RMCP Vol. 13 Num. 2 (2022): April-June [english version]

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Edición Bilingüe Bilingual Edition

Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 2, pp. 323-583, ABRIL-JUNIO-2022

ISSN: 2448-6698

Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 2, pp. 323-583, ABRIL-JUNIO-2022


REVISTA MEXICANA DE CIENCIAS PECUARIAS Volumen 13 Numero 2, Abril-Junio 2022. 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 mayo de 2022. Sistema silvopastoril Alnus acuminata, Morus nigra y ganado vacuno criollo en Bongará, Amazonas, Perú. Fotografía: INDES CES UNTRM-Michel León

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. Jorge Alberto López García, 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).

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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

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REVISTA MEXICANA DE CIENCIAS PECUARIAS

REV. MEX. CIENC. PECU.

VOL. 13 No. 2

ABRIL-JUNIO-2022

CONTENIDO Contents

ARTÍCULOS Articles

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Efecto de extractos naturales sobre la estabilidad oxidativa de hamburguesas de carne de cerdo durante el almacenamiento refrigerado Effect of natural extracts on the oxidative stability of pork hamburgers during refrigerated storage María Josefina Graciano Cristóbal, Javier Germán Rodríguez Carpena, María Teresa Sumaya Martínez, Rosendo Balois Morales, Edgar Iván Jiménez Ruiz, Pedro Ulises Bautista Rosales …………………………….………………………………………………………………………………………………………….323 Diagnóstico de la calidad sanitaria de queserías artesanales en Salinas, San Luis Potosí Diagnosis of the health quality of artisanal cheese dairies in Salinas, San Luis Potosí Rocío Rodríguez-Gallegos, Gregorio Álvarez-Fuentes, Juan Antonio Rendón-Huerta, Juan Ángel Morales-Rueda, Juan Carlos García-López, Luis Alberto Olvera-Vargas …………………..340 Perspectivas sobre la continuidad, calidad de leche y entorno en unidades de producción de leche en el estado de Aguascalientes, México Perspectives on continuity, milk quality and environment in milk production units in the state of Aguascalientes, Mexico Carlos Eduardo Romo-Bacco, Neftali Parga-Montoya, Arturo Gerardo Valdivia-Flores, Rodrigo Gabriel Carranza-Trinidad, María del Carmen Montoya Landeros, Abril Areli Llamas-Martínez, María Mayela Aguilar Romero ……………………………………………………………………..……………………………….357 Actividad antimicrobiana de plantas nativas de Sonora, México, contra bacterias patógenas aisladas de leche de vacas diagnosticadas con mastitis Antimicrobial activity of plants native to Sonora, Mexico, against pathogenic bacteria isolated from milk from cows diagnosed with mastitis Jesús Sosa-Castañeda, Carmen Guadalupe Manzanarez-Quin, Ramón Dolores Valdez-Domínguez, Cristina Ibarra-Zazueta, Reyna Fabiola Osuna-Chávez, Edgar Omar Rueda-Puente, Carlos Gabriel Hernández-Moreno, Alejandro Santos-Espinosa, Alejandro Epigmenio-Chávez, Claudia Vanessa García-Baldenegro, Tania Elisa González-Soto, Ana Dolores Armenta-Calderón, Priscilia Yazmín Heredia Castro …………………………………………………………………………………..……………………………375

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Pharmacokinetic analysis of intraarticular injection of insulin and its effect on IGF-1 expression in synovial fluid of healthy horses Análisis farmacocinético de la inyección intraarticular de insulina y su efecto sobre la expresión del IGF-1 en el líquido sinovial de caballos sanos Fernando García-Lacy, Lilia Gutiérrez-Olvera, María Bernad, Lisa Fortier, Francisco Trigo-Talavera, Margarita Gómez-Chavarín, Alejandro Rodríguez-Monterde …………….…….………………………………391 Productive performance of sheep fed buffel grass silage in replacement of corn silage Desempeño productivo de ovinos alimentados con ensilaje de pasto buffel en sustitución de ensilaje de maíz Tiara Millena Barros e Silva, Gherman Garcia Leal de Araújo, Tadeu Vinhas Voltolini, Mário Adriano Ávila Queiroz, Sandra Mari Yamamoto, Fábio Nunes Lista, Glayciane Costa Gois, Salete Alves de Moraes, Fleming Sena Campos, Madriano Christilis da Rocha Santos ………………..…………………….408

REVISIONES DE LITERATURA Reviews Función ovárica y respuesta a la sincronización del estro en ganado Criollo en México. Revisión Ovarian function and response to estrus synchronization in Creole cattle in Mexico. Review Elizabeth Pérez-Ruiz, Andrés Quezada- Casasola, José Maria Carrera-Chávez, Alan Álvarez-Holguín, Jesús Manuel Ochoa-Rivero, Manuel Gustavo Chávez-Ruiz, Sergio Iván Román-Ponce ………..……422 Ultrasonography and physiological description of essential events for reproductive management in dairy cattle. Review Ultrasonografía y descripción fisiológica de eventos esenciales para el manejo reproductivo en ganado lechero. Revisión María Elena Torres-Lechuga, Juan González-Maldonado ……………………………………….……………….452 Demi-embryo reconstitution, a factor to consider for the success of embryo bisection. Review La reconstitución de demi-embriones: un factor a considerar para el buen éxito de la bisección de embriones. Revisión Alfredo Lorenzo-Torres, Raymundo Rangel-Santos, Agustín Ruíz-Flores, Demetrio Alonso AmbrízGarcía …………………………………………………………………….………………………………………………………..473 Estrés por calor en ganado lechero con énfasis en la producción de leche y los hábitos de consumo de alimento y agua. Revisión Heat stress in dairy cattle with emphasis on milk production and feed and water intake habits. Review Abelardo Correa-Calderón, Leonel Avendaño-Reyes, M. Ángeles López-Baca, Ulises Macías-Cruz ………………………………………………………………………………..……………………………..……………………….488

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Concentrado de proteína de papa: una posible alternativa al uso de antibióticos en las dietas para lechones destetados. Revisión Potato protein concentrate: a possible alternative to the use of antibiotics in diets for weaned piglets. Review Erick Alejandro Parra Alarcón, Teresita de Jesús Hijuitl Valeriano, Gerardo Mariscal Landín, Tércia Cesária Reis de Souza …………………………………..………………………………………………………….510

Apis mellifera en México: producción de miel, flora melífera y aspectos de polinización. Revisión

Apis mellifera in Mexico: honey production, melliferous flora and pollination aspects. Review Fernanda Baena-Díaz, Estrella Chévez, Fortunato Ruiz de la Merced, Luciana Porter-Bolland……..525

NOTAS DE INVESTIGACIÓN Technical notes Induced lactation in Holstein cattle with no exogenous progesterone supplementation and with reduced doses of estradiol benzoate Inducción de la lactancia en ganado Holstein con dosis reducidas de benzoato de estradiol y sin suplementar progesterona exógena Juan González-Maldonado, Raymundo Rangel-Santos, Gustavo Ramírez-Valverde, Jaime GallegosSánchez, Lorenzo Beunabad-Carrasco, Javier-Antillón Ruiz …………………………….………………..….549 Frecuencia y puntaje de jadeo en bovinos productores de carne en finalización intensiva durante el verano Panting frequency and score in beef cattle in intensive finishing during summer in the dry tropics Ana Mireya Romo Valdez, Jesús José Portillo Loera, Jesús David Urías Estrada, Alfredo Estrada Angulo, Beatriz Isabel Castro Pérez, Francisco Gerardo Ríos Rincón ……………………………………….559 Arreglos silvopastoriles con Alnus acuminata y su efecto sobre parámetros productivos y nutricionales del componente forrajero Silvopastoral arrangements with Alnus acuminata and their effect on productive and nutritional parameters of the forage component José Américo Saucedo-Uriarte, Segundo Manuel Oliva-Cruz, Jorge Luis Maicelo-Quintana, Jegnes Benjamín Meléndez-Mori, Roicer Collazos-Silva ……………………………………………….…………573

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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.

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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.

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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.

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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.

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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.

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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).

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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.

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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:

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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


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.

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

xg

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

vs

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.

IX


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.

2.

3.

4.

5.

6.

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.

7.

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).

8.

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.

9.

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

X


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

XI


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)

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.

XI)

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.

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.

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)

vs

versus

xg

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.v13i2.5759 Article

Effect of natural extracts on the oxidative stability of pork hamburgers during refrigerated storage

María Josefina Graciano Cristóbal a Javier Germán Rodríguez Carpena b* María Teresa Sumaya Martínez a Rosendo Balois Morales a Edgar Iván Jiménez Ruiz a Pedro Ulises Bautista Rosales a

a

Universidad Autónoma de Nayarit. Secretaría de Investigación y Posgrado. Laboratorio de Tecnología de Alimentos. Ciudad de la Cultura “Amado Nervo” S/N. 63155. Tepic, Nayarit, México. b

Centro de Investigación y Transferencia Tecnológica. Laboratorio de Ciencia y Tecnología de la Carne. Tepic, Nayarit, México.

*Corresponding author: german.rc@uan.edu.mx

Abstract: The effect of three natural extracts made from culinary spices with antioxidant activity on the oxidative stability of lipids and proteins, color changes, and sensory quality of cooked pork during 12 days of refrigerated storage was evaluated. Hamburger-type model systems were made with the Longissimus thoracis et lomborum muscle, dorsal fat, salt, water and the corresponding extract. The antioxidant activity of the extracts was determined by the DPPH and ABTS+ methods, while the oxidation of lipids and proteins by TBA-RS and DNPH, respectively. For the color evaluation, the parameters of luminosity (L*) and Hue angle (°h) were used. The sensory analysis was carried out with an untrained panel, which evaluated the attributes of taste, color, smell and texture. The statistical processing of the data obtained 323


Rev Mex Cienc Pecu 2022;13(2):323-339

on antioxidant activity, lipid and protein oxidation, as well as color, was performed by an analysis of variance. The sensory evaluation was processed with nonparametric statistics. Extract two had the highest antioxidant activity (P≤0.05); the three extracts managed to inhibit (P≤0.05) lipid oxidation in the hamburgers (P≤0.05); however, none of the three extracts managed to inhibit protein oxidation. There were also no differences (P≥0.05) with respect to the L* parameter, while the values of °h showed that the three extracts managed to preserve the color of the cooked hamburgers during refrigerated storage. Finally, the sensory evaluation showed that none of the three extracts altered the organoleptic quality of the hamburgers. Key words: Natural Extracts, Culinary Spices, Antioxidants, Lipid Oxidation, Protein Oxidation.

Received: 12/08/2020 Accepted: 05/07/2021

Introduction Meat is especially prone to oxidation processes due to its complex structures and composition, including lipids, unsaturated fatty acids and myofibril systems(1). Meat lipids are chemically unstable and easy to oxidize, especially during handling, cooking and postmortem storage(2). Changes associated with lipid oxidation include rancid odor, discoloration, loss of nutritional value, decrease in shelf life and the formation of toxic compounds, which can be harmful to the health of consumers(2). Likewise, protein oxidation implies the loss of nutritional value in meat, causing a decrease in protein bioavailability, a change in amino acid composition, a decrease in protein solubility, loss of proteolytic activity and protein digestibility(3). Recently, with the outbreak of the coronavirus 2019 (COVID-19) pandemic, some researchers have evaluated the changes in the pattern of purchasing behavior of consumers, reporting an increase in the tendency to change habits, especially food and nutritional ones, towards the consumption of functional and nutraceutical foods, with a tendency towards healthier types of meals, made with natural and homemade preservatives(4,5). Antioxidant strategies based on the use of natural sources may be a viable option to enrich meat with bioactive compounds that promote health and, in turn, would prevent degradation

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Rev Mex Cienc Pecu 2022;13(2):323-339

due to oxidation. Antioxidant phytochemicals can be applied through the formulation of foods or dietary strategies(6). The inclusion of natural antioxidants in meat products has been reported by different authors with a positive effect in terms of control of oxidative processes(7,8). Culinary spices, such as cinnamon, cloves, coriander, onion, black pepper, garlic, oregano, bay leaves, turmeric, among others, are an important source of bioactive compounds with antioxidant activity(9,10). Few studies report the inclusion of culinary spice mixtures in pork products(11,12). Therefore, the objective of this study was to evaluate the antioxidant protection of proteins and lipids in processed pork through natural extracts made from mixtures of culinary spices.

Material and methods Preparation of extracts

A total of three extracts were prepared, considered as treatments #1 (200 g of onion, 20 g of coriander, 15 g of oregano), #2 (200 g of onion, 20 g of coriander, 15 g of bay leaves) and #3 (200 g of onion, 20 g of coriander, 2 g of black pepper, 18 g of green chili, 14 g of garlic, 6 g of salt, 8 g of calyces of green roselle (variety UAN-4) with the different culinary spices, plus 50 ml of a base two-spice mixture and 75 ml of lemon juice. Their preparation consisted of grinding each of the ingredients at the same time with the help of a conventional blender until obtaining a pasty consistency. Subsequently, the paste was placed in 50 ml Falcon tubes for its centrifugation (5,000 rpm, 5 min). The supernatant was used as the extract to be mixed with the meat that was used to prepare the model systems.

Preparation of the base two-spice mixture

The preparation of the base two-spice mixture consisted of macerating 5 g of cinnamon and 5 g of clove powder (separately) in 10 ml of rum (40 % of alcohol) for 24 h. The supernatant obtained from the two macerations was mixed to form the base two-spice mixture.

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Rev Mex Cienc Pecu 2022;13(2):323-339

Preparation of hamburger-type model systems

Hamburger-type model systems composed of 80 % pork (Longissimus thoracis et lomborum muscle), 10 % dorsal fat, 1 % salt, 9 % water (for control hamburgers), and 4.5 % water and 4.5 % extract in the treated hamburgers, were prepared. The preparation of the hamburgers consisted of grinding the meat and fat in a meat grinder with a 1/8” sieve (Torrey® brand, model M12-FS), once the meat was ground, the salt, water and extract (if applicable) were mixed until a homogeneous mixture was obtained. The mixture was packaged under high vacuum to remove any internal air bubbles that might form. From the mixture, portions of 60 g were weighed and with the help of a metal ring of 8 cm in diameter, the hamburger-type model systems were made. The hamburgers were previously cooked at a temperature of 230 °C for 5 min on each side on an Oster Bioceramic® grill From each treatment (without extract, extract #1, #2 and #3), the hamburger-type model systems (three replications per treatment and per each day of sampling) were made to evaluate the oxidative stability of color, lipids and proteins. The hamburgers were placed in polystyrene trays and covered with transparent oxygen-permeable film paper (14μm thick and 10,445 ml/m2/24 h) and stored in refrigeration at 4 ± 2 °C with white fluorescent light (1,620 lux) 24 h. The samplings were carried out on days 0, 3, 6, 9 and 12.

Determination of the antioxidant activity Total phenolic compounds were determined by the method of Stintzing et al(13). The antioxidant activity based on the 1,1-diphenyl-2-picrilhidrazil (DPPH) method was evaluated according to the procedure reported by Morales and Jiménez-Pérez(14). The antioxidant activity based on the ABTS+ method was evaluated according to the procedure developed by Kuskoski et al(15). The TBA-RS technique (thiobarbituric acid reactive substances) was evaluated according to the technique described by Ganhão et al(16), which allows the quantitative determination of secondary metabolites of lipid oxidation. The determination of the total carbonyls that are generated during the oxidative processes of meat proteins was based on the DNPH technique described by Ganhão et al(17).

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Evaluation of the color of the hamburgers

To determine the deterioration of the color of the meat due to storage over time, color evaluations were carried out by instrumental measurement(18) on the surface of the hamburgers treated with the extracts, during the days of storage. A Minolta® colorimeter model CR-410 was used. The measurements were made in three different randomly chosen zones and at room temperature (≈ 25 °C). The CIE-L*a*b* color measurement system was used and the Hue angle (°h) (tone) was calculated as indicated by García-Tejeda et al(19): °h=tan-1(b*/a*), when a*>0 and b*≥0 or °h=180 + tan-1 (b*/a*) when a*<0. The total color difference (ΔE) was calculated to evaluate the total color changes suffered by the hamburgers as a result of the refrigerated storage days. Therefore, ΔEC-T was calculated between the samples of the control group (C) and the treated group (T) using the CIE-L*a*b* color scale for each measurement day as follows: ΔEC-T = [(L*T - L*C)² + (a*T - a*C)² + (b*T – b*C)²]1/2

Sensory analysis

For sensory evaluation, each hamburger cooked under the conditions described above was cut into four parts to offer it to an untrained panel of 35 people. The panelists were instructed to evaluate the attributes of smell, color, taste and texture, marking with an “X” the rating they considered appropriate to assign to each sample, using a hedonic test(20) with a sevenpoint scale where the value of 1 corresponded to “I dislike it very much”, the 2 to “I dislike it a lot”, 3 to “I dislike it a little”, 4 to “I neither like it nor dislike it”, 5 to “I like it a little”, 6 to “I like it a lot” and 7 to “I like it very much”.

Statistical analysis

Data on TPC content, antioxidant activity, color determination and inhibition of lipid and protein oxidation were processed using an analysis of variance under a completely randomized design. When the analysis was significant (P≤0.05), a Tukey mean comparison test was performed. The results of total color difference and sensory analysis were performed using the Kruskal-Wallis Hypothesis test (P≤0.05). Pearson’s correlation coefficients were calculated to establish linear associations between variables of interest. The statistical package used was Minitab v.16.0.

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Results and discussion Content of total phenolic compounds and antioxidant activity

The three extracts prepared had a high content of TPC (Figure 1) and good antioxidant activity determined by the DPPH (Figure 2) and ABTS+ methods. The ABTS+ test showed the same antioxidant activity behavior as with the DPPH technique. When analyzing the TPC, we can observe that extracts #2 and #3 were the ones that had the highest TPC content, and the highest antioxidant activity is observed with treatment #2, followed by treatment #3. This may be mainly due to the ingredients that differentiate each of these two extracts, the greater antioxidant activity can be attributed mainly to the derivatives of catechin and procyanidins (cinnamtannin B1) and flavonic heterosides derived from kaempferol that have been reported as the majority in the bay leaves present in extract #2(21) and to chlorogenic acid and its isomers, caffeic acid and protocatechuic acid derivatives reported as the majority and with high antioxidant activity in green roselle calyces in extract #3(22). These ingredients could exert a greater synergistic effect with the rest of the spices, potentiating their antioxidant activity. So far, the use of green roselle calyces to inhibit lipid and protein oxidation has not been reported, but their antioxidant activity has already been reported(23). Figure 1: Content of total phenolic compounds of three extracts

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Means with a different superscript denote a significant difference (P<0.05).

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Figure 2: Antioxidant activity of three extracts by the DPPH method

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Means with a different superscript denote a significant difference (P<0.05).

The extracts prepared showed a significant correlation (P≤0.05) between TPC and antioxidant activity by the DPPH and ABTS+ methods of r= 0.798 and 0.751, respectively. Therefore, it can be said, according to this analysis, that the antioxidant activity of the extracts is a function of the total phenolic compounds.

Determination of thiobarbituric acid reactive substances The results of TBA-RS (Figure 3) showed a significant difference (P≤0.05) between the samples. The three extracts were able to decrease the concentration of malonaldehyde (MDA) with respect to the hamburgers without extract throughout the 12 d of storage, with extracts #1 and #2 being the ones that achieved a significantly better effect in relation to extract #3.

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Figure 3: Effect of extracts on MDA concentration of pork hamburgers cooked and stored at 4 °C for 12 d

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Means with a different superscript denote a significant difference (P<0.05).

TBA-RS values above 0.5 mg MDA/kg of sample are critical as they indicate a level of products of lipid oxidation that produce a rancid odor and taste that can be easily detected by consumers(24). This level of rancidity was reached after cooking in the hamburgers without extract, increasing its values during subsequent refrigerated storage, indicating that the cooking process may be able to accelerate lipid oxidation rates. The intense antioxidant activity shown by the extracts in the in vitro tests (Figure 2) meant an efficient protective effect of the extracts on lipids in real meat products. Other authors(12,25) have obtained similar results, managing to reduce the concentration of TBA-RS by using onion and garlic in pork, as well as tocopherols and ascorbic acid in chicken liver pâté, respectively. However, few studies have attempted to demonstrate the efficacy of natural antioxidant mixtures against lipid oxidation(26). In one of them(11), they used a mixture of essential oils of garlic, cinnamon, cloves and rosemary, and obtained favorable results by inhibiting lipid oxidation in Iberian hams. Some substances, such as MDA, have been reported as compounds with toxic and mutagenic potential for humans(27). So, extracts #1, #2 and #3 can be an efficient strategy to avoid the increase in adverse effects caused by lipid oxidation in cooked pork hamburgers.

Determination of total protein carbonyls

The results of carbonyls in cooked hamburgers (Figure 4) presented a significant difference (P≤0.05) between the samples only on d 6 of storage, with extracts #1 and #2 showing a reduction in carbonyls with respect to the hamburgers without extract. However, this effect

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was not effective on the other sampling days. So, it could be considered that none of the three extracts applied achieved an efficient inhibitory action on protein oxidation in cooked hamburgers. It is known that the increased susceptibility of cooked meats to protein carbonylation can be attributed to the disruption of myofibrillar tissues as a result of high temperatures, which in turn leads to the release of non-heme (non-protein) iron and a greater incorporation of oxygen into the system. Non-heme iron has been recognized as a major promoter of the formation of carbonyl residues from myofibrillar proteins(28). Compared to the results of the present study, other researchers(29) also found high levels of protein carbonyls in pork subjected to cooking and subsequent cold storage, which reveals the impact of high temperatures on the oxidative stability of muscle proteins. Figure 4: Effect of extracts on carbonyl concentration of pork hamburgers cooked and stored at 4 °C for 12 d

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Means with a different superscript denote a significant difference (P<0.05).

According to the results of the present study, other authors have also reported that certain antioxidant strategies with proven efficacy against lipid oxidation were not effective against protein oxidation(30). On the other hand, it is known that the formation of lipid oxidation in meat systems occurs more rapidly than the oxidative degradation of myofibrillar proteins(31). The positive correlation (r=0.560; P=0.000) found in the present study between protein and lipid oxidation in cooked hamburgers supports the theory that lipid and protein oxidation are coupled in food meat systems. In fact, some studies have reported such an interaction between lipids and proteins(32,33), which supports the theory that reactive oxygen species (ROS) formed during the early stages of lipid oxidation can bind to susceptible amino acid residues to trigger their oxidative degradation(34). In contrast to the results of lipid oxidation, the differences between the treatments with respect to protein oxidation were not so clear, possibly due to the complex

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structural composition of the proteins that comes to provide some protection and their degradation does not follow a logical pattern, which coincides with other authors(35,36).

Color evaluation

The results of the luminosity parameter (Figure 5) indicate a slight loss of brightness for all the samples, since the values between them showed a difference of less than 3 points, which ranged from 72.44 to 69.67 during the 12 d of storage, with a significant difference (P≤0.05) between the samples during the three first days of storage, where the hamburgers with extract had a greater luminosity with respect to the hamburgers without it. However, at the end of the storage period (d 12), all hamburgers with extract lost luminosity significantly (P≤0.05) with respect to hamburgers without extract. Figure 5: Effect of extracts on the luminosity or brightness of pork hamburgers cooked and stored at 4 °C for 12 d

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Means with a different superscript denote a significant difference (P<0.05).

For the values obtained of the Hue angle (Figure 6), it can be observed that the addition of the studied extracts had a significant effect (P<0.05) with respect to the hamburgers without extract, since the extracts had a faint orange color with slight touches of brown as a result of the extraction of pigments from the spices and the base two-spice mixture used. The pigments were probably transferred to the hamburgers during their preparation, causing the modification of their color and intensifying it after the cooking process, thus causing a brown coloration with slight touches of gold, or in other words a toasted shade. During the initial

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day of storage, the hamburgers without extract showed significantly (P≤0.05) a lower brown or toasted shade. During the third day of storage, the hamburgers without extract matched the color with respect to the treated hamburgers. However, from d 6 of storage, the hamburgers without extract significantly increased (P≤0.05) the Hue angle value, with an upward trend standing out, and showing a yellow shade with greenish touches with respect to the hamburgers with extracts. It is highlighted that hamburgers with extract #1 better protect toasted coloration throughout the 12 d of storage. Figure 6: Effect of extracts on the color of pork hamburgers cooked and stored at 4 °C for 12 d

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Means with a different superscript denote a significant difference (P<0.05).

The protection given by the extracts may be due to their antioxidant defense, which may be responsible for protecting heme pigments against oxidative processes in cooked hamburgers, which is confirmed by the significant correlation of r=0.690 (P=0.001) between color and lipid oxidation. That is, the bioactive compounds present in the extracts could possibly inhibit the formation of primary products of lipid oxidation (mainly hydroperoxides), which oxidize ferrous iron (Fe2+) of oxymyoglobin to its ferric form (Fe3+) present in metmyoglobin (responsible for discoloration)(37), thus inhibiting the discoloration of hamburgers. Table 1 shows the total numerical color difference (ΔE) between hamburgers without extract and treated hamburgers (with extract #1, #2 and #3) during d 0, 3, 6, 9 and 12 of refrigerated storage. According to some authors(38), color modifications measured instrumentally between two given meat samples can be considered as notable visual changes when ΔE values are greater than 2. In the case of cooked hamburgers, a ΔE greater than 2 was found for hamburgers with extract #1 on d 12 of storage. However, statistically, there was a significant difference (P≤0.05) in the ΔE between the hamburgers treated from d 3 of storage, where the hamburgers with extract #1 showed the greatest color differential during d 6, 9 and 12 of storage. This coincides with the results obtained of the Hue angle of the cooked hamburgers, 333


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where, from d 6 of storage, all the hamburgers with extract presented a significant difference with respect to the hamburgers without it, being precisely the hamburgers with extract #1 the ones that presented the greatest difference. That is, extract #1 showed significantly greater efficacy (P≤ 0.05) in preserving the toasted color of cooked hamburgers throughout the 12 d of storage. Table 1: Total color difference (ΔE) between the sample without extract and the samples treated in pork hamburgers cooked and stored at 4 °C for 12 d Days Samples 0 3 6 9 12 a c a a Extract #1 1.51 0.56 1.35 1.95 2.32a Extract #2 1.57a 1.10a 0.94b 1.36b 1.49c Extract #3 1.80a 1.09b 0.76c 1.09c 1.50b Means with a different superscript within one day of storage denote a significant difference between extracts (P≤0.05).

Sensory analysis

The results of the sensory evaluation (Figure 7) indicate that, in relation to the attributes of smell and color, there was no significant difference (P˃0.05) between the treatments, that is, the application of the extracts did not perceptively modify the smell or the color of a cooked meat. In the evaluation corresponding to the attribute of taste, there were significant differences (P≤0.05) between the different sources of variation used in the experimental design, with hamburgers with extract #3 being the ones that had the highest preference among the panelists, surpassing the hamburgers without extract, while the ones with the lowest acceptance were the hamburgers with extract #1. Regarding the attribute of texture, there were statistically significant differences between treatments, being the hamburgers added with extract # 3 the ones that showed greater acceptability for this attribute and equalizing the texture of the hamburgers that did not have it. While the hamburgers that were added with extract #1 were the ones that had the least acceptance among the tasters. In general, the addition of the three extracts to the hamburgers did not have a negative effect on the preference of the tasters, since the results of the four attributes (smell, color, taste and texture) evaluated were on the scale of 4 to 6, which ranges from “I neither like it nor dislike it” to “I like it very much”, with the preference for hamburgers with extract #3 among diners standing out.

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Figure 7: Sensory evaluation of the attributes of smell, color, texture and taste of cooked pork hamburgers

Conclusions and implications The protective effect of extracts #1, #2 and #3 on lipid oxidation and color deterioration in pork hamburgers coked and stored in refrigeration can be attributed to the phenolic compounds present in the culinary spices present in the extracts, which showed antioxidant activity. The three extracts can be an efficient strategy that causes an increase in the shelf life of meat products without causing damage to nutritional attributes, and without presenting anomalous alterations in sensory perceptions by consumers.

Acknowledgements

To the staff of the Nayarit Center for Innovation and Technology Transfer, especially Dr. Javier Germán Rodríguez Carpena and M. Sc. María Elena Luna Castañeda, as well as M. Sc. Gibrán López Nahuatt, for all the support provided during the realization of this research.

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https://doi.org/10.22319/rmcp.v13i2.5729 Article

Diagnosis of the health quality of artisanal cheese dairies in Salinas, San Luis Potosí

Rocío Rodríguez-Gallegos a Gregorio Álvarez-Fuentes b Juan Antonio Rendón-Huerta a* Juan Ángel Morales-Rueda a Juan Carlos García-López b Luis Alberto Olvera-Vargas c

a

Universidad Autónoma de San Luis Potosí. Coordinación Académica Región Altiplano Oeste. Carretera Salinas-Santo Domingo #200, 78600, Salinas de Hidalgo, San Luis Potosí, México. b

Universidad Autónoma de San Luis Potosí. Instituto de Investigación de Zonas Desérticas. San Luis Potosí. México. c

Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C. Jalisco, México.

*Corresponding author: antonio.rendon@uaslp.mx

Abstract: The objective of this work was to evaluate the microbiological load of total coliforms (TCs), Staphylococcus aureus and Salmonella spp. in milk and fresh cheeses as indicators of process practices carried out in cheese dairies of different localities in the region of Salinas, San Luis Potosí. Fifteen establishments were sampled, milk and cheese samples were obtained, and they underwent a milk composition and microbiological analysis. Sixty-five percent of the production units do not pasteurize the milk, or they use natural rennet. The highest counts in

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milk were 42 x 109 and 40 x 109 CFU/ml for S. aureus and TCs, respectively. For cheese, the counts were 32 x 109 and 26 x 109 CFU/g for S. aureus and TCs, respectively. In addition, the presence of Salmonella was detected in milk and cheese. The lack of hygiene in the utensils and equipment in which the cheeses are made, as well as the use of natural rennet, can be a risk to the health of the consumer. Key words: Zoonosis, Bacterium, Public health, Cow’s milk, Goat’s milk.

Received: 10/07/2020 Accepted: 24/09/2021

Introduction Milk, due to its nutritional characteristics, is one of the foods of animal origin with the highest demand in the world. In Mexico in 2018, milk production was 12,008,239 thousand liters, of which about 73 % was destined to the production of dairy products and derivatives (8,784,055 thousand liters)(1). The region of the Western Altiplano of the state of San Luis Potosí is characterized by being a milk-producing region, in greater proportion of cattle and in less of goats, in which most of the milk is destined to the production of artisanal fresh cheese. Traditional artisanal cheeses are important, not only for their nutritional and gustatory benefits, but also for their ability to generate and keep rural employment that involves some agents of the agro-industrial milk chain: farmers, cheesemakers and traders(2). However, organization among producers is required to, for example, create an “artisanal denomination” with a level of regulation and protection that guarantees the preservation of dairy production systems based on traditional practices and their respective socioeconomic benefits(3). On the other hand, in these production units (PUs) of the Western Altiplano of San Luis Potosí, cheese is made from raw milk, with the use of non-standardized traditional methods, scarce technification and in inappropriate facilities (e.g., they use the kitchens of the producers’ homes). In this regard, most producers do not know about the concept of good manufacturing practices and the pasteurization process, which is a thermal process that allows controlling pathogenic bacteria currently found in milk, such as Mycobacterium tuberculosis, Listeria monocytogenes, as well as some species of Campylobacter, Salmonella, Escherichia coli and other fecal coliforms(4). In addition, cross-contamination of milk after pasteurization must be controlled by applying strict cleaning and disinfection rules(5). Also, in this type of PU, non-sterile natural rennet is sometimes used in the production of cheese, its production consists of drying extended fragments of abomasum of newborn calves in clotheslines under the sun for up to three days, later they are introduced

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into a plastic drum with whey, it is covered and left to ferment for at least five days for later use; this practice can be an important point of microbial contamination. Gastrointestinal diseases due to the consumption of contaminated foods can occur at any time of the year, but the risk increases in the hot season. The most frequent clinical conditions are fever, vomiting, abdominal pain and moderate or severe diarrhea(6). For health institutions such as the Mexican Institute of Social Security (IMSS, for its acronym in Spanish), these diseases are: brucellosis, cholera, typhoid, gastroenteritis, shigellosis, salmonellosis and diarrhea, which represent a severe public health problem for San Luis Potosi state and the country(7,8). Therefore, dairy products must comply with the sanitary provisions and specifications of NOM 243(9). Which indicates that Salmonella spp. must be absent in 25 g of cheese or milliliter of milk, a maximum of 100 colony-forming units per gram or milliliter (CFU/g or ml) is allowed for total coliforms and a maximum of 1,000 CFU/g is allowed for Staphylococcus aureus. Therefore, the objective of the present work was to evaluate the microbiological load of total coliforms, S. aureus and Salmonella spp. in milk and cheeses as indicators of the process practices carried out in the cheese dairies of different localities of the region of Salinas, San Luis Potosí, and to be able to recommend good manufacturing practices that help improve the health conditions of production, without altering the characteristics of the dairy product.

Material and methods Sampling site The study was carried out from January to April 2018 in a part of the Western Altiplano region of the state, in some localities of the municipality of Salinas, San Luis Potosí and Villa González Ortega, Zacatecas, which are located between the following coordinates: 101°43” W and 22°38” N, with an altitude of 2,070 m asl, their climate is dry and their average temperature is 18 °C(10). Field visits were made to various localities of the region that produce cow and goat cheeses. Following the recommendations of the NOM 109(11) project, samples of milk from the first milking of the day and ground fresh cheese were taken in 15 different PUs. Three aliquots of 10 ml of the accumulated milk of the day were taken from the tank or bottle (40 L) and placed in sterile bottles. Three samples of approximately 50 g of different cheeses were placed in individual hermetic bags of the Ziploc type. The milk and cheese samples were placed in a plastic cooler, previously sanitized and disinfected with alcohol to avoid crosscontamination, which contained ice for temperature control. Immediately afterwards, they were transferred to the laboratory for their compositional and microbiological analysis.

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Two samples of commercial milk and two samples of commercial cheese were used as control treatment. In addition, a survey was applied at the sampled sites to obtain information on the conditions under which dairy products are produced and processed.

Milk composition Milk samples were analyzed with a lactoscan equipment (Milkotester, Master Eco) with ultrasonic sensors to determine percentage content of fat, protein, lactose, non-fat solids, salts, freezing point, density and added water.

Microbiological analysis The agars RVBA (red-violet-bile-lactose, BD-BIOXONTM), Salmonella-Shigella (BDBIOXONTM), Baird Parker (BD-BIOXONTM) were prepared according to the sterility instructions indicated by each of the containers, for the growth of specific microorganisms such as total coliforms, Salmonella spp. and Staphylococcus aureus, respectively. In the laboratory, milk and cheese samples were prepared using the method of dilutions in peptone water in accordance with NOM 110(12). For solid samples, a sample of 1.0 g of cheese was taken, diluted and mixed homogeneously in 9 ml of lactose broth or 0.1 % peptone water to crush the solid, this constituted the first dilution of the sample (101). Subsequently, 10 tubes of 20 ml per each sample were prepared, 10 ml of the mixture was placed in the first tube and the tubes from two to ten contained 9.0 ml of 0.1 % peptone water, then 1.0 ml of the initial dilution of the first tube was taken and mixed homogeneously in the second tube, the procedure was repeated up to tube nine (109). For liquid samples (milk), 10 previously sterilized tubes of 20 ml were prepared, a sample of 10 ml milk was taken and emptied into the first tube (100), the tubes from two to ten contained 9.0 ml of 0.1 % peptone water, then 1.0 ml of milk was taken from the first tube and mixed homogeneously in the second tube, the procedure was repeated up to tube nine (109). Once the dilutions were finished, the technique described by Miles and Misra, modified by Slack and Wheldon(13), was used, in which 20 μl of each dilution were deposited on the surface of an agar plate for plate count, performing the drip from a height of 2.5 cm and depositing three drops per each dilution. The presence of Salmonella in cheese and milk, a sample of 25 ml of milk or 25 g of cheese was taken, diluted and mixed homogeneously in 225 ml of lactose broth or 0.1 % peptone water to crush it and it was incubated for 18 h at 36 °C, this constituted the first dilution of the sample (101). It was verified with NOM 114(14) in Salmonella-Shigella agar and placed in an incubator (Yamato IN 804) for 24 h at 35 °C, translucent colonies, occasionally opaque, some colonies with black dots in the center. To determine the presence of S. aureus, the guidelines of NOM 115(15) were followed, using Baird Parker agar

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(+Potassium tellurite) and incubating at 35 °C for 48 h, plates with between 15 and 150 black colonies are selected and the presence of total coliforms was analyzed with NOM 113(16), using red-violet-bile-lactose agar (RVBA) and they were incubated at 35°C for 24 h, plates with between 15 and 150 dark red colonies are selected.

Statistical analysis The descriptive statistics for milk quality parameters resulting from the physicochemical analysis were obtained; the production units were classified by means of a cluster analysis with the hclust function that groups similar and dissimilar farms; to compare the resulting cluster groups, an analysis of variance was performed for a completely randomized design; when this indicated that there was a treatment effect (P<0.05), a Tukey mean test with a significance of P<0.05 was performed. Descriptive statistics of the data from the microbiological analysis CFU/g or ml of cheese or milk that contained Salmonella spp., total coliforms and S. aureus were obtained. The statistical software R Core Team(17) was used to analyze the data.

Results Characteristics of production units Of the fifteen production units (PUs), only three have goats and the rest have cows as described in Table 1. The PUs with cows have on average 17.3 ± 14.2 heads, of which 7.5 ± 6 are milking cows, with a production of 7.08 ± 2.71 L/d. The three production units with goats have on average 45 ± 5 heads, of which 25 ± 5 are milking goats, with an average production of 1.0 ± 0.5 L/d. Milking is done manually twice a day, in the morning and in the afternoon.

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Table 1: Localities sampled, number of cows and goats by farm, milking and daily milk production Production Locality Total, Milking Production Species unit animals animals (L/animal*d) 1

Punteros*

35

25

4.0

Cow

2

El Potro*

8

8

11.0

Cow

3

El Potro*

11

7

8.0

Cow

4

El Potro*

13

10

10.0

Cow

5

El Alegre*

40

20

1.5

Goat

6

El Alegre*

50

30

0.7

Goat

7

El Alegre*

45

25

0.5

Goat

8

Salinas*

8

7

5.0

Cow

9

Salinas*

6

4

8.0

Cow

10

El Refugio **

50

7

6.0

Cow

11

El Refugio **

2

1

4.0

Cow

12

El Refugio **

30

3

5.0

Cow

13

El Refugio **

9

5

4.0

Cow

14

Zumpango**

20

8

10.0

Cow

15

Zumpango**

15

5

10.0

Cow

*Salinas de Hidalgo; ** Villa González Ortega, Zacatecas.

Milk composition Four groups were identified as a result of the cluster analysis (Figure 1), which are detailed in Table 2, resulting in four groups, separating in group I where the PUs that have goats, mainly of the Saanen breed, are, this grouping is based on the similar content of nutrients in milk. In percentage of fat, cluster I showed a statistical difference (P<0.05) in contrast to the other groups (cow’s milk samples), which show some similarity between them. The percentage of protein, lactose, non-fat solids and salts showed no difference (P>0.05) between groups. In the variable total solids, there were significant differences between clusters (P<0.05), the highest value was obtained in cluster I (14.5 %) because it was goat’s milk, and the lowest value was shown by cluster IV (10.8 %). The variable density showed

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statistical differences, the groups of cow’s milk samples (cluster II, III and IV) obtained the highest value (1,024 kg/m3) and were statistically the same, in contrast, cluster I had an average value of 1,019 kg/m3. Finally, the cryoscopic point in cluster I decreased as a result of a higher content of lactose and salts in milk. Figure 1: Dendrogram of the physicochemical analysis of cow’s milk and goat’s milk

Table 2: Nutritional composition of milk samples PVariable Cluster I Cluster II Cluster III Cluster IV value a b bc c Fat, % 6.3 ± 0.71 4.3 ± 0.53 3.3 ± 0.25 2.7 ± 0.65 *** Protein, % 3.2 ± 0.07 3.1 ± 0.12 3.1 ± 0.21 2.9 ± 0.22 N.S. Lactose, % 5.0 ± 0.07 4.7 ± 0.23 4.5 ± 0.19 4.5 ± 0.30 N.S. a b c d Total solids, % 14.5 ± 0.57 13.0 ± 0.15 11.7 ± 0.26 10.8 ± 0.48 *** Non-fat solids, % 9.0 ± 0.13 8.7 ± 0.4 8.5 ± 0.12 8.4 ± 0.51 N.S. 3 b a a a Density, kg/m 1018.8 ± 1.1 1024.8 ± 1.2 1024.3 ± 0.2 1023.5 ± 0.8 *** Salts, % 0.7 ± 0.0 0.67 ± 0.06 0.69 ± 0.04 0.64 ± 0.05 N.S. b Cryoscopic point, °C -0.62 ± 0.01 -0.55 ± 0.03a -0.53 ± 0.02a -0.53 ± 0.04a ** abc

Mean values with different literals are statistically different (P<0.05) by row. N.S. = not significant; ** = P<0.001; *** = P<0.0001.

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Microbiological analysis Milk The microbiological quality specifications of dairy products established by NOM 243(7) are the same for cow’s, sheep’s and goat’s milk products. The results of the microbiological analysis of the milk samples can be seen in Table 3. It is evident that those producers who carry out a thermal process to avoid the decomposition of milk, which consists of boiling the milk, letting it cool at room temperature and then refrigerating it (pasteurization without control), show the lowest microbial loads, compared to those who do not perform a thermal process. Table 3: Microbiological analysis (CFU/ml) of milk samples in different cheese dairies in Salinas, S.L.P. and Villa González, Zacatecas They Salmonella Production unit S. aureus TCs pasteurize spp. milk 1

10x101±1x10

12x101±1x10

Absent

No

2

Absent

Absent

Absent

Yes

3

77x102±1x102

29x101±3x10

Present

Yes

4

43x102±5x101

13x103±2x102

Absent

Yes

5

36x104±5x103

Absent

Present

Yes

6

40x104±1x103

21x105±7x104

Present

No

7

36x101±1x10

24x105±4x104

Present

No

8

32x108±4x107

Absent

Present

No

9

43x107±5x106

12x108±2x107

Present

No

10

40x101±1x10

80x102±1x102

Present

No

11

67x102±5x101

37x106±1x106

Present

No

12

12x103±2x102

40x109±1x108

Present

No

13

72x107±1x107

24 x105±6x104

Present

No

14

42x109±4x108

57 x108±5x107

Present

No

15

13x103±2x102

25 x103±2x102

Present

No

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T1

Absent

Absent

Absent

Yes

T2

Absent

Absent

Absent

Yes

TCs = Total coliforms; T1, T2: Commercial samples considered as controls. Values express means and standard deviation CFU/g.

Regarding the analysis and identification of S. aureus, it was observed that most of the samples have values higher than those allowed; the highest data recorded was in PU 14 with an average value of 42 x 109 CFU/ml, PUs #1, #7 and #10 showed the lowest values, 10 x 101, 36 x 101 and 40 x 101 CFU/ml, respectively. This bacterium is absent in the two control samples and in PU #2. The highest load of total coliform (TCs) was observed in PU #12, with a value of 40 x 109 CFU/ml and the lowest load in PUs #1 and #3, with values of 12 x 101 and 29 x 101 CFU/ml, respectively. In contrast, this microorganism was absent in the control samples and PUs #2, #5 and #8. The results of the analysis of Salmonella spp. show that this bacterium was present in most of the samples, mainly in those where there is no pasteurization of milk. On the other hand, there was an absence of this microorganism in PUs #1, #2 and #4 and the control samples. Dairy products (cheese) The results of the microbiological analysis of the cheese samples are shown in Table 4. Initially, it is important to mention that the presence of S. aureus was detected in most of the samples collected in the PUs visited. The PUs where the highest load of this microorganism was recorded were #13 and #15, where the microbial load corresponded to 32 x 109 and 63 x 109 CFU/g in cheese made with cow’s milk and artificial rennet. In contrast, the lowest microbial load, with values of 23 x 101 and 32 x101 CFU/g, was obtained in PUs #1 and #10. In addition, the control samples and PU # 5 had absence of this bacterium. The analysis of TCs, PUs #6 and #15 had the highest values, 13 x 109 and 26 x 109 CFU/g, respectively, where the highlight of these results is that the first PU uses artificial rennet and the second natural rennet. In contrast, PU #10 had the lowest data on TC load, 74 x 101 CFU/g. In addition, in the samples of control cheese and PU # 5, they presented absence of TCs.

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Table 4: Microbiological analysis of cheese samples and type of rennet used in the production units Production Type of S. aureus TCs Salmonella spp. unit rennet 1

23x101±6x10

32x103±1x102

Present

Artificial

2

83x105±2x104

70x103±9x102

Present

Natural

3

18x103±1x102

16x105±6x104

Present

Natural

4

21x102±1x10

24x108±1x108

Present

Natural

5

Absent

Absent

Present

Artificial

6

41x103±1x102

13x109±1x108

Present

Artificial

7

60x104±2x104

28x103±2x102

Present

Natural

8

87x103±6x102

38x103±3x103

Present

Artificial

9

31x104±1x103

17x104±1x103

Present

Natural

10

32x101±8x10

74x101±5x10

Present

Natural

11

10x102±1x101

32x105±2x104

Present

Natural

12

12x102±1x101

49x105±1x104

Present

Natural

13

32x109±2x108

48x106±2x105

Present

Natural

14

19x105±3x104

11x102±1x101

Present

Natural

15

63x109±1x108

26x109±3x108

Present

Natural

T1

Absent

Absent

Absent

Artificial

T2

Absent

Absent

Absent

Artificial

TCs = total coliforms; T1, T2= commercial samples considered as controls. Values express means and standard deviation CFU/g.

In the determination of Salmonella spp., presence was observed in all samples of the PUs analyzed, as shown in Table 4. Finally, only commercial cheese samples showed an absence of Salmonella spp.

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Discussion The results of the physicochemical analysis in cluster I (goat’s milk samples), the percentage values are very similar to those reported for Saanen goats in Mexico(18). In addition, SalinasGonzález et al(19) point out that these values vary with time, being higher in September. Studies on the factors that affect the stability of the freezing point of milk indicate that the decrease in the cryoscopic point is related to a higher content of lactose and salts (calcium, phosphorus, magnesium), as well as to the passage of time (days) after birth(20,21). Regarding the physicochemical quality of milk from clusters II, III and IV (milk samples), the three groups are within the reference data of NOM 155(22). Álvarez-Fuentes et al(23) mention that small-scale dairy farms located in the south of Mexico City face the challenge of producing milk in quantity and quality; in this regard, the authors point out that the quality of milk is different according to the time of year (dry, rainy and winter periods), the results of milk quality (fat, protein, lactose and total solids) that they recorded in the dry season are very similar to those obtained in this work. In this study, milk and cheese samples were taken from local cheese dairies to analyze pathogenic microbial loads. In addition, a request and comparison of records of the diseases caused by the consumption of dairy products of 2018 of the Basic Community Hospital of Salinas was made (personal communication, Dr. Sugey Bastidas Gastelum, director), and the response was that the hospital only follows up on cases of brucellosis (4 cases in that year) because it is considered as primordial by Epidemiological Surveillance, other diseases possibly caused by Escherichia coli, Staphylococcus aureus and Salmonella in dairy products and other foods are not analyzed because they do not have their own laboratory for sample processing. For fluid milk, NOM 243(9) indicates that the maximum allowable loads for total coliforms and S. aureus must be ≤10 CFU/ml by direct seeding, while Salmonella must be absent. The low counts of microorganisms in PUs 1, 5 and 10 may be due to the fact that these units were the only ones that have exclusive facilities for the handling of milk and cheese making process, and maintain hygiene measures (use of cap, masks, boots and cleaning of equipment, floor and utensils); despite these practices, the facilities do not have hermetic sealing doors that protect from the entry of dust. Table 3 shows that the regulations are not complied with in several PUs, this may be due to the fact that the milk is not pasteurized due to the lack of equipment, knowledge of pasteurization times and temperatures and the influence on the state of health of the animals, cleaning during milking, milking utensils and place of storage of milk. In a study conducted by Fuentes-Coto et al(24), the microbial load of organic milk was analyzed, the presence of mesophilic and coliform bacteria was detected in milk and dairy products, where the amounts of CFU/ml were above the allowed limit.

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On the other hand, Álvarez-Fuentes et al(23) mention that the presence of mesophilic bacteria and the somatic cell count are related to the type of cleaning performed on the udders (traditional, partial and complete); however, regarding the microbial load, even though the work describes low counts of CFU/ml, the species of microorganisms are not specified. Although the microbiological analysis in the milk reveals that the microbial loads are low in some farms, in some cases, the samples of fresh cheese had microbial loads higher than allowed by NOM 243(9), it establishes a maximum of 100 CFU/g for TCs, a maximum allowed of 1,000 CFU/g for S. aureus, and Salmonella spp. must be absent in 25 g of sample. One of the factors of the high microbial loads detected in this study is probably due to the fact that, in PU # 15, natural rennet fermented in non-sterile conditions is used as a coagulating agent. In the interviews that were carried out with the producers, they comment that the use of natural rennet is mainly due to the fact that it impregnates flavors and aromas desirable by consumers of fresh cheese. The results of Table 4 are similar to those reported in other cheese-producing areas in Mexico, some authors(25) report counts of 9.27 log10 CFU/g for TCs and Salmonella present in fresh cheese. The aro cheese that is marketed in the municipality of Teotitlán de Flores Magón, Oaxaca, Mexico, had counts of 6.94 for TCs, 6.74 for E. coli, 5.76 log10 CFU/g for S. aureus and Salmonella was present, consequently, no sample analyzed complies with the regulations(26). Also, artisanal botanero cheese from the northwest of the state of Mexico has serious deficiencies in its microbiological quality, since the counts exceeded the permitted limits of pathogens(27). In the findings of this work, only the results of the control samples comply with the regulations. On the other hand, PU #5 partially complies with the counts of TCs and S. aureus. However, there is the presence of Salmonella colonies, which is associated with the use of unpasteurized milk(25). In other geographical regions, such as Cajamarca, Peru, industrial fresh cheese from six companies was analyzed, under the guidelines described in the Sanitary Standard of microbiological criteria of sanitary quality and safety for foods and beverages for human consumption in that country. In the results, it is highlighted that all companies comply with what is established by the standard for Salmonella spp., since all the samples showed absence, with respect to other pathogens, only one company presents better microbiological conditions for the production of fresh cheese(28). The same happens in Egypt and Middle Eastern countries with soft cheeses (Domiati), where they also have loads of aerobic bacteria S. aureus, TCs, E. coli and yeasts(29). In the north of Iran, Kurdish cheese, named after the region where it is produced (Kurdistan, Iran), is a cheese prepared with cow’s or sheep’s raw milk, the loads of pathogenic microorganisms in the first days are very similar to the loads found in this study; the presence of Salmonella, E. coli of 5.27 log10 CFU/g and 8.22 log10 CFU/g for TCs was found. In addition, they point out that the maturation of cheese (60 d), the loads decrease significantly due to the presence of lactic acid bacteria(30).

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The presence of pathogenic microorganisms in dairy farms is due to several factors, for example, Salmonella is present in other domestic animal species such as pigs and poultry, which can become infected because they are within the production unit, in the environment due to the management of manure and its importance lies in the fact that it can survive for prolonged periods of time in the environment and cause salmonellosis in humans(31,32). On the other hand, Staphylococcus aureus is a very common pathogen that causes mastitis in dairy cows, in this regard, if milk is not pasteurized, this bacterium can be found even in milk storage tanks(33). In the case of coliforms, Van Kessel et al(34) point out that these enteropathogenic microorganisms are very persistent in dairy farms; their origin is the intestine of animals, as well as their feces and water contaminated with manure, with a high risk of contamination of milk and storage tanks. The authors described agree in pointing out that the poor sanitary quality of dairy products throughout the milk and cheese production chain is a health problem, since these can be a vehicle for the transmission of food diseases, due to their high content of: E. coli, Listeria monocytogenes, S. aureus, Salmonella and possibly Brucella spp.(26,35,36). Therefore, they recommend improving the quality at the farm level with hygienic milking measures, establishment of a cold chain, adequate transport and good sanitary measures of milk, such as: hygiene of the facilities where the cheeses are made, use of appropriate clothing, use of drinking water, washing of utensils, tables and hand disinfection(25).

Conclusions and implications According to the results of the analysis of milk and cheese samples, most production units do not meet quality standards in dairy products, because they do not have good hygiene practices throughout the entire production process, so it is necessary to implement actions that make producers aware of taking better health measures to reduce possible sources of contamination and that it does not represent a risk to the health of consumers, causing possible gastrointestinal diseases, mainly. It is necessary to conduct more studies on the process of making fresh cheese, such as taking samples of utensils, analysis of natural rennet and carrying out some process of sterilization of natural rennet by physical means. As well as to hold workshops of good practices in the production of dairy products for producers. Acknowledgements The authors thank the producers for providing us with the samples and the Research Support Fund C18-FAI-05-57.57 UASLP.

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https://doi.org/10.22319/rmcp.v13i2.5744 Article

Perspectives on continuity, milk quality and environment in milk production units in the state of Aguascalientes, Mexico

Carlos Eduardo Romo-Bacco a* Neftali Parga-Montoya a Arturo Gerardo Valdivia-Flores b Rodrigo Gabriel Carranza-Trinidad c María del Carmen Montoya Landeros d Abril Areli Llamas-Martínez a María Mayela Aguilar Romero b

a

Universidad Autónoma de Aguascalientes. Centro de Ciencias Empresariales. Av. Prolongación Mahatma Gandhi #6601, Col. El Gigante, Ejido Arellano, 20340, Aguascalientes, Aguascalientes, México. b

Universidad Autónoma de Aguascalientes. Centro de Ciencias Agropecuarias. Jesús María, Aguascalientes, México. c

Instituto Nacional de Estadística y Geografía (INEGI). Dirección General de Estadísticas Económicas. Aguascalientes, Aguascalientes, México. d

Universidad Autónoma de Aguascalientes. Centro de Ciencias Básicas. Aguascalientes, Aguascalientes, México.

*Correspondng author: ceromo@correo.uaa.mx

Abstract: The objective was to evaluate the productivity, the sale price of milk, the size and the perceptions of their owners about their environment, quality and permanence in milk 357


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production farms in the state of Aguascalientes. Forty milk production units, with similar conditions of age (30 years), zootechnical management, availability of inputs and customers, were evaluated. The productive characteristics of the farms in relation to the herd size factor were compared through a MANOVA. A structural model was formulated to evaluate the effect of environmental factors on milk quality and farmers’ intention to continue production units in the dairy activity. A positive influence was found on the productive scale of dairy farms, the obtaining of higher daily productivity per cow, better perception of quality and the sale price of milk. In the model, environmental factors were significantly associated with the assessment of milk quality by producers and their permanence in the dairy activity (14.2 and 22.7 %, respectively). This confirms that the perception of environmental factors could be considered as a crucial variable to increase milk quality, productivity and for the meeting between the interests of producers and the agribusiness, as well as to favor the performance and integration of the different links in the dairy production chain and boost the global competitiveness of the Mexican agri-food sector. Key words: Competitiveness, Profitability, Agri-food production chain, Milk market.

Received: 24/07/2020 Accepted:16/06/2021

Introduction The consumption of fluid bovine milk has remained relatively stable in different countries, however, milk production has increased markedly(1); this suggests that the dairy industry has diversified its offer with the creation of new products, which give greater added value to milk. The quality, price and characteristics of each dairy product, as well as their availability in a timely and appropriate manner, are criteria associated with the competitiveness of the dairy sector in the Mexican altiplano(2); also, the integration of producers in organizations for the collective purchase of inputs and for the insertion of products in the markets has shown the potential to promote economies of scale and improve their economic profitability(3). Nevertheless, decision-making by representatives of some dairy organizations is complex and negotiations with the agro-industrial sector focus on ensuring the sale of raw milk, as well as meeting the demands of the agribusiness, especially in terms of quality and opportunity(4,5). Some variables that are not directly associated with the productive management of dairy herds, such as the schooling of the producer, the size of the herd or the use of qualified 358


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technical assistance(6), have been shown to have an influence on the productivity in Milk Production Units (MPU), so they are considered important in the evaluation of economic results(7). Milk production in Mexico is carried out under different production systems; the characteristics that identify them are the use of the resources available for production, such as the labor used, the technification of dairy farms, the size of the area, the destination of milk, the number of milking cows, among others(8,9,10). As part of the strategies for the consolidation of milk producers, especially small MPUs(11), the importance of promoting trust between the different actors in the production chains in order to integrate to achieve improvements in milk quality and competitiveness in the sector has been recognized(12). The relationships of trust between the different actors of the agri-food production chains are made evident through commercial exchanges that generate development, well-being of the environment and increase in social capital(13). In this sense, producer organizations that have favorable social capital have been identified in the state of Aguascalientes(14,15); this implies greater advantages for the development of organizations with greater possibilities of success for the achievement of common objectives, both for the consolidated purchase of inputs for production and for the sale of milk(16,17). It has been proposed(11,18,19) that, in order to meet the requirements of consumers, the different actors in the dairy production chain should have economic incentives proportional to the quality of their dairy products; this would have a positive impact on the stability and the possibly of growth of dairy farms, as well as on the structure of the dairy market, and would allow clarifying the challenges and strategies to reduce uncertainty about the outcome of the confluence of forces prevailing in the dairy industry(20). Porter’s model has been used in several industries to propose competitive corporate strategies(21,22); this model proposes(23,24) the competition between five forces that favor or harm the competitiveness of a sector that goes to the product market: 1) bargaining power of suppliers; 2) bargaining power of customers; 3) threat of substitutes; 4) threat of new participants; and 5) rivalry between existing companies(23,25). This model presupposes that the market is attractive to a company or organization when its structure is profitable for the actors present in the productive activities, so it influences its behavior and defines its competitive strategy; therefore, the success of each actor is conditioned by the structure of the market and by the interaction between the actors in the chain(26,27). However, the effect of these forces on the development of companies comparable in age and productive characteristics has not been empirically demonstrated. Therefore, the objective of this work was to evaluate the productivity, the sale price of milk, the size and the perceptions of their owners about their environment, quality and permanence in milk production farms in the state of Aguascalientes. 359


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Material and methods Study design

The study was located in an area specialized in milk production in the municipality of Aguascalientes(5). The total population (40 MPUs) of the register of members of a milk producers’ organization, constituted since 1988 by a group of producers organized for the local and regional production and commercialization of bovine milk(28), was analyzed; this group settled in the same agricultural area, near the city of Aguascalientes, and had, since its inception, herds of comparable genetic quality and equivalent financial support(29), as well as other similar productive conditions and opportunities for the acquisition of inputs. The study conducted in 2018 showed that the group had a total of 5,693 cows, with an average daily production of 23.14 ± 6.9 liters per cow and an annual income from milk sales of US $ 17.7 million. The owners or people in charge of the farms who gave their consent to obtain the information and productive data of each of the production units were interviewed. The questionnaire used to identify the characteristics of the MPUs included variables about the age and experience of the producer, sale price of milk, size, herd structure, predominant use of labor of hired personnel, as well as their perception of quality, the continuity of the MPU and the agents external to production; as well as other variables not used for this study, such as area, value of infrastructure, production and food costs, among others.

Variables

The category quality assessment was determined, for which producers were questioned about the economic incentives and penalties they receive for not producing milk with the optimal quality expected by the milk-receiving agribusiness. This category also included knowledge of the milk quality parameters demanded by customers, awareness of the possibility and benefits of producing quality milk(30). In the same way, the variable continuity in the activity was determined, where the producers were questioned about their willingness to remain in the dairy activity. To explore agents external to production, Porter’s model(23) was adapted to evaluate the competitive forces of the agribusiness based on variables with a five-level Likert scale. The degree of agreement or disagreement of producers on the bargaining power of customers and

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suppliers, competition between producers, facilities for the creation of substitute products, as well as the ease of entry to new competitors in the dairy activity were considered. Hypotheses about the effects of the competitive forces of the agribusiness on different variables of dairy farms were also proposed. H1: The competitive forces of the agribusiness have a significant positive influence on quality assessment. H2: The competitive forces of the agribusiness have a significant positive influence on the continuity of dairy activity. H3: A larger herd size positively influences the sale price of milk.

Statistical analysis

For the analysis of the productive characteristics of the MPUs, a statistical software was used(31). A multivariate analysis of variance (MANOVA) was performed to determine if the means of the variables evaluated (age of the producer, hired labor, milking cows, productivity per cow (liter/day), sale price of milk) differed jointly between the different sizes of dairy farms (<50, 50-250 and >250 milking cows)(32). For herd size, a previously proposed scale was used(33). Likewise, an ANOVA(34) was performed to determine the differences of the means for each variable analyzed (age of the producer, milking cows, productivity per cow (l/d), sale price of milk) according to the size of the farm. When the assumptions of the ANOVA (normality and homoscedasticity) were not met, the equivalent nonparametric Kruskal-Wallis test was applied for the comparison of their respective medians. The Chisquare independence test was performed to evaluate the variables of hired personnel and continuity in the dairy activity in relation to the size of the farms. In all cases, a significance level of 5 % was used. The variable of continuity was evaluated through a binary logistic model (35) with a significance level of 5 % to determine the degree of association with the other variables analyzed (size, age, milking cows, price, productivity, quality assessment and competitive forces of the agribusiness).

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𝑝=

𝑒 𝑏0 +𝑏1 𝑥1 +𝑏2𝑥2 + … 1 + 𝑒 𝑏0 +𝑏1𝑥1 +𝑏2 𝑥2 + …

Where: p = probability of continuing in the dairy activity b0 = constant b1,2,… = coefficients associated with each variable x1,2,… = variables evaluated (size, age, … ) The logistic model, once the previous equation was linearized, was given as: 𝑝 𝑙𝑜𝑔 ( ) = 𝑏0 + 𝑏1 𝑥1 + 𝑏2 𝑥2 + … 1−𝑝 The proposed hypotheses were also tested based on a model of structural equations using the partial least squares method (PLS-SEM)(36-39). The internal consistency of the group of variables that influence the competitive forces of the agribusiness and the assessment of quality was evaluated; when the variables were correlated with each other, it was considered that there was Reliability; in addition, the existence of Validity was considered when the correct measurement of the variables was verified with the partial least squares (PLS) method(40,41). For the evaluation of the categories of the model, the following variables were included: bargaining power of suppliers, bargaining power of customers, threat of substitutes, threat of new entrants and rivalry between existing companies for the category of competitive forces of the agribusiness and, for the category of assessment of milk quality: the economic incentives and penalties they receive for producing poor quality milk, knowledge of the milk quality parameters demanded by customers, awareness of the possibility of producing better quality milk and the benefits of producing quality milk. In the analysis, only the variables that were significant were selected so that the model had satisfactory goodness-of-fit test indices(40,42). Table 1 shows the variables included in the final structural model, for the competitive forces of the agribusiness: the entry of rival producers into the market, the threat of new products and substitutes for dairy products; as well as those that were considered in the assessment of milk quality: knowledge of quality parameters and penalties for not producing quality milk. Both categories were considered latent or reflective because their evaluation was made from the individual measurements of the included variables, so their covariance was evaluated to validate each category(40,43).

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Table 1: Consistency and measurement of indicators for category validity Validity convergent ALC RI T-value AVE CRI(>0.7) Variable Category (>0.700) (>0.5) (>2.57) (>0.5) Forces of the Competitors agribusiness New products Substitute products Assessment Parameters1 of quality Penalty2

0.767 0.628 0.766

0.588 0.394 0.587

3.280 1.741 3.497

0.522

0.765

0.794 0.725

0.510 0.356

1.802 1.362

0.578

0.732

ALC= average loads of the category; RI= reliability indicator; AVE= average variance extracted index; CRI= composite reliability index. 1 Knowledge of milk quality parameters; 2 Knowledge about the penalties for not producing quality milk.

The composite reliability index (CRI) was also considered to measure internal consistency(43,44); this index took into account the factorial loads of each indicator and was obtained by calculating the square of the sum of factorial loads and the sum of the variance of the error terms for each category, arguing that if this criterion is satisfied, there will be consistency and reliability. The estimated CRI was 0.765 and 0.732 for the competitive forces of the agribusiness and quality assessment, respectively, which exceeded the recommended value of 0.708(45). The average extracted variance Index (AVE) was also calculated, which represented the mean value of the square of the loads or factors associated with each category(46). To assess the internal consistency of the measuring instrument and of the variables in each category, Cronbach’s alpha coefficient was calculated; it was also used to measure the reliability of the scales and the affinity that exists in the category, as well as to have an evaluation sensitive to the number of items on the measurement scale(47). Finally, to measure the discriminant validity of the categories, the Fornell-Larcker criterion(46) was calculated and it was validated that each category shared more variance with its corresponding variables than with the variables of the other category, that is, that the AVE of each category was greater than the square of the correlation with the other category of the structural model. A correlation between categories of 0.377 and AVEs of 0.522 and 0.578 for competitive forces of the agribusiness and for the assessment of quality, respectively, were obtained.

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In the analysis of cross-loadings, discrimination between the variables was observed, considering that those that showed the highest factorial load were closely associated with the corresponding category(39,43,48). The hypotheses proposed were evaluated with the structural model using the Bootstrapping technique (500 cases), in order to obtain sufficient evidence to adequately estimate the confidence intervals and increase the accuracy of the parameters(49).

Results and discussion With the structural model proposed, it was found that the competitive forces of the agribusiness had a significant effect on the categories and crucial variables of a group of MPUs developed with similarity of age, zootechnical resources and market situation, in such a way that the MPUs that reached the best price per liter of milk are those with larger and more productive herds; which, if generalized, could be having a positive impact on the development of the Mexican agri-food sector. The main characteristics of the dairy farms evaluated reflected the heterogeneity of intensive dairy production in the Mexican Altiplano, however, the productive system used in most of the MPUs was the stabled one, where most of the producers surveyed said they preferred the use of herd confinement facilities for milk production; the above could be, in part, a reflection of the climatological characteristics of the state of Aguascalientes (average annual temperature of 18.3 °C and average annual rainfall of 530.3 mm)(50), as well as the product of the conformation of the group of producers surveyed, who migrated in the 80s from the urban limits for the establishment of specialized MPUs(29). For the variable average age of the producers surveyed, which was 52.65 ± 12.15 yr, significant differences were found (P<0.05); other studies(51) mention that small-scale milk producer groups have favorable conditions in the MPUs to generate greater added value to production when the owners are older. In the present study, it was observed that only a little more than a third of the MPUs evaluated had the support of family members to carry out the work of milk production, which could suggest a change in the structure of dairy organizations of similar size, or that this type of organizational structure finds greater advantages in salaried labor, since the use of family labor to support the performance of the different productive activities does not prevail(8,52,53). It was found that not only the MPUs with the highest number of milking cows have mostly hired personnel, this characteristic was also identified in the MPUs with the lowest number of milking cows (P>0.05); this coincides with other studies(54) where the use of (unpaid) family labor is not the key factor that determines the economic success of dairy farms.

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The average daily productivity per cow for the MPUs evaluated was 23.14 ± 6.9 L, there were significant differences (P<0.05) for the different herd sizes, with the sizes with the highest number of milking cows being the ones that obtain the highest number of liters per cow per day. The productivity per cow per day reported in this study was higher in relation to other results previously shown(33,55); this suggests that the efficiency in the use of the resources available in the MPUs by dairy farmers has increased. Compared to small MPUs, those with larger herds (>250 milking cows) showed higher productivity per cow and better sale price of milk (P<0.05) (Table 2); this coincides with what was established in other studies(11,33), where the scale in milk production units plays a determining role in economic or quality characteristics that could grant advantages to producers. On the other hand, 41.6 % of the producers with the lowest number of milking cows indicated that their relatives intended to give continuity to the dairy activity of the MPU, however, as the size of the herd grew, the positive response increased, the size of the groups and the dispersion of the response did not allow ensuring the significance of this effect (P=0.116). This suggests that there may be endogenous and exogenous elements in the MPUs that contribute to owners projecting their continuity, such as the market, economic profitability and expectations of growth and improvement. Table 2: Main characteristics of milk production units (MPU) by farm size <50 milking 51 to 250 > 250 milking Variable/Category P-value cows milking cows cows MPU 12 21 7 AB B Age of the owner, years 52.5 (39– 8) 63 (56–64) 45 (38–52)A 0.012* 1 Hired labor (yes/no) 7/5 12/9 5/2 0.792 a b c Milking cows, No. 35.8 ± 10.5 131.6 ± 64.6 357.1 ± 59.1 0.000*** a a b Productivity per cow, L/d 21.9 ± 9.6 21.8 ± 4.6 28.9 ± 5.1 0.019* A B C Sale price, $/L 6.3 (6.25–6.4) 6.4 (6.3–6.4) 6.5 (6.5– 6.7) 0.001** Willingness to continue in 5/7 11/10 6/1 0.166 the dairy activity (yes/no)1 Assessment of quality2 0.312 0.310 0.392 0.938 Competitive forces of the agribusiness3

3.05

3.13

a-c

3.42

0.558

Mean ± standard deviation, by row, those with different superscripts differ (P<0.05). A-C Median, by row, those with different superscripts differ (P<0.05). * P<0.05, ** P<0.01, *** P<0.001. 1 Chi-square with two degrees of freedom. 2 Average number of mentions of any of the 4 factors evaluated in the assessment of milk quality. 3 Average of the degree of agreement in the Competitive Forces of the Agribusiness with Likert scale (1-5).

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Regarding the binary logistic model evaluated, it was determined that the competitive forces of the agribusiness and the price had a significant impact (P<0.05) with the willingness to continue in dairy farms; by observing the coefficients of the model, it was established that continuity in the MPUs is more likely as the price of milk or the influence of the competitive forces of the agribusiness increases. Previous studies mention that agribusiness has control over the primary sector in Mexico, even that it has had a positive impact on the permanence of milk producers(5), this suggest that continuity in the MPUs is influenced by favorable interactions with other participants in the production chains. Regarding the assessment of the hypotheses proposed in this study, it was estimated that the effects of the competitive forces of the agribusiness explained 14.2 % of the variation in the assessment of milk quality (t ≥ 1.96; P≤0.05) and explained 22.7 % of the continuity in the dairy activity (t ≥ 2.57; P≤0. 01) (Table 3), which is considered to have a high impact in socioeconomic studies(43,48). To measure the total influence of the category of competitive forces of the agribusiness, this category was excluded from the analysis and with this, the size of its real effect on the structural model was determined. In the case of the size of the effect on the category of quality assessment and on the variable of possibility of continuity in the dairy activity, a significant f2 effect of medium size was found (>0.15)(42,56); this determines a model where the effects of the competitive forces of the agribusiness are not affected by the other variables involved in the final structural model. The quality of dairy products found in the markets is closely related to the quality of raw milk(57), therefore, the importance of properly attending the processes within the MPUs in order to contribute to ensuring the quality of milk and its derivatives is reaffirmed.

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Table 3: Results of the tests of the hypotheses proposed with the structural model Standardized THypothesis Relationship R² 𝒇𝟐 coefficient β value H1: The competitive forces of the agribusiness have a significant positive influence on quality assessment.

Competitive forces of the agribusiness 0.377** → Quality assessment

2.383

0.166

0.142

H2: The competitive forces of the agribusiness have a significant positive influence on the willingness to continue in dairy activity.

Competitive forces of the agribusiness → Willingness to 0.476*** continue in the dairy activity

4.285

0.292

0.227

H3: A larger herd size positively influences the sale price of milk.

Herd size → Sale 0.541*** price of milk

4.153

0.433

0.293

𝑓 2 Effect size: >0.02= small effect; >0.15= medium effect; >0.35 big effect (Cohen, 1988). R2: >0.20 = Weak; >0.33 Moderate; >0.67 = Substantial (Chin, 1998). ** P<0.01, *** = P<0.001.

It has been mentioned(19) that milk producers should be aware of the risk factors that may arise in milk production because it is a perishable product, this would favor the improvement of the quality of the product, especially due to the use of cooling tanks for milk collection; which suggests that institutional and market measures that are unrelated to production could act as an entry barrier for new competitors in the dairy agribusiness, and have effects on the competitiveness of the actors in the production chain, reducing the possibility of incorporating new technological advances(58), this would indirectly affect the generational transition in primary production units. The positive influence of herd size on the sale price of milk was 29.3 % (t ≥ 2.57; P≤0.01). To evaluate the size of the effect of the variable in the model, it was excluded from it and it was found that the size of the herd had a significant f2 effect, which can be considered as strong (>0.35)(42,56) (Table 3); this coincides with studies that mention that the scale of production positively affects competitiveness and has an impact on the production processes of dairy farms(33); in this way, the efficient use of resources in dairy farms would result in greater development of the sector.

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Continuity in the MPUs has been valued as a factor associated with successful productive characteristics(59); in this study, the producers recognized that the conditions of low productive efficiency were not a trigger for the immediate abandonment of dairy activity. However, producers with better use of their resources expressed their willingness to remain in the activity in the face of price fluctuations in the markets for inputs and dairy products(60,61). Coincidentally, as a strategy for the continuity of dairy farms, it has been shown that the efficient use of the resources available in production units is key to carrying out improvements in production processes, seeking to reduce costs(62,63). In this study, it was found that producers identified the assessment of the success of organizations as the situation that occurs when positive economic indicators are achieved, especially profitability(55). In addition, they recognized that the integration of producers with other actors involved in the production chain could increase their chances of success(64). It has been pointed out that horizontal integration, in some cases, facilitates access to the raw materials involved in production(65,66); in this sense, alternatives to increase the value of primary production would promote the increase in the profitability of the MPUs and would contribute to the obtaining of social benefits of the actors involved in the dairy production chain(14,15,67). Similarly, the vertical integration of producers through formal linkage mechanisms established with the industry could avoid the vulnerability of dairy production systems(68). In this study, it was identified that the competitive forces of the agribusiness could impact on the consolidation of organizations in the primary sector; the associated producers who managed to adapt to their environment show favorable conditions for achieving greater growth and economic success.

Conclusions and implications As formulated in the proposed hypotheses, the competitive forces of the agribusiness had a significant positive effect on the characteristics of milk production units, especially on the importance that producers attribute to the attention of crucial variables such as milk quality and permanence in the dairy activity. This permanence is more likely as the price of milk increases and as they have a favorable perception about the competitive environment of the production unit. This suggests that the implementation of strategies by farmers and authorities that promote the increase in the productivity of dairy farms will have beneficial effects on the Mexican agri-food sector, especially when they are oriented towards the production of quality milk, and that the latter contributes to satisfy markets that demand genuine dairy products. The meeting point between the interests of producers and agroindustrialists can converge in strategies, promoted by the State, that promote the production and development of the Mexican agri-food sector.

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Acknowledgements

Thanks to Lic. Jesús Azuara, leader of the organized group Agroindustrial Fátima S. P. R. de R. L. for the facilities for obtaining data, as well as to all the producers who participated in the study. To the Institute of Agricultural Research of Mabegondo, A Coruña, Spain Project financed with PRODEP resources (Release document: DSA/103.5/16/10627) and with extraordinary resources to support UAA researchers (PIAL16-1N). Literatura citada: 1.

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19. Naing YW, Wai SS, Lin TN, Thu WP, Htun LL, Bawm S, et al. Bacterial content and associated risk factors influencing the quality of bulk tank milk collected from dairy cattle farms in Mandalay Region. Food Sci Nutr 2019;7(3):1063-1071. 20. Kovaleva S, de Vries N. Competitive Strategies, Perceived Competition and Firm Performance of Micro Firms: The Case of Trento. In: Bögenhold D, et al, editors. Competitive strategies, perceived competition and firm performance of micro firms: the case of trento. Cham: Springer International Publishing; 2016:75-93. 21. Porter ME. Competitive advantage of nations: creating and sustaining superior performance. Simon and Schuster; 2011. 22. Porter M, Kramer M. La creación de valor compartido. Harv Bus Rev 2011;89(1):3249. 23. Porter M. Ventaja competitiva. Creación y sostenimiento de un desempeño superior. México: Patria; 2013. 24. Delgado M, Porter M, Scott S. Clusters, Convergence, and Economic Performance. NBER Working Paper Series 2012. Accessed Nov16, 2012. 25. Hove P, Masocha R. Interaction of technological marketing and porter’s five competitive forces on SME competitiveness in South Africa. 2014. 26. Dälken F. Are porter’s five competitive forces still applicable? a critical examination concerning the relevance for today’s business. University of Twente; 2014. 27. Cuevas-Vargas H, Parga-Montoya N, Fernández-Escobedo R. Effects of entrepreneurial orientation on business performance: the mediating role of customer satisfaction—a formative–reflective model analysis. SAGE Open 2019;9(2):2158244019859088. 28. García FG. Investigación comercial. Fourth ed. España: Esic Editorial; 2016. 29. Padilla LF. Expansión urbana e incorporación de colonias periféricas. En Aguascalientes: la Colonia Fátima. Expansión urbana e incorporación de colonias periféricas. XXVII Congreso de la Asociación Latinoamericana de Sociología. VIII Jornadas de Sociología de la Universidad de Buenos Aires, Argentina: Acta Académica; 2009. 30. Vargas MLM. Sobre el concepto de percepción. Alteridades 1994;4(8):47-53. 31. Ryan BF, Joiner BL, Cryer JD. MINITAB Handbook: Update for Release. Cengage Learning; 2012.

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47. Nunnally J. Teoría Psicométrica. México: Trillas; 2009. 48. Chin WW. The partial least squares approach to structural equation modeling. In: Marcoulides editor. The partial least squares approach to structural equation modeling. New Jersey, USA: Lawrence Erlbaum Associates; 1998:295-336. 49. Mooney CZ, Duval RD, Duvall R. Bootstrapping: A nonparametric approach to statistical inference. Sage; 1993. 50. Servicio Metereológico Nacional. Normales Climatológicas. Normales Climatológicas. México: SMN; 2010. 51. Sánchez GRA, Zegbe DJA, Gutiérrez BH. Tipificación de un sistema integral de lechería familiar en Zacatecas, México. Rev Mex Cienc Pecu 2015;6:349-359. 52. Castro L, Sánchez G, Iruegas L, Saucedo G. Tendencias y oportunidades de desarrollo de la red leche en México. FIRA Boletín Informativo 2001;33(317):1-137. 53. SAGARPA. Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación. Situación actual y perspectiva de la producción de leche bovino en México. México. 2005. 54. Jiménez JR, Ortiz V, Soler FD. El costo de oportunidad de la mano de obra familiar en la economía de la producción lechera de Michoacán, México. RIAA 2014;5:47. 55. Romo BCE, Valdivia FAG, Carranza TRG, Cámara CJ, Zavala AMP, Flores AE, et al. Brechas de rentabilidad económica en pequeñas unidades de producción de leche en el altiplano central mexicano. Rev Mex Cienc Pecu 2014;5(3):273-289. 56. Cohen J. Statistical power analysis for the behavioral sciences Lawrence Earlbaum Associates. Hillsdale, NJ. 1988:20-26. 57. Skeie SB, Håland M, Thorsen IM, Narvhus J, Porcellato D. Bulk tank raw milk microbiota differs within and between farms: A moving goalpost challenging quality control. J Dairy Sci 2019;102(3):1959-1971. 58. Gargiulo JI, Eastwood CR, Garcia SC, Lyons NA. Dairy farmers with larger herd sizes adopt more precision dairy technologies. J Dairy Sci 2018;101(6):5466-5473. 59. Albarrán-Portillo B, Rebollar-Rebollar S, García-Martínez A, Rojo-Rubio R, AvilésNova F, Arriaga-Jordán CM. Socioeconomic and productive characterization of dualpurpose farms oriented to milk production in a subtropical region of Mexico. Trop Anim Health Prod 2015;47(3):519-523.

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60. Pieralli S, Hüttel S, Odening M. Abandonment of milk production under uncertainty and inefficiency: the case of western German Farms. Eur Rev Agric Econ 2017;44(3):425454. 61. Tauer LW. When to get in and out of dairy farming: a real option analysis. Agric Econ Res Rev 2006;35(2):339-347. 62. Westbrooke V, Nuthall P. Why small farms persist? The influence of farmers’ characteristics on farm growth and development. The case of smaller dairy farmers in NZ. Aust J Agric Resour Econ 2017;61(4):663-684. 63. Hanrahan L, McHugh N, Hennessy T, Moran B, Kearney R, Wallace M, et al. Factors associated with profitability in pasture-based systems of milk production. J Dairy Sci 2018;101(6):5474-5485. 64. De Los Rios-Carmenado I, Becerril-Hernandez H, Rivera M. La agricultura ecológica y su influencia en la prosperidad rural: visión desde una sociedad agraria (Murcia, España). Agrociencia 2016;50(3):375-389. 65. Carranza TRG, Valdivia FAG. Supply chain: an input-output perspective. An example of application in the dairy products industry. IJSCOR 2018;3:236. 66. García Cáceres RG, Vergara CL, Ortiz Rodríguez OO. Characterization of the supply and value chains of the Colombian potato agribusiness Sector. Espacios 2018;39(48):2442. 67. Olarte Calsina S, Olarte Daza U. La producción de leche orgánica en la región Puno: una alternativa de desarrollo sostenible. Mundo Agrar 2013;13(26). 68. Martinez Borrego E. La lechería en el Estado de México: sistema productivo, cambio tecnológico y pequeños productores familiares en la región de Jilotepec. UNAMInstituto de Investigaciones Sociales/Bonilla Artigas Editores; 2009.

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https://doi.org/10.22319/rmcp.v13i2.6017 Article

Antimicrobial activity of plants native to Sonora, Mexico, against pathogenic bacteria isolated from milk from cows diagnosed with mastitis

Jesús Sosa-Castañeda a Carmen Guadalupe Manzanarez-Quin b Ramón Dolores Valdez-Domínguez a Cristina Ibarra-Zazueta a Reyna Fabiola Osuna-Chávez a Edgar Omar Rueda-Puente a Carlos Gabriel Hernández-Moreno a Alejandro Santos-Espinosa c Alejandro Epigmenio-Chávez c Claudia Vanessa García-Baldenegro c Tania Elisa González-Soto c Ana Dolores Armenta-Calderón c Priscilia Yazmín Heredia Castro c*

a

Universidad de Sonora. Departamento de Agricultura y Ganadería. Carretera 100 a Bahía Kino km 21, 83000. Sonora, México. b

Centro de Investigación en Alimentación y Desarrollo. Tecnología de Alimentos de Origen Animal. Sonora, México. c

Universidad Estatal de Sonora. Unidad Hermosillo. Ingeniería en Horticultura. Sonora, México.

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*Corresponding author: priscilia.heredia@ues.mx

Abstract: Bovine mastitis is a disease caused by pathogenic bacteria that infect the mammary gland of dairy cattle, which generates significant economic losses, in addition, due to the excessive use of antibiotics to treat this disease, microorganisms have created resistance to these drugs, therefore, new alternatives are sought for this purpose. The objective was to evaluate the antimicrobial effect of extracts of plant native to Sonora against pathogenic bacteria isolated from cows diagnosed with mastitis. Seventeen ethanolic extracts were obtained from plants native to Sonora, and their antimicrobial activity was evaluated by the agar diffusion method against seven pathogens isolated from milk from cows with mastitis, using a concentration of 50 mg/ml of each extract. The content of total phenols and flavonoids was determined by spectrophotometry. The results showed that extracts of Ibervillea sonorae (wereke, tuber), Populus alba (poplar, leaves), Ambrosia ambrosioides (chicura, stems), Krameria sonorae (cosahui, roots) and Prosopis velutina (mesquite, leaves) were effective in eliminating S. aureus, Streptococcus spp., E. coli, Enterobacter spp., Proteus spp., Shigella spp. and Citrobacter spp. (P<0.05). In addition, extracts high in total phenols and flavonoids (wereke, poplar, chicura, cosahui and mesquite) showed an inverse correlation with respect to pH (r= -0.94, r= -0.92, respectively) (P<0.05) and had greater antimicrobial activity against the tested pathogens. Therefore, the extracts of plants from Sonoran could represent an alternative for the control of Gram (+) and Gram (-) pathogens that infect the mammary gland of dairy cattle. Key words: Mastitis, Pathogens, Antimicrobial, Plant Extracts, Natural Alternative, Phenols, Flavonoids.

Received: 11/07/2021 Accepted: 20/10/2021

Introduction Mastitis is the main infectious disease that occurs in dairy cattle. The origin of this disease is multifactorial and may depend on the management, production system and environmental conditions in which the cattle are found, and occurs as a response to the infection of a great 376


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biodiversity of microorganisms, such as mycoplasmas, yeasts, fungi, viruses and bacteria(1), and usually manifests as an inflammation in the mammary gland, which, depending on the severity of the infection, can generate fibrosis, mammary edema, atrophy of mammary tissue, abscesses or gangrene; in addition, it can alter the physical and chemical properties of milk, increasing the number of somatic cells and the microbial load in the milk, which can lower the pH of the milk and alter the taste and smell. Likewise, the milk from cows with mastitis has less lactose, fat and caseins, which decreases its technological properties for the food industry(2,3). Sometimes mastitis can be detected clinically, that is, when the presence of pus or blood in the mammary gland and milk is physically observed; whereas, on other occasions, mastitis occurs subclinically and is usually more difficult to detect, since inflammation in the udder is not visible and milk shows a normal physical appearance(4,5). Some of the pathogenic bacteria responsible for this disease are Staphylococcus aureus, Streptococcus agalactiae, Corynebacterium bovis and Mycoplasma bovis, which can cause significant damage to the mammary gland, such as lesions, and in more severe cases, they can generate necrosis in the tissue. Generally, infection with these bacteria occurs at the time of milking(3,6), although, on other occasions, infection may also occur through contact with other bacteria present in the environment, such as Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, Streptococcus uberis, Streptococcus dysgalactiae, among others(5). Although these bacteria are considered pathogenic, they are usually less aggressive at the time of infection, in addition, milk with the presence of these microorganisms cannot be marketed(5,6). Today, mastitis remains one of the biggest challenges in dairy farms, since it is estimated to represent 70 % of dairy farmers’ expenses, which generates annual economic losses of approximately $35 billion dollars worldwide and $2 billion dollars in the United States(7,8,9). In Mexico, losses of $2 and a half million pesos are estimated, which represents between 20 and 30 % of clinical mastitis, so the losses could still be greater due to the other 70-80 % of animals that present subclinical mastitis(10). In Sonora, studies of bovine mastitis are very scarce, however, in a dairy farm in Santa Ana, Sonora, the presence of subclinical mastitis was found in 18.3 % of the animals, while the incidence of clinical mastitis was 5.35 %, where the average monthly cost of each animal with mastitis was $185.40 and the total cost was $30,966.34, $12,470.75 (40.3 %) corresponding to subclinical mastitis and $18,459.59 (59.7 %) to clinical mastitis(11). Today, mastitis is considered the most expensive disease within dairy farms due to the decrease in milk production, waste of contaminated milk, replacement of animals and use of medicines(10). In this context, the excessive use of therapies with antibiotics to prevent or treat this disease has caused some microorganisms to adapt and acquire resistance to these drugs, for example, S. aureus has shown 59.5 % and 49.6 % resistance to penicillin and ampicillin, respectively; while some strains of Streptococcus spp. have reported 40 %, 80 % and 73 % resistance against erythromycin, oxytetracycline and penicillin, respectively; in 377


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addition, some strains of E. coli have shown 88.24 % resistance against erythromycin, oxytetracycline, penicillin and streptomycin and 70.59 % resistance against gentamicin, therefore, one of the great challenges of the Health Sector is to reduce the use of antibiotics in animals and humans(12-15). In this context, the use of natural chemical compounds derived from plants to treat diseases in humans and animals has been increasing in recent decades(16,17). In Mexico, it is estimated that there are around 26,000 species of plants, of which around 4,000 species are used to treat diseases in a traditional way(18,19). Although it has been reported that some plants native to northwestern Pakistan have been effective in eliminating bacteria associated with bovine mastitis(9), the use of plants from Sonora, Mexico, for this purpose has not been reported. However, their antimicrobial potential against the bacteria of the collection Helicobacter pylori ATCC 43504, Mycobacterium tuberculosis H37Rv, Escherichia coli ATCC 35219 and 25922, Shigella flexneri ATCC 12022 and Salmonella typhimorium ATCC 14028(19,20,21) has been evidenced. Considering that Sonora has a great biodiversity of native plants, of which around 400 are used by local ethnic groups to treat diseases(20), and that some of these plants have also shown antimicrobial potential, it is interesting to evaluate the antimicrobial effect of extracts of plants native to Sonora against pathogenic bacteria isolated from cows diagnosed with mastitis.

Material and methods Preparation of ethanolic extracts

The extracts were obtained from 17 plants native to the state of Sonora, Mexico (Table 1), which were harvested in the Botanical Garden of the Department of Agriculture and Livestock (DAG, for its acronym in Spanish) of the University of Sonora (UNISON, for its acronym in Spanish) and identified in the Herbarium of the DAG. Each plant was dehydrated at 34 °C in a hot air oven (Thelco, Precision Science, model 28, USA) and then pulverized in a mill (Pulvex Mini 100, MX) until obtaining a particle size of 100 microns. Subsequently, 100 g of dry matter was placed, and 100 ml of 99 % pure ethanol (Sigma-Aldrich, St. Louis MO) was added in a hermetically sealed glass bottle, which were stored for 5 days in the dark at 25 °C(22). The extracts were filtered with Whatman No. 41 filter paper and the plant material was dehydrated again. The difference in weight of the plant material before and after its storage was considered as the amount of soluble chemical compounds extracted from plants(23). The ethanolic extracts were then concentrated in a rotary evaporator (Yamato RE300) at 40 °C and adjusted to 50 mg/ml with a 20 % dimethyl sulfoxide (DMSO) solution. Finally, the extracts were stored in the dark at 4 °C until use.

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Key E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 E13 E14 E15 E16 E17

Table 1: Identification and parts of the plants used in the ethanolic extracts Common Family Scientific name Part name Poplar Salicaceae Populus alba Leaves Batamote Asteraceae Baccharis glutinosa Stems Ambrosia Chicura Asteraceae Stems ambrosioides Cosahui Krameriaceae Krameria sonorae Root Leucaena Guaje Fabaceae Leaves leucocephala Guamúchil Fabaceae Pithecellobium dulce Bark Simmondsia Jojoba Simmondsiaceae Leaves chinensis Mesquite Fabaceae Prosopis velutina Leaves Parkinsonia Stems and Palo verde Fabaceae microphylla leaves Stems and Palo verde azul Fabaceae Cercidium floridum leaves Rama blanca Asteraceae Encelia farinosa Leaves Jatropha Sangregado Euphorbiaceae Stems cardiophylla Tepehuaje Fabaceae Lysiloma watsonii Leaves Torote Burseraceae Bursera microphylla Leaves Vinorama Fabaceae Acacia constricta Leaves Wereke Cucurbitaceae Ibervillea sonorae Tuber Coursetia Zamota Fabaceae Stems glandulosa Plants harvested in the Botanical Garden of the DAG of UNISON.

Determination of total phenols

One milligram of extract was used and mixed with 0.5 ml of Folin-Ciocalteu reagent. Then, 10 ml of distilled water and 1 ml of saturated Na2CO3 were added and homogenized for 3 min. Finally, the mixture was measured to 25 ml with distilled water and left to stand for 1 h in a place free of light. Absorbance was measured at a wavelength of 750 nm on a spectrophotometer (Spectro Max MD, EU) and total phenol content was expressed as milligrams of gallic acid equivalents per gram of extract(24).

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Determination of total flavonoids

Zero point two five milligrams of extract were used and mixed with 5 ml of distilled water. Afterwards, 0.3 ml of a 5 % NaNO2 solution was added, and the mixture was left to stand in the dark for 6 min. Subsequently, 0.6 ml of a 10 % AlCl3⋅6H2O solution was added and left to stand until the reaction was complete. Finally, 2 ml of NaOH (1 M) was added and the mixture was measured to 10 ml with distilled water. Absorbance was measured at a wavelength of 510 nm in a spectrophotometer (Spectro Max MD, EU) and the total flavonoid content was expressed as milligrams of quercetin per gram of extract(24).

Place of study and sample collection

The samples were taken from two farms located on the outskirts of the city of Hermosillo, Sonora, Mexico. Site-1 belongs to the ejido La Yesca, located southwest of the city 10 km away, while Site-2 belongs to the ejido Los Bajotes, located northwest of the city 12 km away. The cows were selected according to the technique of the California mastitis test(25). The samples were collected over a year and taken from the 4 quarts of 15 cows from Site-1 and 15 from Site-2. Prior to sample collection, the teats were immersed in a 1 % iodine-based solution for 30 sec, and subsequently the excess of iodine was removed with a disposable towel. Then 10 ml of milk was collected from each quarter in a sterile bottle with a screw cap and each teat was again immersed in the disinfectant solution. Finally, the samples were transported at 4 °C and processed two hours after their collection(26).

Isolation and identification of bacteria associated with mastitis in milk

For the isolation and identification of the bacteria from the milk from cows diagnosed with mastitis, the methodology reported by Rodríguez and Muñoz(27) was used. The samples were seeded by stria in Columbia agar base added with 5 % of ram’s blood (BD Difco, Sparks, MD) and MacConkey agar (BD Difco, Sparks, MD) and incubated at 37 °C for 48 h. Afterwards, a Gram stain, coagulase test, oxidase test (Kovacs reagent) and catalase test (3 % H2O2) were performed to differentiate the isolated colonies. The selected colonies were purified three times by subsequent cultures under the conditions stated above. The isolated bacteria were identified by the commercial kits API20E, API Staph and API Strep (BioMérieux, Marcy, France), following the instructions of the manufacturers. The identified bacteria were stored at -80 °C in 80 % glycerol (v/v) until their use.

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Antimicrobial activity of ethanolic extracts

Bacteria isolated from milk from cows with mastitis were propagated in the BHI broth culture medium (brain-heart infusion, BD Difco, Sparks, MD). Subsequently, three plates were prepared with BHI agar (brain-heart infusion, BD Difco, Sparks, MD) for each of the pathogenic bacteria and four sterile discs of Whatman No. 41 filter paper of 6 mm in diameter were placed in each plate, where 20 μL of each ethanolic extract (50 mg/ml) were added. Finally, the plates were incubated at 37 °C for 24 h. Halos greater than 3 mm were considered as inhibition according to the criteria used by Heredia-Castro(28).

Statistical analysis

A completely randomized one-way experimental design at 95 % confidence was used, with three repetitions per treatment. The mean comparison test was performed by Tukey-Kramer at a significance level of 0.05 %, and the correlation analysis was performed with 95 % reliability. The statistical software used was NCSS version 11.

Results and discussion The chemical compounds responsible for the antimicrobial activity of plants are synthesized in the cytoplasm of cells, and within these compounds are flavonoids, which are part of a group of chemical compounds called phenols(29). Table 2 shows the chemical analyses and yield of ethanolic extracts. The results showed that the pH of the ethanolic extracts varied in a range of 4.35 to 6.22, with extract E5 being the most acidic and E11 being the least acidic (P<0.05). Likewise, extract E5 had the highest concentration of total phenols (143.68 ± 0.04 mg) and total flavonoids (95.10 ± 0.05 mg), while extract E11 had the lowest values for total phenols (56.28 ± 0.05 mg) and total flavonoids (30.08 ± 0.90 mg) (P<0.05). The pH of the extracts may be due to the acidic nature of the total phenols and flavonoids, or to the presence of other polar compounds such as tannins, benzoic, oleic, stearic and lignoceric acids, among others(30). In this context, Al-rifai et al(24) studied two medicinal plants from Saudi Arabia (Convolvulus austroaegyptiacus and Convolvulus pilosellifolius) and reported that the total phenol and flavonoid content of the ethanolic extracts was similar to those found in this study. In addition, in the ethanolic extracts of Vernonia amygdalina and Tephrosia purpurea, the presence of total phenols and flavonoids was also found(31,32), however, Bitchagno et al(33) did not find the presence of flavonoids in the ethanolic extract of the fruit of Tectona grandis.

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This suggests that the chemical compounds present may vary from one part of the plant tissue to another.

Extract

Table 2: Chemical analysis and yield of ethanolic extracts pH Total phenols Total flavonoids Yield (%)

E1 E2

4.86 5.22

130.26 ± 0.05d 115.45 ± 0.03f

89.23 ± 0.08d 80.06 ± 0.03h

6.33 5.54

E3

5.15

120.33 ± 0.06e

85.09± 0.07f

5.45

E4

4.82

135.03 ± 0.06c

91.06 ± 0.02c

4.51

E5

4.35

143.68 ± 0.04a

95.10 ± 0.05a

7.82

E6

5.34

110.03 ± 0.04i

70.06 ± 0.08k

4.02

E7

4.4

140.65 ± 0.07b

93.05 ± 0.07b

8.39

E8

5.34

112.12 ± 0.03g

74.05 ± 0.80i

6.49

E9

5.43

95.23 ± 0.08j

71.05 ± 0.03j

6.72

E10

5.6

85.24 ± 0.06k

70.09 ± 0.04k

6.55

E11

6.22

56.28 ± 0.05l

30.08 ± 0.90l

7.44

E12

5.22

115.02 ± 0.04f

81.18 ± 0.06g

5.23

E13 E14 E15

4.57 5.3 5.41

130.14 ± 0.09d 111.56 ± 0.02h 110.09 ± 0.06i

93.78 ± 0.05b 79.28 ± 0.20h 73.29 ± 0.07i

8.62 8.35 6.65

E16 E17

5.55 5.11

109.89 ± 0.07i 120.02 ± 0.04e

70.47 ± 0.80k 87.55 ± 0.40e

9.32 5.85

Total phenols= mg of gallic acid/g of extract; Total flavonoids= mg of quercetin/g of extract. Different literal indicates a significant difference between the data of the same column (P<0.05).

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On the other hand, the yield of the extracts was variable, with extract E16 showing the highest yield (9.32 %) and extract E6 being the one with the lowest yield (4.02 %). In agreement with this study, variations in the yield of extracts obtained with plants from Pakistan were also reported(34). In addition, similar results were reported by Mostafa et al(35), where Punica granatum had the highest yield (9.74 %), while Cuminum cyminum had the lowest yield (3.12 %). Likewise, the yield of ethanolic extracts of 49 medicinal plants from Indonesia was evaluated in another study, and it was found that the highest yield was for the fruit of Salacca zalacca (77.89 %), while the lowest yield was reported in the root of Plectranthus scutellarioides (3.07 %). Additionally, the authors reported that extracts made with leaves showed higher yield compared to extracts where roots or woodier parts of the plants were used(36), which coincides with what was found in this study. This suggests that the amount of ethanol-soluble compounds depends on each plant and the part used. 382


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On the other hand, mastitis is an infectious disease of bacterial origin and is usually very persistent inside dairy farms. Table 3 shows the bacteria identified by biochemical tests and the frequency with which they appear in the milk from cows diagnosed with mastitis. The results showed that, at Site-1, E. coli occurred most frequently in 58 of 60 samples analyzed, followed by S. aureus with 35, Proteus spp. with 6 and Enterobacter spp. with 4. On the other hand, at Site-2, E. coli was found in 43.8 % of the samples analyzed, followed by S. aureus with 32.85 %, Streptococcus spp. with 10.95 %, Shigella spp. with 8.76 % and Citrobacter spp. with 3.65 %. In this study, E. coli and S. aureus were the most representative pathogens for both herds, since their presence was found in 82.50 % of the total samples analyzed. Other authors have mentioned that bacteria of the genus Streptococcus, as well as E. coli and S. aureus, are common microorganisms in cows diagnosed with mastitis(5,37,38), which coincides with what was found in this research. The microorganisms that cause mastitis varied between Site-1 and Site-2, which suggests that the environment and animal management may influence the biodiversity of the pathogenic microorganisms that cause mastitis(5). Table 3: Bacteria identified by biochemical tests and frequency of pathogens present in the milk from cows diagnosed with mastitis Place Bacteria Frequency Percentage Site-1 Staphylococcus aureus 35 33.98 Escherichia coli 58 56.31 Proteus spp. 6 5.83 Enterobacter spp. 4 3.88 Total 103 100.00 Site-2 Staphylococcus aureus 45 32.85 Streptococcus spp. 15 10.95 Escherichia coli 60 43.8 Shigella spp. 12 8.76 Citrobacter spp. 5 3.65 Total 137 100.00 Frequency= number of times the same pathogen occurs in different samples; Percentage= Frequency * 100/Total.

Table 4 shows the antimicrobial activity of ethanolic extracts against pathogenic bacteria isolated from cows diagnosed with mastitis. The results showed that extract E16 presented the highest antimicrobial activity against S. aureus (20.50 ± 1.70 mm) (P<0.05), while extracts E9 and E10 had the lowest activity (5.50 ± 0.70 and 5.50 ± 0.70) (P<0.05). On the other hand, extracts E1 and E16 (Populus alba and Ibervillaea sonorae) showed the highest activity against E. coli (13.00 ± 1.51 mm and 13.00 ± 1.40 mm) (P<0.05), while extract E13 383


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had the lowest activity (3.00 ± 0.70 mm) (P<0.05) and extract E10 did not present activity against that pathogen (P>0.05). Likewise, extract E16 had the highest activity against Enterobacter spp. (16.00 ± 2.40 mm) (P<0.05), while extract E9 showed the lowest activity (5.50 ± 0.70 mm) (P<0.05) and extracts E2, E10, E15 and E17 had no antimicrobial activity against the same pathogen (P>0.05). Extracts E8 and E16 showed the highest activity against Proteus spp. (13.50 ± 1.11 mm and 13.50 ± 2.70 mm) (P<0.05), while extracts E2, E9 and E10 had the lowest antimicrobial activity (5.50 ± 0.60 mm, 5.50 ± 0.70 mm and 5.50 ± 0.70 mm) (P<0.05), and extracts E5 and E13 showed no activity against this pathogen (P>0.05). Similarly, extract E16 presented the highest antimicrobial activity against Streptococcus spp., whereas extracts E12 and E17 had the lowest activity (5.00 ± 0.41 mm and 5.00 ± 0.50 mm) (P<0.05), and extracts E5 and E13 were not efficient against this pathogen (P>0.05). On the other hand, extracts E2 and E8 had the highest antimicrobial activity against Shigella spp. (15.50 ± 2.32 mm and 16.00 ± 1.40 mm) (P<0.05), while extracts E3, E9 and E14 showed the lowest activity (5.50 ± 0.68 mm, 5.50 ± 0.70 mm and 5.0 ± 1.41 mm) (P<0.05), and extract E15 showed no activity against that pathogen (P>0.05). Finally, extract E16 presented the highest antimicrobial activity against Citrobacter spp. (17.00 ± 2.4 mm) (P<0.05), while extract E14 showed the lowest activity (4.5 ± 1.41 mm) (P<0.05), and extracts E2, E9 and E17 were not shown to be effective against this bacterium (P>0.05). Similar results have been reported in S. aureus isolated from cows with mastitis using the extracts of Piptadenia viridiflora and Schinopsis brasiliensis(39), likewise, it has been reported that the extracts of Calpurinia aurea, Croton macrostachyus and Nicotiana tabacum were efficient in inhibiting the growth of S. aureus, which causes mastitis in ruminants(40). It has also been reported that S. aureus, Staphylococcus epidermidis and Streptococcus agalactiae isolated from cows with mastitis were susceptible to the extract of Zingiber officinale Roscoe(41) and the extract of Sanguisorba officinalis was efficient in inhibiting the formation of the biofilm of S. aureus isolated from cows with mastitis. This is favorable since the biofilm is a protective barrier of the bacteria, and by inhibiting its formation, the bacterium is exposed to the natural protection of the host(42). Finally, the effect of purified compounds extracted from plants (trans-cinnamaldehyde, eugenol, carvacrol and thymol) demonstrated their efficacy by inhibiting the growth of S. aureus, E. coli, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis isolated from cows with mastitis(43).

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Table 4: Antimicrobial activity of ethanolic extracts against pathogenic bacteria isolated from milk from cows with mastitis EXT E1 E2 E3 E4 E5 E8 E9 E10 E12 E13 E14 E15 E16 E17

S. aureus 9.00 1.21e 6.00 0.75gh 11.50 2.16cd 10.50 1.11d 12.50 2.32bc 12.00 1.31b 5.50 0.70h 5.50 0.70h 5.00 1.2h 7.50 1.10f 9.00 1.71e 8.50 1.12f 20.50 1.70ª 6.50 0.70g

± ± ± ± ± ± ± ± ± ± ± ± ± ±

Streptococcus spp. 9.00 ± 1.41d 8.50 ± 0.90d 12.50 ± 2.12c 8.50 ± 2.42d n.p 12.00 ± 1.81c 14.50 ± 1.70b 8.50 ± 1.20d 5.00 ± 0.41f n.p 7.00 ± 1.22e 15.50 ± 1.62b 19.50 ± 1.90a 5.00 ± 0.50f

Enterobacter spp.

E. coli 13.00 1.51ª 8.00 1.16d 10.50 0.80c 10.50 1.15c 11.00 2.34bc 12.00 1.33ab 7.00 0.80de

± ± ± ± ± ± ±

n.p 4.50 0.50g 3.00 0.70h 6.0 1.40ef 10.50 0.70c 13.00 1.40a 5.50 0.70fg

10.00 ± 1.21e n.p 15.00 ± 0.70ab 12.00 ± 2.62d 8.50 ± 0.92f 10.50 ± 1.41e 5.50 ± 0.70g n.p

± ± ± ± ± ±

8.50 ± 0.60f 13.50 ± 2.70c 14.0 ± 1.41bc n.p 16.00 ± 2.40a n.p

Proteus spp. 8.00 ± 0.92f 5.50 ± 0.60g 9.50 ± 1.10d 11.50 ± 1.19b n.p 13.50 1.11a 5.50 0.70g 5.50 0.70g 7.00 1.31f

± ± ± ±

n.p 10.00 2.22c 11.50 1.12b 13.50 2.70ª 8.50 0.70de

± ±

Shigella spp. 8.00 ± 0.61d 15.50 ± 2.32a 5.50 ± 0.68f 11.00 ± 1.15c 12.50 ± 2.15b 16.00 ± 1.40a 5.50 ± 0.70f 8.00 ± 1.41d 11.50 ± 1.70bc 6.50 ± 0.70e 5.0 ± f 1.41 n.p

± 8.00 1.4d ± 6.50 0.70e

Citrobacter spp. 10.00 ± c 1.22 n.p 6.50 0.70d 14.00 2.00b 5.00 0.12e 10.00 1.31c

± ± ±

n.p 9.00 1.40c 15.00 2.70b 9.50 0.70c

± ± ±

4.5 ± 1.41f

6.50 0.70d ± 17.00 2.4a ± n.p

EXT= extract; (extracts 6,7 y 11 did not present any activity). Results expressed in mm of inhibition halos; Concentration of extracts= 50 mg/ml; n.p.= it did not present activity. abcde Different literal indicates a significant difference between the data in the same column (P<0.05).

It is interesting to mention that the pH of the extracts showed an inverse correlation with the concentration of total phenols (r= -0.94, P<0.05) and total flavonoids (r= -0.92, P<0.05), that is, the extracts with the lowest pH had higher concentrations of total phenols and flavonoids. In addition, extracts with the highest content of these compounds had greater antimicrobial activity, which suggests that this effect could be associated with the amount of total phenols and flavonoids present in the extracts, since it has been suggested that flavonoids have a hydroxyl (-OH) functional group that increases hydroxylation reactions on the surface of bacteria, altering their functionality and decreasing the thickness of the lipid bilayer, altering 385

±

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the fluidity of the cell membrane and increasing its permeability, causing the outflow of ions and intracellular proteins, causing the death of bacteria, or they can modify the metabolism of bacteria by altering the synthesis of DNA and proteins, which can cause the death of bacteria(44,45).

Conclusions and implications The ethanolic extract of Ibervillea sonorae (wereke) was the most efficient in eliminating pathogenic bacteria isolated from the milk from cows diagnosed with mastitis. However, extracts of Populus alba (poplar), Ambrosia ambrosioides (chicura), Krameria sonorae (cosahui) and Prosopis velutina (mesquite) also exhibited significant antimicrobial activity. In addition, antimicrobial activity was related to the content of total phenols and flavonoids present in the extracts. Therefore, extracts of plant native to Sonora, Mexico, can be considered in in vivo tests as an alternative and natural treatment to control infections in the mammary gland caused by different microorganisms in dairy cattle.

Acknowledgements

To the support of the State University of Sonora and the University of Sonora for the use of materials and facilities, as well as to Lic. Gerardo Reyna Cañez for his technical support. This research work was supported by the project UES-PII-20-UAH-IH-02. Literature cited: 1. Miranda S, Albuja C, Tríbulo H. Asociación entre la mastitis subclínica con la pérdida temprana de gestación en un hato de vacas lecheras. La granja Rev Ciencias la Vida 2019;30(2):48-56. 2. Andrade RM, Espinoza MM, Rojas JA, Tirado PO, Salas RG, Falcón VV. Mastitis bovina y su repercusión en la calidad de la leche. Rev Electrón Vet 2017;18(11):1-16. 3. Ruegg PL. A 100-year review: Mastitis detection, management, and prevention. J Dairy Sci 2017;100(12):10381-10397. 4. Calderón A, Rodríguez VCR. Prevalencia de mastitis bovina y su etiología infecciosa en sistemas especializados en producción de leche en el altiplano cundiboyacense (Colombia). Rev Colomb Cienc Pecu 2008;21(4):582-589.

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5. Klaas IC, Zadoks RN. An update on environmental mastitis: Challenging perceptions. Transbound Emerg Dis 2018;65:166-185. 6. Abebe R, Hatiya H, Abera M, Megersa B, Asmare K. Bovine mastitis: prevalence, risk factors and isolation of Staphylococcus aureus in dairy herds at Hawassa milk shed, South Ethiopia. BMC Vet Res 2016;12(1):1-11. 7. Steeneveld W, van Werven T, Barkema HW, Hogeveen H. Cow-specific treatment of clinical mastitis: An economic approach. J Dairy Sci 2011;94(1):174-188. 8. Guimarães JL, Brito MA, Lange CC, Silva MR, Ribeiro JB, Mendonça LC, et al. Estimate of the economic impact of mastitis: A case study in a Holstein dairy herd under tropical conditions. Prev Vet Med 2017;142:46-50. 9. Amber R, Adnan M, Tariq A, Khan SN, Mussarat S, Hashem A, et al. Antibacterial activity of selected medicinal plants of northwest Pakistan traditionally used against mastitis in livestock. Saudi J Biol Sci 2018;25(1):154-161. 10. Bedolla CC, de León MP. Pérdidas económicas ocasionadas por la mastitis bovina en la industria lechera. Rev Electrón Vet 2008;9(4):1-26. 11. Gerlach BFA, Ayala AF, Denogean BFG, Moreno MS, Gerlach BLE. Incidencia y costo de la mastitis en un establo del municipio de Santa Ana, Sonora. Rev Mex Agroneg 2009;24(8):789-796. 12. Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 2010;74(3):417-433. 13. Oliver SP, Murinda SE, Jayarao BM. Impact of antibiotic use in adult dairy cows on antimicrobial resistance of veterinary and human pathogens: a comprehensive review. Foodborne Pathog Dis 2011;8(3):337-355. 14. Oliver SP, Murinda SE. Antimicrobial resistance of mastitis pathogens. Vet Clin Food Anim Pract 2012;28(2):165-185. 15. Cameron A, McAllister TA. Antimicrobial usage and resistance in beef production. J Anim Sci Biotechnol 2016;7(1):1-22. 16. Mushtaq S, Shah AM, Shah A, Lone SA, Hussain A, Hassan QP, et al. Bovine mastitis: An appraisal of its alternative herbal cure. Microb Pathog 2018;114:357-361. 17. Ginovyan M, Petrosyan M, Trchounian A. Antimicrobial activity of some plant materials used in Armenian traditional medicine. BMC Compl Alternative Med 2017;17(1):1-9.

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39. Ribeiro ICDO, Mariano EGA, Careli RT, Morais-Costa F, de Sant’Anna FM, Pinto MS, et al. Plants of the Cerrado with antimicrobial effects against Staphylococcus spp. and Escherichia coli from cattle. BMC Vet Res 2018;14(1):1-10. 40. Kalayo S, Haileselassie M, Gebre-egziabher G, Tiku'e T, Sahle S, Taddele H, et al. In vitro antimicrobial activity screening of some ethnoveterinary medicinal plants traditionally used against mastitis, wound and gastrointestinal tract complication in Tigray Region, Ethiopia. Asian Pac J Trop Biomed 2012;2(7):516-522. 41. Poeloengan M. The effect of red ginger (Zingiber officinale Roscoe) extract on the growth of mastitis causing bacterial isolates. Afr J Microbiol Res 2011;5(4):382-389. 42. Chen X, Shang F, Meng Y, Li L, Cui Y, Zhang M, et al. Ethanol extract of Sanguisorba officinalis L. inhibits biofilm formation of methicillin-resistant Staphylococcus aureus in an ica-dependent manner. J Dairy Sci 2015;98(12):8486-8491. 43. Baskaran SA, Kazmer GW, Hinckley L, Andrew SM, Venkitanarayanan K. Antibacterial effect of plant-derived antimicrobials on major bacterial mastitis pathogens in vitro. J Dairy Sci 2009;92(4):1423-1429. 44. Radulovic NS, Blagojevic PD, Stojanovic-Radic ZZ, Stojanovic NM. Antimicrobial plant metabolites: structural diversity and mechanism of action. Curr Med Chem 2013;20(7):932-952. 45. Mickymaray S. Efficacy and mechanism of traditional medicinal plants and bioactive compounds against clinically important pathogens. Antibiotics 2019;8(257):1-57

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https://doi.org/10.22319/rmcp.v13i2.5942 Article

Pharmacokinetic analysis of intraarticular injection of insulin and its effect on IGF-1 expression in synovial fluid of healthy horses

Fernando García-Lacy a* Lilia Gutiérrez-Olvera b María Bernad c Lisa Fortier d Francisco Trigo-Tavera e Margarita Gómez-Chavarín f Alejandro Rodríguez-Monterde g

a

Universidad Nacional Autónoma de México (UNAM). Facultad de Medicina Veterinaria y Zootecnia (FMVZ), Ciudad de México, México. b

UNAM. FMVZ, Departamento de Fisiología y Farmacología, Ciudad de México, México. c

UNAM. Facultad de Química, Departamento de Farmacia, Ciudad de México, México.

d

Cornell University. Ithaca, USA.

e

UNAM. FMVZ, Departamento de Patología, Ciudad de México, México.

f

UNAM. Depto. Fisiología, Facultad de Medicina, Ciudad de México, México.

g

UNAM. FMVZ, Secretario de Medicina, Ciudad de México, México.

*Corresponding author: f_garcialacy@hotmail.com

Abstract: Insulin induces mitosis on equine chondrocytes in vitro and enhances production of type II collagen. Insulin, when administered intra-articularly, changes the composition of synovial fluid, including the concentrations of glucose, insulin and glycemia levels. The

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concentration of insulin in the joint was measured by using high-performance liquid chromatography (HPLC), which provided pharmacokinetic values. Six mixed breed horses were administered three different doses of insulin into one antebrachiocarpal joint (10, 15 and 20 IU) and isotonic saline was administered into the contralateral joint. The blood glucose concentration significantly changed through time for all three doses (P<0.0001). No significant differences in protein concentration and cell count in synovial fluid were found between treated and control joints (P>0.05), no significant difference in synovial glucose concentrations was found between treated and control joints (P>0.05). Values obtained by HPLC analyzed with PkAnalyst program revealed that the pharmacokinetic values were dose dependent, there was no significant difference in concentration of blood glucose between the three different doses (P>0.05). ELISA for IGF-1 (insulin-like growth factor 1) revealed a significant difference between treated and control joints (P<0.001). Insulin used in this study proved to be innocuous to the equine joint, no more than 20 IU for a 350-400 kg horse should be administered. Key words: Insulin, Intraarticular, Horse, IGF-1, HPLC.

Received: 15/02/2021 Accepted: 11/08/2021

Introduction Different therapeutic options have been used to treat horses for synovitis. The aims of intra-articular (IA) treatment to ameliorate signs of osteoarthritis are to decrease the inflammatory mediators and biochemical processes of inflammation, relieve pain, avoid destruction of the joint, and normalize function of the joint to allow the horse to resume its normal activities. Corticosteroids, polysulphated glycosaminoglycans, antibiotics, hyaluronic acid, DMSO (dimethylsulfoxide), and parasympathicolythic drugs, such as atropine, have been used intra-articularly by clinicians for the purpose of reducing joint pain and inflammation. Intra-articular administration of corticosteroids has fallen into abuse because, when administered intra-articularly, corticosteroids offer a prompt analgesia and remarkable improvement in the performance of the horse. A corticosteroid can be detected in the joint fluid or synovium for at least 21 d after injection, and so, this treatment, if unaccompanied by an appropriate period of rest and physiotherapy, can result in damage to articular cartilage, fibrosis, and dystrophic calcification of soft tissues of the joint, particularly the synovium. Synoviocytes become replaced by fibrous tissue, resulting in impairment of production of the normal elements comprising synovial fluid, resulting in suboptimal functioning of the joint(1-3).

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There is in vitro evidence that insulin, in concentrations up to 50 ng/mL, in cell cultures (75 cm2 monolayers) induces mitosis of chondrocytes and production of type II collagen(4). The doses of insulin instilled intraarticularly in this study were adapted from the surface of the cell culture plate to be applied in vivo. In addition, the total surface area of the articular cartilage of the antebrachiocarpal joint of a recently euthanized adult Warmblood gelding was measured to help determine the dose of insulin to be injected in this particular joint (92.642 cm2), by using Rhinoceros 4.0® software (Data not shown). Significant changes in synovial fluid composition of all measurable components or in serum glucose concentrations were determined. As far as it is known, pharmacokinetic studies of insulin instilled into the joints of human beings or animals have not been conducted. There is in vivo evidence that intra-articular IGF-1 and 2 increase mitosis of chondrocytes and production of type II collagen by chondrocytes(5). Anecdotally, 20 to 40 IU of insulin has been instilled into joints of 500 kg horses with osteoarthritis without evidence of adverse effects. Whether or not these doses are effective in stimulating mitosis of chondrocytes and production of type II collagen in vitro is not known. There are no commercial, purified products of IGF-1 that can be instilled intraarticularly to treat horses for osteochondral defects. Though recombinant IGF-1, it is available only for in vitro use.

Material and methods Six mixed-breed horses (3 mares, 2 geldings and 1 stallion) with a mean age of 5.08 yr old, and a mean weight of 361.11 kg, were used in this study. All were in a moderate to good body condition(6). The horses were divided randomly into Groups 1, 2, and 3, each group being composed of two horses. Each horse was confined to an individual stall (4x4m) during the study, all were fed 7-8 kg of oat hay three times daily, and water was provided ad libitum. Lameness and radiographic (MinXray® HFX 90V. Illinois, USA) evaluations of the carpal region were performed for every horse. Each horse was evaluated for clinical signs of equine metabolic syndrome (EMS), such as obesity (body condition ≥7/9), abnormal fat deposits (cresty neck), preputial or mammary gland inflammation, and signs of laminitis, such as diverging growth rings on the hoof capsules. To ensure that no horse suffered from insulin resistance, each horse underwent an oral sugar test(7). To perform this test, horses were fasted for 12 h, after which the basal blood

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glucose concentration was measured. Corn syrup (Karo® made for ACH Foods Mexico, S. De R. L. De C. V.) was then administered orally, using a 60 ml syringe, at a dose of 15 ml/100 kg (150 mg/kg). Blood glucose concentration was measured at 60, 75, and 90 min after oral administration of the corn syrup. In order to determine the insulin concentration that is sufficient to induce mitosis in articular cartilage chondrocytes, we considered the area of cell culture plates (75 cm2) in which mitosis has been observed and then, extrapolated it to the whole joint surface(4). This was determined by measuring the surface area of the cartilage of the antebrachiocarpal joint of a recently euthanized adult Warmblood gelding using Rhinoceros 7.0 software. In this study, doses of 10, 15, and 20 IU of recombinant insulin [Humalog Lispro® (100 UI/ml), Eli Lilly and Company. Indianapolis, USA] were instilled into the antebrachiocarpal joint. These three doses were calculated by considering the systemic dose of insulin used to treat horses for hypoglycemia, and the concentration needed to achieve mitosis in cell culture plates; also considering the normal synovial fluid volume of the antebrachiocarpal joint(8). Sterile 0.9 % saline solution was added to the insulin to increase the volume injected to 1 ml. The left antebrachiocarpal joint of each horse was treated with insulin, and at the same time, the right antebrachiocarpal joint was injected with 0.9 % saline solution as control. The volume injected in the control joint was identical to that injected into the treated joint (i.e., 1 ml). Synoviocentesis was performed aseptically using a dorsal approach as previously described(9). All horses were walked in hand for 10 min twice daily, the day of injection and for the entire duration of the study. The concentration of glucose in blood, obtained from the jugular vein with a 25-ga needle, was measured by using a portable glucometer (Accu-chek Performa®, Roche Laboratories, USA). Blood was collected at 10 and 30 min and then hourly until 8 h, and then, every 6 h until 74 h post intra-articular injection of the insulin and isotonic saline solution. The concentration of glucose in at least 1 mL of synovial fluid collected from the antebrachiocarpal joint at 2, 4, and 6 h post intra-articular injection of insulin was determined by using the same glucometer. Synovial fluid was assessed for its color, appearance viscosity, protein concentration, quality of its mucin clot, and types of cells contained within it before intra-articular injection of insulin and isotonic saline solution and at 2, 4, and 6 h post injection. These samples were taken by using the naive pooled-data analysis(10). Insulin concentration in the joint was determined by HPLC, on samples obtained at 30 min, 1, 2-, 4-, 6-, 8-, and 12-h post-injection. IGF-1 concentration in synovial fluid was determined by ELISA (Horse IGF-1 ELISA kit, #MBS017382, MyBio-Source®) on samples obtained at 2, 4, and 6 h post intra-articular injection of two different doses of insulin, 15 IU and 20 IU.

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All samplings for the different evaluations made this a cross-over study of one factor (insulin) and three levels (dosages).

High-performance liquid chromatography (HPLC)

Synovial fluid, at least 1 mL, obtained from the antebrachiocarpal joints at 30 min, 1, 2, 3, 4, and 6 h post intra-articular injection of insulin and saline solution or saline solution alone was examined using HPLC. Samples were stored in Eppendorf tubes and analyzed by following the HPLC technique previously described(11). Using this technique, the samples were deproteinized with acetonitrile:propanol (1:1); the mobile phase was with water and acetonitrile (1:1) and 1% trifluoroacetic acid. A C18 (5µm 4.6 x 250 mm) column was used, providing a recovery of 98 % of the elution volume, with a variation coefficient of 5 %. The limit of detection of this analysis was 0.39 µg/mL, and the limit of quantification was 0.39 µg/mL. The elution time was 20 min. The serum concentrations of insulin vs time relationships were analyzed using compartmental pharmacokinetics through the software from PKAnalyst (MicroMath. Scientific Software, Salt Lake City, Utah, USA, 1995). With the results from the PkAnalyst program, a chi square was made to establish if there was a significant difference between the following measurements: half-life (KAE_half), time of maximum concentration (TConc_Max), and time of residence (residence_time), for the three different dosages of insulin.

Results Clinical evaluations before injection of insulin

None of the six horses used in this study had any clinical signs of equine metabolic syndrome (EMS) or insulin resistance, such as hoof deformities or abnormal adipose tissue deposits (Table 1).

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Table 1: Physical characteristics of all horses. Hoof deformities, cresty neck and corporal condition were considered clinical signs of EMS. Cresty Corporal Height Weight Sex* 1 2 neck scale condition (m) (kg) Group 1 Horse 1 1/5 4/9 1.69 465 M Horse 2 2/5 5/9 1.43 322.5 G Group 2 Horse 3 2/5 6/9 1.43 323 G Horse 4 2/5 6/9 1.46 351.2 M Group 3 Horse 5 2/5 5/9 1.53 388.5 S Horse 6 2/5 5/9 1.45 316.5 G Average 1.83/5 5.1/9 1.49 361.11 1

Carter RA, et al. 2009, 2Henneke DR, et al. 1983.

S= Stallion; M= Mare; G= Gelding.

It was not observed signs of lameness in any horse during the evaluation, which was performed with the horses trotting in a straight line on a hard surface and in a circle on soft surface, no signs were observed off lameness after each carpus was flexed for 1 min. No pathological abnormality was observed during radiographic evaluation of the carpi. None of the six horses displayed any clinically evident reaction to intra-articular instillation of insulin or isotonic saline solution (Table 2). Table 2: Evaluation of circumference of the carpi of all 6 horses, 24-h post injection of insulin (cm) Basal joint Joint Basal joint Joint perimeter perimeter perimeter perimeter Horse Control Control Treated Treated (24h) (24 h) 1 34.5 34.5 34.5 34.5 2 28.0 28.0 28.0 28.0 3 28.5 28.5 28.5 28.5 4 28.5 28.5 28.5 28.5 5 30.0 30.0 30.0 30.0 6 28.0 28.0 28.0 28.0 The synovial fluid was evaluated for changes in color, appearance, cellularity, and protein concentration in both treated and control joints, with no visible changes between the different samples(12).

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The oral sugar test revealed that none of the six horses had a concentration of glucose in the blood greater than 115 mg/dL (Figure 1). The concentration of glucose in the blood of all horses for all three doses, was significantly different (P<0.001) from the pretreatment concentration at 30 min and 1 h after intra-articular injection of insulin (Figure 2). Figure 1: Oral sugar test results, showing glycemia concentration for all 6 horses

Blood samples were taken at 60, 75 and 90 minutes after oral administration of corn syrup.

Figure 2: Average concentration of blood glucose of all 6 horses after each horse was administered

A) 10 IU black circles; B) 15 IU gray circles; C) 20 IU white circles of insulin into the antebrachiocarpal joint.

A post hoc (Holm-Sidak, Tukey) test was used to determine if the difference was significant for all three doses in all blood samples taken from the 1-h sample, because the lowest blood concentration was seen at that sampling time in all three groups. The oral

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sugar test revealed that none of the six horses had a concentration of glucose in the blood greater than 115 mg/dL(7). There was no significant association between the changes seen in the concentration of blood glucose and the dose of insulin administered into the antebrachiocarpal joint, not when compared between all three different doses, 10 vs 15 vs 20 IU (P= 0.372) and nor when compared between different (paired) doses, 10 vs 15 IU (P= 0.369) and 15 vs 20 IU (P= 0.318).

Synovial glucose The concentration of glucose within the antebrachiocarpal joint dropped at 2 and 6 h’ post-injection after 15 IU of insulin was administered into that joint, whereas the concentration of glucose within the antebrachiocarpal joint failed to drop at 2 and 6 h after isotonic saline solution was administered into that joint (Figure 3). Figure 3: Concentration of glucose in the synovial fluid harvested from joints treated with 15 IU and 20 IU of insulin and joints treated with isotonic saline solution

The concentration of glucose within the antebrachiocarpal joint dropped at 4 h’ postinjection after 20 IU of insulin was administered into that joint, whereas the concentration of glucose within the antebrachiocarpal joint failed to drop at 4 hours after isotonic saline solution was administered into that joint (Figure 3). The differences in concentration of glucose in the treated antebrachiocarpal joint did not differ significantly from that in the control antebrachiocarpal joint, however, when the dose of insulin injected into the treatment joint was 15 IU (P= 0.5945) or 20 IU (P= 0.235).

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High performance liquid chromatography (HPLC) Insulin could not be detected in the synovial fluid in the treated joints at 8 and 12 h’ postinjection. Insulin in synovial fluid obtained at 2 and 6 h post injection from the antebrachiocarpal joint of all six horses treated with 15 IU of insulin and insulin in synovial fluid obtained at 1 and 4 h post injection with 20 IU of insulin taken at 30 min, showed a detection peak at 16 ± 0.5 min (Figure 4). Figure 4: Chromatogram of an absorbance peak of insulin at 16 min in every sample where insulin was detected

To establish a proper elimination curve, the synovial fluid sampling was set to obtain for 3 doses of insulin (10, 15 and 20 IU) from 7 samples in a 6-hour period, with a timeline of 0.5, 1, 1.5, 2, 3, 4, and 6 h.

Insulin concentration within the treated joint varied through time and was dose dependent (Figure 5). Figure 5: Concentrations of insulin in the synovial fluid of all horses, determined by HPLC, after injecting 10, 15, or 20 IU of insulin into the antebrachiocarpal joint

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Pharmacokinetic analyses

The results obtained by HPLC were analyzed by using the program Micromath PkAnalyst®, using the program option for model: #5 one compartment, and first order kinetics. This model provides the withdrawal, or absorption, constants, which in this case were the same, and the relationship between volume/dose found in the different sampling of concentration within the compartment (joint). It also provides the area under the curve (AUC) the half-life of elimination (T½), the time of maximum concentration (TConc_Max), and the residence time (Residence_Time). The mean values of the pharmacokinetic variables of the synovial fluid concentrations of insulin from the three groups (10, 15, and 20 UI), were compared using a non-parametric Kruskall–Wallis analysis, through the software package JMP (JMP Statistic Mode Visual 1989–1995 SAS Institute). As the data showed a non-normal distribution, individual pharmacokinetic values were compared using Dunn tests, after a Kruskall–Wallis analysis. The 10 IU group could not be compared pharmacokinetically since only had a one point of concentration and immediately it was found below the quantifiable limits. The group was considered statistically different from the other two groups. The independent variable was time, and the dependent variable was insulin concentration. The values were volume, dose, and the withdrawal constant KAE. The differences in these variables between the 15 and 20 IU doses were slight (Table 3). Table 3: Calculated kinetic values for the 15 and 20 IU dose of insulin

Variable

15 UI Insulin Result Time (min) half-life 0.199927931* 11.99

Withdrawal (KAE_half) Maximum concentration 0.288435035* 17.30 time (TConc_Max) NA** Area under the curve 5.15211030

20 UI Insulin Result Time (min) 0.232681513* 13.8 0.335688466*

19.8

7.37122310

NA**

0.671376931*

40.2

(AUC) Resident time

0.576870070* 34.61

A chi square test revealed, however, that there was no significant difference between the half-life of withdrawal or absorption (KAE_half), the time of maximum concentration (TConc_Max), and the residence time (Residence_Time) for the 15 and 20 IU doses (P=0.9851).

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Synovial fluid analyses for IGF-1 concentration

To determine if there was correlation between the synovial concentration of exogenous insulin and synovial concentration IGF-1, it was measured IGF-1 concentration at 2, 4, and 6 h post intra-articular injection of insulin. It was evaluated only the groups of horses treated with 15 and 20 IU of insulin and observed significant differences between the basal concentrations of IGF-1 at the different sampling times (P<0.001); the 10 IU group was not considered for this analysis because the concentration parameters for this dose were too low to be measured by HPLC (Figure 6). Figure 6: IGF-1 concentration in synovial fluid of horses treated intra-articularly with A) 15 IU and B) 20 IU of IGF-1 concentration in synovial fluid of horses treated intraarticularly. C) Positive correlation of the 2 distinct groups, between IGF-1 concentration and time of synovial fluid sampling

Filled circles represent horses treated intra-articularly with 20 IU and blank circles represent horses treated intra-articularly with 15 IU of insulin. *P<0.05; **P<0.01; ***P<0.001.

Discussion Previous clinical evaluation

A basic clinical evaluation suggested that none of the six horses displayed clinical signs of Equine Metabolic Syndrome (EMS) or Insulin Resistance (IR), each had a body condition score below that established to be a risk factor for these conditions, and none had a cresty neck scale greater than 2 (a score greater 3 being a risk factor for EMS)(6,7,13). An oral sugar test provided objective evidence that none of the horses had EMS or IR. No horse used in this study had a concentration of glucose in the blood greater than 115 mg/dL, the maximum concentration of normality as previously described(7).

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Clinical examination and the oral sugar test were both necessary to eliminate a metabolic abnormality that could alter glucose metabolism and, therefore, alter the degree of glycemia, caused by injecting insulin into a joint. The concentration of glucose in the blood sugar may have been altered had the horse been insulin resistant. It was not conducted more specific examinations for EMS or IR, such as determining the concentration of cortisol and/or ACTH in the blood, the intravenous glucose tolerance test, and the glucose insulin response test, and nor it did tested for pituitary pars intermedia dysfunction (PPID), because the aim of this paper was to determine the pharmacokinetic values of insulin instilled into the horse’s joint, its systemic and local effects, such as the analysis of variations of IGF-1 concentration on synovial fluid. The results obtained from the oral sugar test, along with the absence of clinical signs that could suggest risk factors to IR or EMS, were considered, by the authors, to be sufficient to include these horses in the study.

Lameness exam (clinical and radiographic)

Post treatment clinical exam

The circumference of the carpus at the level of the antebrachiocarpal joint did not differ between the control and treated carpi, though this measurement was made with a measuring tape scaled in centimeters rather than millimeters. Based on the failure to detect a difference in circumference of treated and control carpi and failure of the horse to become lame on the treated limb, it was concluded that insulin injected into the antebrachiocarpal joints caused no clinical signs of inflammation.

Synovial analyses (Physical/Chemical)

Abnormal changes were seen in the synovial fluid obtained from treated and control joints, but the synovial fluid of the treated joints did not differ in color, appearance, protein concentration, and number of cells from that of the control joints. Changes seen in the treatment and control joints included a reddish discoloration, which was attributed to hemarthrosis caused by trauma to the synovium inflected during arthrocentesis(12). The incidence of develop septic synovitis post intra-synovial injection is quite high (34.1 %), however, if a proper aseptic technique is used, this is low. In a retrospective study of 192 horses admitted for treatment for septic arthritis or tenosynovitis, 22 % of infections were caused by synovial injection. In another retrospective study of 13 horses treated for tarsocrural joint infection, infection of 9 occurred after arthrocentesis of that joint. Because the concentration of protein in each treated joint was < 25 g/L and because 402


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only a small number of inflammatory cells (no bacteria) were observed during cytological examination of fluid harvested from the joint, it was concluded that instilling insulin to the joint causes no adverse reaction(14,15,16). In general, horses with septic arthritis have a cell count from 30-90 or 100x109/L, and a protein concentration of more than 25 g/L, even until 60 g/L. The protein values are associated with the pathologic condition that the joint goes by, it could be secondary to trauma or infection, where the values could be from 20-40 and >40 g/L respectively(17). None of the samples obtained in this study were near to the ones that are referred, which is why there was no infection or adverse reaction to insulin injected intra-articularly.

Statistical analyses

The amount of insulin instilled into the antebrachiocarpal joint significantly affected the concentration of blood glucose. For each of the three doses of insulin administered, the concentration of blood glucose was lowest at 1 h. It was found no association between the dose of insulin instilled into the joint and the concentration of glucose in the blood. The concentration of glucose in the synovial fluid did not differ significantly between treated and control joints when 15 or 20 IU of insulin was instilled into the treated joint. A power study to determined how many samples of joint fluid showed that at least 30 samples would have to be tested to produce more reliable results.

High performance liquid chromatography (HPLC)

With an initial assay for the synovial fluid sampling by HPLC, it was not possible to analyze these results with the pkAnalyst program, because the elimination curve of the insulin instilled in the joint, could not be calculated, probably because the concentration of insulin was less than that, that could be detected by the equipment. Consequently, we had to modify the synovial fluid samplings to 30 min, 1 hour, 1.5, 2, 3, 4, and 6 h post intra-articular injection of all three doses of insulin for all horses. The pharmacokinetics for the 15- and 20-IU doses were linear, but it was unable to establish a pharmacokinetic curve for the 10-IU dose, because the analytical technique was not sensitive enough to detect the concentration of insulin in the synovial fluid at 1 h. The curves for the 15- and 20-IU doses appeared similar, but was found slight differences in the pharmacokinetic values. For instance, for the 15-IU dose, the residence time of insulin, the maximum concentration time, and the area under the curve were slightly, but not significantly different from that of the 20-IU dose. For both doses (15 and 20-IU), the maximum concentration time was between 17-20 min, which correlates

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with the drop-in concentration of glucose in the blood seen at 30 min post intra-articular injection. Studies are needed to evaluate the direct effect of insulin on chondrocytes, because the blood glucose concentration in treated joints did not differ significantly from that in the control joints, suggesting that the insulin does bind to its receptor causing the concentration of glucose in the synovial fluid to drop. Studies are needed to determine when the synovial concentration of glucose returns to normal. The time of action of the insulin in the joint or its pharmacokinetic effects could vary from the ones observed in this study, if a similar study was performed using a larger number of horses (e.g., n>30), but this study provides data for comparison for future studies. Based on the results of this study, it suggests that the horse should be observed for one hour after injecting insulin intra-articularly, to ensure that drop in concentration of blood glucose does not cause clinical signs of hypoglycemia. The chromatograms obtained with HPLC were like the ones previously observed after instilling insulin to a joint, observed an insulin peak at 15.8 min(11). In this study these peaks were observed at 16 ± 0.5 min. Because the residence time, half-life time, and maximum concentration time were higher with the highest dose of insulin (20 IU), it suggests using this dose when treating a horse for osteoarthritis by instilling insulin intraarticularly, because at this dosage, the insulin’s effect in the joint was more durable than were the lower doses. Growth factors are a group of proteins, that play an important role in tissue repair by enhancing cellular proliferation, survival, division, growth, and differentiation. IGF-1 is the most important and powerful growth factor for cartilage repair(18). Insulin and IGF-1 have a very similar structure; they share a homologous sequence, they have a similar three-dimensional structure, and they have weakly overlapping biological activity(19). IGF-1 folds into two thermodynamically stable disulfur isomers (via disulfur bond swipe), whereas insulin folds into one unique stable tertiary structure. This is due to post translation processing, that can achieve two different structures with the same aminoacidic sequence. The disulfur bond swipe of IGF-1 allows the protein to change its affinity for receptor bonding. It appears to be able to bond insulin and its own receptor(19). It hypothesizes that insulin can alter this affinity and in some cases bond to the IGF-1 receptor, activating its signaling pathway, since this growth factor has autocrine, paracrine and endocrine functions, it serves as its own positive feedback for activating MAPK/AKT signaling pathways. Instilling insulin into a joint, as a treatment for osteoarthritis of that joint, may be an option for horses suffering from osteoarthritis, because IGF-1 is not available commercially for use in joints of horses(18,20). 404


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Conclusions and implications It was unable to detect any adverse local or systemic reaction to insulin instilled intraarticularly. The residence time of the insulin within the antebrachiocarpal joint was relatively short and proportional to the dose. None of the three doses used in this study [i.e., a low dose (10 IU), a medium dose (15 IU), and a high dose (20 IU)], significantly altered the concentration of glucose in the synovial fluid and blood. Intra-articular administration of exogenous insulin enhanced IGF-1 expression in synovial fluid, and this expression seemed to be dose and time dependent. Studies are needed to clarify the mechanism by which this expression of IGF-1 is enhanced in the equine joint. As far as it is known, there are no studies of insulin used intra-articularly in human or veterinary medicine, therefore this is the first in vivo study. This is a descriptive study, and believe it to be the first in vivo study demonstrating that insulin can be injected safely into a joint. This study could be a base for other studies examining the efficacy of insulin in ameliorating the clinical signs of osteoarthritis in horses. Literature cited: 1. Celeste C, Ionescu M, Poole RA. Repeated intra-articular injections of triamcinolone acetonide alter cartilage matrix metabolism measured by biomarkers in synovial fluid. J Orthop Res 2005;(23):602-610. doi: 10.1016/j.orthres.2004.10.003. 2. Gotoh S, Onaya J, Abe M, Miyazaki K, Hamai A, Horie K, Tokuyasu K. Effects of the molecular weight of hyaluronic acid and its action mechanisms on experimental joint pain in rats. Ann Rheum Dis 1993;(52):817-822. doi: 10.1136/ard.52.11.817. 3. Henson FMD, Davenport C, Butler L, Moran I, Shingleton WD, Jeffcott LB, Schofield PN. Effects of insulin and insulin-like growth factors I and II on the growth of equine fetal and neonatal chondrocytes. Eq Vet J 1997;(29):441-447. doi: 10.1111/j.20423306.1997.tb03156.x. 4. Schumacher HR. Aspiration and injection therapies for joints. Arthitis Rheumatol 2003;(49):413-420. doi: 10.1002/art.11056. 5. Davenport C, Boston R, Richardson DW. Effects of insulin-like growth factor-II on the mitogenic and metabolic activities of equine articular cartilage with and without interleukin 1-β. Am Vet Res 2004;(65):238-244. doi: 10.2460/ajvr.2004.65.238. 6. Henneke DR, Potter GD, Kreider JL, Yeates BF. Relationship between condition score, physical measurements and body fat percentage in mares. Eq Vet J 1983;15,371372. doi: 10.1111/j.2042-3306.1983.tb01826.x

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7. Frank N. Equine metabolic syndrome. Vet Clin North Am Equine Pract 2011;(27):7392. doi: 10.1016/j.cveq.2010.12.004. 8. Smith JS, Ratzlaff MH, Grant BD, Frank FL. The synovial fluid volume of the radiocarpal, intercarpal and tibiotarsal joints of a horse. J Eq Med Surg 1979;(3):479483. 9. Moyer W, Schumacher J. A guide to equine joint and regional anesthesia. 4th ed . USA: Academic Veterinary Solutions, LLC; 2007. 10. Mahmood I. Naive pooled-data approach for pharmacokinetic studies in pediatrics with a very small sample size. Am J Ther 2014;(21):269-74. doi:10.1097/MJT.0b013e31824ddee3. 11. Hafiz Mohd MJ, Affandi MMR, Ah K, Sepria L. A simple and sensitive method for the determination of insulin in rat plasma and its application in pharmacokinetic study. Int J Pharm Pharm Sci 2013;(5):133-137. 12. Meyer DJ, Harvey JW. Evaluations of fluids: Effusions, synovial fluid, cerebrospinal fluid. In Veterinary laboratory medicine interpretation and diagnosis. 3rd ed. Saunders, USA. 2004;245-250. 13. Carter RA, Geor RJ, Burton SW, Cubbit TA, Harris PA. Apparent adiposity assessed by standardized scoring systems and morphometric measurements in horses and ponies. Vet J 2009;(179):204-210. doi: 10.1016/j.tvjl.2008.02.029. 14. Bertone AL, Cohen JM. Infectious arthritis and fungal infections arthritis. In: Ross MW, Dyson SJ. Diagnosis and management of lameness in the horse. 2nd ed. USA: Saunders; 2011;677-687. 15. Olds AM, Stewart AA, Freeman DE, Schaeffer DJ. Evaluation of the rate of development of septic arthritis after elective arthroscopy in horses: 7 cases. J Am Vet Med Ass 2006;(229):1949-1954. doi: 10.2460/javma.229.12.1949. 16. Adams SB. How to avoid complications following joint injections I: Site preparation and selection of needles. AAEP Proc. 2012;58. 17. Taylor FG, Hillyer MH. Enfermedades musculoesqueléticas. In: Taylor FG, Hillyer MH. Técnicas diagnósticas en medicina equina. Aribia, España. 1997;245-284. 18. Fortier LA, Strauss EJ, Cole BJ. The role of growth factors in cartilage repair. Clin Orthop Relat Res 2011;(469):2706-2715. doi: 10.1007/s11999-011-1857-3. 19. Yun CH, Tang YH, Feng YM, An XM, Chang WR, Liang DC. 1.42 Å crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure. Biochem Biophys Res Commun 2005;(326):52-59. doi: 10.1016/j.bbrc.2004.10.203 406


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20.

National Center for Biotechnology https://www.ncbi.nlm.nih.gov/ .

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information

database.

2020.


https://doi.org/10.22319/rmcp.v13i2.5381 Article

Productive performance of sheep fed buffel grass silage in replacement of corn silage

Tiara Millena Barros e Silva a Gherman Garcia Leal de Araújo b Tadeu Vinhas Voltolini b Mário Adriano Ávila Queiroz a Sandra Mari Yamamoto a Fábio Nunes Lista a Glayciane Costa Gois a* Salete Alves de Moraes b Fleming Sena Campos b Madriano Christilis da Rocha Santos a

a

Universidade Federal do Vale do São Francisco (UNIVASF), Campus de Ciências Agrárias, Pernambuco, Brazil. b

Empresa Brasileira de Pesquisa Agropecuária (Embrapa Semiárido), Rodovia BR-428, Km 152, s/n – Zona Rural, 56302-970, Pernambuco, Brazil.

*Corresponding author: glayciane_gois@yahoo.com.br

Abstract: This study aimed to evaluate the productive performance and nutritional status of crossbred sheep fed a diet containing buffel grass silage (BGS) in substitution of corn silage (CS). Thirty-two male Santa Inês sheep with an average body weight of 20.09 ± 2.0 kg were distributed in a completely randomized block design with four treatments (0, 33.3, 66.6, and 100 % of substitution of corn silage with buffel grass silage) and eight animals per treatment. Dry matter intake, apparent nutrient digestibility, water balance,

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nitrogen balance, and productive performance of animals were evaluated. The different levels of substitution of corn silage by buffel grass silage promoted a linear decrease in the consumption of ether extract (P=0.001) and non-fibrous carbohydrate (P<0.001), apparent digestibility of non-fibrous carbohydrate (P=0.019), water intake via food (P<0.001), total water intake (P=0.008), water excretion via urine (P=0.004), total water excretion (P=0.001), among which the highest values were observed in animals fed 100% corn silage. Water excretion via feces (P=0.017) and nitrogen balance (P=0.047) showed a quadratic function, increasing as substitution with buffel grass silage increased from 0 to 33.3 %. No significant differences (P>0.05) were observed in sheep productive performance, with an average daily weight gain of 140.16 g/d. The replacement of 66.6 % of CS for BGS provides satisfactory results for dry matter and nutrients intake, and water intake, with weight gain of up to 155 g/d for Santa Inês crossbred sheep in Brazilian semi-arid. Key words: Animal nutrition, Confined sheep, Santa Inês, Semiarid.

Received: 15/05/2019 Accepted: 28/09/2021

Introduction In semi-arid regions, the dry season is a major obstacle to animal production due to food shortages and reduced nutritional value of available pastures(1). Caatinga is an important food source for ruminant herds in the semi-arid regions of Brazil(2). However, in most cases, native vegetation is not sufficient to meet the nutritional requirements of the animals, resulting in poor animal performance(3). Thus, the planning of food production has a significant impact on the animal production in these regions. Confinement systems are used to increase herd productivity and improve the quality and supply of products in the off-season. However, the success of the intensive confinement of ruminants depends on the availability and cost of the feed. Alternative dietary strategies are necessary to obtain satisfactory results and make this activity more profitable because the animal feed is the costliest component of production and affects profitability(3). The use of forage plants adapted to semi-arid regions by efficiently using water combined with silage production increase food supply, especially in the dry season, making sheep farming sustainable(4). Corn is the standard crop for silage in the Northeast region of Brazil, being one of the main agricultural products in the region, due to its tradition in cultivation, productivity, and nutritional value. The use of corn cultivars, well adapted

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and of high productivity, as is the case of Caatingueiro, Gorutuba, and São Francisco, is important to increase the improvement of the activity yield in the semiarid (5). However, alternative food crops with a lower cost and stable production under adverse climatic conditions have been proposed(6,7). In this respect, buffel grass (Cenchrus ciliaris L.) outperforms other cultivars because of its easy adaptation to adverse climatic conditions, good silage production, and maintenance of the productive capacity even after long periods of drought(8). However, buffel grass is rarely exploited for silage production despite the presence of large cultivated areas; therefore, further studies on the use of this grass as ruminant feed are necessary(9). The objective of this study was to evaluate the productive performance and nutritional status of crossbred sheep fed a diet containing buffel grass silage (BGS) in replacement of corn silage (CS) in a semi-arid region of Brazil.

Material and methods Study site

The study was carried out in the Caatinga Experimental Station, in the Animal Metabolism Unit of Embrapa Semiárido, located in Petrolina, Pernambuco State, Brazil. The local mean annual rainfall is 433 mm, and averages of maximum and minimum annual temperatures are 33.46 and 26.96 ºC, respectively. This study was approved by the Research Ethics and Deontology Committee of the Federal University of Vale do São Francisco (UNIVASF), under Protocol No. 0007/131014.

Animals, treatments, and experimental diets

Thirty-two (32) non-castrated male Santa Inês sheep (6-mo-old and 20.09 ± 2.0 kg of body weight) were distributed in individual stalls (1.2 × 0.8 m) equipped with feeding and drinking troughs for the diets and water supply. The experimental design was a completely randomized block design with four treatments and eight animals per treatment. The initial bodyweight was used to define the blocks.

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The experiment lasted 71 d, comprising 10 d of animal adaptation to the experimental diet and treatments. At the beginning of the adaptation period, the animals were identified, weighed, treated against endo- and ectoparasites, and randomly allocated to the stalls previously identified according to the treatment. The silages of corn (Zea mays; variety Caatingueiro, around 90 d of maturity) and buffel grass (Cenchrus ciliaris L.; variety Biloela, aged about 120 d regrowth) were made in barrel silos with a 200 kg capacity and presented an average density of 113.94 kg and 65.97 kg, respectively. Forage material was processed through a PP-35 forage harvester to an average particle size of about 2.0 cm and then ensiled. Four diets were formulated by substituting corn silage (CS) by buffelgrass silage (BGS) at increasing levels: 1) 100% CS and 0% BGS, 2) 66.6% CS and 33.3% BGS, 3) 33.3% CS and 66.6% BGS, and 4) 0% CS and 100% BGS. These diets were formulated with a roughage: concentrate ratio of 60:40 based on dry matter and composed of corn silage, buffelgrass silage, ground corn, soybean meal, limestone, common salt, and mineral supplement (Tables 1, 2), being balanced for allowing an average weight gain of 200 g/d, as National Research Council(10) recommendations. Table 1: Chemical composition of ingredients used in experimental diets (g/kg DM) Ingredients Item Ground Soybean CS BGS C1 C2 C3 C4 corn meal Dry mattera 225.6 506.4 893.7 908.5 889.7 889.4 890.2 892.9 Organic matter 857.8 844.7 975.3 919.9 946.1 946.4 946.3 942.4 Mineral matter 142.2 155.3 24.7 80.1 53.9 53.6 53.7 57.6 Ether extract 19.0 15.3 64.2 17.9 38.6 38.4 34.0 34.0 Crude protein 60.7 53.0 99.3 498.9 290.9 310.0 320.8 352.5 NDF 533.6 688.1 213.9 186.1 216.3 218.9 219.0 221.8 ADF 292.3 419.9 37.9 135.8 96.1 100.2 103.2 111.3 NFC 248.2 84.6 597.3 217.0 400.3 379.1 372.5 334.1 TDN 621.7 531.9 800.8 731.9 759.8 757.0 754.8 749.1 CS= corn silage; BGS= buffel grass silage; C1, C2, C3 and C4= diets concentrate. a In g/kg fresh matter. NDF= neutral detergent fiber; ADF= acid detergent fiber; NFC= Non-fibrous carbohydrates; TDN= Total digestible nutrientes.

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Table 2: Proportion of ingredients and chemical composition of experimental diets in dry matter basis Substitution of corn silage by Proportion of ingredients in the diets buffel grass silage (%) (%) 0 33.3 66.6 100 Corn silage Buffel grass silage Ground corn Soybean meal Limestone Common salt Premix mineral1

60.00 39.99 19.99 0.00 0.00 19.99 39.99 60.00 24.00 22.40 20.90 19.40 15.90 17.50 19.00 20.50 0.03 0.03 0.03 0.03 0.05 0.05 0.05 0.05 0.02 0.02 0.02 0.02 Chemical composition (% dry matter) 49.12 54.72 60.37 66.10 89.31 89.04 88.78 88.38 10.69 10.94 11.20 11.62 2.46 2.53 2.43 2.50 15.28 15.89 15.16 15.28 40.67 43.85 46.94 50.16 21.38 24.09 26.76 29.65 30.90 26.78 23.24 18.44 67.70 65.77 63.89 61.88 10.245 9.856 11.794 11.422

Dry matter, % fresh matter Organic matter Mineral matter Ether extract Crude protein Neutral detergent fiber Acid detergent fiber Non-fibrous carbohydrates Total digestible nutrients Metabolizable energy, MJ/kg

¹Premix mineral: phosphorus – 45 g; calcium – 90 g; chloro-240 g; sodium-156 g; sulfur – 10 g; magnesium – 8 g; zinc-2,800 mg; iron-1,300 mg; manganese – 2,300 mg; copper -150 mg; iodine – 40 mg; cobalt – 35 mg; selenium – 15 mg and fluorine – 450 mg.

Food was offered daily at 0830 and 1530 h. The amount of food offered was calculated according t o the consumption of the previous day, not allowing leftovers higher than 10 % of the offered quantity. Weekly samples of foods offered and leftovers were collected for chemical analyses.

Nutrient intake and digestibility

The daily dry matter intake (DMI) was obtained by the difference between the total DM of the consumed diet and the total DM present in the leftovers. Nutrient intake was determined as the difference between the total nutrients present in the consumed diet and the total nutrients present in the leftovers, on a total DM basis. A digestibility test was performed in the final third of the experimental period, with a duration of 10 days, 5 d for adaptation followed by 5 d for collection. The animals were distributed in metabolism cages provided with feeders and drinking fountains. Feces were sampled using collection bags fixed to the animals, which were attached to the animals

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before the sampling period. The bags were weighed and emptied twice daily and a subsample of 10 % of the total amount was collected for further analysis.

Nitrogen balance

Urine was collected and weighed using plastic buckets containing 100 mL of 2 N hydrochloric acid in order to avoid nitrogen volatilization and sampled for nitrogen content determination. Nitrogen balance (NB) was determined according to the method described by Silva & Leão(11).

Water balance

Water intake was evaluated daily. The water was supplied in buckets and weighed before being supplied and again 24 h later. Three water-filled containers were placed close to the cages to measure daily evaporation. Water balance was evaluated using the following equations: total water intake = (supplied water – evaporated water) + dietary water; total water excretion = water excreted in the urine + water excreted in the feces; water balance = total water intake – total water excretion(12).

Productive performance

The animals were weighed every 15 d after a solid-feed deprivation period of 12 h (with access to water) to obtain the initial body weight, final body weight, total weight gain (TWG= final body weight at fasting − initial body weight at fasting), and daily weight gain (DWG= total weight gain/experimental period). At the end of the experimental period, the feed conversion (FC) was calculated by the following equation: FC= dry matter intake/mean daily gain.

Laboratory tests

Feed, leftover, and fecal samples were pre-dried in a forced ventilation oven at 55 °C for 72 h and ground to 1 mm particles (Wiley Mill, Marconi, MA-580, Piracicaba, Brazil). Laboratory analyses were performed using the methods described by Association of Official Analytical Chemists(13) for dry matter (DM; Method No. 967.03), mineral matter (MM; Method No. 942.05), crude protein (CP, Method No. 981.10) and ether extract (EE,

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Method No. 920.29). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined as described by Van Soest et al(14). Total carbohydrates (TC) were measured using the equation proposed by Sniffen et al(15), as follows: TC (% DM) = 100 – (CP + EE + ash). Non-fibrous carbohydrate (NFC) content was measured as proposed by Hall(16): NFC = %TC – %NDF. The apparent digestibility coefficient (ADC) of nutrients was calculated as described by Silva & Leão(11): ADC = {[Nutrient intake (kg) – nutrients excreted in the feces (kg)]/nutrient intake (kg)} * 100. Total digestible nutrient (TDN) was measured using the equation of Harlan et al(17), as follows: 82.75 – (0.704 × ADF). TDN intake was estimated using the following formula: %TDN= (TDN intake/DM intake) * 100. TDN of the diet was converted into metabolizable energy (ME) using the following equation proposed by NRC(18): Digestible energy (DE)= (TDN/100) x 4.409, metabolizable energy= DE x 0.82

Statistical analysis

The data were submitted to Shapiro-Wilk and Levene tests to verify the normality of the residues and homogeneity of the variances, respectively. Once that the assumptions were met, the data were submitted to analysis of variance (ANOVA) using the PROC GLM (General Linear Models). Linear and quadratic regression analyzes were performed using PROC REG. Probability values less than 0.05 (P<0.05) were considered statistically significant. Statistical analysis was performed using SAS version 9.0 (SAS Institute, Cary, NC, USA). The following statistical model was used: Yij = µ + Ti + βj + Eij, Where: Yij= value observed for the study variable referring to the i-th treatment in the j-th repetition; µ= general constant; Ti= effect of the level of substitution of corn silage by buffel grass silage in the diet; βj= block effect (i = 1, 2, 3, 4); Eij= random error associated with the Eij observation.

Results EE (P=0.001) and NFC (P<0.001) intakes decreased linearly due to the substitution levels of CS with BGS. However, no effect of the diets was found on the intake (in grams/day) of DM (P=0.180), CP (P=0.111), NDF (P=0.078), ADF (P=0.221), TC (P=0.220), and

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TDN (P=0.267) (Table 2). The digestibility coefficient of NFC (P=0.019) was influenced by the diets, with a linear decreasing effect according to inclusion levels of BGS. No significant differences were observed in the digestibility coefficients of DM (P=0.425), OM (P=0.637), CP (P=0.715), EE (P=0.065), NDF (P=0.536), and TC (P=0.112) among the treatments (Table 3). Table 3: Daily intake of nutritional components and apparent digestibility of nutrients in sheep fed buffel grass silage in replacement of corn silage Substitution of corn silage by buffel grass silage (%) P SE1 value 0 33.3 66.6 100 Variables Intake(g/d) Dry matter 768.04 878.06 835.61 701.02 50.88 0.180 Crude protein 168.52 195.30 184.83 161.75 10.25 0.111 2 Ether extract 26.30 25.27 21.77 18.53 1.35 0.001 Neutral detergent fiber 236.99 305.93 311.44 284.06 20.85 0.078 Acid detergent fiber 144.10 161.29 177.29 171.81 11.60 0.221 Total carbohydrates 529.59 604.47 601.26 437.65 31.91 0.220 3 Non-fibrous carbohydrates 314.75 273.98 216.30 154.66 11.44 <0.001 Total digestible nutrients 649.68 724.64 740.58 538.92 56.81 0.267 Digestibility (%) Dry matter 71.39 73.74 73.95 68.41 1.80 0.425 Organic matter 72.04 74.92 75.33 69.03 1.73 0.637 Crude protein 65.43 72.06 72.47 71.25 1.99 0.715 Ether extract 72.23 76.65 73.34 65.37 2.07 0.065 Neutral detergent fiber 57.10 61.87 61.93 59.85 2.63 0.536 Total carbohydrates 74.19 75.35 75.92 68.38 1.70 0.112 4 Non-fibrous carbohydrates 93.43 91.67 88.17 87.35 1.08 0.019 SE= standard error of the mean; Equations: 2Ŷ=29.66– 2.68x (SED=0.60, R2=0.26, P=0.0001); 3 Ŷ=374.41–53.79x (SED=5.12, R2=0.99, P<0.001); 4Ŷ=77.67–1.68x (SED=0.76, R2=0.95; P=0.038). Significant at the 5% probability level. 1

As BGS increased in the diet, a linear decreasing effect was observed in water intake via food (P<0.001), total water intake (P=0.008), water excretion via urine (P=0.004), total water excretion (P=0.001). While water excretion via feces showed a quadratic response (P=0.017) as substitution levels increased from 0 to 33.3 %, and then decreased, with the lowest values in diets with 100 % BGS (Table 3). No significant effect of substitution of CS with BGS was found on water intake via drinking fountain (P=0.266) and water balance (P=0.900) (Table 4).

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Table 4: Water balance and nitrogen balance in sheep fed buffel grass silage in replacement of corn silage (g/day) Substitution of corn silage P SEM1 by buffel grass silage (%) Variables value 0 33.3 66.6 100 Water intake via drinking fountain 3078 2748 2716 2587 153.51 0.266 2 Water intake via food 951 726 548 359 0.07 <0.001 3 Total water intake 4029 3474 3264 2946 177.95 0.008 4 Water excretion via urine 2054 974 921 849 258.25 0.004 5 Water excretion via feces 487 510 420 335 47.36 0.017 6 Total water excretion 2542 1484 1341 1184 138.79 0.001 Water balance 1811 2227 2072 1808 196.67 0.900 Nitrogen ingested 27.51 32.13 29.57 25.96 2.91 0.385 Nitrogen excreted via feces 6.27 5.47 5.67 6.77 0.32 0.529 Nitrogen excreted via urine 6.87 7.34 8.20 8.57 0.66 0.360 7 Nitrogen balance 14.36 19.31 15.68 10.62 2.98 0.047 SEM - Standard error of the mean; Equations: 2Ŷ=1.58-0.29x (SED=0.032, R2=0.99, P<0.001); Ŷ=6.35-0.78x (SED=0.206, R²=0.84, P=0.001); 4Ŷ=2028.62-331.35x (SED=115.49, R2=0.56, P=0.009); 5Ŷ=440.96+80.11x-26.96x2 (SED=23.68, R2=0.96, P=0.018); 6Ŷ=2.60-0.39x (SED=0.123, R2=0.67, P=0.005); 7Ŷ=-2.50x2+11.03x+6.19 (SED=1.22, R2=0.93, P=0.008). Significant at the 5% probability level. 1

3

Nitrogen balance showed a quadratic response (P=0.047) as substitution levels increased from 0 to 33.3 % BGS and then decreased, with the lowest values in diets with 100% BGS (Table 3). The amounts of nitrogen ingested (P=0.568), nitrogen excreted via feces (P=0.529) or via urine (P=0.360), were not affected by the substitution levels of BGS in diet (Table 4). No significant differences were encountered on final weight (P=0206), daily weight gain (P=0.513), total weight gain (P=0.513), and feed conversion (P=0.605) of Santa Inês sheep fed different levels of BGS, and the mean daily weight gain was 140.16 g/d (Table 5). Table 5: Productive performance of sheep fed buffel grass silage in replacement of corn silage Substitution of corn silage by Variables buffel grass silage (%) SE P value 0 33.3 66.6 100 Initial weight, kg 20.54 21.29 20.64 18.09 0.62 0.179 Final weight, kg 28.69 29.95 29.94 25.62 0.95 0.206 Daily weight gain, g/d 135.83 144.40 155.00 125.42 8.66 0.513 Total weight gain, kg 8.15 8.66 9.30 7.53 0.52 0.513 Feed conversion 5.93 6.27 5.47 5.73 0.24 0.605 SE= standard error of the mean; Significant at the 5% probability level.

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Discussion One of the main influencing factors for the productive efficiency of animals is nutrient intake. The observed mean DMI was 795.68 g/animal/d, that was lower than that recommended (1 kg/animal/d) by the NRC(10) for animals of 20 kg of body weight. Low dry matter intake directly affected daily gain. The animals showed, on average, a of 140.16 g/d, reaching 70.1 % of the expected daily gain, according to the recommendations of the NRC(10). A greater control of DMI and lower values of weight gain found in the treatment with 100 % BGS, in relation to the other diets tested, seem to be related to the greater amount of ADF present in this diet. Mertens(19) reports that DMI is controlled by physiological, physical and psychogenic factors. When high quality diets are offered, the animal consumes to meet its nutritional demand, this consumption being limited by its genetic potential to use the nutrients absorbed. However, when low quality diets are offered (high fiber and low soluble carbohydrates content), feed intake occurs until reaching the maximum level of gastrointestinal capacity(19). The decrease in EE and NFC intakes and in the digestibility coefficient of NFC can be explained by the lower concentration of these components associated with high concentration of NDF and ADF in BGS compared to CS (Table 1). The higher concentration of fibrous fractions provided lower energy concentration to the diets with increasing levels of BGS. According to Allen(20), the use of fiber-rich foods limits DMI, as a consequence of the amount of indigestible material that occupies space inside the rumen, causing physical distension of the rumen. Ruminal microorganisms depend on fermentable energy and nitrogen sources for their metabolic activity, strongly influencing ruminal digestibility and nutrient flow(21). Synchronization between energy and protein is essential in order to maximize microbial efficiency, promoting an improvement in dry matter digestibility(22). The increase in BGS levels in diets had no significant effect on TDN intake. Even the mean value was higher than that recommended by the NRC(10), that is 0.55 kg TDN/d for an animal gaining 200 g/d. This finding can be explained by the absence of significant differences in DMI and NDF intake, within the recommended standards, maintaining the ingestion of TDN and meeting animal requirements (Table 2). The linear decreasing response of water intake via food and total water intake in this study, probably it is due to the increase of the dry matter content according to the increase in BGS levels in diets (Table 2) allied to the low DMI that the animals presented. Thus, the low DMI reduced water intake via food and the water excretion via feces; so the animals sought to hydrate themselves through the water intake via drinking fountain, thus increasing the water excretion via urine. This suggests that the animals tried to maintain a certain level of water flow for metabolic functions to balance the lower physiological demand for water caused by lower dietary intake(23). Souza et al(24) in a study with Biloela

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buffel grass silages offered to sheep in the Brazilian semiarid region, found that the animals had lower DMI and this influenced the reduced water intake via food and water excretion via feces. The reduction in DMI and water intake via food was also observed by Carvalho et al(8) when offering buffel grass silage in diets for sheep in the Brazilian Semiarid Region. These results corroborate our findings. The increase in NB was higher in the replacement of corn silage by buffel grass silage at the level of 33.3 % (19.31 g/d) and 66 % (15.68 g/d) in the sheep diets, which indicates that the animals retained dietary protein achieving the main objective of nutritional planning. According to Tosto et al(25), this result can be explained by the efficient recycling of N performed by ruminants, under conditions of low protein availability in the diet. According to Lindberg(26) the levels of N excreted via urine should be on average up to 45 % of the N ingested. In the present study, large amounts of urinary N were not eliminated, which demonstrates that the use of N was efficient by the ruminal microorganisms converting to digestible microbial protein(27), which can be observed in the CP digestibility values (Table 3) of the formulated diets. Thus, the results of NB obtained demonstrate that there is synchronism between the supply protein and energy in diets that possibly used ammonia, promoting greater microbial protein synthesis and greater use of N supplied(25).

Conclusions and implications Under the experimental conditions, the replacement of 66.6 % of corn silage for buffel grass silage provides satisfactory results for dry matter and nutrients intake, and water intake, with weight gain of up to 155 g/d for Santa Inês crossbred sheep in Brazilian semiarid.

Acknowledgements

The National Council of Scientific and Technological Development (CNPq - Public Call. Ministry of Science, Technology, Innovations and Communications - MCTIC/ CNPq No. 14/2012 - Universal) for the financial support given to the project "Silages of varieties of buffelgrass as new alternatives of bulks for diets of sheep in confinement in the Brazilian semiarid"

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Literature cited: 1. Mudzengi CP, Dahwa E, Kapembeza CS. Livestock feeds and feeding in Semi-Arid areas of Southern Africa, 6. In: Abubakar M editor. Livestock health and farming. 1rst ed. London: IntechOpen; 2020:1–13. 2. Araújo AR, Rodriguez NM, Rogério MCP, Borges I, Saliba EOS, Santos SA, et al. Nutritional evaluation and productivity of supplemented sheep grazing in semiarid rangeland of northeastern Brazil. Trop Anim Health Prod 2019;51(4):957–966. https://doi.org/10.1007/s11250-018-1781-6. 3. Silva, MJS, Silva, DKA, Magalhães, ALR, Pereira, KP, Silva, ECL, Cordeiro, FSB, et al. Influence of the period of year on the chemical composition and digestibility of pasture and fodder selected by goats in caatinga. Rev Bras Saúde Prod Anim 2017;18(3):402-416. https://doi.org/10.1590/s1519-99402017000300001. 4. Campos FS, Carvalho GGP, Santos EM, Araújo GGL, Gois GC, Rebouças RA, et al.

Influence of diets with silage from forage plants adapted to the semi-arid conditions on lamb quality and sensory atributes. Meat Sci 2017;124(1):61-68. https://doi.org/10.1016/j.meatsci.2016.10.011. 5. Santos RD, Pereira LGR, Neves ALA, Azevêdo JAG, Moraes SA, Costa CTF.

Agronomic characteristics of maize varieties for silage production in the submédio São Francisco River valley. Acta Scient Anim Sci 2010;32(4):367-373. https://doi.org/10.4025/actascianimsci.v32i4.9299. 6. Kumar D, Kalita P. Reducing postharvest losses during storage of grain crops to strengthen food security in developing countries. Foods 2017;6(1):1-22. https://doi.org/10.3390/foods6010008. 7. Martin NP, Russelle MP, Powell JM, Sniffen CJ, Smith SI, Tricarico JM, et al. Invited review: Sustainable forage and grain crop production for the US dairy industry. J Dairy Sci 2017;100(12):9479-9494. https://doi.org/10.3168/jds.2017-13080. 8. Carvalho GGP, Rebouças RA, Campos FS, Santos EM, Araújo GGL, Gois GC, et al. Intake, digestibility, performance, and feeding behavior of lambs fed diets containing silages of different tropical forage species. Anim Feed Sci Tech 2017;228:140-148. https://doi.org/10.1016/j.anifeedsci.2017.04.006. 9. Voltolini TV, Araújo GGL, Souza RA. Silagem de capim-buffel: Alternativa para a alimentação de ruminantes na região semiárida. Petrolina: Embrapa Semiárido, (Embrapa Semiárido. Documentos, 259). 2014;34pp. https://ainfo.cnptia.embrapa.br/digital/bitstream/item/114740/1/SDC259.pdf. 10. NRC. Nutrient requirements of small ruminants: Sheep, goats, cervids, and new world camelids. National Research Council. The National Academy Press, Washington, DC. 2007.

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11. Silva JFC, Leão MI. Fundamentos de nutrição de ruminantes. Livroceres: Piracicaba;1979. 12. Church DC. Digestive physiology and nutrition of ruminants: Digestive physiology, 2nd ed. O & B Books Publishing, Corvallis; 1976. 13. AOAC. Official methods of analysis of AOAC International. Ed., Latimer Jr., G.W. 20th ed. Washington DC; 2016. 14. Van Soest PJ, Robertson JB, Lewis BA, Methods for dietary fiber, neutral detergent fiber, and nonstarch polyssacharides in relation to animal nutrition. J Dairy Sci 1991;74(10):3583–3597. https://doi.org/10.3168/jds.S0022-0302(91)78551-2. 15. Sniffen CJ, O’Connor JD, Van Soest PJ. A net carbohydrate and protein system for evaluating cattle diets: II. Carbohydrate and protein availability. J Anim Sci 1992;70(11):3562–3577. https://www.ncbi.nlm.nih.gov/pubmed/1459919. 16. Hall MB. Challenges with non-fiber carbohydrate methods. J Anim Sci 2003;81(12):3226–3232. https://doi.org/10.2527/2003.81123226x. 17. Harlan DW, Holter JB, Hayes HH. Detergent fiber traits to predict productive energy of forages fed free choice to non-lactating dairy cattle. J Dairy Sci 1991;74(4):13371353. https://doi.org/10.3168/jds.S0022-0302(91)78289-1. 18. NRC. Nutrient requirements of dairy cattle. National Research Council. 7th ed. The National Academy Press, Washington, DC; 2001. 19. Mertens DR. Regulation of forage intake, 11. In: Fahey JR editor. Forage quality, evaluation, and utilization. 1rst ed. Madison, WI, USA: American Society of Agronomy 1994:450-493. 20. Allen MS. Effects of diet on short-term regulation of feed intake by lactating dairy cattle. J Dairy Sci 2000;83(7):1598-1624. https://doi.org/10.3168/jds.S00220302(00)75030-2. 21. Castillo-González AR, Burrola-Barraza ME, Domínguez-Viveros J, Chávez-Martínez A. Rumen microorganisms and fermentation microorganismos y fermentación ruminal. Arch Med Vet 2014;46(1):349-361 https://scielo.conicyt.cl/pdf/amv/v46n3/art03.pdf. 22. Yang JY, Seo J, Kim HJ, Seo S, Ha JK. Nutrient synchrony: Is it a suitable strategy to improve nitrogen utilization and animal performance? Asian-Aust J Anim Sci 2010;23(7):972–979. https://doi.org/10.5713/ajas.2010.r.04. 23. Gordon DG. Fundamentals of applied animal nutrition. 1rst ed. CABI, Australia; 2021.

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24. Souza RA, Voltolini TV, Araújo GGL, Pereira LGR, Moraes SA, Mistura C, Belem KVJ, Moreno GMB. Consumo, digestibilidade aparente de nutrientes e balanços de nitrogênio e hídrico de ovinos alimentados com silagens de cultivares de capimbúfel. Arq Bras Med Vet Zootec 2013;65(2):526-536. https://www.scielo.br/j/abmvz/a/89rGRdJ59SJqzzzzJVZZzSv/?lang=pt&format=p df 25. Tosto MSL, Araújo GGL, Pereira LGR, Carvalho GGP, Ribeiro CVM, Cirne, LGA. Intake, digestibility, nitrogen balance and performance of crossbreed Boer goats fed with diets containing saltbush (Atriplex nummularia L.) and spineless cactus (Opuntia ficus‐indica). Trop Anim Health Prod 2021;53(3):1-10. https://doi.org/10.1007/s11250-021-02783-3. 26. Lindberg JE. Nitrogen metabolism and urinary excretion of purines in goat kids. Brit J Nut 1989;61(2):309-321. http://dx.doi.org/10.1079/bjn19890119. 27. Mertens DR. Analysis of fiber and its uses in feed evaluation and ration formulation. Proc Inter Ruminal Symp. Lavras 1992:1–32.

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https://doi.org/10.22319/rmcp.v13i2.6032 Review

Ovarian function and response to estrus synchronization in Creole cattle in Mexico. Review

Elizabeth Pérez-Ruiz a Andrés Quezada- Casasola b José María Carrera-Chávez b Alan Álvarez-Holguín a Jesús Manuel Ochoa-Rivero a Manuel Gustavo Chávez-Ruiz a Sergio Iván Román-Ponce a*

a

INIFAP. Centro de Investigación Regional Norte-Centro. CE La Campana. Km 33.5 Carr. Chihuahua – Ojinaga. 32910. Aldama, Chihuahua. México. b

Universidad Autónoma de Ciudad Juárez. Departamento de Ciencias Veterinarias. Ciudad Juárez, Chihuahua, México.

*Corresponding author: roman.sergio@inifap.gob.mx

Abstract: Nowadays, reproductive biotechnologies have made it possible to conserve and use animal genetic resources. One of these technologies is the estrus synchronization programs, which allow programming the time for mating according to the availability of fodder or the birth of calves for commercial purposes. Another application is the reduction of the calving- first ovulation interval through protocols that facilitate the use of artificial insemination. Creole cattle are a valuable genetic resource due to their hardiness and adaptability to difficult environmental conditions; they are resistant to parasites, take advantage of available forage resources and reproduce in systems with little or no supplementation. In Mexico, the first

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studies of synchronization of Creole cattle suggest that Creole cows do not respond adequately to hormonal protocols and gestation percentages lower than those obtained in other breeds are obtained. The foregoing gave rise to a series of studies on reproductive physiology and the use of biotechnologies in Creole cattle. The objective of this review is to collect existing information on the use of estrus and ovulation synchronization protocols in Creole cattle from Mexico; in order to be able to identify the lines of research necessary for the development of estrus and ovulation synchronization protocols suitable for Creole cattle. Key words: Beef cattle, Animal genetic resources, Artificial insemination, Ovulation.

Received: 11/08/2021 Accepted: 03/01/2022

Introduction Creole cattle are the descendants of Iberian cattle transported to the Americas during the colonization of these countries by the Spanish and Portuguese(1). One of the most important traits of Creole cattle is their adaptability to difficult environmental conditions; they reproduce in systems with extreme fluctuations in ambient temperature(2,3), are resistant to parasites(4) and take advantage of a variety of herbaceous plants, in addition to alternating between grazing and browsing(1). The adaptability of a breed to variable environments, called phenotypic plasticity, is a quality of Creole cattle, which can be used in current selection and reproduction systems(1). Therefore, these cattle are a genetic resource that could contribute to improving productivity in a challenging environment(1,5). Regarding animal genetic resources, in March 2018, 7,745 breeds of the 8,803 breeds registered by FAO were classified as local breeds (i.e., reported to be present in only one country) and a total of 594 local breeds became extinct. Among existing local breeds, 26 % were classified as at risk of extinction, 7 % are not at risk and 67 % as unknown(6). One of the possible causes of the decrease in the population of local breeds is because they are considered unproductive, compared to specialized breeds (European and Zebu). In general, Creole cattle are small animals and can hardly compete with other breeds of cattle specialized in the production of meat or milk, so many farmers have chosen to make crosses with these, or replace Creoles with other cattle breeds(7,8).

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Reproductive biotechnologies are useful tools to open the possibility of conserving and exploiting animal genetic resources(9). With the application of estrus synchronization programs, it is possible to choose the best time for mating (according to the availability of fodder), reduce the interval from calving to first ovulation (CFO), facilitate the use of artificial insemination (AI) and implement genetic improvement programs(10). The manipulation of the estrous cycle and the induction of ovulation through the different synchronization protocols in conjunction with AI have numerous additional advantages, such as the production of calves in homogeneous batches and the possibility of increasing the sale price, as well as facilitating the nutritional and health management of the herd(11). However, the use of reproductive biotechnologies, such as estrus and ovulation synchronization, is little used in Creole cattle, and with variable results(12,13). In Mexico, the first studies related to the synchronization of Creole cattle were carried out in the 1990s, and it was mentioned in them that Creole cows do not respond adequately to hormonal protocols(12). Therefore, the objective of this review is to collect the existing information on the use of estrus synchronization protocols in Creole cattle from Mexico and in this way, to be able to identify the lines of research necessary for the development of estrus and ovulation synchronization protocols suitable for Creole cattle, which will allow directing research and development efforts.

Fixed-time artificial insemination in cattle

With the use of exogenous hormones and their analogues, it is sought to control follicular development, regression of the corpus luteum (CL) and ovulation, to later perform AI at detected estrus or fixed-time AI (FTAI). With the hormonal protocols for FTAI, the following is achieved: 1) reduce the number and frequency of cattle handling, and 2) eliminate the need for estrus detection to perform AI, which is why they are the most used in beef cattle(14). Estrus synchronization treatments are mainly based on the use of two types of hormones, progestogens (mainly progesterone, P4) and prostaglandin F2α (PGF2α) analogues. In ovulation synchronization protocols, estradiol (E2) analogues and gonadotropin-releasing hormone (GnRH)(15) are additionally used. Preference in the use of these treatments for FTAI may be limited in countries where there are restrictions on the use of E2(16), such as the United States of America, Australia, the United Kingdom, among others. The percentage of gestation that can be obtained with these FTAI protocols varies in a range of 40-60 %(15). The establishment of gestation depends to a large extent on the correct function of the CL, the adequate signaling of the embryo for the maternal recognition of gestation and the oviductal and uterine environment that favors the development of the embryo. These factors are modified in part by the preovulatory conditions, that is, there is an effect of the steroidogenic

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capacity of the follicle, and the consequent competence of the oocyte(17). Other critical factors for the success of synchronization protocols are the physiological state of the animals (prepubertal heifers, cyclic or anestric cows), body condition (BC)(18), nutrition, semen quality, inseminator dexterity(11) and time of insemination after hormone treatment(19). For example, females with low BC (usually ≤ 4 on scale from 1 to 9)(11) tend to be in anestrus and, therefore, have low gestation percentages compared to females with better BC (18). Additionally, first-calving heifers are more sensitive to weight loss and low BC, compared to multiparous cows(18). Nutrient intake and energy balance, before and after calving, affect the duration of the postpartum anestrus and the interval from calving to conception, as well as the percentage of pregnancy(11). When AI is performed, the efficiency of the inseminator is influenced by the quality and handling of the semen, as well as by their technical ability to deposit the semen in the right place(11). In addition, the correct handling and evaluation of semen straws is essential to ensure the quality of the material used, as it directly influences the fertilization rate and, therefore, the pregnancy rate. Prior to AI, it is recommended to evaluate the semen that will be used, according to the most important semen characteristics(11). Therefore, it is imperative that inseminator technicians are sufficiently trained to guide the AI gun through the cervix, to deposit the semen completely at the entrance of the uterine body(11).

Conventional protocols for FTAI based on estradiol and progesterone in cattle

The protocol based on the use of P4 and E2 is known as conventional. P4 simulates the function of CL, inhibiting the release of GnRH/LH pulses. E2, applied at the beginning of the treatment with P4, causes ovulation of the possible existing dominant follicle and atresia of the rest of the subordinate follicles. With the emergence of a new follicular wave, between three and five days later, the presence of a new dominant follicle and a viable oocyte at the end of the treatment with P4 are sought(20). The administration of E2 at the end of the treatment with P4 induces, in the same way as the first application, a positive feedback on the hypothalamus for the release of GnRH and the consequent increase in the frequency of LH pulses, which synchronizes and reduces the time in which the ovulation occurs, to perform the FTAI(20). The conventional protocol consists of the insertion of a CIDR and the administration of EB (at a total dose of 2 mg, via IM) on the day of the beginning of treatment with P4 (day 0)(16). Estradiol 17 β (E-17β) has a shorter half-life than EB, so the latter has been shown to be more efficient in FTAI protocols(20). Additionally, PGF2α is administered at the time of removal of

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the CIDR, to ensure the regression of the CL that has formed after ovulation after the first application of E2 (d 7, 8 or 9, in case of existence of a CL)(21). To synchronize ovulation, a total dose of 1 mg of EB can be applied 24 h after the end of the treatment with P4, and the FTAI can be performed 30 to 34 h after applying the second dose of EB(22). A modification to this protocol consists of administering a total dose of 0.5 or 1 mg of EC at the time of removing the device with P4, which simplifies the handling of cattle, with FTAI 48 to 56 h later(15,23). EC has a longer half-life than EB, so its use allows reducing the number of manipulations carried out on cattle(23). Another option is to apply a dose of 100 μg of GnRH 54 h after removal of the CIDR (at the time of performing the FTAI), which induces ovulation of the new dominant follicle, in case it has not ovulated spontaneously(14). One more alternative to this protocol is the administration of equine chorionic gonadotropin (eCG) at the time of finishing the treatment with P4(24) (Figure 1). The eCG binds to the FSH and LH receptors, causing the increase in the growth rate of the dominant follicle, stimulates the expression of steroidogenic enzymes, mainly follicular E2, with the consequent occurrence of the preovulatory peak of LH. Additionally, eCG produces an increase in the diameter of the CL and increases the production of P4 after AI(25). This effect of eCG is more marked in females with low BC (and that gain weight during the time of mating) and in postpartum (PP) anestrus (24). Doses of 300-400 IU of eCG are used in beef cattle, these doses are the most used both in conventional protocols(21) and in GnRH-based protocols(26,27). The eCG can promote the growth of one or more follicles in the same wave, so not only a larger follicular diameter is achieved, but also the increase in the ovulation rate in cows treated with this hormone, when high doses (400-600 IU)(28) are used. The occurrence of multiple births in beef cattle is considered undesirable, so the increase in the ovulation rate due to the effect of eCG is controversial(27). These eCG effects are most evident during PP anestrus, in cows that are lactating and with low BC(24), because the secretion of GnRH/LH is decreased during this stage(28). However, in cows with good BC, a beneficial effect is not observed(26).

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Figure 1: Schematic representation of synchronization protocols based on progesterone and estradiol

In CE (estradiol cypionate), GnRH (gonadotropin-releasing hormone) and eCG (equine chorionic gonadotropin) treatments, the second dose of BE (estradiol benzoate) is replaced with the respective hormone, at the time of removal of the CIDR (intravaginal progesterone releasing device), to simplify the handling of cattle.

Protocols for FTAI based on GnRH and PGF2α

The initial application of a GnRH analogue causes the release of LH in the form of a preovulatory peak and, therefore, the ovulation of a possible dominant follicle present, with the subsequent appearance of a new follicular wave approximately two days later(29). The administration of PGF2α 7 d after the application of GnRH induces the regression of the possible CL formed after the application of GnRH and a second dose of GnRH will again cause the release of the preovulatory peak of LH, producing the ovulation of a new dominant follicle in a synchronized way(30). The most commonly used protocol for FTAI in dairy cattle is known as Ovsynch (ovulation synchronization)(29). This protocol requires the handling of cattle three times to apply hormones and a fourth time to perform the FTAI (Figure 2), so it is impractical for its use in beef cattle(31,32). The alternative for this type of cattle is the CO-Synch protocol(33) (the second dose of GnRH is applied at the time of the FTAI), in which the number of times in which cattle are handled is reduced(32). However, with this protocol, 5 to 20 % of females in PP anestrus have estrus before or immediately after the application of PGF2α, so a lower pregnancy rate is obtained than with the Ovsynch protocol(34). The insertion of a CIDR, between the first administration of GnRH and the application of PGF2α (Figure 2), increases 427


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the pregnancy rate with this protocol(34,35). The use of the CIDR prevents ovulation before and after the application of PGF2α, caused by spontaneous luteolysis of the CL, and as a result, estruses more synchronized with the moment of the FTAI are obtained, therefore, higher pregnancy rates are obtained with the CO-Synch + CIDR protocol(34,35). The success of this protocol depends largely on the percentage of females that ovulate after the first dose of GnRH(14). If the induction of the ovulation of the dominant follicle is achieved, the emergence of the next follicular wave and ovulation will be synchronized(22). In dairy cattle, one more alternative to increase the percentage of pregnant cows in the Ovsynch protocols is the pre-synchronization with one or two doses of PGF2α(36), with a difference of 14 d between each dose, and the application of the first dose of GnRH 12 to 14 d after the second dose of PGF2α, this protocol is known as Pre-Synch (Figure 3). The objective of this pre-synchronization is that the cows are between d 5 and 12 of the cycle at the time of starting treatment with GnRH(36,37). In beef cattle, pre-synchronization is impractical, since this protocol involves handling the cattle a greater number of times and does not increase the percentage of pregnant females after FTAI(37). As a result of the stress produced when introducing beef cows into pens and handling sleeves, animals experience a fight-or-flight response, which activates the adrenal axis and the release of stress hormones (catecholamines and glucocorticoids), which have a negative effect on the reproductive axis(38). Figure 2: Schematic representation of the Ovsynch, Co-Synch and CO-Synch+CIDR protocols for FTAI (GnRH: gonadotropin-releasing hormone; PGF2α: prostaglandin F2α; IATF: fixed-time artificial insemination)

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Figure 3: Schematic representation of the Pre-Synch protocols used in dairy cattle and Pre CO-Synch developed for beef cattle (GnRH: gonadotropin-releasing hormone; PGF2α: prostaglandin F2α; IATF: fixed-time artificial insemination)

5-day Co-Synch protocol

The 5-day Co-Synch protocol with FTAI 72 h later (Figure 4) is based on the idea that increasing the period in which the dominant follicle develops in the presence of gonadotropins can increase the percentage of pregnant females after the hormone treatment(39). The main changes in this protocol are: 1) the reduction of the treatment with P4 from 7 to 5 d, to avoid adverse effects of persistent follicles on the fertility of cows that do not ovulate with the first dose of the GnRH analogue (total dose of 100 μg of gonadorelin, IM), and 2) prolong the period from removal of P4 to administration of GnRH, to increase exposure to circulating E2 concentrations before ovulation(17). With this protocol, a greater diameter of the dominant follicle, an increase in the concentration of E2, as well as a greater production of P4 after ovulation, necessary for gestation(40), are observed.

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Figure 4: 5-day CO-Synch and J-Synch protocols

In these protocols, proestrus is prolonged (increase of the period between the end of treatment with Progesterone (P4) and IATF (PGF2α= prostaglandin F2α; GnRH= gonadotropin-releasing hormone; BE= estradiol benzoate; IATF: fixed-time artificial insemination)

J-Synch protocol

This protocol is based on the use of P4 and EB, with a longer duration of proestrus than with conventional treatment(41). The protocol begins with the administration of a total dose of 2 mg of EB at the time of insertion of a device with P4, which is removed 6 d later. At the time of removal of the device with P4, a single dose of PGF2α is applied. Additionally, 100 μg of GnRH is applied at the time of FTAI, 72 h after (d 9, Figure 4). Similar to the 5-day COSynch protocol, with the prolongation of the proestrus (95-97 h), an increase in the concentrations of E2 (before ovulation) and P4 (after ovulation) is observed, as well as a higher percentage of gestation (when females are in good BC), compared to the conventional Co-Synch protocol(42). Recent studies indicate that the length of the proestrus is decisive for the establishment of gestation. With a longer proestrus, E2 production increases before ovulation. Among the functions of E2 are the modification of cell morphology, secretion and regulation of steroid receptors, which favor the implantation of the conceptus in the uterus(43). Table 1 shows some results obtained for the percentage of pregnancy, with different synchronization protocols.

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Table 1: Percentage of gestation obtained with different protocols for FTAI in cattle Type of protocol† Breed type‡ Pregnancy (%) Reference Based on P4 and E2 Conventional (EB) Conventional (EC) J-Synch Conventional EB + eCG Conventional EC + eCG J-Synch + eCG Based on GnRH and PGF2α Ovsynch Pre-Synch CO-Synch CO-Synch + CIDR 5-d CO-Synch 5-d CO-Synch + CIDR CO-Synch + CIDR + eCG 5-d CO-Synch + CIDR + eCG

BI, BT BI, BT BI/BT, BT BI BI, BT BI/BT, BT

30.0-40.6 34.7-54.0 47.0-67.9 36.8-57.5 50.3-61.8 53.0-60.4

(25, 44-46)

BT BT BT BT BT BT BT BT

32.5-57.0 27.0-49.6 26.7-53.3 50.0-55.1 44.4-59.7 48.0-63.9 43.0 42.9

(33, 36, 37, 54, 55)

(16, 42, 44, 47, 48) (16, 41, 42, 47, 49) (25, 46, 50, 51) (16, 28, 43, 48, 52) (16, 43, 47, 53)

(36, 37, 55-57) (33- 35, 54, 58) (26, 34, 35, 58-60) (61-63) (59, 60, 63-66) (26) (67)

†CIDR= intravaginal progesterone releasing device; P4=progesterone; E2= estradiol; PGF2α= prostaglandin F2α; GnRH= gonadotropin-releasing hormone; EB= estradiol benzoate; eCG= equine chorionic gonadotropin; EC= estradiol cypionate. ‡ BT: Bos taurus taurus; BI: Bos taurus indicus.

Creole cattle in Mexico Creole cattle in Mexico descend from the first specimens brought by the Spanish during the Colonial Period in the sixteenth century(68). These cattle developed qualities of adaptation to the environment in isolated and hard-to-reach areas, which contributed to the formation of breed groups(69,70). In Mexico, of the 53 breeds of cattle recorded in the database of the biodiversity of domestic animals published by FAO(71), the following stand out as local breeds: the Chinampo, from Baja California(72,73); the Coreño, from the Sierra Madre Occidental(74,75); the Creole from the Northern Mountains, also called rodeo Creole or Rarámuri Creole(70); the Mixteco(76); the Creole from the Gulf(77); the Creole from the central region of Chiapas(78) and the Nunkiníen Creole, from the Yucatán Peninsula(79). From 1965, individuals of the tropical dairy Creole and Romosinuano breeds, from the United States and Costa Rica, respectively, were also introduced(80).

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The Rarámuri Creole (RC) bovine, also called “Corriente”, is adapted to the northern region of the country, mainly in the Sierra Tarahumara, in the state of Chihuahua(70). They are animals of small size and large horns; their main zootechnical purpose is for rodeo sports activities, so it is exported in large quantities to the United States(7). The reproductive physiology of this breed of Creole cattle from Mexico is the most researched so far, this includes the characterization of the estrous cycle, ovarian activity, estrous behavior, hormonal profiles(81-83) and development, and evaluation of synchronization and AI protocols (12,13) .

Estrus and ovulation synchronization protocols in Rarámuri Creole cattle

In one of the first studies in RC cattle, a conventional protocol for FTAI was used, with the use of a CIDR (with 1.9 mg of P4) for 7 d, plus the application of 1 mg of β-estradiol at the beginning of treatment with P4; administration of 30 mg of PGF2α, via IM, when removing the CIDR; and 24 h after removing the CIDR, another dose of 1 mg of β-estradiol was applied. The FTAI was performed 54 h after removal of the CIDR. With this protocol, a percentage of gestation of 9.09 % was observed, despite the fact that 100 % of the cows showed signs of estrus. In this study, administering a dose of 50 mg of P4, via IM, at the time of removal the CIDR, in conjunction with the application of β-estradiol, did not improve the percentage of gestation (9.09 % was obtained with both treatments). The low percentage of pregnancy obtained with this protocol was attributed to a variation in the time to ovulation(12). The cows showed estrus between 36 and 43 h after the application of PGF2α. The authors mention that letting so much time pass between the beginning of estrus and AI was a determining factor. It should be noted that the cows that had estrus between 36 and 37 h after removal of the CIDR did not become pregnant, while all the cows that showed estrus around 43 h after removal of the CIDR did become pregnant. It is also mentioned that anovulatory estruses occur in RC cows, so this contributes to a low percentage of gestation(12). In another study with RC cows, the time from the beginning of the PGF2α-induced estrus to ovulation was 46.2 ± 8.2 h and 37.6 ± 6.0 h in a natural estrus (all cows had estrus within 24 to 60 h), with a significant proportion of cows that ovulated in the range of 24-35 h in natural estrus (8 cows out of a total of 22). In both types of estrus, the highest percentage of cows ovulated in the range of 36-47 h (12 cows in natural estrus and 11 cows in induced estrus)(82). This difference in the times from the beginning of estrus to ovulation, in RC females, with respect to Bos taurus taurus cattle, should be considered to improve the percentage of gestation with FTAI protocols, or consider the possibility of using the “AM-PM” rule to program AI, as mentioned by Zárate-Martínez et al(12). However, performing this type of handling, which requires estrus detection, is impractical for RC cattle farmers.

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The growth pattern of ovarian follicles is in waves; one to four waves may occur in each estrous cycle(84-86). The number of follicular waves in each cycle is variable, with two to three waves being the most common(85,86). In RC females, there is a higher percentage of cows with two follicular waves (77.3 %)(81). Females that have two follicular waves per cycle ovulate on average 6.2 h earlier than cows with three waves(83). In addition, the follicular growth rate in the RC breed (0.6 ± 0.2 mm d-1)(82) is lower than in Bos taurus indicus cattle (0.9 ± 0.1 mm d-1) and other Bos taurus taurus breeds (1.1 ± 0.1 mm d-1)(87). In this regard, Quezada et al(82) mention that, in order to optimize the response to synchronization protocols in RC cows, it is necessary to modify the time between hormone treatment and AI, so they suggest performing AI ~28 h after the beginning of estrus(82).

Use of eCG in Creole cattle

In Bos taurus taurus cattle, the application of eCG in protocol for FTAI helps to increase the follicular growth rate and diameter, as well as the production of P4, in females with low BC(24). When evaluating the use of a dose of 400 IU of eCG at the time of FTAI (56 h after removal of the CIDR) in RC cows, with the use of a 8-d CO-Synch protocol (application of a dose of 100 μg of GnRH when inserting the CIDR + 25 mg of PGF2α when removing the CIDR, on d 8 of the protocol + 400 IU of eCG, at the time of FTAI), the percentage of gestation (31.5 vs 46.6 %) was not improved compared to the same protocol without eCG (in this treatment, 100 μg of GnRH was applied at the time of FTAI). The authors mention that the females were in an acceptable BC (4.5 ± 0.2; on a scale from 1 to 9) and received a good diet during reproductive management, so no positive effect of eCG was observed(88). In this same study, supplementation with concentrate, selenium (0.95 mg Se/50 kg LW) or Ca propionate (100 g), did not modify the percentage of gestation either(88). In another study in which the use of a dose of 500 IU of eCG in RC cows was evaluated, a percentage of gestation of 60 % of the total number of females in the treatment (and 75 % of gestation compared to those that showed signs of estrus) was obtained(13). The hormonal protocol used in this study consisted of the use of an CIDR for 7 d + 2.76 mg of EB at the start of treatment with P4 (d 0) + 25 mg PGF2α (d 7) + 1 mg of EC or 500 IU of eCG 24 h after removing the CIDR (d 9); the AI was performed 12 h after the beginning of estrus. The percentage of gestation in the group of cows treated with EC was 27.3 %. It should be noted that, in this study, 100 % of cows treated with EC and 80 % of those treated with eCG showed signs of estrus in response to the AI protocol and all females in both treatments ovulated. The response of the cows to both treatments was similar for the variables: response to estrus, percentage of ovulation and maximum follicular diameter(13). When evaluating these same

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protocols in RC heifers, the response to estrus was lower in both treatments, 89.5 % and 25.0 % for EC and eCG treatments, respectively. The percentage of heifers that had silent estruses was 10.5 % for heifers in the group treated with EC and 75.0 % for those treated with eCG. The occurrence of silent estruses was attributed to a lower follicular growth than that observed with the use of EC. Because only females that showed signs of estrus were inseminated, the percentage of gestation with the use of eCG was only 10 %, compared to the total in the group (40 % of those that had estrus and were inseminated)(13). However, 100 % of the heifers treated with eCG ovulated. In this study, in both cows and heifers, estrus occurred in a shorter time with EC (cows 24.9 ± 2.8 h and heifers 25.8 ± 2.9 h) compared to the treatment of eCG (31.5 ± 2.8 and 30.6 ± 2.9). Additionally, the authors mention the possibility of using FTAI successfully, in multiparous cows with the use of eCG, since, with this protocol, the beginning of estrus grouped between 24 and 36 h after removing the CIDR (31.5 ± 2.8 h on average) and the highest percentage of cows were inseminated 36 to 48 h after finishing the treatment with P4. In addition, the results of response to estrus and percentage of gestation obtained in this study are higher than those obtained by Zárate et al(12) and Sánchez-Arciniega et al(88) in this same breed of cattle.

Restart of postpartum ovulatory/cyclic activity

Postpartum (PP) anestrus is characterized by the absence of ovulations after calving. In this period, the ovarian follicles begin to grow, but none is able to ovulate, at least during the first weeks(89). This is partly due to the absence of LH, and often, the first ovulation is not preceded by the manifestation of signs of estrus; the CL may have a reduced mean life, smaller size and limited steroidogenic activity(90). To reduce the negative effect of suckling, it has been proposed to perform early weaning (EW, a few days after calving); controlled or restricted suckling (RS, it consists of allowing suckling in short periods of the day); or temporary weaning (TW, separating the calf from the mother for a few days)(91). The RS technique increases the proportion of cows that show signs of estrus during the first 100 d postpartum and reduces the calving-first ovulation interval, without affecting the growth of calves(91). In RC cattle, with the RS strategy (beginning on day 76 PP), prior to the hormonal protocol for FTAI, 81.4 % of the cows ovulated within an observation period of 22 d after starting the RS, but only 11.1 % of them showed signs of estrus, that is, they had silent ovulations. In this study, the manifestation of estrous behavior was only observed in females with better BC (4-5, regular to good), while cows with poor BC (2-3, on a scale of 1-9) did not ovulate before the synchronization treatment(12).

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In another study in RC cattle, when assessing weight loss during lactation, cows that were treated with an EW scheme from 68 ± 3.8 d PP lost less weight than those that remained with the offspring (normal weaning at 180 ± 10.2 days) during the evaluation period. In females with EW, weight loss was 4.8 kg, while females in the group of normal weaning lost 18.9 kg during the evaluation period (68-180 d PP)(92). As mentioned, as in other specialized bovine breeds(11), BC in RC cattle is a limitation to re-establish reproductive activity after calving, and to establish gestation(92). RC cattle are rarely supplemented, so there is no control over their body condition(88). Therefore, performing strategies such as RS or EW in Creole cattle, in conjunction with the implementation of protocols for FTAI, could be useful to improve the percentage of gestation.

Estrus synchronization in other breeds of Creole cattle in Mexico

Information on the reproductive performance in response to protocols of estrus synchronization and AI in other breeds of Creole cattle in Mexico is limited. In Coreño Creole cows from Nayarit, with synchronization with a Norgestomet implant for 9 d + 280 IU of eCG, 80 % response to estrus and a percentage of gestation of 60 %(93) were obtained. In the Chinampo breed, the use of two doses of PGF2α (11 d apart between each application) to induce estrus and evaluate the estrous behavior in the presence of the bull was evaluated(94). In the presence of the bull, greater interaction was found between 30 and 60 h after the second dose of PGF2α. Cows exposed to the bull started estrus in less time and had a shorter estrus length than those that remained isolated from the bull (10.7 ± 1.1 h vs 16.3 ± 2.6 h). In purebred and crossbred heifers of the tropical dairy Creole breed, an estrus response of 94.1 % and a percentage of gestation of 68.8 % were found; with the addition of a dose of 500 IU of eCG, on day 10, of a protocol with a subcutaneous implant (with 3 mg of norgestomet, for 12 d + 5 mg of estradiol valerate, EV, via IM on d 0). While with a dose of 0.25 mg of GnRH, 24 h after removal of the implant (implant with 3 mg of norgestomet, for 12 d + 5 mg of EV, via IM on d 0), 76.4 % of females showed estrus and 46.2 % of them became pregnant(95). In this protocol, a dose of 15 mg of PGF2α was applied, via IM, 10 d before placing the subcutaneous implant; to homogenize the estrous cycle. Table 2 summarizes the results obtained in Creole cattle, with the use of various estrous and AI synchronization protocols.

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Table 2: Summary of gestation percentages obtained with hormonal protocols in Creole cattle in Mexico Breed† Cows

RC

Cows

RC

Cows

RC

Cows

RC

Cows

RC

Cows

RC

Cows

Coreño

Heifers

RC

Heifers

RC

Heifers

TDC

Heifers

TDC

Protocol‡

Gestation (%) CIDR for 7 d + 1 mg E2 + 50 mg P4 (d 0) + 9.1 30 mg PGF2α (d 7) + 1 mg E2 (d 8) CIDR for 7 d-1 + 1 mg E2 (d 0) + 30 mg PGF2α 9.1 (d 7) + 1 mg E2 (d 8) CIDR for 7 d-1, 100 μg GnRH + 25 mg PGF2α 31.5 (d 8) + 400 IU 56 h after removal of the CIDR CIDR for 7 d-1, 100 μg GnRH + 25 mg PGF2α 46.6 (d 8) + 100 μg 56 h after removal of the CIDR CIDR for 7 d + 2.76 mg EB (d 0) + 25 mg 27.3 PGF2α (d 7) + 1 mg EC (d 9) CIDR for 7 d-1 + 2.76 mg EB (d 0) + 25 mg 60.0 PGF2α (d 7) + 500 IU eCG (d 9) Norgestomet implant for 9 days + 280 IU 60.0 eCG CIDR for 7 d + 2.76 mg EB (d 0) + 25 mg 27.3 PGF2α (d 7) + 1 mg EC (d 9) CIDR for 7 d + 2.76 mg EB (d 0) + 25 mg 60.0 PGF2α (day 7) + 500 IU eCG (d 9) 15 mg PGF2α (d-10) + Subcutaneous implant 68.8 (3 mg norgestomet) for 12 d-1 + 5 mg EV, 500 IU eCG (d 10) 15 mg PGF2α (d-10) + Subcutaneous implant 46.2 (3 mg norgestomet) for 12 d-1 + 5 mg EV (d 0) + 0.25 mg GnRH (d 10)

Reference (12)

(12)

(88)

(88)

(13)

(13)

(93)

(13)

(13)

(95)

(95)

†RC= Ráramuri Creole. TDC= tropical dairy Creole. ‡CIDR= intravaginal progesterone release device; E2= estradiol; EV: estradiol valerate; PGF2α= prostaglandin F2α; GnRH= gonadotropin-releasing hormone; IU= international units; EB= estradiol benzoate; eCG= equine chorionic gonadotropin; EC= estradiol cypionate.

Factors associated with the reproductive response in Creole cattle

There are physiological differences between Creole cattle and European cattle specialized in beef production, as well as with zebu cattle. Among these, those inherent to ovarian functioning stand out: number of follicular waves, follicular growth rate, diameter of the ovulatory follicle and of the CL; and concentration of reproductive hormones and periods between events of the estrous cycle (length of luteal phase vs follicular phase; time to ovulation, length of estrus)(82,83,85). Additionally, external factors such as nutritional status, 436


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handling, social and hierarchical relationships(38) influence the response of Creole cattle to hormonal protocols for FTAI.

Reproductive behavior, ovarian function and endocrinology in the Creole female

The estrous cycle in RC cows has an average length of 21.1 ± 1.2 d (range of 19-23 d), with a follicular phase of 6-9 d and a luteal phase of 12-16 d(88). Follicular growth in cattle occurs in a pattern of follicular waves, and the number of follicular waves in each estrous cycle is variable, but it can range from two to four waves. In RC cows, there is a higher percentage of females with two follicular waves (77.3 %), and a smaller percentage of females with three follicular waves per cycle (22.7 %)(82). In Bos taurus taurus females(84), Bos taurus indicus females(85), Thai Creole females(86), a higher percentage of females with two follicular waves per cycle has also been observed. Contrary to these findings, in Caqueteño Creole cattle from Colombia(9), in Creole heifers with dairy tendency in Ecuador(96), and Creole cattle from the high Andean zone of Peru(86), there is a higher percentage of females with three follicular waves (Table 3). However, so far there is no published information on follicular dynamics in other breeds of Creole cattle from Mexico. Table 3: Number of follicular waves in the estrous cycle in Creole cattle, Bos taurus taurus and Bos taurus indicus Breed Number of follicular waves (%) 2 3 4 (82) Rarámuri from Chihuahua, Mexico 77.3 22.7 (9) Caqueteño from Colombia 33.3 66.6 (86) Creole from the high Andean zone of Peru 16.0 78.0 6.0 (96) Creole from the highlands of Ecuador 44.4 55.6 (97) Native Thai 70.0 30.0 (85) Nelore 83.3 16.6 (84) Holstein 81.0 19.0 The characterization of the estrous cycle and follicular dynamics in different cattle breeds has allowed observing differences and similarities between them (Table 4). In Caqueteño and Nelore Creole cattle, females with three follicular waves have longer estrous cycles than females with two waves(9,85). In RC cows, the length of the estrous cycle, follicular phase and luteal phase is similar between females with two and three follicular waves, but females with two follicular waves ovulate 6.2 h before than those with three follicular waves(82,83). The ovulatory follicles of RC females with two waves grow at a lower rate than those with three

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waves (0.5 ± 0.04 vs 0.9 ± 0.08, respectively), while the maximum diameter of the ovulatory follicle is similar between both growth patterns (10.5 ± 0.2 vs 10.0 ± 0.4, respectively)(83). The maximum diameter of the CL in RC cows with two and three follicular waves is similar (13.0 ± 1.0 vs 13.2 ± 1.7 mm, respectively)(83). The size of the CL and the production of P4 in RC cows are also smaller than those of other cattle breeds, and it is possible that these differences are adaptations to the environmental and nutritional conditions that Creole cattle have developed to survive in difficult environments(83).

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Table 4: Follicular and ovulatory dynamics in cattle with two and three follicular waves

Number of waves Estrous cycle length, days First wave (non-ovulatory) Emergence, days † Maximum DF diameter‡, mm Growth rate, mm day-1 Regression, days Second wave (non-ovulatory) Emergence, days Maximum DF diameter, mm Growth rate, mm day-1

Breed Rarámuri Mexico (83) 2 3

from

Creole Ecuador (96) 2 3

from

Caqueteño from Colombia (9) 2 3

Native Thai (97)

Holstein (84) 1

2

2

3

21.1±0.3

21.4±0.6

20.3±0.0

23.6±0.0

20±0.6

22±0.5

18.60±0.1

20.38±0.1

−0.5±0.2

−0.4±0.2

1

1

3

4

1.53±0.1

1.54±0.1

-2.0+0-1

-0.5±0.3

8.0±0.1 0.5±0.03 11.0±0.8

7.6±0.3 0.7±0.0 7.8±0.4

13.2±2.2 1.0±0.1 -

12.2±1.7 1.2±0.0 -

8.4±1.3 11

11.7±3.3 10.0±0.5

7.63±0.1 0.65±0.0 8.34±0.1

7.67±0.1 0.75±0.1 8.79±0.1

17.1±0.5 13.0+0.4

16.0±0.4 12.2±0.5

-

9.6±0.5

-

6.6±0.0

-

10.0

-

8.38±0.1

9.0

-

7.2±0.2

-

10.2±0.0

-

12.2±4.4

-

6.79±0.2

12.9±0.7

-

0.4±0.08

-

1.1±0.1

-

-

-

0.75±0.1

-

14.8±0.8

-

-

17.0±0.5

-

12.58±0.1

11.2±0.8

14.8±0.8

7.8±1.6

13,2±1.3

11.0

17.0

11.02±0.1

11.33±0.1

9.6±0.2

16.0±1.1

10.5±0.2

10.0±0.4

15.3±0.0

13.8±1.4

7.5±1.1

13.8±3.6

8.81±0.2

8.14±0.2

16.5±0.4

13.9±0.4

0.5±0.04

0.9±0.08

0.9±0.1

1.1±0.2

-

-

1.07±0.0

1.48±0.1

-

-

22.0

22.5

20.0

23.0

20.0±0.6

22.0±0.5

19.44±0.1

21.13±0.2

20.4+0.3

22.8+0.6

Maximum CL diameter, mm Maximum P4 concentration, ng/ml

13.0±1.0

13.2±1.7

21.7±1.4

23.5±0.6

11.3±4.3

11.2±3.2

13.55±0.1

15.14±0.1

-

-

6.5±0.1

6.5±0.2

20.6±5.4

20.6±3.1

-

-

4.13±0.1

4.25±0.1

-

-

Regression, days

16.3±1.6

16.8±1.1

18.0

20.0

17.0±1

19±0.96

17.11±0.1

19.29±0.1

16.5±0.4

19.2±0.5

Regression, days Ovulatory wave Emergence, days Maximum DF diameter, mm Growth rate, mm day-1 Ovulation, days Corpus luteum (CL)

-

19

† The moment of ovulation (day 0) was used as a reference to determine the beginning of the cycle, so it is possible to observe the emergence of the next follicular wave before ovulation. In the Rarámuri, the evaluation of both ovaries was performed every 8 h after the start of estrus (day 0 = day of ovulation). ‡DF: Dominant follicle.

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Social and hierarchical interaction of the RC

In RC cattle, the existence of dominance relationships between females of higher rank over those of lower hierarchical rank has been observed(12). The social dominance in this type of cattle is not only determined by age, but also by the type of horns (as mentioned, this type of cattle has large horns in relation to the size of the body, which is why they are used for rodeo). Females with horn defects have difficulty defending themselves and their offspring during suckling, so they end up escaping or giving up space(12). Another factor that can influence the percentage of gestation is the temperament of animals; animals with aggressive temperament tend to have a lower reproductive performance than animals with a docile temperament(98). Aggressive temperament disrupts physiological events necessary for reproduction, is associated with increased synthesis and circulating concentrations of adrenocorticotropin (ACTH) and cortisol, which can alter the key physiological events necessary to achieve puberty and the release of the preovulatory wave of GnRH/LH(99). On the contrary, a calmer temperament leads to higher rates of estrus occurrence and gestation, as well as fewer embryonic losses(100). Additionally, the social system of cattle is very hierarchical. Hierarchy influences the intake of feed and social behavior(101). In addition, low-ranking cows adopt a passive attitude as a behavioral strategy to reduce stress. Hierarchy also influences reproduction: stressed and low-ranking mothers produce less LH, which interferes with ovulation and estrous behavior(38).

Conclusions The use of reproductive biotechnologies such as estrus and AI synchronization is limited in Creole cattle and the results obtained are variable. Among the factors that modify the response to synchronization are the reproductive physiology of the Creole cattle, body condition and hierarchical relationships. The review of the existing information on the response in Creole cattle to the use of synchronization protocols allows proposing the lines of research to be developed, and they should be focused on the comparative study of reproductive biology, adaptation of hormonal protocols considering the particularities of Creole cattle and the nutrition, handling and reproduction interaction. Finally, reproductive biotechnologies adapted to Creole cattle may be incorporated into programs of conservation and rational use of this animal genetic resource.

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93. Moreno-Flores LA, Macías-Coronel H, Martínez-Velázquez G, Guerrero-Bustamante JJ. Aspectos reproductivos del bovino Criollo Coreño y sus cruzas en el trópico. Abanico Vet 2012;2(1):32–40. 94. Espinoza-Villavicencio JL, López-Amador R, Palacios-Espinosa A, Ortega-Pérez R, Ávila-Serrano N, Murillo-Amador B. Efecto del toro sobre el comportamiento estral de vacas Chinampas (Bos taurus) en una región tropical seca. Zootecnia Trop 2007;25(1):19–28. 95. Rosendo-Ponce A, Rosales-Martínez F, Cruz-Reyes L, Canseco-Sedano R, GallegosSánchez J, Becerril-Pérez CM. Sincronización de estro en vaquillas criollas Lechero Tropical puras y mestizas. Zootecnia Trop 2017;35:35–44. 96. Ayala LE, Pesantez JL, Rodas ER, Dután JB, Calle JR, Murillo YA, et al. Dinámica folicular de vaquillas Criollas al pastoreo en el altiplano ecuatoriano. Arch Zootec 2019;68(262):184–90. doi.org/10.21071/az.v68i262.4135.

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99. Cooke RF, Moriel P, Cappellozza BI, Miranda VFB, Batista LFD, Colombo EA, et al. Effects of temperament on growth, plasma cortisol concentrations and puberty attainment in Nelore beef heifers. Animal 2019;13(6):1208–1213. doi:10.1017/S1751731118002628. 100. Kasimanickam R, Asay M, Schroeder S, Kasimanickam V, Gay JM, Kastelic JP, et al. Calm temperament improves reproductive performance of beef cows. Reprod Dom Anim 2014;49(6):1063–1067. doi: 10.1111 / rda.12436. 101. Solano J, Galindo F, Orihuela A, Galina CS. The effect of social rank on the physiological response during repeated stressful handling in Zebu cattle (Bos indicus). Physiol Behav 2004;82(4):679–83. doi: 10.1016 / j.physbeh.2004.06.005.

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https://doi.org/10.22319/rmcp.v13i2.5789 Review

Ultrasonography and physiological description of essential events for reproductive management in dairy cattle. Review

María Elena Torres-Lechuga a Juan González-Maldonado b*

a

Former CONACYT fellow.

b

Universidad Autónoma de Baja California, Instituto de Ciencias Agrícolas, Carretera a Delta S/N, C.P. 21705, Ejido Nuevo León, Baja California, México.

*Corresponding

author: jugomauabc@gmail.com

Abstract: The ultrasound allows to visualize the female reproductive tract and helps to understand some of the most relevant reproductive events such as follicular and corpus luteum development, ovulation, pregnancy diagnosis, uterine infections, embryo and fetal growth, among others. Nowadays, there is a massive amount of information regarding the physiology and ultrasonography of the reproductive events mentioned above. However, the overwhelming number of available papers review technical aspects of ultrasonography, physiology and reproductive management separately. Therefore, the objective of the present review is to merge a physiological description with reproductive management and technical aspects of original ultrasound pictures of the most relevant reproductive events in dairy cattle to promote ultrasound use during dairy cattle reproductive management by practitioners and researchers. Key words: Corpus luteum, Embryo, Follicle, Fetus.

Received: 02/10/2020 Accepted: 04/08/2021 452


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Introduction The economic profit of a dairy farm increases as reproductive efficiency improves and the ultrasound is a tool that helps to improve decision-making regarding reproductive management in dairy and beef herds(1). Ultrasonography images may provide information regarding the physiological stage of the main components of the reproductive tract (ovary, follicle, corpus luteum (CL) and uterus) (Figures 1 to 9), which is valuable for accurate work decisions of animal reproduction practitioners and researchers. In addition, ultrasound allows to diagnose pathological conditions of the reproductive tract in real-time on field conditions in a highly reliable way. There are outstanding reviews discussing technical and practical aspects about ultrasound applications in dairy cattle reproductive management(2-7). A search including only “Mexico” and using the key words “cattle, follicle and corpus luteum size” in SCOPUS reveals that during the period from 2015 to 2019 only nine scientific articles were wrote by Mexican researchers, which is lower than the 95 scientific articles found when the search was restricted to the Unites States of America, during the same period. The reasons for this might include a lower number of researchers in Mexico than in the Unites States of America, the restricted access to guiding information to empower new ultrasound practitioners and the economic investment needed to obtain an ultrasound(6). The professional guidance is always recommended when adopting new technologies, but when factors such as time and geographical distance limit the access to professional expertise, then the self-training is the best option. However, the first encounters with the vast amount of scientific literature can be overwhelming to new ultrasound practitioners, because not only the revision of literature related to ultrasonography is mandatory, but also the reproductive physiology and management of dairy cattle must be attended. Despite the available papers reviewing the ultrasound applications to dairy cattle reproductive management, there is still space to enrich the existent literature by merging field experiences performing ultrasonography with cattle reproductive physiology and management. Therefore, the objective is to merge a physiological description with reproductive management and technical aspects of original ultrasound pictures of the most relevant reproductive events in dairy cattle to promote ultrasound use during dairy cattle reproductive management by practitioners and researchers.

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Follicular events The ovarian follicles grow in cyclic and organized events known as follicular waves (Figure 1). A follicular wave comprises three fixed (recruitment, selection and dominance) and two conditional (atresia or ovulation) stages(8). During the fixed stages a dominant follicle is selected from a cohort of growing follicles, while the unselected (subordinate) follicles suffer atresia. A growing follicle must have access to IGF-I, express LH receptors and synthesize estradiol under low FSH concentrations conditions in order to reach the stage of dominance(9). After dominance, the selected follicle will undergo atresia or ovulation depending on the hormonal environment. Ovulation will occur if the follicle reaches the dominance stage under declining blood progesterone concentrations, otherwise it will suffer atresia and a new wave will emerge. Follicle wave emerging is characterized by the appearance of several follicles between 3-4 mm(10). Two or three waves are commonly observed during the estrous cycle in cattle(11). Figure 1: Pictures depicting follicle wave growth in dairy cattle from estrus (day 0) to day 11 of the estrous cycle

Ultrasound allows to visualize only the selection and dominance phase of the follicular wave, but not the recruitment phase because it takes place at very early stage of follicular development. The preovulatory follicle at day zero and the subsequent corpus luteum were located in the left ovary, but only pictures of the right ovary are shown because is where the follicle wave emerged. a) a small antral follicle is encircled with a 454


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dotted line at day zero (the anechogenic characteristics of the follicular fluid makes the follicle appears as dark circle), gray arrows are indicating the presence of other two antral follicles. b) the follicle wave has emerged, the four follicles are encircled with dotted lines at day one, three of them were previously observed at day zero (gray arrows). c) the gray arrows are pointing to the three follicles observed since day one, the increase in its size is easily observed at day two. The growth of the three follicles (dotted lines) observed since day zero is depicted in Pictures c) and d) (day three and four). e) the dominant follicle of the follicular wave is selected (selection of the dominant follicle occurs when the largest follicle reaches approximately 8.5 mm in diameter(11)), this marks the end of the selection and the commencement of the dominance phase. The growth of the dominant follicle (dashed arrows) and the atresia of the subordinate follicles (white lines) are depicted in Pictures g) to i). OS: ovary stroma. White arrow: day of the estrous cycle. Pictures were taken using a 7.5 MHz probe.

The preovulatory follicle is the dominant follicle that naturally emerges from the last follicle wave of the estrous cycle in cyclic non-pregnant cows and, under normal circumstances, is destined to ovulate. The estradiol synthesized by the preovulatoy follicle is responsible for the characteristic signs of estrus in cattle and induces the GnRH/LH surge, which triggers ovulation. Researchers have tried to establish a relationship between preovulatory follicle size and pregnancy success after artificial insemination. In this regard, follicles reach ovulatory capacity in response to GnRH at 10 mm size(12). However, GnRH induced ovulation of small (≤ 11 mm) or large (> 20 mm) follicles negatively affects fertility and increases the probability of pregnancy loss(13,14). Explanatory reasons for the aforementioned include a reduced oocyte competence and an impaired luteal function when small or large follicles are induced to ovulate(14,15). Contrary, preovulatory follicle size does not compromise pregnancy rate when ovulation occurred spontaneously(13). Therefore, the measuring of follicle to ensure an adequate size is advisable during protocols that involve ovulation induction. It is common to observe a wide variation within and between new ultrasound practitioners when measuring follicle and CL size. To avoid this variation, it is recommended to always explore the entire ovary and then capture the largest view of the desired ovarian structure. In addition, physical pressure on the preovulatory follicle should be minimized because of the risk of shape deformation and rupture, which might occur when the ovary is mishandled. The cow normally ovulates one follicle between 28 to 31 h after the onset of estrus(16,17), but occasionally more than one follicle ovulates (Figure 2), increasing the incidence of twin pregnancies. Pregnancies carrying more than one product are undesired because it impairs reproductive performance and reduce the productive life span of the dam(18). Unfortunately, the incidence of twin pregnancies has increased over the years. The improvement in nutrition, management practices and the genetic progress to increase milk production predispose to the augmented incidence of twin pregnancies(19). In addition, cows carrying the Trio allele are more likely to carry twins(20). Fortunately, the incidence of twin pregnancy can be prevented by ablation of multiple follicles before artificial insemination, leaving only one preovulatory follicle(21). 455


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Figure 2: Pictures depicting two (a) and three preovulatory follicles (PF, b) at estrus

The follicular fluid is anechogenic, which make the PF appears as a dark circle. The ovulation of two PF will produce two corpora lutea (CL, c). The CL is usually darker (hypoechogenic) than the ovary stroma (OS). Pictures were taken using a 7.5 MHz probe at random stage of the estrous cycle.

Ovulation is expected to occur after estrus. However, 3.4 and 12.4 % of cows did not ovulate after estrus during the cool and warm season in a study carried out in Spain(22). Ovulation failure (anovulation) completely blocks the opportunity of pregnancy. Therefore, disappearance of the preovulatory follicle (ovulation) should be confirmed by ultrasound after estrus (Figure 4). The causes of anovulatory condition include a low LH pulse frequency after estrus(23) and the formation of follicle cyst (Figure 3). A follicle cyst is a large follicle that fails to ovulate and persist for an abnormal period of time in the absence of a CL, causing a recurrent estrus behavior. A sub-luteal progesterone concentration(24) that allows a rise of LH pulse frequency to sustain follicle growth(25), but not a preovulatory LH surge to induce ovulation(26), favors follicle cyst formation. In addition, the induction of a preovulatory LH surge without subsequent progesterone exposure is also effective to induce follicle cyst formation(27).

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Figure 3: Ovarian cyst in dairy cattle. a) picture depicting two normal pre-ovulatory (PF) follicles and one large follicular cyst (FC)

The anechogenic follicular fluid in the PF and FC makes them appear as a dark circle. Pictures b and c depict a FC after induced luteinization by GnRH injection, notice the increment on the thickness of the follicular wall (c). Pictures were taken using a 7.5 MHz probe.

The follicle cyst appears as a large ovarian structure (> 25 mm in diameter) with a thin wall (< 3 mm), a non-echogenic antrum, a large estradiol: progesterone ratio and it can rupture if mishandled during palpation (Figure 3). Another type of cyst is the follicle-luteal, also known as luteinized follicle or luteinized cyst. The luteinized follicle cyst has a thick wall (> 3 mm), a reduced cavity, a small estradiol: progesterone ratio and it will not rupture during palpation (Figure 3)(28,29). A luteinized follicle cyst might appear after treatment of follicle cyst with GnRH injections. Ovulation and release of the oocyte from the preovulatory follicle are paramount events that open the possibility for pregnancy. After these events, the next expected ovarian structure to develop is the CL.

Corpus luteum development and regression The CL is a transient endocrine ovary gland that regulates the length of the estrous cycle and produces progesterone to create a suitable uterine environment for pregnancy. The CL originates from the transformation of granulosa and theca follicular cells into large and small luteal cells triggered by the preovulatory LH peak(30). The tracking of the growth of the CL during the estrous cycle might begin at 12 to 24 h after ovulation (Figure 4)(31). The CL reaches its maximum size between days nine and 10 of the estrous cycle(32,33). It appears as a semi-circle solid shape structure or with a central cavity (Figure 5). The cavity is filled with a serous transudate or blood(34). The incidence of CL with cavities might reach up to 79%(31). The ovulation of larger follicle predisposes to cavities formation(35), without compromising progesterone production or pregnancy rate in Holstein cattle(35,36). 457


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Figure 4: Growth of the corpus luteum (CL) after ovulation in Holstein dairy cattle

Pictures were taken at approximately every 24 h from day before to 7 d after ovulation. a) preovulatory follicle (PF). b) early CL after ovulation of the PF, notice the presences of luteal cavity (*), the CL and the ovary stroma (OS) are almost isoechogenic until 2 d after ovulation (b-d), but the CL becomes darker (hypoechogenic) than the OS as it ages (c-i). e) the CL is well differentiated from the OS at 3 d after ovulation. f-i) pictures depict a growing CL, the luteal cavity is clearly identified at this stage (4-7 d after ovulation). i) the CL has almost triple its area (+) since ovulation day (b). Pictures were taken using a 7.5 MHz probe.

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Figure 5: Pictures depicting different corpus luteum (CL) shapes at nine days after estrus in Holstein dairy cattle

a, c, d and e) CL with luteal cavity (*) of different sizes. b and f) CL without luteal cavity. OS= ovary stroma; += area of the corpus luteum. Pictures were taken using a 7.5 MHz probe.

The measurement of the CL after insemination is relevant because blood progesterone concentration at mid-luteal phase depends on its size(37) and a positive association between progesterone concentrations and area of the growing CL has been reported(38). In addition, dairy cattle with good genetic merit for fertility traits have greater blood progesterone concentrations and bigger CL than those with poor genetic merit(39,40). The CL grows faster in pregnant than in non-pregnant cows from day six to nine of the estrous cycle(41), a reason to explain the difference in CL growth rate is unknown. However, a faster growth rate might be associated with a healthier corpus luteum. The analysis of CL ultrasound images has been performed to predict the stage of the estrous cycle and its functional status (growing or regressing). During the diestrus, the CL is darker, and its echotexture is more homogenous than during the metestrus and proestrus(34). However, the functional assessment of the CL is difficult to determine by ultrasound images(33,42). Corpora lutea at diestrus stage are easily identified, but to differentiate between those in metestrus (growing CL) and proestrus (regressing CL) is hard because of its morphological similitudes (Figures 4 and 7). In addition, when the area of the CL is between 1.3 and 3.2 cm2, it also difficult to determine if functional CL exists(43). Therefore, to establish the functional status of the CL, it is necessary to perform multiple progesterone concentration analysis, to review the animal reproductive record or to carry on more than one CL measurement by ultrasound at least 2 d apart. In addition, the assessment of luteal blood flow by doppler ultrasound can be used to stablish corpus luteum functionality(44). 459


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The CL undergoes regression if pregnancy is not established. Regression of the CL is under the uterine prostaglandin (F2α) command. A loss of progesterone synthesis capacity and reduction in the size of the CL is observed during the stage of regression (Figure 6). The CL begins to shrink after d 14 of the estrous cycle(33) or 3.2 d before the onset of estrus in nonpregnant cows(32), but becomes sensitive to the luteolytic effects of prostaglandins at d 5 of the estrous cycle(45). Prostaglandin reduces the size of the CL (23 to 47 %) within one to four days(46) and decreases blood concentration of progesterone within 4 h post-injection by ceasing steroidogenic enzymes activity(47) and by inducing luteal cell death(48). In addition, the CL is colonized by immune cells(49) that remove dead cells from the ovarian stroma during regression(48), contributing to CL reduction in size at this stage. After regression of the CL, a preovulatory follicle and estrus will appear, remaining only trace of the former CL on the ovary (Figure 7). However, if pregnancy is established, uterine prostaglandin (F2α) synthesis is blocked. Therefore, regression does not occur and the CL extends its life span until the end of the next reproductive event (pregnancy). Figure 6: Pictures depicting corpus luteum (CL) regression after prostaglandin injection in Holstein dairy cattle

Pictures were taken at approximately 12 h interval after prostaglandin injection until estrus detection using a 7.5 MHz probe. a) CL just before prostaglandin injection. b) CL 10 h after prostaglandin injection, notice the reduction in its size (+). c) CL showing 39 % reduction in its size 25 h after prostaglandin injection. d) CL showing 52 % reduction in its size 34 h after prostaglandin injection. e) picture depicting the regressed CL 48 h after prostaglandin injection, notice that the CL and the ovary stroma (OS) are almost isoechogenic. The picture was captured a few hours after the cow was detected in estrus, the preovulatory follicle was located in the opposite ovary and it is not shown.

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Figure 7: Remains of the corpus luteum at estrus in Holstein dairy cattle

An ovary and regressed corpus luteum (CL) are encircled with + and x. The pre-ovulatory follicle (PF) is the black circle in the top center of the ovary. OS= ovary stroma. Pictures were taken using a 7.5 MHz probe.

Pregnancy diagnoses A land-mark in the pathway to pregnancy is the production of interferon tau by the bovine conceptus, which prevents CL regression induced by prostaglandin F2α and promotes pregnancy recognition(50). The presence of the bovine embryonic vesicle, CL and uterine fluid are indications of pregnancy (51), but the gold standard to diagnose a pregnancy is by the observation of an embryo with a heartbeat, which is rapidly detectable at 30 d after artificial insemination (Figure 8)(52). The pregnancy diagnoses should be performed with care to avoid physical damage to the embryo. The entire uterus and uterine horns should be revised to give an accurate diagnosis. A common mistake is to diagnose a cow as pregnant only by the visualization of uterine fluid, which is also observed in cows during the estrous stage.

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Figure 8: Pictures depicting embryo development in Holstein dairy cattle from 34 to 55 d of gestation

White arrow is pointing at days of gestation. a) 34-d old embryo. b) 41-d old embryo. c) 48-d old embryo. e) 55-d old embryo. *= embryo; A= amnion; AF= allantoic fluid; += size of the embryo; H= head; L= limbs. Pictures were taken using a 7.5 MHz probe. Pictures correspond to the same embryo.

The primary goal of early pregnancy diagnosis (28-30 d after artificial insemination) is to differentiate between pregnant and nonpregnant cows as soon as possible after artificial insemination. After diagnosis, the nonpregnant cows are prepared to rebreed, while the risk of pregnancy loss during the first 60 d of gestation obligates to scheduled pregnant cows to confirmatory diagnosis (40-60 d after artificial insemination) by ultrasound or hand palpation(53,54). Causes of pregnancy loss during this period of time include the selection to increase milk production, impaired uterine environment, poor oocyte and embryo quality(55). The pregnancy diagnosis is followed by two optional events, which are fetus age estimation and sexing. Modern dairy farms keep accurate records of artificial insemination dates, avoiding the need for fetal age estimation. However, the measurement of the crown-rump, head length, trunk diameter and placentoma size might be carried out to estimate the age of the fetus(56,57). The gender of the fetus is determined between 55 to 111 d of gestation(58). However, fetal sexing is not a common practice in dairy farms, probably because ultrasound services are mostly required for early pregnancy diagnosis. In addition, artificial insemination of heifers with sexed semen ensures the born of calves with the desired sex in approximately 90 % of the inseminated animals. 462


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Postpartum reproductive events The next reproductive events after pregnancy diagnosis are calving, shedding of fetal membranes, uterine involution, the establishment of cyclical ovarian activity and diagnosis of uterine health before the first insemination post-partum. Even though all mentioned events must be supervised, the establishment of cyclical ovarian activity and diagnosis of uterine health before the first insemination might be the most relevant for ultrasound practitioners. The achievement of a rapid presentation of ovulation and cyclical estrus after calving are the primary goals for good reproductive management. Ultrasound examination of the reproductive tract, at least every 2 wk after 20 d of lactation, is advisable. The presence of a corpus luteum is the best indicator that ovulation had successfully occurred. After ovulation and CL formation, the observation or data revision from computer software to detect signs of estrus is mandatory. Once cyclical estrus activity has been achieved, the eradication of subclinical uterine infections is necessary to establish a healthy uterine environment capable to host a pregnancy. The clinical uterine infections (metritis and endometritis) are easily detected by observation, smell and palpation of classical signs such as enlarged uterus, vaginal discharge, dullness, and fetid smell. However, signs of subclinical uterine infection (subclinical endometritis) are hidden from simple observation, smell or palpation. A subclinical endometritis is an inflammation of the endometrium, without purulent vaginal discharge and external signs of illness, and it is diagnosed by an increased number of neutrophils in the endometrium using cytology (cytobrush)(59). Subclinical endometritis does not compromise the life of a cow, but fertility might be decreased. A reduced fertilization rate and embryo quality has been reported in superovulated cow with subclinical endometritis(60), which partly explained the low pregnancy rate found in cows suffering the same malady(61). Contrary, others have failed to establish a relationship between subclinical endometritis and poor reproductive performance in cattle(62,63). Origin of the controversy among studies is not well understood because all of them used the same instrumental (cytobrush) to discern between healthy cows and those with subclinical endometritis. The nature of this controversy might be related to the number and intrauterine locations where the samples for cytology were obtained. It is recommended to take at least two samples in two different intrauterine locations, preferably in the horns, to accurate diagnose subclinical endometritis(64). The mentioned studies(61-63) only took one sample and only one of them did it in the uterine horns(61). The cytobrush is one of the most common methods to diagnose subclinical endometritis. However, the uterine lavage, leukocyte esterase test trips and uterine biopsy have been used to successfully diagnose subclinical endometritis in cattle(65,66). The main disadvantage of these methods is that they are time consuming and invasive, requiring a sample of endometrial fluid/cells taken from uterine 463


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horns. Ultrasound is an alternative, non-invasive and reliable tool to diagnose subclinical endometritis, by identification of uterine fluid at 20-47 d in milk(66,67). However, the presences of pus in the uterus might be also considered as indication of subclinical endometritis. The pus is composed by neutrophils and it is a sign of inflammation(68), and it is commonly observed along the uterine horns (Figure 9), normally in such small quantities that it is not observed as vaginal discharge, which comply with the definition of subclinical endometritis(59). Figure 9: Pictures depicting the presence of pus (subclinical endometritis) in the uterus of Holstein dairy cattle at random stages of lactation

White arrows indicating the location of pus (the pus is observed as bright and white spots of different shapes and sizes). White lines are delimiting the uterine horns. Pictures were taken using a 7.5 MHz probe.

Further considerations and areas of opportunity The reason that justified the use of ultrasound must be known before to actually perform any ultrasonographic exploration in the cow. In addition, the safety of the practitioner, the animal and the equipment must be guaranteed at all times during the procedure. It is also advisable to gather as much information as possible about the clinical, nutritional, management, productive and reproductive history of the cows before carrying any physical exploration. The latter will help to make an accurate diagnosis and to provide the most suitable treatment. The selection of experimental units during reproductive research should be done at least 21 d before the study commence. Their reproductive tract must be revised by ultrasonography to ensure that all animals are in the desired condition (healthy: cyclic state, without ovarian cyst and free of uterine infection; unhealthy: with uterine infections or ovarian cysts). 464


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However, if the number of experimental units is limited to perform the research, then there will be at least 21 d to treat cows that require reproductive assistance. The oocyte competence and embryo quality are factors that significantly affect the chances of achieving pregnancy status in a cow after artificial insemination. It will be advantageous to measure specific and predictive characteristics related to those factors on field condition by ultrasonography.

Conclusions The application of ultrasound is mandatory to better understand some of the most relevant reproductive events and to support decision-making during dairy cattle reproductive management. It is important that students and researchers embrace ultrasound as a routine tool for research and field work.

Acknowledgements

The authors thank Dra. Ana Laura Lara Rivera, Dr. Ulises Macias Cruz and Dr. Lorenzo Buenabad Carrasco for their comments to the manuscript.

Conflict of interest statement

The authors declare that they have no conflict of interest. Literature cited: 1. Cabrera VE. Economics of fertility in high-yielding dairy cows on confined TMR systems. Animal 2014;8(S1):211-221. doi:10.1017/S1751731114000512. 2. Kähn W, Leidl W. In: Taverne MAM, Willemse AH. Diagnostic ultrasound and animal reproduction. Dordrecht, Netherlands; 1989:53-65. 3. Rajamahendran R,Ambrose DJ, Burton B. Clinical and research applications of real-time ultrasonography in bovine reproduction: a review. Can Vet J 1994;35(9):563-572. 4. Ribadu AY, Nakao T. Bovine reproductive ultrasonography: a review. J Reprod Dev; 1994;45:13-28. https://doi.org/10.1262/jrd.45.13.

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26. Kaneko H, Todoroki J, Noguchi J, Kikuchi K, Mizoshita K, Kubota C. Yamakuchi H. Perturbation of estradiol-feedback control of luteinizing hormone secretion by immunoneutralization induces development of follicular cysts in cattle. Biol Reprod 2002;67(6):1840-1845. https://doi.org/10.1095/biolreprod.102.007591. 27. Gümen A, Sartori R, Costa FM, Wiltbank MC. A GnRH/LH surge without subsequent progesterone exposure can induce development of follicular cysts. J Dairy Sci 2002;85(1):43-50. https://doi.org/10.3168/jds.S0022-0302(02)74051-4. 28. Douthwaite R, Dobson H. Comparison of different methods of diagnosis cyst ovarian disease in cattle and an assessment of its treatment with a progesterone-releasing intravaginal device. Vet Rec 2000;147(13):355-359. http://dx.doi.org/10.1136/vr.147.13.355. 29. Ambrose DJ, Schmitt EJP, Lopes FL, Mattos RC, Thatcher WW. Ovarian and endocrine responses associated with the treatment of cystic ovarian follicles in dairy cows with gonadotropin releasing hormone and prostaglandin F2α, with or without exogenous progesterone. Can Vet J 2004;45(11):931-937. 30. Abedel-Majed MA, Romereim SM, Davis JS, Cupp AS. Perturbations in lineage specification of granulosa and theca cells may alter corpus luteum formation and function. Front Endocrinol 2019;10:1-10. 10.3389/fendo.2019.00832. 31. Kastelic JP, Pierson RA, Ginther OJ. Ultrasonic morphology of corpora lutea and central luteal cavities during the estrous cycle and early pregnancy in heifers. Theriogenology 1990;34(3): 487-498. https://doi.org/10.1016/0093-691X(90)90006-F. 32. Taylor C, Rajamahendran R. Follicular dynamics and corpus luteum growth and function in pregnant versus nonpregnant cows. J Dairy Sci 1991;74(1):115-123. https://doi.org/10.3168/jds.S0022-0302(91)78151-4. 33. Siqueira LG, Torres CA, Amorim LS, Souza ED, Camargo LS, Fernandes CA, Viana JHM. Interrelationships among morphology, echotexture, and function of the bovine corpus luteum during the estrous cycle. Anim Reprod Sci 2009;115(1-4):18-28. https://doi.org/10.1016/j.anireprosci.2008.11.009. 34. Singh J, Pierson RA, Adams GP. Ultrasound image attributes of the bovine corpus luteum: structural and functional correlates. J Reprod Fertil 1997;109(1):35-44. 10.1530/jrf.0.1090035. 35. Perez-Marin C. Formation of corpora lutea and central luteal cavities and their relationship with plasma progesterone levels and other metabolic parameters in dairy cattle. Reprod Domest Anim 2009;44:384-389. 10.1111/j.1439-0531.2007.01021.x

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36. Kito S, Okuda K, Miyazawa K, Sato K. Study on the appearance of the cavity in the corpus luteum of cows by using ultrasonic scanning. Theriogenology 1986;25(2):325333. https://doi.org/10.1016/0093-691X(86)90068-3. 37. Lüttgenau J, Ulbrich SE, Beindorff N, Honnens A, Herzog K, Bollwein H. Plasma progesterone concentrations in the mid-luteal phase are dependent on luteal size, but independent of luteal blood flow and gene expression in lactating dairy cow. Anim Reprod Sci 2011(1-4);125:20-29. https://doi.org/10.1016/j.anireprosci.2011.02.002. 38. Rizos D, Scully S, Kelly AK, Ealy AD, Moros R, Duffy P, Al-Naib A, Forde N, Lonergan P. Effects of human chorionic gonadotrophin administration on day 5 after oestrus on corpus luteum characteristics, circulating progesterone and conceptus elongation in cattle. Reprod Fertil Dev 2012;24(3):472-481. 10.1071/RD11139. 39. Cummins SB, Lonergan P, Evans AC, Butler ST. Genetic merit of fertility traits in Holstein cows: II. Ovarian follicular and corpus luteum dynamics, reproductive hormones, and estrus behavior. J Dairy Sci 2012;95(7):3698-36710. https://doi.org/10.3168/jds.2011-4976. 40. Moore SG, Scully S, Browne JA, Fair T, Butler ST. Genetic merit for fertility traits in Holstein cows: V. Factors affecting circulating progesterone concentrations. J Dairy Sci 2014;97(2-3):5543-5557. https://doi.org/10.3168/jds.2014-8133. 41. Gómez-Seco C, Alegre B, Martínez-Pastor F, Prieto JG, González-Montaña JR, Alonso ME, Domínguez JC. Evolution of the corpus luteum volume determined ultrasonographically and its relation to the plasma progesterone concentration after artificial insemination in pregnant and non-pregnant dairy cows. Vet Res Commun 2017;41(3):183-188. 10.1007/s11259-017-9685-x. 42. Battocchio M, Gabai G, Mollo A, Veronesi MC, Soldano F, Bono G. Cairoli F. Agreement between ultrasonographic classification of the CL and plasma progesterone concentration in dairy cows. Theriogenology 1999;51(6):1059-1069. https://doi.org/10.1016/S0093-691X(99)80011-9. 43. Kaneko K, Takagi N. Accurate ultrasonographic prediction of progesterone concentrations greater than 1 ng/ml in Holstein lactating dairy cows. Reprod Domest Anim.2014;49(6):985-988. https://doi.org/10.1017/S0022029908003610. 44. Hassan M, Arshad U, Bilal M, Sattar A, Avais M, Bollwein H, Ahmad N. Luteal blood flow measured by Doppler ultrasonography during the first three weeks after artificial insemination in pregnant and non-pregnant Bos indicus dairy cows. J Reprod Dev 2019;65(1):29-36. 10.1262/jrd.2018-084.

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https://doi.org/10.22319/rmcp.v13i2.5922 Review

Demi-embryo reconstitution, a factor to consider for the success of embryo bisection. Review

Alfredo Lorenzo-Torres a Raymundo Rangel-Santos a* Agustín Ruíz-Flores a Demetrio Alonso Ambríz-García b

a

Universidad Autónoma Chapingo, Posgrado en Producción Animal. Carretera MéxicoTexcoco, Km 38.5, 56230. Texcoco, Estado de México, México. b

Universidad Autónoma Metropolitana, Departamento de Biología de la Reproducción, Ciudad de México, México.

* Corresponding author: rangelsr@correo.chapingo.mx

Abstract: For many years it has been sought to increase the reproductive efficiency of livestock using biotechnologies such as embryo bisection. However, despite its potential in livestock, its level of adoption is limited. The present work reviews the importance of demi-embryo reconstitution, after bisection, and the main factors that limit its success in livestock. It is possible to increase its level of adoption if it is possible to increase the efficiency currently obtained with this technique, this can be achieved by making a more precise selection of the embryos subjected to bisection. Embryo quality is one of the most important factors related to the potential to reconstitute into viable demi-embryos after bisection, which can be used with greater reliability in embryo transfer programs. Key words: Embryo bisection, Demi-embryo reconstitution, Embryo development.

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Received: 07/01/2021 Accepted: 15/07/2021

Introduction Embryo bisection is a reproductive biotechnology that allows obtaining identical demiembryos, to be used in research(1) or the livestock industry(2). The aim of embryo bisection is to increase the number of semi-embryos available for transfer and therefore, the offspring of genetically superior animals(1,3,4). This technique can be applied to embryos developed in the morula or blastocyst stage and consists of obtaining two similar halves by mechanical bisection(5,6,7). Morula bisection can be carried out in any position of the embryo, due to its symmetric morphology(8). In the case of blastocysts is important the symmetrical orientation of the embryo to obtain a proportional distribution of the inner cell mass (ICM) and trophectoderm (TE) in the resulting demi-embryos(9). Embryo bisection is performed in embryos of several species(10,11,12), in order to increase embryo availability(12), pregnancy rate(13), and the number of offspring(5,14,15). However, there are studies where the survival of demi-embryos was low(16,17), even lower compared to the use of whole embryos(18). This could be associated with the fact that embryo bisection is an invasive technique(6) and the procedure causes cellular damage(19). Therefore, the success of the technique could be influenced by factors associated with the original embryo(20,21,22) and its ability to reconstitute itself in the resulting demi-embryos(23). The objective of this review is to highlight the importance of demi-embryo reconstitution in embryo bisection programs, as well as to discuss the main factors that influence its success.

Importance of embryo bisection in livestock Embryo bisection has been carried out in different livestock species of interest, such as rabbits(19), sheep(24), bovines(12), goats(10), equines(25), swine(26), and even in humans(22). Despite it is an invasive technique, it is practical and it does not require cell reprogramming like cloning(6). Embryo bisection in livestock allows to produce identical twins for experimental use(14), reducing the number of animals needed per treatment for comparison tests(27) or to increase the availability of transferred embryos(28). In addition, obtaining identical twins facilitates the evaluation of sires or maternal trait tests(29) and, lastly, to maintain desirable characteristics in cattle(3). 474


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Embryo bisection has allowed to increase pregnancy rate(29) and the number of offspring(13), compared to the transfer of whole embryos(13,24). Most authors have reported a higher number of semi-embryos available for transfer in relation to the number of bisected embryos, thus increasing the efficiency in the number of offspring (Table 1). However, there is a great variation (75 to 118 %, efficiency), which could be mainly associated with factors related to the original embryo. In ewes, the pregnancy rate was 64 % when they received airs of demiembryos, obtaining 118 % efficiency in offspring(14). Likewise, there was a higher percentage of embryo survival when transferring two demi-embryos per recipient ewe (101 %, 710/705), compared to the transfer of two whole embryos (62 %, 771/1252) considering the number of original embryos(24). Further, 30 % more lambs were born after the transfer of pairs of demiembryos, compared with whole embryos (85 vs 55 %, P<0.05)(30). Table 1: Efficiency of embryo bisection in the production of offspring in relation to the number of bisected embryos Number of Bisected Efficiency Species offspring Reference embryos (%) born Bovine

36

27

75

13

Bovine

50

61*

105

15

Bovine

11

12*

109

5

Sheep

40

34

85

30

Sheep

24

21**

88

16

Sheep

705

710*

101

24

Sheep

16

17

106

31

Sheep

39

46

118

14

* Number of fetuses diagnosed by ultrasound between 30 and 80 d of gestation or ** by sacrifice surgery after slaughter. Efficiency (%)= Number of offspring born / embryos bisected.

On the other hand, in some studies, low efficiency was obtained with embryo bisection (16,32,33) . It has been reported that a lower percentage of lambs were born after the transfer of bisected sheep embryos, compared to whole embryos (27 vs 52 %, P<0.05)(34). However, in beef cattle, despite the problems being associated with twin gestation(4), implementing embryo bisection represents an economic benefit once the number of offspring is increased(2,17,35). Therefore, embryo bisection can be implemented in embryo transfer programs(2,24).

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Embryonic reconstitution after bisection Embryo reconstitution is an indicator of the ability of demi-embryos to become offspring after their transfer to a recipient female(18,29). In adult tissues, stem cells are responsible for repairing injuries and regenerating tissues(23,36) due to aging or diseases(37). In the case of embryos, something similar happens, through the replacement of specialized cells that have been lost due to some alteration(23). Embryos are capable of repairing their injuries, adapting to environmental conditions to survive after their reconstitution(23). Embryonic cells in early stages can adapt both in mitotic rate and differentiation process(38), due to the plasticity they present(39,40). In addition, it has been proven that embryonic cell conglomerates in the early stages of development can become living organisms through cell reorganization, such as the case of blastocyst splitting(41). Thus, a group of cells has properties that exceed the potency of any of the individual cells within the group for cell reconstitution, which could be a joint effect(23). The extruded cells or even cell debris observed after embryo bisection could contain enough viable cells to proliferate and reorganize, originating another functional embryo(8). At the time of embryo bisection, the resulting halves can be cultured in vitro for 2 to 48 h(5,26). In each demi-embryo, the ICM is reorganized and the reconstitution of the blastocele begins immediately(5,29,42). The union of the edges of trophoblastic cells during bisection is responsible for the trophoblast's ability to reconstitute itself(5) since this group of cells secretes fluid into the blastocele, a process regulated by genes(43,44). This allows the spherical formation of demiembryos(42), being the trophoblastic cells important for embryo implantation(45). Likewise, an embryo, even if it has lost half of its cells, it is still an organism and a characteristic of organisms is to repair their lesions, regenerating them to continue their development(23). In the laboratory, it was bisected sheep expanded blastocysts produced in vitro with a microblade (Figure 1a), using the procedure called scratched bottom dish(46) (Figure 1b) and it was observed a completely spherical reconstitution (70 % the size of the original embryo) 12 h after in vitro culture (Figure 1c).

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Figure 1: Embryo bisection process and reconstitution of the demi-embryos after 12 h of in vitro culture

a) Expanded blastocyst oriented symmetrically with respect to the bisecting microblade, b) resulting demiembryos, and c) reconstituted demi-embryos. ×200.

Several authors have reported a reconstitution percentage ranging from 90 to 178 % (Table 2). Table 2: Reconstitution efficiency of bovine demi-embryos after in vitro culture for 2-48 h post-bisection Bisected Number of demiReconstitution* Reference embryos embryos (%) 29 21 19 90 5 11 16 145 12 176 268 152 47 19 30 158 28 230 408 178 *Reconstitution (%) = Number of demi-embryos / bisected embryos. The evaluation of embryo reconstitution could allow the selection of viable demi-embryos and it can be a useful tool in embryo transfer programs(18).

Factors that affect demi-embryo reconstitution Embryo bisection technique

Embryo bisection is a technique that allows the production of identical twins in embryo transfer programs(5). In the 80s, the technique required up to six embryo manipulation and

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bisection instruments(5). However, over time, different methodologies have been developed to simplify the technique(1), since the procedure required up to 15 min to bisect an embryo(48). In addition, the use of cutting instruments, such as microblade(5,25,49) or glass needle(50,51,52), have been extensively studied, with the objective of minimizing cellular damage at the time of cutting(53). Thus, the success of the technique depends on the minimum damage produced to the embryos(19), since the procedure generates 10 to 13% of cell loss(47,54). On this regard, the microblade embryo bisection method has proven to be practical and with an application under field conditions(1). The implementation of this technique has been simplified by means of vertical pressure at the time of embryo bisection, using a microblade adapted to a single micromanipulator, without the use of an embryo holding micropipette(12,15,34). In the laboratory, has been observed that the use of the scratched bottom dish technique(46) with 50 µL of a commercial bisecting medium, facilitates embryonic fixation and prevents cell adhesion to bisecting and culture materials. This makes it possible to bisect groups of five embryos in approximately 3 min, making the application of this biotechnology more practical and without subjecting the embryos to prolonged stress. On the other hand, there is evidence showing that the technician who performs the embryo bisection influences the productive response. In sheep, it has been evaluated the effect of the technician at the time of bisection, finding a significant difference between the two technicians in pregnancy rate (66 vs 75 %, P<0.05) and demi-embryo survival (51 vs 44 %, P<0.01)(55). Thus, proper training of the technician should be considered before implementing embryonic bisection.

Developmental stage

The stage of embryonic development, morula, early blastocyst, or expanded blastocyst at the time of bisection, is one of the most important factors that affect the pregnancy rate of transferred embryos(8). After the bisection of mouse embryos, it was found that, in the morula stage, the demi-embryos are reconstituted in a lower percentage than in the blastocyst stage (74 vs 90 %, P=0.001) after in vitro culture for 24 h(45). In cattle, no significant differences were reported when transferring demi-embryos from morulae, early blastocysts, and expanded blastocysts, on pregnancy rate (51-65 %, P>0.1)(53). In another study, there was a lower pregnancy rate using bisected morulae (7/44, 16 %) compared to early blastocysts (58/96, 60 %), P <0.01(8). Likewise, it has been reported a higher percentage of viable fetuses after blastocyst bisection compared to morulae, 91 (10/11) vs 30 % (3/10), P<0.05, at d 70 of gestation(47). Finally, in sheep, six pairs of identical twins were obtained from blastocyst bisection, without success in morula bisection (P<0.05)(16).

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In a practical way, it appears that bisecting morulae is easier due to the morphological symmetry they present, however, in the laboratory, it was observed that bisecting expanded blastocysts and even hatched blastocysts was easier once the ICM and TE were clearly identified. Furthermore, some authors have reported a higher pregnancy rate using blastocysts compared to morulae (Table 3), perhaps because they are more tolerant to manipulation and are less affected by the loss of the zona pellucida(8,54). This could be due to the fact that embryogenesis is strictly regulated in time(22) and the more developed the embryos are, the more tolerant they are. Table 3: Effect of the developmental stage of the whole embryo on the pregnancy rate of transferred semi-embryos Developmental stage, % (n) Species Reference Expanded Morula Blastocyst blastocyst 10 Caprine 0 (5) 33 (9) 55 (11)* 16 Ovine 60 (20) 88 (24) 8 Bovine 48 (162) 60 (96) 54 (28) 53 Bovine 51 (71) 64 (61) 58 (12) 12 Bovine 39 (139) 36 (33) 30 (10) %= Pregnancy rate; n= Number of recipient females; *Hatched blastocyst.

Embryo quality

There is wide evidence for the use of excellent quality embryos for bisection purposes(12,40,56,57). The embryos selected for bisection must meet certain morphological criteria, from which the success of demi-embryo reconstitution will depend(28) and, consequently, the pregnancy rate(12,51). The quality of the embryos must be excellent or good, depending on the morphological criteria(58) because when they are of low quality (fair and bad) are more vulnerable to the bisection process(12,47). In bovines, when bisecting morulae, a higher percentage of survival was obtained in the group of excellent and good quality, compared to morulae of regular and poor quality, 167 (20/12) vs 75% (9/12), P<0.001(47). On the other hand, 42 % pregnancy was found in cows after thawing and transferring excellent quality demi-embryos, while when transferring demi-embryos from low-quality embryos, no female became pregnant(51). Likewise, it has been reported a higher percentage of development in pairs of demi-embryos, when bisecting bovine embryos of excellent quality, compared to those of good quality (76 vs 40 %, P<0.05)(12). Therefore, evaluation of embryo quality subjected to bisection should be

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considered to obtain positive results. However, the morphological evaluation of embryos subjected to bisection is a subjective aspect, based on the experience of the researcher. In the experience in manipulating sheep embryos of this research team, was found a higher percentage of demi-embryo reconstitution (145 %, approximately), when bisecting embryos of excellent quality and with a diameter greater than 230 µm. In the resulting demi-embryos, after 12 h of in vitro culture, was found that when they were reconstituted, they had an average diameter of 176 ± 10.03 µm (Figure 2), similar to that reported in quality two porcine demi-embryos (161.6 ± 25.7 µm)(26), but higher than that reported in human demi-embryos (121 µm)(22). Therefore, the embryonic diameter has been proposed as an indicator of quality(25,59) since the size of the embryo is important in maternal recognition(60). On the other hand, the size of the embryo is also associated with the number of cells(61) and, consequently, with the embryo quality. In poor quality embryos, there is a deficiency in the rate of cell division, mitosis, and consequently in the number of cells(47). Thus, the size of the embryo is proportional to the number of cells used for its reconstitution(22). There is a 50 % recovery of cells from the original embryos in the resulting demi-embryos depending on the quality and uniformity of the bisection process(26,28). This suggests that bisecting bigger size embryos will produce semi-embryos with more ICM and TE cells, thus increasing their survival. In the laboratory, was obtained an average of 68 ± 11.3 cells in reconstituted demiembryos, after 12 h of in vitro culture (Figure 3), from embryos with 122 ± 6.6 cells. In this sense, active cell proliferation can be a criterion of embryonic quality(62). Based on the above, the diameter of the embryo could be an objective criterion to select embryos for bisection, in order to achieve the greatest possible success in demi-embryos reconstitution. Figure 2. In vitro reconstitution of demi-embryos after 12 h of culture. ×200

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Figure 3. Cell staining (Hoechst) of in vitro demi-embryos after 12 h of culture. ×200

In vitro or in vivo produced embryos

There are differences between embryos produced in vitro or in vivo, both in morphology and in molecular components(63), where in vivo produced embryos are of better quality. Nevertheless, a high percentage of in vitro survival of ovine demi-embryos has been reported after bisection (80-85 %), and 33 % pregnancy after transferring pairs of demi-embryos to recipient ewes (5/15)(34). On the other hand, for embryos produced in vivo, some authors reported greater pregnancy rates. In bisected bovine embryos produced in vivo and cultured the demi-embryos in vitro, a high percentage of reconstitution was reported (47/60 , 78.3 %)(15). In bovines, there was 53 % (18/34) pregnancy when transferring embryos produced in vivo, bisected, and cultured in vitro(18). Thus, the efficiency of embryo bisection in relation to the origin of the embryo seems to be lower in embryos from in vitro conditions. This could be due to the low quality and efficiency of in vitro production(63). Therefore, it is necessary to improve efficiency in both embryo production procedures, once the two of them are focused on improving productivity in livestock production(64).

Effect of breed and age of embryo donors for bisection

There are other little-studied factors that could affect the embryo's fate under the bisection process. In sheep, the effect of the breed was evaluated, without finding significant differences in pregnancy rate and survival of demi-embryos between embryos from Gotland

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and Finnish Texel (69 vs 50 % and 42 vs 26 %, respectively, P>0.05) and between Danish Texel and Finnish Texel breeds (74 vs 74 % and 50 vs 50 %, respectively, P>0.05)(55). In addition, the effect of the donor age has been reported, without finding differences in pregnancy rate between embryos generated from adult females (24 months of age) and young (approximately 10 mo of age) (74 vs 74 %, P>0.05), however, demi-embryo survival (51 vs 47, P<0.05) and percentage of identical twins was higher in adult ewes than in young (38 vs 27, P<0.01)(55). This could be related to a lower survival capacity of whole embryos from young ewes(65,66,67), which is confirmed after transferring demi-embryos(55).

Conclusions Demi-embryos reconstitution is a key factor for the success of embryo bisection and the highest efficiency is obtained by selecting excellent quality embryos regardless of their developmental stage. The results of the literature show the potential of the bisection technique; therefore, its application should be considered to improve the efficiency of embryo transfer programs. Literature cited: 1. Godke RA, Sansinena M, Youngs CR. Assisted reproductive technologies and embryo culture methods for farm animals. In: Pinkert CA editor. Transgenic Animal Technology: A Laboratory handbook. 3rd ed. Amsterdam: Elsevier; 2014:581-638. 2.

Praharani L. Factors affecting twinning and the impacts of twinning in cattle. WARTAZOA Indones Bull Anim Vet Sci 2019;29(1):13-24.

3.

Yang X, Tian XC, Kubota C, Page R, Xu J, Cibelli J, et al. Risk assessment of meat and milk from cloned animals. Nat Biotechnol 2007;25(1):77-83.

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Wakchaure R, Ganguly S. Twinning in cattle: A Review. ARC J Gynecol Obstet 2016;1(4):1-3.

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Ozil JP. Production of identical twins by bisection of blastocysts in the cow. Reproduction 1983;69(2):463-468.

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Escriba MJ, Valbuena D, Remohı́ J, Pellicer A, Simon C. New techniques on embryo manipulation. J Reprod Immunol 2002;55(1-2):149-161.

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Tang HH, Tsai YC, Kuo CT. Embryo splitting can increase the quantity but not the quality of blastocysts. Taiwan J Obstet Gynecol 2012;51(2):236-239.

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8.

Williams TJ, Elsden RP, Seidel Jr GE. Pregnancy rates with bisected bovine embryos. Theriogenology 1984;22(5):521-531.

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Illmensee K, Levanduski M. Embryo splitting. Middle East Fertil Soc J 2010;15(2):5763.

10. Tsunoda Y, Tokunaga T, Sugie T, Katsumata M. Production of monozygotic twins following the transfer of bisected embryos in the goats. Theriogenology 1985;24(3):337343. 11. Ramón UJ, Meza VV, Deneb CP, Domínguez RA, Quintal FJ. Bisection and embryo transfer in hair sheep. Biotechnology Summit 2012, Mérida, Yucatán, México 2012;1221(3):138-142. 12. Hashiyada Y. The contribution of efficient production of monozygotic twins to beef cattle breeding. J Reprod Develop 2017;63(6):527-538. 13. Dahlen CR, DiCostanzo A, Spell AR, Lamb GC. Use of embryo transfer seven days after artificial insemination or transferring identical demi-embryos to increase twinning in beef cattle. J Anim Sci 2012;90(13):4823-4832. 14. Chesne P, Colas G, Cognie Y, Guerin Y, Sévellec C. Lamb production using superovulation, embryo bisection, and transfer. Theriogenology 1987;27(5):751-757. 15. Saito S, Niemann H. In vitro and in vivo survival of bovine demi-embryos following simplified bisection and transfer of one or two halves per recipient. J Reprod Develop 1993;39(3):251-258. 16. Shelton JN. Factors affecting viability of fresh and frozen-thawed sheep demi-embryos. Theriogenology 1992;37(3):713-721. 17. Echternkamp SE, Gregory KE. Reproductive, growth, feedlot, and carcass traits of twin vs single births in cattle. J Anim Sci 2002;80:E64-E73. 18. Alvarez RH, Pires RML, Campanha A, Oba E. Short-term culture of bovine bisected embryos. Effects on pregnancy rates, sex ratio and birth weight of calves. B Indústr Anim 2008;65(3):191-196. 19. Skrzyszowska M, Smorag Z, Katska L. Demi-embryo production from hatching of zonadrilled bovine and rabbit blastocysts. Theriogenology 1997;48(4):551-557. 20. Reichenbach HD, Schwartz J, Wolf E, Brem G. Effects of embryo developmental stage, quality and short-term culture on the efficiency of bovine embryo splitting. Theriogenology 1998;1(49):224.

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21. Kawachiya S, Bodri D, Shimada N, Kato K, Takehara Y, Kato O. Blastocyst culture is associated with an elevated incidence of monozygotic twinning after single embryo transfer. Fertil Steril 2011;95(6):2140-2142. 22. Noli L, Ogilvie C, Khalaf Y, Ilic D. Potential of human twin embryos generated by embryo splitting in assisted reproduction and research. Hum Reprod 2017;23(2):156165. 23. Condic ML. Totipotency: what it is and what it is not. Stem Cells Dev 2014;23(8):796812. 24. Vivanco HW, Rangel SR, Lynch P, Rhodes A. Large scale commercial application of bisection of sheep embryos. Theriogenology 1991;35(1):292. 25. McKinnon AO, Carnevale EM, Squires EL, Carney NJ, Seidel Jr GE. Bisection of equine embryos. Equine Vet J 1989;21(Suppl 8):129-133. 26. Reichelt B, Niemann H. Generation of identical twin piglets following bisection of embryos at the morula and blastocyst stage. Reproduction 1994;100(1):163-172. 27. Yang X, Anderson GB. Micromanipulation of mammalian embryos: principles, progress and future possibilities. Theriogenology 1992;38(2):315-335. 28. Rho GJ, Johnson WH, Betteridge KJ. Cellular composition and viability of demi-and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro. Theriogenology 1998;50(6):885-895. 29. Baker RD, Shea BF. Commercial splitting of bovine embryos. Theriogenology 1985;23(1):3-12. 30. Vintila I, Benscik I, Pacala N, Corin N, Babusik I, Kulickova L. Embryo splitting- a way to increase the efficiency of embryo-transfer in sheep. Stočarstvo: Časopis za unapređenje stočarstva 1995;49(9-12):349-353. 31. Széll A, Hudson RHH. Factors affecting the survival of bisected sheep embryos in vivo. Theriogenology 1991;36(3):379-387. 32. Shelton JN, Szell A. Survival of sheep demi-embryos in vivo and in vitro. Theriogenology 1988;30(5):855-863. 33. Harkness UF, Crombleholme TM. Twin–twin transfusion syndrome: where do we go from here? Semin Perinato 2005;29(5):296-304.

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34. Morton KM, Rowe AM, Maxwell WC, Evans G. In vitro and in vivo survival of bisected sheep embryos derived from frozen-thawed unsorted, and frozen-thawed sex-sorted and refrozen-thawed ram spermatozoa. Theriogenology 2006;65(7):1333-1345. 35. De Rose EP, Wilton JW. Productivity and profitability of twin births in beef cattle. J Anim Sci 1991;69(8):3085-3093. 36. Kenyon J, Gerson SL. The role of DNA damage repair in aging of adult stem cells. Nucleic Acids Res 2007;35(22):7557-7565. 37. Maynard S, Fang EF, Scheibye-Knudsen M, Croteau DL, Bohr VA. DNA damage, DNA repair, aging, and neurodegeneration. Cold Spring Harb Perspect Med 2015;5(10):a025130. 38. Deuchar EM. Regeneration of amputated limb-buds in early rat embryos. Development 1976;35(2):345-354. 39. Cenariu M, Pall E, Cernea C, Groza I. Evaluation of bovine embryo biopsy techniques according to their ability to preserve embryo viability. J Biomed Biotechnol 2012;2012. 40. De Sousa RV, da Silva Cardoso CR, Butzke G, Dode MAN, Rumpf R, Franco MM. Biopsy of bovine embryos produced in vivo and in vitro does not affect pregnancy rates. Theriogenology 2017;90:25-31. 41. Mitalipov S, Wolf D. Totipotency, pluripotency and nuclear reprogramming. In: Martin U. editor. Engineering of stem cells. Heidelberg, Berlín, Alemania: Springer; 2009;114:185-199. 42. Daniel Jr JC. Some kinetics of blastocyst formation as studied by the process of reconstitution. J Exp Zool 1963;154(2):231-237. 43. Watson AJ, Barcroft LC. Regulation of blastocyst formation. Front Biosci 2001;6:D708D730. 44. Watson AJ, Natale DR, Barcroft LC. Molecular regulation of blastocyst formation. Anim Reprod Sci 2004;82:583-592. 45. Wang ZJ, Trounson A, Dziadek M. Developmental capacity of mechanically bisected mouse morulae and blastocysts. Reprod Fertil Dev 1990;2(6):683-691. 46. Bredbacka P. Biopsy of morulae and blastocysts. Reprod Domest Anim 1991;26(2):8284.

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47. McEvoy TG, Sreenan JM. Effect of embryo quality and stage of development on the survival of zona pellucida-free cattle demi-embryos. Theriogenology 1990;33(6):12451253. 48. Yang X, Foote RH. Production of identical twin rabbits by micromanipulation of embryos. Biol Reprod 1987;37(4):1007-1014. 49. Lopes RFF, Forell F, Oliveira ATD, Rodrigues JL. Splitting and biopsy for bovine embryo sexing under field conditions. Theriogenology 2001;56(9):1383-1392. 50. Willadsen SM, Godke RA. A simple procedure for the production of identical sheep twins. Vet Rec 1984;114(10):240-243. 51. Niemann H, Brem G, Sacher B, Smidt D, Kräusslich H. An approach to successful freezing of demi-embryos derived from day-7 bovine embryos. Theriogenology 1986;25(4):519-524. 52. Seike N, Saeki K, Utaka K, Sakai M, Takakura R, Nagao Y, et al. Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae. Theriogenology 1989;32(2):211-220. 53. Kippax IS, Christie WB, Rowan TG. Effects of method of splitting, stage of development and presence or absence of zone pellucida on foetal survival in commercial bovine embryo transfer of bisected embryos. Theriogenology 1991;35(1):25-35. 54. Skrzyszowska M, Smorag Z. Cell loss in bisected mouse, sheep and cow embryos. Theriogenology 1989;32(1):115-122. 55. Rangel-Santos R. Investigations into procedures for the implementation of a multiple ovulation and embryo transfer scheme using ewe lambs [PhD thesis]. Wellington, New Zealand: Massey University; 1991. 56. Shea BF. Determining the sex of bovine embryos using polymerase chain reaction results: a six-year retrospective study. Theriogenology 1999;51(4):841-854. 57. Lopatarova M, Cech S, Krontorad P, Holy L, Hlavicova J, Dolezel R. Sex determination in bisected bovine embryos and conception rate after the transfer of female demiembryos. Vet Med 2008;53(11):595-603. 58. Stringfellow DA, Seidel G. Manual of the International Embryo Transfer Society. 2nd. IETS 1990;19. 59. Mori M, Otoi T, Suzuki T. Correlation between the cell number and diameter in bovine embryos produced in vitro. Reprod Domest Anim 2002;37(3):181-184.

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60. Goff AK. Embryonic signals and survival. Reprod Domest Anim 2002;37(3):133-139. 61. O'Hara L, Forde N, Kelly AK, Lonergan P. Effect of bovine blastocyst size at embryo transfer on Day 7 on conceptus length on Day 14: can supplementary progesterone rescue small embryos? Theriogenology 2014;81(8):1123-1128. 62. Makarevich AV, Markkula M. Apoptosis and cell proliferation potential of bovine embryos stimulated with insulin-like growth factor I during in vitro maturation and culture. Biol Reprod 2002;66(2):386-392. 63. Camargo LSDA, Viana JHM, Sá WFD, Ferreira ADM, Ramos ADA, Vale Filho VR. Factors influencing in vitro embryo production. Anim Reprod 2006;3(1):19-28. 64. Paramio MT. In vivo and in vitro embryo production in goats. Small Ruminant Res 2010;89(2-3):144-148. 65. Quirke JF, Hanrahan JP. Comparison of the survival in the uteri of adult ewes of cleaved ova from adult ewes and ewe lambs. Reproduction 1977;51(2):487-489. 66. McMillan WH, McDonald MF. Survival of fertilized ova from ewe lambs and adult ewes in the uteri of ewe lambs. Anim Reprod Sci 1985;8(3):235-240. 67. Morton KM. Developmental capabilities of embryos produced in vitro from prepubertal lamb oocytes. Reprod Domest Anim 2008;43(Suppl 2):137-143.

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https://doi.org/10.22319/rmcp.v13i2.5832 Review

Heat stress in dairy cattle with emphasis on milk production and feed and water intake habits. Review

Abelardo Correa-Calderón a Leonel Avendaño-Reyes a M. Ángeles López-Baca a Ulises Macías-Cruz a*

a

Universidad Autónoma de Baja California. Instituto de Ciencias Agrícolas, 21751, Valle de Mexicali, B.C. México.

*Corresponding author: ulisesmacias1988@hotmail.com, umacias@uabc.edu.mx

Abstract: The negative impact of heat stress (HS) in dairy cattle results in considerable economic losses at world level, as it reduces the milk production, reproductive efficiency, and productive life in the cows. In addition, the continuous genetic improvement results in highly productive cows, which are, however, less tolerant to HS because they produce greater metabolic heat. This, together with global warming, will turn HS into a hard-to-control challenge for the daily industry. In response dependent on the degree of HS, the dairy cattle carry out a series of physiological, metabolic and behavioral adjustments as thermoregulatory mechanisms for removing excess body heat and reducing the endogenous production of body heat, in order to maintain the normothermia. However, fertility and milk secretion decrease as a direct effect of hyperthermia and an indirect effect of lower dietary nutrient intake. Food and water intake are closely associated to the reduction of the productivity of dairy cattle exposed to HS. Notably, the impact of HS on productivity of dairy cattle varies among breeds, among which Bos taurus, particularly the Holstein breed, are less tolerant to HS. The identification of genes associated to thermotolerance utilized in selection programs using genetic markers to breed high milk-producing cows in warm climates is currently being studied. Therefore,

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the purpose of this review is to carry out a comprehensive analysis of the effects of HS on milk production, activation of thermoregulatory mechanisms and feed intake behavior in dairy cattle. Keywords: Holstein cattle, Thermoregulation, Hyperthermia, Climate change, Feed intake behavior.

Received: 27/10/2020 Accepted: 12/07/2021

Introduction Heat stress (HS) is defined as the sum of external environmental forces that act on the animal causing an increase in body temperature, and the activation of physiological and behavioral adjustments in the first place(1). In dairy cattle, these adjustments represent adaptation mechanisms activated to attempt of maintaining a balanced body homeostasis(2). As global warming continues to augment, the prevalence of HS in dairy cattle will also increase in frequency, duration, and severity(3,4). Therefore, mitigating the negative effects of HS on the productivity of dairy herds has become a challenge for the dairy industry worldwide(5). The HS problem is higher in warm geographical areas where it can start during the late spring and extend beyond the summer, and consequently low fertility and milk production in cows can be prolonged until the fall(2,6). The magnitude of the HS is determined by the combined effects among ambient temperature (AT), relative humidity (RH), solar radiation, and wind speed(1,2). In the particular case of dairy cattle, the temperature-humidity index (THI) is estimated based on AT and RH, first, in order to determine the degree of HS to which the animals are exposed and, secondly, for the purpose of making herd management decisions during the hot season(1,7). Bouraoui et al(5) reported that milk production diminished by 21 % when the THI increased from 68 to 78 units, or when the AT exceeded 27 °C, regardless of the age or lactation stage. The negative impact of HS on fertility and milk production has given rise to much research in the last 50 years, and today a deep knowledge of the physiological, metabolic, endocrine, and molecular mechanisms has been attained, leading to a reduction of the productivity of the cattle resulting from HS. However, several of these mechanisms are triggered by two key factors: the reduction in feed intake and the increase in water intake(8). In fact, milk

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production is closely related to the dry matter (DM) and water intake in milk producing cows subjected to chronic HS(9). The first physiological and intake behavior adjustments in dairy cattle are considered to be homeostatic mechanisms(8), whose main purpose is to increase evaporative heat losses (i.e., breath and sweat) and to reduce the production of endogenous heat in order to maintain normothermia(10-12). It should be noted that the genotype plays an important role in defining the impact of HS on the dairy cattle production (13,14), among which Holstein is one of the most susceptible breeds, while the pure native cattle of warm regions, or their cross with introduced breeds, are more tolerant(15-18). Thus, the objective of the present review is to carry out a comprehensive analysis of the effects of HS on milk production, activation of thermoregulatory mechanisms, and intake behavior of dairy cattle.

Climate change and the future of dairy production The production of greenhouse gases such as steam, carbon dioxide, methane, and nitrous oxide, among others, alter the permeability of the atmosphere, letting the sun rays through while preventing the release of the heat radiated by the Earth’s surface(19). Thus, these gases cause global warming and, therefore, climate change, which tends to increase the amount of land surface with warm climate and, in extreme cases, desertification(20). Studies by climatologists suggest that the AT may increase by more than 2 °C by the year 2050(19). Today, the profitability of the dairy industry is endangered by the climate change, which has reduced the availability of natural resources, pasturelands, and production of grains and fodder, while it has favored the cattle losses due to natural disasters, as well as the prevalence of parasites and unforeseen diseases(2,21). The increase of agroecological areas with high AT as a result of climate change has also decreased milk production and increased production costs(8,22). In fact, HS is expected to increase in severity, duration, and presence in the near future(21). In the United States of America, economic losses in the dairy industry due to HS were estimated in 897 million dollars(22), and they recently projected economic losses of 0.4 and 1.2 % by 2050 and 2100, respectively, in the dairy basin in the northeast of that country(21). In Mexico, there are no reports of losses generated by HS in dairy cattle; however, such losses are evident, based on the seasonality of the milk production of dairy basins in warm weather(6,23). It is worth mentioning that the world food safety is jeopardized by climate change, considering that the demand of meat and milk will increase by 73 and 58 %, respectively, by 2050 in relation to the 2010 production levels(24).

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Thermoregulation and metabolic heat production in dairy cattle Bovines, like any other farm animal, are homeothermal and, therefore, have the ability to maintain a relatively stable internal body temperature regardless of the environmental conditions of their surroundings(25). In absence of thermal insults or in presence of fever, the body temperature of the dairy cattle ranges between 38.0 and 38.5 °C and is the combined reflex of the balance between environmental heat gains and metabolic heat production in a homeostasis state(1). In fact, in a thermoneutral environment and without milk production, cows can maintain their normothermia because they use mainly non-evaporative means (conduction, convection, and radiation), which do not entail an increase in the energetic cost associated to thermoregulation(26). If the rectal temperature (RT) is ≥42 °C, the body homeostasis balance can be disrupted, resulting in the death of the animal(22). It should be noted that the body temperature is not stable and exhibits a circadian rhythm which varies by approximately 1°C between maximum and minimum temperatures registered through the day(1). The body temperature reaches its maximum peak between 8 and 10 h after the AT has reached its maximum level; however, dairy cows can adapt to changes in AT and RH throughout the year(27,28). This is because its thermoneutral zone is very broad, ranging between -0.5 and 20.0 °C(1,2,10). According to certain studies, the maximum critical AT at which the Holstein cattle can maintain a stable body temperature in the range between 24 and 27° C(29). On the other hand, the milk synthesis in mammary gland of cows demands a great amount of metabolic activity, and therefore, the endogenous heat production increases in proportion to the level of milk production(25,27). It should be noted that the genetic improvement of dairy cattle has focused on increasing the milk production parameters, which has turn cows into metabolic heat producing machines, making them highly susceptible to warm climates(17,30,31). Cows that produce 18 to 31 L/day generate 28 to 48 % more metabolic heat than dry cows(32). A cow weighing 700 kg and producing 60 kg/d of milk generates approximately 44,171 kcal of heat per day; the same cow produces 25,782 kcal of heat per day at the end of the lactation period (20 kg/d)(27). Dairy cows that convert feed into milk more efficiently produce less metabolic heat and have a lower skin temperature(33). Thus, cows with a high milk production will have to make a greater effort in order to regulate their internal temperature under thermoneutral conditions, while under AT conditions they will be less tolerant to HS.

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Physiological thermoregulation of dairy cattle under heat stress High AT´s combined with a high percentage of RH further limit the ability of dairy cows to dissipate heat, and, in consequence, they experience HS and the activation of physiological thermoregulation mechanisms for dispelling the heat load(2,5,26). Certain climate indexes have been developed in order to predict the level of HS in dairy cattle; however, the THI proposed by Hahn(34) is the most commonly used worldwide. This index combines the climate values of AT and RH into an equation (THI = [0.81 × AT] + [RH / 100] * [AT – 14.4] + 46.4), and dairy cows are considered to begin to suffer from HS when they exhibit 72 units (1). Armstrong(35) classified HS into three types according to the THI: mild (72 to 78 units), moderate (79 to 89 units), and severe (90 to 98 units) (Figure 1). However, recent studies(11,36) have reported that a THI of 68 is already a critical value at which dairy cows will begin to exhibit symptoms of HS. Another study(37) found HS symptoms in highly producing dairy cows when the daily average THI was above 68 units or when the minimum THI was above 65 units. The climate in which the dairy cattle develop also plays an important role in defining the onset of HS based on the THI, given that under tropical conditions the negative effects of HS began at a THI > 70 units(1), and at a THI >60 under temperate conditions(9). This suggests that the pressure caused by genetic selection to improve dairy production counters tolerance to HS in dairy cattle(17). An imminent response to presence of HS in dairy cattle is an increase in the RT due to excessive body heat accumulation from the environment. This activates body heat losses through both evaporative and non-evaporative means(6,10,26). However, the activation sequence of physiological thermoregulation mechanisms will depend upon the HS type (acute or chronic) and degree (slight, moderate, or severe)(11). A more severe or more chronic HS will determine the reduction of body heat loss through non-evaporative means, while evaporative heat losses due to sweat and panting will increase considerably(8). Maia et al(26) observed 85 % body heat losses due to skin evaporation, and only 15 % through the respiratory tract in Holstein cows exposed to AT >30 ºC.

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Figure 1: Classification of heat stress according to the temperature-humidity index (THI) in dairy cattle (based on Armstrong, 1994).

A high RH reduces the effectiveness of body heat losses through evaporative means (10). The results obtained by Bonmanova et al(7) showed that RH was the limiting factor for HS in humid climates, while in dry climates the limiting factor was AT. Furthermore, the activation of these evaporative thermoregulatory mechanisms entails additional energy expenditure unlike with non-evaporative mechanisms, whereby there is a greater energetic expenditure for maintenance in heat-stressed dairy cows. An increased respiratory frequency (RF) 493


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requires an additional energy expenditure of 7 to 25 % due to movement of the muscles of the respiratory tract(38), and the water vaporization process through the skin and respiratory tract demands an energy expenditure of 2.43 J/ml of evaporated water(1). For this reason, the activation of heat losses through evaporative means is an option for the animal when nonevaporative losses are unable to release the body heat load under HS. In this sense, the RF increases with a THI above 73 units, while the RT increases with a THI >80 units(39). On the other hand, diurnal variations in climatic conditions and the changes in animal behavior improve the physiological thermoregulatory capacity of heat-stressed dairy cattle. An AT ≤21°C during the nighttime, with a duration of 3 to 6 h, favors the total body heat loss accumulated in the daytime, as the animal promotes a redistribution of blood flow toward the skin, allowing an efficient release of the body heat load throughout non-evaporative means (radiation and convection)(40). A wind speed between 1.8 and 2.8 m/s also promotes body heat losses through the skin(28). Likewise, cows adopt a standing position in order to reduce the body area exposed to sun rays and the heat gain from contact with the ground(41). When under HS, they also reduce their feed intake per day and eat at those times of the day when AT is lowest(8). This allows their daily water intake to increase(42).

Heat stress and feed intake The reduction in milk production by heat stressed dairy cattle has traditionally been associated to a reduction in feed intake(8), and the degree at which it diminishes depends on the duration and severity of the prevalent HS, as well as on the adaptation level of the breed(14,18,25). Thus, the AT has an inverse relationship with DM intake under chronic HS, while, under acute HS, feedd intake is rapidly reduced one day after the exposure(43). In fact, feed intake begins to decline at an AT of 26 °C in lactating cows(1), and its reduction can reach up to 40 % at an AT≥40 °C(44). Given that the presence of HS is determined based on the estimation of THI in dairy cattle and that this climatic parameter is easier to obtain than a daily measurement of feed intake, certain researchers opted to establish the association between them, and results showed a reduction of 0.51 kg in DM intake per unit which increases THI within the range of 72 to 84 units(8). Another study found that the variation in minimum THI has a greater association with the changes in feed intake than the variation in maximum THI(45). Besides the reduction in feed intake, heat stressed cows also modify their feed intake schedules in order to reduce their endogenous heat production during the hottest hours of the day. Metabolic heat associated to ruminal fermentation represents 3 to 8 % of the total

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endogenous heat produced by bovines, depending on the dietary fiber level(12). Dairy cows have a daily natural feed consumption pattern, being high after receiving fresh diet, as well as in the afternoon and evening(46,47). However, dairy cattle under HS prefer to eat very early in the morning and at nightfall, as digestion and metabolic heat production reach their peak 3 to 4 hours after feed intake, just before the hottest period of the day(32). It is worth noting that HS in dairy cattle also impacts the ruminal kinetic of the ingested feed, as it decreases the motility of the gastrointestinal tract, changes the patterns of fermentation and production of volatile fatty acids, and increases the retention time of feed in rumen and its digestibility(38,47). In order to mitigate the reduction of DM intake and sustain milk production during the warm season of the year, certain cattle breeders modify the dietary chemical composition to increase the energy level, which involves increasing the content of grains and reducing the amount of dietary fiber(12,39). This nutritional strategy benefits the cattle by allowing them to meet their nutritional requirements with a lower feed intake. However, the modification in the dietary ingredients can have negative consequences if too many adjustments are made; for instance, a high blood urea concentration due to a high protein digestion in rumen increases the RT(48), or a high concentrate diet can contribute to potentiate the acidosis issue generated by HS in cows(12).

Heat stress and water intake An insufficient availability of drinking water has adverse effects on the productivity and welfare of dairy cattle under any environmental conditions, but it becomes more critical at high AT(42). Regardless of the climate, lactating cows require larger amounts of water, as both the body and the milk are composed of more than 90 % water(38,49). Nevertheless, these cows can vary their water intake according to the amount of DM consumed in the diet and daily kilograms of milk produced(50). A restriction in water intake can reduce milk production by up to 26%, and this reduction can be even greater in HS conditions(51). Dairy cows satisfy their water requirements from three sources: direct water intake, water from foods, and water derived from the tissue oxidative metabolism(42). However, the main source of water for cattle is direct intake under any climate condition (83 %)(49). Water intake is affected by such factors as: DM intake, milk production, and intake of potassium, sodium, and nitrogen(52). Cows under thermoneutral conditions can consume 14 to 171 kg/d of water, with a daily average of 82 kg, and, based on the milk production and dietary sodium intake, water intake is estimated to be 4 to 4.5 L/kg of milk produced(42). On the other hand, a water intake of 146 g per gram of ingested sodium(52) was found in absence of a thermal insult. In

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dry cows without heat stress, water intake is significantly lower (15 to 61 L/d)(53), but increases by 27 % with the onset of milk production during the early lactation(54). Dairy cows can experience severe dehydration when they lose 12 % of their body weight due to lack of water intake; such dehydration can be lethal if the total body water loss reaches approximately 20 %(55). Given that HS increases water loss in ruminants considerably, its intake at adequate levels becomes critical for dairy cows(54). In warm areas, water intake can increase between 10 and 20 % during the summer, when even a cow classified as low producing can drink more than 100 L/d(32). A recent study reported that the water intake can increase by up to 50 % as the THI approaches 80 units (56). A cow is estimated to increase its water intake by 1.52 kg for each Celsius degree that the AT increases(42). Furthermore, the time devoted every day to water intake increases from 0.26 to 0.5 h as the THI changes from 56.2 to 73.8 units(57) or the AT increases from 15 to 33 °C(58). The greater water intake under HS conditions is attributed to an increase in the urine volume (25 %), evaporation through the respiratory tract (panting), and sweating(1), which are thermoregulatory mechanisms that help dissipate excess body heat load in this type of environments(59). On the other hand, as previously stated, water intakes in dairy cattle are very high under thermoneutral conditions and even higher under HS, which leads this species to have different water intake behaviors compared to other species. Under thermoneutral conditions, Holstein cows with a medium milk production drink water approximately eight times a day, consuming an average of 12.9 L on each occasion(60), while high producing Holstein cows drink water more frequently (12 to 16 times) but consume a smaller amount (5.4 to 6.0 L) on each occasion(61). In the absence of HS, these cows prefer to drink 97 % of their total daily intake during the daytime, rather than at night(60). Conversely, when under HS, the water intake behavior among these animals becomes highly variable at AT above 30 °C(62). This may be due to individual differences in tolerance to heat and, therefore, in milk production. In general, heat stressed cows resort to the water trough more frequently than unstressed cows, but they prefer to drink water at times when solar radiation is at its lowest or absent(63). The reason for this low water intake behavior during the daytime, particularly during the hottest hours of the day (on three to five occasions), is that cows prefer to remain motionless, rather than gaining heat by seeking this nutrient. Therefore, the construction design of pens or grazing areas is crucial for the cow’s decision to taken a drink of water under hot weather. The water troughs located at a large distance from the feed troughs, particularly when these have no shade, causes that heat stressed cows have to choose whether to move toward the shaded area or toward the water trough after eating(56). So, it is recommended to strategically distribute water troughs both in pasturelands and pens in order to prevent cows having to walk more than 250 m(32); likewise, these animals prefer high and long water troughs(64). The cleanliness and temperature of the water are factors that largely determine intake in sufficient amounts by cows exposed to high AT. Like any other species, dairy cattle avoids drinking 496


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foul-smelling water, being necessary to continually wash the water troughs under any climate(60). Cows also prefer to drink their warm water (≥24 ºC), rather than cool (15 a 20 ºC) or cold (≤10 ºC), under both thermoneutral(65) and HS(66) conditions. It is noteworthy that this preference of heat stressed cows to drink warm rather than cold water is contradictory, as it has been proven that the cold or cool water intake reduces both RT and RF(67). Given the importance of this nutrient, it is essential to measure the water intake of dairy cattle, whether heat stressed or not, in order to maintain an optimal milk production and the good health of cows(42,50).

Impact of heat stress on milk production Dairy cows reach their optimal milk production at a temperature range of 5 to 25 °C, which is considered as the comfort zone for lactating cows(1). Exposure of cows to ATs above the upper limit of this comfort zone may reduce milk production by 10 to 40 %(38). However, the impact of HS on milk production depends on the stage of lactation, intensity of the heat, and cow’s genetic potential to synthesize milk. Thus, in presence of HS, high producing dairy cows experience a greater reduction in milk production than medium or low producing cows(12,25). Medium-lactation dairy cows decrease their milk production by 35 % in response to HS, while early-lactation cows reduce it only by 14 %(25). High-producing dairy cows tend to exhibit a larger bone structure and gastrointestinal tract capacity, which allows them to ingest and digest more food; however, metabolic heat production also increases and reduces their ability to maintain normothermia under HS conditions(1). In fact, an increase in milk production from 35 to 45 kg/day reduces by 5 °C the AT at which HS begins(68). Milk production starts to diminish at an AT of 27 °C, regardless of the cow’s age or lactation stage(1); this negative effect becomes evident 24 to 48 h after the onset of any type of HS(43). A study carried out on Israeli Holstein cows of 3rd and 4th lactation found a reduction of 0.38 kg/cow/day in milk production for every Celsius degree that the AT increased during the summer(69). As expected, there is a close negative association (r= - 0.76) between the THI and milk production(5); the same applies to the AT. Therefore, this production parameter decreases by 0.41 kg/cow/day for every unit of THI above 68(40). Nevertheless, there are studies reporting a lower reduction in milk production (0.2 kg/cow/d) per unit of THI above 72(70). Variations in milk production level and AT may explain this discrepancy between studies. Reduction in milk production occurs by direct and indirect effect of HS. Direct effects include adjustments in energy and protein metabolism in order to prioritize the availability of nutrients for the cows’ thermoregulation processes instead of sending them to the mammary

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gland for milk synthesis(71). Likewise, HS increases the presence of oxidative stress, which alters the cellular metabolic and molecular activity in the mammary secretory tissue, reducing the cellular efficiency for synthesis of milk components(30). Indirectly, the mechanism is associated with a reduction in DM intake, since maintenance energy requirements increase as a result of the activation of physiological thermoregulation mechanisms in a scenario where availability of body nutrients is limited due to low feed intake(3,38). A reduction in the efficiency of energy use for milk production by 30 to 50 % has been reported for dairy cows in warm climates(1). The HS negatively affects not only milk production but also its composition(56). A reduction in fat and protein content combined with an increase in somatic cell count (SCC) are results commonly associated to HS(5). The results of a study contrasting spring thermoneutral conditions versus severe summer HS showed that fat (3.58 %) and protein (2.96 %) contents in milk decreased by 30 and 23 %, respectively, during the summer(5). The same study detected an increase in SCC by 110 % due to summer HS. Another study reported a 40 % decrease in milk fat content of Holstein cows under moderate to severe HS, as well as a 17 % decrease in protein content(1). Bernabucci et al(72) observed a slightly lower casein concentration in milk during the summer compared to winter (2.18 vs 2.58 %). The reduction in the casein percentage may account the low cheese production during that season(73).

Adaptive responses of dairy breeds to heat stress Holstein cattle is the genotype par excellence for the global dairy industry; however, they have little resistance to HS. Genetic improvement programs for this breed have focused on selecting traits associated to increase milk quality and production, whose effects are contrary to their ability to tolerate high AT´s(28). Thus, the heritability index for milk production in Holstein cows can diminish due to HS and, consequently, the selection response based on this trait may not be as expected in warm climates(17). In spite of this, Holstein cattle continues to be used in warm regions because it maintains a larger average milk production than other genotypes adapted to the region, mainly in technified and semi-technified production systems(13,18). Although Holstein cattle has proven to maintain a higher milk production than such breeds as Simmental (20.4 vs 28.1 L)(13) and Jersey (26.6 vs 34.2 L)(16) under tropical HS conditions, their milk is characterized by having a high SCC and low fat and protein content. In addition, the reduction in milk production as a result of HS is more pronounced in Holstein cows; this is an unmistakable symptom of lower tolerance to high AT´s(13,18). Therefore, the use of the Holstein breed in warm climates, particularly in tropical and subtropical regions, cannot be

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justified based on the milk production level, given that HS conditions reduce the quality of this product and compromise the health and welfare of cows. The use of animal genetic resources of the region, including native and introduced cattle breeds that are highly adapted to the warm-humid climate(31,74), is recommended for milk production in tropical climates. Some studies also suggest crossbreeding between the Holstein and zebu breeds in order to generate cattle with a dairy genotype tolerant to warm climates(10,15). Thus, dairy cattle breeds adapted to HS exhibit greater tolerance to these environmental conditions because, phenotypically, they have thinner and less dense hair, as well as a higher density of sweat glands in the skin, which allows them to more easily release their body heat load by convection and radiation(14,75). In addition, this type of cattle under HS relatively maintain their feed intake and thyroid gland activity, which explains the lower variation in milk production compared to non-adapted cattle(75). The main breeds used in the Mexican tropics for milk production are Brown Swiss, Jersey, Holstein, Gyr, and Brahman, as well as their crosses; however, other management and feeding factors, along with the climate conditions, lead to a low productive and reproductive efficiency(76). On the other hand, the cells of HS-adapted cattle activate heat shock transcription factors to coordinate protective mechanisms in response to the thermal insult, including expression of genes, activation of heat shock proteins (HSP), and carbohydrate metabolism (77). In HS tolerant bovines (Bos indicus) compared to HS intolerant cattle (Bos taurus), cells exhibit a greater HSP expression (mainly of 70 kDa(78) and 90 kDa(79)) in response to HS, which improves their viability and immune activity(80). However, it has been proven that dairy cattle mononuclear cells may exhibit an HSP-72 overexpression in response to hyperthermia, which is counter-productive for their immune response and tolerance to heat(81). This became evident in a study in which mononuclear cells of Holstein and Brown Swiss cattle were cultivated in vitro at 39 (normal), 40, 41, 42, and 43 ºC, and results showed that hyperthermia conditions triggered excessive mRNA production for HSP-72 and low DNA synthesis in Brown Swiss cattle compared to Holstein(81). Contrary to the predictions, these findings suggest that Holstein cattle resists better hyperthermia than Brown Swiss cattle, although the latter can maintain an adequate productive efficiency under HS as long as their thermoregulatory capacity is not compromised and the core temperature does not rise above 40 ºC(81).

Genetic markers associated to thermotolerance in dairy cattle A large number of studies have focused on identifying the gene expression associated to thermotolerance in HS-adapted dairy cattle, and this information is being utilized to establish

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marker-assisted genetic selection programs(82). Likewise, work is being carried out to identify males expressing thermotolerance genes, which are then selected as sires in order to transmit these characteristics to their offspring(28). Special attention is being given to the search for these genes in high-producing dairy cattle, given the interest of the dairy industry in having HS tolerant genotypes that will produce large volumes of high quality milk. In Mexico, there is little research on the identification of genes linked to thermotolerance in dairy cattle. Hernández-Corderos et al(83) identified seven genes (AVPR1A, Furin, IGFBP5, IGFBP6, PMCH, PRLR, and STAT5B) associated with milk production in heat-stressed Holstein cattle reared in the Yaqui Valley, Sonora; specifically, a single-nucleotide polymorphism (SNP) within each gene related to the prolactin and somatotropin pathway. Another study(84) reported the identification of six genes associated with milk production and its fat and protein contents (SFXN1, LOC781028, ANKRD31, LOC100296562, LOC107131388, and WDR41) in a genome-wide association analysis conducted in Holstein cattle reared in the desert region of Mexicali, Baja California, Mexico. Further studies of Mexican Holstein cattle located in warm climate are required to identify other genes that may be directly associated with thermoregulatory traits, which, in combination with those already known to be related to milk production, may serve as genetic markers to recognize HS tolerant cattle. In the United States, a recent study(82) identified three genomic regions (BTA5, BTA14, and BTA15) which account a large part of the variation in milk production of heat-stressed Holstein cows. Ten genes were identified within these regions as associated to thermotolerance, given that they are involved in various cellular processes in response to HS, such as activation of HSP (PEX16, HSF1, EEF1D, and VPS28), reduction of oxidative stress, modulation of the apoptosis process (MAPK81P1, CREB3L1), DNA maintenance (TONSL), and thermotolerance (CRY2). Other genomic regions associated with the presence of thermotolerance genes in American Holstein cattle are BTA-24(85) and BTA-26(86), which are related to RT and milk production, respectively. Regarding the Chinese Holstein cattle, there is also evidence that they carry SNPs associated to thermotolerance genes such as ATP1A1(87), HSP90AA1(88), HSF1, HSP70A1A, and HSPB1(28). Certain HS-thermotolerant genes have also been identified in other dairy cattle breeds. The SLICK haplotype gene has been identified in Senepol cattle and has been introduced into the Holstein breed with positive results for HS tolerance without compromising milk production(89). The genes HSP90AB1 and HSPB8 have been found in Sahiwal dairy cattle, native breed to India(90). It is worth noting that, seemingly, dairy breeds of the Mexican tropic have not been typified to detect genes associated with thermotolerance at high AT´s; this, therefore, remains a topic for future research.

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Conclusions and implications Heat stress is a key factor that conditions the milk production level in cattle, being more noticeable in those breeds that are sensitive to high AT´s. Dairy cattle start to experience HS when environmental conditions promote a THI of 72 units, although high-producing dairy breeds can begin to experience it at 68 units. Facing the HS environment, the cattle make physiological and metabolic adjustments to reduce endogenous heat production while releasing excess body heat load; however, the activation of thermoregulation mechanisms decreases the capacity of milk synthesis and secretion of cows. At least 40 % of the reduction in milk production is associated with low DM intake, and the rest, with direct effects on the general metabolism and cellular thermo-resistance. Today, the high pressure to select traits associated to milk production has resulted in cattle with a lower tolerance to HS; this genetic improvement strategy must therefore be modified by taking into account the presence of thermotolerant genes. In this sense, research on HS in dairy cattle must focus mainly on HS mitigation strategies that help reduce the energetic cost resulting from the activation of thermoregulatory mechanisms, as well as increase the daily feed intake. Literature cited: 1. Kadzere CT, Murphy MR. Silanikove N, Maltz E. Heat stress in lactating dairy cows: a review. Livest Prod Sci 2002;77:59-91. 2. Herbut P, Angrecka S, Walczak J. Environmental parameters to assessing of heat stress in dairy cattle—a review. Int J Biometeorol 2018;62(12):2089-2097. 3. Min L, Zhao S, Tian H, Zhou X, Zhang Y, Li S, et al. Metabolic responses and “omics” technologies for elucidating the effects of heat stress in dairy cows. Int J Biometeorol 2017;61(6):1149-1158. 4. Theusme C, Avendaño-Reyes L, Macías-Cruz U, Correa-Calderón U, García-Cueto RO, Mellado M, Vargas-Villamil L, et al. Climate change vulnerability of confined livestock systems predicted using bioclimatic indexes in an arid region of México. Sci Total Environ 2021;751:141779. 5. Bouraoui R, Lahmar M, Majdou A, Djemali M, Belyea R. The relationship of temperaturehumidity index with milk production of dairy cows in a mediterranean climate. Anim Res 2002;51:479-491.

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6.Hernández-Rivera JA, Avendaño-Reyes L. Efecto de época del año (verano vs. invierno) en variables fisiológicas, producción de leche y capacidad antioxidante de vacas Holstein en una zona árida del noroeste de México. Arch Med Vet 2015;47(1):15-20. 7. Bohmanova J, Misztal I, Colef JB. Temperature-humidity indices as indicators of milk production losses due to heat stress. J Dairy Sci 2007;90:1947-1956. 8. Anzures-Olvera F, Macías-Cruz U, Álvarez-Valenzuela FD, Correa-Calderón A, DíazMolina R, West JW. Effects of heat-stress on production in dairy cattle. J Dairy Sci 2003;86:2131-2144. 9. Gorniak T, Meyer U, Südekum KH, Dänicke S. Impact of mild heat stress on dry matter intake, milk yield and milk composition in mid-lactation Holstein dairy cows in a temperate climate. Arch Anim Nutrit 2014;68:358-369. 10. Berman A. Are adaptations present to support dairy cattle productivity in warm climates? J Dairy Sci 2011;94:2147-2158. 11. Avendaño-Reyes L. Heat stress management for milk production in arid zones. In: Narongsak Chaiyabutr editor. Milk production - An up-to-date overview of animal nutrition, management and health. Intech Open, London UK; 2012:165-184. 12. Min L, Li D, Tong X, Nan X, Ding D, Xu B, et al. Nutritional strategies for alleviating the detrimental effects of heat stress in dairy cows: a review. Int J Biometeorol 2019;63:1283-1302. 13. Gantner V, Bobic T, Gantner R, Gregic M, Kuterovac K, Novakovic J, et al. Differences in response to heat stress due to production level and breed of dairy cows. Int J Biometeorol 2017;61:1675-1685. 14. Polsky L, von Keyserlingk MAG. Invited review: Effects of heat stress on dairy cattle welfare. J Dairy Sci 2017;100(11):8645-8657. 15. Pereira AM, Baccari F Jr, Titto EA, Almeida JA. Effect of thermal stress on physiological parameters, feed intake and plasma thyroid hormones concentration in Alentejana, Mertolenga, Frisian and Limousine cattle breeds. Int J Biometeorol 2008;52(3):199-208. 16. Smith DL, Smith T, Rude BJ, Ward SH. Short communication: comparison of the effects of heat stress on milk and component yields and somatic cell score in Holstein and Jersey cows. J Dairy Sci 2013;96(5):3028-33.

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52. Spek JW, Bannink A, Gort G, Hendriks WH, Dijkstra J. Effect of sodium chloride intake on urine volume, urinary urea excretion, and milk urea concentration in lactating dairy cattle. J Dairy Sci 2012;95:7288-7298. 53. Holter J, Urban W. Water partitioning and intake prediction in dry and lactating Holstein cows. J Dairy Sci 1992;75:1472-1479. 54. Ammer S, Lambertz C, von Soosten D, Zimmer K, Meyer U, Dänicke S, et al. Impact of diet composition and temperature-humidity index on water and dry matter intake of high-yielding dairy cows. J Anim Physiol Anim Nutr 2018;102(1):103-113. 55. Roussel AJ. Fluid therapy in mature cattle. Vet Clin North Am Food Anim Pract 1999;15: 545-557. 56. Pawar MM, Srivastaba AK, Chauhan HD, Damor SV. Nutritional strategies to alleviate heat stress in dairy animals – A Review. Int J Livest Res 2018;8(1):8-18. 57. Cook NB, Mentink RL, Bennet TB, Burgui K. The effect of heat stress and lameness on time budget of lactating dairy cows. J Dairy Sci 2007;90:1674-1682. 58. Beatty DT, Barnes A, Taylor E, Pethick D, McCarthy M, Maloney SK. Physiological responses of Bos taurus and Bos indicus cattle to prolonged, continuous heat and humidity. J Anim Sci 2006;84:972-985. 59. Ji B, Banhazi T, Perano K, Ghahramani A, Bowtell L, Wang C, et al. A review of measuring, assessing and mitigating heat stress in dairy cattle. Biosyst Eng 2020;199:426. 60. Cardot V, Le Roux Y, Jurjanz S. Drinking behavior of lactating dairy cows and prediction of their water intake. J Dairy Sci 2008;91:2257-2264. 61. Dado RG, Allen MS. Intake limitations, feeding behavior, and rumen function of cows challenged with rumen fill from dietary fiber or inert bulk. J Dairy Sci 1995;78:118-133. 62. Brscic MF, Gottardo A, Mazzenga A, Cozzi G. Behavioral response to different climatic conditions of beef cattle in intensive rearing systems. Poljoprivreda 2007;13(1):1-5. 63. Jago JG, Roche JR, Kolver ES, Woolford MW. The drinking behavior of dairy cows in late lactation. Proc NZ Soc Anim Prod 2005;65:2209-2014. 64. Pinheiro MFLC, Teixeira DL, Weary DM, vonKeyserlingk MAG, Hötzel MJ. Designing better water troughs: dairy cows prefer and drink more from larger troughs. Appl Anim Behav Sci 2004;89:185-193.

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78. Bhanuprakash V, Singh U, Sengar G, Sajjanar B, Bhusan B, Raja TV, et al. Differential effect of thermal stress on HSP70 expression, nitric oxide production and cell proliferation among native and crossbred dairy cattle. J Thermal Biol 2016;59:18-25. 79. Deb R, Sajjanar B, Singh U, Kumar S, Singh R, Sengar G, et al. Effect of heat stress on the expression profile of Hsp90 among Sahiwal (Bos indicus) and Friswal (Bos indicus x Bos taurus) breed of cattle: A comparative study. Gene 2014;536:435-440. 80. Bhanuprakash V, Singh U, Sengar GS, Raja TV, Sajjanar B, Alex R, et al. Comparative expression profile of NOD1/2 and certain acute inflammatory cytokines in thermalstressed cell culture model of native and crossbred cattle. Int J Biometeorol 2017;61:931–941. 81. Lacetera N, Bernabucci U, Scalia D, Basiricò L, Morera P, Nardone A. Heat stress elicits different responses in peripheral blood mononuclear cells from Brown Swiss and Holstein cows. J Dairy Sci 2006;89(12):4606-4612. 82. Sigdel A, Abdollahl-Arpanahl R, Aguilar I, Peñagaricano F. Whole genome mapping reveals novel genes and pathways involved in milk production under heat stress in US Holstein cows. Front Genet 2019;10:928. 83. Hernández-Cordero AI, Sánchez-Castro MA, Zamorano-Algandar R, Luna-Nevárez P, Rincón G, Medrano JF, et al. Genotypes within the prolactin and growth hormone insulin-like growth factor-I pathways associated with milk production in heat stressed Holstein cattle: Genotypes and milk yield in heat stressed Holstein cows. Genet Mol Res 2017;16(4):gmr16039821. 84. González ME, González VM, Montaño MF, Medina GE, Mahadevan P, Villa C, et al. Genome-wide association analysis of body conformation traits in Mexican Holstein cattle using a mix of sampled and imputed SNP genotypes. Genet Mol Res 2017;16(2):gmr16029597. 85. Dikmen S, Cole JB, Null DJ, Hansen PJ. Genome-wide association mapping for identification of quantitative trait loci for rectal temperature during heat stress in Holstein cattle. PLoS One 2013;8:e69202. 86. Macciotta NPP, Biffani S, Bernabucci U, Lacetera N, Vitali A, Ajmone-Marsan P, et al. Derivation and genome-wide association study of a principal component-based measure of heat tolerance in dairy cattle. J Dairy Sci 2013;100:4683-4697. 87. Liu Y, Li H, Zhou X, Wang G. A novel SNP of the ATP1A1 gene is associated with heat tolerance traits in dairy cows. Mol Biol Rep 2011;38:83-88.

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88. Badri TM, Chen KL, Alsiddig MA, Li L, Cai Y, Wang GL. Genetic polymorphism in Hsp90AA1 gene is associated with the thermotolerance in Chinese Holstein cows. Cell Stress Chaperones 2018;23:639-651. 89. Dikmen S, Khan FA, Huson HJ, Sonstegard TS, Moss JI, Dahl GE, Hansen PJ. The SLICK hair locus derived from Senepol cattle confers thermotolerance to intensively managed lactating Holstein cows. J Dairy Sci 2014;97(9):5508-5520. 90.Verma N, Gupta ID, Verma A, Kumar R, Das R, Vineeth MR. Novel SNPs in HSPB8 gene and their association with heat tolerance traits in Sahiwal indigenous cattle. Trop Anim Health Prod 2016;48:175-180.

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https://doi.org/10.22319/rmcp.v13i2.5980 Review

Potato protein concentrate: a possible alternative to the use of antibiotics in diets for weaned piglets. Review

Erick Alejandro Parra Alarcón a Teresita de Jesús Hijuitl Valeriano b Gerardo Mariscal Landín a,b,c Tércia Cesária Reis de Souza a,b*

a

Universidad Nacional Autónoma de México. Facultad de Medicina Veterinaria y Zootecnia Maestría en Ciencias de la Producción y de la Salud Animal. Ciudad de México, México. b

Universidad Autónoma de Querétaro. Facultad de Ciencias Naturales. Maestría en Salud y Producción Animal Sustentable. Av. De las Ciencias s/n. Querétaro, Querétaro, México. c

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Centro Nacional de Investigación Disciplinaria en Fisiología y Mejoramiento Animal. Ajuchitlán, Querétaro, México.

*

Corresponding author: tercia@uaq.mx

Abstract: The weaning period is critical in the life of piglets and can cause gastrointestinal disorders and low growth, which are lessened with the use of antibiotics in starter feeds. However, due to the need to eliminate antibiotics from animal nutrition, some possible alternatives to their use are mentioned. In this literature review, antimicrobial peptides and plant-derived protease inhibitor compounds are described, especially those from potatoes, which have traditionally been recognized for their potential biomedical application and activity against pathogenic bacteria and fungi. The characteristics and applications of potato protein concentrate (PPC) from the starch industry, which is distinguished by its amino acid profile and high digestibility, were reviewed. Molecules that are present in the protein fraction and that can

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contribute to the intestinal health of piglets stand out in PPC, so it is emerging as an ingredient with potential to be used in antibiotic-free diets. However, it is necessary to have more bibliographic information on PPC to verify whether the health response is consistent or not, and to recommend its inclusion in starter diets for newly weaned piglets as an alternative to antibiotics. Key words: Piglets, Weaning, Antimicrobial peptides, Protease inhibitors, Potato.

Received: 07/04/2021 Accepted: 26/08/2021

Introduction The newborn piglet has a low intestinal capacity to digest and absorb solid feeds, especially those of vegetable origin, so its digestive system must mature quickly to ensure its survival(1,2). Enteral nutrition (colostrum and milk) plays a fundamental role in the maturity of the piglet(1); however, milk soon ceases to cover the nutritional demand of the piglet and it begins a gradual consumption of other feeds, allowing the gradual maturity of the nervous, immune and digestive systems. This maturation process of the digestive tract is stimulated by the colonization of different bacterial genera(3.4), which use some nutrients and produce enzymes(2,5) and, through competitive exclusion, prevent the adhesion of pathogens(4). Under natural conditions, piglet weaning occurs between weeks 10 to 22 of life(6,7). However, under commercial conditions, weaning is carried out between 21 and 28 d of age in order to allow the maximum productive efficiency of the female; higher number of births and piglets per sow per year, reduce the cost of facilities, etc.(6). Commercial weaning, unlike natural weaning, is not gradual but an abrupt and sudden event, which is extremely stressful for the still immature piglet. This fact is characterized by the separation of the mother, the environmental change and from a dairy diet to a solid one (mainly composed of cereals). All this, added to the presence of new pathogens, causes neuroendocrine, immunological and digestive complications(2,7), with piglets showing in the first 24 to 48 h after weaning a low, and even zero, feed consumption, weight loss, atrophy of the intestinal structure and with it of the digestive and absorption capacity, as well as an increase in the incidence of postweaning diarrheas(6,8). The presence of post-weaning diarrheas is widely related to the sudden change in diet, as well as gastrointestinal infections. Both factors promote rapid dysbiosis, that is, an imbalance 511


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in the composition of bacterial populations, with an increase in E. coli, which contributes to the loss of intestinal structure(9), as well as an abrupt reduction of Lactobacillus spp. The incidence of these post-weaning digestive disorders generates great economic losses(10). To combat these gastrointestinal complications, antibiotics have been used as growth promoters (GPAs) in diets in recent years, since their use in animal feed favors the growth rate and reduces the incidence of diseases and mortality(11,12). These molecules decrease the number of pathogens and with it the atrophy of the intestinal villi and hypertrophy of the crypts are prevented. This leads to a greater and early consumption of feed, adequate digestive capacity and better feed efficiency, thus increasing the retention of nitrogen and energy from the diet(13,14). However, in recent years, the inclusion of GPAs in animal diets has been questioned, and even banned in some countries, since they represent a serious problem for public health, due to the development of bacterial resistance to antibiotics, which can potentially reduce the treatment of diseases in animals and possibly in humans. Therefore, the search for substitutes or alternatives to antibiotics is of great relevance for pig farming(15,16). With the growing human population, the demand for safe food resources by the pig industry has increased dramatically in recent years(17). Due to its nutritional and possibly therapeutic characteristics, potato protein concentrate (PPC), obtained after starch extraction, seems to be a good choice as a protein source. Therefore, the present work aims to review some characteristics of this protein ingredient, which place it as a nutritional alternative with potential to improve the intestinal health of newly weaned piglets.

Alternatives to growth-promoting antibiotics (GPAs)

The crisis of bacterial resistance to antibiotics shows no signs of a solution in the short term and the lack of new antimicrobial drugs, as well as the few companies that invest in this area, threatens the ability to treat and prevent infections. One reason for the shortage of new antibiotics is that typical points of action, such as cell wall and protein synthesis, as well as that of DNA/RNA, have perhaps been overexploited. Currently, thanks to access to complete bacterial genomes, strategies based on new molecular targets are sought; however, this approach has not been fully developed(18). However, the antibiotic crisis demands measures that contemplate a new approach and different therapeutic strategies(18). There are many literature reviews on the different alternatives to GPAs. Among the most studied alternatives the following stand out: plant extracts, chicken egg antibodies, organic acids and enzymes(12), as well as essential oils(10),

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probiotics(19), prebiotics(20), minerals such as copper and zinc(11), as well as animal plasma(21) and plant proteins such as potato protein concentrate(22). Among the most successful alternatives for the control of post-weaning digestive disorders, animal proteins stand out, such as animal plasma and fishmeal, which, due to their high digestibility and amino acid profile, favor feed consumption and growth rate(23,24). However, ingredients of animal origin are expensive and inadequate handling during storage could favor the transmission of some pathogens. Fishmeal is the essential protein in piglet feeds, but marine overfishing has caused a vertiginous increase in prices, reducing its availability. The results of including these ingredients in piglet diets can sometimes be inconsistent and hardly able to match the effect of antibiotics in productive terms, however, benefits to intestinal health are reported, which should be considered when using antibiotic-free diets. Plant-based antimicrobial peptides (AMPs) are an alternative to GPAs that have shown potential use(12).

Plant-based antimicrobial peptides

AMPs are developed by different plants and tubers as a defense mechanism and in response to microbial aggressions and infections(25-27). These peptides are expressed and stored in different plant tissues(25). AMPs are encoded by genes that have a wide range of activity against Gram-negative, Gram-positive bacteria, fungi and bacilli of the genus Mycobacterium. They have been isolated and characterized from tissues and organisms that represent practically all kingdoms and phyla(28). These peptides play an important role in the mechanisms responsible for eliminating or preventing the growth of pathogens, both inside and outside plant organisms(29). AMPs exist in different molecular forms, the most common are linear, although there are also cyclic forms. Most AMPs have 2 to 6 cysteine residues(26), which give them high thermal, enzymatic and chemical stability(30). They are polypeptides with less than 200 amino acids (AAs), commonly less than 50 AAs, of low molecular weight (approximately 10 kDa), basic character and are generally cations at physiological pH due to their residues loaded with arginine and lysine(25). Cystine-rich AMPs are classified into families according to their sequence similarity, cysteine motifs, that is, the cysteine combinations that accumulate in the tertiary structure of the peptide and disulfide bond patterns. Families of cystine-rich plant AMPs include thionines, defensins, hevein-type peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinines and snakins(25,26). It should be clarified that there are AMPs rich in other amino acids (glycine, histidine). The ability of plant AMPs to organize into

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families with conserved structural characteristics allows the variation of the sequence of residues that are not cysteine in the same structure within a particular family to perform multiple functions(26). Plant AMPs have activity against bacteria, fungi, viruses and parasites. The mechanism of action of AMP is generally believed to be related to membrane lysis or peptide penetration followed by attack on intracellular targets. The cationic nature and amphipathic capacity of AMPs allows interaction with the (anionic) wall and phospholipid membrane of microorganisms(25,26). Mechanisms of action such as pore formation and membrane depolarization, disruption of bacterial energy metabolism and interference with biosynthetic pathways for the antimicrobial activity of several AMPs that contain disulfide bridges (31) have been suggested. The aforementioned characteristics profile them as an important alternative for the development of antibiotic and anti-inflammatory molecules(25). These characteristics, in addition to cysteine residues, are classic of the families of thionines and defensins. Other families of AMPs such as hevein-type peptides bind to chitins, and lipid transfer proteins bind to cell membrane lipids to disrupt microbial penetration into cells(26). Some authors(28,32,33) suggest that PPC may be an alternative to feeds medicated with antibiotics, because it showed antimicrobial activity by effectively reducing the population of coliform bacteria. It is suggested(33) that potato protein may have an additional potential advantage over antibiotics by selectively inhibiting the in vitro growth of pathogenic bacteria (Staphylococcus aureus, Salmonella gallinarum and E. coli). The explanation of the antimicrobial effects of PPC could be related to the action of certain antimicrobial peptides that can be found in the protein of the tuber Solanum tuberosum(34), since peptides with antimicrobial activity are produced by the potato in its defense against pathogens.

Antimicrobial potato peptides

Potato proteins are divided into three groups: patatin, protease inhibitors (PIs) and other proteins that are also involved in the defense of the potato, as they all have antifungal or antimicrobial actions(35). PIs represent a high proportion of the total potato protein(36), and are a structurally heterogeneous group (Table 1) with a wide range of antifungal and antimicrobial activities(35).

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Protease inhibitor Protease inhibitor l Protease inhibitors ll

Table 1: Potato protease inhibitors(35) Molecular Isoelectric point Inhibited mass SU enzyme (kDa) 7.68 - 7.87 5.1–6.3–7.2–7.8 5 T, Q 5.5–5.8–5.9–6.0–6.1–6.5– 20.02 - 20.68 2 T, Q 6.9

Aspartate protease inhibitor

19.87 - 22.03 6.2–7.5–8.2–8.4–8.6–8.7

1

Cysteine protease inhibitor

20.1 - 22.7

Kunitz-type protease inhibitor

20.19 - 20.24 8.0–9.0

1

Other serine protease inhibitors

21.03 - 21.80 7.5-8.8

1-2 T, Q

Carboxypolypeptidase inhibitor

4.20

1

5.8–6.6–6.7–7.1–8.0–8.3– 1 >9

not determined

T, Q, CD T, Q, Pap. T, Q

CA

SU= subunits. T = trypsin; Q= chymotrypsin; CD= cathepsin D; Pap= papain; CA= carboxypolypeptidase A.

In the past, PIs were only considered antinutritional factors; however, they have recently aroused interest because they have multiple biological activities. Potato PIs have been studied for their antimicrobial effect, anticancer activity and regulation of feed consumption related to the modulation of cholecystokinin through trypsin inhibition(35). The high stability of antifungal activity of potato PIs I and II at high temperatures opens a new market for starch producers, due to the potential use of these peptides in the food, pharmaceutical or agricultural industry(27). In some experiments(35,37), potato PIs I and II reduced the growth of several fungi; while members of the Kunitz family (proteins Potide-G, AFP-J, Potamin-1 and PG-2) inhibited pathogenic bacteria (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli or Candida albicans). In an in vitro study(37), Potamin-1 inhibited the growth of different plant pathogens, and also had inhibitory activity against the enzymes trypsin, chymotrypsin and papain. The peptide Potamin-1 is currently the most mentioned in the literature to explain the mechanism of action of PPC in animals(38).

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Other potato proteins Within the group called “other potato proteins”, cysteine-rich peptides such as thionines, defensins, lectins and snakins are included(39). Thionines base their antimicrobial activity on the interaction with the phospholipid membrane of pathogenic microorganisms. Defensins seem to act on specific membrane receptors, however, information on these peptides is limited(35). Snakin peptides are part of an independent group. They are within the classification of other potato proteins due to their practically zero similarity to any other type of potato peptide(35,40). Snakins are involved in biological processes such as cell division, elongation, and growth and signaling in potato defense. Two types of snakin peptides are found [snakin-1 (SN1) and snakin-2 (SN-2)] and although they only share 38 % of their structure, they have similar functions. Both are peptides rich in cysteine residues with reported activity against Gramnegative and Gram-positive bacteria (C. michiganensis, R. solanacearum, E. chrysanthemi and R. meliloti), as well as against some fungi(35,39). Their spectrum of antimicrobial activity against bacterial and fungal pathogens is quite similar to each other and different from that of defensin peptides from the same tissues. However, the expression of the SN2 gene is induced in the potato by a local wound and shows differential responses to infection by pathogens. The expression patterns and antimicrobial activities of SN2 are consistent with its participation in the constitutive and inducible defense barriers of the potato(41).

Potato protein concentrate in the feed of weaned piglets There are about 5,000 varieties of potato, originating mainly from the Andes, with variants in size, shape, color, texture, as well as nutritional profile. About 10 varieties are cultivated, and the most cultivated variety worldwide is the species Solanum tuberosum. China is responsible for 80 % of the world’s production of this variety. The chemical composition of the potato is modulated by different factors such as geographical area and cultivation practices; its protein content in the fresh state varies between 0.49 and 2.7 %(42,43). In addition to consumption in fresh form, the potato is also used to obtain starch, fiber and juice. In the European Union alone, 8 million tonnes of potatoes are processed annually. PPC is a coproduct of the starch industry, by recovering the liquid fraction that remains after its extraction(44). The process is based on thermal coagulation followed by protein separation and drying(27,45). Potato proteins are typically classified according to their molecular mass and electrophoretic separation into three large groups: patatins, protease inhibitors and other proteins(35). Patatins represent about half of potato proteins and are glycoproteins with

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molecular weights of 39 to 43 kDa, with enzymatic activities (hydrolases, phospholipases and glucanases)(35). The potato has some antinutritional factors such as glycoalkaloids of solanines (927 – 2,632 mg/kg) and trypsin inhibitors (0.97 - 3.70 mg/kg), where the concentration can be variable according to the conditions of processing(44). Thermal processing (100°/15 min) before drying is capable of inactivating up to 48 % of protease inhibitors and up to 89 % of glycoalkaloids(46). PPC is an ingredient that contains adequate amounts of essential amino acids, which can replace animal protein in piglet diets(47), as it has been characterized by a highly balanced amino acid profile and is especially rich in lysine, methionine, threonine, tryptophan and valine. In piglets that received a diet with PPC with the same level of inclusion as fishmeal, an improvement in the growth rate of piglets was observed(48), which can be attributed to the quality of its amino acid profile. Thus, the nutritional value of PPC is related to the concentration and availability of amino acids since it has a profile similar to that of soybean(49) and some animal proteins(50). The proportion of eight essential amino acids (threonine, valine, methionine, isoleucine, leucine, phenylalanine, lysine and tryptophan) corresponds to 40.7 % of the PPC protein(42). The amino acid profile of potato protein completes or exceeds the ideal protein profile, except for tryptophan and lysine with 64.5 and 89.75 % of the requirement, respectively. In 2008 and 2009, three articles were published (28,32,33) in which a refined potato protein (RPP or PP) purified at laboratory level from a special variety of potato (Solanum tuberosum L. cv. Golden valley) was used. This protein fraction showed an inhibitory effect on the in vitro growth of pathogenic bacteria, so they studied the incorporation of RPP or PP at different levels in the diet of newly weaned piglets and compared it with a control diet with antibiotics. Results varied between studies, and an advantage of using the diet with antibiotics was observed in the three studies. When the authors(33) used diets with 0, 200, 400 and 600 ppm of RPP, it was reported that the increase in levels of inclusion in the diets linearly improved the productive performance and reduced the populations of total bacteria, coliforms and Staphylococcus spp. in the contents of the colon and rectum and in feces. The apparent fecal digestibility of dry matter and crude protein, as well as apparent ileal digestibility of amino acids, did not differ between pigs fed the control diet (0 ppm) with antibiotics and diets with RPP(33). With higher levels of inclusion (0.0, 2.5, 5.00 and 7.50 g PP/kg of diet), researchers(28) also observed a linear improvement in feed efficiency during the 28 experimental days and an increase in the apparent fecal digestibility of dry matter in phase II (0 to 14 days postweaning) with the increase in the level of inclusion of PP. A linear decrease in fecal bacteria 517


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was also observed on days 21 and 28 with the increase in the level of inclusion of PP. Piglets that consumed the diets with PP had a linear reduction of total bacteria, coliforms and Staphylococcus spp. in the cecum and rectum. The apparent ileal digestibility of amino acids and the morphology of intestinal villi and crypts were not affected by the consumption of experimental diets(28). In another study(32) with piglets fed diets with 0.0, 0.25, 0.50 or 0.75 % potato protein (PP) during phase I (0 to 14 d post-weaning) and phase II (14 to 28 days post-weaning), the authors observed that increasing levels of PP linearly improved daily weight gain, daily feed consumption and feed efficiency in both phases (I and II); in addition to increasing the digestibility of the dry matter in phase II. Consumption of diets with increasing levels of PP linearly reduced bacterial populations in feces and contents of the cecum, colon and rectum. The height of the villi and the depth of the intestinal crypts did not vary with the increase in the level of PP in the diet. These three studies(28,32,33) opened an area of opportunity for the use of potato protein concentrate generated in the starch industry in animal feed. The use of PPC of commercial origin(22) in growing pigs (25 kg live weight) showed a high ileal digestibility of nitrogen, both standardized (93.0 %) and apparent (85.8 %). The apparent ileal digestibility and the digestibility of most AAs were similar to the digestibility of soybean protein concentrate and isolate, with the highest standardized and apparent ileal digestibility of leucine (96.3 and 94.7 %, respectively) and threonine (94.7 % and 86.9 %, respectively) standing out in PPC(22). In another study(47) with growing pigs (21 kg live weight), the inclusion in the diet of 17.5 % PPC with a low concentration of glycoalkaloids and low trypsin inhibitory activity did not affect daily weight gain, daily feed consumption or the relative weight of stomach, duodenum and jejunum, as well as other aspects of intestinal morphology. Protein digestibility was lower in pigs fed the diet with PPC than with the diet with casein, however, the apparent ileal digestibility of fat was better in piglets with PPC. The authors conclude that the diet with a high level of PPC was well used by growing pigs(47). The good results to the consumption of PPC observed in growing pigs are probably related to its nutritional characteristics; however, due to the reported antimicrobial properties, the use of PPC is more recommended for its inclusion in diets for newly weaned piglets. In 2005, it had already been observed(48) that piglets fed diets with 6 % PPC had a greater daily weight gain in the first 1-21 and 21-50 d post-weaning than piglets fed diets in which they included fishmeal, sunflower meal and gluten meal in similar amounts. Mortality in the period was low (about 4 %) and the severity of diarrheas was slight (1.6 points) in all piglets(48).

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Some authors(51), using PPC with a low level of glycoalkaloids (PPCLG) in diets of newly weaned pigs, found no differences in the productive behavior with respect to diets with animal plasma. The authors observed a quadratic response in weight gain and feed consumption in weaned piglets when using increasing levels of inclusion of PPCLG, replacing 25, 50, 75 and 100 % of animal plasma in the diet. Feed efficiency improved linearly with the inclusion of PPCLG. It was concluded that PPCLG may be an effective substitute for a part of the pig plasma in diets for weaned piglets(51). During the first week after weaning(52), it was observed that daily weight gain and feed efficiency were also not different between piglets that consumed dehydrated porcine plasma or PPC in their diets. During the second week post-weaning and in the total experimental period, the daily feed consumption was similar among all animals. The apparent ileal digestibility of crude protein was higher in piglets fed antibiotic and PPC. The apparent total digestibility of dry matter and energy was higher for piglets fed PPC than the other diets. The severity index of diarrheas in piglets fed the PPC diet was similar among piglets fed the control diet with antibiotic(52). These results demonstrate a potential use of PPC for newly weaned piglets.

Conclusions In recent years, the search for alternatives to the use of antibiotics in feeds as promoters of growth and intestinal health in newly weaned piglets has increased. The potato is some food rich in antimicrobial peptides (AMPs) and other proteins that are involved in its defense system, which have already been purified at the laboratory level. The industry of starch extraction from the potato generates a large amount of a protein concentrate, which probably preserves these AMPs. In this context, potato protein concentrate could exert a positive effect on the productive development of pigs due to its nutritional value, in addition to the possible benefits of its AMPs on intestinal health. However, for it to be considered as an alternative to the use of antibiotics in starter diets, it is necessary to have more bibliographic evidence on the presence of these peptides in commercial products based on potato protein concentrate available on the market, and if their beneficial effects persist at the gastrointestinal level, decreasing post-weaning diarrheas.

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37. Kim JY, Park SC, Kim MH, Lim HT, Park Y, Hahm KS. Antimicrobial activity studies on a trypsin–chymotrypsin protease inhibitor obtained from potato. Biochem Biophys Res Commun 2005;330(3): 921– 927. 38. Cisneros JS, Cotabarren J, Parisi MG, Vasconcelos MW, Obregón WD. Purification and characterization of a novel trypsin inhibitor from Solanum tuberosum subsp. andigenum var. overa: Study of the expression levels and preliminary evaluation of its antimicrobial activity. Int J Biol Macromol 2020;158(1):1279-1287. 39. Kovalskaya N, Hammond RW. Expression and functional characterization of the plant antimicrobial snakin-1 and defensin recombinant proteins. Protein Expr Purif 2009;63(1):12–17. 40. Segura A, Moreno M, Madueño F, Molina A, García-Olmedo F. Snakin-1, a peptide from potato that is active against plant pathogens. Mol Plant Microbe Interact 1999;12(1):1623. 41. Berrocal-Lobo M, Segura A, Moreno M, López G, Garcıa-Olmedo F, Molina A. Snakin2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection. Plant Physiol 2002;128(3):951-961. 42. Mu TH, Tan SS, Xue YL. The amino acid composition, solubility and emulsifying properties of sweet potato protein. Food Chem 2009;112(4):1002–1005. 43. Wijesinha-Bettoni R, Mouillé B. The contribution of potatoes to global food security, nutrition and healthy diets. Am J Potato Res 2019;96(2):139–149. 44. Taciak M, Tuśnio A, Pastuszewska B. The effects of feeding diets containing potato protein concentrate on reproductive performance of rats and quality of the offspring. J Anim Physiol Anim Nutr 2011;95(5):556-563. 45. Pastuszewska B, Tuśnio A, Taciak M, Mazurczyk W. Variability in the composition of potato protein concentrate produced in different starch factories—A preliminary survey. Anim Feed Sci Tech 2009;154(3-4):260–264. 46. Wojnowska I, Poznanski S y Bednarski W. Processing of potato protein concentrates and their properties. J Food Sci 1982;47(1):167-172. 47. Tuśnio A, Pastuszewska B, Święch, Taciak M. Response of young pigs to feeding potato protein and potato fibre - nutritional, physiological and biochemical parameters. J Anim Feed Sci 2011;20(3):361–378. 48. Sardi L, Paganelli R, Parisini P, Simioli M, Martelli G. The replacement of fishmeal by plant proteins in piglet production. Ital J Anim Sci 2005;4(suppl. 2):449-451.

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https://doi.org/10.22319/rmcp.v13i2.5960 Review

Apis mellifera in Mexico: honey production, melliferous flora and pollination aspects. Review

Fernanda Baena-Díaz

a*

Estrella Chévez b Fortunato Ruiz de la Merced a Luciana Porter-Bolland a

a

Instituto de Ecología, AC. Departamento de Ecología Funcional, Carretera Antigua a Coatepec No. 351, El Haya, 91070, Xalapa, Veracruz. México. b

Universidad Nacional Autónoma de México. Posgrado Ciencias de la Sostenibilidad, Ciudad de México, México.

*Corresponding author: fernanda.baena@inecol.mx

Abstract: The honeybee, Apis mellifera, is a species that, since its introduction to Mexico, has had great social, cultural and economic importance, representing an important source of income for thousands of families who are engaged in beekeeping. However, in the context of the so-called “pollinator crisis”, it is considered that we do not know how this phenomenon affects A. mellifera in Mexico. In review, it is analyzed and discussed the information about A. mellifera in Mexico related to the phenomena that affect its distribution, honey production and its ecology, including interactions with the local flora. In general, it is considered that there is a need for an integration of data on beekeeping at the national level, and that there are few studies on the ecology of A. mellifera in Mexico, from the flora they visit, their efficiency as a pollinator and competition with other native bee species. It is believed that increasing studies on A. mellifera will help to predict changes in honey production as well as understand and address threats to these pollinators, contributing to

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generate better management practices and establish better pollinator conservation strategies that include the presence of A. mellifera. Key words: Apis mellifera, Beekeeping, Pollination, Honey, Hives.

Received: 09/03/2021 Accepted: 03/09/2021

Introduction From the identification of the so-called “pollinator crisis”(1,2), which identifies the collapse of different groups of pollinators in various parts of the world, especially in North America and Europe, much interest has arisen to understand the role of the honeybee, Apis mellifera, in the different ecosystems where it lives. This interest results from the fact that A. mellifera is a species of great importance for humans for providing goods such as honey, wax, pollen, propolis and other derivatives of the colony(3,4), as well as for its role as a crop pollinator(5). Currently, the commercial, cultural, nutritious and medicinal value of honey has caused that, of the eleven species existing in the genus Apis, the species A. mellifera (Apidae: Apini), known as honeybee or in some localities as swarms or European bee, due to its origin, is the most valued worldwide(6). In Mexico, A. mellifera, despite being an introduced species, has a great cultural and commercial value, heir to the importance that the Mesoamerican peoples gave to bees because they were part of their traditional activities(7). Given the environmental crisis of pollinators, the phenomenon of collapse in Latin American countries seems less accentuated, and the effects on bee populations respond to processes related to the type of beekeeping management, land use change and the type of agricultural practices(8). In this context, it is necessary to review the current state of knowledge about this species in our country, in order to know its role in the productive aspects, its insertion in ecosystems, and analyze the threats to which they are subject. Likewise, identify the information gaps that exist and should be reviewed in the context of the “pollinator crisis” and climate change, as phenomena that represent threats to natural and managed populations of pollinators. Identifying these points would help establish better management and action strategies at the national level to support this important activity. This paper aims to assess what is known about A. mellifera in Mexico from an ecological and socioeconomic perspective from a review of the literature and official government data.

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A bit of history

The presence of the honeybee, A. mellifera, in Mexico, and its importance as an ecological and social element within the country is the result of different processes that take to back to Mesoamerican cultures and the time of the Spanish colony, when, around 1760 and 1770, A. mellifera was introduced(9,10). The management of meliponines or stingless bees (Apidae: Meliponini) represented an activity of important cultural value in different Mesoamerican peoples (e.g., Mayans, Nahuas and Totonacs;(9-11). This biocultural relationship with stingless bees was also transferred to A. mellifera, replacing the products obtained from the colonies of stingless bees, although it did not completely displace them(12). Despite its presence since colonial times, beekeeping began as an activity of economic relevance until the mid-twentieth century(13). Since then, different varieties of A. mellifera have inhabit practically the entire continent, and, in the warm areas, they are almost all Africanized, a process that has happened over more than half a century since their arrival on the continent in 1956(14). Due to the above and due to the use of the different floral resources where beekeepers move or keep their bees, A. mellifera is currently established in most of the ecosystems of this country(15) (Figure 1). Figure 1: Records of the presence of Apis mellifera in Mexico from data obtained in the Global Biodiversity Information Facility, GBIF (without duplicate data) and changes in beehives per state

The colors of each state indicate whether in the period from 2009 to 2018 (SIAP) there has been a decrease (red), increase (green) or no change (yellow) in the number of hives recorded (negative binomial analysis)

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Abundance and distribution of colonies

The success in the introduction of Apis in most of the world is because it is a generalist or polylectic species, that is, it can visit a great diversity of flowering plants to collect nectar, pollen and resins, and produces large amounts of honey that can be used by people. Considering that A. mellifera is an exotic species and of great economic importance in Mexico, its geographical distribution and abundance in the different ecosystems, both managed and wild colonies, still lacks precise information to analyze its spatiotemporal distribution. A review of the official records obtained from the database of the National Commission for the Use and Knowledge of Biodiversity (CONABIO, for its acronym in Spanish) and the GBIF (Global Biodiversity Information Facility), shows us that, for Mexico, there are only 1900 records of A. mellifera (without duplicate data; Figure 1). This contrasts with the data from beekeepers’ associations and SAGARPA data, which, up to 2016, estimated around 45 thousand beekeepers who manage around 1.9 million hives throughout the country(6,13), and 2018 censuses estimate around 2.172 million hives(16). This discrepancy highlights the need to integrate productive information with biological information to have a better understanding of the situation of A. mellifera in Mexico, not only of its distribution in apiaries but also of those colonies that have escaped management. In a review of the national agricultural survey developed by INEGI in 2016, was found that there are 7,080 apiaries with at least one hive, and they estimate an area of approximately 613,090.22 ha of land with apiaries, however, they do not report the number of hives that are kept per apiary, nor is the foraging area of bees clear. Despite having data on the number of hives and an approximation of the number of apiaries, it is still necessary to integrate the geographical information of the location of apiaries, both those that are moved to take advantage of the different flowerings and those with sedentary management. If the estimated area is divided by the number of hives reported by SAGARPA and SIAP (1.9 or 2.17 M), the information suggests that there are between 3 and 3.5 hives per hectare nationwide, which represents a low number. However, it is known know that there are five main beekeeping regions in Mexico (Altiplano, Pacific Coast, North, Gulf and Yucatán Peninsula), and that they do not have the same national representation in honey production, so it is necessary to better integrate the data on the number of hives by territory (region, state, municipality) as well as the foraging area of bees. Likewise, the saturation of hives is influenced by the types of flowering, whether natural or from crops, for example, orange or orange blossom honey that is produced in citrus-growing areas or butter honey from the Mexican plateau and have a greater number of associated colonies(6). Therefore, the nonhomogeneous spatial distribution of hives, as well as the interest of beekeepers in

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seeking certain flowerings to increase the value of honey, results in regions with a higher density of bees and others that are underutilized. In the context of land use change and high levels of deforestation in the national territory(17), it is of vital importance to have information on the ecological quality of the foraging territories that support bee populations, both A. mellifera and native bees. This information could be very valuable to inform the policy that regulates beekeeping by knowing which territories are more favorable to locate apiaries and manage the movement of hives, while promoting better coordination between the associations of the different beekeeping areas of the country. This is particularly for those beekeepers who seek organic certification, which is increasingly demanded by the market, and therefore has been increasing and requires particular environmental conditions for the foraging of bees. On the other hand, very little is known about the wild colonies of A. mellifera, and it is not known if CONABIO records include this type of colonies, which in general belong to Africanized colonies that have escaped human management and have become feral(18,19). The loss of hives due to different factors such as diseases, pesticides, climatic phenomena such as frosts, hurricanes, lack of food due to alterations in flowering, as well as absconding (when bees leave the nest and migrate elsewhere) and swarming (when the colony divides and a large part of the bees leave the nest to form a new one) processes, is a problem little studied in Mexico, despite being commonly mentioned, mainly in the media. A study conducted by Medina-Flores(20), who interviewed 196 beekeepers from 14 states of the republic, revealed that during the winter of 2015-2016, of the total of 41,907 hives they managed, about 33 % were lost. The reasons for this loss were attributed to bad weather, diseases, pesticide use, absconding and swarming. On the other hand, this paper analyzes the official data on the number of hives by state from 2009 to 2018(16) (negative binomial model: number of hives by state ~ year) in order to know if there is evidence of a significant reduction in hives. The result of the analysis revealed that, on the contrary, for 16 states there is a significant increase in the number of hives (Figure 1), while a significant decrease was observed only in 9 states and no change is observed in 7 states. The states with the highest number of hives coincide with the states with the highest honey production (Yucatán, Campeche, Quintana Roo; Table 1). Although beekeeping has grown in some regions and has remained stable in others, this does not necessarily mean that there have been no decreases in the number of colonies, but rather that these could have been replaced or that there is an increase in the number of beekeepers.

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Table 1: Results from negative binomial analyses per state evaluating the increase or decrease of beehives in ten years (2009, 2018; SIAP) SATE Aguascalientes Baja_California Baja_California_Sur Campeche Coahuila Colima Chiapas Chihuahua CDMX Durango Guanajuato Guerrero Hidalgo Jalisco México Michoacán Morelos Nayarit Nuevo_León Oaxaca Puebla Querétaro Quintana_Roo San_Luis_Potosí Sinaloa Sonora Tabasco Tamaulipas Tlaxcala Veracruz Yucatán Zacatecas

Deviance # Hives~Year

P (Chi-square)

0.08456 7.8445 11.114 5.9368 12.716 24.87 36.623 0.85191 0.59024 12.886 19.71 15.982 15.921 5.5714 12.899 0.44123 111.34 5.887 13.428 49.825 3.509 0.029103 83.473 143.68 64.141 38.018 88.249 25.088 0.26805 20.788 0.090502 26.363

0.000212 0.005097 0.0008569 0.01483 0.0003626 6.13E-07 1.43E-09 0.356 0.4423 0.000331 9.01E-06 6.39E-05 6.60E-05 0.01826 0.0003287 0.5065 2.20E-16 0.01525 0.0002478 1.68E-12 0.06104 0.8645 2.20E-16 2.20E-16 1.16E-15 7.01E-10 2.20E-16 5.48E-07 0.6046 5.13E-06 0.7635 2.83E-07

*** ** *** * *** *** ***

*** *** *** *** * *** *** * *** ***

*** *** *** *** *** *** *** ***

Analyses were performed in R 3.5 (R Development Core Team, 2011) with MASS package (Venables and Ripley; 2002+). Asterisks indicate significant effect of year over the number of beehives. +Venables WN, Ripley BD (2002). Modern Applied Statistics with S, Fourth edition. Springer, New York. ISBN 0-387-95457-0,

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Honey production in Mexico Current beekeeping is present to a greater or lesser degree in the 32 states that Mexico comprises, according to data from the Secretariat of Agricultural, Rural Development and Fisheries (15). The benign climate in much of Mexico makes it possible for the colonies of A. mellifera remain active throughout the year(14), as well as the great diversity of plants and ecosystems that allow a great variation in the quantity and quality of the honey that is produced, which sometimes gives an added value to this product(21). Due to the diversity of these ecosystems and the socioeconomic characteristics of beekeepers, the activity is carried out under two schemes 1) Fixed or sedentary beekeeping, where the apiaries that contain the hives are kept in the same place throughout the year, and 2) Transhumance or mobile beekeeping. In this, the apiaries are moved to different sites throughout the year, according to the flowerings of interest of the beekeeper(22). Fixed beekeeping is favored in places where floral resources remain more or less constant throughout the year or in cases where beekeepers decide to make fewer annual harvests, of a smaller scale. Transhumance is favored in places with greater seasonality or fewer floral resources and is a strategy used to increase the number of annual harvests(22). In any of its two forms of use of nectarpolliniferous resources, the beekeeper learns to know the behavior of the flowering seasons and schedules the harvest times, so that, depending on the place and management techniques used, one, two or even three or more annual harvests can be achieved(23). Thus, the amount of honey produced per hive depends in part on the nectar-polliniferous resources present in the different beekeeping areas of the country, although there are other factors such as the time of year, the ecosystem, diseases, as well as the investment capital of beekeepers(24). For approximately 30 years, the management of the Apis colonies has led to Mexico being among the ten most important countries in honey production worldwide(25). Among the most important regions in terms of honey production are the Yucatán Peninsula (Campeche, Yucatán and Quintana Roo), Jalisco and Veracruz(16). Historical data on honey production indicate that the number of hives and total honey production increased substantially from the 60s and were on the rise before 1986, when the Africanized bee was first recorded in the south of our country (a hybrid of European varieties with African varieties;(26,27); (Figure 2). Nevertheless, Mexico ranked third worldwide in 1991, with 63,886 t(26). However, honey production has been decreasing since 1986 and Mexico ranked ninth in honey production in 2017, with 51,066 t(25) (Figure 3).

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Figure 2: Historical data on the number of hives, total honey production per year (tons) and honey production per hive (kg) in Mexico from 1961 to 2018 (FAOSTAT, 2020)

The red line indicates the year that was recorded as the beginning of the process of Africanization of the European bee in Mexico.

Figure 3: Honey production in Mexico in the last 31 years (1986-2017). Data from (FAOSTAT, 2020)

On the other hand, honey exports before 1990 represented a very low percentage of total production (between 21-33 %). However, as of 1990, the average export of honey increased to 52.5 %, with around 30,333 t per year between 1991 and 2017(25) (Figure 4). Thus, in some years, exports represented around 40 % of total production, while 2015, which was one of the best years for export, represented 68.1 % of total production(25).

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Figure 4: Honey production and export from 1986 to 2017 (FAOSTAT, 2020)

The dotted line indicates the year in which the record of Africanization began in Mexico

This despite the increase in restrictions and demands of the international market, and the market fluctuations that are affected by different aspects, both internal and external(28). The main importing countries of Mexican honey have been Germany, the United States and the United Kingdom, countries with a long tradition of consumption. In contrast, domestic consumption of honey is very low(28), and the percentage of losses for beekeepers from production that is not exported and not consumed locally is unknown. In summary, despite Africanization, Mexico has positioned itself as one of the largest producers of honey and its production has increased compared to decades prior to the entry of African bees. However, the decrease in hives and the declines in honey production in recent decades have been attributed to multiple reasons that together have affected beekeeping activity(6,13). One of the main reasons was the arrival of Africanized bees, which produce less honey, swarm easily, and having a more defensive behavior have generated economic losses due to damage caused, which caused many beekeepers to abandon the activity(27). Despite this, Africanization has not had the same effects in all regions and, in some, it could even have benefited beekeeping, because they are better adapted to tropical environments and because they became a source of colonies (collected from the field) for beekeepers(29). Since the Africanization process began in the country, in the 1980s, the National Program for the Control of the African Bee was developed to counteract these negative effects and integrate its presence into management(30). On the other hand, the decrease in production in the mid-90s coincides with the presence of the mite Varroa destructor, first reported in 1992 in Mexican territory(31). This parasite 533


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infests the hives and feeds on the hemolymph of the bees, promoting the entry of other diseases associated with different viruses(32), affecting the reproduction and population of the colony, which means a lower production of honey and, in extreme cases, its death(33). This disease is currently controlled with the application of drugs based on components such as thymol, oxalic acid and formic acid, among others(34). However, there are controversies regarding their use and abuse, and even organic certification measures limit the type of drugs that can be used(23,35). Another recent problem has been the proliferation of the small hive beetle (SHB) Aethina tumida Murray 1867, since it was first reported in Mexico in 2007(36) or the case of fungi of the genus Nosema, which have been found in the country since 1965(37). The consequences of such diseases should not only be measured in the context of the loss of colonies of A. mellifera, but they could have ecological consequences when transmitted to other pollinating insects. Another aspect that may be relevant in honey production in Mexico is the frequent presence of natural phenomena, such as hurricanes and storms, which can be further altered by the effects of climate change(38,39). Changes in flowering times, resulting from drought events or alterations in rainfall patterns, are another destabilizing factor for beekeeping(40); because beekeepers need to coordinate and anticipate the flowering time to ensure that hives are ready for honey harvest time(23). Among the regions with the highest incidence of these phenomena, the Yucatán Peninsula and Veracruz stand out, which together represented 45 % of the total honey production in 2018, and the Pacific coasts, especially the states of Jalisco, Guerrero, Michoacán, Chiapas and Oaxaca, that represented 26 % of production. Future analyses should try to consider how these climatic phenomena alter beekeeping activity in the country. Finally, the availability of resources for honey production depends on the vegetation cover in the different ecosystems. The change of land use towards agricultural uses with intensive and industrial management and the consequent loss of floral diversity mean fewer floral resources for bees(41). In addition to the problems with the use of toxic agricultural chemicals and even the use of genetically modified organisms that affect the health of bees (42). However, the interaction of forest cover loss or change and its effects on both native and introduced bees is another situation that has been little studied in Mexico(43,44). All these factors contribute to the fluctuations in production and number of hives reported in this study. In addition, the variation in the quality of honey, its floral origin, associated with the variation in honey production, are directly related to its commercialization, since the standards in the regulation of honey for export must be met (see NOM-004-SAG/GAN2018). According to Soto-Muciño and collaborators(6), in Mexico, beekeepers with high commercial power have decreased and beekeepers with small and medium production have increased. This could represent a window of opportunity to promote beekeeping in regions where the management of Apis mellifera is a complementary activity within the agricultural 534


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and livestock practice, as is already the case in the north of the country, as well as to promote beekeeping, especially under agroecological management, and that it is a source of income and family employment. An important part to promote the care of bee colonies is to improve production conditions and have a better assessment of the state of beekeeping in Mexico and have better data on the number of hives, the socioeconomic characteristics of beekeepers in different regions, management statistics, and identify their threats in different contexts. Likewise, knowledge about apiary distribution areas, flowering calendars, and coordination between beekeepers to avoid oversaturation of foraging areas are required.

Characterization of honeys and botanical origin

Despite the commercial value of Mexican honey, which is recognized in the Official Mexican Standard (NOM-004-SAG/GAN-2018), and that there is a general characterization of some honeys at the regional level (e.g., multifloral honeys from coffee plantations, or monofloral such as that of orange blossom from citrus flowering (Citrus sp.)), there are relatively few studies that evaluate the organoleptic characteristics of honeys (color, taste, smell, etc.)(45), as well as the botanical composition of honeys(46,47) (Table 1). These studies are important not only to provide commercial added value to honey in each of the regions, but also to know the interactions of A. mellifera with the plants it visits and its possible role in ecosystems. From a non-exhaustive review of the literature on the melliferous and polliniferous floras of A. mellifera, considering papers, book chapters, theses and publications in congresses, about 30 studies that characterize the diversity of plants visited by honeybees were found for Mexico (Table 2). Many of these studies are based on melissopalynological studies, that is, the analysis of pollen grains contained in honey to determine the species used by bees. However, some works are based on literature and direct observations of visits and interviews with beekeepers to learn about the plants in the region that bees visit to obtain nectar and pollen. From this review, it can be said that A. mellifera visits an average of 43 plant species per locality, for which the diversity of plant families varies in a range of 16-60 families. Although the number of species visited seems to be high, still not known what percentage it represents of the total native flora in each region and whether honeybees provide them with an effective pollination service. Only for the Yucatán Peninsula, it is estimated that Apis visits 40 % of the total flora(48). Another important aspect is that A. mellifera does not always collect pollen from the plants visited and only goes for nectar, so nectariferous plants may be underrepresented in melissopalynological studies(49).

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Table 2: Records of studies on melliferous floras in different states of Mexico for Apis mellifera from 1994 to 2019 State QR QR CAM

TAM COL VDM MOR YUC, CAM, QR YUC, CAM QR

Method Palynological analysis of nectar Analysis of pollen taken from the hive Interviews with beekeepers and field observations It does not describe methodology (the whole state) Literature review and observations of visit Melissopalynological Melissopalynological Melissopalynological Melissopalynological

Local (n) 1

GUER GUER DUR BC TAM Páztcuaro, MICH Hopelchén, CAM

Melissopalynological

Family (n)

Species /types (n)

Ref.

-

148

(49)

2

206

41

168

(50)

1

-

35

146

(51)

1

-

-

50

(52)

1

-

45

140

(53)

2 3 40 (total)

2 3

15 23

19 41

(54) (55)

78

15

250

(56)

168

36

238

(57)

56

26

92

56

19

66

55

36

80

39

29

64

(58)

36 32

129 63

(59) (60)

53

143

(61)

27 16 33 60

43 22 150 215

(62) (63) (64) (65)

33

93

(66)

26

56

(67)

17 (total) 10 YUC) 5 (CAM) 2 (QR)

YUC CAM QR OAX TAB TAB

Sample (n) 44 (22 and 22)

4

Melissopalynological 4 40 Melissopalynological 3 12 Literature review and observations of visit Melissopalynological 2 12 Melissopalynological 3 3 Melissopalynological 13 52 Melissopalynological 11 27 Direct observation of 1 4 visits and interviews Interview and 40 1 Herbarium review produces Local= municipality, region, state.

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The role of A. mellifera as a pollinator

Although A. mellifera is very productive in terms of its establishment and honey production, it turns out that, from the perspective of plants, it is not necessarily the most efficient pollinator(1,68). This means that, despite transporting pollen from one flower to another, the amount and place where it deposits it is not necessarily the most suitable for the plant to maximize seed production, and they can even be nectar robbers, that is, they take the nectar without making contact with the androecium and gynoecium of the flower. Despite this, it has been shown that A. mellifera is one of the most important crop pollinators so that millions of bees are managed for this purpose globally(69). Perhaps one of the best-known examples is that of almond cultivation in California(68), where Apis is used as the main pollinator. It is for this reason that the importance of studying and knowing the efficiency of A. mellifera as a pollinator of both the local flora and the crops is highlighted. In Mexico, the Beekeeping Pollination Manual(70) includes recommendations for the use of Apis for pollination of different crops (citrus, cucurbits, cotton, etc.). Despite being a species widely used in crops, its efficiency as a pollinator compared to other pollinators is unknown. In Mexico, studies on the efficiency of pollination by A. mellifera in crops are still limited(71-77). Some studies have even excluded the Apis data because it is very abundant, to focus on native bees, so its role in pollination is not known(78). The studies reviewed reveal that, for some crops, A. mellifera is not the most effective pollinator, as in the case of tomatoes and habanero peppers(73) or as in coffee(79). In the case of the squash species Cucurbita moschata (Cucurbitaceae), although Apis is not the best pollinator (at each visit), its effectiveness is compensated by being very abundant(71). In addition, it was found to be a very important pollinator during the time of year where the main pollinator is absent (71). However, another study on pollination networks in other Cucurbitaceae species (melon, squash, cucumber and watermelon) did not report any visits by Apis(80), indicating that the role of Apis as a pollinator is variable. In some cases, Apis turns out to be a pollinator as efficient as other native bee species(81), and in others a very important one, as in avocado(72), or even more efficient than other pollinators(77); while in other crops it is irrelevant, as is the case of rambutan, where no visits by Apis were observed(74). In addition, little is known about the effect of A. mellifera on species of economic importance that are not cultivated. For example, in different species of Agave, it was found that A. mellifera is a nectar stealer, that is, it consumes the nectar of the flower but does not pollinate, and in other species it turns out to be a secondary pollinator during the hours of the day when there is less production of pollen and nectar(82). Finally, it can be said that the efficiency of Apis as an effective pollinator in native species of no commercial importance has been evaluated very little. An example is the work on Kallstroemia grandiflora, where it was found that Apis is as efficient as native pollinators(83). Although there is information in the literature about the

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visit of Apis to non-cultivated plants, particularly studies on plant reproductive biology, this information deserves to be reviewed to complement the knowledge about the interaction of Apis with native plants, but it has not been the subject of this review. Studies at the landscape level and its effects on pollination indicate that Apis mellifera can take advantage of landscapes modified for agricultural or urban uses. A study that compared the effect of shade and sun coffee plantations on bee diversity found that shade coffee plantations harbor greater diversity of bee species, including A. mellifera, but that it substantially preferred sun coffee plantations, which have less plant diversity and where the diversity of native bees was lower(84). In European and South American landscapes, studies suggest that, due to its characteristics, Apis seems to adapt and is abundant in highly transformed landscapes, including urban areas and semi-natural forests or with little plant diversity(85-87). Finally, studies on pollination networks are important because they help to understand more comprehensively the role of A. mellifera as a floral visitor of different species and its possible interaction with other pollinators. Nevertheless, in Mexico, studies on pollination networks are very few(80,88-92). These studies reveal that A. mellifera is a very abundant species and that it has a large number of connections within the network (91). Given this evidence, there is a need for more studies to evaluate the role of A. mellifera in the different ecosystems of Mexico, in its role as a pollinator and in that of its interactions.

Competition with native bees

One aspect that should not be forgotten is that Apis is an introduced species and can therefore have negative effects on local fauna, particularly on other bees with which it might be competing for resources. This competence can occur in different ways, and at least seven have been described(93). Of these, the most studied are the reduction of pollen and nectar in a community due to the presence of Apis; the exclusion of native bees due to prolonged foraging times in floral patches, forcing native bees to travel further in search for resources; the active movement of native bees, mainly in the case of Africanized bees and the transmission of parasites of Apis to native bees(93). In Mexico, there are still relatively few studies on the matter(12,79,83,94,95) and of which the work of Villanueva-Gutiérrez et al(12) in Quintana Roo stands out, which showed that A. mellifera and the native bee Melipona beecheii, in a context of abundant floral resources, avoided competition by diversifying resources, that is, they avoided visiting the same plants. A similar result was found in studies of competition between Apis and three other native bees, among them Partamona bilineata (a stingless bee), in squash and watermelon crops in Yucatán(95). Another study suggests

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that Apis displaces native pollinators in coffee plantations because it was found that the greater the presence of A. mellifera, the lower the richness of other bee species(79). Several studies in other parts of the world have shown that Apis mellifera is capable of displacing native bees; however, critics of these studies argue that competition has not been demonstrated because these studies have not explored the effects on the adequacy of native bees(93). However, evaluating the success of native bees against interaction with Apis is very complicated if one considers that for most native bee species is know practically nothing about their natural history, even for most species only females have been described [personal communication from experts in the area(96)]. Finally, the arrival of the Africanized variety of Apis mellifera could have altered the relationship with other native bees, since these varieties are more aggressive when it comes to defending their hive and floral resources, in addition to the fact that the Africanized bee is more adapted to become feral than European varieties(13,20). Future studies should evaluate the role of feral colonies of A. mellifera on pollination and on other native bees and insects. Competition with other species is mainly for floral resources, although it is not dismissed that they also compete for nesting sites. Studies on this subject are needed. Considering the transformation of the landscape and the reduction of the floral supply in transformed or impoverished landscapes, and the high densities of European bees in some areas of Mexico, it wonders if there would be greater competition between native bees and A. mellifera for plant resources, or if in landscapes with greater floral diversity the competition is less. This point is relevant to the regulations regarding the management of A. mellifera, as well as to the conservation of native fauna and of which there is no evidence.

Conclusions The evidence presented indicates that although A. mellifera is an extremely important species both culturally and economically for thousands of Mexican families, and that, compared to native bees, it has been much more studied, there is still a need for studies that address both productive aspects from the generation of better databases on production, management, diseases, etc., and ecological aspects, such as its interaction with local fauna. On the other hand, and under the current scenario of global change, including climate change, land use changes and pollution, among other aspects, there is a need to have more ecological studies on A. mellifera in Mexico. These studies will help to predict changes in honey production as well as understand and address threats to these pollinators, contributing to the generation of better management practices. On the other hand, it will help to

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understand more about its interactions with other bee and plant species, and to be able to have better conservation strategies that include the presence of A. mellifera. Likewise, it will provide valuable information that contributes to the management of other native bees in order to improve agricultural practices by considering the pollination efficiency of different pollinators, including A. mellifera. Based on this review, it is concluded that studies of pollination ecology that integrate the role of A. mellifera not only in species of economic importance but in other groups of plants are very relevant. Likewise, more studies on the various threats to pollinators in general are needed. Although there are global patterns of the role of changes in the landscape on the loss of pollinators and the service of pollination, which can be extrapolated to the country, the ecological, orographic, and cultural complexity of Mexico demand a better characterization of the current state of pollinators and particularly of A. mellifera, due to its economic and biocultural importance.

Acknowledgements

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https://doi.org/10.22319/rmcp.v13i2.5819 Technical note

Induced lactation in Holstein cattle with no exogenous progesterone supplementation and with reduced doses of estradiol benzoate

Juan González-Maldonado a* Raymundo Rangel-Santos a Gustavo Ramírez-Valverde b Jaime Gallegos-Sánchez c Lorenzo Beunabad-Carrasco d Javier-Antillón Ruiz d

a

Universidad Autónoma Chapingo. Posgrado en Producción Animal. Carretera MéxicoTexcoco. 56230. Chapingo, Estado de México. México. b c

Colegio de Postgraduados. Departamento de Estadística, Estado de México, México.

Colegio de Postgraduados. Departamento de Ganadería, Estado de México, México.

d

Universidad Autónoma de Chihuahua. Facultad de Zootecnia y Ecología, Chihuahua, México.

*Corresponding

author: jugomauabc@gmail.com

Abstract: Lactation induction in cattle is an alternative way to increase the life span of cows with reproductive and productive issues that would otherwise be culled. An alternative lactation induction protocol is proposed. This 21-d protocol takes advantage of endogenous progesterone production and uses low doses of estradiol to stimulate udder growth and function. Holstein cows (n= 5) and heifers (n= 4) were injected with 500 mg of somatotropin on days one, eight, fourteen, and twenty-one. Estradiol benzoate (3-10 mg d-1) was applied

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from day one to fourteen. Cloprostenol (500 µg) was administered to the animals am and pm on d 1, and from d 18 to 21, they were injected dexamethasone (5 mg d-1). The cows and heifers were milked for the first time on d 22. Exogenous progesterone was not supplemented, but lactation protocol began in the diestrus phase of the estrous cycle and GnRH was injected to induce formation of an accessory corpus luteum. Lactation induction was successful in all animals, milk production ranged from 18.3 to 25.5 kg d-1. In conclusion, endogenous progesterone production in combination with reduced doses of estradiol benzoate is sufficient to induce lactation in Holstein cattle. Key words: Bovine, Lifespan, Hormones, Milk.

Received: 02/10/2020 Accepted: 06/08/2021

Artificial lactation induction is achieved by exogenous hormone administration to cows that fail to initiate a new lactation after a dry period(1). The decision to artificially induce a cow to produce milk might be a subject of debate. It is necessary to take into consideration that cows induced to produce milk very often suffer reproductive disorders due to the large number of hormones required to stimulate udder growth and function(2). On the other hand, lactation induction means another opportunity for a cow to avoid culling and to stay productive on the dairy farm for a longer time than would be expected(3). Progesterone and estradiol are supplemented to cows for several days to stimulate udder alveoli and duct growth(4). Progesterone is supplemented to cows by injections and intravaginal devices. Formation of an accessory corpus luteum is an alternative way to increase endogenous progesterone concentration(5). Erb and colleagues reported concentrations of blood progesterone in cows induced to lactation using progesterone injections similar to those reported in cows with an accessory corpus luteum(5,6). Therefore, it seems logical that extra endogenous progesterone produced by an accessory corpus luteum would be enough to sustain udder development during lactation induction. However, what is known, this approach has never been tested in a lactation induction protocol. Also, there are not information of the specific dose requirement of estradiol to induce lactation. Therefore, the tested hypothesis was that lactation induction in cows can be accomplished without exogenous progesterone supplementation and with reduced doses of estradiol. The study was conducted in two phases. Phase one was a pilot test to gather information about the effectiveness of the protocol to induce lactation. This phase was carried out using two 6- to 7-yr-old Holstein cows with at least two calving, and one year off milking. The

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recurrent abortions were the common cause of reproductive failure in both cows, but they were not culled because of their high genetic value. A modification of the methods previously described for lactation induction was carried out in the two cows(7,8). The reproductive cycle of the cows was synchronized by two injections of 500 µg of cloprostenol (Celosil®, MSD Animal Health) administered at 14-d intervals. The cows were monitored every 6 h by visual observation to detect standing heat after the second injection of cloprostenol. A cow was declared in standing heat when she stood to be mounted by another cow. The cows’ ovaries were scanned on d-5 after detecting standing heat by ultrasonography (Aloka Prosund 2, 7.5 MHz transducer, Hitachi Aloka Medical, Ltd Japan). After the presence of a corpus luteum was confirmed, location of the largest follicle was registered, and an injection of 250 µg of gonadorelin acetate (GnRH®, Sanfer) was administered to cows. The ovaries were scanned every 12 h after GnRH injection until disappearance of the previously registered follicle. Once the largest follicle disappeared (within 48 h after GnRH injection), formation of an accessory corpus luteum was assumed(9), and the lactation protocol began. The recombinant bovine somatotropin (Lactotropina®, Elanco), estradiol benzoate (Benzoato de estradiol®, Syntex), cloprostenol, and dexamethasone ((Lapicor®, Lapisa) were used to induce lactation. The protocol lasted 21 d. The cows were injected with 500 mg of somatotropin on days one, eight, fourteen, and twenty-one. Estradiol benzoate (7.5 mg d-1) was applied from d-1 to 14. The udder of each animal was massaged for periods of one to two minutes in the morning and afternoon from d 16 to 20. The cloprostenol (500 µg) was administered to cows in the morning and afternoon on d 18. Dexamethasone (5 mg d-1) was injected from days 18 to 21. The cows were milked for the first time on d 22. The somatotropin was further administered to cows every 14 d after the first milking. The cows (n= 3) had at least two calving and one year off milking, but they were not culled due to their high genetic value. The heifers (n= 4) were inseminated at least five times without pregnancy success. The common cause of reproductive failure in cows was recurrent abortions, while a closed cervix that prevent the delivery of semen into the uterus, and persistent uterine infections were observed in heifers. The reproductive management of animals was the same as in phase I. The same protocol used to induce lactation in phase I was applied to cattle in phase II, but the dose of estradiol benzoate was modified. The animals were assigned to one of four different groups (GI-GIV). Group GI, a 2-yr-old heifer (671 kg), was injected with 3 mg d-1 of estradiol benzoate. Group GII, a 4-yr-old heifer (651 kg) and an 8-yr-old cow (580 kg), were treated with 5 mg d-1 of estradiol benzoate. Group GIII, a 4-yr-old heifer (737 kg) and an 8-yr-old cow (844 kg), were treated with 7.5 mg d-1 of estradiol benzoate. Group IV, a 3-yr-old heifer (753 kg) and an 8yr-old cow (691 kg), were injected with 10 mg day-1 of estradiol benzoate. 551


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The animals in phase I and II were fed corn silage ad libitum and 4 kg of commercial concentrate a day (Ganadero 18, Productos Agropecuarios Tepexpan, S.A. de C.V. (protein 18%, fat 4%, fiber 12%) before the first milking. After the first milking, the cows and heifers were located in the same pen and fed with a mixed ration through the experimental period (21.9 kg of fresh alfalfa, 21.9 kg of corn silage and 7.7 kg of the commercial concentrate as fed once a day to each cow). The measured response variable was daily milk production, which was measured once a week from the first day to day 328 of milking. However, measurements were stalled at 221 d in milk for a heifer in GI due to low milk production. The phase one was a pilot test and their data is not included in the statistical analysis. However, a descriptive analysis of the data from his phase is shown in the results section. An experiment with repeated measures was used in a randomized complete block design, assuming a first-order autoregressive structure among times for the variance-covariance matrix. Only two blocks were used, one made up of heifers (never calving) and the other of cows with more than two calving. The statistical analysis was performed using the statistical package Infostat (2018). Lactation was induced in cows of phase I. Average milk production of each cow was 25.5 ± 1.33 and 24.6 ± 1.18 kg d-1. The milk production pattern across lactation for both cows is shown in Figure 1.

Milk production (kg)

Figure 1: Milk production of cows induced to lactation without exogenous progesterone supplementation 40 35 30 25 20 15 10 5 0 0

17

36

72

86 100 129 154 168 182 238 252 305 342 Days of lactation

Cow 1 (solid line) and cow 2 (dotted line)

Milk production of cow and heifer in GIV was lower (P≤0.05) than that of cows and heifers in groups GII and GIII. Average milk production was 20.06 ± 0.73, 19.54 ± 0.71 and 18.36 ± 0.72 kg d-1 for cows and heifers in GII, GIII, and GIV, respectively. The differences in milk production between GII and GIII were not significant (P˃0.05). The milk production pattern

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of groups GII-GIV is depicted in Figure 2. Figure 3 depicts milk production of the heifer in GI. Statistical comparison between this and the rest of the groups was not possible due to the lack of replications. Figure 2: Milk production of Holstein cows and heifers induced to lactation without exogenous progesterone supplementation and injected with different doses of estradiol benzoate (black line: 5, gray lines: 7.5 and dotted line: 10 mg d-1) for 14 d.

Milk production (kg)

30 25 20 15 10 5

0 8 19 26 33 40 47 54 61 68 89 110 117 125 131 138 166 184 194 221 270 284 291 328

0 Days of lactation

Figure 3: Milk production in a Holstein heifer induced to lactation without exogenous progesterone supplementation and injected with 3 mg d-1 of estradiol benzoate for 14 d

Milk production (kg)

25.00 20.00 15.00 10.00 5.00 0.00 0

8

19 26 33 40 47 54 61 68 89 110 117 125 131 138 166 184 194 221

Days of lactation

A modification of the lactation induction methods described by Mellado et al. and Freitas et al. was proposed(7,8). The main modifications are that no exogenous progesterone is applied, and lower doses of estradiol benzoate and glucocorticoids are used. In addition, hormones (estradiol and glucocorticoids) are prescribed by day and not according to animal live weight. 553


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The results of the present study show that lactation induction can be achieved without exogenous progesterone supplementation in combination with reduced doses of estradiol benzoate. The ovarian follicles and corpus luteum are the main sources of estradiol and progesterone in non-pregnant cattle. Therefore, manipulation of endogenous production of these hormones to favor udder development during lactation induction is reasonable. In this regard, Mohan et al mentioned that milk production was higher in heifers induced to lactation after estrus synchronization protocol than in those induced to lactation on random days of the estrous cycle(10), probably due to a synergic effect of the steroid hormones naturally produced by the new follicles and corpus luteum after estrus synchronization with those injected. The injections of estradiol and progesterone are necessary to induce lactation in cattle(11). An attempt to induce lactation using only estradiol benzoate has been successfully carried out in heifers, but with poor results in cows(12). In addition, average milk production was low (3.4 to 9.6 L d-1), regardless of whether estradiol benzoate was injected alone or in combination with progesterone. The lack of bovine somatotropin in Harnes and colleague's lactation induction protocol might explain the obtained results in milk production. Previous studies reported milk production in cows (18.9-28.4 kg d-1) similar to that found in the present study, but cows were injected with progesterone and with higher doses of estradiol benzoate(7,8). Therefore, based on the results, it seems that endogenous progesterone is sufficient to stimulate udder development and milk production. Estradiol benzoate is prescribed to cattle according to animal live weight(13) or by day(14), as in the present study, to induce lactation. To date, there is no estradiol benzoate prescription to stimulate udder growth and milk production. In the present study, 3 to 10 mg d-1 of estradiol were effective to induce lactation. Researches induced lactation in cattle by injecting 2.4 and 3.3 mg d-1 of estradiol benzoate, which is similar to the lower dose used in this study, but milk production was low (< 7 L d-1)(15,16). The differences in milk production might due to the use of bovine somatotropin. The injection of estradiol can cause a follicular cyst(17). Follicular cysts cause an undesired and recurrent standing heat behavior in cattle. This kind of behavior has been reported in lactation induced cattle(15,18). The heifers and cows used in the present study only showed standing heat behavior once during execution of the lactation induction protocol, but never in an abnormal recurrent way after its termination. Therefore, it can be assumed that reproductive behavior is not altered by the proposed lactation induction protocol. Today, the implementation of induced lactation protocols on dairy farms may seem unnecessary because of the large supply of heifers, which makes the decision of culling cows with productive or reproductive issues easier. However, the easiest decision may not be the most adequate. Reasons for culling cows may be related to reduced animal welfare, which makes man nor the cow itself mainly responsible for shortening the lifespan of high 554


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producing cows on dairy farms(3). Therefore, artificially induced lactation is another chance to improve general management to ensure a larger life span of cows. On the other hand, hormone traces in the milk of lactation-induced cows are a concern for human health(19). However, estradiol concentrations return to basal concentrations 8 d after the last injection, and not differences have been found in estradiol blood concentrations between lactating heifers 17 d after calving and lactation-induced heifers(20,21). Thus, after a period of 8 to 17 d, milk from lactation-induced cows can be used to feed humans or calves. The lack of exogenous progesterone injections and the reduced doses of estradiol benzoate used in the proposed lactation induction protocol is affordable and especially favors small farm holders, who might find it difficult or too expensive to raise or buy a replacement heifer. The disappearance of the largest follicle at d-5 of the estrous cycle and after GnRH injection was taken as a sign for accessory CL formation(9). However, the actual formation of an accessory CL was not confirmed and blood progesterone concentrations were not measured, which stop the authors to conclude that an increment in blood progesterone concentrations occurred in the animals. However, since the lactation induction was successful in cows and heifer, it can be safely state that endogenous progesterone produced by a single or multiple corpus luteum is sufficient to stimulate udder growth during a lactation induction protocol in cattle. The milk yield during lactation in cattle is determined by several factors, such as genetics, season, and parity(22-24). The effect of parity and season on milk yield has been studied in cows induced to lactation, the highest milk production was observed at the fifth lactation, and during the fall and winter(24). An unsuccessful approach to study the effect of genetics on milk production in lactation-induced animals was carried out in ewes(25), it was observed that the number of ewes with a positive response to the lactation induction protocol was affected by breed. In the present study, the cows and heifers were induced to lactation during the same season and they were subjected to the same management practices and nutrition (animals were allocated in the same pen during the entire experiment). In addition, the animals belong to the same breed and the effect of parity was considered during the statistical analysis of the information. Therefore, it is suggested that the differences in milk production among experimental groups could be related to different responses of the animals to the hormonal treatment(25) and the genetic merit for milk production(26). Endogenous progesterone production in combination with reduced doses of estradiol benzoate is sufficient to induce lactation in Holstein cattle. However, the small sample size used in the present study do not allow to fully guaranty the effectiveness of the proposed protocol at commercial level.

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Literature cited: 1. Maffi AS, Luz GB, Gasperin BG, Mattos RF, Xavier EG, Corrêa MN, et al. Induction of lactation: an economic study of tool for dairy heifers with successive reproductive failures. Ciênc Rural 2019;49(12):1-8. https://doi.org/10.1590/0103-8478cr20180661 . 2. Luz GB, Maffi AS, Xavier EG, Correa MN, Gasperin BG, Brauner CC. Endocrine profile and reproductive performance in heifers induced to lactation. Acta Sci Vet 2019;47: 1658. 10.22456/1679-9216.92095. 3. De Vries A, Marcondes MI. Review: Overview of factors affecting productive lifespan of dairy cows. Animal 2020;14(S1): S155–64. 10.1017/S1751731119003264. 4. Magliaro AL, Kensinger RS, Ford SA, O’Connor ML, Muller LD, Graboski R. Induced lactation in nonpregnant cows: profitability and response to bovine somatotropin. J Dairy Sci 2004;87(10): 3290-3297. https://doi.org/10.3168/jds.S0022-0302(04)734657. 5. Howard JM, Manzo R, Dalton JC, Frago F, Ahmadzadeh A. Conception rates and serum progesterone concentration in dairy cattle administered gonadotropin releasing hormone 5 days after artificial insemination. Anim Reprod Sci 2006;95(3–4):224–33. 10.1016/j.anireprosci.2005.10.010. 6. Erb RE. Harmonal control of mammogenesis and onset of lactation in cows–A Review. J Dairy Sci 1977;60(2):155–169. 10.3168/jds.s0022-0302(77)83849-6. 7. Freitas PRC, Coelho SG, Rabelo E, Lana ÂMQ, Artunduaga MAT, Saturnino HM. Artificial induction of lactation in cattle. Rev Bras Zootec 2010;39(10):2268–2272. http://dx.doi.org/10.1590/S1516-35982010001000024. 8. Mellado M, Nazarre E, Olivares L, Pastor F, Estrada A. Milk production and reproductive performance of cows induced into lactation and treated with bovine somatotropin. Anim Sci 2006;82(4):555–559. https://doi.org/10.1079/ASC200656. 9. Schmitt ÉJP, Diaz T, Barros CM, De La Sota RL, Drost M, Fredriksson EW, et al. Differential response of the luteal phase and fertility in cattle following ovulation of the first-wave follicle with human chorionic gonadotropin or an agonist of gonadotropinreleasing hormone. J Anim Sci 1996;74(5):1074–83. 10.2527/1996.7451074x. 10. Mohan K, Shridhar NB, Honnappa TG, Ramachandra SG, Nirmala GC, Jayakumar K. Induction of lactation in repeat breeding crossbred heifers. Indian J Anim Sci 2009;79: 379-380.

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11. Paiano RB, Lahr FC, Poit DAS, Costa AGBVB, Birgel DB, Birgel EH. Biochemical profile in dairy cows with artificial induction of lactation. Pesq Vet Bras 2018;38(12): 2289-2292. 10.1590/1678-5150-PVB-5951. 12. Harness JR, Anderson RR, Thompson LJ, Early DM, Younis AK. Induction of lactation by two techniques: success rate, milk composition, estrogen and progesterone in serum and milk, and ovarian effects. J Dairy Sci 1978;61(12):1725–35. 10.3168/jds.S00220302(78)83794-1. 13. Stark A, Wellnitz O, Dechow C, Bruckmaier R, Baumrucker C. Colostrogenesis during an induced lactation in dairy cattle. J Anim Physiol Anim Nutr 2015;99(2):356-366. 10.1111/jpn.12205. 14. Luz GB, Maffi AS, Xavier EG, Correa MN, Gasperin BG, Brauner CC. Induction of lactation in dairy heifers: milk production, inflammatory and metabolic aspects. Arq Bras Med Vet Zootec 2020;72(2): 371-378. https://doi.org/10.1590/1678-4162-11246 . 15. Fulkerson WH, McDowell GH. Artificial induction of lactation in cattle by use of dexamethasone trimethylacetate. Aust J Biol Sci 1975;28(2):183–187. 10.1071/bi9750183. 16. Fulkerson WJ. Artificial Induction of Lactation: A comparative study in heifers. Aust J Biol Sci 1978;31(1):65–72. 17. Gümen A, Sartori R, Costa FMJ, Wiltbank MC. A GnRH/LH surge whitout subsecuente progesterone exposure can induce development of follicular cysts. J Dairy Sci 2002;85(1):43-50. 10.3168/jds.S0022-0302(02)74051-4. 18. Lembowicz K, Rabek A, Skrzeczkowski L. Hormonal induction of lactation in the cow. Br Vet J 1982;138(3):203–8. https://doi.org/10.1016/S0007-1935(17)31083-7. 19. Malekinejad H, Rezabakhsh A. Hormones in dairy foods and their impact on public health- A narrative review article. Iran J Public Health 2015;44(6):742–58. 20. Delouis C, Djiane J, Kann G, Terqui M, Head HH. Induced lactation in cows and heifers by short-term treatment with steroid hormones. Ann Biol Anim Biochim Biophys 1978;18(3):721–34. https://doi.org/10.1051/rnd:19780410. 21. Narendran R, Hacker RR, Smith VG, Lun A. Hormonal induction of lactation: estrogen and progesterone in milk. J Dairy Sci 1979;62(7):1069–1075. 10.3168/jds.S00220302(79)83376-7 22. Lee JY, Kim IH. Advancing parity is associated with high milk production at the cost of body condition and increased periparturient disorders in dairy herds. J Vet Sci 2006;7(2):161–166. 10.4142/jvs.2006.7.2.161 557


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23. Strucken EM, Laurenson YCSM, Brockmann GA. Go with the flow-biology and genetics of the lactation cycle. Front Genet 2015;6:1–11. 10.3389/fgene.2015.00118. 24. Mellado M, Antonio-Chirino E, Meza-Herrera C, Veliz FG, Arevalo JR, Mellado J, et al. Effect of lactation number, year, and season of initiation of lactation on milk yield of cows hormonally induced into lactation and treated with recombinant bovine somatotropin. J Dairy Sci 2011;94(9):4524–4530. 10.3168/jds.2011-4152. 25. Ramírez AB, Salama AAK, Caja G, Castillo V, Albanell E, Such X. Response to lactation induction differs by season of year and breed of dairy ewes. J Dairy Sci 2008;91(6):2299–2306. 10.3168/jds.2007-0687. 26. Kennedy J, Dillon P, O’Sullivan K, Buckley F, Rath M. The effect of genetic merit for milk production and concentrate feeding level on the reproductive performance of Holstein-Friesian cows in a grass-based system. Anim Sci 2003;76(2):297–308. https://doi.org/10.1017/S1357729800053546.

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https://doi.org/10.22319/rmcp.v13i2.5977 Technical note

Panting frequency and score in beef cattle in intensive finishing during summer in the dry tropics

Ana Mireya Romo Valdez a Jesús José Portillo Loera a Jesús David Urías Estrada a Alfredo Estrada Angulo a Beatriz Isabel Castro Pérez a Francisco Gerardo Ríos Rincón a*

a

Universidad Autónoma de Sinaloa. Facultad de Medicina Veterinaria y Zootecnia. México.

*Corresponding author: fgrios@uas.edu.mx

Abstract: Panting frequency and score are indicative of cattle well-being in intensive finishing feedlots. A six-week-long descriptive observational study was done of intensive finishing beef cattle during summer in the dry tropics in northwest Mexico. Data were collected on pen type, observation time (0800, 1200 and 1600 hours), panting frequency and score, environmental temperature, relative humidity and animal phenotypic predominance (i.e. Bos taurus or Bos indicus). The temperature and humidity index (THI) was calculated. Panting frequency was recorded as the number of animals exhibiting this behavior per pen, and panting scored on a five-point scale. Three categories of phenotype were assigned (no hump, medium hump and large hump), greater hump size being indicative of greater B. indicus influence. Panting frequency was highest at 1200 and 1600 h (P<0.01), when THI values consistently exceeded 84 units. Panting score tended to be higher (P<0.01) in pens with less space for individual animals, at later observation times, and in animals with no hump. Panting frequency and

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score in beef cattle in intensive feedlots was influenced by time of day, pen design and a predominantly B. taurus phenotype. Key words: Heat stress, Bovines, Intensive finishing.

Received: 06/04/2021 Accepted: 14/08/2021

Industrial beef production focuses on efficiency and production unit profitability which depend on reproductive management strategies, genetic improvement technologies, exogenous growth promoters, vaccines and antibiotics, processed feed, and ration formulation(1). In Mexico, beef production is the main livestock activity, contributing 29.8 % of the value of national livestock production in 2018, equivalent to 134.4 billion Mexican pesos(2). In intensive beef cattle finishing pens, several common factors concur which affect animal thermal balance. These include animal genotype, body condition, fat cover, coat color and degree of adaptation to the environment(3). This concurrence needs to be considered when assessing animal welfare indicators and their relationship to beef productive parameters in intensive finishing(4). For example, animal production indicators can decline sharply in warm regions due mainly to animal stress caused by high temperatures coupled with high relative humidity(5). In cattle, heat stress occurs when environmental temperature exceeds animals’ thermoneutral zone, preventing them from dissipating excess heat(6). Optimum cattle performance occurs in a thermoneutral zone of 20 °C, which can vary from 10 to 26 °C; for example, in young animals the comfort zone ranges from 7 to 26 °C while the range for mature cows and heavy cattle is from -17 °C in winter to 23 °C in summer. Most cattle types have difficulty tolerating temperatures above 27 °C, especially if relative humidity (RH) exceeds 40 %(7). Ruminants are homeothermic animals. Maintaining homeostasis in temperatures outside a species’ thermoneutral zone requires physiological and behavioral changes. When environmental temperature rises, the immediate physiological response in cattle manifests as increased respiratory rate, decreased feed intake and greater water intake(6). Under extreme conditions, cattle can die from heat stress(8). Both respiratory rate and panting are appropriate indicators for measuring heat stress intensity in cattle(8,9). In addition, highly productive cattle have a high metabolic rate, making them more susceptible to heat stress(6). The present study objective was to quantify panting frequencies and scores in beef cattle in intensive finishing during summer in the dry tropics. 560


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This descriptive observational study was done in a Livestock Production Unit (LPU), in Los Becos, south of the city of Culiacán, Sinaloa, Mexico (24° 38’ 58” N; 107° 17’ 10” W; average altitude= 70 m amsl). According to the Köppen classification, climate in the Culiacán Valley is classified as BS1(h')w(w)e. The “B” refers to dry climates, and the “BS1” classification indicates a type of dry steppe climate with moderate precipitation; this classification is between very arid climates (BW) and humid ones (A or C). The symbol “(h')” indicates a warm climate with an average annual temperature above 22 °C and an average temperature of 18 °C in the coldest month. The “w(w)” means rainfall occurs during the summer months; indeed, in the wettest month of the warmest season rainfall is 10 times greater than in the driest month, and only 5 % of annual rainfall occurs in the winter. Finally, the symbol “e” indicates the presence of extreme thermal oscillation, from 7 to 14 °C, between average annual monthly temperatures. In summary, BS1(h')w(w)e means the study area is in a very hot semi-dry climate with extreme summer rains, and winter precipitation representing less than 5 % than the annual total(10,11). The experimental period was in the summer. Based on historical climatological records for the Culiacán Valley(12), a six-week-long period was selected spanning from the seventh to the twelfth week of summer, that is, 4 wk in August and 2 wk in September. These months have the highest average rainfall of the year (209.2 and 141.6 mm), high average RH (75 and 75 %) and extreme average temperature (34.8 and 34.4 ° C). The cattle were housed in pens of three different dimensions used for beef production at the LPU. For study purposes these were labeled as type 1, 2 and 3 pens (Table 1). Table 1: Characteristics of intensive finishing pens at LPU in the dry tropics Pen type Characteristics 1 2 3 No. of pens 4 4 4 Animals 67 71 89 Dimensions, m 22.67 x 38.98 22.92 x 40.36 32.1 x 41.60 Available surface, m2 883.67 925.03 1335.07 2 Living space, m /animal 13.14 13.13 15.16 2 Available shade, m 134.75 137.57 192.32 Available shade, % 15.29 14.87 14.40 2 Shade area, m /animal 2.01 1.95 2.18 Shade orientation N-S N-S E-W Feeder length, m 22.39 22.90 32.05 Feeder availability, cm/head 33.4 32.2 36.0 Water trough length, m 6.0 6.0 6.0 Water trough availability, cm/head 8.9 8.5 6.7 LPU= Livestock production unit. 561


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Temperature and RH were measured using two digital thermohygrometers (Model No. VAEDT- 1-55ª, Avaly Taylor, Mexico City, Mexico) placed in the feeding area of each pen type. The temperature and humidity index (THI) was calculated using the formula: THI = [0.8 × T] + [(RH ÷ 100) × (T − 14.4)] + 46.4, where T is environmental temperature in degreees Celsius and RH is relative humidity expressed as a percentage(13). All the documented pens contained only uncastrated males (average LW= 450 kg). The animals were fed the finishing program diet utilized at this LPU. Four pens of each of the three sizes (n= 12) were included in the study. Observation visits were made to the pens from Monday to Friday throughout the study period. Observations were randomized within each pen type such that six pens were observed daily (two per type). All observations were made by a single previously-trained person. Environmental temperature and RH were recorded at 0800, 1200 and 1600 h. Panting frequency was recorded as the number of panting animals in a pen at the time of observation. An established methodology using a 0-4 scale was applied to score panting severity in individual animals (Table 2, Figure 1)(13,14). Table 2: Scale for scoring panting in beef cattle in intensive finishing pens Score

RR/min

Description

0

Max 40

Normal respiration, no panting

41 - 70

Light panting, mouth closed, no saliva; chest movement easily observed

71 - 120

Panting, open-mouth, some salivation. Neck extended and head generally up

121 - 160

Panting, open-mouth, some salivation. Neck extended and head generally up

>160

Mouth open, tongue completely extended, long periods of salivation

1 2 3 4

Source: Mader et al (2002) and Mader et al (2006). RR= respiration rate.

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Figure 1: Panting score in beef cattle in intensive finishing pens. A) 0; B) 1; C) 2; D) 3; E) 4.

Photo credit: Ana Mireya Romo Valdez.

Animals were assigned to one of three estimated phenotypic predominance categories generally suggestive of their predominant species (Bos taurus or Bos indicus). Classifications were made based on the presence of a hump and its size: no hump, medium hump (8-14 cm) and large hump (>15 cm)(15). Based on the number of animals in each phenotype category, panting was recorded in six animals per pen, divided proportionally among the categories present at the time of observation in each pen. The panting frequency observation unit was the pen and recorded values were converted to rates using the formula(16): 𝑎 ( )𝑘 𝑎+𝑏 Where 𝑎 = frequency of the event during a specific period; 𝑎 + 𝑏 = number of animals exposed to risk of the event during the same period; 𝑘 = 100.

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The transformed rates did not meet normality criteria. The technique of Herrera and Barreras(17), using the RANK procedure(18), was applied to calculate ranges and an ANOVA applied to these with the GLM procedure, stated in this general linear model: 𝑌𝑖𝑗𝑘 = 𝜇 + 𝐶𝑖 + 𝐻𝑗 + 𝐶𝐻𝑖𝑗 + 𝜀𝑖𝑗𝑘 Where: 𝐘𝐢𝐣𝐤= Rate ranges for behavioral variable; 𝛍= General mean; 𝐂𝐢 = Effect of i-th pen type; 𝐇𝐣 = Effect of j-th observation time; 𝐂𝐇𝐢𝐣 = Effect of i-th pen type and j-th observation time interaction; 𝜺𝒊𝒋𝒌 = Random error. Comparison of means for pen type and observation time was done with a Dunn test (Bonferroni)(18). Panting score was analyzed using a χ2 test, with 5x3 contingency tables: five panting score levels (0 to 4); three pen types (1, 2 and 3); three observation times (0800, 1200 and 1600 h); and three phenotype categories (no hump, medium hump, and large hump). Highly significant statistical differences resulted, therefore, χ2 tests were run for each panting score and each factor. The results are expressed as the percentage of observations per column and the number of total observations in parentheses. During the experimental period, average temperature was 34.6 °C, average RH was 67.3 % and the average THI value was 87.3 units (Table 3). The latter indicates that throughout the experimental period the animals were in the Emergency category (THI > 84 units)(13). Table 3: Average temperature, relative humidity and temperature/humidity index values during experimental period (summer) Temperature °C RH1, % THI2 Week3 1 2 3 4 5 6 Overall4 1

Min. 25.0 27.6 26.6 23.9 24.4 27.1 23.9

Max. 40.6 41.2 43.1 45.8 43.1 46.3 46.3

Mean 33.4 34.5 35.2 33.9 35.2 35.3 34.6

Min. 50 51 48 52 54 60 48

Max. 85 81 81 85 93 81 93

Mean 66.8 66.0 64.6 66.3 71.6 69.3 67.3

Min. 75 79 77 74 75 78 74

Max. 98 96 99 101 100 103 103

Mean 85.4 87.1 87.6 86.1 89.0 88.9 87.3

Category Emergency Emergency Emergency Emergency Emergency Emergency Emergency

RH = Relative humidity; 2 THI= Temperature/humidity index; Min.= minimum; Max.= maximum. 3 Per week, n=90. 4Overall, n=540.

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Results for average temperature and THI per observation time in each pen type show that these parameters varied (P<0.01) in response to pen type and observation time (Table 4). The pen type/observation time interaction also affected temperature and THI values (P<0.01), indicating the presence of microclimates within pens that affect animal physiology. These values also highlight the persistently high temperatures and THI that constitute the Emergency category, and the consequent daily cumulative caloric load experienced by animals in the finishing pens. Table 4: Average environmental temperature and temperature/relative humidity index (THI) values per observation time and pen type Observation time Pen type Temperature, °C THI 0800

1200

1600

1 2 3 1 2 3 1 2 3

31.1 31.4 28.8 37.9 37.2 35.2 36.0 35.6 35.5 0.39 0.01 0.01 0.01

SME Time Pen type Time x Pen type

84.2 84.7 80.8 90.9 89.9 87.0 89.0 88.4 88.1 0.55 0.01 0.01 0.01

SME= Standard mean error (n= 60).

Recording and interpretation of temperature and THI values are used as a way of quantifying the degree of heat stress experienced by cattle in a feedlot(19); this is logical since heat stress severity depends largely on diurnal fluctuations in environmental temperature(20). The maximum temperature values recorded throughout the day in the present study exceeded the high critical temperature limit for cattle (Table 4). In addition, observation time clearly affected panting frequency during the present study, with much higher frequencies (P<0.01) at 1200 and 1600 h than at 0800 h (Figure 5). This coincides with THI values in excess of 84 units, the lower limit for the Emergency category. These results are vital to evaluating animal well-being because conditions outside the thermoneutral zone compromise cattle thermal comfort in intensive finishing systems(21). Cattle physically suffer from loss of thermal balance which can undermine productive function because an animal’s physiological response is restricted in unfavorable climatic conditions during intensive confinement(5,6,8). This negatively affects beef cattle well-being in intensive finishing environments.

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Figure 5: Panting frequency in finisher beef cattle during summer in a dry tropical climate, by observation time.

ab

Different lowercase letters indicate significant difference (P≤0.01).

Among domestic animals, cattle are particularly vulnerable to environment thermal conditions, a situation exacerbated under feedlot conditions(5). Tropical regions characterized by high temperature and high RH also pose considerable challenges to animal thermoregulation(22). The present results confirm previous reports of the relationship between environmental conditions and heat stress in cattle. In a study of native South African beef cattle in finishing feedlots during the summer months of February and March at temperatures ranging from 17 to 38 °C, panting frequency increased between 1200 and 1400 h, indicating that the cattle were under heat stress(23). Environmental temperature and RH can also affect respiratory rate in cattle, providing another indicator of heat stress. One study of Angus × Charolais cattle found fluctuating respiratory rates when average environmental temperature was reduced from 23.7 to 14.5 °C, and average RH increased from 57.5 to 60.5% in a feedlot between July and October(24). A study done in the tropics of Peru, with 34.1 °C maximum temperature and 81.2 % RH, reported that respiratory rate indicated heat stress in Nellore and Nellore x Swiss Brown cattle(25). In four breeds studied in Mexico (Criollo, Holstein, Jersey and Charolais x Brahman) during the warm months of May to September, respiratory rate increased when environmental temperature rose from 36.2 to 40.3 °C and RH from 70 to 85 %, and THI values were greater than or equal to 72 units(26). Increased panting has also been reported in Angus cattle in a feedlot as high temperature and RH humidity increased during the year (27).

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Panting is an indicator of caloric load and is associated with heat stress and declining animal well-being in feedlots. Breeds derived from B. taurus can be more susceptible to thermal stress, while B. indicus breeds are more thermotolerant and therefore more adaptable to hot climates(28). Intensive beef cattle production in Mexico involves various breeds, resulting in the coexistence of a diversity of phenotypes in finishing pens. For this reason, data on phenotypic expressions (i.e. hump size) were included in the panting score (PS) evaluation (Table 5). Table 5: Panting score of beef cattle (n) in intensive finishing in summer by pen type, observation time and hump size Panting score P 0 1 2 3 4 Pen type 32.3ab 1 33.3 (545) 36.5a (62) 31.8 (27) 45.0a (36) 0.009 (413) 2 31.0b (396) 33.0 (540) 39.4a (67) 30.6 (26) 33.7ab (27) 3 36.7a (469) 33.7 (550) 24.1b (41) 37.6 (32) 21.3b (17) Observation time 0800 63.1a (806) 17.0c (277) 2.9b (5) 5.9b (5) 8.7c (7) 0.0001 c a a a b 1200 11.0 (141) 45.6 (746) 51.2 (87) 45.9 (39) 31.3 (25) 1600 25.9b (331) 37.4b (612) 45.9a (78) 48.2a (41) 60.0a (48) Hump size 0.0001 No hump 24.8b (317) 29.7b (486) 55.9a (95) 49.4a (42) 62.5a (50) a a b a b Medium 48.8 (624) 51.4 (840) 40.0 (68) 42.4 (36) 32.5 (26) Large 26.4b (337) 18.9c (309) 4.1c (7) 8.2b (7) 5.0c (4) abc

Different letter superscripts within pen type, observation time and hump size, and within each panting score column indicate significant difference (P<0.05).

Of the total number of animals with a PS of 4, a higher proportion (P<0.01) were in type 1 pens (45 %) than in type 3 pens (21.3 %). In contrast, most (P<0.01) of the animals with a PS of 2 were housed in type 1 (36.5 %) and 2 (39.4 %) pens, with fewer in in type 3 pens (24.1 %). Finally, animals with a PS of 0 were more frequent (P<0.01) in type 3 pens (36.7 %) than in type 2 pens (31.0 %). In terms of numbers of panting animals, there was no difference between pen types. However, THI values in type 3 pens were generally lower than in type 1 and 2 pens (Table 4). The slightly lower occurrence of high PS scores, and lower THI values, in type 3 pens suggest that these pens provide somewhat better microclimatic conditions than type 1 and 2 pens. This may be related to their greater shaded area (2.18 [type 3] vs 2.01 [type 2] vs 1.95 [type 1] m2/animal), and greater living area (15.16 [type 3] vs 13.14 [type 2] vs 13.13 [type 1] m2/animal), as well as their East-West orientation.

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Cattle can withstand adverse weather conditions, but shading intensive finishing feedlots reduces the impact of heat stress, mitigating its negative physiological effects and improving cattle production performance(6,8). Previous reports have found panting rate can be reduced by providing adequate shade (3 - 5 m2/head for animals > 400 kg LW, in intensive finishing) and living space (15 - 19 m2/animal), consequently improving panting scores and well-being in feedlot cattle(24,27,29). This is especially the case in geographical areas with extreme weather conditions where heat is most intense from 1200 h onwards(9). In terms of observation time, the highest frequency (63.1 %) of animals with a PS of 0 was recorded at 0800 h, which was higher (P<0.01) that at 1600 h (25.9 %) and 1200 h (11.1 %). Animals with a PS of 1 were more frequent (P<0.01) at 1200 h (45.6%) than at 16:00 h (37.4 %) and 8:00 h (17.0 %). In the PS 2 and 3 categories no difference was observed in panting frequency between 1200 h and 1600 h, although it was lower (P<0.01) at 0800 h. Animals with a PS of 4 were more frequent (P<0.01) at 1600 h (60.0 %) than at 1200 h (31.3 %) and 0800 h (8.7 %). In the present results, higher THI values (which increase throughout the day) were clearly related to higher panting scores. Since exposure of beef cattle in intensive finishing systems to extreme heat and humidity can reduce animal productivity and well-being(26), the present results highlight the need for adequate shade and living space areas in feedlots. Phenotype, as a proxy for cattle species, did effect animal response to the evaluated environmental conditions. At low panting scores (PS 0 and 1), animals with a medium hump were more frequent (P<0.01) than those with no hump or a large hump. In the PS 2, 3 and 4 categories, animals with a large hump were much less frequent (P<0.01) than those with a medium or no hump. Under the evaluated conditions animals with a substantial B. taurus genetic predominance (i.e. no hump or medium hump) exhibited physiological difficulties in adapting to the rainy summer conditions in the dry tropics with THI values far in excess of 75 units. In contrast, the B. indicus animals (i.e. large hump) were more tolerant of these conditions, manifesting much lower panting scores. These results support reports that genetic differences between breeds influence heat tolerance and can notably modify animal behavior under adverse physiological parameters(30). Time of day, pen type and a phenotypic predominance of B. taurus affected panting frequency and panting score in beef cattle housed in intensive finishing pens. The persistently high THI values in the summer in the study region compromised physiological stability of the evaluated finisher cattle. Animals with a predominantly B. taurus phenotype were most strongly affected, but housing characteristics (e.g. living space, shaded area and drinker availability per animal) had a greater capacity to exacerbate the adverse conditions during summer, worsening the already negative impacts of extreme heat conditions on animal wellbeing.

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Literature cited: 1

Drouillard JS. Current situation and future trends for beef production in the United States of America — A review. Asian-Australas J Anim Sci 2018;37(7):1007-1016. doi: 10.5713/ajas.18.0428.

2

FIRA. Panorama Agroalimentario. Carne de bovino. 2019.: https://www.fira.gob.mx/InvYEvalEcon/EvaluacionIF. Consultado Nov 30, 2020.

3

Lees AM, Sejian V, Wallage AL, Steel CC, Mader TL, Lees JC, Gaughan JB. The impact of heat load on cattle: review. Animals 2019;9(6):322. doi: 10.3390/ani9060322.

4

Temple D, Manteca X. Animal welfare in extensive production system in still an area of concern. Front Sustain Food Syst 2020;4:545902. doi: 10.3389/fsufs.2020.545902.

5

Renaudeau D, Collin A, Yahav S, Basilio V, Gourdine JL, Collier RJ. Adaptation to hot climate and strategies to alleviate heat stress in livestock production. Animal 2012;6(5):707-728. doi:10.1017/S1751731111002448.

6

Bernabucci U, Lacerera N, Baumgard LH, Rhoads RP, Ronchi B, Nardone A. Metabolic and hormonal acclimation to heat stress in domesticated ruminants. Animal 2010;4(7):1167-1183. doi: 10.1017/S175173111000090X.

7

Mader T, Griffin D, Hahn L. Managing feedlot heat stress. Institute of Agriculture and Natural Resourcer. University of Nebraska, Lincoln, USA: 2007. http://extensionpublications.unl.edu/assets/html/g1409/build/g1409.htm.

8

Brown-Brandl TM, Eigenberg RA, Nienaber JA, Hahn GL. Dynamic response indicators of heat stress in shaded and non-shaded feedlot cattle, part 1: Analyses of indicators. Biosyst Eng 2005;90(4):451-462. doi: 10.1016/j.biosystemseng.2004.12.006.

9

Gaughan JB, Mader TL. Body temperature and respiratory dynamics in un-shade beef cattle. Int J Biometeorol 2014;58(7):1443-1450. doi: 10.1007/s00484-013-0746-8

10 García E. Modificaciones al sistema de clasificación climática de Köppen. Universidad Nacional Autónoma de México. México. 2004; ISBN: 970-32-1010-4.

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11 Estación climatológica de la Escuela de Biología de la Universidad Autónoma de Sinaloa. Universidad Autónoma de Sinaloa.: http://www.uas.edu.mx/servicios/clima/. Consultado Sept 22, 2019.

12 CONAGUA. Comisión Nacional del Agua. Servicio Meteorológico Nacional. Normales climatológicas del Estado de Sinaloa. Periodo 1951-2010.: https://smn.conagua.gob.mx/es/informacion-climatologica-por-estado?estado=sin

13 Mader TL, Davis MS, Brown-Brandl T. Environmental factors influencing heat stress in feedlot cattle. J Anim Sci 2006;84(1):712-719. doi: 10.2527/2006.843712x . 14 Mader TL, Holt SM, Hahn GL, Davis MS, Spiers DE. Feeding strategies for managing heat load in feedlot cattle. J Anim Sci 2002;80(9):2373-2382. doi: 10.1093/ansci/80.9.2373. 15 Méndez RD, Meza CO, Berruecos JM, Garcés P, Delgado EJ, Rubio MS. A survey of beef carcass quality and quantity attributes in Mexico. J Anim Sci 2009;87(11):37823790. doi:10.2527/jas.2009-1889. 16 Daniel WW. Bioestadística base para el análisis de las ciencias de la salud. Cuarta ed., México, DF: Editorial Limusa S.A. de C.V. 2002. ISBN: 968-18-6164-7. 17 Herrera HJG, Barreras SA. Manual de procedimientos: Análisis estadístico de experimentos pecuarios (utilizando el Programa SAS). Segunda ed. Colegio de Postgraduados, Campus Montecillo. México. 2005. ISBN: 968-839-300-2. 18 SAS Institute. Statistical Analysis Software. SAS/STAT System for Windows 9.0. Cary, NC, USA. SAS Institute Inc. 2002. ISBN: 978-1-60764-599-3. http://www.sas.com/en_us/software/analytics/stat.html# 19 Gaughan JB, Mader TL, Holt SM, Lisle A. A new heat index for feedlot cattle. J Anim Sci 2008;86(1):226-234. ISSN: 1525-3163 doi: 10.2527/jas.2007-0305.

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20 Silanikove N. Effects of heat stress on the welfare of extensively managed domestic ruminants. Livest Prod Sci 2000;67(1):1-18. doi: 10.1016/S0301-6226(00)00162-7. 21 Arias RA, Mader TL, Escobar PC. Factores climáticos que afectan el desempeño productivo del ganado bovino de carne y leche. Arch Med Vet 2008;40(1):7-22. ISSN: 0301-732X doi: 10.4067/S0301-732X2008000100002. 22 Da Silva RG, Guilhermino MM, De Morais DAEF. Thermal radiation absorbed by dairy cows in pasture. Int J Biometeorol 2010;54(1):5–11. doi 10.1007/s00484-009-0244-1. 23 Blaine KL, Nsahlai IV. The effects of shade on performance, carcass classes and behaviour of heat-stressed feedlot cattle at the finisher phase. Trop Anim Health Prod 2011;43(3):609-615. doi: 10.1007/s11250-010-9740-x. 24 Miltlöhner FM, Morrow JL, Dailey JW, Wilson SC, Galyean ML, Miller MF, McGlone JJ. Shade and water misting effects on behavior, physiology, performance, and carcass traits of heat-stredded feedlot cattle. J Anim Sci 2001;79(9):2327-2335. doi: 10.2527 / 2001.7992327x. 25 Unchupaico PI, Bazán AL, Quispe EC, Ancco GE. Temperatura ambiental y su efecto sobre parámetros fisiológicos en vacas Nellore y cruces bajo condiciones del trópico peruano. Rev Inv Vet Perú 2020;31(1):e17549 http://dx.doi.org/10.15381/rivep.v31i1.17549. 26 Espinoza VJ, Ortega PR, Palacios EA, Guillén TA. Tolerance to heat and atmospheric humidity of different breeds groups of cattle. Rev MVZ Córdoba 2011;16(2):2302-2309. doi:10.21897/rmvz.288. 27 Sullivan ML, Cawdell-Smith AJ, Mader TL, Gaughan JB. Effect of shade area on performance and welfare of short-fed feedlot cattle. J Anim Sci 2011;89(9):2911-2925. doi: 10.2527/jas.2010-3152. 28 Hansen PJ. Physiological and cellular adaptations of zebu cattle to thermal stress. Anim Reprod Sci 2004;82–83:349–360. doi:10.1016/j.anireprosci.2004.04.011. 29 Gaughan JB, Bonner S, Loxton I, Mader TL, Lisle A, Lawrence R. Effect of shade on body temperature and performance of feedlot steers. J Anim Sci 2010;88(12):40564067. doi:10.2527/jas.2010-2987.

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30 Valente ÉEL, Chizzotti ML, Ribeiro OCV, Castlho GM, Domingues SS, Castro R A, Machado LM. Intake, physiological parameters and behavior of Angus and Nellore bulls subjected to heat stress. Semina: Ciências Agrárias, Londrina 2015;36(6):4565-4574. doi: 10.5433/1679-0359.2015v36n6Supl2p4565.

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https://doi.org/10.22319/rmcp.v13i2.5007 Technical note

Silvopastoral arrangements with Alnus acuminata and their effect on productive and nutritional parameters of the forage component

José Américo Saucedo-Uriarte a Segundo Manuel Oliva-Cruz a* Jorge Luis Maicelo-Quintana a Jegnes Benjamín Meléndez-Mori a Roicer Collazos-Silva a

a

Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas. Instituto de Investigación para el Desarrollo Sustentable de Ceja de Selva, Campus Universitario: Calle Universitaria N° 304, Chachapoyas, Perú.

*Corresponding author: soliva@indes-ces.edu.pe

Abstract: Silvopastoral systems (SPS) are an alternative for sustainable livestock production. For this reason, the present study was developed with the aim of evaluating productive and nutritional parameters of the forage component (FC) in different silvopastoral arrangements with Alnus acuminata and their comparison with open field systems. A randomized complete block design was established, for which 16 plots with characteristics of homogeneity in age and type of FC were selected. The floristic composition, functional classification of herbaceous species, biomass, dry matter and nutritional composition were evaluated. The results obtained recorded the presence of 22 species, with the family Poaceae (8 species) predominating, it was also found that silvopastoral arrangements have the highest percentage of desirable species, a situation contrary to what happened in open field systems. On the other hand, the productive and nutritional parameters showed significant differences (P<0.05) between the production systems, being the arrangement with trees in alleys the one that registered better yields of biomass (16.60 t /ha), dry matter (3.65 t/ha), crude fiber (27.23 %), total protein (17.39 %) and gross energy (4,864 kcal/kg).

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Key words: Floristic composition, Nutritional composition, Species desirability, Forage yield, Silvopastoral system.

Received: 02/08/2018 Accepted:30/08/2021 In Peru, the rate of deforestation increased by 2,672,554 ha(1,2) during the years 1975 to 2000, with the increase in the agricultural sector (extensive production)(3) being the main cause of this discouraging panorama, followed by mining activity, fires and illegal logging of forests(2,4); this situation is further aggravated by limited land use and tenure policies, as well as by a lack of knowledge of new sustainable production systems(5). In 2012, the Peruvian agricultural area amounted to 38,742,000 ha, of which 46.5 % represent natural pastures(6), characterized by being an open field productive system, that is, without the presence of tree cover. The lack of trees and tree cover in general cause several ecological problems, such as extreme weather events, soil erosion, water pollution, decrease in biodiversity(7), and consequently economic problems(8), due to low productivity because of the limited soil fertility(9). However, the negative impacts associated with extensive livestock production can be reduced if livestock farming is focused on systems that increase productivity, improve sustainability and provide ecological services to the ecosystem(8,10). In this sense, studies demonstrate the importance of pastures associated with trees for the conservation of biodiversity(11,12). Thus, silvopastoral systems are an option for the exploitation of ruminants, since they diversify the products (milk, meat, wood, poles and firewood), provide shade, improve the diet of animals and reduce the use of external inputs(13,14). Therefore, the objective of this study was to evaluate the floristic composition and functional classification of herbaceous species, as well as the productive and nutritional parameters of the forage component established under silvopastoral arrangements. The study was conducted in the district of Molinopampa, specifically in the localities of Molinopampa, Santa Cruz del Tingo, Pumahermana and Ocol; located at an altitude above 2,421 masl, between the coordinates 06°12’20” south latitude and 77°40’06” west longitude. They have a slightly humid and warm temperate climate, with an average annual temperature of 14.5 °C and an average annual rainfall of 1,200 mm(15). Four silvopastoral arrangements (SPAs) [living fences (LFs), trees scattered in the paddock (TSP), trees in alleys (TIAs) and open field system (OFS)] were studied, which were selected

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due to the homogeneity in the forage component, age of the trees and area between 1 to 2 ha. In each SPA, the floristic composition was evaluated through the transect method (16), which consisted of stretching a 50-m rope with marks between (01) meters (contact point) for sampling with a census ring (four transects per each SPA). The functional classification was determined according to the degree of preference of herbaceous species [desirable species (DS), less desirable species (LDS) and undesirable species (US)](17,18). The biomass of the forage component (BFC) was determined by the square meter method(19), for which 40 samples per each SPA (10 per each locality or repetition) were weighed. For the dry matter (DM) content, the 40 samples obtained from the biomass evaluation were mixed, then 100 g of forage from each SPA was weighed and placed at 65 °C in a BINDER FD 115 forced-air circulation oven (BINDER GmbH, Germany). The nutritional composition of FC: total protein (TP), ethereal extract (EE), crude fiber (CF), ash (A) and crude energy (GE), was quantified in 1 kg of forage (obtained from the mixture of the 40 samples collected per each SPA) using the guidelines established by the AOAC(20). It should be noted that the analysis was conducted over a period of 12 months, considering two periods for sample collection: rainy season (November 2016 to April 2017) and dry season (May to October 2017). For the statistical analysis, a randomized complete block design consisting of four treatments (OFS, LFs, TSP and TIAs) was used, in four localities (Molinopampa, Santa Cruz del Tingo, Pumahermana and Ocol) or replicates considered as blocks. The results were processed using the statistical software SPSS 15.0, in which they were subjected to the analysis of normality and homogeneity of variances with the Shapiro-Wilk and Levene tests. Nutritional composition data were processed using an analysis of variance with a confidence level of 95 % (P<0.05) and the Tukey test for multiple comparisons. The BFC was analyzed with the Mann-Whitney U test(21). The results of the joint study of the productive systems (OFS, TIAs, TSP and LFs) recorded the presence of 22 species, grouped into 11 families. The greatest richness was found in the family Poaceae (8 species), with Lolium multiflorum being the most representative species, with a presence between 15 and 32 % within each production system. On the other hand, species such as Equisetum giganteum, Ageratina azangaroensis and Verbena litoralis were the least abundant, being found only in the OFSs (Table 1).

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Table 1: Herbaceous species recorded in different grass production systems (%) Floristic composition OFS TSP TIAs LFs Poaceae Brachiaria brizantha 9.09 2.29 2.30 6.17 Lolium multiflorum 15.78 19.08 31.12 21.08 Paspalum penicillatum 1.87 Dactylis glomerata 3.74 6.36 7.65 6.17 Sporobolus indicus 3.74 Pennisetum clandestinum 0.80 16.03 11.48 16.45 Paspalum bonplandianum 8.14 1.28 8.23 Setaria sphacelata 3.05 1.28 4.88 Asteraceae Taraxacum officinale 6.95 1.02 0.51 Ageratina azangaroensis 0.80 Philoglossa mimuloides 8.82 6.62 4.59 7.20 Fabaceae Trifolium repens 7.49 12.72 11.73 8.48 Trifolium pratense 3.31 2.55 1.54 Cyperaceae Cyperus sp. 4.01 2.80 2.81 2.06 Eleocharis geniculata 7.22 2.29 2.30 2.06 Polygonaceae Rumex obtusifolius 12.03 5.85 5.87 5.91 Plantagnaceae Plantago lanceolata 4.01 2.29 7.40 3.60 Equisetaceae Equisetum giganteum 4.01 Primulaceae Anagallis arvensis 0.80 3.56 3.57 0.51 Araliaceae Hydrocotyle vulgaris 3.48 2.04 2.04 0.51 Verbenaceae Verbena litoralis 0.80 Thelypteridaceae Thelypteris sp. 4.55 2.54 1.53 5.14 OFS= open field system; TSP= trees scattered in paddock; TIAs= trees in alleys; LFs = living fences.

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The functional classification indicates that SPAs report a higher abundance of DS (Trifolium repens, Taraxacum officinale, Lolium multiflorum, Dactylis glomerata, Pennisetum clandestinum, Setaria sphacelata and Trifolium pratense), with a percentage that ranges from 58.0 % to 67.0 %; on the other hand, the highest percentage of LDS (Brachiaria brizantha, Rumex obtusifolius, Paspalum penicillatum, Sporobolus indicus, Philoglossa mimuloides and Paspalum bonplandianum), as well as that of US (Cyperus sp., Plantago lanceolata, Equisetum giganteum, Anagallis arvensis, Hydrocotyle vulgaris, Ageratina azangaroensis, Verbena litoralis, Eleocharis geniculata, and Thelypteris sp.) were reported in OFSs with 33.0 % and 28.0 %, respectively (Figure 1). Among the 22 species recorded, the most prominent within the DS group belong to the family Poaceae, with L. multiflorum and P. clandestinum being the most representative of the group. Figure 1: Functional classification of species

ED= desirable species (DS); EDP= less desirable species (LDS); EI= undesirable species (US); SCA= open field system (OFS); ADP= trees scattered in paddock (TSP); AEC= trees in alleys (TIAs); CV = living fences (LFs).

Regarding BFC production, evaluations during the rainy and dry seasons showed significant differences (P<0.05) between SPAs and OFS. In this sense, the SPA that reached the highest yield during the rainy and dry seasons was TIAs, and the OFS was the one that registered the lowest level of this parameter. Regarding the analysis of DM, the productive systems (SPA and OFS) showed significant differences (P<0.05) both in the rainy and dry season, evidencing that the SPA with TIAs registered better levels in both evaluation periods (Figure 2).

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Figure 2: Yield of the forage component A

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The nutritional components (A, EE, CF, TP and GE) recorded in the rainy and dry seasons were significantly different (P<0.05) between the production systems (SPA and OFS), except for the CF content recorded during the dry season since it showed no statistical difference. The results of both seasons show that the content of A and TP was higher in the arrangement with TIAs, and that the levels of CF ranged from 24 to 30 %. The highest level of GE during the rainy season was recorded in the system with TIAs, on the contrary, during the dry season, the highest value was reached in the OFS (Figure 3). Figure 3: Nutritional composition of the forage component A

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A) Evaluation in rainy season. B) Evaluation in dry season. C= ash (A), EE= ethereal extract, FC= crude fiber (CF), PT= total protein (TP), EB= gross energy (GE); AEC= trees in alleys (TIAs); ADP= trees scattered in paddock (TSP); CV= living fences (LFs); SCA= open field system (OFS). 578


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The species with the greatest dominance in the FC of the productive systems belong to the following families: Poaceae, Fabaceae and Asteraceae; among them, the latter is more present in the OFSs and may be related to the modification of seeds typical of this family, thus allowing their easy dissemination favored by the free circulation of air flow. The results are similar to those reported in the basin of the Ilo River (Moquegua), where it was found that the greatest species richness belongs to Asteraceae and Poaceae(16), which indicates the wide distribution of these families in Peru. The SPAs had the highest abundance of DS, but an opposite case occurs in the OFSs, where low soil fertility and the high presence of weeds are limiting to pasture development (7). In general, the results of the present study agree with the report for grasslands of the high Andean areas of Cusco (Peru), where the percentage of DS was higher (approximately 70.0 %) compared to species of another functional classification(22). On the contrary, they differ from the reports for grasslands of the high Andean areas of Pasco (Peru), where the presence of LDS (34.0 %) and US (54.7 %) exceeded that of DS (11.3 %)(17). The yields recorded in the different production systems (in rainy and dry seasons) allow demonstrating the positive impact of SPAs on grass production, as confirmed by the results of a study, in which an SPA reached a yield of 12.78 t GF/ha, while the OFS only reached 6.79 t GF/ha(23). The presence of trees can increase the productivity of FC because it influences soil fertility by increasing the content of organic matter, as a result of the decomposition of the tree, shrub and herbaceous strata(24,25). In addition, trees take advantage of nutrients from the deepest layers, and these in turn can be used in grasslands due to recycling effects(26,27). The influence of trees can be even more pronounced when using species that can increase the availability of nitrogen in the soil, such as A. acuminata. Regarding the content of DM, the highest yields of this study were obtained in the SPAs, however they are below what was reported for an SPA of A. acuminata associated with P. clandestinum, where the percentage of DM reached was 29.5 % for the SPA and 28 % for the OFS(28), demonstrating that the yield is also influenced by the forage species that makes up the silvopastoral system. The EE levels recorded in this study (between 2.48 % and 5.52 %) were higher than the report made in an SPA of Leucaena leucocephala with Cynodon nlemfuensis (1.28 %) and an OFS of C. nlemfuensis (1.13 %)(29). On the other hand, an SPA of L. leucocephala with improved pastures and an OFS with grasses reported 2.74 % and 1.72 % of EE, respectively(30). The variation in the results of these studies suggests that the energy values represented by the EE are influenced by the cultivated forage species, but not by the production system. In this study, the ash level recorded in the arrangement with TIAs (during the rainy season) exceeded the reports for an SPA of L. leucocephala with C. nlemfuensis (9.35 %) and an OFS of C. nlemfuensis (9.02 %)(29). The ash content is related to the availability of minerals that 579


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fulfill an electrolytic function, which are involved in osmotic pressure, balance and permeability of membranes and tissues, as well as catalytic functions(31), so it is important that grasses show an adequate level for the diet of cattle. Compared to the OFS, the level of CF recorded in the SPAs was slightly lower. A similar behavior was reported for the stem and leaf fractions of C. nlemfuensis grown under OFS and in association with L. leucocephala(29). These results may be related to the shade effect produced by treetops, which can reduce evaporation and improve nutrient dynamics (32). In addition, silvopastoral systems provide better quality and easy-to-digest fiber, reducing methane emissions by 30 % to 40 % compared to the OFS(33). On the other hand, the high levels of TP reported for SPAs suggest that A. acuminata trees perform symbiosis with nitrogen-fixing microorganisms, allowing improving the protein and nutritional content of FC(34). These results are similar to the report for an SPA of P. clandestinum with Sambucus nigra (16.6 %), since it was higher than the record of OFS (13.9 %)(28). On the other hand, another study did not show a marked difference between the systems, reporting 15.61 % crude protein in the SPAs (A. acuminata with P. clandestinum) and 15.51 % in the OFS(35). The level of GE reported in the rainy season shows that the SPAs (except in TSP) reached values higher than that of OFS (4,555 kcal/kg). Results with a similar trend were described in an SPA with Buddleja incana, Buddleja coriaceae and Polylepis racemosa, where grasses reached GE of 4,182.78, 4,179.11 and 4,182 kcal/kg, respectively, being higher than the value reported in the OFS (3,838.56 kcal/kg)(36). Finally, it is worth mentioning that, in the dry season, the level of GE in the OFS (4,462 kcal/kg) was higher than the GE in the SPA. In conclusion, the open field system had the largest number of botanical families, but most desirable species for grazing animals were found in silvopastoral arrangements with trees in alleys. The families with the greatest importance for the forage component of the productive systems were: Poaceae, Fabaceae and Asteraceae. Levels of productivity, dry matter and nutritional composition (total protein and gross energy) were higher in all silvopastoral arrangements, especially in the area of trees in alleys, this being important for dairy cattle production. Literature cited: 1. Malleux J. Mapa forestal del Perú. Memoria explicativa. Universidad Nacional Agraria; 1975. 2.

MINAM. Mapa de deforestación de la Amazonía Peruana: Causas de la deforestación. Ministerio del Ambiente. Lima, Perú; 2000.

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Dancé J. Tendencias de la deforestación con fines agropecuarios en la Amazonía Peruana. Rev For Perú 1981;10(1-2):1-8.

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Aldy JE, Hrubovcak J, Vasavada U. The role of technology in sustaining agriculture and the environment. Ecol Econ 1998;26(1):81-96.

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INEI. IV Censo Nacional Agropecuario: Resultados definitivos. Instituto Nacional de Estadística e Informática. Lima, Perú; 2012. http://censos.inei.gob.pe/cenagro/tabulados/ Consultado 20 Feb, 2018.

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Edición Bilingüe Bilingual Edition

Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 2, pp. 323-583, ABRIL-JUNIO-2022

ISSN: 2448-6698

CONTENIDO CONTENTS ARTÍCULOS

Pags.

Effect of natural extracts on the oxidative stability of pork hamburgers during refrigerated storage María Josefina Graciano Cristóbal, Javier Germán Rodríguez Carpena, María Teresa Sumaya Mar�nez, Rosendo Balois Morales, Edgar Iván Jiménez Ruiz, Pedro Ulises Bau�sta Rosales ………………….……………………………………………………………………………………………………........……………….....………...........323

Diagnóstico de la calidad sanitaria de queserías artesanales en Salinas San Luis Potosí

Diagnosis of the health quality of artisanal cheese dairies in Salinas, San Luis Potosí Rocío Rodríguez-Gallegos, Gregorio Álvarez-Fuentes, Juan Antonio Rendón-Huerta, Juan Ángel Morales-Rueda, Juan Carlos García-López, Luis Alberto Olvera-Vargas …………………………..............................…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....................340

Perspectivas sobre la continuidad, calidad de leche y entorno en unidades de producción de leche en el estado de Aguascalientes, México

Perspectives on continuity, milk quality and environment in milk production units in the state of Aguascalientes, Mexico Carlos Eduardo Romo-Bacco, Ne�ali Parga-Montoya, Arturo Gerardo Valdivia-Flores, Rodrigo Gabriel Carranza-Trinidad, María del Carmen Montoya Landeros, Abril Areli Llamas-Mar�nez, María Mayela Aguilar Romero ………………………….....……..…....……..…......……..…….....…….....…….....…….....……..…....……..…....……..…....…….357

Actividad antimicrobiana de plantas nativas de Sonora, México, contra bacterias patógenas aisladas de leche de vacas diagnosticadas con mastitis

Antimicrobial activity of plants native to Sonora, Mexico, against pathogenic bacteria isolated from milk from cows diagnosed with mastitis Jesús Sosa-Castañeda, Carmen Guadalupe Manzanarez-Quin, Ramón Dolores Valdez-Domínguez, Cris�na Ibarra-Zazueta, Reyna Fabiola Osuna-Chávez, Edgar Omar Rueda-Puente, Carlos Gabriel Hernández-Moreno, Alejandro Santos-Espinosa, Alejandro Epigmenio-Chávez, Claudia Vanessa García-Baldenegro, Tania Elisa González-Soto, Ana Dolores Armenta-Calderón, Priscilia Yazmín Heredia Castro…………….….....…...……….....…….....…….....…….....…..…….......……...................…….....…….....…….....…….....……..........…375

Pharmacokinetic analysis of intraarticular injection of insulin and its effect on IGF-1 expression in synovial fluid of healthy horses

Análisis farmacocinético de la inyección intraarticular de insulina y su efecto sobre la expresión del IGF-1 en el líquido sinovial de caballos sanos Fernando García-Lacy, Lilia Gu�érrez-Olvera, María Bernad, Lisa For�er, Francisco Trigo-Talavera, Margarita Gómez-Chavarín, Alejandro Rodríguez-Monterde……….……………….................………….……..…..391

Productive performance of sheep fed buffel grass silage in replacement of corn silage

Desempeño productivo de ovinos alimentados con ensilaje de pasto buffel en sustitución de ensilaje de maíz Minerva Jaurez-Espinosa, Pedro Abel Hernández-García, Amada Isabel Osorio-Terán, Germán David Mendoza-Mar�nez, Tiara Millena Barros e Silva, Gherman Garcia Leal de Araújo, Tadeu Vinhas Voltolini, Mário Adriano Ávila Queiroz, Sandra Mari Yamamoto, Fábio Nunes Lista, Glayciane Costa Gois, Salete Alves de Moraes, Fleming Sena Campos, Madriano Chris�lis da Rocha Santos………………………………………………………………………………….…...……..............…….....…….....…….....…….....…….....……..........…..408

REVISIONES DE LITERATURA Función ovárica y respuesta a la sincronización del estro en ganado Criollo en México. Revisión

Ovarian function and response to estrus synchronization in Creole cattle in Mexico. Review Elizabeth Pérez-Ruiz, Andrés Quezada- Casasola, José Maria Carrera-Chávez, Alan Álvarez-Holguín, Jesús Manuel Ochoa-Rivero, Manuel Gustavo Chávez-Ruiz, Sergio Iván Román-Ponce……………...…......422

Ultrasonography and physiological description of essential events for reproductive management in dairy cattle. Review

Ultrasonografía y descripción fisiológica de eventos esenciales para el manejo reproductivo en ganado lechero. Revisión María Elena Torres-Lechuga, Juan González-Maldonado…………......…………………………………………………………………….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....……...……..…..452

Demi-embryo reconstitution, a factor to consider for the success of embryo bisection. Review

La reconstitución de demi-embriones: un factor a considerar para el buen éxito de la bisección de embriones. Revisión Alfredo Lorenzo-Torres, Raymundo Rangel-Santos, Agus�n Ruíz-Flores, Demetrio Alonso Ambríz- García………………………………………..…….....…….....…….....……....…….....…….....…….....…….......….………………….473

Estrés por calor en ganado lechero con énfasis en la producción de leche y los hábitos de consumo de alimento y agua. Revisión

Heat stress in dairy cattle with emphasis on milk production and feed and water intake habits. Review Abelardo Correa-Calderón, Leonel Avendaño-Reyes, M. Ángeles López-Baca, Ulises Macías-Cruz……………….…………………………………………………………………………………………….....…….....…….....……...........…......488

Concentrado de proteína de papa: una posible alternativa al uso de antibióticos en las dietas para lechones destetados. Revisión

Potato protein concentrate: a possible alternative to the use of antibiotics in diets for weaned piglets. Review Erick Alejandro Parra Alarcón, Teresita de Jesús Hijuitl Valeriano, Gerardo Mariscal Landín, Tércia Cesária Reis de Souza ……………………………………...………….....…….....…….....…….......….....…….....…….....……...510

Apis mellifera en México: producción de miel, flora melífera y aspectos de polinización. Revisión

Apis mellifera in Mexico: honey production, melliferous flora and pollination aspects. Review Fernanda Baena-Díaz, Estrella Chévez, Fortunato Ruiz de la Merced, Luciana Porter-Bolland…….. ……..………..……….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….........…525

NOTAS DE INVESTIGACIÓN Induced lactation in Holstein cattle with no exogenous progesterone supplementation and with reduced doses of estradiol benzoate

Inducción de la lactancia en ganado Holstein con dosis reducidas de benzoato de estradiol y sin suplementar progesterona exógena Juan González-Maldonado, Raymundo Rangel-Santos, Gustavo Ramírez-Valverde, Jaime Gallegos- Sánchez, Lorenzo Beunabad-Carrasco, Javier-An�llón Ruiz……………………………………....…………………......549

Frecuencia y puntaje de jadeo en bovinos productores de carne en finalización intensiva durante el verano

Panting frequency and score in beef cattle in intensive finishing during summer in the dry tropics Ana Mireya Romo Valdez, Jesús José Por�llo Loera, Jesús David Urías Estrada, Alfredo Estrada Angulo, Beatriz Isabel Castro Pérez, Francisco Gerardo Ríos Rincón……………………………………………....…..……559

Arreglos silvopastoriles con Alnus acuminata y su efecto sobre parámetros productivos y nutricionales del componente forrajero

Silvopastoral arrangements with Alnus acuminata and their effect on productive and nutritional parameters of the forage component José Américo Saucedo-Uriarte, Segundo Manuel Oliva-Cruz, Jorge Luis Maicelo-Quintana, Jegnes Benjamín Meléndez-Mori, Roicer Collazos-Silva…………..................................……………………………..…..…..573

Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 2, pp. 323-583, ABRIL-JUNIO-2022

Efecto de extractos naturales sobre la estabilidad oxidativa de hamburguesas de carne de cerdo durante el almacenamiento refrigerado

Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 2, pp. 323-583, ABRIL-JUNIO-2022