health, mucin production and function have played a relevant role because changes in the amount or composition of mucus can lead to impaired nutrient absorption or a reduction in the protective function of mucins(50). Intestinal mucin 2 (MUC2), one of the major gel-forming mucins, represents the primary component of the mucus layer barrier, is a site where secretory IgA resides and is the first line of defense that limits epithelial contact or penetration of microbiota and other potentially dangerous antigens into the body(51). Changes in mucin secretion and the resulting inflammatory response at weaning increase the susceptibility of the piglet to bacterial infections. The immune system of the weaned pig is immature; therefore, its response to the presence of antigens and pathogens is deficient. Examples of this are seen in the limited function of B and T lymphocytes during the first weeks of life, and the poor antigen-specific responses in pigs less than six weeks old compared to older animals. T lymphocytes are divided into CD4 (helper) and CD8 (cytotoxic), whose function is to establish and maximize the capabilities 87

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of the immune system; CD4 cells appear in the intestinal mucosa between the third and fourth week of life, while CD8 T cells begin to appear in the epithelium at the age of four to six weeks. In pigs at weaning, CD4+ and CD8+ T lymphocytes are increased, and the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) is more prominent in the jejunum two days after weaning, due to transient intestinal inflammation. The intestinal microbiota in pigs is very dynamic and subject to change over time, especially in early life. In the newborn pig, the microbiota is modulated by sow's milk, and contains a higher abundance of lactic acid type bacteria(39,52). However, at weaning, opportunistic pathogens have been reported to be present in the small and large intestine of piglets, and, during this critical period, this reservoir of pathogens can trigger infections leading to severe diarrhea(53). Immediately after weaning, the relative abundance of Lactobacilli is reduced, and other types of bacteria proliferate such as Clostridium spp., Prevotella spp., Proteobacteriaceae, and E. coli, resulting in a loss of microbial diversity as a consequence of the cessation of milk intake and the initiation of solid feed intake(39,52). The composition and diversity of the intestinal microbiota of pigs after weaning can be modified by the levels and sources of proteins, sugars or dietary fibers present in the starter feed, in addition to management, environmental and sanitary status of the farm(44,45,54). Therefore, in order to avoid severe alterations in the integrity of the mucosa, the immune response capacity and the intestinal microbiota, it is necessary to design special diets that will provide the amounts and proportions of nutrients appropriate to the age and weight of the pigs at weaning, including highly digestible ingredients but also conventional ingredients that favor an adequate maturation of the digestive epithelium, and that facilitate the transition to the use of less complex diets. Also, at this stage, additives can be included in the diet to improve nutrient assimilation, increase the speed and effectiveness of the immune response, as well as to modulate changes in the dynamics of microbial populations during the period of milk consumption to solid feed change. Nevertheless, these recommendations will be successful to the extent that they are accompanied by feeding strategies and programs that ensure adequate food intake in the pre- and post-weaning stages. Contributions by INIFAP The relevance of maintaining high feed intakes at weaning through strategies that induce the maturation of digestive capacities in a gradual manner, and based on the presence of adequate substrates, is given priority, to the induction of early maturation of digestive enzymatic activity by pharmacological means(55,56). For this reason, different strategies have been evaluated to induce early solid feed intake in lactating piglets and in the immediate postweaning period. During lactation, the introduction of a pre starter feed, starting at 14 d of age, serves as a stimulus for pigs to become sensitized to the odor and texture of feed other than milk. Although solid feed intake is low and variable in suckling piglets, the use of 88

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ingredients such as molasses and oil, especially if they are also used in sow feeds, can be a good way to reduce the amount of solid feed intake by sows(57), in addition to milk byproducts, can facilitate and accelerate feed recognition at weaning. Also during lactation and the first days immediately after weaning, it is recommended to offer feed in small quantities, but frequently, in order to simulate the suckling habits of piglets that on average suckle every 50 minutes(58). The greatest benefit of feeding "little and often" at weaning is observed during the first week post-weaning and, in particular, it is an effective practice in the adaptation to the consumption of simple diets(59,60). In order to design feeds that provide the appropriate amounts and proportions of nutrients for the age and weight of the pigs at weaning, a methodology for ileal cannulation of pigs was developed to evaluate ileal digestibility, as well as the impact of different ingredients on endogenous nutrient losses(61). This methodology has been used to determine the ileal digestibility of dry matter, protein and energy of ingredients such as beef tallow and coconut oil(62), and different sources of lactose(63), cereals such as oats, corn and sorghums high and low in tannins(64), protein sources such as soybean concentrate, soy isolate and whey(65), and sesame(66). Apparent ileal digestibility of amino acids has been obtained in soybean meal and sesame meal(66,67) canola meal(68), casein(69, and sorghum(70). In addition, endogenous ileal losses of nitrogen and amino acids have been determined using casein as a reference(69,71). Post-weaning morphophysiological changes, such as villus histology and digestive enzyme activity, associated with the use of different feed ingredients such as cereals, including corn, oats and sorghum hybrids(72), low and high tannin sorghums(73), protein sources such as sesame and soybean meal(74), and soy concentrate, soy isolate and whey(75) have also been evaluated. Another important area of study is the addition of probiotics and prebiotics to feeds, and the design of these. The use of low protein diets combined with Bacillus (B). subtilis and B. licheniformis in pigs at weaning allowed changing the fermentative pattern of the intestinal microbiota increasing the concentration of acetic, propionic and butyric acid, and reducing the concentration of ammonia in the small intestine and cecum, reducing the incidence and severity of diarrhea, and improving the productive parameters compared to pigs that received high protein diets plus antibiotic (76). The addition of benzoic acid and a mixture of B. subtilis and B. licheniformis administered to piglets at weaning maintained productive parameters, reduced coliform counts, and ammonia release in wastewater, compared to responses of pigs at weaning fed diets containing antibiotics(77). Supplementation with Saccharomyces (S.) cerevisiae, and S. boulardii improved villus height and reduced pro-inflammatory cytosine concentrations in the intestinal epithelium; control of intestinal inflammation and mucosal preservation was more effective with the use of S. boulardii in weaning piglets(52,78). Also, the use of potato protein concentrate in piglets consuming antibiotic-free diets reduced the severity of diarrhea and maintained productive parameters compared to piglets that received antibiotic-added feed(79). 89

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Feeding of growing-finishing pigs

Current situation Modern swine feed formulation, as far as macronutrient intake is concerned, is based on the use of three concepts developed in the second half of last century. The first is ideal protein, which refers to a protein in which all essential amino acids are co-limiting to the pig's productive performance. That is, the supply of amino acids coincides exactly with their requirement and is based on the first limiting amino acid, which in pigs is lysine. This theoretical concept was challenged experimentally by the English group of Reading(80). Currently, it has been modified by several researchers, refining the profile for the different production stages. The second is the use in the formulation of rations of the net energy content of raw materials, optimizing the use of nutrients by the animal by reducing the use of dietary protein as a source of energy; the development of this concept was carried out by Noblet and his team at INRA(81). The third is the use of nutrient digestibility. Ileal digestibility of amino acids had its apogee in the last quarter of the last century(82) and the type of coefficient chosen to be use in formulation was the standardized ileal digestibility coefficient, which has two essential characteristics: the first is its additivity, an essential aspect when formulating a ration, since it allows the best estimation of the amount of the amino acid that will be metabolically used by the animal. The other characteristic is that its value is not influenced by the protein level of the diet used in its determination, an important aspect especially for cereals and low-protein raw materials. The use of digestible phosphorus content in the formulation of rations begins with the commercial use of the enzyme phytase, since its use allows reducing the use of mineral phosphorus in the diet(83); the practical implementation of this concept has been carried out by various research groups. At the beginning of this century, these three formulation principles were integrated into models capable of predicting nutritional requirements(84,85), this has allowed the approach to "Precision Nutrition", a concept that applies the results of research in nutrition and related sciences, using large databases through computer science to predict and provide the nutrient requirements as accurately as possible, seeking a safe and high quality efficient production, in addition to ensuring the least possible impact on the environment. The joint use of these concepts opened up the possibility of using new raw materials and byproducts of the industry, since it potentiates their use by estimating both their nutritional contribution and their effect on the growth and conformation of the pig carcass.

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Contributions by INIFAP The Swine Nutrition research program first compiled data on the standardized ileal digestibility coefficients of various raw materials(86); but later INIFAP contributed to the methodology for the study of ileal digestibility of amino acids(61), as well as to the estimation of endogenous basal losses of nitrogen and amino acids in both piglets and growing pigs(69,71) which made it possible to generate information on the standardized ileal digestibility coefficients of the amino acids of the main cereals used in Mexico ―sorghum (87-89), corn(90) ―, and from various protein sources: sesame meal(67), safflower meal(91), canola meal(68,92), gluten feed(90). Regarding the use of digestible phosphorus, INIFAP has conducted research on the impact of the use of the enzyme phytase on the increase in phosphorus digestibility of various raw materials(66,93,94). Studies were also carried out on the use of crystalline amino acids(95-98) and on the nutritional adequacy to be achieved when using metabolic modulators(99-101); all the aforementioned research was focused on generating the necessary information to optimize the use of the most widely used raw materials in the country, which in some cases are scarcely researched at the international level. Regarding the use of dietary energy, INIFAP's contribution consisted of studies of metabolizable energy balances(102,103). Currently, the Institute has respiratory chambers to determine the Net Energy content of diets in pigs, which opens the possibility of conducting studies on this subject.

Carcass conformation and meat quality

The generation of pig carcasses with a good conformation and quality begins with the selection of parents(104), of proper handling from weaning to finishing, including proper procedures during slaughter and post-mortem meat handling. In this regard, in some countries there are grading systems for pig carcasses, such as the USDA Pork Carcass Grading System, in the US; the Pork Carcass Classification and Grading, in Canada; (EC) REGULATION No 1249/2008, in the European Union, and the Mexican Norm (NMX-FF-081-SCFI-2003) for the evaluation of Pork Meat in Carcass-Quality of Meat-Classification, in Mexico(105). The purpose of these systems is to facilitate trade and provide producers with a system to grading and paying a fair price for the carcass. The generation of the NMX norm(106) was the beginning of the objective evaluation of pork carcasses in Mexico. In general, grading systems require the evaluation of complete strains, through the subjective and objective evaluation of the primary cuts (after dissection), and their correlation with the kilograms produced as live or carcass weight (amount of lean meat)

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to generate mathematical models(107). Such factors as fat-free lean, fat content, ultrasonography measurements in the dorsal region, and hot carcass weight have been included in the models(108). In the 1990s, information was generated on the quality of meat obtained from pure breeds and from certain crossbreeds(109). In these studies, the Duroc breed predominantly showed greater productive potential (daily weight gain) and meat quality (lower back fat thickness, greater infiltrated fat, lower resistance to cutting, greater juiciness and tenderness), which is why it is currently one of the most commonly used breeds in terminal crossbreeding. However, the evaluation of meat quality at the industrial level is complicated by the fact that, when looking for the least damage to carcasses and primal cuts, measurements are taken at sites that are not representative of the existing variability, and therefore they lead to deficient studies, for example, when evaluating the ventral surface of the loins, for which the correct procedure should be to make a transverse cut to the muscle at the P2 point, at the level of the tenth rib. INIFAP’s National Center for Disciplinary Research in Animal Physiology and Improvement (CENID Fisiología y Mejoramiento Animal) has generated a series of technical-scientific documents to help perform objective evaluations, which can be requested at the following e-mail address: lopez.lhumberto@inifap.gob.mx There are studies that reviewed the key aspects for producing quality pork, taking into account the interaction of metabolic pathways (gluconeogenesis, glycogenolysis, betaoxidation and adipogenesis) and endogenous-exogenous antioxidant systems in the animal(110-112). Specifically, the impact of micronutrients and the ante-mortem (fasting, handling), slaughter (desensitization) and post-mortem procedures(113) will have an effect on meat quality. During the transformation of muscle to meat, anaerobic glycolysis plays a fundamental role; therefore, the study of the glycolytic potential(114) is crucial to explain the final pH of the meat and its relationship, in populations with the Halothane and Rendement Napole genes(104,115), with a high susceptibility to generate pale soft exudative (PSE) meat and sour meat in pigs, respectively. Meat with the PSE condition is characterized by low pH (<5.5), low water holding capacity (WHC) and a pale color as a consequence of surface dewetting. WHC is a property that describes the technological aptitude of the meat and PSE meat is a direct consequence of an oxidative imbalance at membrane level(116). In order to ensure a high WHC, several lines of research were generated at the CENID Fisiología y Mejoramiento Animal, in which trace minerals(117), functioning as cofactors of endogenous antioxidant enzymes ―selenium in glutathione peroxidase (Gpx); iron in catalase, and copper in superoxide dismutase―, partially reduced the damage caused by oxidative stress by reducing drip weight loss and improving the color. However, the pro-oxidant factors could be greater, and, therefore, alternatives were sought, using vitamin E (exogenous antioxidant) to supplement the diet. Vitamin E significantly reduces oxidation reactions measured mainly by the TBARS (2-Thiobarbituric Acid Reactive Substances) technique, which affect meat 92

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color and shelf life(117,118). The use of adequate levels of trace minerals and vitamin E has been shown to have positive effects on meat quality(119). In Mexico, research conducted at the CENID Fisiología y Mejoramiento Animal, recommends reducing the dose of trace minerals from chelated sources(120) and administering 120 IU of vitamin E (alpha-tocopherol acetate) per kilogram of feed in the last 42 d of fattening(121,122), a dosage without detriment to meat productivity and quality. Other micronutrients of benefit have been found as well; such is the case of 25hydroxycholecalciferol, a secondary metabolite of vitamin D3 with lower toxicity, which, when added to the diet, affects muscle hyperplasia during embryo development with possible effects on meat quality(123); it also reduces leg issues in pigs of average to high weight, favoring productive performance by lessening the number of low-yielding animals, with positive effects on meat color, WHC, and meat texture(124,125). Enrichment of meat Meat enrichment is a finite quality, intrinsic to genetics and to the anatomical region, but strongly influenced by nutrition, especially during the finishing stage. The widely studied molecules for enriching meat are long-chain fatty acids, vitamins (mainly fat-soluble) and trace minerals. In addition, through surveys and some sampling in the last decade, the National Health and Nutrition Survey ENSANUT(126) has identified among the Mexican population vitamin and mineral deficiencies for which animal products are the recommended solution. Today, obtaining foods of animal origin enriched with good quality fats, such as long-chain omega 3 and 6 fatty acids, is necessary for human nutrition. In pork, the lipid profile deposited in the meat and fat will differentiate products enriched with these fatty acids from those not enriched(127). The use of highly oxidizable lipids together with other pro-oxidant factors, such as high concentration of certain minerals, can cause a delay in pig growth(128); therefore, low doses of minerals are recommended, which can be attained by using chelated sources(120). In this regard, INIFAP's research on pork fortification in recent years has focused on the modification of the lipid profile (linoleic, linolenic, docosahexaenoic acid)(129-131), concentration of vitamins (25-OHD3 and vitamin E)(125,132,133) and trace minerals (Se, Zn, Cu, Mn, Mg, and Fe)(134,135).

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Impacts of INIFAP It is difficult to quantify the impact that INIFAP has had on swine production and on Mexican society through research in swine nutrition, since the improvements in production observed in the last 35 yr are the result of several factors such as genetics, feeding-nutrition, reproduction, health and management. However, it is worth noting that INIFAP's research generated the first Mexican carcass quality standard and quality criteria for pork produced in Mexico. It also generated information on non-conventional ingredients and the digestibility of several of the most commonly used raw materials in the country, which has allowed their inclusion in swine feeds. However, it is considered that the greatest impact has been achieved through the agreement with the National Autonomous University of Mexico (Universidad Nacional Autónoma de México, UNAM), in which more than 170 nutritionists have been trained and are currently working in the industry, and specialized training courses have been provided to a large number of agents of change; in addition to the leading role that its researchers have played and continue to play in the trade associations that affect the sector, such as the Mexican Association of Animal Nutrition Specialists (Asociación Mexicana de Especialistas en Nutrición Animal, AMENA) and the Mexican Association of Veterinarians Specialists in Swine (Asociación Mexicana de Veterinarios Especialistas en Cerdos, AMVEC).

Challenges The current world population is almost 7.5 billion, and it is expected that by 2050 there will be more than 9 billion people; therefore, FAO estimates that the world will have to produce approximately 60 to 70 % more food in the next 30 yr. It is also predicted that animal protein production should increase at least threefold and meat production should double by 2050(136). Thus, the challenge for animal production in general and pig farming in particular in the 21st century will be to sustainably produce food of animal origin in adequate quantity and quality. The challenge will be to produce that amount of food with diminishing resources year after year, and in a sustainable manner. Therefore, the issues that become relevant and need to be researched are: The use of alternative ingredients: The incorporation of cereals and protein sources in animal nutrition, which are also consumed by humans, generates increasing pressure on the livestock industry to create or use raw materials that do not compete directly with humans. This situation favors the use of by-products and new products such as protein sources from larvae, worms, algae, etc. The rational use of these raw materials will depend on the generation of

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information through research on the composition and digestibility of their protein, phosphorus and net energy intake, as well as on the knowledge of their anti-nutritional or toxic factors. Nutritional requirements: Selection and continuous genetic improvement produce animals with higher growth speed and animals with a higher growth rate and protein deposition capacity, traits that modify their nutritional requirements. This area of research has traditionally been carried out in developed countries, where pig farming takes place in closed facilities with controlled climate, which means that the knowledge generated for these conditions is not necessarily repeatable in the country, given that in Mexico the growthfattening phase is carried out in open facilities, with important climatic variations in certain seasons of the year, which are capable of modifying the productive behavior of the animals. For this reason, research will be required in this aspect in order to be able to adapt the nutritional requirements to the environmental situation of the country. Intestinal health: Weaning is associated with social, environmental and nutritional stress factors, which can lead to reduced digestive capacity and stunted growth of piglets. Antibiotics have been used in sub-therapeutic doses in starter diets to reduce the occurrence of severe diarrhea and the negative impact of weaning. However, their use as growth promoters has been banned, as they favor the presence of antibiotic-resistant bacteria. Therefore, it is necessary to have alternatives to its use in the weaning phase when animals are more susceptible(79). Climate impact mitigation: Pig production is estimated to issue 668 million tons of CO2eq/yr, or 9 % of total livestock emissions(137). Theoretically, this amount can be reduced by improving feed efficiency through the use of the above formulation criteria (ideal protein, net energy, digestible amino acids, and phosphorus) and translating them into precision nutrition. Studies show that the use of these concepts in the formulation of the diet and feeding program can reduce by up to 22 % the excretion of nitrogen, which is the main pollutant in swine excreta, and from which nitrates, ammonia and nitrous oxide ― the main greenhouse gas produced by pig farming― are formed. New approaches to nutrition: The metabolic interaction between nutrients and how they affect the expression of specific genes that can influence feed intake, preferential use of nutrients towards protein deposition, and the metabolic interaction between nutrients and protein deposition have become increasingly important. From a nutritional point of view, the impact of the diet on the intestinal microbiota and the manner in which the nutrientmicrobiota relationship modulates the animal's metabolism, particularly to reduce the presence of post-weaning diarrhea in the case of piglets, has also become important. Likewise, there has been increased research on the use of nutrients by the organism that impact pig production, such as the functional use of some amino acids. Another approach 95

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that has gained importance is to promote animal welfare by reducing stress and stereotyped behaviors, especially in the breeding herd and in weaned piglets. Biotechnology products: Biotechnology has been defined as "any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for a specific use". More and more biotechnological products are becoming available for animal feeding, such as: pro- and prebiotics, crystalline amino acids, exogenous enzymes, growth hormone, vitamins, chelated trace minerals, etc. The use of which has allowed to increase production efficiency, as well as to reduce the environmental impact of swine farming. Regarding the breeding herd With respect to the challenges and perspectives in the short, medium and long term, in a survey conducted in 2015 in Canada, USA, Mexico, Brazil, Ecuador and Chile, to determine the main factors affecting pig production, the predominant factors up to 2015 were found to be "Nutrition and Feeding Strategies, Biosecurity, Health, as well as increasing the Volume of Production", the main factors in pig production being feed efficiency and piglets weaned per female per year (138). Feeding strategies employed during the growth of replacement sows can influence their short- and long-term reproductive performance. For example, in the short term it is possible to manipulate the ovulatory rate and, in the long term, the amount and type of body reserves, which can have a major impact on subsequent reproductive efficiency and sow longevity. In particular, the feeding strategies used during gestation and lactation are important factors that influence the productive performance of the sow and can interact, to a greater or lesser extent, with the feed received by the sows during their growth.

Conclusions Due to the great diversity of topics to be researched and the breadth and diversity of knowledge areas involved (nutrition, reproduction, immunology, microbiology, proteomics, metabolomics, etc.), as well as the costs associated with the development of research, it is imperative to establish bonds with different institutions and research groups both nationally (UNAM, State Universities, College of Postgraduates, etc.) and internationally, working on topics of common interest. Strengthening should be two-fold: firstly, by strengthening joint research through the formation of interdisciplinary and interinstitutional groups, including the incorporation of new researchers to the program. And secondly, by strengthening the physical infrastructure of INIFAP, which is concentrated in the CENID Fisiología y Mejoramiento Animal (experimental farm and metabolic unit), as well as the institutional

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laboratories of Nutrition, Meat Quality, Molecular Biology, Proteomics, and through the creation of the Metabolomics laboratory, which would favor frontier research in swine farming. Obituary: The authors wish to express our posthumous recognition to Dr. José Antonio Rentería Flores, DVSc, who passed away on December 17, 2020; and who was well known for his lifelong dedication to his work and for his interest in research, as well as for his active participation in the development of this publication. May he rest in peace! Literature cited: 1. USDA. Ask USDA. Date acces: September 8th, 2020. https://ask.usda.gov/s/article/What-is-the-most-consumed-meat-in-theworld#:~:text=According%20to%20the%20United%20Nations,goats%2Fsheep%20 (5%25). 2.

Consejo Mexicano de la Carne. Compendio estadístico 2018. Consejo Mexicano de la Carne, Cd. de México. 2018.

Rutherford KMD, Baxter EM, Birgitte A, Peer P, D’Eath RB, Jarvis S, et al. The ethical and welfare implications of large litter size in the domestic pig: challenges and solutions. Danish Centre for Bioethics and Risk Assessment (CeBRA). 2011

Nielsen NP. Productivity is vital for sustainable pig production-the Danish experience. Adv Pork Prod. 2019.

Ek-Mex JE, Segura-Correa JC, Alzina-López A. Efecto de la reducción o incremento del número de cerdos nacidos vivos en el segundo parto en la vida productiva de las cerdas en el sureste de México. Arch Med Vet 2016;48:243-246.

Pérez CJE. Cómo influye la nutrición en la estrategia para una producción eficiente de reemplazos porcinos. AMENA. 2020. 24 de agosto,. https://www.youtube.com/c/AMENAAC.

Lucia T, Dial GD, Marsh WE. Lifetime reproductive performance in female pigs having distinct reasons for removal. Livest Prod Sci 2000;63:213-222.

Mejía GCA, Cuarón IJA, Rentería FJA, Braña VD, Mariscal LG, Gómez RS. Alimentación del hato reproductor porcino. Colon, Qro., México: INIFAPSAGARPA, Libro científico No.1 2007.

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Mejía-Guadarrama CA, Prunier A, Quesnel H. Dietary protein intake during oestrus cycle does not alter the ovulation rate in gilts. Repro Fert Develop 2004;16:589-597.

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127. Woods VB, Fearon AM. Dietary sources of unsaturated fatty acids for animals and their transfer into meat, milk and eggs: A review. Livest Sci 2009;126:1-20. 128. Dove CR, Ewan RC. Effect of excess dietary copper, iron or zinc on the tocopherol and selenium status of growing pigs. J Anim Sci 1990;68:2407-2413. 129. Valdés-Reyez J. Impacto de la calidad de la grasa en la dieta de las cerdas reproductoras y sus consecuencias en producción. [tesis maestría]. Cd. de México: UNAM; 2016. 130. Sanchez-Piñeyro S. Uso de una fuente de dha proveniente de algas marinas en la alimentación de cerdos en crecimiento y finalización: evaluación de la calidad de carne y grasa. [tesis licenciatura]. Cd. de México: UNAM; 2018. 131. Calderón M, López H, Morales R, Castañeda G, Cuarón IJA. Aplicación de un suplemento natural de metabolitos de colina como aditivo en dietas para cerdos en crecimiento y finalización. VII Congreso Latinoamericano de Nutrición Animal. Pto. Vallarta, Jalisco. 2016. 132. Guerrero-Huerta D. Resultado de un ajuste a los niveles de vitaminas en dietas para cerdos en crecimiento. [tesis licenciatura]. Cd. de México: UNAM; 2018. 133. Garrido-Monroy B. Efecto de la suplementación de vitamina D3 y 25hidroxicolecalciferol en la calidad microbiológica y estabilidad oxidativa de carne de cerdo. [tesis licenciatura]. UNAM; 2013. 134. González-Mendoza M. Efecto de la fuente de minerales en la protección antioxidante de la carne y la regulación de proteínas asociadas a estrés durante el sacrificio y la transformación de músculo a carne en cerdos. [tesis maestría]. Cd. de México: UNAM; 2018. 135. López-Rios F. Contenido y biodisponibilidad de minerales traza (Cu, Fe, Mn y Zn) en la carne de cerdo. [tesis licenciatura]. Cd. de México: UNAM; 2018. 136. Babinszky L, Verstegen MWA, Hendriks WH. Challenges in the 21st century in pig and poultry nutrition and the future of animal nutrition. In: Hendriks WH, et al, editors. Poultry and pig nutrition, challenges of the 21st century. First ed. Wageningen, The Netherlands: Wageningen Academic Publishers; 2019:17-37. 137. McAuliffe GA, Takahashi T, Mogensen L, Hermansen JE, Sage CL, Chapman DV, et al. Environmental trade-offs of pig production systems under varied operational efficiencies. J Cleaner Prod 2017;165:1163-1173.

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138. Córdoba DJ. Tendencias hacia el año 2025 sobre los principales factores que inciden en la producción en la industria en América. Congreso AMVEC- Veracruz, México. 2016.

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https://doi.org/10.22319/rmcp.v12s3.5848 Review

Background and perspectives of certain priority diseases affecting cattle farming in Mexico

Carmen Rojas Martínez a Elizabeth Loza Rubio b Sergio Darío Rodríguez Camarillo a Julio Vicente Figueroa Millán a Francisco Aguilar Romero b Rodolfo Esteban Lagunes Quintanilla a José Francisco Morales Álvarez b Marco Antonio Santillán Flores b Guadalupe Asunción Socci Escatell b Jesús Antonio Álvarez Martínez a*

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. CENID Salud Animal e Inocuidad, Carretera Cuernavaca-Cuautla 8534. Col. Progreso Jiutepec, 62574 Morelos. México. b

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. CENID Salud Animal e Inocuidad. Ciudad de México. México.

*Corresponding author: alvarez.jesus@inifap.gob.mx

Abstract: The review focused on concisely presenting the contributions that INIFAP researchers have developed, directly or in collaboration with researchers from other institutions, on different 111

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aspects of the diseases that affect cattle farming in Mexico. It describes the research on viral diseases such as rabies and bovine viral diarrhea; bacterial diseases such as anaplasmosis, brucellosis, tuberculosis, paratuberculosis, leptospirosis and bovine respiratory disease, and among parasitic diseases, tick infestation and babesiosis. It identifies potential lines of research that can help mitigate the impact of diseases on production. It considers contributions on the development or adaptation of serological and molecular diagnostic techniques and the diagnosis of resistance to ixodicides. In addition, it indicates epidemiological parameters of the diseases and makes reference to the biologics generated, which include vaccines against rabies, anaplasmosis and babesiosis; bacterin against leptospirosis, and a bacterin-toxoid against pneumonia. It also discusses the evaluations of the use of BCG against tuberculosis and a new generation vaccine against brucellosis. The review concludes that the research of INIFAP in animal health must necessarily have the omic sciences as a perspective. This is the only way to complement the understanding of disease mechanisms, the development of new diagnostic techniques and the design of effective and safe vaccines. Therefore, the great challenge will be the involvement of the animal health area in the concept of "One Health". Key words: Diseases, Vaccines, Prevention, Control.

Received: 11/11/2020 Accepted: 08/03/2021

Introduction The purpose of livestock production is to produce quality food that is affordable for society and obtained in a sustainable environment, which is difficult in the face of a growing need for meat and milk. INIFAP researchers maintain a constant attention to the demands of producers, through the generation of scientific knowledge and technological innovation in animal health problems. In Mexico, the inventory is slightly more than 34 million cattle(1), which are exposed to viral, bacterial or parasitic pathogens, which often behave as co-infections or complexes. The distribution and frequency of diseases vary according to the interactions between the pathogen, the bovine host and the ecological conditions. Its occurrence causes different rates of morbidity, mortality and low productivity, with a detrimental effect on the use of the production potential, and inherently generates trade restrictions at both the national and 112

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international levels. It has been estimated that a disease outbreak can affect 20% of the commercial activities related to the herd(2). Diseases involve a wide variability in the costbenefit ratio of prevention and control programs, which results in underestimates of the impact on production, and, consequently, in inconsistency in the information on losses. It is also important to note that certain bovine diseases affect the human population(3). Each disease has a different economic burden that is determined through direct costs, indirect costs of consumption or loss of resources; in general terms, it includes human, structural and economic resources. The objective of this review is to present in a concise manner the contributions that INIFAP researchers have made, directly or in collaboration with researchers from other institutions, on different aspects of the main diseases affecting cattle in Mexico. At the same time, the aim is to identify lines of research to mitigate the impact of diseases on production.

Bovine paralytic rabies Bovine paralytic rabies (BPR), also known in Spanish as derriengue, is an encephalitis caused by a negative-strand RNA virus of the Rhabdoviridae family and of the genus Lyssavirus. In Mexico, the main transmitter is the chiropteran Desmodus rotundus, a hematophagous bat, distributed in Latin America from the coasts of Mexico to the north of Argentina(4). In Mexico, BPR is a frequent disease: 284 positive cases were diagnosed in 2019(5).

Contributions by INIFAP

Diagnosis. In endemic countries such as Mexico, diagnosis is critical for the prevention and control of rabies. The reference test is direct immunofluorescence (DIF); however, in tropical climates, the brain tissue frequently exhibits decomposition when handled, which makes it impossible to perform the diagnosis or leads to false negative results. Therefore, a real-time polymerase chain reaction test (RT-PCR) was developed based on the sequences of 40 virus isolates from different reservoirs and geographical areas of the country. For this purpose, primers were designed for the N gene, which is the most conserved gene of the virus. With the application of the test, sensitivity, specificity and predictive value rates of 86, 91 and 96 %, respectively, were obtained(6). The virus was also detected in samples stored at 27 °C for 23 days. Thus, RT-PCR is currently accepted as an excellent alternative for virus diagnosis(7).

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Molecular Epidemiology. INIFAP researchers have pioneered the antigenic and molecular characterization of the rabies virus. They performed the detection of antigenic variants using a panel of monoclonal antibodies obtained from the Pasteur Institute in Paris, France(8). Subsequently, using monoclonal antibodies from the U.S. Center for Disease Control, they achieved molecular characterization of samples from humans and domestic and wild animals collected from 1990 to 1995. Thus, they recognized a new cycle, called "hypervariables", circulating in skunks in Baja California Sur. At the same time, antigenic and molecular variants circulating in vampires and other wildlife were identified(9,10). In a collaborative study with researchers from the Pasteur Institute, the main epidemiological cycles of rabies in Mexico were determined using the Restriction Fragment Length Polymorphism (RFLP) technique(8). In other research, using a portion of the P gene, it was discovered that a variant of the virus that circulates in cats also circulates in the bat Tadarida brasiliensis(11,12). Vaccination. The use of gamma radiation with a Cobalt-60 source made it possible to maintain the potency, safety, stability, and shelf life of traditional vaccines(13-16). In the application of a gene vaccine in dogs, it was possible to successfully replace the gene gun with an insulin syringe(17). Edible rabies vaccines were generated using the N gene expressed in the tomato; however, a low level of immunogenicity was obtained(18). In contrast to the G protein expressed in carrot embryogenic callus, it provided a 60% protection in mice(19). Subsequently, an antirabies vaccine was produced in corn, whereby the protection was increased to 80% in sheep in the face of a challenge with a lethal virus(4). Recent collaborative research has uncovered differences in Toll-like receptors (TLR) between chiropterans and terrestrial mammals(20). The Nature series has published the hologenome of the vampire, and it has been inferred that the hematophagous bat has adapted to blood through a close relationship between its genome and the gut microbiome(21). Perspectives. In tropical conditions, the maintenance of the cold chain is a serious inconvenience; therefore, a thermostable vaccine must be generated for massive use. Also, mass testing for neutralizing antibodies associated with protection must be carried out in order to evaluate the effectiveness of vaccines. In addition, it is essential to produce a good quality conjugate that will allow high sensitivity, specificity and lower cost of the test.

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Bovine viral diarrhea Bovine viral diarrhea (BVD) is a globally distributed disease that causes significant losses to livestock. The causal agent is a Pestivirus of the Flaviviridae family, which has an immunosuppressive effect that facilitates secondary or concomitant infections. It affects the digestive, respiratory and reproductive systems, and is a component of the bovine respiratory complex(22). The virus has high genetic variability and is classified into two genotypes and several subgenotypes.

Contributions by INIFAP

Epidemiology. In Mexico, the first description of BVD was made in 1975, specifically in cattle with reproductive problems in which circulating antibodies were detected(23). INIFAP studies have been limited to understanding the epidemiology and measurement of risk factors. However, the presence of subgenotypes 1a, 1b, 1c and 2a has been demonstrated in Mexico(24). A report describes a sampling of dairy cattle in different states of the country, in which a seroprevalence of 78.8% was determined. In the same study, the significant risk factors were herd size, pens, intensive production, and long inter-calving periods(25). Perspectives. The high prevalence of BVD suggests the opportunity to create lines of basic and applied research for the prevention and control. The ideal challenge would be the elimination of BVD, for which a vaccine should be developed with Mexican isolates representing the subgenotypes present. It would be desirable to develop new generation vaccines, as well as diagnostic techniques with high sensitivity and specificity for recognizing concomitant infections.

Bovine Anaplasmosis Bovine anaplasmosis is a disease caused by the Gram-negative bacterium Anaplasma marginale, which affects mostly grazing cattle in tropical areas where the largest livestock populations are concentrated in extensive farms in Mexico. The disease can cause up to 25% of the total death losses of animals moved to the tropics for breeding programs(26). The clinical form is manifested by anemia, jaundice, lack of appetite, loss of weight and milk 115

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production, miscarriage in the third third, and death. At INIFAP, the Anaplasmosis Unit was founded by Dr. Ramón Aboytes Torres in 1994, where research on diagnosis, epidemiology, bovine immune response, in vitro culture of the bacteria, and the generation of vaccines is carried out.

Contributions by INIFAP

Diagnosis and epidemiology. Serological and molecular studies have been performed, and prevalence rates of 50% have been estimated in northern Veracruz(27). Serological diagnosis was improved with the development of an indirect enzyme-linked immunosorbent assay (ELISAi), which has been adopted by SENASICA(28). In molecular epidemiology studies using the msp1 and msp4 genes as markers, Mexican strains have been observed to have a distribution that allows to assume their migration. Several strains of A. marginale present in Mexico were also found to be more similar to those characterized in Brazil than to U.S. strains(29). More than 20 strains are stored in the laboratory, having been collected in different states of the Republic and used for testing conserved antigens(30,31). Immunity and vaccines against anaplasmosis. A. marginale infects mature erythrocytes; this makes it behave as an extracellular bacterium, since it does not infect nucleated cells and, therefore, does not induce a typical Tc response with CD8+ cells, but prompts a Th1 response. This Th1-type immune response model had been previously postulated(32); at INIFAP, it was tested in calves that normally establish infection but resist the clinical occurrence of the disease. The model was also corroborated in adult cattle, in which a Th1type response was observed, associated with the presence of IgG2, Interferon- and CD4+ T-helper (Th) lymphocytes, which is essential for resistance to the clinical occurrence of anaplasmosis(33). An inactivated immunogen was developed to induce protective immunity to a homologous challenge(28,34,35). In the search for broad-spectrum alternatives, a strain of A. marginale from the state of Yucatan was identified, which was named "Tizimín" and characterized as a strain of low natural virulence(36). This strain was used as a vaccine and was shown to protect against a heterologous challenge in cattle vaccinated with doses of 1x104-1x1010(37). The inactivated immunogen has been used to vaccinate both local and imported animals in Veracruz and Tamaulipas, thus contributing to reduce the morbidity and mortality due to anaplasmosis. On the other hand, the use of live immunogen has been limited due to the difficulty to maintain it in liquid nitrogen. In Mexico, the most important biological vector of A. marginale is known to be the Rhipicephalus microplus tick(38,39). Trans-ovarian transmission was demonstrated in the laboratory this was done with R. microplus larvae

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that were fed on infected cattle and which subsequently transmitted the infection to susceptible cattle(40). Genome studies. The first complete genome of A. marginale was published in 2005(41), revolutionizing the study of potential vaccine candidates against this bacterium. Today, there are 23 complete sequences, including seven Mexican strains(42,43). Membrane proteins with vaccine potential have been analyzed for the development of immunogens(44), and trials have been conducted with recombinant proteins or synthetic peptides(31). However, there is still no immunogen capable of fully protecting experimentally or naturally challenged cattle against this bacterium. Currently, in the Anaplasmosis Unit, studies focus on these sequences in order to include proteins associated with transport, signaling or metabolic pathways in the design of vaccines(30). Perspectives. After the publication of the 23 genomes of A. marginale, the sequences are to be analyzed by bioinformatics procedures in order to establish criteria for the identification of vaccine candidates linked to vital or virulence functions. Currently, there are examples of multi-epitope vaccines and reverse vaccinology strategies; thus, INIFAP research group according is making use of these tools to design new vaccines against A. marginale(30). It is very likely that proteins other than those already studied will be identified for inclusion in a vaccine. This may take place within a period of five years, at which time an immunogen will be widely and safely used.

Brucellosis Brucellosis is an infectious disease caused by bacteria of the genus Brucella that affects different domestic species such as cattle, sheep and goats. The most important species that affects cattle is Brucella abortus(45). In Mexico, brucellosis is the main zoonosis of bacterial origin. In cattle, the most notorious clinical signs are reproductive, including miscarriage and reduction of milk production, which have a high impact on cattle farming. For the purpose of controlling the disease in the country, there is a National Campaign against Brucellosis in animals, which applies the NOM-041-ZOO-1995 standard based on diagnosis and vaccination. Nationally, B. abortus strains S19 and RB51 are used for immunization of cattle. S19 induces the presence of antibodies in serum and milk, but interferes with official diagnostic tests; therefore, the alternative is RB51(46-49). For diagnosis, the most commonly used serological methods are the 8% card test and the rivanol test(50); these tests detect antibodies against the components of the outer membrane of Brucella, directed against the O-chain of 117

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the lipopolysaccharide (LPS), which is the most antigenic structure of the smooth strains(51). Despite the efforts made in the campaign, brucellosis in Mexico continues to have an unfavorable effect on animal and human health. Prevalence in production units is higher than 20%; in humans, an average of 3,000 new cases are reported each year according to Ministry of Health of Mexico (Secretaría de Salud), CENAPRECE 2013-2018).

Contributions by INIFAP

At INIFAP, researchers have made relevant contributions to the campaign in multiple aspects. The diagnostic tests that are applied directly or indirectly have been the result of its research, and are endorsed by the Mexican Official Standard. The tests utilized are Rose Bengal, rivanol, complement fixation and ring in milk. However, with the use of these tests, it is difficult to differentiate between vaccinated and infected animals, especially in those that are revaccinated; this issue has been solved with the radial immunodiffusion (RID) test(52). In turn, this test facilitated the development of other tests with greater sensitivity and specificity, such as ELISA and polarized fluorescence, in which the polysaccharide known as native hapten (NH) is used as antigen(49,53). In relation to the pathogenesis of brucellosis, it was studied the survival and intracellular trafficking of the vaccine strain RB51 vs. field strains in phagocytic cells. Thus, a shorter survival time of the vaccine strain was observed, and a lower probability of causing disease was inferred(54). The effect of revaccination and the management of infected herds were evaluated in order to demonstrate the effectiveness of the vaccines used in the campaign. Trials have also been carried out with new generation vaccines such as rfbK mutants(47,55,56). The RB51 vaccine exposed the potential risk to public health, as it was shown to be eliminated in the milk and vaginal secretions of cows(57). Although the RB51 vaccine strain displaced Strain 19, its real protective potential is still unknown, although its efficacy in eliminating reagent animals to conventional tests has been demonstrated. Vaccination per se has not been sufficient to reduce the high incidence of the bacterium in infected herds(46,47). Vaccination with the RB51 strain has been described as not interfering with official diagnostic tests because it lacks the "O" chain. While some positive "outlier" responses have been observed, these have been attributed to contact with field strains that occurred during the studies(58,59). The use of the rough mutants RB51 and rfbK as vaccines has been described as inducing adequate protection in a herd under medium prevalence conditions(56).

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Perspectives. Despite the existence of the national campaign for the control of brucellosis, the prevalence and incidence of the disease remain at a level that has economic and social repercussions. Therefore, the prevention and control of brucellosis could be approached under the concept of "One Health"; this would involve producers and authorities in charge of animal and human health. Technically, projects should be continued to improve the efficacy and safety of existing vaccines and to develop new types of vaccines.

Tuberculosis Bovine tuberculosis is a chronic disease caused by the mycobacterium Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. M. bovis affects a wide variety of species, including humans. In Mexico, tuberculosis is the second most important zoonosis of bacterial origin after brucellosis(60). Control depends on the application of the Mexican Official Standard NOM-031-ZOO-1995 of the National Campaign Against Bovine Tuberculosis (Mycobacterium bovis)(61), whose strategy is based on the diagnosis and elimination of reagents. The diagnosis is performed with the tuberculin test, using as antigen the bovine purified protein derivative (PPD) made with M. bovis strain AN5. Bovine PPD is applied in the caudal fold or at par with avian PPD made with M. avium strain D4, in a comparative cervical test(62). Animals positive to this test are sent to the slaughterhouse; the diagnosis is confirmed by specific bacteriological analysis and by histopathology of granulomatous lesions, which is established in NOM-031-ZOO-1995(61). In Mexico, the prevalence is usually above 2.5 % in milk production units; it is lower in beef cattle, but in both systems it affects the commercialization of cattle. More than 15,000 new cases of tuberculosis are reported in humans each year (Secretaría de Salud, CENAPRECE 2017).

Contributions by INIFAP

INIFAP, through its researchers, has contributed to the development and application of the different diagnostic techniques applied in the campaign, which are endorsed by the Mexican Official Standard. An outstanding contribution is a study that proved that the tuberculin test does not identify animals in the terminal stages of tuberculosis. Therefore, complementary tests such as ELISA, Interferon- and spoligotyping were implemented to improve the reliability of the diagnosis and identify these anergic animals(62).

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The use of sodium tetraborate in the isolation of mycobacteria was established as a routine procedure for the optimal preservation of samples for up to 90 days; this is also a contribution made by INIFAP researchers. Another contribution was the use of PCR and histopathological analysis with Ziehl-Neelsen staining, which improved the sensitivity and specificity of bacteriological culture(63). A major achievement was the development of endpoint PCR and Multiplex PCR tests, with which it is possible to differentiate between animals vaccinated with BCG and those infected with field strains(64,65). In Mexico, there is no authorized vaccine to prevent tuberculosis in animals; however, INIFAP has conducted studies of the BCG vaccine used in humans to evaluate its protective capacity in animals. Laboratory animals have been used as models and preliminary tests have been carried out in cattle. In a study of calves vaccinated with BCG and challenged with a pathogenic strain of M. bovis, a marked reduction in granulomatous lesions was demonstrated. Therefore, its use has been suggested for the control of tuberculosis in high prevalence areas(66). Perspectives. The scientific information that has been generated, in association with with the existence of a campaign for the control of tuberculosis with an Official Standard, suggests that an efficient control of bovine tuberculosis is feasible. However, the suitability of the use of BCG vaccine, which is currently the only vaccine in existence to prevent tuberculosis in cattle, must be solidly demonstrated. At the same time, an alternative line of research should be established for another vaccine that will not interfere with discrimination between vaccinated and infected animals, which would reduce the prevalence and allow efficient control of bovine tuberculosis.

Paratuberculosis Paratuberculosis is a chronic infectious disease affecting cattle, sheep and goats. It is caused by Mycobacterium avium subspecies paratuberculosis (Map); it is characterized by granulomatous lesions in the small intestine. This disease causes nutrient malabsorption syndrome, loss of physical condition in infected animals, and a reduction of productive capacity. The etiological agent is eliminated in feces; therefore, the animals become infected by ingesting contaminated colostrum, milk, feed, or water. The slow spread of the disease and its chronic course cause periodic economic losses(67).

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Contributions by INIFAP

Researchers at former CENID-Microbiología obtained a protoplasmic antigen from a strain called Map 3065, derived from a clinical case of a sheep. This antigen was used to standardize agar immunodiffusion techniques (IDGA) and enzyme-linked immunosorbent assay (ELISA)(68). In Mexico, epidemiological indicators have been determined in production units (PU) in the states of Chihuahua, Coahuila, Sinaloa, Durango, San Luis Potosí, Jalisco, Aguascalientes, Guanajuato, Querétaro, Hidalgo, Puebla, Chiapas, and Veracruz. Prevalences ranged from 1.0 to 32.37 % in the different states; in each individual PU, prevalences ranged from 1.0 to 88.87 %. In another epidemiological study, the presence of paratuberculosis was associated with the sanitary conditions of each PU, which allowed the issuance of sanitary management recommendations for the control of the disease(67,69,70,71). Another technique that was implemented was the fluorescence polarization assay (FPA), which improved the epidemiological sensitivity and specificity rates(72). A PCR was also implemented, in which DNA is extracted from feces, milk, cheese, or tissues with lesions. Using this technique, cases of serology-negative animals are confirmed, which, if they remain in the herd, would be the main source of infection. Thus, PCR is useful as a confirmatory test for the disease. In addition, a nested PCR (nPCR) has been standardized, for which primers were designed to amplify a region of the IS900 insertion sequence gene specific for Map. With nPCR, results are obtained in a shorter time, and high sensitivity and specificity are attained. It should be contrasted with bacteriological isolation, which regularly requires 16 weeks(73). Perspectives. In order to understand the processes of humoral and cellular immunity of cattle to M. avium subspecies paratuberculosis, it is necessary to generate a line of research, and the challenge will be to develop an effective immunogen for the prevention of the disease.

Bovine respiratory disease This is a multifactorial disease involving exposure to viral, bacterial, environmental and physiological stressors affecting the cattle. It has been described as the most common and costly disease afflicting cattle worldwide. Clinical manifestations include fever (>40°C), 121

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nasal and ocular discharge, dyspnea, poor appetite, depression, prostration, and death. The economic impact due to morbidity, mortality, treatment costs, and lower production is substantial. Bovine respiratory disease (BRD) involves infectious bovine rhinotracheitis (IBR), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), parainfluenza3(PI3), and bovine herpes virus type 1 (BHV1). Viruses create conditions conducive to the colonization and replication of bacteria, facilitating their adhesion to infected cells. Thus, in cattle with viral infections and subjected to stressful conditions, severe respiratory infections associated with bacteria are present. The most frequent are Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni; these are normally part of the microbiota of the upper respiratory tract. They possess various virulence factors, M. haemolytica produces a leukotoxin that affects ruminant leukocytes; P. multocida has an antiphagocytic capsule and lipopolysaccharides; H. somni can survive intracellularly and is capable of producing biofilm(74). The complex is capable of altering the functions of alveolar macrophages, suppressing lymphocyte proliferation, inducing apoptosis, modifying cytokine expression, and triggering an inflammatory process(75).

Contributions by INIFAP

A collaborative project called "Pneumonic Complex in Ruminants”, with the aim of determining the bacterial genera involved in BRD, their serotypes and resistance to chemotherapeutics, was developed between INIFAP former CENID-Microbiología and UNAM. This project allowed the isolation of H. somnus (H. somni)(76), P. haemolytica (M. haemolytica), and P. multocida. These bacteria were also serotyped and characterized for resistance to chemotherapeutics. Likewise, it was found that most of the M. haemolytica serotypes belonged to type A1, and those of P. multocida, to type A(77,78,79); specifically, resistance to penicillin, ampicillin, and streptomycin was detected(80,81). Subsequently, virulence factors such as leucotoxin were detected in M. haemolytica(82), and biofilm formation was evidenced in P. multocida, M. haemolytica, and H. somni(83). In addition, P. multocida was observed to produce vesicles on the outer membrane(84). From the isolates, strains were generated with which it was possible to formulate a bacterin-toxoid vaccine for the prevention of BRD, which was evaluated in ovine models(85,86,87). The biologic generated is currently produced at INIFAP and is used in some ovine health programs. Perspectives. The priority will be to develop biologics with domestic strains of IBR, BRSV and PI3 viruses, and combine them with live attenuated strains or subunits of M. 122

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haemolytica, P. multocida, and H. somni to confer effective protection against BRD. In order to improve diagnosis, it will be necessary to initiate metabolomics studies to monitor metabolites during the course of the disease. It would also be desirable to create a line of research on the genetic resistance of cattle to BRD. Collaterally, transcriptomics would be a very useful tool to try to make a genetic selection and form BRD-resistant herds.

Leptospirosis Leptospirosis is a zoonosis of worldwide distribution; it is caused by bacteria of the genus Leptospira. According to the DNA analysis, this genus includes 10 pathogenic, 5 intermediate, and 7 saprophytic species(88). Serology recognizes more than 300 serovars(89). Worldwide, hardjo is the most commonly detected serovar in cattle(90). Small mammals are the main reservoirs of the bacterium, large herbivores are a source of infection, and humans can be accidental hosts(91). Cattle are renal carriers of Leptospira spp. and therefore eliminate the bacteria through urine, contaminating the environment(92). Leptospirosis causes reproductive disorders such as miscarriages, stillbirths, weak premature calves, and reduced milk production, resulting in considerable economic losses(93). In Mexico, the first descriptions of leptospirosis in humans and cattle were made in 1928 and 1930, respectively(94).

Contributions by INIFAP

A collaborative work between INIFAP, UAM and UAEM reported the situation of bovine leptospirosis in Mexico. Prevalence rates were determined in different ecological zones of the country; in the arid and semi-arid zones, the prevalence was 37.8%; in the dry tropics, 45.9%; in the humid tropics, 63.8%, and in the temperate zone, 39.4%. The presence of the hardjo, wolffi, and tarassovi serovars was demonstrated in all regions. In the temperate region, the icterohaemorrhagiae, portland-Vere, bratislava, pyrogenes, canicola, and pomona serovars were detected(95). The grippotyphosa, mini, and tarassovi serovars were isolated; this had not been done in Mexico(96,97). In other epidemiological studies, the same serovars were identified, but prevalences varied widely from 31 to 91 %(98-103). For diagnosis, INIFAP implemented the PCR technique, which allowed the detection of bacteria in urine collected from cattle with a history of reproductive problems(100).

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Bacterins were generated to prevent the disease: one was added with adjuvant using liposoluble vitamins, which yielded satisfactory results(104). Another bacterin was made with serovars isolated in the state of Chiapas that were not contained in commercial bacterins; an excellent level of protection was observed in susceptible cattle with this homologous biologic. INIFAP currently has a bacterin that has been validated in dairy herds(105). Perspectives. The endemicity and high prevalence of leptospirosis in Mexico is evident; therefore, it is a real challenge to massify the use of microagglutination, which is the reference test for determining the Leptospira serovars present in the different regions, and then produce homologous bacterins that will effectively prevent leptospirosis. A line of research should be generated to develop molecular vaccines that can be used in any ecological region. It would also be advisable to implement an accurate methodology with high sensitivity and specificity, fast execution, and low cost. This can result in a better diagnosis that will increase the reproductive and productive parameters of cattle.

Ticks Ticks are hematophagous ectoparasites capable of injecting toxins and transmitting to livestock different pathogens such as A. marginale and Babesia spp. with high morbidity and mortality rates. Of the various ticks identified in Mexico, the most important is Rhipicephalus microplus. Today they constitute a global problem due to their great adaptability to different ecological niches, it is considered that 65% of the national territory is infested with this tick.

Contributions by INIFAP

Epidemiology. An epidemiological study involving different states of the country corroborated that the distribution of R. microplus was essentially associated with environmental temperature, rainfall and water vapor(106). In another research, greater efficiency and reproductive fitness was observed in ticks of a native strain collected in the field in Sinaloa, compared to a reference strain from CENID-SAI(107). Biological control. For tick control, this strategy has been well documented in studies conducted at INIFAP. Among the evaluation of techniques for the collection of R. microplus tick larvae, the double-traveled flag technique was selected for various studies. 124

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The effect of the recovery of R. microplus larvae using tropical legumes in the state of Morelos was evaluated(108). Another study evaluated the anti-larvae effect using Stylosanthes humilis, S. hamata, Cenchurus ciliaris, and Andropogon gayanus grasses in artificially infested plots. A favorable effect was observed in S. humilis plots where only 3% of live larvae were recovered(109). Other research using mature plants of S. humilis and S. hamata observed no anti-tick effect(110). On the other hand, when evaluating crops of the legumes Leucaena leucocephala and Macroptilium artropurpureum, S. humilis, and S. hamata, a significant reduction in the number of larvae of R. microplus was observed(111). Based on these findings, certain chemical compounds in S. humilis and S. hamata were identified as possible causes of the repellent effect(112). Similarly, another study using M. minutiflora grass also showed a reduction in larval recovery (113). Other strategies have involved the use of fungi or bacteria for tick control; thus, the use of the entomopathogenic fungus Metarhizium anisopliae demonstrated its ability to infect ticks and induce up to 100 % mortality, which allowed inferring that it could be a potential acaricide for the biological control of R. microplus(114). On the other hand, in engorged adult ticks that were experimentally infected with Staphylococcus saprophyticus bacteria, it was able to induce tick mortality(115). It was reported for the first time that the fungus Aspergillus flavus is capable of infecting 80 % of engorged adult ticks, the ovigerous masses and the larvae that emerge after hatching, under controlled conditions(116). Resistance. This is one of the most studied topics at INIFAP; in one of the first studies it was demonstrated that in R. microplus tick populations resistant to organophosphates, there is an elevated expression of carboxylesterase enzymes(117). Subsequently, some genes coding for esterases were characterized to provide molecular markers for discriminating ixodicide-susceptible and ixodicide-resistant tick strains(118). Genes coding for carboxylesterases B were analyzed by PCR assays in individual R. microplus larvae, detecting polymorphisms upon protein translation(119,120); an esterase was also identified in the "Coatzacoalcos" strain (Cz EST9)(121). Another study sought to identify the association of gene mutations with pyrethroid resistance. Noting that the presence of the mutation is not associated with resistance in the dose-response form(122). Studies on pyrethroid resistance attempted to correlate different diagnostic tests, and it was concluded that resistance is mediated by a mutation in the target gene Kdr(123). The participation of cytochrome P450 has also been studied, and it has been observed to be expressed at high levels in pyrethroid-resistant strains(124). However, a multifactorial process has been evidenced in the resistance of R. microplus to organophosphates and pyrethroids(125). The first case of amitraz resistance was reported(126), and selection pressure with amitraz was described as increasing the level of resistance in field populations(127). In addition, RT-PCR methodology was used to measure the expression of cholinesterase and carboxylesterase in acaricide-resistant ticks(128). 125

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Immunological control. For tick control, immunogenic proteins derived from extracts of R. microplus ovaries obtained from cattle after immunization have been identified (129). Other studies have characterized and evaluated homology to vitellogenin proteins(130) and ATAQ, both as potential vaccine candidates against R. microplus(131,132). Certain immunization experiments against R. microplus and R. annulatus ticks have shown inconclusive results. However, similar studies have continued, such as the use of the protein subolesin, which was described as a potential target for developing a tick vaccine(133,134). Perspectives. There is an undeniable need to place greater emphasis on research into the epidemiology of ticks; especially climate change is a factor that is favoring their greater spatial distribution and, therefore, the infestation of livestock not previously exposed to ticks. It is also imperative to develop molecular techniques for the rapid diagnosis of resistance to the different chemical principles of ixodicides. Collaterally, a line of research on biological control should be maintained, involving the identification and characterization of plants. A line of research on the development of immunogens from conserved proteins associated with vital tick functions should be a priority. The major challenge will be to implement an integrated program for the control of R. microplus.

Babesiosis Bovine babesiosis or piroplasmosis is a parasitic disease caused by protozoa of the genus Babesia that invade the erythrocytes of the bovine host. In Mexico, the recognized species are Babesia bovis and B. bigemina, both transmitted by the R. microplus and R. annulatus ticks(135). Approximately 70% of the country's 35,224 960 head of cattle(136) are permanently exposed to tick infestation. Thus, the prevalence of Babesia spp. varies between 50 and 96 %, which in turn explains the high risk of outbreaks occurring(137). Babesiosis has been identified as the most important arthropod-borne disease of cattle(138). In Mexico territory, losses are estimated at 573.61 million dollars per year due to ticks and the diseases they transmit(139). However, there is no commercial vaccine, and no national production of diagnostic reagents. In addition to the above, the wide distribution of resistance to ixodicides and climate change are major factors contributing to the abundance of vectors and the facilitation of pathogen transmission(140).

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Contributions by INIFAP

Diagnosis and epidemiology. INIFAP has implemented direct methods for the confirmatory diagnosis of babesiosis. Techniques for the identification of intraerythrocytic stages are routinely available. The most common is the peripheral blood smear with which B. bovis and B. bigemina are identified by means of microscopic observation; brain tissue imprints are also made, particularly for the detection of B. bovis(141,142). Histopathological analysis of tissues collected at necropsy can also be performed(143,144,145). Immunologically based indirect methods have been developed to detect circulating anti-B. bovis or anti-B. bigemina antibodies(143,146,147). Defined and characterized parasitic antigens have been obtained for these procedures(148,149). Advantages have been observed when compared to crude antigens with which a low specificity is regularly obtained in diagnostic tests; this occurs due to the similarity of epitopes present between different species of Babesia(146,150,151), and it can also generate cross-reactions with other species(148,151,152). INIFAP research group has also improved the specificity of serological tests. For this purpose, genes coding for immunodominant, species-specific peptides have been cloned, and monoclonal antibodies have been used(148,149,153). Other studies have identified the most conserved antigens for B. bovis(154-158), utilized for developing indirect ELISA tests for both species(159,160), which in turn were tools for serological monitoring of experimentally immunized animals (161,162,163). These tests were also incorporated in seroepidemiological studies of cattle herds located in different cattleraising areas of the country(160,164). On the other hand, there was a notorious advance in direct diagnosis; molecular procedures that detect genetic material of the parasites were reported. These have included the use of nucleic acid probes or nucleic acid amplification techniques(165,166), which have been used in epidemiological studies in different cattle-raising regions of the country(167). Using B. bigemina genomic DNA, a PCR with high analytical sensitivity was developed, for which the amplified product was hybridized with a non-radioactive DNA probe(168,169). A multiple format was also implemented for the simultaneous detection of B. bovis and B. bigemina, to which the diagnosis of A. marginale was added(166,170,171). DNA probes were used in epidemiological studies in Yucatán, Tabasco and Campeche(172,173). They were also used for the monitoring of cattle inoculated with vaccine strains of B. bovis and B. bigemina(149,174); as well as in the monitoring of susceptible animals introduced to endemic areas(175,176). This same methodology proved useful for the detection of pathogen DNA in ticks(177), as well as for the specific identification of B. bovis and B. bigemina in the tick R. microplus(178,179).

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Prevention. So far the best babesiosis prevention strategy in endemic regions is immunization with live attenuated vaccines, which can be derived from subinoculation into splenectomized calves, or from in vitro culture of B. bovis and B. bigemina(180). The application of attenuated vaccines in susceptible cattle has been shown to induce a robust immune response in the face of attacks by highly virulent parasites(181,182). INIFAP researchers have participated in the development and adaptation of in vitro culture of B. bovis and B. bigemina, and today attenuated strains of these protozoans are available in Mexico(183,184). A review of the development in Mexico of the attenuated vaccine from in vitro culture can be carried out based on various studies. These include the demonstration of low virulence of in vitro-culture derived parasite clones that were inoculated into susceptible cattle(185). When using the material as fresh immunogen, the appropriate dose was determined to be 1 x 107 erythrocytes infected with B. bovis or B. bigemina(186,187). Another study showed the need to include both Babesia species to induce successful protection against the disease(188). Similar results were obtained with the vaccination of cattle against a natural challenge in the tropics(189). Subsequently, it was determined that material derived from in vitro culture that was removed from cryopreservation in liquid nitrogen (-196 °C) required increasing the dose to 1 x 108 infected erythrocytes of each species in order to protect cattle from challenge with virulent parasites(190). The use of the vaccine was also evaluated in native cattle kept in farms with high endemicity and enzootic instability, where an excellent level of protection against babesiosis was also demonstrated(191). In another study, the vaccine was spiked with Lactobacillus casei and evaluated against a natural challenge; increased levels of specific IgG1 against B. bovis and B. bigemina; however, the level of protection was analogous to that of the vaccine without the bacteria(192). In vitro culture of B. bovis and B. bigemina is apparently a simple methodology; however, few laboratories in the world do it successfully. After more than 30 years of being established in Mexico, there was a low efficiency in the production of biomass. In recent years, INIFAP has positioned itself as a leading institution at the international level for innovations that have been integrated into the in vitro cultivation of B. bovis and B. bigemina. Bovine serum has been successfully removed from the culture medium and replaced by vital components such as insulin, transferrin, selenite, and putrescine. For the first time, the process was transferred to a perfusion bioreagent, thereby increasing the number of infected erythrocytes by 300%. This implied obtaining a high number of vaccine doses, compared to the traditional procedure(193,194,195). The bioreagent-derived material evaluated as an immunogen conferred to cattle a level of protection above 80% in a field challenge(196). That immunogen without the presence of serum proteins has been proposed to induce a response with greater immunological specificity(197). At the same time, the incorporation of the bioreagent has generated a line of research on the use of soluble antigens derived from the culture supernatant. Recently, in INIFAP laboratory have 128

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achieved for the first time the proliferation of B. bigemina in a culture medium free of animal components, and also successfully transferred it to the bioreagent —a procedure that represents a scale-up of the process for vaccine production—(198). These changes will facilitate the continued development of subunit vaccines(199). Due to their degree of invention, the innovations described above have caused two patents to be granted in favor of INIFAP, and a third one is pending. One of the granted patents is entitled "Serum-free in vitro culture composition for obtaining erythrocytes parasitized with Babesia spp." (Patent No. 347729), and the other is called "Process for the elaboration of vaccinal reagent of erythrocytes parasitized with Babesia spp. Babesia bovis or Babesia bigemina" (Patent No. 337161). Perspectives. There is a need to generate highly sensitive diagnostic tests with the ability to identify Babesia strains resistant or susceptible to antibabesial compounds. It would also be relevant to implement a procedure to discriminate attenuated (vaccine, conventional, genetically modified) or virulent field strains. Dynamic mapping of distribution and frequency is essential for the timely application of babesiosis prevention or control procedures. Live vaccines are now the only way to prevent the disease, but it is imperative to maintain the omics sciences in order to generate more knowledge of the interactions between parasites and cattle. This knowledge will facilitate the development of subunit vaccines that may be safer and more easily scalable.

Conclusions INIFAP has developed and adapted serological and molecular diagnostic tools that have contributed to programs for the prevention and control of cattle diseases. Techniques for the detection of resistance to ixodicides have also been implemented. The distribution and frequency of some of the most important diseases affecting cattle farming in Mexico have been determined. The biologics developed include vaccines against rabies, anaplasmosis and babesiosis, as well as a bacterin against leptospirosis and a bacterin-toxoid against pneumonia. In addition, a BCG vaccine against tuberculosis and a new generation vaccine against brucellosis have been studied. The animal health perspective on zoonotic diseases such as tuberculosis and brucellosis suggests directing scientific and technical efforts toward those diseases elimination. Research on the effect of climate change, especially on vector-borne diseases, should be developed through the protocols and methods of omics sciences, such as genomics, epigenomics, transcriptomics, proteomics, metabolomics and other omics derivatives. It is currently the most appropriate way to understand the mechanisms of disease, and, therefore, it generates more effective vaccines and allows designing more precise diagnostic tools, which will be essential to integral control 129

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programs. Probably the biggest challenge will be to incorporate animal health research at INIFAP into the "One Health" concept. This has been defined as a multi-sectoral and transdisciplinary collaborative process at local, regional, national and global levels, based on the interconnections between humans, animals, plants and the environment(200). Literature cited: 1.

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174. Figueroa MJV, Alvarez MJA, Canto AGJ, Ramos AJA, Mosqueda GJJ, Buening GM. Comparative sensitivity of two tests for the diagnosis of multiple hemoparasite infection of cattle. Ann NY Acad Sci 1996;791(1):117-127. 175. López M, Figueroa MJV, Ramos AJA, Mosqueda GJJ, Rojas, REE, Vega MCA, et al. Infection and seroconversion of susceptible animals introduced into a babesiosis endemic area. Ann NY Acad Sci 2008;1149(1):131-135. 176. Figueroa MVJ, Cantó AGJ, Álvarez MJA, Lona R, Ramos AJA, Vega MCA. Capacidad protectora en bovinos de una cepa de Babesia bigemina derivada del cultivo in vitro. Téc Pecu Méx 1998;(36):95-107. 177. Sparagano OAE, Allsopp MTEP, Mank RA, Rijpkema SGT, Figueroa MJV, Jongejan F. Molecular detection of pathogen DNA in ticks: A review. Exp Applied Acarol 1999;23(12):929-960. 178. Rojas RE, Mosqueda GJJ, Álvarez MJA, Hernández OR, Ramos AJ, Rojas MC, et al. Transmissibility of Babesia bigemina and Babesia bovis attenuated strains by Rhipicephalus microplus ticks. Rev Mex Cienc Pecu 2011;2(3):267-281. 179. Figueroa MJV, Lira JJ, Polanco MDJ, Álvarez MJA, Rojas MC, Bautista GCR. Diferenciación de Babesia bovis y Babesia bigemina mediante el uso de una prueba molecular en ADN extraído de garrapatas repletas. Entomol Mex 2015;(2):706-713. 180. Bock RE, de Vos AJ, Lew A, Kingston TG, Fraser IR. Studies on failure of T strain live Babesia bovis vaccine. Aust Vet J 1995;72(8):296-300. 181. Shkap V, de Vos AJ, Zweygarth E, Jongejan F. Attenuated vaccines for tropical theileriosis, babesiosis and heartwater: the continuing necessity. Trends Parasitol 2007;(23):420-426. 182. Shkap V, Kocan K, Molad T, Mazuz M, Leibovich B, Krigel Y, et al. Experimental transmission of field Anaplasma marginale and the A. centrale vaccine strain by Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus ticks. Vet Microbiol 2009;134(3-4):254-260. 183. Figueroa MJV, Cantó AGJ, Juárez FJ, Ruiz LF. Cultivo in vitro de Babesia bovis: establecimiento y condiciones óptimas de multiplicación. Téc Pecu Méx 1984;(46):4652. 184. Monroy BM, Romero OG, Torres AR, Álvarez MJA, Canto AGJ, Vega MCA. Establecimiento en México del cultivo in vitro de Babesia bigemina. Téc Pecu Méx 1987;(25):141-50.

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185. Hernández OR, Álvarez MJA, Buening GM, Cantó AGJ, Monroy BM, Ramos AJA, et al. Diferencias en la virulencia y en la inducción de protección de aislamientos de Babesia bigemina derivados de cultivo in vitro. Téc Pecu Méx 1990;28(2):51-61. 186. Cantó AGJ, Figueroa MJV, Álvarez MJA, Ramos AJA, Vega MCA. Capacidad inmunoprotectora de una clona irradiada de Babesia bovis derivada del cultivo in vitro. Téc Pecu Méx 1996;34(3):127-135. 187. Figueroa MVJ, Cantó AGJ, Álvarez MJA, Lona R, Ramos AJA, Vega MCA. Capacidad protectora en bovinos de una cepa de Babesia bigemina derivada del cultivo in vitro. Téc Pecu Méx 1998;36:95-107. 188. Vega MCA, Figueroa MJV, Rojas REE, Ramos AJA, Cantó AGJ. Insuficiente inmunidad cruzada en bovinos por Babesia bigemina y/o Babesia bovis derivadas del cultivo in vitro. Téc Pecu Méx 1999;37(1):13-22. 189. Cantó AG, Figueroa MJV, Ramos AJ, Álvarez MJA, Mosqueda GJJ, Vega MC. Evaluación de la patogenicidad y capacidad protectora de un inmunógeno fresco combinado de Babesia bigemina y B. bovis. Vet Méx 1999;30(3):215-20. 190. Alvarez MJA, Ramos AJA, Rojas RE, Mosqueda GJJ, Vega MCA, Olvera MA, et al. Field challenge of cattle vaccinated with a combined Babesia bovis and Babesia bigemina frozen immunogen. Ann NY Acad Sci 2004;1026(1):277-283. 191. Ojeda JJ, Orozco Flores R, Rojas C, Figueroa JV, Alvarez JA. Validation of an attenuated live vaccine against babesiosis in native cattle in an endemic area. Transboun Emer Dis 2010;57(1-2):84-86. 192. Bautista GCR, Lozano AR, Rojas MC, Alvarez MJA, Figueroa MJV, García GR, et al. Co-immunization of cattle with a vaccine against babesiosis and Lactobacillus casei increases specific IgG1 levels to Babesia bovis and B. bigemina. Parasitol Int 2015;64(5):319-323. 193. Rojas MC, Rodriguez VRI, Figueroa MJ, Acosta VKY, Gutiérrez RJ, Alvarez MJ. In vitro culture of Babesia bovis in a bovine serum-free culture medium supplemented with insulin, transferrin, and selenite. Exp Parasitol 2016;(170):214-219. 194. Rojas MC, Rodriguez VRI, Figueroa MJV, Acosta VKY, Gutiérrez REJ, Alvarez MJA. Putrescine: essential factor for in vitro proliferation of Babesia bovis. Exp Parasitol 2017;(175):79-84.

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195. Rojas MC, Rodriguez VRI, Figueroa MJV, Acosta VKY, Gutiérrez REJ, Bautista GCR, et al. Babesia bigemina: advances in continuous in vitro culture using serum free medium, supplemented with insulin, transferrin, selenite and putrescine. Parasitol Int 2018;67(3):294-301. 196. Rojas MC, Rodriguez VRI, Figueroa MJV, Bautista GCR, Castaneda ARO, Lira AJJ, et al. Bovine babesiosis: Cattle protected in the field with a frozen vaccine containing Babesia bovis and Babesia bigemina cultured in vitro with a serum-free medium. Parasitol Int 2018;67(2):190-195. 197. Brown WC, Palmer GH. Designing blood-stage vaccines against Babesia bovis and B. bigemina. Parasitol Today 1999;15(7):275-281. 198. Álvarez, M.J.A, Figueroa, MJV, Ueti, MW, Rojas MC. Innovative alternatives for continuous in vitro culture of Babesia bigemina in medium free of components of animal origin. Pathogens 2020;9(5):343. 199. Alvarez MJA, Rojas MC, Figueroa MJV. An Overview of current knowledge on in vitro Babesia cultivation for production of live attenuated vaccines for bovine babesiosis in Mexico. Front Vet Sci 2020;(7):364. doi:10.3389/fvets.2020.00364. 200. Mackenzie SJ, Jeggo M. The one health approach. Why is it important? Trop Med Infect Dis 2019;4(2):88.

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https://doi.org/10.22319/rmcp.v12s3.5879 Review

Swine health: history, challenges and prospects

José Francisco Rivera-Benítez a* Jazmín De la Luz-Armendáriz b Luis Gómez-Núñez a Fernando Diosdado Vargas a Guadalupe Socci Escatell a Elizabeth Ramírez-Medina c,d Lauro Velázquez-Salinas c,e Humberto Ramírez-Mendoza b Maria Antonia Coba Ayala f Catalina Tufiño-Loza a,b Marta Macías García g Víctor Carrera-Aguirre h Rebeca Martínez-Bautista i María José Martínez-Mercado i Gerardo Santos-López j Irma Herrera-Camacho j Ignacio Siañez-Estrada k Manuel Zapata Moreno b

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Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, Km 15. 5 Carretera MéxicoToluca, Palo Alto, Cuajimalpa, CP. 05110, Ciudad de México, México. b

Universidad Nacional Autónoma de México. Facultad de Medicina Veterinaria y Zootecnia, Ciudad de México, México. c

USDA/ARS Plum Island Animal Disease Center. Foreign Animal Disease Research Unit, Greenport NY, USA. d

University of Connecticut. Department of Pathobiology and Veterinary Science, Storrs, CT, USA. e

Kansas State University. College of Veterinary Medicine, Manhattan, KS, USA.

Práctica privada.

LAPISA Salud Animal. La Piedad, Michoacán, México.

SANFER Salud Animal. Ciudad de México, México.

Zoetis Swine, Ciudad de México, México.

Instituto Mexicano del Seguro Social. Centro de Investigación Biomédica de Oriente, Atlixco, Puebla, México. k

Benemérita Universidad Autónoma de Puebla. Centro de Química, Instituto de Ciencias, Puebla, México.

*Corresponding author: rivera.francisco@inifap.gob.mx

Abstract: In swine production systems, one of the critical points that must be strictly attended to is the health of the pigs. Health is a structural component of animal welfare and reflects an optimal state of the animals, which has a direct impact on a higher productive performance and better development conditions. Infectious diseases are one of the greatest threats to the health of pigs and can cause losses of up to 100 % of production; therefore, it requires constant attention and continuous monitoring by the veterinarian and producers, in perfect coordination with the official health authorities. Currently, the implementation of best practices in the production chain is of interest to both producers and consumers. The control of infectious diseases requires collaboration between the various actors in the environment

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and must be considered a public good, since their negative repercussions can range from the local to the global level. This review will address the main infectious diseases that endanger swine health, their impact, the main contributions made by the National Institute for Research in Forestry, Agriculture and Livestock (INIFAP) in its 35 years of life, mainly at the National Center for Disciplinary Research in Animal Health and Safety (CENID-SAI), formerly known as the emblematic CENID-Microbiología or Palo Alto. Key words: Pig Farming, Infectious Diseases, Technology, Innovation.

Received: 24/11/2020 Accepted: 11/03/2021

Introduction Pig farming in Mexico and its global context It is estimated that there are close to 100 million head of pigs in the world, with China, the United States and Brazil being the countries with the largest inventories. In 2018, FAO estimated that the per capita consumption of pork worldwide was 12.3 kg per year, making it the second most consumed meat(1). In Mexico, the states of Jalisco, Sonora and Puebla are the largest producers. In 2020, the Agri-food and Fisheries Information Service (SIAP) reported an estimated production of 134,953 t, and the FAO recorded a per capita consumption of 12.8 kg in Mexico (2018), for which pork is considered the third livestock commodity with the highest economic participation in the country(2). Swine production units in Mexico have been classified by their level of technification and by their production objective; with respect to the level of technification, there are technified, semi-technified and low-scale production units, commonly known as backyard units(3). Technified production units account for 40 to 50 % of the national inventory and 75 % of national pork production(4). Semi-technified production units have a 20 % national share and are production systems that are decreasing. Finally, there are low-scale or backyard production units; this type of production has a 30 % distribution at the national level(3,4). In these three types of swine production, it is important to highlight that, in order for the species to be produced efficiently, it is necessary to comply with animal welfare standards during production, quality parameters during transport and, above all, to control the main critical points during slaughter, in order to obtain the best quality meat to be offered to the final consumer. 151

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Pig farming worldwide has been constantly challenged by several direct and indirect factors. Currently, the Covid-19 global pandemic, generated by the SARS-CoV-2 virus, which is responsible for more than 45 million confirmed cases, including more than one million deaths, as of October 2020, has been identified as the most serious pandemic in the world(5). It has been confirmed that pigs are not susceptible to SARS-CoV-2 infection. However, the pork industry has been affected, as the export and import of pigs has been restricted, and infection is common among workers on farms and in processing plants, decreasing pork production capacity(6). There has been a low consumption of meat products during this period; for this reason, there were farms that had to eliminate the inventory that was destined for the market, due to the lack of sales. In addition, the price of live pigs in Mexico was affected, reaching extremely low prices (15 to 16 pesos per kilo), causing producers to forego some of the health programs used on farms. The global Covid-19 pandemic has altered consumer behavior, distribution, production and market prices. Production setbacks were one of the biggest challenges faced by the meat industry, but the sector's capacity has largely returned to normal in recent months. Another factor affecting pig farming is infectious agents that cause high morbidity and mortality rates. A recent example is African swine fever (ASF), which is a viral disease that causes high mortality rates in domestic pigs. In 2018, outbreaks of this disease were reported in different provinces of China and currently causes outbreaks in Europe and Asia; the implementation of strict biosecurity measures are the tool to prevent the entry of this viral agent and depopulation is the control protocol, until the development of an effective biologic is achieved(7,8). Fortunately, the American continent is still free of this infectious agent and this makes it one of the potential exporters of pork to China. Within this context, Mexican pork exports to China, reported a 929 % growth during January 2020, totaling 4,076 t of meat, versus the 396 t reported in January 2019. At the end of 2019, Mexico exported 30,072 tons of pork to China, which placed the Asian country as the second largest buyer of this type of Mexican meat(9).

Swine health, infectious agents and their repercussions Today, the stability of human society around the world has been affected by various aspects, such as population growth, food security, the need for more efficient and sustainable production methods, and climate change. Population growth is expected to require 70 % more food production than today by 2050(10). This requires more intensive production systems, with larger animal populations, leading to the emergence of emerging and re-emerging diseases, which are a continuous challenge in animal health. The following is a description of the main diseases that must be treated, some of which are exotic, while others are endemic;

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however, all of them have a negative impact on swine production in economic and productive terms.

Bacterial agents Respiratory diseases

Since 1960, respiratory diseases in pigs(11) have been described, and several investigations have been carried out with the aim of identifying the etiological agents involved in them. Different studies in pigs have shown that co-infections between bacteria and viruses lead to an exacerbation of pulmonary lesions, due to an increased immunological reaction characterized by an increase in the production of proinflammatory cytokines(12). Porcine respiratory complex (PRC) related agents can be divided into primary and secondary or opportunistic pathogens(13). Among the primary agents, there are some bacteria with certain serotypes of high virulence of Actinobacillus pleuropneumoniae (App), Mycoplasma hyopneumoniae, and Bordetella bronchiseptica. Among the bacteria included as secondary or opportunistic pathogens are low-virulence strains of App, Glaesserella parasuis (formerly Haemophilus parasuis), Pasteurella multocida, and Streptococcus suis(13). Actinobacillus pleuropneumoniae (App), Gram-negative bacteria causing fibrinous pleuritis, hemorrhagic and necrotic bronchopneumonia, which can lead to increased mortality(14). The most virulent strains of App have tropism for the lower respiratory tract (bronchioles and pneumocytes), their main damage is caused by exotoxins (Apx I, II, III and IV) that produce cell lysis, which results in characteristic lesions(14). Mycoplasma hyopneumoniae, is the cause of enzootic pneumonia(15). Two mechanisms are derived from M. hyopneumoniae and its participation in CRP: i) alteration in the ciliated epithelium cells, with loss of cilia and, therefore, permissiveness to the invasion of secondary pathogens, and ii) alteration of the immune response(15). Infection with M. hyopneumoniae inhibits the phagocytic activity of some cells of the innate immune response, such as macrophages, favoring infections by other pathogens(15,16). An established M. hyopneumoniae infection contributes to the potentiation of viral infections(12,17). In recent years, several efforts have been made to eliminate M. hyopneumoniae, mainly in breeding females(18). The probability that the herd will remain negative for at least one year after culling is 83 %(19).

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Bordetella bronchiseptica, Gram-negative bacteria, which can be considered as primary or secondary pathogens, depending on the time of infection. As a primary pathogen, it can cause necrotic and hemorrhagic bronchopneumonia in piglets. Clinical signs can range from a transient cold to atrophic rhinitis, when associated with another pathogen such as Pasteurella multocida. Most studies on the interactions of CRP pathogens focus on the evaluation of clinical signs and the impact of the disease; however, the mechanisms involved at the molecular level have been little studied(12), which opens up a field of research in this area. Glasserella parasuis, (formerly Haemophilus parasuis), a Gram-negative bacterium causing Glässer's disease, which produces fibrinous polyserositis and septicemia with localization in the brain, joints and/or lungs(13). Mortality can be high, mainly in populations with no previous exposure(18). Streptococcus suis, is an encapsulated Gram-positive coccus(20) that mainly affects pigs from 5 to 10 wk of age. It causes acute death by septicemia, meningitis, polyarthritis, polyserositis, valvular endocarditis, and can also cause damage to the digestive and genital tract; occasionally, pigs may present dyspnea and cyanosis. In healthy pigs, it is commonly found in the tonsils and the upper respiratory tract. It is a zoonotic microorganism that has increased its importance in the last 10 years, of which serotype 2 is the most important for public health(21). S. suis has been classified into 35 serotypes(22), and its distribution depends on the geographical location(23). In the USA and Canada, serotypes 2 and 3 are the most abundant; in the case of Mexico, no data are available, but it can be suggested that they are similar. At CENID-SAI, studies have been conducted to identify the presence of these infectious agents; in 1997, a serological survey was carried out which detected a significant association between bacterial infection with M. hyopneumoniae, App, and infection with Aujeszky's disease (AD) virus(24). In 2008, an end-point PCR test was evaluated and standardized, which identified different strains of App(25). In 2011, M. hyopneumoniae was identified by PCR in early infected pigs with or without the presence of clinical signs(26).

Digestive diseases

In intensive production farms, enteric diseases in pigs cause economic losses due to increased medication costs and stunted growth. Brachyspira hyodysenteriae is considered to be an anaerobic intestinal spirochete, which causes a mucohemorrhagic colitis known as swine dysentery. Swine dysentery affects pigs in the growing and finishing stage, which manifest moderate mucoid diarrhea without

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affecting body condition or, in some cases, hemorrhagic diarrhea with mortality rates of 50 to 90 %(27). In affected herds, swine dysentery causes economic losses due to mortality, decreased growth rates, lower feed conversion, and treatment costs(28). Lawsonia intracellularis is a Gram-negative obligate intracellular bacterium that causes proliferative enteropathy or ileitis. The disease is characterized by a thickening of the intestinal mucosa due to a proliferation of the intestinal crypt epithelium, located mainly in the ileum(29). The disease manifests itself in acute and chronic forms. The acute presentation causes hemorrhagic proliferative enteropathy, with high mortality and bloody diarrhea, affecting pigs in the finishing stage and replacement females. Intestinal adenomatosis is the chronic manifestation of the disease, subclinical and self-limiting in young pigs, although complication by opportunistic bacteria is possible, resulting in necrotic enteritis with presence of fibrinous exudate and necrosis(30). It has a wide distribution in pig farms. Its economic impact is due to the fact that clinical cases result in lower finishing weight and poor feed conversion(31). Salmonella spp. is a ubiquitous bacterium. In the case of pigs, S. typhimurium has an enteric presentation with diarrhea, a consequence of enterocolitis, while S. cholerasuis has a septicemic presentation(32). It is most frequent in animals during the weaning stage up to five months of age. In the superacute form, septicemia causes sudden death, mainly in pigs from two to three months of age, with diffuse hemorrhage in different organs; the acute form presents yellowish diarrhea, fever and emaciation, with ulcers, which can lead to a chronic form, with the presence of botulinous ulcers, intestinal necrosis, and stenosis. Infected animals remain carriers for months and excrete the bacteria intermittently, via feces(33). Escherichia coli, a Gram-negative, facultative anaerobic bacillus, classified within the family Enterobacteriaceae, normally colonizes the intestinal microbiota of domestic animals. However, it causes neonatal diarrhea in piglets and edema disease in the postweaning stage, commonly associated with enterotoxigenic strains, which produce, as a virulence factor, enterotoxins that cause secretion of water and electrolytes into the intestinal lumen, causing diarrhea, dehydration, acidosis, and edema(34). Other virulence factors, related to adherence and infection of epithelial cells, are fimbria and pili, which are identified for a more accurate diagnosis of the type of strain involved in the clinical picture. There are other strains that produce the Shiga toxin (Stx2e) that causes edema disease(35). Colibacillosis has economic implications resulting from mortality rates of 50-90 %, low growth rates, weight loss, treatment costs due to the use of antibiotics, antisecretory or probiotic drugs, and vaccination(36). In 1998, the PCR test was established for the first time in Mexico at the CENID-SAI of INIFAP, with the objective of detecting L. intracellularis(37). The advantages of this methodology are its versatility, speed, high sensitivity and specificity. In 2005, a study was 155

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conducted to determine the frequency of herds infected with L. intracellularis, and it detected 37 % of positive farms(38). With the establishment of this methodology, diagnostic services were provided to private companies, and several studies were carried out on the excretion patterns of L. intracellularis. In 2004, microbial resistance-causing phages were identified in strains of Salmonella spp(39). In recent studies, L. intracellularis, B. hyodisenteriae and Salmonella spp. were detected in 26 %, 11 % and 4 %, respectively. At CENID-SAI, technology was generated and validated based on the simultaneous detection of B. hyodysenteriae, L. intracellularis, and Salmonella spp. by PCR from a single stool sample. Clinical and laboratory diagnosis for these three diseases was difficult, laborious and costly. This technology was transferred to private laboratories, which were able to offer the service to producers in order to confirm the presence of these agents in their herds. This was reflected in a difference in net income of 650 % for users of INIFAP technology compared to a control technology, and an economic benefit of $936,000.00 MXN, derived from the analysis of 900 samples from pigs(40).

Viral agents Endemic diseases

Infection by porcine circovirus type 2 (PCV2) Porcine circovirus (PCV) belongs to the genus Circovirus of the family Circoviridae, viruses with a single-stranded circular DNA genome. To date, four types of porcine circoviruses (PCV1-4) have been reported(41,42). There is a high genetic diversity of PCV2 and eight genotypes (PCV2a-h) have been identified. PCV2 genotypes cannot be identified by conventional serology, as they have high cross antigenicity; this characteristic has maintained the use of available PCV2 vaccines. However, there is no cross antigenicity between PCV2 and PCV3(41,42). To date, PCV1 (contaminant of the PK-15 cell line) is considered nonpathogenic in swine(43,44). In 1997, PCV was associated with a disease affecting weaning pigs known as postweaning multisystemic wasting syndrome (PMWS)(45,46). PMWS is distributed worldwide and is commonly described in pigs at weaning or early fattening in unvaccinated farms. PCV2 seroprevalence within farms ranges from 15 % to 100 %, regardless of the existence of PMWS(46,47). In 2003, the first isolation and detection of antibodies against PCV2 was performed in Mexico. A retrospective study demonstrated the presence of antibodies against PCV2 in Mexico since 1973. This study showed that PCV2 infection has been enzootic in Mexico for many years prior to the first description of PMWS(48). Epidemiological studies have detected up to 98 % seroprevalence in backyard pigs(49).

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Current studies have demonstrated the existence of PCV2a (12.5 %), PCV2b (87.5 %)(50), PCV2d and, recently, PCV3(51). In 2018, 49 % of PCV2-positive cases were identified and co-infection with PRRS virus was confirmed, these results were obtained from standardized and validated molecular tests at CENID-SAI(52). Porcine circovirus type 3 infection (PCV3) In 2015, reproductive problems and swine nephropathy syndrome, pneumonia and swine dermatitis were identified in swine production units in the United States. When molecular diagnosis was performed for the identification of PCV2, the results were negative, so it was decided to perform metagenomic studies, identifying the presence of a new porcine circovirus genogroup, which was named porcine circovirus type 3 (PCV3)(53). In subsequent years it has been identified in Japan(54), China(55,56), the United Kingdom (since 1992)(57), Italy(58), Germany(59), and Sweden(60). As for Latin America, the first report of identification of specific antibodies against PCV3 was in samples obtained from swine production units in Mexico and the U.S.A.; these results were reported in 2016(53). In 2017, the presence of PCV3 was reported in Brazil(61), and the presence of PCV3 was confirmed in the Americas, Europe and Asia. The main clinical signs associated with the infection were post-weaning multisystemic wasting syndrome, nephropathy syndrome, dermatitis and reproductive failure. In Mexico, in 2018, the presence of PCV2a and PCV2b(50) was confirmed, and, therefore, vaccination strategies were implemented that have allowed the control of these clinical signs and of the economic and productive impact. These control strategies had been efficient; however, as of 2013, the appearance of some associated clinical signs was reported, and, upon diagnosis, the presence of PCV2 was ruled out, but the presence of PCV3 was confirmed. In 2017, at CENID-SAI, INIFAP, the complete genome of PCV3, detected in a production unit with reproductive failure and in pigs with clinical signs associated with postweaning multisystemic wasting syndrome, dermatitis and nephropathy, was detected and amplified; the sequences were reported in the global gene bank (GenBank: MH192340.1 and MH192341.1)(51). CENID-SAI has continued studying this disease; in 2019, serum samples obtained between 2012 and 2017 were analyzed; in the states of Guanajuato and Jalisco, the presence of PCV3 was identified since 2012 in both states, with a frequency of 31 %; coinfection PRRSV and PCV2 was also detected. Sequencing, genetic characterization and phylogenetic analysis were performed on the positive samples. In 2020, PCV3 whole genome sequences from serum samples of pigs from the states of Jalisco and Guanajuato were reported; these sequences were submitted to GenBank and are currently under review. These studies confirmed the presence of PCV3 in Mexico and established genetic homologies between strains; however, it is necessary to increase the number of representative sequences from different swine production units in order to establish such control strategies as the design of biologics for vaccination.

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Porcine Reproductive and Respiratory Syndrome (PRRS) Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease caused by a virus that belongs to the family Arteriviridae, genus Arterivirus. It is an enveloped virus with a 15 kb RNA genome containing nine open reading frames(62). PRRS affects pigs of all ages, but the greatest problems occur in pregnant sows and piglets. In females, the clinical picture is characterized by decreased fertility, late abortions, increased repetitions, and a high incidence of stillbirths, mummifications, and weak births. In piglets, it causes mainly respiratory problems. PRRS was first described in 1987 in North Carolina, USA(63). The PRRS virus (PRRSV) was first isolated in 1991 in Lelystad, The Netherlands(64). In the U.S.A. it was isolated in 1992 (strain VR-2332)(65). The PRRSV has a high mutation rate, generating the emergence of various viral strains grouped into two genogroups ―the European strains (EUPRRS1) and the American strains (NA-PRRS2)―, which have a homology of 63%, indicating a high genetic variability(66). Despite the great productive and economic impact, no vaccines have been developed to serve as prevention and control tools for the clinical signs caused by this viral agent(67). PRRSV is one of the most important infectious problems of viral origin, due to the economic impact it causes to the national and international swine industry. Worldwide, annual losses of up to $664 million dollars have been reported. In 2016, the economic expense associated with this virus was estimated in more than 40 farms in Mexico, identifying losses of more than $3,000.00 pesos per year per sow(68). The economic losses in Mexican swine farming due to this disease are estimated at 400 million pesos per year, making it one of the most important diseases in Mexico. For pigs in the production line, the estimated cost is $130 to $260 pesos per animal per year. In Mexico, the first study showing positive serology for PRRSV was carried out in pigs imported from Canada and the United States, and a prevalence of between 2.7 and 13% was identified in the states of Sonora, Jalisco, Guanajuato, and Aguascalientes(69). In 1997, it was reported that 78-84 % of swine production units were positive for the presence of PRRS(70). In 2000, the first viral isolation was performed in Mexico(71). In recent years, epidemiological studies carried out by CENIDSAI have shown that the proportion of farms that have animals with antibodies is high, reaching up to 70 % in the central part of the country. In 2007, a molecular diagnostic test for the detection of PRRSV was developed at CENID-SAI and adopted by the National Animal Health Diagnostic Center (Centro Nacional de Servicios de Diagnóstico en Salud Animal, CENASA) of the General Directorate of Animal Health (Dirección General de Salud Animal, DGSA). Currently in Mexico, antigenic and genetic characterization studies have been carried out with the strains circulating in Mexico, and it has been reported that PRRS strains present antigenic and genetic variations in the same production unit(72). Various groups of researchers are working on the study of the antigenic regions of PRRSV(73), with the aim of identifying prototype strains for the development of diagnostic tools and vaccines, as potential tools for prevention and control in Mexico.

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Blue eye disease Porcine rubulavirus (PRV), the causative agent of swine blue eye disease, was discovered in the early 1980s(74-76). PRV is currently classified as Porcine orthorubulavirus, within the family Paramyxoviridae(77). PRV has been described only in Mexico(78). The disease is characterized by neurological, respiratory and reproductive alterations accompanied by corneal opacity in pigs of different ages(75,79-83). Serological diagnosis can be performed with hemagglutination inhibition, viral neutralization, immunoperoxidase and ELISA tests. The hemagglutination inhibition test is the most commonly used test, although it can frequently give false positives if it is not correctly standardized(84). Detection and quantification of PRV by real-time RT-PCR has been reported(85,86); these tests can be costly if applied to large populations. Therefore, there are areas of opportunity for the development of rapid tests applicable in the field. Control of the disease has not yet been achieved, mainly due to the fact that animals may present subclinical and persistent infections(82). Sequencing of neurovirulent strains that affected the states of Jalisco and Mexico in 2015, as well as other studies, indicate that there are genetic variations from earlier outbreaks(87). These changes in viral proteins can generate antigenic diversity, which would cause antibodies produced against one variant to lose the ability to recognize other variants(88). From the point of view of human health, no zoonoses due to PRV have been reported, although the presence of antibodies against the virus has been demonstrated in veterinary staff(89). It has been suggested that PRV has the potential to cause zoonosis, due to the widespread contact between humans and pigs, as has occurred with other paramyxoviruses infecting animals(90). There are two commercial inactivated virus vaccines on the market. The results of studies suggest that the use of an outdated vaccine strain may generate little protection against circulating PRV strains(88), due to the accumulation of mutations. Therefore, further options have been investigated, e.g., the possibility of using recombinant PVR proteins as antigens to produce a protective response. The use of HN protein expressed in E. coli and Pichia pastoris, which induce the formation of antibodies, has been studied(91,92). Structural and antigenic prediction studies show that, in addition to the HN protein, the F, NP and M proteins potentially induce an immune response. It should be considered that the F protein of paramyxoviruses is widely conserved; in most of the predicted epitopes for PRV, very little or no variation was identified(93). PRV has been circulating in Mexico for at least 40 yr, and the challenge is to eradicate the disease; therefore, it is important to focus on three important issues: first, the development of an effective, rapid and inexpensive diagnostic method that will allow wide use; second, the development of an effective vaccine against different variants of the virus that normally circulate, and third, a molecular epizootiological surveillance program that will allow the updating of both the diagnosis and the vaccine. These points will make an important contribution to the control and eradication of PRV in pig farms in Mexico and, thus, focus efforts on other important conditions in pigs.

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Coronavirus disease Within the family Coronaviridae, there are two subfamilies: on one hand, Coronavirinae, with the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and on the other, Deltacoronavirus, and the subfamily Torovirinae(94). Five coronaviruses have been identified in swine: four belong to the genus Alphacoronavirus, transmissible gastroenteritis virus (TGEV), described in 1946; porcine respiratory coronavirus (PRCov), originated by mutation of the TGEV, isolated in 1984, and porcine epidemic diarrhea virus (PEDV), identified in 1977 and the recently discovered porcine enteric coronavirus (PEC), resulting from the recombination of the S gene of PEDV CV777 and TGEV. Porcine hemagglutinating encephalomyelitis virus (PHEV), isolated in 1962, which belongs to the genus Betacoronavirus; porcine deltacoronavirus (PDCov), of the genus Deltacoronavirus, detected in 2012(95-97). TGEV was described in 1946, in the USA, and was highly prevalent during the 1970s and 1980s. PRCov is the consequence of a natural deletion in the S protein of TEGV that turns its enteric tropism into a respiratory one, causing a subclinical disease in pigs. The emergence and spread of PRCov resulted in a decrease in the impact of TGE in the U.S. and Europe, as PRCov-seropositive farms reduced TGE-attributed mortality through cross-immunity. In contrast to Europe, outbreaks with both TGEV and PEDV were frequently observed in Asian countries, leading to co-infections and the need for differential diagnosis(98). Infection with TEGV, PEDV, PDCov, and SECov affects the gastrointestinal tract of pigs, causing severe clinical signs of diarrhea and vomiting, with high mortality rates attributed to dehydration, especially in newborn piglets(95,98). These pathogens are present in the main swine producing countries because they are highly contagious and because of the international trade of live animals or by-products, which is spreading in countries like China, USA, Canada, South Korea, and Mexico. Immunological pressure and the high passage of the virus between animals generated mutations in the virus, giving rise to highly pathogenic variant strains of PEDV, responsible for the epidemic outbreaks in 2010. In 2013, the first outbreak of PED in the USA (phylogenetically related to strain AH2012) was described with 90-95 % mortality in piglets. Subsequently, strains with lower virulence have been identified that register insertions and deletions (INDELs) in the S(99). According to the sequence of the spike or S protein, PEDV strains have been classified into genogroups G1a, G1b, G2a, and G2b. Group G1a includes the prototype strain CV777 and the attenuated strains historically distributed in Europe and Asia; G1b includes the S-INDEL strains, located in Europe, Asia, and North America. Strains in genogroup G2a are exclusive to the Asian continent, and in G2b is the 2013 U.S. prototype strain. PDCov was identified in 2014 during outbreaks of porcine epidemic diarrhea (PED) co-infection with PEDV in the USA. A retrospective study using samples collected prior to 2014 showed that PDCov was circulating prior to its isolation. The signs are similar to those caused by PEDV; however, the mortality rate is significantly lower(96). The first cases of PED in Mexico occurred in the Bajío region, in Jalisco and Michoacán, in 2013. INIFAP 160

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researchers and collaborators were pioneers in attending to producers concerned about the sanitary situation. In the first cases, diarrhea, vomiting and anorexia were observed in pregnant sows and growing pigs; in piglets, profuse yellowish diarrhea, vomiting and 100% mortality were observed(100). By 2014, the disease was widespread in the states of Jalisco, Michoacán, Guanajuato, Querétaro, Hidalgo, Mexico, Aguascalientes, Puebla, Veracruz, Nuevo León, Tamaulipas, Sinaloa, and Sonora, causing severe economic losses. The presence of the disease was proven by the clinical and epidemiological characteristics of the outbreaks that occurred in 2013 and early 2014(101). In that year, the National Service of Health, Safety and Food Quality (SENASICA) officially recognized the PED in our country(102). The first sequences of the circulating strains in Mexico, in 2013, were reported by INIFAP, in the GenBank global repository. The economic impact analysis revealed a decrease in the swine herd from 16.2 million in 2013 to 16.1 million head in 2014. On the other hand, the annual rate of pork production reported a growth of 1.9 % between 2005 and 2013; however, only 0.5 % growth was registered in 2014. Finally, 8.7 % fewer pigs were processed in 2014 than in 2013(103). In 2016, the 2014 disease report was released, with mortality rates of 100 % in piglets(104). According to the latest report sent to the OIE on February 11, 2016, cases of PED continue, and it is currently considered an endemic disease in Mexico(102,105). In the Virology Laboratory of CENID-SAI, the genetic characterization of PEDV circulating in six states of the Mexican Republic in the period 2013-2016 was carried out, identifying the presence of G2 and INDEL genotypes(106). From the identification of PEDV and PDCov in various states(101), INIFAP has developed technologies to support producers, and two diagnostic methods have been made available: ELISA for antibody detection(107) and realtime RT-PCR for quantification of viral RNA. Research has been carried out to isolate, identify tropism, cell susceptibility and, as part of the innovation process, the development of a recombinant biologic has been proposed which has shown satisfactory results in a second phase of evaluation(108,109). It is currently working on obtaining greater antigenic mass through scaling processes(110) in order to perform tests under farm conditions and seek registration of the product for transfer to interested laboratories in the area. Swine flu Influenza is an emerging and re-emerging acute respiratory disease that affects a wide range of birds and mammals, including humans. Influenza A viruses belong to the family Orthomyxoviridae, have an envelope made up of the glycoproteins hemagglutinin (HA) and neuraminidase (NA), which correspond to surface antigens. These proteins participate in pathogenesis, determine viral subtypes and play a crucial role in the interaction between the virus, the host cell and the pig immune system. Currently, 18 types of HA and 11 types of NA are recognized(111-115). The mechanism of transmission is by air via aerosols or by direct contact with nasal secretions or contaminated objects (fomites). When the virus enters the 161

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mucosa of the upper respiratory tract, NA evades the defensive action of cilia and mucus, and the initiation of virus replication is mediated by HA binding to sialic acid (SA) receptors in respiratory tract epithelial cells. These receptors are primarily linked galactose by an α2,6-linkage (SA α-2,6), present in human tracheal epithelial cells, and by SA α-2,3, present in epithelial cells of the intestinal tract of birds. However, its presence has been demonstrated in respiratory tract cells in humans(116). The pig expresses receptors for human and avian viruses, giving rise to the possibility of generating new viral subtypes(117,118). H1N1, H3N2, and H1N2 subtypes of swine influenza viruses are the most frequently reported(114,119,120). Disease outbreaks are generally observed in the winter season with a morbidity of almost 100 % and mortality close to 1 %(121,122). Because this disease is a zoonosis and therefore has public health importance, early and timely diagnosis of swine influenza virus should be considered(123). Diagnosis should be made by laboratory tests, including viral isolation, RT-PCR, and serological tests. In addition, differential diagnosis should be performed(122). In 2009, the first influenza pandemic of the century occurred, caused by subtype pH1N1(124). It was shown that pigs are susceptible to this subtype(125); in retrospective studies, seropositivity has been recorded since 2009 (126). The origin and genetic and antigenic characteristics of these viruses differ according to the continent or region where they are isolated, due to two phenomena: recombination and genetic drift(115,127). Currently, the disease is widely distributed in all swine-producing countries, and is endemic in Mexico(120). In 2004, a study was conducted to determine the association of PRRS with other viral and bacterial agents, including swine influenza(128). In 2016, an experimental study found that co-infection of H1N1 influenza A virus in conjunction with Porcine rubulavirus exacerbates respiratory disease in growing pigs(129). CENID-SAI is currently working on the validation of molecular and serological diagnostic tests and on the development of a universal biologic that will confer immunity, regardless of the subtype circulating on the farm. Parvovirosis Porcine parvovirus (PPV, recently named Ungulate Protoparvovirus 1) causes reproductive disorders in sows(130). Due to the absence of the immune response in the embryo or fetus in early stages, the virus can replicate, resulting in the death of the products(131). PPV is present in the areas with the highest swine production, being widely described in the United States of America, China, Germany, Europe, Hungary, Mexico, Colombia, and Cuba. A large proportion of first-time females are naturally infected with PPV before entering the breeding herd(131,132). Despite the continued use of vaccines, new strains have recently been described. PPV was considered to have a more conserved genome than other parvoviruses and ssDNA viruses. The first evolutionary analysis was performed in 2011, studying viruses affecting pigs in intensive production(133) and wild boar(134), and high mutation rates (approximately 35 x 10-4) in the VP gene were found. The main divergences have been introduced in the last 162

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10 to 30 yr. This evolutionary history is similar to that of carnivorous and human parvoviruses, suggesting that high mutation rates may be typical of porcine parvoviruses. Studies with strains from clinical events in various countries, including Austria, China, Rumania, and Switzerland, have reported the existence of six genotypes, with new profiles and clusters (A, B and E), exhibiting a predominance of domestic pig strains in Clusters C and D in Europe and Cluster F in China(133-136). Molecular profiles of new capsids with different antigenic properties have been described, including viruses used in commercial vaccines(137). These findings have led to the hypothesis that the emergence of new capsid profiles may be due to viral adaptation to the most commonly used vaccines and, therefore, may represent "escape mutants" in a partially immune population(133,134). The fact that novel porcine parvoviruses have been found in domestic pigs and wild boar suggests active interspecies gene flow(132). As PPV is able to replicate in cells of bovine and human origin, its host range may be broader than commonly thought. In 1991, specific antibodies against porcine parvovirus were identified in sows and rats(138). In 1996, CENID-SAI researchers identified that there is no statistical difference between the immunity conferred by vaccination and the immunity conferred by natural infection and that the use of vaccination does not completely prevent the reproductive problems associated with infection by this virus(139). In 2004, they also conducted a study based on the identification of the association between PRRS virus and other infectious agents and stated that no statistical association was found with parvovirus, since all sows exhibited antibodies against this virus(128). In the CDMX, seroprevalence has been described in backyard pigs during 2000-2009(140). It is necessary to continue monitoring PPV in the various swine producing regions of the country in order to determine the epidemiology of the virus and to have a picture of its distribution at the national level. Actions such as the establishment of efficient diagnostic methods and updating of vaccine strains for PPV will help to strengthen disease control strategies.

Exotic diseases

Classical swine fever One of the biggest sanitary problems in Mexican swine farming in the past decades was classical swine fever (CSF). In 2018, the eradication of this disease was internationally recognized, and the disease-free status has been maintained throughout the national territory. Classical swine fever is caused by a Pestivirus of the family Flaviviridae. It is a highly contagious disease, which causes, as main signs, fever, poor appetite, general weakness, neurological deterioration and hemorrhages. Morbidity and mortality in acute cases can reach

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100 %(141). In 1975, the efforts made by INIP (now INIFAP) through the work carried out by Dr. Pablo Correa in coordination with scientists from Cornell University, U.S.A., resulted in an excellent vaccine, PAV-250 (porcine attenuated virus-passage 250), which proved to have superior characteristics to existing commercial vaccines. Studies identified that the vaccine was safe, had satisfactory potency and did not spread. The technology developed was made available to the National Veterinary Biologics Producer (Productora Nacional de Biológicos Veterinarios, PRONABIVE), and to the private industry (SANFER and Litton Laboratories), which contributed to the success of the National CSF Eradication Campaign. Studies were conducted with the PAV-250 vaccine for the purpose of analyzing the stability of the biological product(142) and potency in the face of challenge with highly virulent strains(143). In the same way, the safety of the vaccine was tested at different stages of production(144,145). With the validation of PAV-250 in field conditions, it was concluded that, when applied in areas with frequent outbreaks of the disease, it was effective and safe. All the work carried out at INIFAP on the PAV-250 vaccine contributed significantly to the eradication of CSF(146). As part of the process, it was of vital importance to have methods and techniques for the diagnosis of the disease. For virus detection, various batches of conjugate were prepared, which proved to be highly specific, of excellent quality, and with a satisfactory titer. This was verified by CENASA, as it was used routinely. It was also marketed to private industry and provided through the UN’s FAO to several Latin American countries. On the other hand, the RT-PCR technique for the detection of the CSF virus was established for the first time in 2003. The test was compared with the official diagnostic tests established by the disease control and eradication campaign, direct immunofluorescence and viral isolation. It was comparable with both techniques, and, therefore, it was recommended for use as a confirmatory test for the disease(147). With the established technology, it was possible to determine the kinetics of the vaccine virus and the characterization of field strains(148). The widespread use of the PAV-250 vaccine led to the eradication of CSF in the country in 2009. It is estimated that the use of this vaccine prevented losses of at least 26 billion pesos during the most critical stages of the campaign to control and eradicate this disease. Aujeszky's disease Aujeszky's disease (AD) was the second swine disease that required the implementation of a national campaign for its control and eradication. At present, it is considered eradicated in Mexico. The etiological agent is porcine alphaherpesvirus 1, which mainly causes severe neurological disease in young pigs; in adult animals, manifestations include respiratory symptoms and reproductive failure(149). In countries where AD is endemic, it causes high economic losses and constitutes a barrier to trade in pigs and their by-products. AD still affects some countries in Europe, Asia and South America. In Mexico, AD was diagnosed for the first time in cattle in 1945(150), and later it was isolated and typed(151). Outbreaks in pigs were observed in the late 1970s. In the early 1990s, epidemiological studies focused on 164

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the sanitary evaluation of pig farm animals and backyard pigs(152-154). These studies helped the animal health authorities to make decisions in the campaign for the benefit of the national pig industry. With the generation of knowledge based on epidemiological studies, evaluation of vaccines, the use of a deleted vaccine, and the ELISA test for the detection of animals infected with the field virus, the country was declared free of AD on June 24, 2015. The vaccine used in the National Campaign against Aujeszky's disease (NOM-007-ZOO-1994), which was the key to this enormous effort, was developed from a strain with gE gene deletion. Previously, different vaccine strains used in Mexico were evaluated in order to identify which strains conferred greater protection(155). In 1997, INIFAP developed and evaluated a dot enzyme-linked immunosorbent assay (Dot-ELISA) proposed as an alternative screening test for the detection of antibodies against AD virus. The study reported a high degree of agreement with the serum neutralization test(156). At the request of CENASA authorities, the polymerase chain reaction (PCR) test for the detection of the AD virus was established in 2012. The test showed high sensitivity and specificity and was recommended as a complementary test to those established in the disease control and eradication campaign(157). Subsequently, the simultaneous molecular diagnostic test for AD and enzootic pneumonia in pigs was generated. This was adopted by Laboratorio de Investigación y Patología S.A. de C.V., located in the municipality of Tepatitlán, Jalisco. The technology adopted allowed producers to detect the infectious agent early, reducing their medication costs by up to 15 % in the development and completion stages, and stunting by 10 %. On the other hand, this technology contributed to the Aujeszky's disease control and eradication campaign through its use as a complementary diagnostic test in the epidemiological surveillance of the region. African swine fever The African swine fever virus (ASFV) is an arbovirus responsible for producing the disease of the same name (ASF) and currently represents one of the main economic threats to swine farming in the world, due to its high morbidity and mortality rate in domestic and wild pigs(158). ASFV is a double-stranded DNA virus and is the only member of the family Asfarviridae(159). The B646L gene sequence has been used to characterize ASFV in 22 genotypes (I-XXII), however, it is not predictive of virulence(160). In terms of virulence, the various strains of ASFV can show contrasting clinical characteristics ranging from acute presentations, associated with hemorrhagic fever and death within a few days of infection, to chronic presentations with a subclinical presentation, the biological mechanisms related to the differences in virulence between strains being currently unknown(161). ASFV was first described in Kenya in 1921; since then, it has remained endemic in a sylvatic cycle among ticks and wild boars, the latter being able to produce viraemia during infection, without developing clinical signs(158). The first reports of ASFV (genotype I) outside the African continent were described between the 1950s and 1980s in Rusia, Spain, Italy, France, Sardinia, Malta, Belgium, the Netherlands, Brazil, Cuba, and the Caribbean islands(158). The last outbreaks in the American continent were recorded in 1984, while ASFV was eradicated 165

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in the mid-1990s in countries outside the African continent, with the exception of Portugal where an isolated outbreak was recorded in 1999, and the island of Sardinia, where the virus has been endemically established until the present day(162,163). In 2007, the ASFV related to genotype II was reported to have emerged in the Republic of Georgia and spread to several countries in Europe and Asia(164). According to the OIE, it was recently reported in wild pigs in Germany, in September 10, 2020. In Europe, 67 % of the outbreaks associated with this genotype were reported between the years 2016 and 2020, mainly in wild pigs. On the other hand, in terms of mortality, Asia represents 82 %, with a total of 6,733,791 dead domestic pigs. The high virulence of strains associated with genotype II has been experimentally demonstrated in domestic pigs and wild boars, and a mortality rate of infected animals of up to 100 % has been identified within 7 to 10 d after infection(165-168). Undoubtedly, one of the most important challenges in terms of ASF control and prevention is the development of an effective vaccine, which does not exist commercially at present. Different strategies have been employed with the aim of obtaining a vaccine against ASF(169), with attenuated vaccines being the most promising candidates(170). In this sense, the development of attenuated vaccine candidates has been based on the selective deletion of ASFV genes(166,167,171-174). One of the most promising vaccine candidates at present is the recombinant ASFV-G/∆I77L virus(167). This recombinant was developed by deletion of the I177L gene of the highly virulent Georgia (genotype II) strain of ASFV. In initial tests, none of the pigs inoculated with different doses (1x102- 1x106 HAD) of the recombinant ASFVG/∆I77L developed clinical signs. Interestingly, 28 days after inoculation, 100% of the animals survived the challenge with the parental strain, producing sterile-type immunity in these animals. The results are promising; however, further research is still needed. Another interesting question, previously raised by other authors(170), is associated with the ability of attenuated ASFVs to become endemically established in regions where this type of vaccine is used, due to the presence of a viraemia phase produced by viruses such as ASFV-G/∆I77L, which could represent a source of virus for ticks, with the potential to produce sylvatic cycles. All these questions reflect the complexity of ASF control and the need for multiple research efforts in the short, medium and long term. Although ASF is a disease not found in the Mexican territory, it is essential to have a diagnostic and prevention system against it. The National Service for Agri-Food Safety and Quality (Servicio Nacional de Seguridad, Inocuidad y Calidad Agroalimentaria, SENASICA), in addition to having a high security level 3 laboratory, also has a network of laboratories throughout Mexico, all of which are managed by the U.S.-Mexico Commission for the Prevention of Foot and Mouth Disease and Other Diseases (CPA). Based on this infrastructure, it is considered that one of the greatest challenges for Mexico is to remain at the forefront in terms of diagnosis and training of those involved in the laboratory and in the field. In this sense, it is possible to suggest interinstitutional collaboration agreements with important laboratories in the region, such as the Plum Island Animal Disease Center, in the United States, and the National Center for Foreign 166

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Animal Disease, in Canada, which are dedicated to the diagnosis and research of multiple viral diseases with economic impact on domestic animals. The creation of a group to harmonize the diagnosis of ASF among the three countries may also be proposed. Finally, it is important to note that the National Producer of Veterinary Biologicals (Productora Nacional de Biológicos Veterinarios, PRONABIVE) has a proactive participation in regard to the possibility of obtaining licenses for the use of different ASFV vaccine candidates and preparedness to provide a rapid response in case of the arrival of this disease in Mexico.

Challenges and perspectives The increasing pressure of pig production, the wide network of imports-exports, the constant evolution of pathogens that allow them to develop new adaptation and diversification mechanisms, and climate change, are some of the challenges faced by the global pork industry. Control protocols based on herd depopulation and restocking have historically been used to curb the damage caused by high impact diseases. At present, the great technological advances in the development of effective biologics, diagnostic tools, and in the development and implementation of biosecurity measures, among others, have contributed positively to the resolution of these challenges, reducing the transmission of diseases and preventing, in some cases, the use of aggressive control methods. It is important that more complete studies on the predominant strains and serotypes be carried out in our country, and that the diagnostic techniques be improved in order to be able to evaluate them using molecular methods with a genetic profile, which will make it possible to determine the properties and virulence of the infectious agents. Infection models require optimization and have the potential to improve knowledge about the pathogenicity of the disease; these models will contribute significantly to the development of new vaccines. In the coming years, when antibiotic restrictions and pork consumption will increase, the use of effective vaccines will be an important factor. Today, autogenous vaccines have shown high effectiveness, and in Europe and the United States their use is being regulated with good manufacturing practices (GMP), although validation through efficacy studies is required. INIFAP will continue to do research focused on the generation of diagnostic tests and vaccines based on biotechnology and molecular biology. The adoption of these technologies will contribute to complement a set of tools aimed at preserving animal health and, consequently, improving the productivity of swine production units. Thus, it will be possible to implement a support program for small and medium-sized producers, aimed at strengthening herd health and, therefore, improving herd productivity in the short and medium term. An important point to consider during the upcoming years is the increase of pork consumption, not only at the national level, but also at the international level. For this

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purpose, must be consider the health in pig farms, since proper management and control of the various pathogens will allow both a higher production and a reduction of costs.

Conclusions Control and eradication strategies should be developed, under the premise that many of the diseases are controllable through good animal husbandry practices. Timely and effective diagnosis should be proposed as a method of control and prevention in the production units, as well as vaccination, encouraging the updating and use of the strains that circulate at the national level. Biosafety measures should be strengthened, and the technification of production units should be encouraged through the dissemination of information and technology transfer to small and medium-sized producers. The application of diagnostic tests in production units to identify the circulation of infectious agents should be promoted in order to establish the prevalence of these in different regions of the country and define control programs. To develop validated, easy to apply diagnostic methods with adequate sensitivity and specificity, using samples collected through non-invasive procedures. Studies should be designed to demonstrate the efficacy of commercially available vaccines in the target population (pregnant sows or their litters). In the innovation process, national biologics should be developed using different strategies and formulations (inactivated and attenuated viruses, subunit vaccines, replicating particles, DNA vaccines, vectored vaccines, etc.) should be promoted, along with the evaluation of safety, efficacy and the best cost-benefit ratio. All these technologies, developed by INIFAP, will benefit producers, allowing them to achieve better yields and profits.

Acknowledgments

The authors are grateful to all CENID-Microbiología researchers who have devoted their professional life to research in swine diseases —especially to Pablo Correa Girón†, MA; Atalo Martínez Lara†, MSc; María Antonia Coba Ayala, MSc; Laura Zapata Salinas, MSc; Antonio Morilla González, PhD—, and to all the technical and support personnel who worked in the past and are currently working at INIFAP, as well as to the project FONSEC SADERCONACYT 2017-06-292826.

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Conflict of interests

The authors declare that they have no conflict of interests with the information presented herein.

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156. Cuevas SC, Guzmán HM, De Paz VO, Colmenares VG, Hernández BE, Pérez GE. Desarrollo y evaluación de un Dot-ELISA como prueba tamiz para el diagnóstico de la enfermedad de Aujeszky en México. Téc Pecu Méx 1997;35(3):170-176. 157. Coba AMA, Socci EG, Zapata SL, Carrera SE, Chávez CE. Polymerase Chain Reaction for Aujeszky disease in Mexico. J Anim Vet Adv 2012;11(22):4217-4220. 158. Gaudreault NN, Madden DW, Wilson WC, Trujillo JD, Richt JA. African Swine Fever Virus: An emerging DNA arbovirus. Front Vet Sci 2020;(7):215. 159. Dixon LK, Sun H, Roberts H. African swine fever. Antiviral Res 2019;165:34-41. 160. Malogolovkin A, Kolbasov D. Genetic and antigenic diversity of African swine fever virus. Virus Res 2019;271:197673. 161. Blome S, Gabriel C, Beer M. Pathogenesis of African swine fever in domestic pigs and European wild boar. Virus Res 2013;173(1):122-130. 162. Boinas FS, Wilson AJ, Hutchings GH, Martins C, Dixon LJ. The persistence of African swine fever virus in field-infected Ornithodoros erraticus during the ASF endemic period in Portugal. PLoS One 2011;6(5):e20383. 163. Laddomada A, Rolesu S, Loi F, Cappai S, Oggiano A, Madrau MP, et al. Surveillance and control of African Swine Fever in free-ranging pigs in Sardinia. Transbound Emerg Dis 2019;66(3):1114-1119. 164. Cwynar P, Stojkov J, Wlazlak K. African Swine Fever status in Europe. Viruses 2019;11(4):310. 165. Gabriel C, Blome S, Malogolovkin A, Parilov S, Kolbasov D, Teifke JP. et al. Characterization of African swine fever virus Caucasus isolate in European wild boars. Emerg Infect Dis 2011;17(12):2342-2345. 166. O'Donnell V, Risatti GR, Holinka LG, Krug PW, Carlson J, Velazquez-Salinas L. et al. Simultaneous deletion of the 9gl and UK genes from the African swine fever virus Georgia 2007 isolate offers increased safety and protection against homologous challenge. J Virol 2016;91(1):e01760-16. 167. Borca MV, Ramirez-Medina E, Silva E, Vuono E, Rai A, Pruitt S. et al. Development of a highly effective African swine fever virus vaccine by deletion of the i177l gene results in sterile immunity against the current epidemic Eurasia strain. J Virol 2020a;94(7):e02017-19.

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168. Ramirez-Medina E, Vuono E, Pruitt S, Rai A, Silva E, Zhu J. et al. X69R is a nonessential gene that, when deleted from African swine fever, does not affect virulence in swine. Viruses 2020a;12(9):918. 169. Gaudreault NN, Richt JA. Subunit vaccine approaches for African Swine Fever Virus. Vaccines (Basel) 2019;7(2):56. 170. Bosch-Camós L, López E, Rodriguez F. African swine fever vaccines: a promising work still in progress. Porcine Health Manag 2020;(6):17. 171. O'Donnell V, Holinka LG, Gladue DP, Sanford B, Krug PW, Lu X, et al. African swine fever virus Georgia isolate harboring deletions of mgf360 and mgf505 genes is attenuated in swine and confers protection against challenge with virulent parental virus. J Virol 2015;89(11):6048-6056. 172. Borca MV, O'Donnell V, Holinka LG, Risatti GR, Ramirez-Medina E, Vuono EA, et al. Deletion of CD2-like gene from the genome of African swine fever virus strain Georgia does not attenuate virulence in swine. Sci Rep 2020b;10(1):494. 173. Ramirez-Medina E, Vuono EA, Rai A, Pruitt S, Silva E, Velazquez-Salinas L, et al. The C962R ORF of African swine fever strain Georgia is non-essential and not required for virulence in swine. Viruses 2020b;12(6):676. 174. Ramirez-Medina E, Vuono E, Pruitt S, Rai A, Silva E, Zhu J, et al. X69R is a nonessential gene that, when deleted from African swine fever, does not affect virulence in swine. Viruses 2020c;12(9):918.

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https://doi.org/10.22319/rmcp.v12s3.5840 Review

Control and prevention of nematodiasis in small ruminants: background, challenges and outlook in Mexico

David Emanuel Reyes-Guerrero a Agustín Olmedo-Juárez a Pedro Mendoza-de Gives a*

Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias. Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad. Unidad de Investigación en Helmintología. Boulevard Paseo Cuauhnahuac No. 8534, Col. Progreso, 62550, Jiutepec, Morelos, México.

*Corresponding author:pedromdgives@yahoo.com; mendoza.pedro@inifap.gob.mx

Abstract: Nematode parasites are an ongoing challenge in livestock production. Pharmaceutical anthelmintics are effective but pose their own risks. This is an overview of nematodiasis in small ruminants in Mexico focusing on the main problems faced by producers to maintain productivity. It includes general information on gastrointestinal nematodes and their effects on animal health and productivity. It also summarizes the main challenges faced by livestock producers in combating these parasites and current control and prevention strategies, including pharmaceuticals, anthelmintic resistance, grazing management, selective deworming, protein nutritional strategy, vaccination, and selection of animals genetically resistant to nematodes. The potential use of plants and compounds with nematocidal activity, and nematophagous fungi as biological control agents are also covered. Research by the Helminthology Department of the CENID-SAI of the INIFAP is highlighted, and a comprehensive nematode control method is proposed that targets different control strategies at specific nematode developmental stages. Controlling nematodiasis in small ruminants is vital to the success of production systems since it negatively affects animal health and producer results. Continued development of new nematode control options holds promise for successful long-term management of this disease. Key words: Nematodiasis, Parasites, Sheep, Goats, Control, Prevention. 186

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Received: 06/11/2020 Accepted: 19/01/2021

Introduction Importance of sheep and goat production in Mexico

Small ruminant production in Mexico represents a significant source of animal protein in human diets(1), and generates approximately 50,000 direct and indirect jobs that benefit as many as 400,000 families(2). However, animal health and producer income are adversely affected by poor quality pastures(3), high feed costs(4), extreme weather driven by climate change(5), and a suite of nematode parasites.

Gastrointestinal nematodes in small ruminants

Gastrointestinal nematodes (GIN) are cylindrical worms that inhabit the digestive tract of ruminants. There are considered significant parasites in the livestock industry, mainly in extensive systems, in both tropical and temperate climates(6). Adult parasites copulate and produce immense quantities of eggs which are released into the environment in the feces. Here they develop into infective larvae (L3) that contaminate pastures. Infection occurs when animals consume grass contaminated with larvae(7). The principal GINs in small ruminants in Mexico are Haemonchus contortus, Trichostrongylus colubriformis, T. axei, Teladorsagia (Ostertagia) circumcincta, Cooperia spp., Oesophagostomum, Trichuris ovis, Strongyloides papillosus and Bunostomum sp.(8,9). They generally occur simultaneously, causing clinical symptoms that can vary in severity, depending largely on animal age and nutritional status(10). Haemonchus contortus is considered one of the most pathogenic nematodes in sheep and goats due to its hematophagous habits and high prolificacy. Infection with H. contortus is known as haemonchosis and results in weight loss, poor appetite, decreased body condition, anemia, weakness, emaciation, edema of lower body regions, susceptibility to other diseases and death in young animals(11). Diseases from GINs occur in countries with tropical and subtropical climates(11), as well as those with temperate climates(12). No matter where they occur, GINs in small ruminants are the cause of substantial losses due to declines in animal productive potential(13). No study has yet been done on the losses generated by GIN in small ruminants in Mexico. However, based on the US$ 445.10 million dollar losses calculated in a study of the

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economic impact of GIN in cattle in Mexico(14), it is probable that they also cause significant losses in goat and sheep production.

Synthetic drugs or anthelmintics

Anthelmintic (AH) drugs are intended for control of livestock parasites. They are classified according to their mode of action: 1) benzimidazoles; 2) imidazothiazoles; and 3) macrocyclic lactones(15). Benzimidazoles (BZ) bind to the alpha subunit of the βtubulin protein, preventing polymerization between the alpha and beta subunits, blocking microtubule formation and causing death in nematodes(16,17). Imidazothiazoles (IMZ) act selectively as cholinergic agonists (nicotinic receptors) on the muscle cell membranes of GIN, resulting in muscle contraction and spastic paralysis(16). Macrocyclic lactone (ML) molecules bind selectively and irreversibly to the subunits of chlorine ion channels activated by different neurotransmitters (e.g. glutamate), causing hyperpolarization of the muscle or neuronal cell membrane, consequent paralysis of the nematode and its expulsion(18).

Anthelmintic resistance

Anthelmintic resistance (AR) occurs when parasite susceptibility declines vis-à-vis a drug dose that would normally eliminate most parasites(19). In Mexico, AR has been reported in sheep herds in the states of Tabasco, Chiapas, Yucatán, Campeche, Tlaxcala, Puebla and Veracruz, and is also known to affect cattle(20,21,22,23). Some GINs are known to have developed anthelmintic detoxification mechanisms(24,25). In nematodes, AR can alter the target protein, as well as transport xenobiotic molecules such as AH via transmembrane proteins (P-glycoproteins, P-gp), both of which play roles in multi-drug resistance(16). In Mexico, changes have been reported in the relative expression of P-gp genes associated with AR in isolates from ivermectin (IVM)-resistant and IVM- susceptible H. contortus. This suggests they may function as an effective reference germplasm in the design of study strategies for AR diagnosis and control methods aimed at maintaining drug toxicity in the field and controlling GIN. Resistance develops in response to the interaction between many factors, including GIN population density, treatment time and weather conditions, among others, which influence selection of resistance genes(17,26).

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Environmental consequences of anthelmintic drug use

Most AH are eliminated in the feces and urine. Some, such as ML, are not fully biotransformed inside the animal and when eliminated into the environment can pose a risk to non-target microorganisms, such as beneficial arthropods(27) or dung beetles(28). They can also pollute groundwater and generate significant imbalances in aquifer ecosystems. Macrocyclic lactones such as abamectin are extremely toxic to the planktonic crustacean Daphnia magna and highly toxic to other daphnids and fish(29). When in soils, they can harm beneficial organisms such as arthropods, including flies(30).

Public health risks from anthelmintic drugs

Excessive use of AH in cattle can contaminate meat, milk and its by-products, constituting a public health risk(31,32). They are widely used and thus pose a serious threat. For instance, in Ireland almost 60 % of dairy herds receive preventative administration of AH(33), while in Brazil 17.8 % of milk samples were reported to contain IVM residues(34). A study of bulk tank milk in Minas Geráis, Brazil, found it to contain aminobenzimidazoles (55.42 %), levamisole (53.57 %), avermectins (60.24 %), thiabendazole (67.47 %), moxidectin (73.49 %), triclabendazole (45.78 %) and benzimidazoles (6.02 %)(35). Research is still needed in Mexico to quantify AH residues in various products and verify their safety(36).

Alternative methods for nematode control in livestock Selective deparasitization (FAMACHA©) The FAMACHA© method is a selective deworming strategy based on degree of animal anemia quantified through the paleness of the lower eye mucus membrane as determined using a reference card. The card shows five colors ranging from intense red to pale or white, representing a 1-to-5 scale, and is used to measure coloration of the mucus membrane(37). When applied in tandem with body condition measurement, stoolparasitological examination, and fecal egg count (FEC), it helps in developing a deworming criterion(38). The FAMACHA© method is very useful in identifying the risk of H. contortus infection in small ruminants(39,40), but must be applied by a trained professional.

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Grazing management

Under tropical conditions, rotational grazing (RG) involves grazing an area for 3.5 d and then letting it rest for 31 d. This considerably reduces GIN in sheep and goats(41). In India, a decrease in FEC of up to 55.52 % has been reported when using RG in comparison to continuous grazing (CG)(42). Another study reported up to a 48.1 % reduction in the L3 population in feces, as well as better weight gain, in animals under a RG scheme compared to those under CG(43).

Protein diet nutritional strategy

Iso-energy and iso-protein diets have been proven to help prevent and control some parasites(44). The protein and energy levels in diets contribute to controlling GIN, and improve macro- and micronutrient quality and quantity(45), consequently strengthening immunity against nematodes(46).

Using plants with anthelminthic activity

Legumes have high contents of secondary metabolites (e.g. condensed and hydrolysable tannins, flavonoids and other groups of polyphenols) which are an alternative for GIN control(47-50). Some legume species in Mexico have shown efficacy against GIN. For example, in vitro and in vivo studies of Leucaena leucocephala show it to have an AH effect against GIN in cattle(51,52). Other legumes such as acacias contain hydroxycinnamic acid derivatives in their leaves, which exert powerful in vitro ovicidal activity against H. contortus, H. placei and Cooperia punctata(53,54). In an in vivo study using acacia leaves, goats artificially infected with H. contortus and administered 10 % dehydrated leaves in their diet exhibited up to a 70 % reduction in elimination of parasite eggs(55). The pods of Acacia farnesina contain flavonoids such as narigenin 7-O-(6″-galloylglucoside), known to be ovicidal and larvicidal against H. contortus(56). Both L. leucocephala and A. farnesina also constitute protein-rich forages for ruminants(57,58). The nuts of the legume Caesalpinia coriaria exhibit antimicrobial and anthelmintic activity in public health and livestock conditions(59,60,61). Gallic acid and a tannin derivative isolated from C. coriaria fruit were found to exercise an AH effect against GIN eggs in cattle(62). When included in complete diets for sheep and goats, this same fruit was found not to affect intake at a diet inclusion level of 2 % for sheep and 10% for goats(63,64). A bio-directed study of the legume tree Prosopis laevigata identified and isolated the flavonoid isorhamnetin which was found to be a potent in vitro nematicide against H. contortus(65).

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Vaccination

An effective alternative treatment for nematodes in ruminants under grazing conditions are antigens (ag) from autochthonous isolates of highly pathogenic nematodes, which can exhibit potential immunoprotective activity(66). For example, analysis of ag from Haemonchus spp. is vital in development of recombinant vaccines against the main GINs(67). Vaccines against GIN are increasingly sought after as research begins to focus on more sustainable approaches to GIN control(68). An outgrowth of this research has been the first vaccine (Barbervax) against H. contortus, which was derived from surface ag isolated from the intestinal lining of nematodes, and provides partial protection against this pathogen. Another study evaluated the proposed immunization of lambs with a recombinant somatic ag (rHC23) versus H. contortus, finding that it reduced egg counts by 70 to 80 %(69). A separate study using goats infected with H. contortus analyzed the efficacy of a protein known as transthyretin, derived from H. contortus excretion and secretion products (HcTTR). Two 500 μg doses of recombinant HcTTR reduced FEC by 63.7 % and postmortem parasite load by 66.4 %(70).

Genetic selection for resistant animals

Genetic resistance (GR) is variation in immune response present in a population of animals with the ability to control an infection or disease. It is highly dependent on the adaptive immune response and has a specific origin linked to an ag(71). Resistance to GIN infections has been reported in various sheep breeds. It is mediated by the adaptive immune response after reinfection with a specific pathogen and is related to the animals’ genetic profile in that it is a trait that can be inherited by offspring from parents(72). Genetic resistance to GIN is therefore a trait that can be pursued in small ruminant production aimed at controlling this problem. The effects of resistance and resilience in this phenotype against GIN infection can be enhanced in future generations by evaluating and selecting breeds and/or crosses of resistant animals for breeding programs(71,73,74). Selection of animals with a resistant phenotype requires evaluation and measurement of various standards relating to parasitological, immunological and pathogenicity parameters. These include determination of hpg, body condition, hematocrit percentage, antibody (IgA, IgE) concentrations, and degree of eosinophilia, among others(71,73,74,75). Once a resistant phenotype has been selected it can function as a reference point for improving progeny resistance in rearing programs. Resistant offspring will harbor fewer adult nematodes, reducing elimination of eggs into the environment and consequently reducing L3 contamination of pastures(73,74). Lower parasitosis rates in a herd will improve production parameters, potentially lessening dependence on AH use and decreasing AH-caused damage to beneficial organisms in pastures(72,76,77). In small ruminants, genetic improvement is an alternative medium-term control strategy for GIN

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parasitosis. Selection of genetic markers and identification of genomic positions (loci) in the chromosomes linked to a resistant phenotype are vital to understanding the mechanisms of the immune response associated with GIN resistance(71,76,77,78).

Biological control

Nematophagous fungi (NF) are among the principal natural enemies of nematodes. In addition to being saprobes, they are parasites or facultative predators of nematodes(79). The most promising NF in cattle nematode control is Duddingtonia flagrans. This fungus produces a large amount of chlamydospores that can be incorporated into feed, or they can be administered orally to animals in an aqueous suspension(80,81,82). They pass through the digestive tract and once in the feces they capture nematode larvae and feed on them, reducing their population by 70-90 %(82-85). Decreasing the larvae population in feces reduces infections and re-infections(86). Studies by INIFAP researchers have shown this strategy to be highly effective in reducing feces larvae populations in cattle and sheep under different production conditions, and in different regions of Mexico. One example is a study of an organic milk production unit in the Malpaso region of the state of Chiapas(80). There are currently two products available based on D. flagrans chlamydospore formulations: BioWorma in Australia(82), and Bioverm in Brazil(87). In Mexico, the CENID-SAI of the INIFAP is currently negotiating an agreement with a company to market a product based on chlamydospores from a Mexican D. flagrans strain for livestock applications.

Comprehensive nematode control

Adequate GIN control requires an understanding of where nematode parasites are found based on their lifecycle. In livestock they are found principally in three areas. In animals they can be found in the gastrointestinal system as histotrophic larvae (L4), pre-adult stages (L5) and adults, in addition to eggs from females. The feces contain eggs, L1 and L2 (pre-infectious) stages, and the L3 (infectious) stage. Soils and pastures harbor L3. Based on this information a comprehensive control strategy can be developed that focuses on these sites (Figure 1) in which different control tools are applied in a coordinated, synergetic approach for more efficient GIN control.

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Figure 1: Diagram representing integrated application of the main gastrointestinal nematode control methods in sheep focused on parasite developmental targets within the endogenous and exogenous phases of the biological cycle

Conclusions Scientifically proven control measures exist that are effective in herd-level nematode infection. When implemented in a comprehensive way they can improve animal health and herd productivity, while avoiding excessive AH use. The comprehensive nematode control method also reduces the occurrence AR, contributing to a sustainable approach to nematode control.

Challenges and outlook for nematode control in livestock in Mexico In the future, parasitologists will face a number of challenges in developing control strategies that move away from widespread AH use. The wide variability in parasite population dynamics largely responds to changes in climate(88). The spread of AR and resulting progressive inefficacy of AH are a growing threat in livestock production systems. Strategies are needed that block or reverse the adaptive genomic mechanisms behind AR(89). New immunoprotective ag´s based on recombinant technologies can be explored to improve animal immune system effectiveness(69,70,90). Sustainable 193

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technologies can also play a role in control strategies(91), especially those involving plants and their metabolites with nematocidal activity(92,93). Application of NF in nematode control in cattle and small ruminants is promising(80,81,82). In Mexico, this method needs to be developed to a point where it can be marketed and then promoted to producers. Nanoparticles and metabolites from NF are also promising possibilities that need more extensive research(94), since they are potentially effective additions to the arsenal of nematode control strategies(95,96).

Contributions to the study of nematodiasis in livestock Researchers in Mexico have contributed to better understanding and addressing nematode infection in livestock. One area of particular emphasis has been anthelmintic resistance, including the use of molecular tools for identification of resistance marker genes against anthelmintic drugs(17,21,82). Nematode transcriptomes have also been explored as part of a new perspective on the possible reversal of anthelmintic resistance in parasites, as have genetic and molecular detection of animals resistant to parasites(78,80). Important research is also being done on plants, and metabolites derived from them, with nematocidal activity against livestock parasites. This has generated data that will help to establish the use of plants with antiparasitic activity in livestock production(61,66,72). A sustainable method of nematode control in ruminants has been developed using a Mexican strain (FTHO-8) of the NF Duddingtonia flagrans, a natural predator of nematodes. Resistance spores, or chlamydospores, from this NF have been incorporated into “cookies” or “pellets” for cattle. When ingested they pass through the digestive tract to the feces where they germinate, colonize the feces and form mycelia traps to capture, kill and feed on nematodes, thus interrupting the biological cycle of nematodes(92). This is another sustainable method that has been successfully tested under different environmental and animal handling conditions(87,88,91,93). Cutting-edge research is also in progress on the antiparasitic properties of edible fungi, with promising results such as identification of bioactive metabolites that control nematodes(97).

Acknowledgments

The authors thank the INIFAP and CONACYT for the support provided our research projects.

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Conflicts of interest

The authors declare no conflict of interest.

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90. Shamim A, Sajid MK, Imran M, Saqib MN. Peptides isolation from crude somatic antigens of Haemonchus contortus through SDS- PAGE. Indian J Ani Res 2017;52(914-916).doi: https://doi.org/10.18805/ijar.v0iOF.8473. 91. Powell K, Foster C, Evans S. Environmental dangers of veterinary antiparasitic agents. Vet Rec 2018;183(19):599–600. doi:10.1136/vr.k4690. 92. Githiori JB, Höglund J, Waller PJ. Ethnoveterinary plant preparations as livestock dewormers: practices, popular beliefs, pitfalls and prospects for the future. Anim Health Res Rev 2005;6(01):91–103. doi:10.1079/ahr2005099. 93. Minho PA, Domingues FL, Gainza AY, Figueiredo A, Boligon A, Domingues R, et al. In vitro screening of plant extract on Haemonchus contortus and Rhipicephalus (Boophilus) microplus. J Essential Oil Res 2020. doi: 10.1080/10412905.2020.1746414. 94. Magri-Ferraz C, Pinheiro CSL, Elias-de-Freitas SF, Oliveira-Souza RL, Tobias LF, Victor-de-Araújo J, et al. Effect of silver nanoparticles (AgNP’s) from Duddingtonia flagranson cyathostomins larvae (subfamily: cyathostominae). J Invertebr Pathol 2020;107395. doi:10.1016/j.jip.2020.107395. 95. Degenkolb T, Vilcinskas A. Metabolites from nematophagous fungi and nematicidal natural products from fungi as an alternative for biological control. Part I: metabolites from nematophagous ascomycetes. Appl Microbiol Biot 2016;100(9):3799-3812. 96. Ocampo-Gutiérrez AY, Hernández-Velázquez VM, Aguilar-Marcelino L, CardosoTaketa A, Zamilpa A, López-Arellano ME, et al. Morphological and molecular characterization, predatory behaviour and effect of organic extracts of four nematophagous fungi from Mexico, Fungal Ecol 2021;49(101004). https://doi.org/10.1016/j.funeco.2020.101004. 97. Cruz-Arévalo J, Sánchez JE, González-Cortazar M, Zamilpa A, Andrade-Gallegos HR, Mendoza-de-Gives P, et al. Chemical composition of an anthelmintic fraction of Pleurotus eryngii against eggs and infective larvae (L3) of Haemonchus contortus. BioMed Res Int Hindawi 2020;2020:4138950. doi: https://doi.org/10.1155/2020/4138950.

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https://doi.org/10.22319/rmcp.v12s3.5801 Review

Important infectious diseases in goat production in Mexico: history, challenges and outlook

Gabriela Palomares Reséndiz a Francisco Aguilar Romero a Carlos Flores Pérez b Luis Gómez Núñez a José Gutiérrez Hernández a Enrique Herrera López a Magdalena Limón González b Francisco Morales Álvarez a Francisco Pastor López c Efrén Díaz Aparicio a*

Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias (INIFAP). CENID Salud Animal e Inocuidad. Carretera México –Toluca, colonia Palo Alto, 05110. Ciudad de México. México. b

Universidad Autónoma de México. Programa de Maestría y Doctorado en Ciencias de la Producción y de la Salud Animal. Ciudad de México, México. c

INIFAP. CE La Laguna, Matamoros, Coahuila, México.

*Corresponding author: efredia@yahoo.com

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Abstract: Goat production in Mexico is concentrated in poorer areas in arid and semi-arid areas of the country’s north and center, particularly in the Mixtec region of Oaxaca, Guerrero and Puebla. Because goats can survive in near desert conditions their production of milk and animals is a valuable nutritional contribution to human diets in these areas. Disease in goats in Mexico has generally received scant attention, however researchers at the INIFAP have studied this species and its pathologies. This review focuses on the main diseases affecting goats in Mexico and the research in this area by the INIFAP. In most of the goat herds studied in Mexico reproductive disorders are often caused by diseases such as brucellosis, leptospirosis and chlamydia, all considered to be endemic and potentially zoonotic. Respiratory and gastrointestinal disorders are the main ailments in kids. High frequencies of arthritic encephalitis, a disease caused by infection with small ruminant lentiviruses, and paratuberculosis and caseous lymphadenitis, both bacterial in origin, have been documented in productive-age goats throughout Mexico. All three are chronic, causing producers to mistakenly assume they have no major impact on productivity. Q fever, a known zoonotic, is currently considered exotic in Mexico, but in other countries is frequently associated with reproductive disorders, abortions and occasional respiratory problems in goats. The INIFAP has addressed all the above diseases. It was instrumental in diagnosing and controlling brucellosis, the principal bacterial zoonosis in Mexico. Researchers at INIFAP have also helped to determine that diseases previously considered exotic in Mexico have become endemic, and then developing the tools needed for their diagnosis. The INIFAP has made vital contributions to understanding the national disease panorama in goats and transferring diagnostic and treatment technologies to livestock laboratories nationwide. Key words: Goats, Diseases, Research.

Received: 14/09/2020 Accepted: 16/02/2021

The importance of goat farming in Mexico The goat population in Mexico is currently 8,791,894(1), most of which is distributed in the states of Zacatecas, San Luis Potosí, Coahuila, Puebla and Oaxaca. All have a traditional cuisine that includes goat meat in dishes such as birria, cabrito, mole de caderas and barbacoa. Specialized meat production or dual-purpose breeds, such as Boer and Nubia, have

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been developed, but most of the goat population continues to be Creole or local animals, which are descendants of Spanish breeds. Specialized dairy breeds, such as Saanen, French Alpine, Toggenburg and Nubia, are found mainly in the states of Coahuila, Guanajuato, Durango, Jalisco and Chihuahua(1); cheese, cajeta (caramel) and candys are produced from goat’s milk in these states(2). Goat farmers in the Bajío region of central Mexico and those in northern Mexico have technified production more than those of the south, such as in the Mixtec and Guerrero regions. This is probably due to the primary focus of production being milk to supply several companies that produce goat dairy products. Even so, regional production systems in Mexico are predominantly heterogeneous, utilize traditional technology, face myriad animal health problems, and suffer from minimal organization among producers and throughout the value chain(3). Goats’ importance as producers of food and valued-added derivatives is more pronounced in arid and semi-arid areas, which is also where human populations tend to be poorer. Goats are capable of utilizing the vegetation found in these areas, making it the preferred livestock for its ability to adapt and produce, even in challenging desert conditions(4).

Brucellosis Brucellosis melitensis is the principal etiological agent of brucellosis in goats. It is also the main species of the genus and is considered to be one of the causative agents of human brucellosis, known as Malta fever(5). Transmission of B. melitensis is principally oral, normally through intake of food or water contaminated with vaginal secretions or the remains of abortions from infected animals. For smooth phenotype brucellae, venereal transmission is not generally accepted as a main route of infection. However, B. melitensis is excreted in the milk and colostrum, suggesting that most latent infections may be contracted through their consumption(5). Infected cattle manifest clinical signs that can have substantial financial impacts. In sexually mature females it causes reduced fertility, abortion and decreased milk production. In males it colonizes the reproductive system, causing orchitis and epididymitis. Cases of arthritis have also been reported(6). Serological diagnosis of brucellosis in goats is most commonly done with the card test using 3 % B. abortus antigen. Its sensitivity is near 100 %, and it is also simple, inexpensive and practical. However, this test cannot be used in goats vaccinated with Rev 1 at any dose until

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8 mo post vaccination. Before eight months the test cannot differentiate between antibodies generated in response to the vaccine and those generated in response to infection. In these cases, techniques such as radial immunodiffusion (IDR) with native hapten are used because they allow differentiation between vaccinated and infected animals(7). In Mexico. Brucellosis is the main bacterial zoonosis in the country. Its control relies on diagnosis, identification of infected animals, their elimination, and especially vaccination. Goat farmers (especially in poorer goat farming communities), veterinarians, and slaughterhouse workers are the most vulnerable human populations, but laboratory personnel and consumers of unpasteurized dairy products are also at risk(5).

Contributions from research at INIFAP

The National Campaign against Animal Brucellosis was restarted in Mexico in the late 1990s. At this time there was still limited scientific data on the protection conferred by the Rev 1 vaccine in goats. To date, this has been the only available option worldwide for preventing brucellosis in small ruminants. Using experimental challenge techniques, INIFAP researchers have evaluated the protection conferred against brucellae. They found that application of reduced doses of Rev 1 were sufficient to protect vaccinated goats in endemic areas for at least 5 yr after immunization. These results are the first of their kind worldwide; they scientifically support that the Rev 1 vaccine protects vaccinated goats throughout their life and that revaccination is therefore unnecessary(8,9). Based on these results a reduced dose of Rev 1 was implemented during the National Campaign against Animal Brucellosis. There has also been a notorious lack of scientific information on the sensitivity and specificity of caprine brucellosis diagnosis tests. In their evaluations of diagnostic tests INIFAP researchers have found that techniques used with success in cattle, such as the milk ring and rivanol tests, did not generate effective diagnosis of brucellosis in goats. This research found that the card test, which is both basic and an important screening technique for serological diagnosis of brucellosis in animals, should be used at a 3 % cell concentration, a modification which increased its sensitivity. In goats, this screening test exhibits 98 % sensitivity and 100 % specificity as determined by Official Mexican Standards (Norma Oficial Mexicana - NOM). The antigen preparation methodology developed by INIFAP was transferred to the National Veterinary Biological Production Company (Productora Nacional de Biológicos Veterinarios - PRONABIVE)(10,11). In an effort to test a widely held dogma that male goats cannot be vaccinated against brucellosis due to development of lesions in the reproductive organs from bacterial tropism,

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researchers from the INIFAP and the UNAM conducted a study with 48 six-mo-old kids from brucellosis-free herds. They found that both the tested vaccines exhibited a low colonization capacity in the reproductive tract, and that this dogma was a myth(12).

Challenges and outlook in Mexico

Over the short-term, changes and updates can be expected in the National Campaign against Brucellosis in Animals (Official Mexican Standard, NOM-041-ZOO-1995). Over the medium-term, disease control programs need to be implemented and adapted to the particular conditions of the goat farming regions throughout Mexico. If the short- and medium-term goals are met then over the long-term, it is possible that caprine brucellosis could be controlled.

Chlamydiosis Chlamydiosis is an infectious-contagious and zoonotic disease caused by bacteria of the genus Chlamydia. The species that most affects goats is C. abortus, an obligate intracellular bacterium with an asynchronous multimorphic development cycle(13). It has been reported to affect pregnant women, mainly after exposure to infected goats(13). In goats, chlamydiosis is characterized as causing abortions in the last two or three weeks of gestation, or birth of weak offspring(13). Abortion generally occurs without previous signs, although behavioral changes and vaginal discharges containing a large number of elementary bodies can occur between 24 to 48 h beforehand. Placental lesions develop initially in the hilum of the placentaloma and extend to the intercotyledonary membranes. This leads to destruction of placental tissue which affects nutrient acquisition and hormonal regulation, resulting in premature expulsion of the fetus. Histological changes in the placenta and appearance of lesions typically occur after 90 d gestation(13). In Mexico. The first report of isolation of chlamydiosis’ etiological agent in goats was published in 1997(14). Initially considered exotic in Mexico, C. abortus is now viewed as common in goats based on increasing evidence. For example, a 2015 study was aimed at isolating C. abortus in dairy goats from herds with abortion problems in the state of Guanajuato, and developing adequate diagnostic tests for its detection(15). Analysis by ELISA of serum samples and vaginal swabs taken from six goat herds found that 9.60 % of the evaluated animals were seropositive for C. abortus. A PCR analysis using vaginal exudate

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DNA identified 23.8 % positive animals, and Chlamydia spp. was isolated in 26.98 % of the sampled animals. These three diagnostic methods were complementary, and all were applied in areas where Chlamydia is suspected of causing abortions. The combined results confirmed that this pathogen is present in dairy goat herds in Mexico(15). In a 2016 study, 1,307 serological samples were collected from goats distributed in 14 municipalities in Guanajuato, and analyzed using indirect ELISA. Frequency in females with a history of abortion was 46.62 %, whereas it was 27.13 % in females with at least one parturition that were clinically healthy but lived with females that tended to abort(16). A study done in Culiacán, in the state of Sinaloa, identified the presence of C. abortus in a goat herd with abortion problems, further supporting its presence in the country(17).

Contributions from research at INIFAP

The first isolation of C. abortus in goats in Mexico was achieved as part of a collaboration between researchers from the then INIP and the UNAM(14). From 2012 to 2013, a total of 186 samples were collected from 49 herds in the states of Coahuila, Jalisco, Puebla, Veracruz and Querétaro. After bacterial isolation, PCR and sequencing, analysis of the amplification products found 99% homology with four C. abortus strains: A.22, FAS, S26, EBA and VPG(18). In an analysis of goats from various municipalities in Guanajuato, C. abortus was identified in goats from herds with abortion problems, even in herds administered tetracyclines in an attempt to prevent it. Analysis of samples from organs and vaginal exudate corroborated that the treatment does not prevent infection, and may or may not cause abortion(19).

Challenges and outlook in Mexico

Regional livestock laboratories need to have the ability to run end-point PCR and real-time PCR to correctly diagnosis chlamydiosis, generate data on the Chlamydia species affecting goats and thus establish effective prevention and control measures within herds(17,19). Improved diagnosis will require development of routine techniques, such as serological tests like ELISA(20).

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Colibacillosis Colibacillosis is caused by Escherichia coli, and one of the main conditions caused by this bacterium is diarrhea in goat kids. Two main forms of the disease are recognized: enteric and systemic colibacillosis. The enteric form affects animals 2 to 8 d of age in which it causes diarrhea that is usually yellowish white in color, and creamy to almost liquid in consistency, while the kid exhibits weakness, cachexia and dehydration. If not quickly and adequately treated, infected animals can die within 12 h of clinical onset. The systemic, or septicemic, form affects animals between two to 6 wk of age. The bacteria crosses the intestinal or respiratory mucosa and enters the circulatory stream, causing animals to exhibit higher rectal temperature, meningitis and arthritis, but without diarrhea. Infected animals are the most important source of transmission, the most common route being fecal-oral. Reducing exposure to E. coli is attained through proper hygiene and handling practices. Lack of colostrum makes kids more susceptible to diarrhea(21). In Mexico. One of the two main causes of mortality in goat kids is diarrhea(22). Little research has been done on the pathogenic mechanisms and identification of virulence genes involved in E. coli diarrhea in goats. Most of the strains isolated from animals have been enterotoxigenic E. coli (ETEC), which express adhesins and enterotoxins as pathogenicity factors, and cause neonatal enteritis. Other pathogens isolated from diseased animals include enteropathogenic E. coli (EPEC), which also cause diarrhea, and enterohemorrhagic E. coli (EHEC), a known pathogen in humans(23). The EHEC pathotype O157:H7 infects humans and its reservoirs are ruminants. Fecal contamination of water and other foods, as well as cross contamination during food preparation (with contaminated meat products, surfaces and kitchen utensils), are common infection routes(24).

Contributions from research at INIFAP

A study done in 2014 characterized E. coli isolates from diarrhea collected from kids in some of Mexico’s goat-producing regions. Most of the isolates were found to belong to the B1 phylogenetic group of the O25:H8 serotype. This group and serotype have a wide variety of virulence genes, particularly st and stx2, which combine in the EHEC and ETEC pathotypes, as well as exhibiting low resistance to pharmaceutical treatment(25,26). Using three of the isolated strains, a bacterin was developed to control the disease. When administered to pregnant goats it provided passive immunity to kids through colostrum, with only 2.31 % of the vaccinated females producing kids with diarrhea. In addition, administration of the

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bacterin raised antibody levels in vaccinated females and their offspring. The protection provided the animals was identified as a serotype specifically against purified O25 LPS (27).

Challenges and outlook in Mexico

Research is still needed into the behavior of the virulence genes involved in E. coli diarrhea to develop an exact diagnosis that would allow identifying and developing specific preventive measures. The bacterin developed at INIFAP for E. coli diarrhea prevention in kids is promising and should be included in the development of immunization protocols.

Respiratory disease complex (RDC) The causes of RDC in goats include the environment, animal condition and the presence of infectious agents such as viruses and bacteria. Kids are particularly susceptible to RDC. The adverse environmental conditions that predispose goats to respiratory problems are sudden changes in climate, overcrowding of animals, inadequate pens, lack of ventilation, accumulation of dust and ammonia, poor protection against drafts and stress from transportation(28). Among infectious agents, some viruses are known to be primary agents, particularly the parainfluenza-3 and respiratory syncytial viruses(29). The bacteria that can generate RDC include Pasteurella multocida, Mannheimia haemolytica, and Mycoplasma spp., all commonly found in the upper respiratory tract of healthy animals(30). When immune system mechanisms become depressed these bacteria colonize the lungs, causing RDC. Various host-related factors can contribute to development of RDC or pneumonia. These include deficient colostrum intake in kids, damage from viruses in the mucociliary clearance system (which causes insufficient bacterial clearance from airways), a depressed immune system, malnutrition, parasitosis, dehydration, etc. When pneumonia occurs, symptoms can range from 40 to 41 ºC fevers, cough, shortness of breath, lack of appetite, mucopurulent nasal and ocular discharge, depression, prostration and death(31). In Mexico. Goat producers in Mexico recognize pneumonia as one of the most frequent health problems in herds, but rarely is a diagnosis made or a prevention program implemented(22).

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Contributions of research carried out at INIFAP

The INIFAP began research on this problem by identifying and characterizing the virulence factors of the bacteria participating in RDC, finding P. multocida (biotype D:3) to be present in goats. A valuable complement to this study would be to isolate lesion microorganisms directly from the lungs of diseased individuals to confirm that this strain (and/or others) is the cause of pneumonia(32-35).

Challenges and outlook in Mexico

Controlling RDC in goats in Mexico will require creation of a vaccination program and producer education. An important aim is formulation of a M. haemolytica toxoid from isolates collected in Mexico and its evaluation in kids, together with the bacterin already developed. The results would contribute to implementing a vaccination program to prevent RDC. Training courses are needed for goat farmers that focus on improving RDC prevention. These can use a comprehensive approach that reviews and corrects problems leading to RDC factors, such as management practices that generate excessive stress, while promoting adequate hygiene protocols, reviewing nutritional status, identifying parasitosis status, and improving production installations (e.g. redesign to prevent sudden temperature changes, and provide proper ventilation).

Q fever A worldwide zoonotic disease, Q fever is caused by the bacterium Coxiella burnetii. Its hosts are myriad, and range from domestic (cows, sheep, goats, dogs, cats, rabbits) to wild animals (small rodents, foxes). Most hosts are chronic carriers and do not suffer from the disease, but they excrete the bacteria via urine, feces, milk, and birth by-products such as amniotic fluid, the placenta and abortions. These secretions form aerosols that can transmit the microorganism by air to susceptible human populations(36). In Mexico. Coxiella burnetii is an exotic species in Mexico, and as such has been considered by SADER as a notifiable disease since 1994(37). The first serological evidence of this bacterium in animals was reported in the state of Baja California in 1990(38), and the first reports of it in humans were from the Comarca Lagunera region(39).

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Contributions from research at INIFAP

In a study done from 2018 to 2019, researchers from the INIFAP and the Ministry of Health identified C. burnetii in goats that exhibited a tendency to abort. Using endpoint PCR, they identified the IS1111 insertion sequence; this was the first molecular evidence that goats in Mexico can be reservoirs of C. burnetii(40).

Challenges and outlook in Mexico

Over the short-term, C. burnetii will probably transition from being an exotic to an endemic disease. Research will also be needed to develop diagnostic tests. Over the medium-term, high sensitivity and specificity diagnostic tests need to be established in laboratories to detect Q fever in both animals and humans, and programs developed and implemented to control the disease in animals.

Small ruminant lentivirus Caprine arthritis encephalitis virus (CAEV) and Maedi-Visna virus show genetic, structural and pathogenic similarities; consequently, they have been reclassified as small ruminant lentiviruses (SRLV)(41). In goat production, SRLV can have negative financial impacts directly related to the presence of multisystemic and incurable chronic-degenerative infections. In adult goats, arthritis and mastitis occur, while in kids the viruses manifest in the nervous system during the first months of age(42). Replication of lentiviruses in mammary gland epithelial cells plays an important role in viral particle transmission. Mononuclear cells and infected macrophages can also be shed through colostrum and milk. Direct contact with aerosolized respiratory secretions, urine, and feces from infected animals are considered sources of infection that can become exacerbated by overcrowding. Water and feed, as well as inadequate disinfection of facilities, machinery and milking equipment, allow the spread of SRLV(43). In Mexico. A seroprevalence study of CAEV in Mexico’s goat-producing regions in 1985 determined that the disease entered the country through import of live breeding animals from the United States(44). The SAGARPA subsequently classified CAEV as an endemic disease and required mandatory monthly reporting. It was isolated, and sequencing done of the entire genome of a recombinant SRLV belonging to the B1 subtype(45).

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Contributions from research at INIFAP INIFAP researchers are developing SRLV diagnostic techniques based on the genetic and antigenic characteristics of strains that circulate naturally in Mexico and which affect goat production. Expression of a recombinant antigen of the CAEV p25 protein has been achieved in the E. coli system. The generated antigens are considered to be excellent candidates for establishing an ELISA-type serological test for CAEV diagnosis(46). In addition, a real-time PCR test is being standardized to identify the presence of provirus in SRLV and the production of the recombinant proteins CA, p25 and MA, p16 in CAEV.

Challenges and outlooks in Mexico

Further research is needed to understand the circulation of other SRLV genetic groups in Mexico and to identify their genetic diversity. To date, the B1 genogroup has been identified and found capable of infecting both sheep and goats. Tools and techniques have been developed for serological and molecular diagnosis of subtype B1 (ELISA and PCR). No serological or molecular test has yet been developed that is capable of detecting all existing SRLV groups or subtypes. Results have been most promising when SRLV diversity is characterized and this data used to complement or adapt tests developed to meet current or future needs in each country. Breed stock and registered cattle producers are also advised to participate in government programs for SRLV-free herd certification, while producers with fewer resources are reached through government social programs. An important long-term goal is to certify Mexico’s main goat-producing regions as SRLV-free, and establish a culture among producers of animal health based on timely diagnosis that allows increasing productivity.

Leptospirosis Leptospirosis is an infectious disease caused by bacteria belonging to the genus Leptospira. It is distributed worldwide in rural and urban areas with specific climatological and orographic characteristics, natural drainage networks, extensive agricultural areas and seasonal rainfall; these conditions favor the spread of Leptospira spp. Infection with Leptospira affects domestic and wild mammals differently, and cases can range from asymptomatic to acute or chronic infection. This microorganism is eliminated in the urine of infected animals continuously or intermittently, thus contaminating the environment. Acute leptospirosis in goats can cause symptoms such as an increase in body temperature, anorexia,

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depression, jaundice, and anemia. In its chronic form it causes abortions, mummifications, infertility, premature births and neonatal mortality, all resulting in financial losses(47). Humans can occasionally host Leptospira spp. When infected they can manifest pathological disorders such as fever, headache, muscle and joint pain, cough, stroke, nausea, vomiting, jaundice, and kidney and liver disorders(48). In Mexico. In a study in the state of Veracruz, an analysis of 873 serum samples identified the most frequent serovars to be Wolffi (45.58 %) and Icterohaemorragiae (45.13 %)(49). In the Lagunera region, an analysis of 802 serum samples showed 60.1% to be seropositive for leptospirosis(50). In goat herds from the state of Guerrero, 64.26 % of a set of serum samples had antibody titers against at least one Leptospira interrogans serovar(51).

Contributions from research at INIFAP

A 2016 study by INIFAP researchers of the serological frequency of antibodies against Leptospira spp. in the main goat-producing region of Guanajuato, found a leptospirosis frequency of 37.90 %(52). In 2018, also in Guanajuato, a study evaluated the seroprevalence of Leptospira spp. serovars in goats, their geographic distribution and co-exposure patterns. By analyzing 1,640 samples with the microscopic agglutination test, total prevalence was found to be 45.5 %, that of Icterohaemorrhagiae was 34.16 %, that of Hardjo was 6.77 %, and the remaining serovars represented less than 5%. All the identified serovars exhibited an aggregation pattern that suggests risk areas and transmission vectors. Analysis of antibody co-occurrence showed Icterohaemorrhagiae to be dominant over the other identified serovars(53). A 2019 study did a serological diagnosis of the main abortive diseases in goats from Guanajuato using samples from dairy farms with different levels of technology, breeds and management practices; Leptospira spp. seropositivity was confirmed(54).

Challenges and outlook in Mexico

Leptospirosis prevention and control measures are needed in herds throughout Mexico. Immunization should be implemented using Leptospira spp. serovars previously identified in goats in an effort to reduce problems caused by the disease, such as abortions, mummifications, premature births and infertility.

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Caseous lymphadenitis Caseous lymphadenitis is a chronic infectious disease affecting goats caused by the Grampositive bacterium Corynebacterium pseudotuberculosis, a facultative intracellular pathogen capable of replicating and surviving inside phagocytes. The disease is characterized by presence of suppurative lesions in the lymph nodes, lungs and other internal organs. At a subclinical level the disease manifests in the viscera in the form of abscesses in internal organs (e.g. lungs, liver and mesenteric lymph nodes), and occasionally animals may exhibit chronic pneumonia and progressive weight loss(55).

Contributions from research at INIFAP

INIFAP researchers have contributed to development of caseous lymphadenitis diagnostic tests, and genetic characterization of C. pseudotuberculosis strains from sheep, goats and horses(56). Another study established a multiple PCR test for diagnosis of caseous lymphadenitis in goats, showing it to be an efficient technique based on clinical samples which can also differentiate between biovars ovis and equi of C. pseudotuberculosis(57). An initial study of mutant C. pseudotuberculosis strains as candidates for development of immunogens found that a mutant strain failed to protect Balb/c mice after an experimental challenge, and did not demonstrate adequate humoral or cellular immune responses in this murine model(58).

Challenges and outlook in Mexico

Further research is needed to better understand the prevalence and distribution of C. pseudotuberculosis in Mexico. This in turn would contribute to development of an immunogen capable of reducing morbidity in goat production and its consequent financial impacts in Mexico.

Paratuberculosis Caused by the bacterium Mycobacterium avium subspecies paratuberculosis, paratuberculosis is an infection characterized by chronic regional inflammation in the small 217

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intestine of ruminants. Its most frequent signs in goats are a decrease in body condition and weight, declining milk production, loss of stool consistency and diarrhea (only in the terminal phase)(59). This bacterium has been associated with Crohn’s disease, a chronic disease leading to inflammation and ulceration of the gastrointestinal tract (mainly in the ileum and colon)(60), although it is not recognized as the main etiological agent. In Mexico. Paratuberculosis was first identified in goats in a 1983 study that reported a clear clinical picture of the disease, including intestinal lesions such as enteritis and granulomas in mesenteric lymph nodes, presence of M. avium subsp. paratuberculosis in the injured tissues, and antibodies in blood serum(61). Various reports have since addressed seroprevalence, isolation and detection of M. avium subsp. paratuberculosis genetic material in infected animals in various states in Mexico(59,61,62).

Contributions from research at INIFAP

Although paratuberculosis prevalence in goats is variable, research at INIFAP helped establish the risk factors for transmission in Mexico, which include high population density, introduction of infected animals to herds, permanent coexistence with other species, permanence of infected animals and poor hygiene conditions(63).

Challenges and prospects in Mexico

Over the short-term, strategies are needed to differentiate the clinical manifestations of paratuberculosis (e.g. low body condition and declining milk production) from other conditions common in herds throughout Mexico, such as poor nutrition from lack of good quality food (especially frequent in areas where community grazing is common). In the medium-term, monitoring of herds in the primary goat-producing areas of Mexico is needed to assess paratuberculosis’ health, productive and financial impacts on a national level. Longterm challenges include substantially reducing paratuberculosis prevalence and incidence, guaranteeing food safety, increasing productivity and thus opening new markets. This will be vital because countries such as the United States and the European Union produce diseasefree goats the derivatives of which are consequently more valuable and marketable. Literature cited: 1. Servicio de Información Agroalimentaria y Pesquera [SIAP]. Inventario 2019 caprino. www.gob.mx.SIAP.

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Barrera POT, Sagarnaga VLM, Salas GJM, Leos RJA, Santos LR. Viabilidad económica y financiera de la ganadería caprina extensiva en San Luis Potosí, México. Mundo Agrario 2018;19:40.

Blasco JM. Control and eradication strategies for Brucella melitensis infection in sheep and goats. Prilozi 2010;31(1):145-165.

Lebre A, Velez J, Seixas D, Rabado E, Oliveira J, Saraiva da Cunha J, Silvestre AM. Brucellar spondylodiscitis: case series of the last 25 years. Acta Med Port 2014;27(2):204-210.

Díaz-Aparicio E, Marín C, Alonso B, Aragón V, Pardo M, Blasco JM, Díaz R, Moriyón I. Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats. J Clin Microbiol 1994;32:1159-1165.

Mancera A, Díaz AE, Vázquez NJ, Velázquez F, Suárez GF, Flores CR. Vacunación de cabras con la cepa Rev 1 de Brucella. melitensis en diferentes dosis: Evaluación serológica y desafío. Vet Méx 1992;2:117-123.

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10. Díaz AE, Blasco MJM, Suárez GF. Prueba de tarjeta modificada para el diagnóstico de la brucelosis caprina. Vet Méx1999;30(4):307-311. 11. Díaz-Aparicio E, Marin C, Alonso-Urmeneta B, Aragón V, Pérez-Ortiz S et al. Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats. J Clin Microbiol 1994;32:1159-1165. 12. López VIA. Efecto del eritritol en la colonización de Brucella melitensis (Rev1 eryCD), en el tracto reproductor de machos cabríos [tesis maestría]. CDMX: Universidad Nacional Autónoma de México; 2020. 13. Essig A, Longbottom D. Chlamydia abortus: New aspects of infectious abortion in sheep and potential risk for pregnant women. Curr Clin Micro 2015; Rpt 2;22–34.

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14. Escalante-Ochoa C, Diaz-Aparicio E, Segundo-Zaragoza C, Suarez-Guemes F. Isolation of Chlamydia psittaci involved in abortion of goats in Mexico: first report. Rev Latinoam Microbiol 1997;39:117-121. 15. Mora-Díaz J, Díaz-Aparicio E, Herrera-López E, Suárez-Güemez F, Escalante-Ochoa C, Jaimes-Villareal S, Arellano-Reynoso B. Isolation of Chlamdia abortus; in dairy goat herds and its relation to abortion in Guanajuato, Mexico. Veterinaria México OA 2015;2(1). 16. García LX. Frecuencia de clamidiosis en casos de cabras que presentaron aborto en el estado de Guanajuato [tesis maestría] CDMX: Universidad Nacional Autónoma de México; 2019. 17. Romero JA. Diagnóstico de Chlamydia abortus, mediante cultivo celular y PCR de un rebaño caprino con problemas de abortos en Culiacán, Sinaloa [tesina licenciatura] CDMX: Universidad Autónoma Metropolitana; 2020. 18. Sánchez RL. Presencia de Chlamydia abortus en cabras de México [tesis maestría] México, DF: Universidad Nacional Autónoma de México; 2014. 19. Hernández RP. Presencia de genes de resistencia (tet(C)-tetR(C)) a tetraciclina en aislamientos de Chlamydia abortus [tesis maestría] CDMX: Universidad Nacional Autónoma de México; 2020. 20. Santiago BC. Expresion de OmpA recombinante deChlamydia abortus en Escherichia coli [tesis maestría] CDMX: Universidad Nacional Autónoma de México; 2019. 21. García De Jalón CJA. Diarreas en corderos y cabritos. PR 2000;1(1):8-14. 22. Cuellar OJA, Tortora PJ, Trejo GA, Román RP. La producción caprina mexicana, particularidades y complejidades. 1era ed. UNAM, México: Aridana; 2012. 23. Matthew AC, Robyn JL. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol 2013;26(4):822-880. 24. Organización Mundial de la Salud [OMS]. E. coli. 2018. 25. Martínez FRI. Identificación de genes de virulencia en aislados de E. coli de origen caprino [tesis maestría] DF: Universidad Nacional Autónoma de México; 2014. 26. Yáñez VA. Determinación de factores de virulencia y clonalidad de cepas de E. coli procedentes de diarrea de cabritos [tesis maestría] DF: Universidad Nacional Autónoma de México; 2016.

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27. Limón GMM. Desarrollo y evaluación en campo de una bacterina de E. coli, en caprinos [tesis maestría] México: Universidad Nacional Autónoma de México; 2017. 28. Rahal A, Ahmad AH, Prakash A, Mandil R, Kumar AT. Environmental attributes to respiratory diseases of small ruminants. Vet Med Int 2014;10. 29. Lamontagne L, Descoteaux JP, Roy R. Epizootiological survey of Parainfluenza-3, Reovirus-3, Respiratory Syncytial and Infectious Bovine Rhinotracheitis Viral antibodies in sheep and goat flocks in Quebec. Can J Comp Med 1985;49:424-428. 30. Ponnusamy P, Masilamoni BS, Ranjith KM, Manickam R. Isolation, identification and antibiogram of Mannheimia hemolytica associated with caprine pneumonia in the Cauvery Delta Region of Tamil Nadu, India. Int J Curr Microbiol Appl Sci 2017;6(9): 3118-3122. 31. Blanco VFJ, Trigo TFJ, Jaramillo ML, Aguilar RF, Tapia PG, Suárez GF. Serotipos de Pasteurella multocida y Pasteurella haemolytca aislados a partir de pulmones con lesiones inflamatorias en ovinos y caprinos. Vet Mex 1993;24(2):107-112. 32. Pérez-Romero N, Aguilar-Romero F, Arellano-Reynoso B. Isolation of Histophilus somni from the nasal exudates of a clinically healthy adult goat. Trop Anim Health Prod 2011;43:901–903. 33. Soriano VE, Vega SV, Zamora EJL, Aguilar RF, Negrete AE. Identification of Pasteurella multocida capsular types isolated from rabbits and other domestic animals in Mexico with respiratory diseases.Trop Anim Health Prod 2012;44, 935–937. 34. Martínez RI. Aislamiento, identificación y caracterización de Mannheimia haemolytica y Pasteurella multocida aisladas de caprinos [tesis maestría] México DF: Universidad Nacional Autónoma de México; 2013. 35. Rojas-Fernández M, Vaca S, Reyes-López M, de la Garza M, Aguilar-Romero F, Zenteno E, Soriano VE, Negrete-Abascal E. Outer membrane vesicles of Pasteurella multocida contain virulence factors. Microbiologyopen 2014;3(5):711-717. 36. Hartzell JD, Wood-Morris RN, Martinez LJ, Trotta RF. Q fever: epidemiology, diagnosis, and treatment. Clinic Proc 2008;83(5):574-579. 37. ACUERDO mediante el que se da a conocer en los Estados Unidos Mexicanos las enfermedades y plagas exóticas y endémicas de notificación obligatoria de los animales terrestres y acuáticos. DOF publicado el 29/11/2018. 38. Salman MD, Hernández JA, Braun Y. A seroepidemiological study of five bovine diseases in dairy farms of the coastal region of Baja California, Mexico. Prev Vet Med 1990;9(2):143-153. 221

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39. Silva R. Fiebre Q en México. Méd Rev Méx 1950;61(7):493-497. 40. Flores PCF. Identificación de Coxiella burnetii, bacteria exótica en México, mediante PCR, en caprinos y bovinos que presentaron aborto [tesis maestría] CDMX Universidad Nacional Autónoma de México; 2020. 41. Gómez-Lucia E, Barquero N, Domenech A. Maedi-Visna virus: current perspectives. Vet Med 2018;9:11-21. 42. Straub OC. Maedi-Visna virus infection in sheep. History and present knowledge. Comp Immunol Microbiol Infect Dis 2004;27(1):1-5. 43. Blacklaws BA, Berriatua E, Torsteinsdottir S, Watt NJ, de Andres D, Klein D, Harkiss GD. Transmission of small ruminant lentiviruses. Vet Microbiol 2004;101(3):199-208. 44. Nazara SJ, Trigo FJ, Suberbie E, Madrigal V. Estudio serológico de la artritisencefalitis caprina en México. Tec Pecu Mex 1985;48:96-101. 45. Ramírez H, Glaria I, de Andrés X, Martínez HA, Hernández MM, Reina R, et al. Recombinant small ruminant lentivirus subtype B1 in goats and sheep of imported breeds in Mexico. J Vet 2011;190(1):169-172. 46. Valladares RB. Generación y caracterización de la proteína p25 del Lentivirus de pequeños rumiantes (LvPR), expresada en Escherichia coli [tesis maestría] México: Universidad Nacional Autónoma de México; 2016. 47. Suwancharoen D, Chaisakdanugull Y, Thanapongtharm W, Yoshida S. Serological survey of leptospirosis in livestock in Thailand. Epidemiol Infect 2013;141(11). 48. Gamage CD, Koizumi N, Perera AK, Muto M, Nwafor-Okoli C, Ranasinghe S, et al. Carrier status of leptospirosis among cattle in Sri Lanka: a zoonotic threat to public health. Transbound Emerg Dis 2014;61(1):91-96. 49. Fernández TAI, Herrera LE, Díaz AE, Barradas PF, Cristóbal CO, Limón GMM. Leptospirosis caprina en diferentes municipios del estado de Veracruz. Congreso Nacional de Buiatria. Acapulco, Guerrero. 2013:694-697. 50. García GN. Estudio Epidemiológico de Leptospirosis Caprina en la Región Lagunera del Estado de Coahuila [tesis licenciatura]. Torreón, Coahuila: Universidad Autónoma Agraria “Antonio Narro”, 2011. 51. López HA. Diagnóstico serológico de Leptospira spp y de C. abortus en las principales zonas de producción caprina del estado de Guerrero, México. [tesis licenciatura] México, DF: Universidad Nacional Autónoma de México; 2011.

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52. Flores PP. Diagnóstico serológico de Leptospira interrogans y Brucella melitensis en rebaños caprinos en el estado de Guanajuato [tesis licenciatura] CDMX: Universidad Nacional Autónoma de México; 2016. 53. Gaytán CF. Seroprevalencia, distribución geográfica y co-exposición de serovariedades de Leptospira spp. en rebaños caprinos pertenecientes a grupos ganaderos de validación y transferencia de tecnología del estado de Guanajuato [tesis licenciatura] CDMX: Universidad Nacional Autónoma de México; 2018. 54. Rueda GY. Seroprevalencia de enfermedades abortivas en cabras de unidades de producción pertenecientes a la región centro-oriente de Guanajuato [tesis especialidad] Querétaro: Universidad Autónoma de Querétaro; 2019. 55. Windsor PA. Control of caseosus lyphadenitis. Vet Clin Food Anim 2011;27:193-202. 56. Parise D, Parise MTD, Viana MVC, Muñoz-Bucio AV, Cortés-Pérez YA, ArellanoReynoso B, et al. First genome sequencing and comparative analyses of Corynebacterium pseudotuberculosis strains from Mexico. Standards in Genomic Sci 2018;13:21. 57. Quiroga VDB. Evaluación de una técnica de reacción en cadena de la polimerasa para diagnóstico de linfadenitis caseosa de los pequeños rumiantes. [tesis licenciatura] Cuautitlán Universidad Nacional Autónoma de México; 2019. 58. Ibarra ZC, Arellano RB, Hernández CR, Palomares REG, Díaz AE. Evaluation of the aroA mutant of Corynebacterium pseudotuberculosis in cellular and murine models. Vet Méx 2016;3:4:1-16. 59. Velázquez MJ, et al. Detection of Mycobacterium avium subsp. Paratuberculosis in reproductive tissue and semen of naturally infected rams. Animal Rep 2019;( 4):930937. 60. Yamamoto FJK. Crohn´s disease: diagnosis and treatment. Rev Gastroenterol Mex 2013;78(Supl 1): 68-70. 61. Ramírez PC, Ramírez CIC, Valero EG, Trigo TE. Paratuberculosis en cabras en México. Tec Pecu Mex 1983;45:104-106. 62. Gallaga MEP, Arellano RB, Santillán FMA, Favila HLC, Córdova LD, Morales JR, Díaz AE, Situación epidemiológica de la paratuberculosis en las principales regiones caprinas del estado de Puebla, México. Quehacer Científico en Chiapas 2017;12(1). 63. Ruiz CCG, Flores MAS, López DC. Prevalence and possible risk factors for caprine paratuberculosis in intensive dairy production units in Guanajuato, Mexico. J Vet Med Anim Health 2016;8(11):156-162. 223

https://doi.org/10.22319/rmcp.v12s3.5919 Review

Results and impact of research on honeybee genetics and breeding conducted by INIFAP in Mexico

Miguel Enrique Arechavaleta-Velasco a* Claudia García-Figueroa a Laura Yavarik Alvarado-Avila a Francisco Javier Ramírez-Ramírez a Karla Itzel Alcalá-Escamilla a

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Centro Nacional de Investigación Disciplinaria en Fisiología y Mejoramiento Animal. km 1 Carretera a Colón; Ajuchitlán, Colón, Querétaro. México.

*Corresponding author: arechavaleta.miguel@inifap.gob.mx

Abstract: In Mexico, beekeeping is an activity of economic, social and ecological importance that faces various problems; two of the most important problems are the high defensive behavior of honeybee colonies (Apis mellifera L.) caused by the africanization and varroosis caused by the mite Varroa destructor. The high defensive behavior of the colonies has made beekeeping more complex and less profitable. Varroa destructor affects honey production and is a factor that has been associated with honeybee colony losses worldwide. To address these problems, INIFAP conducts research on honeybee genetics and breeding. The objective of this article was to make a review of the research conducted at INIFAP in honeybee genetics that has created scientific knowledge about the genetic, genomic, and epigenetic factors that regulate the expression of honeybee defensive behavior, guarding behavior, stinging behavior, grooming behavior and hygienic behavior. To review the results of research conducted by INIFAP in honeybee breeding to reduce the defensive behavior of honeybee colonies, this research has created methods to evaluate and to select this trait, and has generated honeybee lines with low defensive behavior, from which queens has been transferred to beekeepers. As well as to

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review the work conducted by INIFAP to preserve genetic material of European origins that has led to the establishment of a honeybee germplasm bank. Key words: Honey bees, Genetics, Breeding, Defensive behavior, Varroa destructor, Grooming behavior.

Received: 04/01/2021 Accepted: 11/03/2021

Introduction Importance of beekeeping in Mexico

Mexico is one of the leading producers and exporters of honey globally; it currently ranks as the tenth producer of honey and the fifth exporter worldwide(1). From 2010 to 2019, the average annual honey production was 58,094 t, with an average annual yield of 29.7 kg per hive(2). During this same period, 34,745 t of honey per year were exported, representing 60% of the production(1). The honey produced in Mexico has a high demand in the international market due to its quality traits. The commercial value of honey production in Mexico for 2010-2019 was 2,924 million pesos per year(1). In Mexico, there are 2’157,866 bee colonies, which belong to approximately 43,000 beekeepers(2). Approximately 70 % of beekeepers are small producers, for whom the sale of honey and wax represents an important part of their income. Around 60 % of the colonies in Mexico belong to this type of beekeepers, who have a low degree of technification and manage 40 hives on average. The remaining colonies (40%) belong to production units of various sizes and degrees of technification(3). In addition to the production of honey, wax, pollen, royal jelly, and propolis, the pollination carried out by honeybees is essential for maintaining the balance of the ecosystems and for food production. Honeybees partially or completely pollinate approximately 70 % of the plants cultivated for human consumption. Thus, humans heavily depend on honeybees for feeding purposes. In Mexico, the value of the pollination carried out by honeybees in cultivated plants is 20 times greater than the value of honey production(4). The estimated value of the pollination of cultivated plants from 2010 to 2019 was 58,480 million pesos per year.

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Mexico is divided into five beekeeping regions: North, Plateau, Pacific Coast, Gulf, and Yucatan Peninsula. These regions are classified based on their climate and vegetation(5). The Yucatan Peninsula, Plateau, and Pacific Coast regions are the major contributors to honey production. These regions produce 36 %, 25 %, and 22 % of the national honey production, respectively, equivalent to 32 %, 27 %, and 24 % of the economic production value. The North and Gulf regions produce 9 % and 8 % of the national honey production, representing 9 % and 8 % of the total economic value, respectively(2).

Main beekeeping problems in Mexico in which INIFAP has conducted research The highly defensive behavior of honeybee colonies caused by Africanization and varroosis caused by Varroa destructor are the two main problems that beekeeping faces in Mexico, for both INIFAP has conducted research in the fields of genetics and breeding to reduce the impact of these problems. The Africanization of honeybee populations that has occurred during the last 60 years in the American continent is considered one of the cases with highest impact of an invasive species in history. Africanized honeybees originated in Brazil from the cross of European (A. mellifera mellifera, A. mellifera ligustica, and A. mellifera carnica) and African honeybees (A. mellifera scutellata), which were introduced into this country in 1956(6). Africanized honeybees have short brood development stages(7) and high reproductive rates, which translates into high swarm production and facilitates the dispersion of these honeybees throughout South America and Central America, reaching Mexico in 1986(8) and the United States of America in 1990(9). Africanization had drastic consequences for beekeeping and even public health in many countries(10). Since 1986, when Africanized honeybees reached the country, Mexican beekeeping has suffered important changes due to Africanization(11). Compared to European honeybees, these honeybees are more defensive, show a greater tendency to swarm and evade, and produce less honey(12-15). The highly defensive behavior of Africanized bee colonies has complicated their management; this has decreased profitability due to the increased production costs derived from the practices implemented by beekeepers to manage this type of bee(11). Africanized honeybees are distributed throughout the country, and the degree of Africanization of bee populations managed by beekeepers is high(16). A study carried out in Yucatan indicated that 61 % of the colonies managed by beekeepers and 87 % of the wild colonies are Africanized(17). Meanwhile, a different study reported that 100 % of the

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analyzed colonies were Africanized(16). In Mexico State, a study estimated that 37 % of the colonies managed by beekeepers were Africanized, and 70 % had some degree of Africanization(18). In Morelos, a study indicated that 65 % of the colonies showed some degree of Africanization(19). Varroosis represents a severe threat to the survival of honeybees and honey production both in Mexico and the world. A study in Mexico indicated that colonies with infestation levels of 6% produced on average 65 % less honey than parasite-free colonies(20). A different study reported that the production of honey decreased by 52.8 g per infestation percentage unit(21). Furthermore, this parasite has been associated with colony collapse disorder (CCD). In recent years, this phenomenon has been responsible for the loss of bee colonies in Mexico(22,23) and other parts of the world(24-27). Varroosis is controlled with synthetic chemical acaricides and organic chemical products. None of the products are 100 % effective against this mite. The synthetic acaricides used in Mexico are flumethrin and fluvalinate, which have a reported efficiency of 98 %. However, several studies in Mexico and in other countries have reported mites resistant to these products; acaricides have an efficiency lower than 50 % against resistant V. destructor(28,29). Furthermore, these products can leave chemical residues on the honey and wax, affecting both honeybees and humans(30-34). The organic chemicals most used in Mexico for mite control are thymol, oxalic acid, and formic acid. Although these products do not generate resistance, their efficacy is 93 % or less(35). Varroa destructor was first detected in Mexico in 1992(36), and this mite is now distributed throughout the country. A study carried out in Zacatecas reported that the prevalence of varroosis is 89 %, with an average level of infestation of 4.85 %(37). In Morelos, a prevalence and average infestation level of 80% and 4.76 %, respectively, were reported(38). Jalisco has a prevalence of 88% and an infestation level of 5.2 %(39). Varroosis is 63 % prevalent in Yucatan, with an infestation level of 1.70 %(40). This article aims to review the results obtained from research studies conducted in Mexico and outside the country by INIFAP or with the participation of INIFAP researchers. This review includes research in genetics and breeding of honeybees related to their defensive behavior and resistance to varroosis.

Results of studies conducted on honeybee defensive behavior genetics The studies that have been carried out in Mexico to know/understand the genetic mechanisms that regulate the defensive behavior expression in honeybees have allowed determining that colony defensive behavior, which is measured by the number of stings

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left by the honeybees of the same colony on a black suede flag waved in front of the beehive for a specific period of time, is regulated by dominant genetic effects; since, when compared between European, Africanized and hybrid genetic groups, defensive behavior has significant differences between European and Africanized groups, and defensive behavior of colonies belonging to the hybrid group is as high as the one of the Africanized group. In addition, these studies have allowed determining that interactions between honeybees belonging to the three genetic groups within the same colony have an influence on the defensive behavior of that colony. This means that if European genotype honeybees interact with honeybees of the African or hybrid groups within the same colony, the defensive behavior of such colony will be as high as the defensive behavior of a colony formed only by hybrid of Africanized honeybees(41,42,43). Mexico was the first country to identify regions within the bee genome that regulate the expression of the defensive behavior of the colonies. In this study, five Quantitative Trait Loci (QTL) were linked to the expression of defensive behavior using a population of 172 colonies formed by a single backcross-derived family of worker bees obtained following a crossbreeding scheme from Africanized and European honeybees(14). The effect of three of the five QTL on the defensive behavior of honeybees was confirmed by two independent studies in Africanized(43) and European honeybees(44). The defensive response of a colony involves two behaviors carried out by the colony's worker honeybees at the individual level, the guard and stinging behaviors(45-51). In Mexico, some studies evaluated the relationship between the guard and stinging behavior during the defensive response of a bee colony. Their results indicate that the guarding bees participate in the defensive response of the colony; thus, their presence, number, and proportion of bees that respond by stinging influence the intensity of the colony's defensive response(44,50,51). Furthermore, researchers in Mexico have evaluated the genetic mechanisms that regulate the expression of the guarding and stinging behaviors at an individual level. These studies are essential to understanding how behaviors that are genetically regulated at an individual level can influence the phenotype of the entire colony. Additionally, previous studies have observed that, at an individual level, the stinging behavior (measured as the time it takes for a bee to sting a piece of black suede after receiving a constant electrical stimulus) of Africanized honeybees is significantly higher than in European honeybees. Thus, the response of Africanized honeybees is faster than that of European honeybees. Furthermore, one of these studies reported that the stinging behavior of bees with guarding behavior at the hive's entrance and bees that respond by trying to sting an intruder was superior compared to the bees that carry out other duties within the colony; this was observed in both Africanized and European colonies(49).

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Arechavaleta-Velasco et al(44) determined that three of the QTL previously associated with the defensive behavior of bees at a colony level(14) affect the stinging behavior of individual honeybees. However, Shorter et al(52) identified two new QTL for this trait, from which four candidate genes could be identified for the expression of the stinging behavior of honeybees. As for the guarding behavior, studies have identified effects of genetic origin for the number of bees that perform guarding behavior in a honeybee colony(44). Arechavaleta-Velasco and Hunt(53) identified seven Binary Trait Loci (BTL) associated with the expression of guarding behavior in worker bees using two reciprocal backcrossderived bee colonies generated from European lines with high and low defensive behavior. A different study determined that one of the QTL previously associated with the defensive behavior of bees at a colony level(14) affects the guarding behavior of individual bees(44). Meanwhile, Shorter et al(52) identified one new QTL for this trait from which three candidate genes could be identified for the expression of the guarding behavior of bees. Guzmán-Novoa et al(54) observed that the defensive behavior of hybrid colonies derived from reciprocal crosses between Africanized and European bees is different. The hybrid colonies derived from the cross between European queens and Africanized drones are significantly more defensive than the hybrid colonies derived from the cross between Africanized queens and European drones. The phenotypic differences between reciprocal hybrid groups are an indicator of epigenetic effects(55,56). Thus, these results suggest that the increased defensive behavior of the colonies whose paternal origin is Africanized is due to the epigenetic effects generated by an imprinting process of paternal origin. A study carried out by Kocher et al(57) analyzed the transcriptome of different tissues of bees with reciprocal hybrid genotypes obtained from a crossbreeding scheme between Africanized and European bees. These researchers reported that the selective expression of genes by their paternal origin is one of the mechanisms by which epigenetic effects occur. The selective expression of genes by their parental origin occurs when the expression level depends on whether an allele is inherited through the mother or father. Gibson et al(58) found differences in the stinging behavior of bees at an individual level between reciprocal hybrid genotypes that originate from crosses between European and Africanized bees. Hybrid bees whose paternal origin was Africanized responded to an electrical stimulus by stinging a piece of black suede much faster than the hybrid bees with a European paternal origin. These observations indicate that, as reported for the defensive behavior, epigenetic effects exist due to an imprinting process of paternal origin.

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The transcriptome of the genes localized in the genomic regions that correspond to the QTL associated with the defensive behavior was analyzed to evaluate if the epigenetic effects detected in the stinging behavior of individual genes(58) result from the selective gene expression due to their parental origin(14,43,44,52,59). This analysis was carried out in bees, for which the stinging behavior had been individually evaluated. The results indicated that this mechanism is involved in the regulation of gene expression in hybrid bees with European maternal origin and Africanized paternal origin. This was apparently due to disturbances in the signaling pathways between nuclear and mitochondrial genes that modulate brain metabolism and defensive behavior in bees(58).

Results of studies and breeding programs to reduce the defensive behavior of honeybees Guzmán-Novoa and Page(59) reported the results of a breeding program that started in November 1991 and concluded in 1996. This program was implemented for four generations of selection in a commercial population of approximately 3,000 bee colonies to improve honey production and reduce the defensive behavior of the population. This study demonstrated that it is possible to genetically improve bee populations in Africanized zones without instrumental insemination of honey-bee queens. The breeding program consisted of conducting mass selection in stages, considering honey production, defensive behavior, laying pattern of bees, and the average length of the forewing of the worker bees in the colony. The results of this program showed that it was possible to maintain honey production, reduce the defensive behavior of the colonies, and increase the average length of the forewing of the worker bees in the population under selection despite the process of Africanization of bees that was occurring at the time in the region of study. These results indicate that the defensive behavior of the population decreases when the relative frequency of colonies with European morphotype and haplotype increases due to the selection process. In 1996, INIFAP started a breeding program to generate lines of bees selected for high honey production and low defensive behavior. The breeding program was developed in Mexico's State; approximately 500 bee colonies were formed with colonies from INIFAP and cooperating beekeepers. The traits included in the program as selection criteria were: honey production, defensive behavior, the average length of the forewing of the worker bees in the colony, colony morphotype (European, hybrid, or Africanized), and colony haplotype (European or African)(60).

In this breeding program, three lines of bees were generated. In various studies, honey production, defensive behavior, the average length of the forewing of worker bees,

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morphotype distribution (European, Hybrid, and Africanized), haplotype distribution (European and African), and the genetic variability of the mitochondrial DNA have been evaluated and compared to European and Africanized populations outside breeding programs(61-65). As for honey production, in two different studies, the genetically improved lines of bees produced significantly more honey than the bees that did not belong to a breeding program(61,63). In one of the studies, INIFAP lines produced on average 34.5% more honey than non-selected Africanized bees(61). In the second study, bees produced on average 27.5 and 40.3 % more honey than non-selected European and Africanized bees, respectively(63). The defensive behavior was measured by counting the number of bee stingers deposited on a black suede flag waved in front of the hive for a specified time. Improved lines were on average 57.4 % less defensive than the non-selected Africanized bees and 44.3 % more defensive than the non-selected European bees(63). Regarding the average length of the forewing of worker bees, a study evaluated two lines of INIFAP bees, one of these lines had longer (9.07 and 9.06 mm) forewings than European (9.03 mm) and Africanized (8.90 mm) bees. The forewing length of the second line was similar (9.02 mm) to that of non-selected European bees, and both groups had longer forewings than non-selected Africanized bees(63). Another study observed that the improved lines of bees had longer forewings (9.05, 9.04, and 9.03 mm) than the nonselected Africanized bees (8.98 mm)(65). Two studies evaluated the morphotype distribution in different colonies. They found that the frequency of bee colonies with European, Hybrid, or Africanized morphotypes in selected lines was significantly different from that observed in the populations of nonselected bees(62,65). One study observed that in the populations of selected bees, the average relative frequencies of colonies with European, Hybrid, and Africanized morphotypes were 0.47, 0.35, and 0.18, respectively. In the population of non-selected colonies, the relative frequency of the European, Hybrid, and Africanized morphotypes were 0.17, 0.43, and 0.40, respectively(62). In a different study, the population of three INIFAP lines showed average relative frequencies of 0.37, 0.42, and 0.21 for the European, Hybrid, and Africanized morphotypes, respectively. The frequencies for the populations of non-selected bees were 0.17, 0.43, and 0.41(65). For the haplotype distribution, the average relative frequencies of the population of selected lines with European or African haplotypes were significantly different from that of the non-selected bee populations. The European and African haplotype frequency was 0.93 and 0.07, respectively, in the population of INIFAP bees, and in the population of non-selected bees, the frequencies were 0.34 and 0.66, respectively(62). Finally, regarding the genetic variability of the mitochondrial DNA, estimated by the Shannon Index, the

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lines of selected bees showed lower variability (IS=0.12) than non-selected populations (IS=0.41); this resulted from the selections process to which bees were subjected(64). Improved lines have been used to develop projects in which INIFAP has transferred the improved genetic material to beekeeper groups in Mexico's State, Hidalgo, Queretaro, and Morelos. These projects have provided 10,000 royal cells, 3,000 queens for free fertilization, and 500 instrumentally inseminated bees as breeding stock. From this genetic material, beekeepers have produced at least 20,000 bees for free fertilization(66).

Results of the studies conducted to preserve European honeybee germplasm In 2004, INIFAP established a Germplasm Bank aimed to preserve the genetic material of European bees(67). This Bank is in the National Center for Disciplinary Research in Animal Physiology and Genetics. This bank includes a population of 100 bee colonies, which are handled following a closed population scheme with instrumental insemination of queen bees(68,69). This approach genetically isolates the population by having total control over mattings; only queen bees and drones from the germplasm are used for the crossings required to produce the queens for the annual queen replacement that has to be carried out in the population. The germplasm is characterized in morphometric, molecular, and behavioral terms. Evaluations of the bee colonies are carried out every year to ensure that the genetic material kept in the bank has European characteristics. These genetic analyses use molecular markers in the mitochondrial DNA that classify bee haplotypes into European or African(70). The length of the forewing of bees is determined through morphometric analyses, which allow classifying colonies into European, Hybrid, or Africanized(71). The defensive behavior of colonies is evaluated by qualitatively determining how much the bees of a colony run on the honeycomb, fly over the hive, and collide and sting the beekeeper during their routine check(60). Finally, the tendency of colonies to swarm and evade is also evaluated. INIFAP's bee germplasm bank has been preserved for 17 generations, and each generation corresponds to an annual beekeeping cycle. The genetic material preserved in the bank has been used to develop research projects and generate, validate, and transfer improved genetic material(66).

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Results of studies conducted on the genetics of honeybee resistance to varroasis In Mexico, studies have evaluated the differences in susceptibility to Varroa destructor of Africanized and European bees(72,73). Guzmán-Novoa et al(72) compared the susceptibility to Varroa destructor of adult bees and broods with Africanized, European, and two reciprocal hybrid genotypes. Their results indicate that the Africanized and two hybrid genotypes in adult bees were equally susceptible but less susceptible than the European genotype. As for the broods, the Africanized genotype was the least susceptible, followed by the European and hybrid genotypes originated from a European mother and an Africanized father; the hybrid originated from an Africanized mother and a European father was the most susceptible. This same study also evaluated the effect of these genotypes on the reproductive capacity of the mite and found similar capacities in European and Africanized genotypes; the highest reproductive capacities were observed in both hybrid genotypes. Guzmán-Nova et al(73) analyzed the results of various studies carried out in Mexico to determine if the susceptibility to Varroa destructor was different between Africanized and European bees. These results indicate that the susceptibility or resistance to the mite does not depend on the genetic group as both groups show variability in their susceptibility to Varroa destructor. Furthermore, these studies found that the environment and the interaction between the genotype and the environment play an important role in the infestation levels of bee colonies. The resistance to Varroa destructor depends on the expression of resistance mechanisms against this mite. These mechanisms include the grooming behavior, the hygienic behavior, the differential attraction of the worker brood and the adult worker bees to the mite, a shorter brood development period after capping, and factors affecting the fertility and reproduction of the mite(73-78). Researchers in Mexico carried out one of the two studies that measured the relative contribution of each one of these mechanisms of resistance to Varroa destructor(75,79). Arechavaleta-Velasco and Guzmán-Novoa(75) determined that, in Mexico, there is variability of genetic origin in the resistance of bee colonies to Varroa destructor and that this resistance is not related to one line or genetic great in particular. In this study, they evaluated how the grooming and hygienic behaviors of bees, the differential attraction of Varroa destructor to the brood, and the effect of the brood on the reproductive capacity of the mite contribute to the resistance of bee colonies to the population growth of this mite. This study indicates that grooming behavior is the primary mechanism that colonies use to resist the population growth of Varroa destructor. Furthermore, susceptible and resistant colonies showed differences in their hygienic behavior; however, the contribution to resistance of this behavior was not clear. On the other hand, Harbo and

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Harris(79) identified the hygienic behavior against Varroa destructor as the primary mechanism of resistance. Arechavaleta-Velasco et al(80) developed a study to identify the regions within the bee genome that regulate the expression of their grooming behavior. This study associated this grooming behavior to a quantitative trait locus (QTL), groom-1, located in chromosome 5 of the bee genome. Twenty-seven genes were identified within the region that corresponded to the 95 % confidence interval for the location of the QTL and reported in the Honeybee genome database; one of these genes was neurexin-1 (AmNrx1). This gene's orthologs are associated with autism and schizophrenia in humans, synapses formation and associative learning in Drosophila and Aplysia, and grooming behavior in mice. The grooming behavior of Neurexin-1-alpha knockout mice is higher compared to wild-type mice. The fact that neurexin-1 influences grooming behavior in mammals and bees is evidence of the effect of this gene on the expression of this trait. The effect of the QTL groom-1 and neurexin-1 (AmNrx1) on the expression of the grooming behavior was confirmed using a bee population different to the one in which the effect of QTL and the gene was first identified(81). As for the hygienic behavior, a study carried out at the level of individual bees identified seven BTL associated with the expression of this trait(82). This number of BTL was similar to the number of QTL detected in the study carried out by Lapidge et al(83) to identify the genomic regions for this trait at the phenotypic level of the entire colony.

Impacts The research developed in INIFAP regarding the genetics of the defensive behavior of honeybees and the mechanisms of resistance of the honeybees to Varroa destructor has had a substantial scientific impact worldwide. These studies have contributed to understanding the genetic, genomic, and epigenetic mechanisms that regulate the expression of the defensive behavior of colonies, the guarding and stinging behaviors of individual bees, and the grooming and hygienic behaviors. As for reducing the defensive behavior of bee colonies, these studies have had a significant scientific and technological impact by generating methods for the evaluation, selection, and breeding for this trait. Honeybee lines with low defensive behavior were obtained by applying the knowledge provided by these studies. From these lines, 500 breeding stock queens and at least 33,000 queens of free fertilization have been transferred to producers, representing a significant social and production impact. The studies developed by INIFAP to establish a Honeybee Germplasm Bank have an important ecologic and technological impact. This bank is the only nucleus of

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conservation of germplasm of European origin in Mexico. There is a high risk of losing the germplasm of European origin because of the high levels of Africanization in Mexico's honeybee populations; therefore, it is essential to preserve the honeybees maintained in this bank as a vital genetic resource for beekeeping. Literature cited: 1. FAO. Organización de las Naciones Unidas para la Alimentación y la Agricultura. Food and agriculture data. 2020. http://www.fao.org/faostat/en/#data/QA Consultado 1 Ago, 2020. 2. SIAP. Servicio de Información Agroalimentaria y Pesquera. Anuario Estadístico de la Producción Ganadera. México. 2010-2019. https://nube.siap.gob.mx/cierre_pecuario/ Consultado 1 Ago, 2020. 3. Vélez IA, Espinosa GJ, Gutiérrez RA, Arechavaleta-Velasco ME. Tipología y caracterización de apicultores del estado de Morelos, México. Rev Méx Cienc Pecu 2016;7(4):507-524. 4. Guzmán-Novoa E. La apicultura en México y Centroamérica. Congreso Iberolatinoamericano Apícola. Mercede, Uruguay. UNA;1996:14-17. 5. García GL, Meza RE. Oportunidades y obstáculos para el desarrollo de la apicultura en Nayarit.http://www.eumed.net Consultado 25 Nov, 2012. 6. Kerr WE. The history of the introduction of Africanized bees to Brazil. S Afr Bee J 1967;39:3-5. 7. Winston ML. The biology and management of the Africanized honey bees. Annu Rev Entomol 1992;37:173-193. 8. Moffett JO, Maki DL, Andre T, Fierro MM. The Africanized bees in Chiapas, Mexico. Am Bee J 1987;127:520-571. 9. Sudgen EA, Williams KR. October 15: the day the bee arrived. Glean Bee Cult 1991;119:18-21. 10. Winston ML. The Biology of the honey bee. Harvard University Press. EUA 1987. 11. Guzmán-Novoa E, Page RE. The impact of Africanized bees on Mexican beekeeping. Ann Bee J 1994;124(2):101-106. 12. Collins AM, Rinderer TE, Harbo J. Colony defense by Africanized and European honey bees. Science 1982;218:72-74.

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13. Otis GW. Population biology of the Africanized honey bee. In: Jaisson P, editor. Social insects in the tropics. 1982;1:209-219. 14. Hunt G, Guzmán-Novoa E, Fondrk MK, Page RE. Quantitative trait loci for honey bee stinging behavior and body size. Genetics 1998;149:1203-1213. 15. Guzmán-Novoa, Uribe-Rubio. Honey production by european, africanized and hybrid honey bee (Apis mellifera) colonies in Mexico. Am Bee J 2004;144:318-320. 16. Domínguez-Ayala R, Moo-Valle H, May-Itza W, Medina-Peralta S, Quezada-Euán J. Stock composition of northern neotropical honey bees: mitotype and morphotype diversity in Mexico (Hymenoptera: Apidae). Apodologie 2016;47:642-652. 17. Clarke K, Oldroyd B, Quezada-Euán J, Rinderer T. Origin of honeybees (Apis mellifera L.) from the Yucatán peninsula inferred from mitochondrial DNA analysis. Mol Ecol 2001;10:1347-1355. 18. Arechavaleta VM, Robles RC, Espinosa SA. Caracterización molecular y morfométrica de las poblaciones de colonias de abejas del Estado de México. Reunión Nacional de Investigación Pecuaria. Mérida, Yucatán. 2008:176. 19. Arechavaleta VM, Vázquez PS, Ramírez RF, Camacho RC, Robles RC, Amaro GR. Distribución de los morfotipos europeo, africanizado e híbrido en poblaciones de colonias de abejas de Morelos. Reunión Nacional de Investigación Pecuaria. Veracruz. 2013:456. 20. Arechavaleta-Velasco ME, Guzmán-Novoa E. Producción de miel de colonias de abejas (Apis mellifera L.) tratadas y no tratadas con un acaricida contra Varroa jacobsoni Oudemans en Valle de Bravo, Estado de México. Rev Vet Méx 2000;31:381–384. 21. Medina-Flores CA, Guzmán-Novoa E, Aréchiga-Flores CF, Aguilera-Soto JI, Gutiérrez-Piña FJ. Efecto del nivel de infestación de Varroa destructor sobre la producción de miel de colonias de Apis mellifera en el altiplano semiárido de México. Rev Mex Cienc Pecu 2011;2(3):313-317. 22. Medina-Flores CA, Esquivel-Marín NH, López-Carlos M, Medina-Cuellar SE, Aguilera-Soto JI. Estimación de la pérdida de colonias de abejas melíferas en el altiplano y el norte de México. Exosist Recur Agropec 2018;5(14):365-371. 23. Arechavaleta VM, Velez IA, Espinosa GJ, Amaro GR, Alcalá EB, Vázquez PS, Ramírez RF. Presencia del síndrome del colapso de las colonias de abejas (CCD) en México. Reunión Nacional de Investigación Pecuaria. Mérida, Yucatán. 2014:82.

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24. Genersch E, Von Der OH, Kaatz H, Schroeder A, Otten C, Büchler R, et al. The German bee monitoring project: a long term study to understand periodically high winter losses of honey bee colonies. Apidologie 2010;41(3):332-352. 25. Currie RW, Pernal SF, Guzmán-Novoa E. Honey bee colony losses in Canada. J Apic Res 2010;49:104–106. 26. Dahle B. The role of Varroa destructor for honey bee colony losses in Norway. J Apic Res 2010;49:124–125. 27. Peterson M, Gray A, Teale A. Colony losses in Scotland in 2004–2006 from a sample survey. J Apic Res 2010;48:145–146. 28. Rodríguez-Dehaibes SR, Otero-Colina G, Villanueva-Jiménez, Corcuera MRP, Chávez VC, Lagunas ZR. Resistencia de Varroa destructor a los plaguicidas usados para su control en las regiones Golfo y Centro-Altiplano, México. Congreso Internacional de Actualización Apícola; 2007 mayo 16-18; Boca del Río, Veracruz. 2007:40-44. 29. Arechavaleta VM, Torres NG, Robles RC, Correa BA. Identificación de poblaciones de Varroa destructor resistentes al fluvalinato en colonias de abejas en el Estado de México. Cong Int Actualiz Apícola. Boca del Río, Veracruz. 2007:113-116. 30. Slabezki Y, Gal H, Lensky Y. The effect of fluvalinate application in bee colonies on population levels of Varroa jacobsoni and honey bees (Apis mellifera L.) and on residues in honey and wax. Bee Sci 1991;1(4):189-195. 31. Lodesani M, Pellacani A, Bergomi S, Carpana E, Rabitti T, Lasagni P. Residue determination for some products used against Varroa infestation in bees. Apidologie 1992;23(3):257-272. 32. Wallner K. Varroacides and their residues in bee products. Apidologie 1999;30:235248. 33. Mathieu L, Faucon JP. Changes in the response time for Varroa jacobsoni exposed to amitraz. J Apicult Res 2010;39(3-4):155-158. 34. Pettis JS, Collins AM, Wilbanks R, Feldlaufer MF. Effects of coumaphos on queen rearing in the honey bee, Apis mellifera. Apidologie 2004;35(6):605-610. 35. Guzmán NE, Emsen B, Gashout H, Rodríguez MF, Correa BA. Eficacia de productos orgánicos y de diferentes métodos de aplicación en el control del ácaro Varroa destructor y su inocuidad en las abejas melíferas. 14° Congreso Internacional de Actualización Apícola; 2007 mayo 16-18; Boca del Río, Veracruz. 2007:45-51.

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36. Chihu AD, Rojas AM, Rodríguez DS. Presencia en Veracruz, México del ácaro Varroa jacobsoni, causante de la varroasis de la abeja melífera (Apis mellifera L.). Tec Pecu Méx 1992;30(2):133-135. 37. Medina-Flores CA, Guzmán-Novoa E, Hamiduzzaman MM, Aréchiga-Flores CF, López-Carlos MA. Africanized honey bees (Apis mellifera) have low infestation levels of the mite Varroa destructor in different ecological regions in Mexico. Genet Mol Res 2014;13(3):7282-7293. 38. Vázquez PS, Arechavaleta VME, Ramírez RFJ, Amaro GR, Robles RC. Prevalencia y niveles de infestación de Varroa destructor en colonias de abejas del Estado de Morelos. Reunión Nacional de Investigación Pecuaria. Veracruz. 2013:66. 39. Tapia-González JM, Alcazar-Oceguera G, Macías-Macías JO, Contreras-Escareño F, Tapia-Rivera JC, Petukhova T, Guzmán-Novoa E. Varroosis en las abejas melíferas en diferentes condiciones ambientales y regiones de Jalisco, México. Ecosist Recur Agropec 2019;6(17):243-251. 40. Martínez PJ, Medina ML, Catzin VG. Frecuencia de Varroa destructor, Nosema apis y Acarapis woodi en colonias manejadas y enjambres silvestres de abejas (Apis mellifera) en Mérida, Yucatán, México. Rev Mex Cienc Pecu 2011;2(1):25-38. 41. Guzmán-Novoa E, Page RE. Backcrossing Africanized honey bee queens to European drones reduces colony defensive behavior. Ann Entomol Soc Am 1993;86:352-355. 42. Guzmán-Novoa E, Page RE. Genetic dominance and worker interactions affect honey bee colony defense. Behav Ecol 1994;5:91-97. 43. Guzmán-Novoa E, Hunt GJ, Uribe JL, Smith C, Arechavaleta-Velasco ME. Confirmation of QTL effects and evidence of genetic dominance of honeybee defensive behavior: results of colony and individual behavioral assays. Behav Genet 2002;32:95-105. 44. Arechavaleta-Velasco ME, Hunt GJ, Emore C. Quantitative trait loci that influence the expression of guarding and stinging behaviors of individual honey bees. Behav Genet 2003;33:357-364. 45. Moore AJ, Brees MD. Morr MJ. The guard honey bee-ontogeny and behavioral variability of workers performing a specialized task. Anim Behav 1987;35:11591167. 46. Breed DM, Robinson GE, Page RE. Division of labor during honey-bee colony defense. Behav Ecol Sociobiol 1990;27:395-401.

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47. Breed DM, Guzmán-Novoa E, Hunt GJ. Defensive behavior of honey bees: organization, genetics, and comparisons with other bees. Annu Rev Entomol 2004;49:271-298. 48. Guzmán-Novoa E, Hunt GJ, Uribe-Rubio JL, Prieto-Merlos D. Genotypic effects of honey bee (Apis mellifera) defensive behavior: results of colony and individual behavioral assays. Behav Genet 2004;32:95-102. 49. Uribe-Rubio JL, Guzmán-Novoa E, Vasquez-Pelaez CG, Hunt GJ. Genotype task specialization, and nest environment influence the stinging response thresholds of individual Africanized and European honeybees to electrical stimulation. Behav Genet 2008;38:93-100. 50. Hunt GJ, Guzmán-Novoa E, Uribe-Rubio JL, Prieto-Merlos D. Genotype environment interactions in honeybee guarding behavior. Anim Behav 2003;66:459467. 51.Arechavaleta-Velasco ME, Hunt GJ. Genotypic variation in the expression of guarding behavior and the role of guards in the defensive response of honey bee colonies. Apidologie 2003;34:439-447. 52. Shorter JR, Arechavaleta-Velasco ME, Robles- Ríos C, Hunt GJ. A genetic analysis of the stinging and guarding behaviors of the honeybee. Behav Genet 2012;42:663674. 53. Arechavaleta-Velasco ME, Hunt GJ. Binary trait loci that influence honey bee (Hymenoptera: Apidae) guarding behavior. Ann Entomol Soc Am 2004;97:177-183. 54. Guzmán-Novoa E, Hunt GJ, Page RE, Uribe-Rubio JL, Prieto-Merlos D, BecerraGuzmán F. Paternal effects of the defensive behavior of honeybees. J Hered 2005;96:376-380. 55. Queller DC. Theory of genomic imprinting conflict in social insects. BMC Evol Biol 2003;15(3):1-23. 56. Ferguson-Smith AC. Genomic imprinting: the emergence of an epigenetic paradigm. Nature Reviews. Genetics 2011;12:565-575. 57. Kocher SD, Tsuruda JM, Gibson JD, Emore CM, Arechavaleta-Velasco ME, Queller D, et al. A search for parent of origin effects on honey bee gene expression. G3 2015;5:1657-1662. 58. Gibson JD, Arechavaleta-Velasco ME, Tsuruda JM, Hunt GJ. Biased allele expression and aggression in hybrid honeybees may be influenced by inappropriate nuclear-cytoplasmic signaling. Front Genet 2015;6:343.

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59. Guzmán-Novoa E, Page Jr RE. Selective breeding of honey bees (Hymenoptera: Apidae) in africanized areas. J Ecom Entomol 1999;92(3):521-525. 60. Arechavaleta-Velasco ME. Mejoramiento genético de las abejas melíferas. Folleto Técnico N° 17 INIFAP-CENID Fisiología y Mejoramiento Animal. 2012. 61. Guzmán NE, Uribe RJL, Prieto MD. Producción de miel de tres estirpes de abejas seleccionadas en el altiplano mexicano. Reunión Nacional de Investigación Pecuaria. CDMX. 2003:227. 62. Arechavaleta VME, Robles RCA, Uribe RJL, Guzmán NE. Distribución de mitotipos y morfotipos en poblaciones de colonias de abejas melíferas seleccionadas y no seleccionadas. Reunión Nacional de Investigación Pecuaria. Veracruz. 2006:199. 63. Arechavaleta VME, Robles RCA, García FF, Noriega G, Uribe RJL. Producción de miel, comportamiento defensivo y longitud promedio del ala anterior de colonias de abejas desarrolladas bajo dos métodos de selección. Reunión Nacional de Investigación Pecuaria. Sinaloa. 2007:205. 64. García FF, Arechavaleta VME, Robles RCA. Variabilidad genética del ADN mitocondrial en poblaciones de abejas melíferas seleccionadas y no seleccionadas. Reunión Nacional de Investigación Pecuaria. Saltillo, Coahuila. 2009:98. 65. Alcalá EKI, Arechavaleta VME, Robles RCA. Niveles de africanización en poblaciones de colonias de abejas desarrolladas bajo dos métodos de selección en zonas africanizadas. Reunión Nacional de Investigación Pecuaria. Campeche, Campeche. 2010:208. 66. Arechavaleta VM, Guzmán-Novoa E, Uribe RJ, García FC, Ramírez RF. Mejoramiento genético para el manejo de la africanización de colonias de abejas melíferas. In: Cruz Cruz E, Reyes-Muro L compiladores. Aportaciones del INIFAP al campo mexicano en 35 años. Libro Técnico N° 1 INIFAP 2020. 67. GRIN. Animal Germplasm Resources Information Network. Centro Nacional de Recursos Genéticos-INIFAP. https://agrin.ars.usda.gov/database_collaboration_page_dev Consultado 17 Mar, 2021. 68. Page Jr RE, Laidlaw Jr HH. Closed population honeybee breeding. Bee world 1985;66(2):63-72. 69. Cobey S, Lawrence T. Commercial application and practical use of the Page-Laidlaw closed population breeding program. Am Bee J 1988;128(5):341-344.

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70. Nielsen DI, Ebert PR, Page RE, Hunt GJ, Guzmán-Novoa E. Improved polymerase chain reaction based mitochondrial genotype assay for identification of the africanized honey bee (Hymenoptera: Apidae). Ann Entomol Soc Am 2000;93:1-6. 71. Sylvester HA, Rinderer TE. Fast africanized bee identification system. Ame Bee J 1987;127(7):511-516. 72. Guzmán-Novoa E, Sánchez A, Page Jr RE, García T. Susceptibility of European and Africanized honeybees (Apis mellifera L.) and their hybrids to Varroa jacobsoni Oud. Apidologie 1996;27(2):93-103. 73. Guzmán-Novoa E, Vandame R, Arechavaleta-Velasco ME. Susceptibility of European and Africanized honeybees (Apis mellifera L.) to Varroa jacobsoni Oud. in México. Apidologie 1999;30:173-182. 74. Moosbeckhofer R. Beobachtungen sum Auftreten beschädigter Varroamilben im natürlichen Totenfall bei Völkern von Apis mellifera carninca. Apidologie 1992;23:523-531. 75. Arechavaleta-Velasco ME, Guzmán-Novoa E. Relative effect of four characteristics that restrain the population growth of the mite Varroa destructor in honey bee (Apis mellifera) colonies. Apidologie 2001;32:157–174. 76. Mondragón L, Spivak M, Vandame R. A multifactorial study of the resistance of honeybees Apis mellifera to the mite Varroa destructor over one year in Mexico. Apidologie 2005;36(3):345-358. 77. Ibrahim A, Spivak M. The relationship between hygienic behavior and suppression of mite reproduction as honey bee (Apis mellifera) mechanisms of resistance to Varroa destructor. Apidologie 2006;37(1):31-40. 78. Ibrahim A, Reuter GS, Spivak M. Field trial of honey bee colonies bred for mechanisms of resistance against Varroa destructor. Apidologie 2007;38(1):67-76. 79. Harbo JR, Harris JW. Selecting honey bees for resistance to Varroa jacobsoni. Apidologie 1999;30(2-3):183-196. 80. Arechavaleta-Velasco ME, Alcala-Escamilla K, Robles-Rios C, Tsuruda JM, Hunt GJ. Fine-scale linkage mapping reveals a small set of candidate genes Influencing honey bee grooming behavior in response to varroa mites. PLOS ONE 2012;7(11):e47269. 81. Hamiduzzaman M, Emsen B, Hunt GJ, Subramanyam S, Williams CE, Tsuruda JM, Guzmán-Novoa E. Differential gene expression associated with honey bee grooming behavior in response to Varroa mites. Behav Genet 2017;47(3):335–344.

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82. Arechavaleta-Velasco ME, Hunt GJ, Spivak M, Camacho-Rea C. Loci de rasgos binarios que influyen en la expresión del comportamiento higiénico de las abejas melíferas. Rev Mex Cienc Pecu 2011;2(3):283-298. 83. Lapidge KL, Oldroyd BP, Spivak M. Seven suggestive quantitative trait loci influence hygienic behavior of honey bees. Naturwissenschaften 2002;89:565-568.

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https://doi.org/10.22319/rmcp.v12s3.5876 Review

Rehabilitation of degraded pastures in the tropics of Mexico

Javier Francisco Enríquez Quiroz a* Valentín Alberto Esqueda Esquivel b Daniel Martínez Méndez c

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Campo Experimental La Posta, Km. 22.5 Carretera Federal Veracruz-Córdoba. 94277, Medellín, Veracruz, México. b

INIFAP. Campo Experimental Cotaxtla, Medellín, Veracruz, México.

Centro de Bachillerato Tecnológico Agropecuario 251. General Felipe Ángeles, Oaxaca, México.

*Corresponding author: enriquez.javier@inifap.gob.mx

Abstract: Extensive livestock production systems are common in Mexico. Overall, livestock production uses about 108.9 million hectares nationally, which represents over half (55.5 %) of the country’s surface area. Approximately one quarter of Mexico is in the tropics and livestock grazing is one of the most important economic activities in this region. In at least 24 states the cattle population is estimated to exceed grassland carrying capacity based on forage production. This situation results in gradual grasslands degradation and a consequent decrease in forage productivity. It also reduces the products and services obtained from them, primarily forage, meat and milk, but also water and recreational space. Grassland rehabilitation research has been active in Mexico for at least ten years, and has mainly focused on weed control by mechanical and chemical means, which provide satisfactory short-term results. However, grassland degradation continues in Mexico due to inadequate pasture management, particularly in the form of animal loads in excess of pasture forage production capacity. This review provides an overview of grassland degradation, mainly in

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Mexico’s tropical regions, summarizes grasslands recovery research by the INIFAP, and analyzes medium- and long-term prospects. Key words: Livestock, Grasses, Soil fertility, Overgrazing, Weed control.

Received: 23/11/2020 Accepted: 25/02/2021

Introduction Constant worldwide population growth drives a need for increasing food production. By 2050 there will be an estimated 9.6 billion inhabitants on the planet (more than 2 billion more than in 2021), and they will have fewer available resources and will need to generate less pollution(1). Over the last 40 yr, world meat production has increased 90 percent, and in the tropics that increase has been as much as 200 %(1). In Mexico, around 108.9 million hectares are used for livestock production, an area representing 55.5 % of the country’s total surface area(2). The national cattle population consists of 32.6 million head, highlighting the importance of this industry(3). Livestock production occurs in all of Mexico’s ecosystems, but is particularly prominent in dry and humid tropical zones. Approximately 40 % of national meat production and 18 % of dairy production occur in these zones(4). Around 56 million hectares are used for livestock production in these regions, of which more than 23 million hectares are for grazing(5). The main source of feed for cattle in these regions is forage produced in pastures, and consumed directly by the animals. This is the most cost effective means of transforming grassland biomass into high nutritional quality food, such as meat and milk. In at least 23 states cattle population exceeds environmental carrying capacity in terms of forage production in pastures; in other words, overgrazing is common(6). Pastures gradually degrade under these circumstances, progressively producing less forage, water and recreational space. As a consequence, meat and milk production decrease. This review summarizes the factors that affect pasture degradation and discusses potential solutions to this problem, in addition to presenting the results of INIFAP research on grasslands rehabilitation in the Mexican tropics.

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Grasslands Grasslands, a vegetation type dominated by grasses, are present on five continents, cover a quarter of the earth’s surface, and contribute to the livelihoods of more than 800 million people(7). The main source of feed in animal production systems involving ruminants is forages produced on native and cultivated grasslands as well as agricultural land(8). Future grassland health will clearly be an essential element when considering how to feed the nine billion people who will inhabit the planet in 2050(9). Grasslands provide numerous environmental services, ranging from ground cover to prevent wind and water erosion, to recreational space and habitat for ornamental and medicinal plant species(10). Grasses are also effective at retaining water(11), especially when in good condition, because they improve soil filtration(12). Grasslands can potentially sequester carbon, particularly when moderately grazed(12), and this capacity is augmented if they are associated with legumes(13).

Grasslands in Mexico The cattle industry in Mexico includes approximately 1.4 million ranches, feedlots, multipurpose companies and other parties(3). Carcass meat production in 2019 was 2.027 million tons, and per capita domestic beef consumption was 14.9 kilograms. In the same year, milk production was 12.275 million liters, which is 16th worldwide, and per capita domestic consumption was 95.1 L. As a percentage of total national production, forage accounts for 42 % and livestock for 8 %. Of these totals, the Northwest region represents 7% of forage production and 6 % of livestock, the Northeast 24 % and 22 %, the Central Western 37 % and 43 %, the Center 11 % and 12 %, and the Southeast 20 and 16 %(2). In Mexico’s tropical regions, more than 50 % of the surface area is used for livestock activities in four states: Tabasco (65.7 %); Tamaulipas (58.2 %); Sinaloa (50.6 %); and Veracruz (50.2 %)(2).

Forage production in Mexico Annual forage production in Mexico is 183 million tons (dry matter). In general terms, 42 % of this total is produced in pastures, 29 % is from native grasslands, 24 % comes from 245

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agricultural waste and 4.9 % from forage crops(14). In other words, pastures and native grasslands account for 71 % (136 million tons) of total forage production. However, if adequate management strategies were employed, only a maximum of 60 % (82 million tons) of pasture and native grasslands production would be used, whereas all forage crop and agricultural waste production (55 million tons) would be used. Under this scenario, therefore, Mexico would produce 137 million tons of usable forage. Currently, approximately 34 million animal units use about 170 million tons of forage annually, meaning that there is an annual forage production deficiency of 33 million tons. These figures suggest that in Mexico an excess of animal units is resulting in overexploitation of grazing lands, with serious consequent damage and deterioration of natural resources(15).

Tropical regions Mexico’s humid tropical regions cover 23.9 million hectares(16). These regions are defined by annual rainfall greater than 1,300 millimeters and an altitude of less than 1,000 meters asl. Dual-purpose and beef cattle ranching is common in these regions and utilizes pastures with a high proportion of introduced or improved grasses(16,17). Dry tropical regions cover 31.7 million hectares. These are defined as having annual rainfall of 600 to 1,300 millimeters, and can range in altitude from sea level to 2,000 m asl. Cattle production in these areas is largely of calves for growing(16,17). In both the humid and dry tropics the area covered by introduced grasses, particularly Brachiaria sp., has been increasing since seeds were first marketed in 1999. Based on the quantity of seed sold in 2004, an estimated 2,616,130 ha were planted with introduced grasses in the tropics(18,19). From 2004 to 2020, new pasture coverage has increased substantially as different species and cultivars are planted. A particularly popular species is Meghatyrsus maximus (Jacq.) BK Simon & SWL Jacobs, of which the Mombasa and Tanzania cultivars are used. Other grasses planted for their vegetal material production include the harvest forages Cenchrus purpureus (Schumach.) Morrone, which has several cultivars apt for intensive planting, pangola grasses (Digitaria eriantha Steud.) and African star grass [Cynodon plectostachyus (K. Schum.) Pilg.]. All these species and cultivars provide higher forage productivity and quality than native grasses, and have contributed to improving livestock productivity in tropical areas.

Issue overview From 2010 to 2050, global consumption of meat is projected to increase by 173 % and that of dairy by 158 %; both increases are expected to be much higher in developing countries(20). 246

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Raising production to meet these increases will require greater availability of animal feed. This could, in turn, drive conversion of high-value biomes into grazing land, exerting ever greater pressure to overgraze in livestock production systems based on native grasslands or cultivated pastures(21). Recent decades have seen a steady degradation of grasslands due to overgrazing, which is the leading cause of damage in all major biomes. Worldwide estimates are that about 20 % of pastures and 73 % of native grasslands have been degraded(22). In Central America, an estimated 50 to 80 % of grassland areas are in an advanced state of degradation, and can only support an animal load 40 % less than more recently established, properly managed pastures(23). Grasslands are degrading at a rate of 12 %, and the renewal rate is 5 %, representing a net loss(23,24). Since overuse of pastures and native grasslands is a major limiting factor in cow-calf and dual-purpose systems, the Forage and Pastures Program of the INIFAP has made rehabilitation of degraded pastures a research priority(25).

Causes of pasture degradation Pasture degradation due to improper management begins with loss of plant vigor, manifested in narrower leaves, low greenness index values and declines in regrowth capacity (Figure 1). Forage species experience a consequent loss in aerial cover, allowing weed growth or leaving bare soil, which favors compaction by animal trampling and erosion(26,27). Figure 1: Causes of degradation in tropical grasslands

Six criteria are used to evaluate a degraded pasture: 1) Decreased forage production and quality; 2) Decreased vegetation cover and plant density; 3) Fewer new plants from natural propagation; 4) Soil erosion from rainfall; 5) Presence of broad- and narrow-leaf weeds not 247

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consumed by animals; and 6) Colonization by native grasses(27). The degree of grassland degradation can be classified into four major categories based on the percentage of area occupied by invasive plant species: 1) Productive grasslands, 0 to 10 % invasive species cover; 2) Mild degradation, 11 to 35 %; 3) Moderate degradation, 36 to 60 %; and 4) Advanced degradation, 61 to 100 %(27). Another four-level classification system is based on the qualitative criterion of plant color, and the quantitative criteria of dead matter, bare soil and weed coverage (%), as well as pasture age (Table 1). Table 1: Four-level pasture degradation scale based on qualitative and quantitative parameters Degradation Level 1 Parameters 2 3 4 None Low Moderate Severe apparent Plant color

Dark green

Light green

Green/yellow

Yellow

Dead matter, %

<10

11-20

21-30

> 30

Bare soil, %

<10

11-20

21-30

> 30

Weeds, %

<10

11-20 narrow-leaf weeds

21-30 wide-leaf weeds

> 30 native grasses

Age, years

1-3

4-6

7-9

> 10

Pastures at level 1 (None apparent) include grasslands of one to three years of age (since establishment), intense green leaves and values less than 10 % of dead matter, bare soil and weeds. At the other extreme, pastures at level 4 (Severe) are older than 10 yr, have a yellow leaf color, greater than 30 % dead matter, bare soil and weeds, as well as high native grass colonization(23). Pasture productivity can decline in response to numerous factors that can cause degradation, including use of species unsuited to environmental conditions; poor grazing management (characterized by overgrazing, especially in low rainfall periods); pest and disease incidence; planting in areas with fragile soils; soil nutrient depletion due to nutrient extraction (higher in improved grass species) and minimal or no fertilizer use; high herbaceous plant and shrubby weeds infestation; and indiscriminate burning(28,29,30). Poor grassland management, especially in the form of low fertilizer use and overgrazing, will eventually result in decreased grass growth rate, mainly due to nitrogen and phosphorus deficiencies in soils(31). Grassland degradation reduces animal production rate and increases costs, making it a financial and ecological problem(27).

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Pasture use-life Grassland use-life varies between countries in response to various factors. After pasture establishment in the Amazon region of Brazil, production gradually decreases under traditional management conditions, and can be characterized in four phases: 1) high productivity (3-5 yr after establishment), loads >1.5 animal units (AU); 2) medium productivity (4-7 yr), loads >1 AU; 3) low productivity (7-10 yr), loads = 0.5 AU; and 4) degraded (7-15 yr), animal load <0.3 AU(32). A study done in Honduras estimated that grasslands have a use-life of ten years, although there were differences caused by grass species; the shortest use-life was nine years with Brachiaria humidicola (Rendle) Schweick and Digitaria swazilandensis Stent, and the longest was twelve years with C. plestostachyus(23). No scientific research has been published on pasture use-life in Mexico, but personal observations and personal communication with producers suggest that a D. eriantha pasture has a use-life of eight to ten years, while one planted with B. humidicola, B. decumbens and/or B. brizantha has one in excess of ten years. The discrepancy between uselifes may be due to differences in soil fertility requirements and pasture management. For instance, D. eriantha requires highly fertile soils, B. brizantha medium fertility soils and B. humidicola can grow in low fertility soils. For optimum productive performance, each species must be planted in soils with the appropriate fertility level. Overall, the use-life of improperly managed tropical grasslands would probably average about eight years.

Rehabilitation strategies for tropical grasslands Various factors must be considered if a grassland is to recover, including soil physicochemical factors, plant species, and how degraded are the grass species to be restored. What recovery treatment is most apt for a degraded pasture and its cost will depend on the degree of pasture degradation. When degradation is not too advanced (e.g. <10 % broadleaf weed cover), techniques can be applied to recover pasture production capacity, but when degradation is severe it is usually most viable to establish a pasture anew. Some of the practices used to increase the population and production of desirable species are agricultural, such as improving soil physical properties, fertilization, weed control and replanting(27). Fertilization is vital to pasture rehabilitation. After many years of grazing or cutting, soils can become depleted, biomass production begins to decline and desired grasses are replaced by other species. One study of a pasture with 20 % B. decumbens cover and native grasses on the remainder evaluated different treatments, including soil preparation or removal systems, and fertilization with only 22 kg ha-1 phosphorus or complete formulas (22 kg ha-1 249

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P, 45 kg ha-1 N, 25 kg ha-1 K-CaO, 28 kg ha-1 MgO and 15 kg ha-1 S)(33). Fertilization increased average B. decumbens coverage up to 72 % while the control without fertilizers reduced B. decumbens to 18 %. Forage production in the control was 844 kg ha-1, with phosphorus only fertilization it was 3,386 kg ha-1, and with complete fertilization it was 4,266 kg ha-1. Soil preparation and removal systems had no effect on pasture recovery, possibly because these techniques function better with stoloniferous plants since their removal encourages replanting of stolons. These results coincide with two other studies. One, of a degraded B. decumbens pasture with over ten years of use, evaluated application of macro- and micronutrients and field tilling. Recovery was best when macro- and micronutrients were applied, while tilling negatively affected root development and dry matter production and had no effect on pasture recovery(34). The negative response was due to destruction of plants during the tillage process, preventing any response to the fertilizers. Another study found that tilling alone has no significant effect on pasture recovery in nutrient-deficient soils, so this practice requires posttillage fertilizer application. In the absence of fertilization, mechanical treatments made no improvement to pasture development or productivity(35). Stoloniferous grasses such as D. eriantha and C. plectostachyus may be the exception since they benefit from tilling as a recovery technique as this results in overseeding. The planting of legumes is a viable technique for rehabilitation of degraded B. brizantha meadows. One study in Brazil found that manual sowing of legumes and fertilization with 50 kg P increased dry matter production(36). Another study of a degraded Hyparrhenia rufa (Nees) Stapf pasture more than 15 yr old and with 60 to 70 % weed cover evaluated three recovery methods (weed control; weed control + P fertilization + legumes; and weed control + planting B. humidicola + legumes), low and high animal loads, and continuous and rotational grazing(37). The most efficient method for recovery or replacement of the degraded H. rufa pastures was weed control + planting B. humidicola + legumes, since this produced a larger quantity of forage with better chemical composition, allowed for a higher animal load and resulted in greater animal weight gain. Because of its aggressiveness and broad adaptation, as well as its association with legumes, B. humidicola produced better quality forage which resulted in higher animal production parameter values than in the other treatments.

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Pasture rehabilitation using chemical weed control

High weed concentrations are characteristic of degraded grasslands. Weeds occupy the spaces left by grasses to the point where competition for water, light and nutrients becomes critical(38). Suites of weed species make more efficient use of these resources than do grasses, because they encompass different species with different needs and abilities in conjunction with non-uniform spatial distribution and development stages(39). These qualities allow them to more efficiently explore the environment in search of the elements essential to growth, thus reducing availability for grasses(40,41). Although both monocotyledonous and dicotyledonous weeds can occur in pastures, the latter are generally more important because they have greater diversity and frequency of appearance(42,43). On occasion grass weeds can become dominant(44). Competition from weeds causes a reduction in pasture grass development and vigor, which is reflected in lower forage yield. For example, three studies evaluated three locations with Aw climates in the center and north of the state of Veracruz, Mexico, with the grasses Digitaria decumbens Stent., Andropogon gayanus Kunth. and C. plectostachyus(45). In different evaluation stages uninterrupted competition from weeds caused reductions in pasture dry biomass production ranging from 54 to 80% in D. decumbens(45), from 61 to 81 % in A. gayanus(46) and from 57 to 84 % in C. plectostachyus(47). In addition, in A. gayanus this competition resulted in a significant reduction in crude protein content after 163 days. In C. plectostachyus, reductions in crude protein content were observed after 155 and 224 d. In a study of Urochloa brizantha grass (Hochst. Ex A. Rich) RD Webster in Mato Grosso, Brazil(48), in which weed competition was allowed for 15 d from emergence, reductions were observed of 30.8 % in grass height and 9.5 % in number of tillers. When the competition period was extended to 60 d, grass height declined by 51.1 % and number of tillers by 35.7 %. Competition also resulted in declines in pasture dry biomass of 50.2 % at 15 d and 69 % at 60 d. In another study done in the same pasture(49), weed competition was found to reduce leaf/stem ratio values in a manner directly proportional to competition period. Furthermore, grass crude protein content declined by 7 to 33 % at periods of 60 d or longer. Of all the effects of weed competition loss of pasture productivity has the most serious impact because it reduces forage availability for livestock. However, some weed species can also cause negative physical effects from stinging thorns or trichomes, or poisoning from intake of bioactive compounds(50,51). The severe agricultural, financial and livestock health problems caused by weeds highlight the need for their timely control before they can affect pasture productivity and quality. 251

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Several factors influence when control should be implemented, including weed species and density, grass variety and agroclimatic conditions, especially temperature and relative humidity. For example, in warm weather conditions in U. brizantha pastures weed control must be done at no later than 9(52), 15(48) or 30(49) days grass-weed coexistence, while in U. ruziziensis (R. Germ. & CM Evrard) Crins pastures it should be done before 22 d coexistence(53). The most widely used methods for weed control in pastures and paddocks are manual or mechanical clearing and application of selective herbicides. Clearing does not completely eliminate weeds, can affect both weeds and grasses and is only a temporary measure. Herbicides are more efficient than clearing because they can completely eliminate weeds without causing significant damage to grasses. The herbicides 2,4-D, picloram, fluroxypyr, aminopyralid and triclopyr are widely used in pastures and grasslands. They are applied alone post-emergence or mixed to function as growth regulators. Metsulfuron-methyl, an amino acid synthesis inhibitor, is also widely used(54). To avoid or minimize their environmental effects, herbicides need to be applied correctly and using the concentrations, periodicity and seasons recommended by the manufacturer. Workers who apply chemical herbicides should wear appropriate protective clothing to reduce the risk of contamination or poisoning. Several cases of herbicide use in the rehabilitation of degraded tropical grasslands have been reported in Mexico. One study evaluated application of herbicides (2,4-D, picloram+2,4-D, metsulfuron-methyl, or aminopyralid+metsulfuron-methyl) and clearing for weed control in a pasture in the municipality of Medellín, in the state of Veracruz, with an initial coverage of 27 % U. brizantha, 15 % other grasses, 56 % weeds and 2 % bare soil. Thirty days after application, the herbicide treatments reduced weed cover an average of 3.8 % and increased U. brizantha cover to 88 %; the latter was as high as 98.3 % after 75 d. In contrast, thirty days after clearing weed coverage was 67%, but dropped to 33% after 75 d, while U. brizantha coverage was 12 % at 30 and 54 % at 75 d. These differences were reflected in average dry biomass U. brizantha production at 75 d, which ranged from 5,475 to 6,381 kg ha-1 in the chemical control treatments but was only 1,448 kg ha-1 in the clearing treatment(55). Another study also done in Medellín evaluated application of herbicides (metsulfuronmethyl, 2,4-D and a formulated mixture of picloram+2,4-D) to a pasture with an initial coverage of 23 % pangola grass (D. eriantha) and 33 % Baltimora recta L.(56). Compared to a control treatment without herbicide application, after 30 d the herbicide treatments had controlled B. recta by more than 90 % and average D. eriantha dry forage yield was 51.9 % higher. In a final example, a study was done in a degraded C. plectostachyus pasture on the effects of three herbicide mixtures (picloram+2,4-D; aminopyralid+2,4-D; and aminopyralid+fluroxypyr-meptil+2,4-D) in controlling three brush species: Sida acuta Burm. F. (66.3 % initial coverage), Sida rhombifolia L. (62.5 %) and Jatropha gossypifolia L. (42.5 %)(57). The best control at 45 days was exhibited with the aminopyralid+fluroxypyrmeptyl+2,4-D mixture, which allowed 22.8 % more average grass dry matter production than the picloram+2,4-D mixture, 15.2 % more than the aminopyralid+2,4-D mixture and 199% 252

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more than in the control with no herbicide application. Chemical weed control is clearly the most effective strategy for tropical grassland rehabilitation since it results in much better control than manual or mechanical methods, with consequently higher forage production and quality.

Challenges in and outlook on pasture rehabilitation in Mexico Short-term (5 years). The expectation is that the data generated on grassland rehabilitation will be well known throughout the tropics and be applied to mitigate the impacts of degradation. This will need to be accompanied by adjustments in animal load and grazing management, the two main causes of pasture degradation. Technicians and producers will require effective training to understand and apply these latter two adjustments. Further research is needed on the economic losses and social costs of pasture degradation on Mexico’s livestock industry. Medium-term (10 years). Massive expansion and opening of new grasslands in the Mexican tropics began with the National Clearing Program implemented by the Ministry of Agriculture and Livestock in the 1970s. In other words, many areas in this region have been under livestock grazing for almost 50 yr. For this reason, research is needed on other factors that affect grassland degradation, such as loss of fertility in soils and their depletion, which result from long-term cutting or grazing of grasslands in the absence of fertilization. Compaction from animal traffic also degrades pastures by reducing the depth to which roots can penetrate the soil, lowering water infiltration rates and generating laminar erosion. To stabilize grasslands and promote a sustainable productive environment, further research is needed to develop rehabilitation methods that involve mechanical means of decompressing soil accompanied by correction of soil nutrient deficiencies, be it via chemical, organic and/or biological means (e.g. legumes). Long-term (20 years). The overall goal for livestock production in Mexico is selfsufficiency, that is, to meet domestic market demand for meat and milk products. Attaining this goal will involve creating safe products, generating greater profits for the livestock industry and preventing environmental degradation. New technologies will become tools to reverse the pasture degradation caused by ongoing poor management. Multidisciplinary research teams and sufficient long-term financial commitments will be needed to reach this goal, as will infrastructure to carry out innovative technological research applicable to conditions in the Mexican tropics.

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Conclusions The degradation of tropical grasslands in Mexico is the consequence of continuous overexploitation. For decades, animal load has far exceeded pasture capacity and no effort has been made to return nutrients to the soil through fertilization. Chemical control of weeds has proven to be the most efficient method for rehabilitating degraded pastures; indeed, pastures recover high forage production capacity after only one to two cycles of selective herbicide application. Once rehabilitated, however, pastures need to be maintained by implementing grazing strategies that acknowledge seasonal forage production patterns, consider animal load, and return nutrients via chemical or organic fertilizers. Numerous facets of grassland productivity remain to be studied, such as maintenance fertilization of grasslands based on grass species or cultivar nutritional requirements; optimal practices in silvopastoral systems, which are promising sustainable animal production systems in the tropics; optimizing production at cattle ranches; and the best training methods for technicians and producers. Comprehensive research approaches will be needed in grasslands management and rehabilitation to work towards a livestock industry that is both financially profitable and ecologically sustainable.

Acknowledgements

The authors thank Dr. Fernando Rivas Pantoja† and Javier Enrique Castillo Huchim, formerly of the INIFAP, for their contribution of research on degraded pasture rehabilitation on the Yucatan Peninsula.

Conflicts of interest

The authors declare no conflict of interest with the research reported herein.

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31. Boddey RM, Macedo R, Tarré RM, Ferreira E, de Oliveira OC, Rezende C de P, et al. Nitrogen cycling in Brachiaria pastures: the key to understanding the process of pasture decline. Agric Ecosyst Environ 2004;103(2):389-403. 32. Serrão SEA. Pastagem em área de floresta no trópico úmido Brasileiro: conhecimentos atuais. In: 1° simposio do trópico úmido. Belem, Pa, Brasil: EMBRAPA-CPATU. 1986:147-174. 33. Arruda GN de, Cantarutti RB, Moreira EM. Tratamentos fisico-mecânicos e fertilização na recuperação de pastagens de Brachiaria decumbens em solos de tabuleiro. Pasturas Trop 1987;9(3):36-39. 34. Soares FCV, Monteriro AF, Corsi M. Recuperação de pastagens degradadas de Brachiaria decumbens. 1. Efeito de diferentes tratamentos de fertilização e manejo. Pasturas Trop 1992;14(2):2-6. 35. Carvalho SID, Vilela L, Spain JM, Karia CT. Recuperação de pastagens degradadas de Brachiaria decumbens cv. Basilisk na região dos Cerrados. Pasturas Trop 1990;12(2):24-28. 36. Costa NL, Towsend CR, Maagalhaes JA. Métodos de introdução de leguminosas em pastagens degradadas de Brachiaria brizantha cv. Marandu. Pasturas Trop 2003;25(3):39-41. 37. Gonçalves CA, Cruz OJR da, Dutra S. Recuperação e manejo de pastagens de capim jaraguá (Hyparrhenia rufa) em Rondônia Brasil. Pasturas Trop 2002;24(2):47-56. 38. Esqueda EVA, Tosquy VOH, Rosales RE. Efectividad de la mezcla picloram y fluroxipir en el control de malezas perennes de pastizales tropicales. Agron Mesoam 2005;16(2):187-192. 39. Kruchelski S, Szymczak LS, Deiss L, Moraes A. Panicum maximum cv. Aries establishment under weed interference with levels of light interception and nitrogen fertilization. Planta Daninha 2019;37:e019188589. 40. Souza Filho APS, Veloso CAC, Gama JRN. Capacidade de absorção de nutrientes do capim-marandu (Brachiaria brizantha) e da planta daninha malva (Urena lobata) em função do pH. Planta Daninha 2000;18(3):443-450. 41. Ruas RAA, Lima JCL, Appelt MF, Dezordi LR. Controle de Brachiaria decumbens Stapf com adição de ureia à calda do glifosato. Pesq Agropec Trop 2012;42(4):455-461.

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42. Guevara S, Meave J, Moreno-Casasola P, Laborde J, Castillo S. Vegetación y flora de potreros en la sierra de Los Tuxtlas, México. Acta Bot Mex 1994;28:1-27. 43. Lara JFR, Macedo JF, Brandão M. Plantas daninhas em pastagens de várzeas no estado de Minas. Planta Daninha 2003;21(1):11-20. 44. Galvão AKL, Silva JF, Albertino SMF, Monteiro GFP, Cavalcante DP. Levantamento fitossociológico em pastagens de várzea no estado do Amazonas. Planta Daninha 2011;29(1):69-75. 45. Esqueda EVA, Tosquy VOH. Efectividad de métodos de control de malezas en la producción de forraje del pasto Pangola (Digitaria decumbens Stent.). Agron Mesoam 2007;18(1):1-10. 46. Esqueda EVA, Montero LM, Juárez LFI. El control de arvenses en la productividad y calidad del pasto Llanero. Agron Mesoam 2010;21(1):145-157. 47. Esqueda EVA, Montero LM, Juárez LFI. Efecto de métodos de control de malezas en la productividad y calidad del pasto Estrella de África (Cynodon plectostachyus (K. Schum.) Pilg.). Trop Subtrop Agroecosys 2009;10(3):393-404. 48. Marchi SR, Bellé JR, Foz CH, Ferri J, Martins D. Weeds alter the establishment of Brachiaria brizantha cv. Marandu. Trop Grassl – Forrajes Trop 2017;5(2):85-93. 49. Bellé JR, Marchi SR, Martins D, Souza AC, Pinheiro GHR. Nutritional value of Marandú palisade grass according to increasing coexistence periods with weeds. Planta Daninha 2018;36:e018170348. 50. Tuffi SLD, Santos IC, Oliveira CH, Santos MV, Ferreira FA, Queiroz DS. Levantamento fitossociológico em pastagens degradadas sob condições de várzea. Planta Daninha 2004;22(3):343-349. 51. Carvalho RM, Pimentel, RM, Fonseca DM, Santos MER. Caracterização de perfilhos em relação à planta daninha no pasto de capim-braquiária. Bol Ind Animal 2016;73(2):103-110. 52. Jakelaitis A, Gil JO, Simões LP, Souza KV, Ludtke J. Efeitos da interferência de plantas daninhas na implantação de pastagem de Brachiaria brizantha. Rev Caatinga 2010;23(1):8-14 53. Lourenço AA, Mota RV, Sanches JL, Marques RF, Marchi SR. Weed interference in the establishment of Urochloa ruziziensis. Planta Daninha 2019;37:e019184957. 259

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54. Enríquez QJF, Esqueda EVA, Rivas PFA, Castillo HJE, Martínez MD, López GI, et al. Rehabilitación y mejoramiento de tierras de pastoreo en el trópico de México. Folleto Técnico Núm. 79. Medellín de Bravo, Ver.: INIFAP. CIRGOC. Campo Experimental La Posta; 2015. 55. Martínez-Méndez D, Enríquez-Quiroz JF, Ortega-Jiménez E, Esqueda-Esquivel VA, Hernández-Garay A, Escalante-Estrada JAS. Rehabilitación de una pradera de pasto Insurgente con diferentes métodos de manejo. REMEXCA 2016;7(8):1787-1800. 56. Enríquez QJF, Martínez MD, Esqueda EVA, Hernández GA. Control químico de maleza para rehabilitación de una pradera de pasto Pangola. In: Toca RJA, et al. compiladores: VI congreso internacional de manejo de pastizales. Durango, Dgo.2015:109-113. 57. Esqueda EVA, Enríquez QJF. Efecto de herbicidas en el control de malezas y la producción de forraje en praderas tropicales. In: Delgado CJC, et al. editores. XXXIX Congreso mexicano de la ciencia de la maleza. Aguascalientes, Ags. 2018:137-141.

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https://doi.org/10.22319/rmcp.v12s3.5875 Review

The grasslands and scrublands of arid and semi-arid zones of Mexico: Current status, challenges and perspectives

Pedro Jurado-Guerra a* Mauricio Velázquez-Martínez b Ricardo Alonso Sánchez-Gutiérrez c Alan Álvarez-Holguín a Pablo Alfredo Domínguez-Martínez d Ramón Gutiérrez-Luna c Rubén Darío Garza-Cedillo e Miguel Luna-Luna f Manuel Gustavo Chávez-Ruiz a

Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias (INIFAP). Centro de Investigación Regional Norte-Centro (CIRNOC), Campo Experimental (CE) La Campana, Km. 33.5 Carretera Chihuahua-Ojinaga, Aldama, Chihuahua, 32910, México. b

INIFAP, CIRNE, CE San Luis, San Luis Potosí, México.

INIFAP, CIRNOC, CE Zacatecas, Zacatecas, México.

INIFAP, CIRNOC, CE Valle del Guadiana, Durango, México.

INIFAP, CIRNE, CE Río Bravo, Tamaulipas, México.

INIFAP, Centro Nacional de Investigación en Agricultura Familiar, Ojuelos, Jalisco, México.

* Corresponding author: jurado.pedro@inifap.gob.mx

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Abstract: The objective was to review the current state of the semi-arid grasslands and arid scrublands of northern and central Mexico, as well as to analyze the challenges and perspectives of the use of these ecosystems. Since the 1950s, INIFAP, in collaboration with other institutions, has generated and transferred knowledge on rangeland management, which has reflected in the use of management practices on cattle ranches in the country. The grasslands and scrublands have suffered disturbances —particularly the opening of land for crops— and are deteriorated mainly from overgrazing. The use of grasslands and scrublands through grazing should include adequate stocking, grazing systems, and strategic grazing distribution practices. Despite the deterioration, there is a great diversity of genetic resources, mainly pastures, which can be used for conservation and seed production for the rehabilitation of grasslands. Although costly and risky, re-seeding is an option for restoring decayed grasslands or shrublands. These ecosystems can provide environmental services, mainly carbon sequestration, to mitigate climate change. The challenges are to generate, transfer, and apply knowledge and technological innovations in order to achieve sustainable management of grasslands and scrublands, despite some threats such as low investment in science and technology, climate change, and human greed. The joint and committed participation of all the actors and institutions involved in the use of these ecosystems is essential to attain this goal. Key words: Range condition, Grazing management, Genetic resources, Seed production, Rangeland reseeding, Soil carbon.

Received: 23/11/2020 Accepted: 26/05/2021

Introduction The grasslands and shrubs of the arid and semi-arid zones of central and northern Mexico are natural resources that comprise approximately 25 % of the national territory(1) and have the ability to provide several products and environmental services to society. One of these products is ruminant meat from extensive livestock farming, while carbon sequestration is one of their most important environmental services. Grasslands and scrublands have undergone major transformations, mainly due to changes in land use, the climate, and overgrazing, which cause a serious deterioration of these resources.

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Grassland rehabilitation is a necessary activity for correcting this deterioration. Fire is an ecological, low-cost alternative for grassland improvement(2,3), while adjusting the stocking rate along with practices for improving grazing distribution are recommended to conserve or improve the condition of arid and semi-arid grasslands(4,5). Re-seeding pastures, although a risky and costly practice due to erratic rainfall and to the high cost of land and seed preparation, is still an option for improving pastures. Studies on the management and rehabilitation of grasslands and scrublands in Mexico began in the 1950s at the La Campana Experimental Ranch of the now extinct National Institute for Research in Livestock (Instituto Nacional de Investigaciones Pecuarias). Subsequently, since the creation, in 1985, of INIFAP —which merged the agricultural, livestock and forestry research institutes—, these studies were intensified in its Livestock Experimental Stations in the north of the country, such as "La Campana" in Chihuahua, "Carbó" in Sonora, "Aldama" in Tamaulipas, and "Vaquerías" in Jalisco. The results of these works are reflected in countless publications and have been transferred to the producers through courses, workshops, and demonstrations. During the 1970s and 1980s, they were supported by livestock organizations, state governments and CONACYT, while in the 1990s and the early XXIst century, research and grassland technology transfer were promoted by the State Produce Foundations. In the last 20 yr, Federal Programs such as SAGARPA's PROGAN have supported INIFAP in training a large number of producers and technicians in rangeland management. In addition, INIFAP has participated in the promotion of grassland management through the co-organization of Symposiums, Conferences, and Forums with different universities in central and northern Mexico and the Mexican Society for Range Management. The socioeconomic and environmental impact of pasture research carried out at INIFAP and other institutions is palpable in the management of cattle ranches. For example, a large part of the ranches in the north and center of the country, including some ejidos, carry out grazing management practices such as deferred grazing or pasture fallowing, adjustment of animal load, and supplementation of livestock during drought. This has allowed the conservation and improvement of the condition of certain grassland and scrubland areas. Today, there is a large amount of knowledge and technological innovations available to achieve sustainable management of grasslands and scrublands, generated by INIFAP, universities, and research centers. This document presents the current status of arid and semi-arid grasslands and shrubs in relation to certain strategic themes, as well as the main challenges and perspectives which must be faced with endeavor for conserve and sustainable exploitation of these ecosystems.

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Grassland and scrubland condition The semi-arid grasslands of Mexico are distributed in a strip from north to southeast of the country, from Sonora to Guanajuato, while the arid scrublands extend widely from Baja California to Oaxaca(6) (Figure 1). These ecosystems have suffered reductions by 14 % in grasslands and 26 % in shrublands as a result of their conversion to agriculture in the last 50 years, so that they currently comprise around 9.77 and 40.95 million ha, respectively(1). Unfortunately, 95 % of the grasslands and 70 % of the shrublands are overgrazed, according to official sources (Figure 1). Figure 1: Intensity of grazing in grasslands and natural shrublands of Mexico. Source: Dirección General de Ordenamiento y Conservación de Ecosistemas, INE, SEMARNAT, México. 2003

The first studies at the national level during the decades of 1950 to 1970 indicate that the semi-arid grasslands of central and northern Mexico consisted mainly of native grasses of low and medium size, such blue grama (Bouteloua gracilis), sideoats grama (B. curtipendula), hairy grama (B. hirsuta), Rothrock’s grama (B. rothrockii), purple grama (B. radicosa), slender grama (B. repens), black grama (B. eriopoda), sprucetop grama (B. chondrosioides), scorpion grama (B. scorpioides), six weeks three awn (Aristida adscencionis), poverty three-awn (A. divaricata), spidergrass (A. ternipes), buffalo grass (B. dactyloides), while in arid or halophytic grasslands the most common grasses included the tobosa grass (Pleuraphis mutica), alkali sacaton (Sporobolus airoides), and saltgrass

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(Distichlis spicata)(6). However, recent studies report some changes in its floristic composition and the appearance of exotic grasses. In northeastern Sonora, semi-arid grasslands are mainly composed of native grasses of the Aristida and Bouteloua genera(7), while in the central plains of the state, approximately 800 thousand hectares of exotic buffel grass (Pennisetum ciliare) are used for cattle raising and that were originally native scrublands(7). Unfortunately, the overgrazing caused by beef cattle industry has degraded and reduced condition of short grass prairies, affecting the economic income of families(8). In the case of the Chihuahua grasslands, the most common grasses are of the Bouteloua and Aristida genera, and among the arid grasslands, the dominant species are the tobosa, alkali sacaton, and alkali lovegrass (Eragrostis obtusiflora)(9). In addition, an invasion of shrubs such as the catclaw mimosa (Mimosa biuncifera) and whitebrush (Aloysia gratissima), as well as exotic grasses such as Lehmann lovegrass (Eragrostis lemahnniana) and natal grass (Melinis repens)(9,10). Local studies confirm a high degree of deterioration, as well as losses of 378 thousand to 2.72 million hectares(11,12), attributed to the opening of land to cultivation, overgrazing(10,12), climate change, human settlements, and inadequate public policies in the grasslands of Chihuahua(12). In Durango, semi-arid grasslands are mainly composed of native grasses of the Aristida, Bouteloua, Elionurus, Eragrostis, and Heteropogon genera, while in arid grasslands, saltgrass, scratch grass (Muhlenbergia asperifolia), alkali grass, chino grama (Bouteloua ramosa), and toboso are the most common(13). However, overexploitation of grasslands, adverse climatic conditions, and the introduction and invasion of exotic species such as the rose natal grass have caused a significant reduction of the state's grasslands(13), as well as the change of an association of Bouteloua - Bothriochloa grasses to another of natal grass/tanglehead (Heteropogon contortus)(14). For the semi-arid grasslands of Zacatecas, the most common associations are Aristida / Bothriochloa / Bouteloua and other exotic grasses such as buffel, weeping lovegrass (Eragrostis curvula), lehmann lovegrass and natal grass, while in arid-halophyte grasslands the most common is the alkali sacaton(15). In addition, most Zacatecas grasslands appeared to be in a moderate health state, although the properties of the soils are acceptable, the vegetation exhibits a high degree of deterioration(16). In the case of the semi-arid grasslands of Aguascalientes, the dominant grasses are sprucetop grama, blue grama, and wolfgrass (Muhlenbergia phleoides), which exhibit a good condition, with 80% of the original vegetation, especially of the Bouteloua genus(17). As for the state of Jalisco, Bouteloua / Microchloa / Aristida are the most prevalent communities in the semiarid grasslands(18), which were in moderate to extreme health conditions in 2002(19), the main problem being the deterioration of the vegetation. In San Luis Potosí, the most common grasses are blue grama, wolfgrass, buffalo grass, and the invaders buffel and natal grass(20). 265

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Unfortunately, a recent study indicates that overgrazing has impacted the composition of grasslands in central Mexico throughout history, but that it is still possible to improve the grasslands(21). In the 1970s, the scrublands of the Sonoran Desert were represented by shrubs such as saguaro (Carnegia gigantea), burro-weed (Ambrosia dumosa), elephant tree (Bursera microphylla), ironwood (Olneya tesota), yellow paloverde (Parkinsonia microphylla), and the creosote bush (Larrea tridentata)(6). Recent studies on the vegetation of Sonora indicate that the predominant shrubs in the central plains region are the ironwood and brittlebush (Encelia farinosa); in the central coast, the elephant tree and Jatropha cinerea; in the highlands, the yellow paloverde, Cylindropuntia , Opuntia, and saguaro, and in the Lower Colorado River Valley, the Larrea / Ambrosia association(7). In the case of the xerophilous shrubs of the Chihuahuan Desert, the dominant species are the creosote, the tarbush (Flourensia cernua), lechugilla (Agave lechuguilla), ocotillo (Fouquieria splendens), Chihuahua whitethorn (Acacia vernicosa), and others of the Dasylirion, Opuntia, and Yucca genera(6). At present, viscid acacia (Acacia neovernicosa), honey mesquite (Prosopis glandulosa), spiny allthorn (Koeberlina spinosa), guajillo (Acacia berlandieri) and Warnock’s snakewood (Condalia warnockii) are also common, while in Coahuila there is also Hechtia spp, candelilla (Euphorbia antisyphilitica), leatherstem (Jatropha dioica) in the north, and Allenrolfea sp., Atriplex sp., Suaeda sp., and mesquite in the south(22). Other dominant shrubs such as cenizo (Leucophyllum frutescens), mariola (Parthenium incanum), creosote bush, mesquite, lechuguilla, catclaw mimosa (Mimosa aculeaticarpa), guajillo, and Gutierrezia microcephala have also been reported in the xerophilous shrub of northern Coahuila(23). For the more arid area of the Chihuahuan Desert in the south of San Luis Potosí, there are shrubs such as the creosote, tarbush, honey mesquite, and chamiso (Atriplex canescens)(22). In Durango, xerophilous bushes are represented by creosote, tarbush, spiny allthorn, honey mesquite, leatherstem, candelilla, and Opuntia, as well as lechuguilla / guapilla (Hechtia glomerata) / Ocotillo associations, the latter species being dominant in the bushes of the north and center of the state of Durango, and the Henricksonia genus standing out as endemic to the Durango and Coahuila scrublands(24). In contrast, the scrublands of Aguascalientes have diverse associations where the most common species are nopal cacti (Opuntia spp), catclaw (Mimosa monancistra), huisache (Acacia schaffneri), and creosote bush / mariola, exhibiting a high degradation and preserving only 20 % of its primary vegetation(17). The availability of practical tools and methodologies is critical for documenting the changes in grasslands and scrublands due to the effects of management and climate. The use of remote sensors has been shown to be a practical tool for estimating forage / productivity and plant cover(25,26) and the extent and fragmentation of grasslands and shrubs(27).

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Grazing management and utilization of grasslands and shrubs

The use of pasture forage resources as primary food is the basis of the cow-calf production system, as well as of the exploitation of goats and sheep under extensive conditions. The alarming deterioration of the grasslands has been largely attributed to the overuse of the resource; therefore, the development of an adequate grazing management program should be considered as preventive. This program is based on a series of principles already established and discussed by numerous authors(28,29), mainly highlighting: a) the use of the optimal stocking rate, b) the determination of the best grazing season, c) the implementation of the most appropriate grazing system, d) the use of the optimal species or combination of animal species, and e) the establishment of practices for a uniform distribution of grazing. Since the 1980s, lines of research have been established to document the aforementioned aspects. The research results obtained during the first 50 yr of the La Campana Experimental Ranch, in the selection of the diet, the voluntary consumption of forage and the ethology of the cattle have been detailed by some authors(30). Table 1 shows the ranges in some components of the diet selected by cattle, with a marked seasonal fluctuation in two types of grasslands and a scrubland that are representative of the state of Chihuahua(30). Other research efforts on the grasslands of Jalisco(31,32) and a Baja California Sur scrub(33) report similar trends that reflect nutritional deficiencies in grasslands and scrubs during the dry season. Likewise, in a study carried out in a medium grassland invaded by catclaw and huisache, the diet of goats showed protein deficiencies during the dormant season of the grasslands of Guanajuato(34). Table 1: Ranges reported in components of the diet of beef cattle in three types of vegetation in the state of Chihuahua Short grass Bunchgrass Creosotebush Component prairie prairie scrublands Crude protein, % 4.9 – 11.5 5.3 – 11.8 6.5 – 12.5 -1 Metabolizable energy, Mcal kg 1.83 – 2.27 1.7 – 2.38 1.58 – 2.22 Digestibility, % 54.1 – 67.2 50.6 – 70.6 46.2 – 66.7 Chávez and González(30).

Within this context, voluntary forage consumption is undoubtedly the most important component in regulating the energy balance of grazing cattle. Likewise, it is an essential variable for a correct estimation of the carrying capacity of grazing lands. Several studies(35,36) have extensively reviewed the factors that control willing forage consumption, inherent to the animal and to the characteristics of the vegetation, justifying studies in this regard. Average consumption values fluctuate between 1.8 and 3.5 % of live weight, depending on the type of vegetation, the time of the year, and the physiological state of the 267

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cattle(30). Close data were obtained in an oak-bunchgrass grassland of Chihuahua, reporting intakes of 2.6 and 3.1 % of live weight, for pregnant and lactating cows, respectively(37). According to several authors, stocking rate is the most critical factor in the implementation of a grazing and utilization management scheme(4,38,39,40). The first utilization studies carried out since 1965 at the La Campana Experimental Ranch, showed an increase in vegetation cover using moderate loads. The interaction between animal load and grazing systems has been evaluated(5), confirming the impact of stocking rate as a primary management factor. In regard to research on grazing systems, some authors have pointed out the statistical complexity and high costs of this type of study(41), which has been a limitation from a methodological and financial point of view. In general, rotational grazing systems have been used as a tool to counteract the unwanted effects of selective grazing(4). However, the evidence generated during the last 60 yr does not indicate a superiority of rotational grazing over continuous grazing, and the supposed advantages of rotational systems are based more on the perception of “anecdotal interpretations” than on an objective evaluation of the experimental evidence(39). Finally, research in grazing systems should not be focused about finding a best method, but to identify and quantify the grazing principles and processes that support an adaptive management and decision making(42). In Jalisco, Deferred Rotational Grazing was found to preserve the condition of the pasture, compared to continuous grazing(43). Short-term grazing systems (STG) have also been the object of evaluation, with not very favorable results. For example, this system exhibited a 49 % reduction in vegetation cover in a Chihuahua grassland(44). This same negative trend for STG was observed after 12 yr of assessment in three grasslands and a Chihuahua scrubland(45), with a 75 % decrease in plant cover, while in continuous grazing (CG), a 124 % increase was obtained. Similar results have been reported in a 7-yr study in a semiarid pasture in Jalisco(46), with biomass production reductions of approximately 72 and 43 % for STG and CG, respectively. In addition, an increase in forage availability has been observed at a greater distance from the water, reaching up to 110 % at 1 km from the center of the grazing cell, suggesting an inefficient distribution of grazing with STG in a Chihuahua grassland(44).

Genetic resources and grass varieties

The deterioration of grasslands has led to the need to conserve germplasm or diversity of forage genetic resources in order to search among them for plant alternatives that stabilize both the ecosystems and the economy of grasslands in Mexico(47). To this end, INIFAP began to collect and evaluate germplasm of native and introduced grasses since the late 1970s, in

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collaboration with germplasm banks from different parts of the world. Some of the native species of greatest interest have been the sideoats and blue grama, and introduced species such as weeping lovegrass, wilman lovegrass (Eragrostis superba) and buffel. The germplasm was established in different states of Mexico, achieving greater persistence in the evaluations in San Luis Potosí, and, as a result, outstanding accessions were identified and later registered with the Seed Inspection and Certification Service (SNICS). In sideoats grama, accessions from different states of the republic have been evaluated, identifying several outstanding ecotypes for forage production such as INIA-235-ZAC, INIA-426COAH, INIA-315-JAL, with values of 3.0 to 3.1 t of dry matter (DM) ha-1, out of a total of 59 accessions(48); while, in San Luis Potosí, 147 accessions from several central states of the country were assessed and, as a result, the variety called “Banderilla Diana”, with average yields in rain-fed conditions of 1.85 t DM ha-1, with crude protein (CP) from 8.6 % in the flowering stage and 3.6 % in maturity(49). The "Cecilia" variety of blue grama was obtained through a selection program, where 53 genotypes from various states in the center of the country were evaluated. This variety can produce up to 0.98 t DM ha-1, with 9.7 % CP in the flowering stage and 3.4 % in maturity(50). Regarding the introduced species, the weeping love grass of the “Imperial” variety was selected and registered, out of 205 evaluated genotypes. This variety can obtain an average yield of 1.2 t DM ha-1, under rain-fed conditions. In addition, it has a CP content of 10.2 % at the beginning of flowering and 4.6 % at maturity(51). In the collection of wilman love grass, the outstanding accession of 14 genotypes was "Hercules", whose average yield in rain fed conditions was 1.2 t DM ha-1, with 10.2 % CP at the beginning of flowering and 4.6 % at maturity(52). In the buffel grass germplasm, of the 78 evaluated accessions, two outstanding genotypes were identified and registered as "Titan" and "Regio". The yields recorded for these varieties were 2.12 and 2.25 t DM ha-1, with 6.1 % and 5.8 % CP in flowering, and 5.8 %, and 4.0 % and 3.3 %, respectively, while in maturity(53). Varieties of buffel grass are available in northeastern Mexico; “Milenio”, in the states of Tamaulipas and Nuevo León(54), and “Zaragoza 115” and “Zaragoza 119”, in the state of Coahuila(55). In recent years, vegetative material collections have been made and morphological, productive and genetic attributes of native grasses have been evaluated. For example, researchers from the College of Postgraduates (Colegio de Postgraduados) managed to identify and register the varieties NDEM-5, NDEM-125, NDEM-303, NDEM-417, and NDEM-LA ZARCA of sideoats grama(56). In Mexico, a wide genetic, morphological and productive diversity of this grass has been reported, with yields from 13.7 g to 1,213 g DM plant-1, from a collection carried out in 13 states from Sonora to Guerrero(56,57). In addition, in a collection of 55 populations of sideoats grama from the state of Chihuahua, values of 4 to 260 g DM plant-1 were found(58). In a germplasm bank established in Zacatecas with 17 ecotypes, yields of 1,320 to 3,337 kg DM ha-1 were found during the rainy period(59). The morphological diversity within this species can be attributed to its great variation in the

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ploidy level(60), since other researches mention that the ploidy level can have effects on the anatomy, morphology, and physiology of plants(61). Regarding blue grama, a great genetic diversity has been found within this species(62), which may be due to the fact that the center of origin of this species is located in central Mexico(63). Likewise, there is the possibility of selecting high yielding blue grama germplasm, since from only 145 ecotypes collected in Chihuahua, a yield range from 0.3 to 48 g DM plant-1 in a single cutting was reported(64). In Zacatecas, yields ranging from 842 to 1,957 kg DM ha-1 were observed in 20 genotypes(59). A great genetic and morphological diversity has been found in Chihuahua in Arizona cottontop (Digitaria californica), reporting two outstanding accessions in forage production, out of 91 ecotypes collected(65). For green sprangletop (Leptochloa dubia), a wide genetic variability was found in 32 genotypes, with yields ranging from 6 to 174 g DM plant-1(66). Finally, it was concluded that there is a wide genetic variation in plains brittlegrass (Setaria macrostachya), and three populations with forage potential were identified, from a collection made in 44 sites(67).

Grass seed production The main value of the production of grass seeds lies in its use for the reseeding or rehabilitation of grasslands, but it also has a great value for the conservation of soils. However, the commercial production of grass seeds for semi-arid zones has been recently developed in Mexico. From 2001 to 2016, the National Catalog of Plant Varieties(68) has documented 27 varieties and hybrids of grasses, 12 of which are suitable for semi-arid areas and belong to five species. Therefore, it can be said that there already exists genetic material to produce qualified seed. Research on production of grass seeds for arid and semi-arid zones date back to the 80s and can be seen in Table 2. These works have mainly determined the volume of production and quality characteristics of the seeds, in some cases with fertilization and irrigation trials. The two most studied species have been buffel grass and sideoats grama.

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Table 2: Maximum seed production of different species of grasses in arid and semi-arid zones of Mexico Seed Especies Variety/Accession production Management Reference -1) (kg ha Panicum Kleingrass 188 Eguiarte and 70.2 Rainfed. Fertilized. coloratum L. González(69) Buffel Zaragoza With irrigation, Hernández et 100 115 Fertilized al.(55) Eguiarte and Buffel Biloela 30.4 Rainfed. Fertilized. González(69)

Pennisetum ciliare (L.)

Bouteloua gracilis H.B.K. (Lag.)

Bouteloua curtipendula (Michx.) Torr.

Buffel Milenio

182

Common buffel

324.0

Buffel Titán

527.0

Buffel Regio

566.2

Cecilia bluegrama

414.0

Alma bluegrama

289.0

(Sideoats grama ecotypes: INIA-365-NL, INIA-315-JAL, INIA-426-COAH, INIA-235-ZAC, INIA-263-ZAC) Chihuahua sideoats grama 75 Sideoats grama AN Selección 75 Diana sideoats grama Niner sideoats grama Diana Sideoats grama

326-374

922 752 998.5 707.0 1,305.1

271

Rainfed

Garza(54)

With irrigation. Fertilized. With irrigation. Fertilized. With irrigation. Fertilized. With irrigation. Fertilized. With irrigation. Fertilized.

Sáenz-Flores et al.(70) Beltrán et al(53) Beltrán et al.(53) Beltrán et al.(50) Sáenz-Flores et al.(70)

Rainfed

Rubio(48)

With irrigation. Fertilized. With irrigation. Fertilized. With irrigation. Fertilized. With irrigation. Fertilized. With irrigation. Fertilized. One crop, second year

González(71) González(71) Beltrán et al.(49) Sáenz-Flores et al.(70) Velázquez et al.(72)

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after establishment. Leptochloa dubia (Kunth) Nees. Eragrostis curvula (Schrad) Nees Eragrostis superba (Peyr).

Van Horn sprangletop

green

670.0

Imperial weeping 685.0 lovegrass Wilman lovegrass

1619.0

Hercules lovegrass

1651.0

wilman

With irrigation. Fertilized. With irrigation. Fertilized. One crop in a year. With irrigation. Fertilized. With irrigation. Fertilized. One crop in a year.

Sáenz-Flores et al.(70) Beltrán et al.(51) Sáenz-Flores et al.(70) Beltrán et al.(52)

Grassland seeding in arid and semi-arid zones Considering the deterioration of grasslands and shrublands, grass seeding is a common practice to reverse this decay, which consists in establishing vegetation through the artificial dissemination of seeds of a single species or in a mixture, using adapted species and appropriate seedbeds. This is done in order to increase the productivity and quality of forage and stocking rate and diversification of diet for livestock, in addition to reducing erosion and invasion of less desired species; however, this practice is expensive and high risk. In general, range seeding is recommended in sites with areas with less than 15 % coverage of native grasses(73,74,75). It is very important to use species adapted to the soil and climate conditions of the area to be rehabilitated. Native species adapt better to different climate and soil conditions and are more persistent; but they are more difficult to establish. Therefore, it is suggested that the origin of the seeds be not more than 300 km from the site where it is going to be seeded. In general, it is recommended to use a mixture of species, preferably native ones. The advantage of mixing is that the diversity of species can make better use of the variability in the soil conditions of the pasture lands(76). The main forage grasses used for sowing are: sideoats grama and blue grama, among the native species, and weeping lovegrass, wilman love grass, and buffel, among the introduced ones(77). The presence or lack of moisture in the soil is the most important variable for the establishment of grass seedlings. Therefore, the land must be prepared for sowing, which is known as "seedbed"; This has the function of loosening the soil, giving it greater porosity,

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so that it retains a greater amount of water, and providing more favorable conditions for the establishment of grasses in these environments(76,77). There are many practices for replanting grasslands, ranging from subsoiling, plowing, disking, level borders, “lister” furrows, and the use of aerator roller, among other options. The latter carries out one of the fastest and most effective processes, especially in desert scrublands, as it increases the water infiltration capacity, reduces soil compaction(78), and has allowed a good grass establishment such as blue grama, sideoats grama, Arizona cottontop, and buffel(76,79,80) in Sonora, Chihuahua, and Coahuila. However, such practices as subsoiling plus plowing and disking also favor coverage with values up to 80 % and a yield of 5.0 to 13.6 t DM ha-1 in buffel grass in the states of Coahuila and Jalisco, respectively(81,82). In the state of Sonora, total clearing has had repercussions on loss of soil and plant diversity; therefore, it should be avoided in the preparation of the seedbed(83). Since the different sowing methods yield different results, it is considered necessary to emphasize that the practices should be adjusted to particular environmental conditions. On the other hand, it is advisable to turn over and break up the soil when wet and harrow it once or twice in order to make a good seedbed(84). However, it is better to sow in wet soil at the beginning of the rainy season in order to increase the opportunity for the seed germination and plant establishment. The ground can be prepared in advance, by "scratching" counter slope or with plowing and disking. Dry sowing is more risky; therefore, it would be advisable to sow one or two weeks before the rainy season in order to prevent loss of seeds(77). After seeding, during the first year the weeds can compete for light, space and soil nutrients, which will reduce the potential of the grasses. It is recommended to control the weed mainly with selective herbicide. From the second year on, in order to keep forage production in good condition, it is recommended to carry out chemical or manual weed control tasks at the beginning of the rainy season, although this is a costly practice(77). However, it is recommended for the control of shrubs when their density increases. Fertilization will depend on the amount of precipitation; in very dry years with rainfall under 200 mm, it is not recommended to fertilize. In the first year, fertilization is carried out with the 20-20-00 formula and only when the moisture condition is favorable. Once the grass is established, it is suggested to fertilize every year(77). However, fertilizers must be applied sparingly due to high costs and to the temporal and spatial variability of rainfall. Other options with good results in seeding native grasses are the application of biofertilizers(85) and organic by-products such as biosolids(86).

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Carbon storage and sequestration in grasslands and scrublands

Like forests, grasslands and shrubs can provide various environmental services for society, including carbon sequestration. Currently, the world's grasslands have good potential for climate change mitigation through carbon sequestration(87,88), which could reach up to 148 Tg CO2 year-1 through the implementation of grazing management strategies(88). In Mexico, the grasslands with moderate grazing contain a greater store of soil carbon (800 g C m-2) compared to grasslands that have been overgrazed (650 g C m-2)(89). Another study(90) found similar results, detecting higher carbon stores in moderately grazed grassland soils with a high cover of forage grasses compared to overgrazed grasslands with a low plant cover, at an average of 34.5 and 24.3 t C ha-1 to 0.3 m in semi-arid and halophyte grasslands, respectively. Considering a soil carbon sequestration capacity of 0.1 t C ha-1 yr-1(91) and an area of 9.77 million ha, the semi-arid grasslands of Mexico could capture about 3.5 Tg CO2 yr-1, with appropriate management of grazing. Regarding the bushlands, no great differences were found with grazing, although the soil carbon stores are low with 21.7 and 23.0 t C ha-1 at 0.3 m deep in the creosotebush and lechuguilla bushes, respectively, in the Chihuahuan Desert(90). Semi-arid areas store more carbon than arid areas of Mexico, and in the grasslands, the soil stores 90 % of carbon, while scrubland soil stores only 45 % of carbon(92). In regard to carbon sequestration, it has been reported that native or natural grasslands are carbon sinks, since they can capture up to 0.054 t CO2 ha-1 d-1(93). Other authors(94) conclude that the carbon sequestration potential of biomass in induced grasslands varies from 0.99 to 1.51 t CO2eq ha-1 yr-1 after 30 and 10 yr of abandonment, respectively. Contrary to expectations, a semi-arid grassland with high plant cover showed to be neutral as a carbon source, and grassland with low plant cover showed to be a carbon sink, while the species composition did not influence the carbon balance(95). Furthermore, another study concludes that semi-arid grasslands could shift from carbon sinks to carbon sources due to the effects of climate change(96). Although digital images and regression models, exhibited a low adjustment, they have a potential for the prediction of soil carbon levels(97). Other authors propose an ecological model for estimating soil carbon in semi-arid grasslands, with variables such as forage grass cover, mean annual rainfall, and soil sand content(98).

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Challenges and perspectives Given the great reduction in the areas of grasslands and scrublands, as well as their high deterioration, it is urgent to stop the advance of the agricultural frontier and avoid overgrazing. Thus, it is urgent to comply with current federal and state laws on conservation and sustainable use of ecosystems, as well as a rural technology transfer program in order to train and educate all ranchers on the sustainable management of grasslands and shrubs in the north-central region of the country. Technologies such as the use of images and geographic information systems are currently available for monitoring and evaluating grasslands and scrublands. However, it is important to continue with the development of technologies that allow finer detection of the processes and structure of grasslands, such as the identification of plants at ground level, in order to facilitate small and large scale decision making. Regarding the use of grasslands, it is urgent to implement grazing management strategies that adapt to the vegetation and climate conditions of each ranch, but, above all, an adjustment of the stocking rate that will allow a moderate use of the grasslands. It is also necessary to reactivate studies of diet quality, selectivity, forage consumption and grazing management strategies to achieve efficient use of resources and the response of vegetation, in a more ecological approach, and in order to maximize animal production and rangeland conservation. In regard to the conservation of genetic resources, more than 1,200 ecotypes of native and introduced grasses have been assessed(48-53,56,57,58). However, up to date, only eight varieties of sideoats grama, eight of buffel grass, and one of each of the blue grama, weeping and wilman love grasses have been generated(99). For this reason, it is important to design financing strategies that will allow to continue performing diversity studies and implementing grass genetic improvement programs. In addition, it is necessary to search for other forage species that grow in arid and semi-arid Mexico, since more than 300 species of grasses have been reported to exist in these regions(13). These actions would allow the generation of new grass varieties, with good potential for establishing and producing forage in the face of the challenges of climate change. Native grass seeds can be produced with currently available technologies at a lower cost than imported seeds although the main challenges are to reduce production costs, and increase seed yield and quality. In the immediate future, more research should be done on the seed vigor, scarification and coating and fixation of products such as nutrients or insecticides in the seeds in order to improve their performance in the field. Subsequently, the generation of technological guides for seed production of different grasses is essential, evaluating the use of agronomic practices and mechanization of seed harvesting and processing.

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For the range seeding, it is recommended to revegetate degraded pastures, preferably with native plants, in order to recover their structure and functioning for the production of goods and services for society. In this activity, the availability of seeds of native species is a great challenge to solve, because, although certain varieties have been released by INIFAP and other institutions, such as blue grama and sideoats grama, the available quantity is insufficient to meet the demand. Establishing seed production lots with cooperating producers is a viable option. Secondly, there is a pressing need to design simple, practical and low-cost equipment for the preparation of land for range seeding. In regard to carbon capture in grasslands and scrublands, the implementation of moderate grazing in “ejidos” and cattle ranches is urgently needed in order to reduce carbon emissions and increase carbon sequestration. Some important challenges are to generate reliable, fast and simple methodologies for the estimation of carbon sequestration and carbon pools, to include the sustainable management of grasslands and shrublands as an option for buyers of carbon credits in official or voluntary markets, and to promote the advantages of grazing management to increase carbon sequestration through sustainable cattle raising. Climate change is already affecting grasslands and will directly and much more dramatically reduce the productivity and contribution of environmental services from the grasslands and scrublands of northern Mexico. This will impact the carrying capacity of rangelands with reductions in meat production, loss of biodiversity, decrease in carbon sequestration capacity, and effects on the hydrological cycle of these ecosystems. The new climatic conditions make it necessary to carry out comprehensive research studies in order to find options for the management and rehabilitation of these ecosystems according to these changes. Literature cited: 1.

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53. Beltrán LS, García DCA, Loredo OC, Urrutia MJ, Hernández AJA, Gámez VHG. "Titán" y "Regio", variedades de pasto buffel (Pennisetum ciliare) (L.) Link para zonas áridas y semiáridas. Rev Mex Cienc Pecu 2017;8(3):291-295. 54. Garza CRD. Producción de semilla de buffel Milenio. En: Tecnologías generadas, validadas o transferidas en los estados de Tamaulipas, San Luis Potosí, Coahuila y Nuevo León en el año 2009. SAGARPA-INIFAP-Campo Experimental Río Bravo. 2010. http://www.inifapcirne.gob.mx/Revistas/Archivos/FichasTecnologicas.pdf. Consultado 12 Oct, 2020. 55. Hernández RP, Cuéllar VEJ, Martínez VJ. Guía para el establecimiento y manejo de zacate buffel Zaragoza 115 para producción de semilla bajo riego. SAGARPA-INIFAPCampo Experimental Zaragoza. 2002. http://www.inifapcirne.gob.mx/Biblioteca/Publicaciones/228.pdf. Consultado 12 Oct, 2020. 56. Morales-Nieto C, Quero A, Le-Blanc O, Hernández A, Pérez J, González S. Caracterización de la diversidad del pasto nativo Bouteloua curtipendula Michx. Torr. mediante marcadores de AFLP. Agrociencia 2006;40(6):711-720. 57. Morales-Nieto CR, Quero A, Pérez J, Hernández A, Le-Blanc O. Caracterización morfológica de poblaciones nativas de pasto banderita [Bouteloua curtipendula (Michx.) Torr.] en México. Agrociencia 2008;42(7):767-775. 58. Morales NCR, Avendaño AC, Melgoza CA, Gil VK, Quero CA, Jurado GP, et al. Caracterización morfológica y molecular de poblaciones de pasto banderita (Bouteloua curtipendula) en Chihuahua, México. Rev Mex Cienc Pecu 2016;7(4):455-469. 59. Rubio-Aguirre FA, Villanueva JF, Sánchez RA. Comportamiento morfológico y productivo de “Colectas base” de gramíneas nativas e introducidas del altiplano de Zacatecas. Publicación especial. Núm. 22. Campo Experimental Zacatecas. CIRNOCINIFAP. Zacatecas. 2016. http://zacatecas.inifap.gob.mx/publicaciones/Publesp222016.pdf. Consultado 19 Sep, 2020. 60. Morales NCR, Quero CAR, Avendaño ACH. Caracterización de la diversidad nativa del zacate banderita [Bouteloua curtipendula (Michx.) Torr.], mediante su nivel de ploidía. Tec Pecu Méx 2007;45:263-278. 61. Comai L. The advantages and disadvantages of being polyploid. Nat Rev Genet 2005;6(11):836-846.

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62. Morales-Nieto CR, Álvarez A, Villarreal F, Corrales R, Pinedo A, Martínez SM. Phenotypic and genetic diversity of blue grama (Bouteloua gracilis) populations from Northern Mexico. Arid Land Res Manage 2019;34(1):83-98. 63. Avendaño-González M, Morales JF, Siqueiros ME. Genetic structure, phylogeography, and migration routes of Bouteloua gracilis (Kunth) Lag. ex Griffiths (Poaceae: Chloridoideae). Mol Phylogenetics Evol 2019;134:50-60. 64. Morales NCR, Madrid PL, Melgoza CA, Martínez SM, Jurado GP, Arévalo GS, et al. Análisis morfológico de la diversidad del pasto navajita [Bouteloua gracilis (Willd. ex Kunth) Lag. ex Steud.], en Chihuahua, México. Téc Pecu Méx 2009;47(3):245-256. 65. Morales NCR, Melgoza CA., Jurado GP, Martínez SM, Avendaño AC. Caracterización fenotípica y molecular de poblaciones de zacate punta blanca (Digitaria californica (Benth.) Henr.). Rev Mex Cienc Pecu 2012;3(2):171-184. 66. Morales-Nieto CR, Rivero O, Melgoza A, Jurado P, Martínez M. Caracterización morfológica y molecular de Leptochloa dubia (Poaceae) en Chihuahua, México. Polibotánica 2013;36:79-94. 67. Morales-Nieto CR, Avendaño AC, Melgoza CA, Martínez SM, Jurado GP. Caracterización morfológica y molecular de poblaciones de zacate tempranero (Setaria macrostachya Kunth) en Chihuahua, México. Phyton 2015;84(1):190-200. 68. SNICS. Servicio Nacional de Inspección y Certificación de Semillas. México. 2018. https://datos.gob.mx/busca/dataset/cnvv/resource/2651402e-8b42-411a-b23b22936176fb42. Consultado 25 Sep, 2020. 69. Eguiarte VJA, González SA. Producción de semilla y forraje de pastos tropicales en el Sur de Jalisco. Tec Pecu Mex 1995;33(2):105-111. 70. Sáenz-Flores E, Saucedo TRA, Morales NCR, Jurado GP, Lara MCR, Melgoza A, et al. Producción y calidad de semilla de pastos forrajeros como respuesta a la fertilización en Aldama, Chihuahua. Tecnocienc Chih 2015;9(2):111-119. 71. González DJR. Producción de semilla de dos variedades de zacate banderilla con diferentes densidades de siembra. Agraria 1988;4(2):137-145. 72. Velázquez-Martínez M, Santiago-Hernández F, Gámez-Vázquez HG, CervantesBecerra JF. Fertilización para la producción de semilla de pasto banderita (Bouteloua curtipendula) en condiciones de riego. En: Simposio “Manejo del pastoreo, Toral en el manejo de pastizales” y “60 aniversario del Rancho Experimental La Campana”. Chihuahua, Chih. 2017:198-201.

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73. Beltrán LS, Loredo O C, García DCA, Hernández AJA, Urrutia MJ, Gámez VHG, et al. Llorón Imperial y Garrapata Hércules Nuevas Variedades de pastos para el altiplano de San Luis Potosí (Establecimiento y producción de semilla). INIFAP-CIRNE Campo Experimental San Luis. (Folleto Técnico No. 36). 2009. 74. Martín GO. Técnicas de refinamiento y recuperación de pastizales. 1a ed. San Miguel de Tucumán: Universidad Nacional de Tucumán. E-Book. ISBN 978-950-554-865-1. 2014. 75. Beltrán LS, Loredo OC. ¿Cuándo sembrar y cómo realizar una siembra de pastos? INIFAP-CIRNE-CE San Luis Potosí (Desplegable Técnico No. 2). 2005. 76. Sierra TJS, Ramírez GH, Gutiérrez RE. Paquete tecnológico para la siembra de pastos en los agostaderos de Chihuahua. INIFAP-CIRNOC Sitio Experimental La Campana. (Folleto Técnico No. 50). 2014. 77. Velázquez MM, Hernández GFJ, Cervantes BJF, Gámez VHG. Establecimiento de pastos nativos e introducidos en zonas semiáridas de México. INIFAP-CIRNE Campo Experimental San Luis. (Folleto para Productores No. 66). 2015. 78. Medina-Guillén R, Cantú-Silva I, González-Rodríguez H, Pando-Moreno M, Kubota T, Gómez-Meza MV. Efectos del rodillo aireador y el fuego en las propiedades físicas e hidrológicas del suelo en matorrales de Coahuila, México. Agrociencia 2017;51:471485. 79. Berlanga RCA. Uso del rodillo aereador para la rehabilitación de pastizales degradados. INIFAP-CIRNE-Campo Experimental Saltillo. (Desplegable Técnico No. 10). 2009. 80. Rivera SFJ, Pérez CJM, Montañez AMP, Lavandera BG. Uso del rodillo aireador en la restauración de pastizales en Agua Prieta, Sonora. Biotecnia 2017; XIX(3):23-28. 81. Olhagaray REC, García EG, Espinoza AJ. Influencia de dos sistemas de labranza mínima y tradicional en la producción de zacate buffel (Cenchrus ciliaris) empleando curvas a nivel con relación 1:1 en el suroeste de Coahuila, México. VI Simposio Internacional de Pastizales. Monterrey, N.L. 2009. 82. Unión Ganadera Regional de Jalisco. Establecimiento de zacate buffel. https://www.ugrj.org.mx/index2.php?option=com_content&do_pdf. Consultado 29 Dic, 2020. 83. Castellanos-Villegas AE, Bravo CL, Koch WG, Llano J, López D et al. Impactos ecológicos por el uso del terreno en el funcionamiento de ecosistemas áridos y semiáridos. En: Molina FF, Van Devender RT, editores. Diversidad Biológica de Sonora. México. UNAM; 2009:157-186.

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84. CEPAB. Campo Experimental Pabellón. Agenda Técnica Agrícola Aguascalientes, México. SAGARPA-INIFAP; 2017. 85. Esqueda CMH, Carrillo R, Sosa M, Melgoza A, Royo MH, Jimenez J. Emergencia y sobrevivencia de gramíneas inoculadas con biofertilizantes en condiciones de invernadero. Tec Pecu Mex 2002;42(3):459-475. 86. Jurado-Guerra P, Sierra JS, Lara MC, Saucedo TR, Morales NC. Establishment of native grasses with biosolids in abandoned croplands in Chihuahua, Mexico. Appl Env Soil Sci 2013; http://dx.doi.org/10.1155/2013/573808. 87. Soussana JF, Tallec T, Blanfort V. Mitigating the greenhouse gas balance of ruminant production systems through carbon sequestration in grasslands. Animal 2010;4(3):334350. 88. Henderson BB, Gerber PJ, Hilinski TE, Falcucci A, Ojima DS, Salvatore M, et al. Greenhouse gas mitigation potential of the world´s grazing lands: modeling soil carbon and nitrogen fluxes of mitigation practices. Agric Ecosyst Environ 2015;207:91-100. 89. Medina-Roldán E, Arredondo JT, Huber-Sannwald E, Chapa L, Olalde V. Grazing effects of fungal root symbionts and carbon and nitrogen storage in a shortgrass steppe in Central Mexico. J Arid Environ 2008;72:546-556. 90. Jurado GP, Saucedo TRA, Morales NCR, Martínez SM. Carbono orgánico del suelo y su relación con la condición en pastizales y matorrales de Chihuahua. En: Paz F, Wong J, Bazán M, Saynes V editores. Estado actual del conocimiento del ciclo del carbono y sus interacciones en México: Síntesis a 2013. Texcoco, Estado de México. PMCColegio de Postgraduados-UACH-ITESM; 2014:62-69. 91. Schuman GE, Janzen HH, Herrick JE. Soil carbon dynamics and potential carbon sequestration by rangelands. Environ Pollut 2002;116:391-396. 92. Montaño NM, Ayala F, Bullock S, Briones O, García F, García R. Almacenes y flujos de carbono en ecosistemas áridos y semiáridos de México: Síntesis y perspectivas. Terra Latinoam 2016;34:39-59. 93. Zermeño-González A, Ríos EJA, Gil MJA, Cadena ZM, Villarreal QJA. Dinámica del flujo de carbono y de energía sobre un pastizal natural del norte de México. Bioagro 2011;23(1):1-13. 94. Yerena YJI, Jiménez PJ, Alanís RE, Aguirre COA, González TMA, Treviño GEJ. Dinámica de la captura de carbono en pastizales abandonados del noreste de México. Trop Subtrop Agroecosyst 2014;17;113-121.

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95. Delgado-Balbuena J, Arredondo JT, Loescher HW, Huber-Sannwald E, Chávez G, Luna M, et al. Differences in plant cover and species composition of semiarid grassland communities of central Mexico and its effects on net ecosystem Exchange. Biogeosci 2013;10:4673-4690. 96. Delgado-Balbuena J, Arredondo JT, Loescher HW, Pineda MLF, Carbajal JN, Vargas R. Seasonal precipitation legacy effects determine the carbon balance of a semiarid grassland. JGR Biogeosci 2019;124:987-1000. DOI:https://10.1029/2018JG004799. 97. Linares-Fleites G, Tenorio MG, Torres E, Oroza HAA. Estimación del carbono orgánico en suelos por teledetección y modelos de regresión. Rev Latinoam Amb Cienc 2017;8(18):26-40. 98. Jurado GP, Juárez MM, Saucedo TRA, Morales NC, Martínez SM. Modelo ecológico de predicción de carbono en pastizales de Chihuahua. En: Paz F, Velázquez A, Rojo M editores. Estado actual del conocimiento del ciclo del carbono y sus interacciones en México: Síntesis a 2018. Texcoco, Estado de México. PMC-ITS; 2018:62-69. 99. Servicio Nacional de Inspección y Certificación de Semillas (SNICS). Catálogo Nacional de Variedades Vegetales. https://datastudio.google.com/u/0/reporting/5b7206ba-e190-48fe-969673523bfccf58/page/itBWB. Consultado 7 Jul, 2020.

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https://doi.org/10.22319/rmcp.v12s3.5846 Review

History and perspectives of the GGAVATT model (Groups for Livestock Technological Validation and Transfer)

Heriberto Román Ponce a Miguel Arcangel Rodríguez Chessani b José Antonio Espinosa García c Tomás Arturo González Orozco d Alejandra Vélez Izquierdo c Juan Prisciliano Zárate Martínez a* Martha Eugenia Valdovinos Terán a Rubén Cristino Aguilera Sosa a Rafael Guarneros Altamirano e Rubén Santos Echeverría f Héctor Macario Bueno Díaz b Ubaldo Aguilar Barradas b

Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias. CIR Golfo Centro, Campo Experimental la Posta. Km 22.5, carretera federal Veracruz-Córdoba, 94277, Paso del Toro, Municipio de Medellín, Veracruz, México. b

Universidad Veracruzana. Facultad de Medicina Veterinaria y Zootecnia, Veracruz, Ver., México. c

Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias. CENID Fisiología, Ajuchitlán, Querétaro, México. d

Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias. CIR Centro, Campo Experimental Bajío, Celaya, Guanajuato, México.

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Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias. CIR Noreste, Sitio Experimental Aldama, Aldama, Tamaulipas, México. f

Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias. CIR Pacífico Sur, Campo Experimental Iguala, Iguala, Guerrero, México.

*Corresponding author: zarate.juan@inifap.gob.mx

Abstract: In this document, the aim was to gather the experiences obtained with the GGAVATT model since its creation. The results, experiences, and impacts of the development and implementation of this model, reported in secondary sources, were evaluated. Five stages were identified: laying the foundations (1970 - 1982), model development (1983 - 1989), model validation (1990 - 1996), national expansion (1997 - 2007), and adaptation and survival (2008 - to date). Here present the results obtained in each of these stages and the participation in different projects associated with official programs in the different Mexican States. In all the projects where the GGAVATT methodology has been appropriately applied, the technical, economic, social, and ecological results have been positive. At this point, it is know the success factors that provide good results. Therefore, it has a strategy that could be very useful in improving the current situation of small and medium-scale livestock producers in Mexico. Key words: Transfer model, Innovation, Livestock, Technology adoption, Impacts.

Received: 10/11/2020 Accepted: 06/01/2021

Introduction Mexico's National Institute for Forestry, Agriculture, and Livestock Research (INIFAP) and higher education institutions have developed technology that could potentially double or triple meat, milk, and honey production in the main agroecological regions in the country. Additionally, INIFAP developed and implemented a technology transfer model called

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Groups for Livestock Technological Validation and Transfer (GGAVATT). This model facilitates the process of technology transfer and thus increases the productivity and profitability of the production units, improving the standard of living and quality of life of the producers and the society in general(1). The specific aims of this model are to group farmers with common objectives; change their attitude, behavior, and aptitude towards technology use and adoption; increase the productivity of the Livestock Production Units (LPU), so that they are profitable, competitive, and sustainable; improve the standard of living of the producers and their families; promote the conservation and optimal use of natural resources; strengthen the integration of livestock value chains; and provide feedback to research and teaching institutions through demands and technological problems(1). This model has proven to facilitate the transfer of livestock technology. Therefore, in this document, we aim to review the experiences obtained with the work of organized groups since 1982. For the latter, a bibliographic review was carried out with a historical perspective, identifying five stages of the GGAVAT model (Figure 1), described below. Figure 1: Timeline of the GGAVATT model

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Laying the foundations: 1970 - 1982

The technological transformation of the farm "Bella Esperanza" (RBE), located in the Huasteca Veracruzana, started in 1970 following the recommendations provided by the Experimental Station "La Posta," in Paso del Toro (ES La Posta), Veracruz(1,2). In the United States(3), the use of bulls with high genetic value, obtained through artificial insemination (AI), had given positive results. Likewise, in the ES La Posta, the Holstein, Brown Swiss, and Jersey breeds could adapt to a semi-intensive system(4). Based on this evidence, in the RBE, Brahman cattle were crossed with Holstein, cattle and paddock management were improved along with genetics, and the systematic record of the herd's productive and reproductive responses was initiated. A determining factor for this technological change's success was the producer's participation, his family, and his workers, who trusted the researchers' recommendations. By methodically adopting and applying the technology, the milk production per cow/day increased from 3.9 kg in 1971 to 6.3 in 1981 and decreased the interpartum period from 475 to 436 days. The positive change in milk production, cattle genetics, paddock conditions, and overall infrastructure motivated the first technicalpractical demonstration in the RBE(5). Several farmers from the Local Livestock Association of Tepetzintla, other farmers, researchers, professors, and authorities of the livestock sector attended this demonstration. After their approval, the integration of the Tepetzintla Livestock Program (PROGATEP) began.

Model formation: 1983 to 1989

Upon learning of the RBE advances, neighboring farmers showed interest in using the same technology; this resulted in the creation of the PROGATEP, with 28 farmers from the Local Livestock Association of Tepetzintla, Veracruz. Farmers were selected based on personal conversations, visits to their farms, their interest in participating in the program, and the importance of milking in their farms. Farmers agreed to a general farm inventory, identifying their animals; record milk production, reproduction, income, and expenses; and attending a monthly work meeting(6). It was suggested that the program should be continuous, with gradual changes according to the producers' economic and operational possibilities and decisions. The technical assistance provided by a Zootechnical Veterinarian from the Rural Development District of Tuxpan was defined. INIFAP took responsibility for the technological innovations; the farmers appointed a Coordinator who served as a liaison between them, the Regional Cattle Union, and the municipal, state, and federal authorities. Subsequently, the transfer model was created; this

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propitiated the interaction between producers, technical consultants, researchers, and government institutions. The animal and paddock activity calendar; the program of periodic visits, the calendar of monthly meetings (first Saturday of every month); the program of technical talks for the monthly meetings; and the annual technical, productive, and economic evaluation (April) were created with the participation of the producers. Producers from all over the region, researchers, academics, facilitators, various service providers, directors of various livestock associations, and government officials were encouraged to participate. Furthermore, the families and workers of the GGAVATT members were also involved(7). In addition to PROGATEP, INIFAP was technically responsible for six groups in the Center of Veracruz: Jamapa Livestock Program, Joachín Livestock Program, Tres Valles Livestock Program, Jilguero Livestock Program, La Tasajera Livestock Program, and Jarocho Pig Livestock Program. In all of them, the results were similar to PROGATEP. In 1989, in a meeting carried out at the offices of the Centro de Investigación Regional del Golfo Centro [Regional Research Center of the Central Gulf], INIFAP researchers discussed and approved that PROGATEP be called GGAVATT since it involves a group for livestock technological validation and transfer. During this meeting, the model and work methodology were also defined(2,8). Tepetzintla is the first GGAVATT in Mexico, and it is known as the "cradle of the GGAVATT."

Model validation: 1990 to 1996

In 1990, the GGAVATT model was made available to all farmers in Veracruz. The PROGATEP, as a group organization, initially adapted its structure and functions according to the official policies indicated by INIFAP and the Rural Development District. Soon, daily work dynamics and the monthly and annual meetings modified the methodology based on the country's changing economic and political circumstances. The GGAVATT model methodology proved to be versatile and adaptable(2). The Tepetzintla group documentation was used to support the promotion, training, and operation of other groups. The promotion was carried out at the local Livestock Associations, municipalities, ejidos, Rural Development Districts. In general, previously formed groups were used to invite interested farmers. Group formation is preceded by the static diagnosis applied directly in the farms of future associates; this is required to schedule the Constituent Assembly, where representatives (president, secretary, and treasurer) are elected. The membership of all partners is also defined during this assembly. Group components are proposed and accepted; this includes the farmer, the professional responsible for providing

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technical consulting, and the institutional component. The latter is composed of the institutions or agencies that coordinate technology generation, validation, and transfer. Finally, the operation is carried out with both group activities, such as the monthly meeting, training, management, outreach, annual evaluation, and the national conference; and individual activities, such as technology validation, application of the actions agreed to in the animal and flock management calendar, keeping productive, reproductive, and economic records, and attend ranch imponderables, such as clinical cases(6). In Veracruz and Tabasco, other research, teaching, and operative institutions related to the federal and state governments were necessary for monitoring the GGAVATT. The Regional Livestock Unions and their corresponding Local Livestock Associations were actively incorporated. In Veracruz, the Unique Program of Groups for Livestock Technological Validation and Transfer (PROGGAVATT) was created to modernize livestock through the application of new technology and by organizing the producers to increase farm production and productivity without deteriorating natural resources and the agroecological environment. In 1990, INIFAP oversaw 11 GGAVATT in Veracruz. With inter-institutional participation, this number increased to 37 in 1991, 67 in 1992, and 79 in 1993. State-level GGAVATT meetings started during this stage. These meetings were carried out at the port of Veracruz from 1990 to 1995. During these meetings, the participants shared their experiences and agreed to continue working in an organized manner. In Tabasco, where they observed similar results to those in Veracruz, the model was validated with two groups of producers in the Huimanguillo municipality. Since1990, the model methodology, so far applied and supervised by INIFAP, was released; this allowed the formation of groups under the direction or technical responsibility of whom the producers decided: Livestock Unions and Associations, Districts, municipalities, Livestock Direction, Universities, College of Zootechnical Veterinarian, and others. Therefore, by 1995, the GGAVATT were consolidated in Veracruz and Tabasco(2,7) and continued at a national level(6).

National extension of the GGAVATT model: 1997 to 2007

The GGAVATT model and its results were intensely communicated in the National Livestock Research Meetings (RNIP) organized by INIFAP. Furthermore, in 1997, the first GGVATT National Meeting was scheduled in the Port of Veracruz; more than 1,000 producers of different Mexican States participated in this event. Simultaneously, State GGAVATT Meetings were also organized, and technological exchange tours were promoted

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all over the country(1). The extension stage of the GGAVATT model started this year when INIFAP created the National Program for Validation and Support of Technology Transfer (PRONAVATT); this was a national strategy that operated in each state. An INIFAP researcher was appointed as head of PRONAVATT. This action aimed to promote the integration of technological, intellectual, material, economic, and political resources between the public, social, and private sectors of the three levels of government. This integration would accelerate and mass technology transfer and promote sustainable development. GGAVATT considered the PRONAVATT a basic tool(9). The Operation Rules (OR) of the Ministry of Agriculture, Livestock, and Rural Development, published in the 1998 Official Journal of the Federation (DOF), included the operation of the DPAI, which states that DPAI technicians should preferably attend to farmers in GGAVATT groups(10); this was decisive for consolidating the model. The GGAVATT model appeared, with some changes, in the OR of the Alianza para el Campo from 1998 to 2006, as one of the models promoted by the Comité Técnico de Ganadería (COTEGAN)(11). At least one GGAVATT was established and operated in each state (except in Estado de México); 1,098 GGAVATT were integrated, and more than 60 State and 10 National meetings were held. Moreover, from 2004 to 2009, through INIFAP's National Network for Validation and Technology Transfer (RENAVATT), change agents continued to be trained in the GGAVATT model(9).

Adaptation and survival of the model: 2008 to date

After the rise of the GGAVATT model, its expansion throughout the country decreased due to either the establishment of new models or the lack of economic and human resources. However, in 2008, SAGARPA, within the Programa Soporte, instructed to consider the GGAVATT model among other models. SAGARPA also designated INIFAP as a Specialized Technical Unit in Livestock Matters (STU), which proposed that the Programa Soporte should apply the GGAVATT model and be trained in its methodology and take courses of Diagnostic Evaluation and Livestock Farms Administration(12). In 2011, SAGARPA implemented the National Strategy to provide quality technical service to the livestock producers in the country; this involved different participants, such as the State Centers for Training and Monitoring of the Quality of Professional Services (CECS). These centers include State institutions or organizations, such as Universities(13). This action by SAGARPA significantly decreased the establishment of GGAVATT in the country; some States continued with the model for their technical assistance strategy, innovation, and training. However, no study concentrated these efforts. From the review of the reports of the

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2010 National Livestock Research Meeting (RNIP)(13) to date, GGAVATT continuity results were found in some states, where the support of the PRODUCE Foundations was decisive, as is the case of Sinaloa and Guanajuato, and the support of the governments of the states of Guerrero, Michoacán, Morelos, Nayarit, Colima, Tamaulipas, Veracruz, Campeche, Nuevo León, and Chihuahua. In 2013, given the concern of organized beef producers, the "Integration of the Beef Cattle Network Cattle Meat of Veracruz" project was established(1). This project was based on the GGAVATT methodology and was financed from 2013 to 2014 by resources from FIRA, SAGARPA (General Livestock Coordination), and the Productores y engordadores de bovinos del Centro de Veracruz, S.A. de C.V. (Grupo Veracarne), and from 2015 to 2016 by the Programa de Innovación, Investigación, Desarrollo Tecnológico y Educación (PIDETEC) of SAGARPA. The project ended in 2017. The aim was to increase productivity, profitability, and sustainability of the production units of small and medium-size producers. This would be attained with technical assistance, training, and financing, integrating these producers into the beef value network. A total of 20 groups participated with their corresponding consultants; 202 farmers were benefited in the entire state, with an average of 12 producers per group. The project used an innovative computer platform (SIGEN-TTP Veracruz) to obtain the static diagnosis at the beginning of work. Subsequently, the productive, reproductive, economic, and use of credit information was also obtained. From 2013 to 2017, the established goals were met. The GGAVATT model operated successfully following the rules of credit institutions and private livestock organizations such as Veracarne and complying with the operating rules of official agencies.

Results of the GGAVATT model as a generator of information

As part of the work methodology, information is generated in the groups from the static diagnosis (social, use of technology, productive and economic). Furthermore, technical, economic, and use of technology records are implemented parallel to the work calendars with the animals and paddocks. From these records, information is generated due to the validation, use, and adoption of technology. Thanks to the data capture directly in the farms, it was possible to know the socioeconomic characteristics of the producers, as well as the estimation of technical-productive and economic indicators.

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Socioeconomic characteristics of the producers

Characterizing producers and groups is important to implement the level of technology transfer that must be applied and to evaluate the activities' future impact. A study of 12 GGAVATT in the central zone of Veracruz(14) reported that the age of producers ranges from 20 to 80 yr, 49 on average. Of the producers, 94 % knew how to read and write, 20 % received professional education, 31 % attended secondary and high school, 43 % only elementary school, and 6 % had no school grade. The production units have an average of 45.5 ha, with an animal load of 46.6 animal units (AU) and without a rotating paddock system. As for group characterization, a previous report indicates that in 1996 there were 79 GGAVATT in Veracruz. These GGAVATT included 1,288 farms, with 54,415 ha and around 54,000 cattle heads. Of the producers, 473 were small owners, and 815 were ejidatarios. Of the groups, 58 were dual-purpose cattle producers; seven were dairy cattle producers; ten were sheep and goat farmers; one focused on pig production; one specialized in beekeeping, and two were poultry producers(1). Furthermore, by 2016 there were 1,165 GGAVATT at a national level; these GGAVATT grouped 17,095 producers and close to 1,000 agents of change. The groups represent the main agri-food chains: dual-purpose cattle, 41.1 %; beef cattle, 22.8 %; goats for meat and milk, 10.7 %; dairy cattle, 10.1 %; poultry, 6.4 %; sheep for meat, 6.1 %; pigs, 2.1 %; family poultry farming, 0.5 %; and aquaculture, 0.2 % (1). The GGAVATT model stimulated technology adoption and increased social networks and interactions. This process was influenced by socioeconomic and technical-productive characteristics(15). Other authors report evidence that the GGAVATT has contributed to developing the technological capacities of the cooperating agro-entrepreneurs. However, the impact of innovation adoption and the profitability of the PU is low or null, suggesting that more time is needed to perceive the benefits(16).

Use of technology

According to the static diagnosis, the producers initially carry out 30 % of the suggested technologies, and in 5 yr, they reach 70 %. More precisely, after one year of working as a group, 126 groups in Veracruz reported a 48 % use of technology, 63 % after three years, and 73 % at the fifth year. In a further analysis of 24 groups, 72 ± 39 % was reported after several years of work(6).

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Moreover, a study that aimed to evaluate the economic and productive impact of the dualpurpose bovine technology in Tabasco and Veracruz collected the annual data (1986-1997) of 139 farms incorporated into the GGAVATT. The results indicated that medium technology outperformed low technology (P<0.05) in milk and meat production. Food supplements benefited 106 vs 44% of milk and 193 vs. 81% of meat, respectively. Breeding of dairy cattle, improved pastures, and livestock management increased milk production by 67 and 69 % and meat production by 15 and 26 % with low and medium technology, respectively. Moreover, producers that use medium technology outperform those with low technology (P<0.05) in milk production, meat production, and benefits over variable costs by 106, 44, and 81 %, respectively(17). In Guanajuato, a different study evaluated the use of innovation in groups from the Programa Soporte from 2010 to 2011. This study considered 21 GGAVATT and 248 producers of family dairy systems(18); three types of producers were compared: 1) 27 with a low technological level that apply less than 33 % of the technologies, 2) 73 with a medium technological level that apply more than 33 % and less than 66 %, and 3) 148 with a high technological level that apply more than 66 %. Results showed that group one was 2 yr old; groups two and three were three years old. The most used practices were those related to health, feeding, and management. In Sinaloa, a study evaluated from 2010 to 2011 the use of innovations in eight GGAVATT from the Programa Soporte(19). Eight GGAVATT and 121 producers were considered, and three strata were compared. In the first, 38% of producers had low practice implementation levels, were 3.7 yr old and used only six proposed innovations. In the second strata, 36.4 % of producers had medium implementation, were 4.5 yr old, and implemented 12.2 innovations. In the third strata, 25.6 % of producers had high levels of implementation, were 6.3 yr old, and used 22.7 innovations. In Michoacán, the use of the technology promoted by the model was evaluated in 15 GGAVATT participating in the Program for the Development of Capacities, Technological Innovation and Rural Extensionism from 2011-2012(20). This study reported that producers carried out on average 77 % of the activities proposed by the technician and 20 % of the technological components presented by INIFAP. The GGAVATT for goat and activities associated with disease prevention and livestock management showed the highest values. The determining factors in adopting new technologies were months with technical assistance by GGAVATT, producer age, number of localities, marginalization index, and number of women per group. In the same state, a different study evaluated the impact on the adoption of innovations. A total of 81 milk producers from the Ciénega de Chapala region were interviewed; 41 of these producers belonged to a GGAVATT from July 2008 to July 2011. The use of 22 innovations was evaluated: five related to administration/organization, four to reproduction and genetics, four to facilities/hygiene, four to nutrition, and five to health. 295

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Results show that the farmers that belong to a GGAVATT apply 51.6 % of these innovations; those that do not belong to a GGAVATT apply 44.9 %. These results are not enough to conclude that there is a difference between both groups(21). In Veracruz, the use of innovations from a GGAVATT constituted in 1989 was evaluated. After 10 yr, three strata of livestock farmers were interviewed: I) active participants in this GGAVATT, II) previous participants, and III) those who have never participated. Seventeen explanatory or predictive variables of a social and productive nature were selected. The response variable, use of innovations, was also selected. Results showed significant differences regarding the use of technology between the years 1989-1990, 1990-1991, and 1993-1994. An increase in innovations was reported, mainly regarding production and financial records, deworming, mastitis and gestation diagnosis, and milk weighing. A significant increase was observed from 1990 to 1991 in milk weighing, artificial insemination, mineral and by-products supplementation, silages, haymaking, supplementation with cut and carry fodder, paddock fertilization, and sowing of cut and carry fodder. During 1993 and 1994, there was an increase in the use of the following innovations: lactation control, supplementation with a balanced diet and by-products, routine use of hormones, organized commercialization, double bucket milking, stool analysis, intensive grazing, and sowing of cut and carry fodder(22). Also, in Veracruz, a study evaluated the use of 17 technological components and the productive efficiency of 86 dual-purpose farms belonging to eight GGAVATT that participated in the Programa Soporte in 2010. Of these GGAVATT, 12 % received technical assistance for three or fewer years, 45 % received assistance for three years, 31 % for four years, and the remaining 12 % had five or more years of assistance. On average, these GGAVATT used 62 % of the 17 technological components evaluated; no significant differences were observed. Artificial insemination and weaning weighting were the least used components; vaccination, deworming, paddock rotation, and mineral supplementation were the most used. Significant differences were reported in milk production per cow per day. Farms that had belonged to a GGAVATT for longer were more efficient and productive(23). In Veracruz, a different study was carried out to evaluate the dynamics of technology adoption as a measure of success of the GGAVATT model from a social perspective. A total of 26 producers belonging to three GGAVATT were interviewed. This study observed that technology adoption was associated with the increase in the social network and interactions, with few changes in the central actors. Technology adoption is positively associated with education, is inversely proportional to the age of the producers, and is influenced by the management and production scale. The authors of this study concluded that the GGAVATT model stimulated technology adoption, increasing the social network and interactions, a process influenced by farmers' socioeconomic and technical-productive characteristics(24).

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Another study evaluated the productive and economic efficiency of dual-purpose (DPS) and family dairy systems (FDS) in Veracruz by applying the case study of four GGAVATT with different technological levels in each production system. The 1DPS GGAVATT had 12 members, 30 yr of existence, and applied 100 % of the technological package. The 2DPS GGAVATT had 16 members, 5 yr of existence, and applied 66 % of the technological package. The 1FDS GGAVATT had 12 members, 5 yr of existence, and applied 57 % of the technological package; 2FDS had nine members, three years of existence, and used 53 % of the technological package. The results reported for the DPS show that the total milk production in GGAVATT 1DPS is 84% higher than GGAVATT 2DPS. This difference results from adopting a series of technological practices with different response times and the time applying the model. Additionally, its income is higher due to the higher price of its animals due to breeding. As for the FDS GGAVATT, no significant differences were reported since both GGAVATT have a similar technological level(25).

Technical, productive, economic, and ecological indicators

In all the GGAVATT of the country, compared to traditional indicators, milk production increased by 100-200%, meat production increased by 50-100 %, reproductive and economic indicators also increased. In Veracruz, associated with the use of 70 % of the technology, 286 ± 44 milking days, 2,159 ± 921 kg of milk per lactation, 7.25 ± 2.3 kg of milk per cow day, 4.7 ± 2.1 kg of milk per day between calving, and 900 ± 396 kg of milk per hectare were reported(6). Tepetzintla exemplifies the possibilities of increase. Farms initially achieved 74 % use of technology and exceeded 1,000 kg per lactation. The use of technology was intensified to 95 % and exceeded the 2,000 kg of milk per lactation. The leadership of producers, technicians, and institutions allowed adopting high, complicated, and expensive technology. Subsequently, although technology use decreased to 74 %, it was possible to exceed 3,000 kg per lactation. Lastly, with 85 % of technology and using high levels of concentrated feed, 4,000 kg per lactation were achieved, and by reducing feed, it stabilized at 3,500 kg(6). In 2006, the economic impact of the use of technological components in animal feeding was evaluated in Sonora. Using the technology generated for the GGAVATT, the dry forage yield of the sorghum crop was 7 versus 3.9 t of DM/ha, which represented an increase of 79 %; milk production per cow per day increased 60 %, and the lactating period increased from 180 days with the traditional system to 210 with INIFAP's technology(26). In Veracruz, the

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averages of 24 dual-purpose cattle GGAVATT were analyzed. A return above variable costs (RAVC) of 85 ± 49 % and a return on investment (ROI) of only 3.5 ± 3.6 %(6) was observed. Since its inception, one of the purposes promoted in the groups has been the care and improvement of natural resources. In the central zone of Veracruz, a study was carried out in 2005 and 2006 in four GGAVATT with family dairy systems (FDS) of the mountainous area and in eight with dual-purpose cattle systems (DPCS). Information for this study was obtained through interviews and field trips to sample living fences and vegetation fragments. A total of 66 tree and shrub species were inventoried. In the living fences, 37 species were reported; 33 species were identified on the banks of rivers and streams (riparian vegetation); 37 pasture species were identified. Farmers also reported introducing exotic species, crops, and fruit trees(15); this shows that the farmers incorporated into the GGAVATT care for vegetation.

Successful examples of the GGAVATT model

In 2005, INIFAP carried out a study to identify how the GGAVATT model contributed to livestock development(27) in 16 states: Veracruz, Guanajuato, Campeche, Coahuila, Tabasco, Guerrero, Sinaloa, Puebla, Yucatán, San Luis Potosí, Durango, Morelos, Sonora, Nayarit, Baja California Sur, Nuevo León. Thirty-three producers from 10 states, 19 GGAVATT advisers, and three researchers involved in developing this model were interviewed. Of the producers, 94 % agreed that the most crucial factor for success was working as an organized group, 57.6 % said technology transfer, 54.5 % mentioned the support achieved as an organized group, and 45% referred to the professional and institutional leadership. Only 27.3 % mentioned the increase in their farm production as an important factor. Of the consultants, 37 % considered the increase in production as one of the main factors, 32 % mentioned the unity of the producers, 26 % the group organization, and 21 % the productive and economic records, the desire to work, and the interest in new technology; 16 % mentioned the application of the methodology, good relationships, and an environment of healthy competition. Only 10 % alluded to the calendar of activities, training, consulting, companionship, understanding of the benefits of adopting technology, static diagnosis, and learning. Researchers agreed that GGAVATT is a fully replicable model that changes all participants. To a lesser extent, they mentioned that this model propitiates institutional leadership, organization, group work, social coexistence, training, obtaining technical-productive and

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financial information, the use of new technology, appropriation of the model by the producers, interest, motivation, friendship, and, overall, humanism. Producers, consultants, and researchers agreed that organized group work, training, productive information, motivation, and interest in the new technology are essential factors for success. In Tepetzintla, the adoption of new technology increased production and income and improved the tangible capital (pastures, livestock, constructions, and equipment), partially fulfilling the initial aim(6). However, after 25 years of work, intangible capitals emerged that motivated an important change in all participants. In principle, the "tacit cognitive capital" of the Huasteca, a region with an important inherited agricultural and livestock culture, was respected. True leaders emerged in favor of technological change: producers, consultants, and researchers. The Huasteca's "cultural capital" remained intact: traditions, myths, beliefs, language (Nahuatl), artistic manifestations; all this favored the development of the "institutional capital." In addition to INIFAP, the state and federal governments, other livestock organizations, research and teaching institutions, credit, training, development, and promotion institutions were also involved. The methodology was inclusive from the beginning. From the producer's point of view, the "psychosocial capital" increased. Thanks to its organization, the group obtained recognition and respect based on values, true friendship, work commitment, fair competition, individual and family participation (women's work stands out), and the ability to specify and solve problems. The development of "social capital" was fostered. The GGAVATT started as an associative figure "to the word," a group of friends, and, based on the circumstances and work versatility, legal and family associative figures were integrated. Without question, the "human capital" development is the most important; it increased the capacity of self-management and the security to communicate technically. A generation of university children was formed and became involved in the group (seven women and seven men, six zootechnical veterinarians among them). Lastly, producers were nationally recognized as "the cradle of GGAVATT." Guanajuato is an example of consistency with the application of the GGAVATT model. Since its first family dairy group, "La Labor," established in the Apaseo el Grande municipality(28), Guanajuato has maintained an average of 77 groups per year: 26 % of dairy cattle, 20.3 % of goats, 19.1 % of beef cattle, 16.3 % of sheep, 10 % of pigs, and 8.3 % of bees. An average of 1,352 livestock farmers per year have benefited from the GGAVATT model. From 1999 to date, the groups had the support of different programs. In 2002, with the support of DEPAI, 74 GGAVATT were established. DEPAI mainly contributed by paying consultants until 2006. From 2007 to 2012, the groups were supported by the Programa Soporte, and from 2012 to 2018, the Extensionism Program supported them. In 2019, the Federation withdrew its support, and the State of Guanajuato paid 100 % of the consultant's salary. Furthermore, from 1998 to 2007, farmers gad the support of the 299

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Fundación Guanajuato Produce (FGP). In addition to the support provided by the programs, the groups also benefited from the researchers from INIFAP, the Universidad de Guanajuato, the Instituto Tecnológico de Roque, pharmaceutical laboratories, and other private companies. Mexican farmers have emerged from a deeply rooted inertia after being part of the GGAVATT model and perceiving improvement. Now they associate a government program with support: thus, producers have privileged technical support and training over equipment, infrastructure, and livestock, as evidenced by an analysis of their change in attitude(29). From 2002 to 2020, 14 State technology transfer meetings have been held, more than 1,000 producers attended each event. Of the information presented in these events, the GGAVATT "United Goat Farmers of Guanajuato" stands out with a production per lactation exceeding 1,100 kg. For the genetic value of their animals, these producers have supplied stallions in government support programs, benefiting hundreds of goat farmers in Guanajuato; this has undoubtedly contributed to the increase of goat milk production in the state. From 2010 to 2019, milk production increased by 68.9 %, going from 24,980 to 42,196 t. As a result, Guanajuato climbed from 3rd to 2nd place in the national goat milk production(30). Sinaloa also applied the GGAVATT model for a considerable period, reaching productive, technological, economic, and social impacts(31). A total of 499 GGAVATT grouped 4,661 producers, which were advised by 185 technicians(31). The priority chain was dual-purpose cattle. They participated in various programs: DEPAI, SINDER, PEAT, UTEP, SOPORTE, in addition to those of the state government. This work continued until 2013-2014(32). Since 2014, without federal support, the Fundación Produce de Sinaloa took responsibility for the work and continuity of the groups. Throughout the national meetings of PRONAVATT, GGAVATT from Sinaloa were present as "successful cases": "Zavala 1" and "El Sacrificio" in 2000 and "Chinobampo" in 2003; all GGAVATT were from dual-purpose systems(31). Veracruz, like the previous states, has worked intensively with the GGAVATT model. A total of 409 groups were reported from 1982 to 2006. Of these groups, 38 % remained integrated for only one year, 17 % were together for four to five years, and 14 % more than five years. The aim is for the groups is to remain together for at least four to five years. Tepetzintla is an exemplary case as it has stayed together for more than 35 yr. Other GGAVATT overcame the 10-yr and even 15-yr barriers. These groups were generally associated with INIFAP as the responsible institution(6). In Tamaulipas, 34 GGAVATT were formed in 2009. Each one included an average of 20 producers, served by their corresponding extensionist. Groups were in 16 of the 43 300

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municipalities (37 %). Mante had seven groups and Aldama four; the rest had one or two. In 2010 and 2011, the STU provided training, supervision, and evaluation to the extensionist. A total of 559 producers of the different species-product participated. Specific surveys were conducted based on the type of producer: beef cattle (16), dual-purpose (7), goats (5), beekeeping (4), and sheep (2). It was possible to register 66 technologies: 18 were incorporated in the Feed and Forage area, 23.7 %; four were included in the area of Genetics, 6.1 %; seven in Animal Reproduction, 10.6 %; in Health 11, 16. 7 %; three in Administration, 4.5 %; three in other complements (use of implants and additives, 4.5 %; and two in honey industrialization, 3.0 %. It may seem easy, but working with producers of five different species-product was challenging(32).

Environmental, economical, productive, and social impacts of the GGAVATT model

As for the environmental impacts, the number of living fences increased in dairy and dualpurpose farms in Veracruz(15), which positively impacted vegetation and animal welfare. In Sinaloa, the use of technological components was promoted in the area of animal feeding: preparation and use of multinutritional blocks, establishment and management of pastures, use of living fences, new options of forage species and silages; decreasing soil and water loss by 88 % and 50 %(26,31). One of the technologies that the GGAVATT model promotes is the use of technical and economic records. Analyzing the information in these records makes it possible to evaluate the productive and economic impact of technology implementation in the production units participating in a GGAVATT. A study analyzed the information of 206 dual-purpose farms in Campeche, Colima, Nayarit, Sinaloa, and Veracruz; these farms received technical assistance and livestock training during 2011 and 2012. Two types of producers were identified: one with a low technological level, in which 76 % of producers apply on average 33 % of the proposed innovations, and another with intermediate technical level, in which 24 % of producers use 66 % of the innovations. After comparing the productivity and profitability variables of these two types of producers, it was found that intermediate producers obtain an additional 1.86 L of milk per cow per day and 8 % more profitability(33). Without a doubt, one of the most significant impacts of the GGAVATT model has been the use of technological innovations and its social implications, in which producers develop technical and organizational abilities. This is demonstrated by the studies carried out with dairy producers of Michoacán(21) and Guanajuato(18) and dual-purpose cattle producers in

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Veracruz(23,24,25) and Sinaloa(19). These studies mention that the production units that participated in the GGAVATT model increased their use of innovations.

Perspectives of the GGAVATT model

The GGAVATT model has been adapted and maintained for more than 30 years, suggesting that the work dynamics between producers, professional experts, and government institutions would resurface under the same conditions and support. This assumption is based on the following: The GGAVATT model has been adapted to the great diversity of programs and projects at the three levels of government(1). Furthermore, INIFAP has played a leading role in most of them. Chronologically, in 1986, the Technical Exchange Groups of the Rural Development Districts (RDD) stand out; INIFAP was responsible for the technological packages. From 1990 to 1995, the Agricultural and Forestry Research and Extension Program (PIEX) was established with RDD, FIRA, and BANRURAL, collateral with FIRCO. In 1995, the National Coordination of Produce Foundations (COFUPRO) was integrated into the states with their corresponding Produce Foundations. In 1996, SAGARPA remained as normative, and operating by the States; the Development Commissions were formed with the participation of INCA Rural, INIFAP, FIRCO, and the Commercialization and Development agency (ASERCA). In this same year, the Programa Alianza para el Campo, the National Training and Extension System (SINDER), and the Elementary Program for Technical Assistance (PEAT) began, with good participation of the Rural INCA; the Sustainable Rural Development Program was also established (DRS). In 1998, very important for the GGAVATT, the Comprehensive Agricultural Project Development Program (DPAI) was established to develop productive basins. In 2001, SINDER and PEAT merged into the Professional Services and Extension Program (PESPRO), which became the Capacity Development Program (PRODESCA). In 2003, all programs merged and formed the Livestock Promotion and Development of Comprehensive Projects (DPAI), in which GGAVATT continues; at the same time, the Livestock Productivity Incentive Program (PROGAN) is established with national coverage. Finally, in 2008, the General Coordination of Livestock invited INIFAP to participate in the National System of Evaluation Centers as a Specialized Technical Unit (STU), through which it continued to support farmer groups with about 1,000 PSP. As previously mentioned, the GGAVATT model was adapted to all the programs and projects, which motivates candidates to continue participating.

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Conclusions The GGAVATT model is a process technology that granted INIFAP researchers the opportunity to participate in the farms of organized producers to validate, transfer and innovate the technologies created in experimental stations. The GGAVATT is a participatory model based on the collaboration between producers and researchers; it increases the productivity and profitability of the LPU, generates tangible and intangible capital, and increases the producers' social, intrinsic, horizontal, and vertical capital. The GGAVATT can be successfully used in any livestock production system and agroecological region of the country; it may be an excellent conduit to enhance natural resources and reduce climate change impact. This model has an adequately documented work methodology; thus, it can be replicated in all Mexican states. The GGAVATT model may be the axis of technological transformation and integral development of national livestock.

Acknowledgments

The authors thank all participating technicians and producers, especially the MVZ Jesús Manuel Pérez Saldaña✝ and the producer Caín Román Ponce✝ for their invaluable commitment to validation, innovation, and technology transfer.

Conflicts of interest

The authors declare that they have no conflicts of interest regarding the work presented in this report. Literature cited: 1. Román PH, Rodríguez ChMA, Aguilera SR, Rivera VGH. La transferencia de tecnología en las regiones tropicales de México. En: Rodríguez RO editor. Estado del arte sobre investigación e innovación tecnológica en ganadería bovina tropical. Segunda ed. Ciudad de México, México: RED GATRO CONACYT; 2018:331-343.

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Revista Mexicana de Ciencias Pecuarias

Edición Bilingüe Bilingual Edition

Rev. Mex. Cienc. Pecu. Vol. 12 Suplemento 3, pp. 1-307, NOVIEMBRE-2021

ISSN: 2448-6698

Pags. Logros, retos y perspectivas de la investigación en mejoramiento genético de bovinos productores de carne en el INIFAP Beef cattle genetic improvement research at the INIFAP: accomplishments, challenges and perspective Ángel Ríos Utrera, Guillermo Mar�nez Velázquez, René Calderón Chagoya, Moisés Montaño Bermúdez, Vicente Eliezer Vega Murillo …..…………..…………..…………..…………..…………..………………………………….…1

El ganado bovino Criollo Coreño del occidente de México en la producción de carne: caracterización, retos y perspectivas

Criollo Coreño cattle in western Mexico: characterization, challenges and outlook Guillermo Mar�nez-Velázquez, Ángel Ríos-Utrera, José Antonio Palacios-Fránquez, Vicente Eliezer Vega-Murillo, Moisés Montaño-Bermúdez …….……….………………………………….………..….......…...………………..23

Biotecnologías reproductivas en el ganado bovino: cinco décadas de investigación en México

Reproductive biotechnologies in beef cattle: five decades of research in Mexico Jorge Víctor Rosete Fernández, Horacio Álvarez Gallardo, David Urbán Duarte, Abraham Fragoso Islas, Marco Antonio Asprón Pelayo, Ángel Ríos Utrera, Sandra Pérez Reynozo, José Fernando De La Torre Sánchez ……..….……………………………..…………..…………..…………..…………..…..………..……..….....……..…………….39

Principales aportes de la investigación del INIFAP a la nutrición porcina en México: retos y perspectivas

Main contributions of INIFAP research to swine nutrition in Mexico: challenges and perspectives José Antonio Rentería Flores, Sergio Gómez Rosales, Luis Humberto López Hernández, Gerardo Ordaz Ochoa, Ana María Anaya Escalera, César Augusto Mejía Guadarrama, Gerardo Mariscal Landín ..........……..…….....……...……..…….....…...……..…….....…...……..…….....…...……..…….....…...……..…….....…...……..…….......………79

Antecedentes y perspectivas de algunas enfermedades prioritarias que afectan a la ganadería bovina en México

Background and perspectives of certain priority diseases affecting cattle farming in Mexico Carmen Rojas Mar�nez, Elizabeth Loza Rubio, Sergio Darío Rodríguez Camarillo, Julio Vicente Figueroa Millán, Francisco Aguilar Romero, Rodolfo Esteban Lagunes Quintanilla, José Francisco Morales Álvarez, Marco Antonio San�llán Flores, Guadalupe Asunción Socci Escatell, Jesús Antonio Álvarez Mar�nez ......…………..……………………….…..111

Salud porcina: historia, retos y perspectivas

Swine health: history, challenges and prospects José Francisco Rivera-Benítez, Jazmín De la Luz-Armendáriz, Luis Gómez-Núñez, Fernando Diosdado Vargas, Guadalupe Socci Escatell, Elizabeth Ramírez-Medina, Lauro Velázquez-Salinas, Humberto Ramírez-Mendoza, Maria Antonia Coba Ayala, Catalina Tuﬁño-Loza, Marta Macías García, Víctor Carrera-Aguirre, Rebeca Mar�nez-Bau�sta, María José Mar�nez-Mercado, Gerardo Santos-López, Irma Herrera-Camacho, Ignacio Siañez-Estrada, Manuel Zapata Moreno …..…………………...……………………...……………………...……………………...……………………………………………………………........…………………...………..149

Control y prevención de nematodosis en pequeños rumiantes: antecedentes, retos y perspectivas en México

Control and prevention of nematodiasis in small ruminants: background, challenges and outlook in Mexico David Emanuel Reyes-Guerrero, Agus�n Olmedo-Juárez, Pedro Mendoza-de Gives……………………………………………………....……………………………………………………………………………………………………...…….…….….......186

Enfermedades infecciosas de relevancia en la producción caprina, historia, retos y perspectivas

Important infectious diseases in goat production in Mexico: history, challenges and outlook Gabriela Palomares Resendiz, Francisco Aguilar Romero, Carlos Flores Pérez, Luis Gómez Núñez, José Gu�érrez Hernández, Enrique Herrera López, Magdalena Limón González, Francisco Morales Álvarez, Francisco Pastor López, Efrén Díaz Aparicio …………………..……..……………………………………….………..205

Resultados e impacto de la investigación en genética y mejoramiento genético de las abejas melíferas desarrollada por el INIFAP en México

Results and impact of research on honeybee genetics and breeding conducted by INIFAP in Mexico Miguel Enrique Arechavaleta-Velasco, Claudia García-Figueroa, Laura Yavarik Alvarado-Avila, Francisco Javier Ramírez-Ramírez, Karla Itzel Alcalá-Escamilla ……………………………………………..…………..…………..224

Rehabilitación de praderas degradadas en el trópico de México

Rehabilitation of degraded pastures in the tropics of Mexico Javier Francisco Enríquez Quiroz, Valen�n Alberto Esqueda Esquivel, Daniel Mar�nez Méndez…………………………..……..……..……..……..……..……..……..……..……………………….……………………………………….…………...243

Los pastizales y matorrales de zonas áridas y semiáridas de México: Estatus actual, retos y perspectivas

The grasslands and scrublands of arid and semi-arid zones of Mexico: Current status, challenges and perspectives Pedro Jurado-Guerra, Mauricio Velázquez-Mar�nez, Ricardo Alonso Sánchez-Gu�érrez, Alan Álvarez-Holguín, Pablo Alfredo Domínguez-Mar�nez, Ramón Gu�érrez-Luna, Rubén Darío Garza-Cedillo, Miguel Luna-Luna, Manuel Gustavo Chávez-Ruiz …………………………………………………………………....…261

Historia y perspectivas del modelo GGAVATT (Grupos Ganaderos de Validación y Transferencia de Tecnología)

History and perspectives of the GGAVATT model (Groups for Livestock Technological Validation and Transfer) Heriberto Román Ponce, Miguel Arcangel Rodríguez Chessani, José Antonio Espinosa García, Tomás Arturo González Orozco, Alejandra Vélez Izquierdo, Juan Prisciliano Zárate Mar�nez, Martha Eugenia Valdovinos Terán, Rubén Cris�no Aguilera Sosa, Rafael Guarneros Altamirano, Rubén Santos Echeverría, Héctor Macario Bueno Díaz, Ubaldo Aguilar Barradas……………………………………..………………....…………………..…………..…………..…………..…………..…………..286

Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 12 Suplemento 3, pp. 1-307, NOVIEMBRE-2021

CONTENIDO CONTENTS

Rev. Mex. Cienc. Pecu. Vol. 12 Suplemento 3, pp. 1-307, NOVIEMBRE-2021