RMCP Vol. 13 Num. 4 (2022): October-December [english version]

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Edición Bilingüe Bilingual Edition

Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 4, pp. 846-1094, OCTUBRE-DICIEMBRE-2022

ISSN: 2448-6698

Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 4, pp. 846-1094, OCTUBRE-DICIEMBRE-2022


REVISTA MEXICANA DE CIENCIAS PECUARIAS Volumen 13 Numero 4, OctubreDiciembre 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 04-2022-033116571100-102. ISSN: 2448-6698, 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 septiembre de 2022. Revisando a las abejas del apiario; Tlalmanalco, Estado de México. Fotografía: Fidel Ávila Ramos

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. 4

OCTUBRE-DICIEMBRE-2022

CONTENIDO Contents ARTÍCULOS Articles

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Evaluation of morphological and yield traits in the populations of Vicia spp. Evaluación de rasgos morfológicos y de rendimiento en las poblaciones de Vicia spp. Hamideh Javadi, Parvin Salehi Shanjani, Leila Falah Hoseini, Masoumeh Ramazani Yeganeh.......846 Efecto de la cobertura del suelo sobre el crecimiento y productividad del zacate buffel (Cenchrus ciliaris L.) en suelos degradados de zonas áridas Effect of soil cover on the growth and productivity of buffel grass ( Cenchrus ciliaris L.) in degraded soils of arid zones Ernesto Herssaín Pedroza-Parga, Aurelio Pedroza-Sandoval, Miguel Agustín Velásquez-Valle, Ignacio Sánchez-Cohen, RicardoTrejo-Calzada, José Alfredo Samaniego-Gaxiola………………………866 Tipología de consumidores de miel con educación universitaria en México Typology of honey consumers with a university education in Mexico Fidel Ávila Ramos, Lizeth Paula Boyso Mancera, Mercedes Borja Bravo, Venancio Cuevas Reyes, Blanca Isabel Sánchez Toledano .............................…………………………………………………………….879 Vertical and spatial price transmission in the Mexican and international cattle and beef market Transmisión vertical y espacial de precios en el mercado mexicano e internacional de ganado vacuno José Luis Jaramillo Villanueva …………………………………………………………………………………………..…894 Exploring bovine fecal bacterial microbiota in the Mapimi Biosphere Reserve, Northern Mexico Explorando la microbiota bacteriana fecal bovina en la Reserva de la Biosfera de Mapimí, norte de México Irene Pacheco-Torres Cristina García-De la Peña, César Alberto Meza-Herrera, Felipe VacaPaniagua, Clara Estela Díaz-Velásquez, Claudia Fabiola Méndez-Catalá, Luis Antonio TarangoArámbula, Luis Manuel Valenzuela-Núñez, Jesús Vásquez-Arroyo ..............................…………..…910

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Perfil fitoquímico, actividad antimicrobiana y antioxidante de extractos de Gnaphalium oxyphyllum y Euphorbia maculata nativas de Sonora, México Phytochemical profile, antimicrobial and antioxidant activity of extracts of Gnaphalium oxyphyllum and Euphorbia maculata native to Sonora, Mexico Priscilia Yazmín Heredia-Castro, Claudia Vanessa García-Baldenegro, Alejandro Santos-Espinosa, Iván de Jesús Tolano-Villaverde, Carmen Guadalupe Manzanarez-Quin, Ramón Dolores Valdez-Domínguez, Cristina Ibarra-Zazueta, Reyna Fabiola Osuna-Chávez, Edgar Omar Rueda-Puente, Carlos Gabriel Hernández-Moreno, Susana Marlene Barrales-Heredia, Jesús Sosa-Castañeda ..........……………………………………………………………………928 Effects of acid whey on the fermentative chemical quality and aerobic stability of rehydrated corn grain silage Efectos del suero ácido sobre la calidad química fermentativa y la estabilidad aeróbica del ensilado de grano de maíz rehidratado Ediane Zanin, Egon Henrique Horst, Caio Abércio Da Silva, Valter Harry Bumbieris Junior.........…943

Growth performance and carcass classification of pure Pelibuey and crossbred lambs raised under an intensive production system in a warm-humid climate Rendimiento productivo y clasificación de canales de corderos Pelibuey puros y cruzados criados bajo un sistema de producción intensivo en un clima cálido-húmedo Miriam Rosas-Rodríguez, Ricardo Serna-Lagunes, Josafhat Salinas-Ruiz, Julio Miguel AyalaRodríguez, Benjamín Alfredo Piña Cárdenas, Juan Salazar-Ortiz ……………………………………………..962 Effect of weight and body condition score from pregnant cows on the carcass characteristics of their progeny: Meta-analysis Efecto del peso y la puntuación de la condición corporal de vacas gestantes en las características de la canal de su progenie: Meta análisis Sander Martinho Adams, John Lenon Klein, Diego Soares Machado, Dari Celestino Alves Filho, Ivan Luiz Brondani, Luiz Angelo Damian Pizzuti ....................................…………………………………981 Factores de riesgo asociados a la seroprevalencia de lentivirus en rebaños ovinos y caprinos del noreste de México Risk factors associated with lentivirus seroprevalence in sheep and goat herds from northeastern Mexico Rogelio Ledezma Torres, José C. Segura Correa, Jesús Francisco Chávez Sánchez, Alejandro José Rodríguez García, Sibilina Cedillo Rosales, Gustavo Moreno Degollado, Ramiro Avalos Ramírez ..................................................................................…………………………………………..995 Caracterización de los sistemas de producción familiar ovina en la Mixteca Oaxaqueña, México Family sheep production systems in the Mixteca region of Oaxaca, Mexico Jorge Hernández Bautista, Héctor Maximino Rodríguez Magadán Teódulo Salinas Rios, Magaly Aquino Cleto, Araceli Mariscal Méndez ..........................................……………….............1009

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REVISIONES DE LITERATURA Reviews

La hipocalcemia en la vaca lechera. Revisión Hypocalcemia in the dairy cow. Review Carlos Fernando Arechiga-Flores, Zimri Cortés-Vidauri, Pedro Hernández-Briano, Renato Raúl Lozano-Domínguez, Marco Antonio López-Carlos, Ulises Macías-Cruz, Leonel Avendaño-Reyes ..............................................................................................................…1025 NOTAS DE INVESTIGACIÓN Technical notes Comportamiento productivo y valor nutricional del pasto Pennisetum purpureum cv Cuba CT-115, a diferente edad de rebrote Productive performance and nutritional value of Pennisetum purpureum cv. Cuba CT-115 grass at different regrowth ages Gloria Esperanza de Dios-León, Jesús Alberto Ramos-Juárez, Francisco Izquierdo-Reyes, Bertín Maurilio Joaquín-Torres, Francisco Meléndez-Nava ........................................………………........1055

Evaluación bacteriana de queso artesanal Zacazonapan madurado bajo condiciones no controladas en dos épocas de producción Bacterial evaluation of Zacazonapan artisanal cheese matured under non-controlled conditions in two production periods Jair Jesús Sánchez-Valdés, Vianey Colín-Navarro, Felipe López-González, Francisca Avilés-Nova, Octavio Alonso Castelán-Ortega, Julieta Gertrudis Estrada Flores..................………………...........1067

Antigen production and standardization of an in-house indirect ELISA for detection of antibodies against Anaplasma marginale Producción de antígenos y estandarización de un ELISA casero indirecto para la detección de anticuerpos contra Anaplasma marginale Elizabeth Salinas Estrella, María Guadalupe Ortega Hernández, Erika Flores Pérez, Natividad Montenegro Cristino, Jesús Francisco Preciado de la Torre, Mayra Elizeth Cobaxin Cárdenas, Sergio D. Rodríguez ....................................................................................………………........1079

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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.

2.

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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.

4.

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.

7.

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).

8.

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.

9.

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.”).

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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).

Key rules for references 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. 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). 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).

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 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. 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 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.

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.v13i4.6125 Article

Evaluation of morphological and yield traits in the populations of Vicia spp. Hamideh Javadi a* Parvin Salehi Shanjani a Leila Falah Hoseini a Masoumeh Ramazani Yeganeh a a

Gene Bank of Research Institute of Forests and Rangelands, Agricultural Research, Education and Extension Organization, Tehran, Iran.

*

Corresponding author: Hjavadim@yahoo.com; Javadi@rifr-ac.ir

Abstract: The study was focused on estimation of genotypic variation for the morphological and forage yield traits of some vetch genotypes to assess their breeding potential. A small-plot trial was carried out in 2018-2020 at the experimental field of the Research Institute of Forests and Rangelands, Alborz province, Iran. Fifty eight (58) vetch genotypes of Vicia spp. from the natural resources gene bank of Iran, were tested. There was significant (P<0.01) genotypic variation among populations, for all the traits measured. V.monantha (32845) produced high plant and large pods, while V. villosa (322) produced more biomass than other accessions. In the shorter growing seasons, the earliness of V. sativa var.angustifollia (4740,7243), V. sativa var.stenophylla (1862), V. villosa (315, 322) resulted in high seed yield. The principal component analysis showed that the variations observed were mainly caused by traits such as days to flowering and seed ripening and seed traits, that their contribution was important in discriminating the populations. Direct selection can also be made for the populations with high biomass yield based on the recorded performance of these populations during the field experiments. A cluster analysis of the tested vetch populations based on measured traits, at 11.49 genetic distance, created five main groups that showed the similarity of members of each group. Generally, vetch species and their populations had different growth features, phenology, forage and seed productivity. The generated information in this study gives a base for genetics variety of genus Vicia L. and could be useful for including in the future breeding programs. Key words: Biomass yield, Morphological traits, Phenology, Seed yield, Vicia spp.

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Rev Mex Cienc Pecu 2022;13(4):846-865

Received: 22/12/2021 Accepted: 21/04/2022

Introduction Vicia L. is a genus with around 232 species in the world and 45 species in Iran, from the legume family, Fabaceae, as an annual and perennial herb. These species have been known by the common name vetches. The genus is primarily found in the Mediterranean and Irano-Turanian regions, such as in Iran, Anatoly, Caucasus, Iraq, Afghanistan, Central Asia, Talesh, Syria, Armenia, Turkmenistan, Jordan, North Africa, Greece, Pakistan, and Palestine(1). Vetches are short-lived forage plants that are highly resistant to cold and dehydration conditions and can be grown in rainfed and irrigated climates. They fix nitrogen in the soil by fixation in root nodes, and help to soil erosion by planting in sloping areas(2,3). As a legume crop, it provides nitrogen to the soil and reduces the incidence of diseases in succeeding non-leguminous crops. Their widespread adaptation and excellent capacities to produce biomass make them very attractive to farmers(4). One attraction of vetch is its versatility, which permits diverse utilization as either ruminant feed or green manure. Because of rapid growth in the first year, different species of Vicia spp. can be used to improve overall livestock, feed quality, improve soils, agriculture for fodder, green manure, human nutrition, and the pharmaceutical industry(5).

Iran is a genetics resources of the genus Vicia and it is widely distributed in different habitats and conditions. Most of the plants in Vicia genus show more variety in morphological traits and sometimes it is difficult to distinguish species of this genus(6,7).

Genetic variation among Vicia genotypes is imperative for their efficient utilization in plant breeding schemes and effective conservation. Diversity studies available in germplasm, collections have been performed on many plant species for Vicia genus from different regions of the world. In comparison to other annual forage legumes, advances in breeding vetches (Vicia spp.) are rather modest. It has been one of the morphological characteristics of the plant reported in V. sativa(8-14), V. faba(15), V. narbonensis(8,10,11,15), V. ervilia(16), V. villosa(10,11), V. atropurpurea(11), V. dasycarpa(8), V. hybrid, V. pannonica, V. lutea, V. peregrine, V. lathyroides and V. grandiflora(11).

There are 335 accessions of 25 Vicia spp. in natural resources gene bank of Iran, that have been collected from different geographical regions of Iran. In this study, it was aimed to determine some morphological characteristics and forage yields of different vetch genotypes by collecting from natural flora of Iran region. The present study was focused on the estimation of genotypic

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Rev Mex Cienc Pecu 2022;13(4):846-865

variation for 12 morphological traits within the V. michauxii, V. michauxii var.stenophylla, V. monantha, V. narbonensis, V. sativa with three varieties: V. sativa var.angustifollia, V. sativa var.cordata, V. sativa var.sativa and V. villosa, to assess their breeding potential and suitability for developing novel common vetch lines with improved agronomic characteristics related to grain production and quality.

Material and methods Germplasm

A total of 58 germplasm populations were evaluated in this study. This consisted of 1 V. michauxii, 1 V. michauxii var.stenophylla, 1 V. monanta, 1 V. narbonensis, 34 V. sativa, 9 V. sativa var.angustifollia, 1 V. sativa var.cordata, 4 V. sativa var.sativa and 6 V. villosa. The populations were acquired from the Natural Resources Genebank of Iran (Table 1).

Table 1: The list of studied 58 vetch (Vicia spp.) populations

Taxon

Code

V. michauxii V. michauxii V. monantha var.stenophylla V. narbonensis

2944 37129 32845 34878 5321 6646 6654 6681 11760 11761 11762 11763 11764 11771 11772 11774 24062 24069 24074 24076 24084 24097 32972 33456 38517 38523 38526 38527 38528 38531

V. sativa

Abbre. code Vmi Vmis Vmo Vn Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs Vs

Origin, province East Azerbaijan, Kaleybar Qom Kermanshah Lorestan, Aleshtar East Azerbaijan Lorestan, Kohdasht Lorestan, Kohdasht Lorestan, Kohdasht Gilan, Rezvanshahr Gilan, Rasht Gilan, Rezvanshahr Gilan, Rasht Gilan, Talesh Gilan, Talesh Gilan, Rezvanshahr Gilan, Rasht Gilan, Astaneh Ashrafiyyeh Gilan, Chabuksar Gilan, Astaneh Ashrafiyyeh Gilan, Chabuksar Gilan, Rahimabad Gilan, Rahimabad Kermanshah, Hersin Hamadan Gilan, Siyahkal Gilan, Talesh Gilan Gilan, Astra Gilan, Rudsar Gilan, Rezvan shahr

848

Longitude

Latitude

47° 50° 47° 48° 46° 33° 33° 33° 37° 36° 37° 37° 37° 37° 37° 37° 37° 36° 37° 36° 37° 37° 34° 47° 49° 49° 48° 48° 50° 49°

38° 34° 34° 33° 37° 47° 47° 47° 49° 49° 49° 49° 45° 48° 49° 49° 49° 50° 50° 50° 50° 50° 47° 34° 36° 37° 37° 38° 36° 37°

02´ 56´ 14´ 10´ 16´ 40´ 17´ 32´ 31´ 51´ 37´ 59´ 32´ 42´ 32´ 11´ 20´ 56´ 19´ 57´ 02´ 01´ 13´ 57´ 57´ 3´ 46´ 58´ 12´ 20´

51´ 11´ 8´ 45´ 54´ 30´ 27´ 37´ 13´ 37´ 07´ 33´ 55´ 55´ 07´ 39´ 47´ 32´ 07´ 35´ 18´ 17´ 25´ 24´ 59´ 36´ 41´ 24´ 48´ 30´

Altitude (m asl) 1500 2482 1338 1495 1750 1200 1130 1260 280 80 280 100 280 150 20 120 25 170 16 210 40 45 1367 1545 342 405 827 21 608 315


Rev Mex Cienc Pecu 2022;13(4):846-865

V. sativa var.angustifollia

V. sativa var.cordata V. sativa var.sativa

V. villosa

38532 38533 38536 40310 40315 40326 40334 43100 38524 38525 38530 38534 38535 38537 4740 7243 38529 34295 1862 24631 29802 32900 315 322 6268 14561 28061 34212

Vs Vs* Vs* Vs Vs Vs Vs Vs Vsa Vsa Vsa Vsa Vsa Vsa Vsa Vsa Vsa Vsc Vss Vss Vss Vss Vv Vv Vv Vv Vv Vv

Gilan, Talesh Gilan Gilan Kermanshah, Salase babajani Kermanshah, Salase babajani Kermanshah, Javanrud Kermanshah, Salase babajani Khozestan, Masjed soliman Gilan, Siahkal Gilan, Talesh Gilan, Talesh Gilan, Rasht Gilan, Rodbar Gilan, Gilan Ilam, Ivan Kohkiloye ve Boyerahmad, Gilan, Rezvan shahr Firozabad Gilan, Rezvan shahr Kermanshah Kermanshah Kohkiloye ve Boyerahmad Kermanshah Alborz, Karaj Karaj Fars, Shiraz, Sepidan, Sheshpir Merkezi, Arak Ardabil Chahar-mahale Bakhtiyari,

49° 38° 36° 34° 34° 34° 34° 31° 50° 48° 48° 49° 49° 49° 46° 52° 49° 49° 47° 47° 30° 34° 35° 35° 30° 34° 38° 31°

4´ 10´ 54´ 49´ 49´ 48´ 51´ 56 14´ 51´ 52´ 35´ 40´ 31´

26´ 5´ 57´ 4´ 06´ 06´ 59´ 16´ 83´ 83´ 25´ 09´ 25´ 46´

37° 48° 49° 46° 46° 46° 46° 49° 36° 37° 37° 37° 36° 36° 33° 28° 37° 37° 34° 34° 51° 46° 51° 51° 51° 49° 48° 50°

37´ 20´ 26´ 05´ 05´ 33´ 01´ 18´ 53´ 41´ 41´ 0´ 46´ 56´ 38´ 86 28´ 36´ 31´ 31´ 07´ 09´ 01´ 01´ 98´ 70´ 29´ 59´

450 600 577 1395 1395 1525 1395 870 670 281 215 137 968 187 1170 1900 307 310 1350 1400 2380 1444 1460 1470 2350 1730 1350 2600

Borujen

Field trial

Seed of all 58 populations were sown in seedling pots (December 2018). Then planting and maintenance operations were carried out in the field at the research field of Research Institute of Forests and Rangelands, Alborz province, Iran (2018–2020). A week before planting, the soil was prepared as a fine seedbed to enhance good seedling establishment. The field experimental layout was a One-way analysis of variance (ANOVA) designed. The row and plant spacings were 100 and 40 cm, respectively. The trial was managed according to previouse experiences (several hand weeding was practiced, the first hand weeding was made 40 d after crop emergence, and then repeated every forty days until the end of the growing season, to minimize yield reduction due to weed competitions for soil nutrients, water and solar radiation). Irrigation was applied during the trial. The populations were harvested for seed during the period July to November 2020, depending on their maturity.

Morphological traits

Ten plants (normal growth, uniform performance, disease- and insect pest-free) of each 58 Vicia populations were evaluated by 12 different quantitative traits including day to spourat (day to germination), days to first flowering, days to total flowering, days to maturity (days to 849


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seed ripening), plant height (at 50 % flowering, cm), internode length (second internode at 50 % flowering, cm), stems number, pod length (cm), pod width (cm), pod index (pod length/width), biomass yield (plant fresh weight) (g), and plant dry weight (g)(17).

Data analysis

Data were subjected to analysis of variance (ANOVA) using the SAS software system(18). Significant differences among the mean values of 12 traits were compared the DMRT Duncan test. Pearson correlation was determined using SPSS v.21. To evaluate the information contained in the collected morphological data, principal component analysis (PCA) was carried out by Minitab software (version 15). PCA was used to identify the most important traits (plant height, internode length, stems number, pod length, pod width, pod index, biomass yield, dry weight, day to sprout, days to first flowering, days to total flowering, days to maturity) in the data set. Mean values populations were used to create a correlation matrix from which the standardized PCA scores were extracted and a Scatter plot on the first two PCA was performed. Cluster analysis was performed using Ward’s methods and Euclidean distance and a dendrogram was calculated.

Results The results of analysis variance revealed significant (P<0.01) variation for eight morphological and yield traits among taxa and populations of Vicia spp. except for pod width trait among populations (Table 2). Table 3 shows the comparison of mean morphological and yield traits in nine taxa of Vicia spp. The value of plant height, internode length and stems number differ between 24.50-150 cm, 3.29-15 cm and 2.81-9, respectively. The highest value of plant height (150 cm), stems number (9) and internode length (15 cm), were shown in V. monantha (Vmo) and V. michauxii var. stenophylla (Vmis), respectively. The variation of pod length between taxa was significant and it differs from1.06 cm in V. sativa var.cordata (Vsc) to 4 cm in V. monantha (Vmo). There was no significant difference in pod width between taxa and they located as two groups (a and b), so two taxa of V. michauxii (Vmi and Vmis) had the widest pod (1.14 and 1.1 cm). Despite the significant differences in biomass yield and dry weight traits, V. villosa (Vv) showed the most value of these traits (biomass yield=60.12 g and dry weight=15.63 g).

Fifty eight (58) populations of Vicia spp. were compared for vegetative and phenology traits (Table 4). There was a wide range of value in plant height from 19 cm in V. sativa var.angustifollia (38534) to 150 cm in V. monantha (32845), also the most value of plant height between populations of species were shown in V. villosa (322) (100.33 cm). V.michuxii

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var.stenophylla (37129) (100 cm), V. sativa (38527) (90 cm) and V. sativa var.cordata (34295) (85.13 cm). The length of the internode was a very differet from 1.83 cm in V. sativa (24062, 40334, 43100) to 15 cm in V. michauxii var.stenophylla (37129). Also, 9.83, 8.69, and 8.28 cm of internode length were shown in V. sativa (38527), V. sativa var.cordata (34295), and V. sativa var.angustifollia (38525), respectively. The highest and lowest number of stems were 2 and 15, which were shown in two different taxa of V. sativa species (Vsa38530 and Vs11774). This trait in populations of V. villosa was no significant different. Four populations of V. sativa (38527, 33456, 24074 and 32972), V. sativa var.angustifollia(38525) and V. monantha (32845), had the largest pod in terms of length (4-4.53 cm) and populations V. michauxii var.stenophylla (37129), and V. sativa (5321) had the largest pod in terms of width (1.1 and 1.06 cm). In compare of yield traits (biomass yield and dry weight), three populations of V. sativa: Vs11761, Vs24062, Vs40326, and two populations of V.villosa:Vv322, Vv6268, had the most values of these traits. The values of these traits in these populations were Vs11761 (83 and 26 g.), Vs24062 (83 and 26 g), Vs40326 (103.67 and 36.33 g), Vv322 (108.33 and 38.60) and Vv6268 (83.50 and 19.73 g).

The results of phenology traits showed that all of the populations based on day to sprout and days to first flowering traits were divided into two groups (a and b). V. narbonesis (34878), V. monantha (32845) and two taxa of V. michauxii (Vmi2944 and Vmis37129) had the same value in day to sprout and days to first flowering traits, but populations V. sativa var.angustifollia (Vsa4740, Vsa7243), V. sativa var.stenophylla (Vss1862, Vss24631) and two populations of V. villosa (Vv315, Vv6268) with 21 and 90 d for day to sprout and first flowering were separated from the rest of populations by earlier germination and flowering. In days to total flowering and maturity traits, populations were divided as four groups (a, b, c and d). Days to total flowering as four groups:125a,120b,115c,107d and seed maturity:167a,162b,158c,150d. Populations in group d (107 and 150 d of flowering and seed maturation) had the shortest time required for full flowering and seed maturation. That is, they reached full flowering and seed maturity earlier than other populations. Populations of V. sativa var.angustifollia (Vsa4740, Vsa7243), V. sativa var.stenophylla (Vss1862, Vss29802, Vss32900) and V. villosa (Vv315, Vv6268), having the shortest day for full flowering and seed maturation (Table 4).

Analysis of the genetic correlations among the mentioned traits in the tested vetch populations revealed the existence of several significant positive coefficients (Table 5), namely between plant height with internode length (rgxy=0.43; P<0.01), stems number (rgxy=0.38; P<0.01) and pod length with internode length (rgxy=0.24; P<0.05), pod width (rgxy =0.23; P<0.05), day to sprout (rgxy=0.28; P<0.05), days to first flowering (rgxy=0.28; P<0.05) and days to maturity (rgxy=0.26; P<0.05), pod index with day to sprout (rgxy =0.23; P<0.05) and days to first flowering (rgxy=0.23; P<0.05). On the other hand, the relationship between pod width with pod index (rgxy =−0.26; P<0.05), biomass yield (rgxy =−0.35; P<0.01), and dry weigth (rgxy =−0.28; P<0.05), internode length with dry weigth (rgxy=−0.38; P<0.01) were negatively and significant.

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Two-dimensional principal component analysis showing the relationship among quantitative traits of studied populations is presented in Figure 1. Populations V. sativa var.angustifollia (4770, 7243), V. sativa var.sativa (1862), V. villosa (315, 6268) were separated partially by PC1; traits related to this separation are mainly phenology traits (day to spourat, days to first flowering, days to total flowering, days to maturity).

A cluster analysis of the tested Vicia spp. populations showed five main groups (Table 6 and Figure 2). Cluster G1 contained five populations, belonging to V.sativa var.angustifollia with two populations (7243, 4740), V. sativa var.sativa one population (1862) and V. villosa with two populations (315, 6268).They are characterized by the lowest values of phenology traits (day to spourat, days to first flowering, total flowering, and seed maturity). Cluster G2 contained 13 populations: 11 populations belonging to V. sativa (6646, 6681, 11761, 24062, 24069, 24074, 32972, 40310, 40315, 40326, 40334), population 38530 of V. sativa var.angustifollia and 322 of V. villosa. They are also characterized by the highest amount of vegetative, seed and yield traits compared to other populations. Cluster G3 included 16 popullations belonging to V. sativa (6654, 11760, 11762, 11771, 11772, 24076, 24084, 24097, 43100), population 38529 of V. sativa var.angustifollia, V. sativa var.sativa (24631, 29802, 32900), 28061, 34212 and 14561 of V. villosa, with high amount of vegetative traits were collected in one group. Cluster G4 contained seven populations: five belong to species V. sativa (11763, 11764, 11774, 38526, 38527), population 34295 of V. sativa var.cordata and V. monantha (32845). These were classified with the highest plant height, stems number and vegetative traits compared with other clusters. Cluster G5 was the largest one with 17 populations, nine from V. sativa (5321, 33456, 38517, 38523, 38528, 38531, 38532, 38533, 38536), five from V. sativa var.angustifollia (38524, 38525, 38534, 38535, 38537), V. michauxii (2944), V. michauxii var.stenophylla (37129) and V. narbonensis (34878).These were classified as highest vegetative and pod traits populations.

The principal component analysis (PCA) of the 12 quantitative traits is summarized in Table 7. The first five PCs had eigenvalues >1 and they explained more than 80 % of the total variation for the vegetative and phenology traits. Day to sprout, days to first flowering, days to total flowering and days to maturity were loaded highly in PC1 and they accounted for 25.7 % of the total variation. In PC2, Biomass yield and dry weight accounted for 21 % of the total variation. In PC3, plant height and internode length accounted for 14.3 % of the total variation. PC4 contributed 11.2 % of the total traits variation in these populations with plant length and stems number loading highly. PC5 accounted for 9.8 % of total variation with length, width and pod index. Generally, for the 12 vegetative and phenology traits studied, PC1 and PC2 constituted more than 46 % of the total traits variation with most phenology traits and yieldrelated traits. This indicated that these traits can be used to classify the populations under study.

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Table 2: Analysis variance of eight morphological traits of 58 vetch (Vicia spp.) populations

Source of Variations

Degrees of freedom (d.f)

Plant height

Internode length

Stem number

Pod length

Pod width

Pod index

Biomass yield

Dry weight

Taxon

8

3770.70**

48.92**

25.93**

5.23**

0.47**

9.19**

3809.63**

223.70**

Population

48

905.59**

12.11**

15.62**

1.89**

0.06 ns

7.76**

1967.42**

202.36**

Error

150

346.70

2.28

2.58

0.39

0.05

1.30

218.67

18.08

35.88

29.18

32.38

22.77

39.08

22.53

44.56

48.83

CV %

*, ** significant at 0.05 and 0.01 levels, respectively;

ns

not significant.

Table 3: Means comparison of 8 traits in different species of Visia spp. Plant height (cm) V. michauxii (Vmi) 63.38 cd V. michauxii var. stenophylla 100.0 b V. monantha (Vmo) 150.0 a (Vmis) V.narbonensis (Vn) 24.50 e V. sativa (Vs) 48.41 de V. sativa var. angustifollia 45.07 de V. sativa var. cordata (Vsc) 85.13 bc (Vsa) V. sativa var. sativa (Vss) 54.67 ce V. villosa (Vv) 63.56 cd Species

Internode length (cm) 6.56 bc 15.0 a 8.0 b 4.75 cd 4.8 cd 6.9 bc 8.69 b 3.29 d 3.36 d

Stems number 2.81 b 5.0 b 9.0 a 3.0 b 5.08 b 4.6 b 8.75 a 4.58 b 4.5 b

853

Pod length (cm)bc 2.54 2.5 bc 4.0 a 3.3 ab 2.88 bc 3.27 ab 1.06 d 2.0 cd 2.17 c

Pod width (cm) 1.14 a 1.1 a 0.6 b 0.65 b 0.56 b 0.60 b 0.29 b 0.5 b 0.51 b

Pod index 4.36 b 2.27 c 6.67 a 5.1 ab 5.33 ab 5.48 ab 3.75 bc 4.0 bc 4.26 b

Biomass yield (g) 9.59 cd 20.0 bd 0.06 d 5.13 cd 35.06 b 19.03 bd 27.92 bc 41.08 ab 60.12 a

Dry weight (g) 5.46 bc 5.0 bc 0.01 c 1.15 c 9.16 ab 5.18 bc 6.92 bc 9.32 ab 15.63 a


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Table 4: Means comparison of 12 traits of 58 populations of different species of Vicia spp. Population Vmi2944 Vmis37129 Vmo32845 Vn34878 Vs5321 Vs6646 Vs6654 Vs6681 Vs11760 Vs11761 Vs11762 Vs11763 Vs11764 Vs11771 Vs11772 Vs11774 Vs24062 Vs24069 Vs24074 Vs24076 Vs24084 Vs24097 Vs32972 Vs33456 Vs38517 Vs38523 Vs38526 Vs38527 Vs38528

Plant Height (cm) 78.56 c-e 100 b 150 a 24.50 i-k 43.88 e-k 44.33 e-k 28.33 h-k 63.00 c-h 56.67 c-k 66.67 b-g 37.67 f-k 80.00 b-e 61.67 c-i 52.33 d-k 65.00 b-h 68.33 b-f 40.00 f-k 41.00 f-k 49.67 d-k 41.33 f-k 55.00 c-k 51.33 d-k 44.67 e-k 43.43 e-k 34.67 f-k 43.33 e-k 86.17 b-d 90.00 bc 53.40 c-k

Internode Length (cm) 7.94 b-e 15 a 8 b-e 4.75 f-p 4.00 i-p 2.83 n-p 2.00 p 3.33 l-p 4.50 g-p 6.17 c-l 3.67 j-p 6.33 c-i 5.17 e-o 4.83 f-p 3.17 m-p 4.33 h-p 1.83 p 2.50 n-p 4.17 h-p 3.50 k-p 5.33 d-o 3.00 n-p 2.83 n-p 6.07 c-m 6.17 c-l 7.33 c-g 6.50 c-j 9.83 b 8.00 b-e

Stems Number 2.67 g-i 5 f-i 9 b-d 3.00 g-i 2.50 hi 4.67 f-i 4.00 f-i 5.00 f-i 4.67 f-i 5.33 f-h 6.00 e-g 11 bc 11.67 b 4.33 f-i 4.67 f-i 15.00 a 4.67 f-i 5.33 f-h 6.00 e-g 4.33 f-i 4.67 f-i 6.00 e-g 3.00 g-i 4.86 f-i 3.33 f-i 3.67 f-i 6.33 f-i 3.00 g-i 6.40 d-f

Pod Length (cm) 2.87 c-j 2.5 f-k 4 a-c 3.30 b-g 2.14 g-l 2.83 c-j 2.00 h-l 3.97 a-d 2.00 h-l 2.50 f-k 2.50 f-k 3.33 b-g 2.67 e-k 2.00 f-k 2.00 f-k 2.67 e-k 2.00 f-k 3.83 a-e 4.00 a-c 2.50 f-k 2.67 e-k 2.00 f-k 4.00 a-c 4.04 a-c 2.50 f-k 3.17 b-h 2.73 e-k 4.53 a 3.63 a-f

Pod Width (cm) 0.82 ab 1.1 a 0.6 bc 0.65 bc 1.06 a 0.50 bc 0.50 bc 0.50 bc 0.50 bc 0.50 bc 0.50 bc 0.50 bc 0.50 bc 0.50 bc 0.50 bc 0.50 bc 0.50 bc 0.50 bc 0.47 bc 0.47 bc 0.50 bc 0.50 bc 0.57 bc 0.64 bc 0.53 bc 0.60 bc 0.40 bc 0.60 bc 0.63 bc

Pod Index

Biomass Yield (g)

3.72 k-p 2.27 op 6.67 a-h 5.10 f-n 2.00 p 5.67 c-k 4.00 j-p 7.93 ab 4.00 j-p 5.00 f-n 5.00 f-n 6.67 a-h 5.33 e-m 4.00 j-p 4.00 j-p 5.33 e-m 4.00 j-p 7.67 a-d 8.67 a 5.50 d-l 5.33 e-m 4.00 j-p 7.11 a-f 6.39 b-i 4.72 g-n 5.28 f-m 6.18 b-j 7.56 a-e 5.75 b-k

10.15 j-n 20.00 h-n 0.06 n 5.13 l-n 2.07 mn 61.83 b-d 42.67 e-i 74.83 bc 53.00 c-g 83.00 ab 19.00 h-n 45.00 e-h 51.17 c-g 56.67 b-f 41.50 e-i 53.00 c-g 83.00 ab 37.17 e-k 44.00 d-h 41.50 e-i 44.00 d-h 45.00 e-h 48.00 c-h 21.29 h-n 11.27 j-n 25.87 g-n 18.92 h-n 20.41 h-n 34.49 e-l

854

Dry Weight (g) 2.66 i-n 5.00 g-n 0.01 n 1.16 k-n 0.55 l-n 14.61c-f 10.83 d-i 18.43 cd 15.27 c-f 26.00 b 5.06 g-n 9.33 e-k 9.03 e-l 11.43 d-h 8.93 e-l 15.27 c-f 26.00 b 10.43 d-j 10.43 d-j 8.93 e-l 10.43 d-j 8.77 e-m 15.10 c-f 5.30 g-n 3.05 h-n 7.17 f-n 5.50 g-n 3.91 h-n 8.91 e-l

Day to Sprout 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a 28 a

Days to first Flowering 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a 95 a

Days to total Flowering 115 c 115 c 115 c 120 b 115 c 115 c 115 c 115 c 115 c 115 c 115 c 120 b 115 c 115 c 120 b 115 c 120 b 115 c 115 c 120 b 115 c 115 c 120 b 115 c 115 c 115 c 115 c 115 c 115 c

Days to Maturity 158 c 158 c 158 c 162 b 158 c 158 c 158 c 158 c 158 c 162 b 158 c 162 b 158 c 158 c 162 b 158 c 162 b 158 c 158 c 162 b 158 c 158 c 162 b 158 c 158 c 158 c 158 c 162 b 158 c


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Days to Days to Internode Pod Pod Dry Stems Day to Days to Pod Biomass total Length Length Width Weight Population first Number Index Yield (g) Sprout Maturity (cm) (cm) (cm) (g) Flowering Flowering Vs38531 6.40 c-k 5.20 f-i 2.84 c-j 0.60 bc 4.68 h-n 13.00 i-n 3.95 h-n 28 a 95 a 115 c 158 c Vs38532 7.60 b-f 4.80 f-i 2.00 f-k 0.64 bc 3.27 l-p 8.35 k-n 2.00 i-n 28 a 95 a 115 c 162 b Vs38533 4.74 f-p 3.40 f-i 3.66 a-f 0.62 bc 5.93 b-k 10.54 j-n 2.29 i-n 28 a 95 a 115 c 158 c Vs38536 7.00 c-h 3.67 f-i 2.23 g-k 0.50 bc 4.47 h-o 3.04 mn 0.83 k-n 28 a 95 a 115 c 158 c Vs40310 3.17 m-p 3.67 f-i 3.33 b-g 0.50 bc 6.67 a-h 55.00 c-g 15.77 c-e 28 a 95 a 115 c 158 c Vs40315 2.50 n-p 3.67 f-i 3.17 b-h 0.50 bc 6.33 b-i 51.33 c-g 14.63 c-f 28 a 95 a 115 c 158 c Vs40326 2.33 op 4.33 f-i 3.50 a-f 0.50 bc 7.00 a-f 103.67 a 36.33 a 28 a 95 a 120 b 162 b Vs40334 1.83 p 4.67 f-i 3.33 b-g 0.43 bc 7.83 a-c 41.50 e-i 8.93 e-l 28 a 95 a 115 c 158 c Vs43100 1.83 p 4.33 f-i 1.50 kl 0.50 bc 3.00 n-p 9.57 j-n 0.33 mn 28 a 95 a 120 b 162 b Vsa38524 7.70 b-f 4.40 f-i 3.16 b-h 0.78 ab 4.07 j-p 7.18 l-n 1.95 j-n 28 a 95 a 115 c 158 c Vsa38525 8.28 b-d 5.56 f-h 4.22 ab 0.61 bc 6.96 a-g 19.92 h-n 5.62 g-n 28 a 95 a 115 c 162 b Vsa38530 6.67 c-i 2.00 i 3.17 b-h 0.53 bc 5.69 c-k 29.33 e-n 10.98 d-h 28 a 95 a 115 c 162 b Vsa38534 5.50 d-n 5.00 f-i 3.25 b-h 0.50 bc 6.50 b-i 26.75 f-n 5.80 g-n 28 a 95 a 115 c 162 b Vsa38535 6.50 c-j 2.50 hi 2.75 d-k 0.65 bc 4.29 i-o 4.55 l-n 1.25 k-n 28 a 95 a 115 c 158 c Vsa38537 8.00 b-e 5.67 f-h 2.93 c-i 0.60 bc 4.89 f-n 4.70 l-n 1.05 k-n 28 a 95 a 115 c 162 b Vsa4740 3.50 k-p 5.00 f-i 2.00 h-l 0.50 bc 4.00 j-p 45.00 f-h 9.33 e-k 21 b 90 b 107 d 150 d Vsa7243 5.17 e-o 4.33 f-i 2.67 e-k 0.50 bc 5.33 e-m 18.67 h-n 5.67 g-n 21 b 90 b 107 d 150 d Vsa38529 4 i-p 3 g-i 0.6 m 0.3 c 2.00 p 3.10 mn 0.60 l-n 28 a 95 a 115 c 158 c Vsc34295 8.69 bc 8.75 c-e 1.06 l 0.29 c 3.75 k-p 27.92 e-n 6.92 f-n 28 a 95 a 115 c 158 c Vss1862 4.33 h-p 4.33 f-i 2.00 0.50 bc 4.00 j-p 31.67 e-m 9.00 e-l 21 b 90 b 107 d 150 d Vss24631 2.33 op 5.00 f-i 1.83 i-l 0.50 bc 3.67 k-p 56.67 b-f 11.43 d-h 21 b 90 b 125 a 167 a Vss29802 3.50 k-p 4.33 f-i 2.50 h-l 0.50 bc 5.00 f-n 41.50 e-i 8.93 e-l 28 a 95 a 107 d 150 d Vss32900 3.00 n-p 4.67 f-i 1.67 j-l 0.50 bc 3.33 l-p 34.50 e-l 7.93 e-n 28 a 95 a 107 d 150 d Vv315 2.83 n-p 4.33 f-i 2.00 h-l 0.50 bc 4.00 j-p 53.83 c-g 13.08 c-g 21 b 90 b 107 d 150 d Vv322 4.00 i-p 4.33 f-i 2.50 f-k 0.50 bc 5.00 f-n 108.33 a 38.60 a 28 a 95 a 115 c 158 c Vv6268 3.17 m-p 4.67 f-i 1.67 j-l 0.53 bc 3.17 m-p 83.50 ab 19.73 bc 21 b 90 b 107 d 150 d Vv14561 4.50 g-p 4.67 f-i 2.67 e-k 0.50 bc 5.33 f-n 57.71 b-e 10.83 d-i 28 a 95 a 115 c 158 c Vv28061 2.00 p 4.33 f-i 2.00 h-l 0.50 bc 4.00 j-p 18.00 h-n 3.73 h-n 28 a 95 a 115 c 158 c Vv34212 3.67 j-p 4.67 f-i 2.17 g-l 0.53 bc 4.06 j-p 39.33 e-i 7.80 e-n 28 a 95 a 115 c 158 c Different letters indicate significant differences among different populations for the same species. P <0.05. V. michauxii (Vmi), V. michauxii var. stenophylla (Vmis), V. monantha (Vmo), V.narbonensis (Vn), V. sativa (Vs), V. sativa var. angustifollia (Vsa), V. sativa var. cordata (Vsc), V. sativa var. sativa (Vss), V. villosa (Vv). Plant Height (cm) 46.00 e-k 29.80 g-k 33.30 f-k 36.33 f-k 33.33 f-k 38.33 f-k 44.67 e-k 31.33 f-k 22.00 j-k 29.60 g-k 50.44 d-k 65.33 b-h 19.00 k 36.50 f-k 43.67 e-k 49.67 d-k 54.33 c-k 30 f-k 85.13 b-d 44.67 e-k 51.00 d-k 67.33 b-g 55.67 c-k 55.00 c-k 100.33 b 47.67 e-k 58.33 c-j 51.67 d-k 68.33 b-f

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Table 5: Simple correlation matrix for the 12 traits of Vicia spp. populations Traits

Internode length Stems number Pod length Pod width Pod index Biomass yield Dry weight Day to sprout Days to first flowering Days to total flowering Days to maturity

Plant height

internode length

Stems number

Pod length

Pod width

Pod index

Biomass yield

Dry weight

Day to sprout

Days to first flowering

0.43** 0.38** 0.13 ns 0.10 ns 0.09 ns 0.11 ns 0.13 ns 0.04 ns 0.04 ns -0.08 ns -0.07 ns

0.11ns 0.24* 0.51 ns -0.06 ns -0.46 ns -0.38** 0.19 ns 0.19 ns -0.03 ns 0.13 ns

0.03 ns -0.22 ns 0.16 ns 0.14 ns 0.07 ns 0.06 ns 0.06 ns 0.05 ns 0.02 ns

0.23* 0.86 ns -0.03 0.03 ns ns 0.28* 0.28* 0.17 ns 0.26*

-0.26* -0.28* 0.11 ns 0.35** 0.11 ns 0.03 ns 0.06 ns

0.20 0.22 ns 0.23* ns 0.23* 0.15 0.21

0.95 ns -0.16 ns -0.16 ns 0.04 ns -0.03 ns

-0.09 -0.09 ns ns 0.07 ns ns 0.02

1 ns 0.46 ns 0.50 ns

0.46 ns 0.50 ns

Days to total flowering

0.92 ns

ns

*, ** significant at 0.05 and 0.01 levels, respectively; ns not significant. ns

Table 6: Means comparison of 12 traits of five vetch groups produced in Figure 2 Groups

Plant height (cm)

Internode length (cm)

Stems number

Pod length (cm)

Pod width (cm)

Pod index

Biomass yield (g)

Dry weight (g)

Day to sprout

G1 G2 G3 G4 G5

50.27 b 50.97 b 49.50 b 88.76 a 43.91 c

3.80 c 3.40 c 3.43 c 6.98 b 7.12 a

4.53 c 4.36 c 4.60 b 9.25 a 4.21 c

2.07 c 3.24 a 2.04 c 3.00 b 3.01 b

0.51 b 0.50 b 0.49 c 0.48 c 0.68 a

4.10 d 6.51 a 4.14 d 5.93 b 4.72 c

46.53 b 63.15 a 37.73 c 30.92 c 13.43 d

11.36 b 18.94 a 8.20 c 7.14 c 3.44 d

21.00 c 28.00 a 27.56 b 28.00 a 28.00 a

abc

Days to Days to Days to first total maturity flowering flowering

90.00 c 95.00 a 94.69 b 95.00 a 95.00 a

Different letters indicate significant differences among different populations for the same species. P<0.05.

856

107.00 c 116.15 a 115.56 b 115.71 b 115.29 b

150.00 c 159.54 a 158.31 b 159.14 a 159.18 a


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Table 7: Eigenvalues, the proportion of variance, and morphological traits that contributed to the first five principal components (PC) Variable Plant height Internode length Stems number Pod length Pod width Pod index Biomass yield Dry weight Day to sprout Days to first flowering Days to total flowering Days to maturity Eigenvalue Proportion Cumulative

PC1

PC2

PC3

PC4

PC5

0.058 0.208 0.050 0.310 0.141 0.230 -0.144 -0.101 0.464 0.464 0.380 0.419 3.340 0.257 0.257

-0.029 0.302 -0.107 -0.154 0.303 -0.332 -0.53 -0.541 -0.017 -0.017 -0.129 -0.100 2.723 0.210 0.467

0.466 0.428 0.176 0.389 0.243 0.265 -0.004 0.043 -0.102 -0.102 -0.338 -0.267 1.856 0.143 0.610

0.441 0.055 0.668 -0.244 -0.301 -0.084 0.062 -0.042 0.045 0.045 0.057 0.014 1.452 0.112 0.721

-0.267 -0.242 0.075 0.378 -0.357 0.533 -0.236 -0.321 -0.075 -0.075 -0.164 -0.151 1.279 0.098 0.820

Figure 1: Two principal components showing the relationship among 12 traits of 58 populations of Vicia spp. 4

Vms37129 Vs5321

3

Vm2944 Vs43100 Vsa38524 Vs38532 Vsa38529 Vsa38535 Vsa38537 Vs38536 Vm32845 Vs38517 Vs38533 Vn34878 Vss32900 Vs38531 Vv28061 Vs11762 Vs38527 Vs38523 Vsc34295 Vs33456 Vv34212 Vsa38534 Vs24097 Vss29802 Vs38526 Vs38528 Vsa38525 Vs11772 Vv14561 Vs24084 Vs6654 Vsa38530 Vs11764 Vs11771 Vs24076 Vs11760 Vss24631 Vs40334 Vs6646 Vs11763 Vs24069 Vs40315 Vs24074 Vs40310 Vs11774 Vs32972 Vs11761

Second Component (21% )

2 1

Vss1862

Vv315

0 -1

Vsa4740 Vsa7243

Vv6268

-2 -3

Vs24062

-4

Vv322

Vs6681

Vs40326

-5 -6

-5

-4

-3

-2 -1 First Component (26% )

1 857

0

1

2

3


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Figure 2: Dendrogram of 58 populations of Vicia spp. explained by complete linkage clustering of 12 traits Vss1862 Vsa7243 Vv6268 Vv315 Vsa4740 Vv322 Vs40326 Vs24062 Vs11761 Vsa38530 Vs32972 Vs40334 Vs24074 Vs24069 Vs6681 Vs40315 Vs40310 Vs6646 Vss24631 Vsa38529 Vs43100 Vss32900 Vss29802 Vs24076 Vs11772 Vv14561 Vs24084 Vv34212 Vs24097 Vs11771 Vv28061 Vs11762 Vs11760 Vs6654 Vsc34295 Vs38526 Vs11774 Vs11764 Vs11763 Vs38527 Vm32845 Vsa38524 Vsa38537 Vs38532 Vs38531 Vs38523 Vsa38535 Vs38536 Vs38517 Vsa38525 Vs38528 Vsa38534 Vs38533 Vs33456 Vn34878 Vms37129 Vs5321 Vm2944 34.48

11.49

22.99

Distance

2858

0.00


Rev Mex Cienc Pecu 2022;13(4):846-865

Discussion In these study, 58 populations of Vicia spp. were investigated for genetic diversity based on morphological and phenology traits. Due to, genetic diversity analysis of germplasms using morphological traits is an initial step for crop improvement(19-22). There was significant (P<0.01) genotypic variation among 58 germplasm accessions of Vicia spp. for all the measured vegetative and yield traits: plant length, internode length, stems number, pod length, pod width, pod index, biomass yield and dry weight.The estimates of genotypic variation and repeatability for these traits indicated the potential genetic variation available among the germplasm accessions within Vicia spp. investigated. Similar results were obtained by the Ebrahimi et al(23) on plant and seed morphology traits of white Bean genotypes, Mikic et al(12) on forage and seed yields of three lines of common vetch and Berhanu and Abera(24) on forage yield of vetch species investigation.

A comparison between taxon (V. sativa: Vs, Vsa, Vsc and Vss, V. mchauxii: Vmi and Vmis, V. monantha: Vmo, V. narbonensis: Vn and V. villosa: Vv) showed V. monantha (Vmo) with high values of plant height, stems number, pod length and V. villosa with high values of biomass yield and dry weight. Berhanu and Abera(24) showed that among the vetch species (V. sativa, V. villosa, V. dasycarpa, and V. bengalensis), V. dasycarpa and V. villosa were the best performing species for forage. Then the vetch species tested in the current study could be used for pasture expansion and forage production, in livestock exclusion areas, in forage strips, as an under-sowing with food crops, or as a backyard forage crop in the pasture of the country.

The populations demonstrated high variation in plant height, internode length, stem number, pod length, biomass yield and dry weight. Populations: Vmo32845, Vv322, Vmis37129 (for plant height), populations: Vmi37129, Vs38527, Vsc34295 (for internode length), populations: Vs11774,Vs11764,Vs11763 (for stems number), populations: Vs38527, Vmo32845, Vsa38525 (for pod length) and populations:Vs11761, Vs24062, Vs40326, Vv322, Vv6268 (for biomass yield and dry weight) showed the highest values of the mentioned traits. However, broadening the genetic base from diverse sources is recommended to include most of the genetic determinants of these traits(25). This variability can be exploited in fodder breeding programs to select an adapted plant material for the arid and semi-arid areas(26). Phenology (earliness and lateness) of vetch species has a great effect on seed yield productivity. Late maturity for forage and seed was recorded at 125 and 167 d, respectively. This could be due to high and extended rainfall at the region of populations that encouraged vegetative growth and delayed forage and seed harvesting stages. The results indicated that for vetch populations tested, 107 to 125 and 150 to 167 d were required after the emergence of the seedlings for total flowering and seed maturity, respectively. On average, the difference in

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harvest forage and seed yield between populations are about 18 and 17 d. This indicates different responses of the tested populations for these important agronomic traits.

According to Getnet et al(27), Vicia narbonensis and Vicia sativa are early maturing and Vicia villosa is late maturing species. But in this study two populations of V. villosa (315 and 6268) and four populations of V.sativa var.angustifollia (4740, 7243), V. sativa var.sativa (1862, 29802, 32900) with 107 and 150 d for flowering and seed maturity is recommended for seed production due to earliness, whereas late-maturing species like V. sativa var.sativa (24631) should not be advisable to grow for seed purpose.

There is a direct relationship between plant length with internode length and the number of stems, this indicates that tall plants produce long internodes and more number stems. Also, the length and width of the pod have a direct relationship with the number of days of sprout, flowering and seed maturation, which means long and wide pods are produced by lateflowering and seed maturation time. Since, in cereals, the correlation between grain yield and plant height is often negative, but in legumes, this correlation is often positive, because legumes have unlimited growth, therefore, with increasing height, more pods are produced, which has a positive effect on performance, so similar results were obtained in the traits of white Bean genotypes where high grain yields were strongly correlated to days to flowering and plant height(23) and Lens spp.(28).

In PCA, since the first component includes changes that are not explained by the second component and the two components are independent of each other, so the two components were intersected vertically and in the form of a biplot diagram to determine the diversity between different genotypes and determine the far and near genotypes to be used. Phenology traits (day to sprout, days to first flowering, days to total flowering, days to maturity) accounted for the variations recorded in the populations in PC1. On the other hand, yield traits (biomass yield and dry weight) accounted for the variation observed in the populations in PC2. The total cumulative variance in the first two PC was more than 46 %, indicating the high degree of diversity among the traits under study. Furthermore, the traits can be used as phenotypic traits in differentiating the populations. In plot PCA (Figure 1), populations, V. sativa var.angustifollia (Vsa7243, Vsa4740), V. sativa var.sativa (1862) and V. villosa (Vv315, Vv6268), separated from other populations and located on the left of X-axis by containing less of phenology traits (important in the first component). So, these populations recommend for areas with short growth periods. Populations V. sativa (40326) and V. villosa (322), for containing high value of biomass and dry weight, located on the bottom of Y-axis (negative effect of biomass and dry weight on the second component). As a result, two populations, V. sativa (40326) and V. villosa (322), produce forage yield more than other populations.

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In the present study, the 58 populations of Vicia spp. were grouped into five clusters using 12 traits.The populations of cluster G1 are characterized by the lowest values of days to sprout, flowering, and seed maturity which are the candidate of further evaluations. Also, these populations had a shoter time for these traits. Members of G1 are similar to the dispersion of these populations in the PCA plot (Figure 1). It is interesting that the population from different climates like Shiraz clustered with populations from Karaj. This pattern of clustering indicates, the diversity of populations within these geographical areas and, the similarity of populations from different geographical areas.

These results agree with the report of Alemayehu and Becker(29) in Brassica carinata. Cluster G2 contained 13 populations belonging to V. sativa and V. villosa species. These populations had a high value of seed, yield and phenology traits. Member of G2 due to having a long time for flowering and seed maturity, produce more seed and forage yield. This is the best factor, that can be used for livestock feeding. Cluster G3 contained mixed 16 populations of V. sativa and V. villosa. with lowest values of seed and forage yield gather together in a group, that they are not important inbreeding. Cluster G4 contained seven populations of V. sativa and V. monantha with high vegetative traits, that recommend livestock feeding and control of erosion. G5 group with 17 populations of V. sativa, V. michauxii and V. narbonensis were classified as later flowering and seed ripening and containing less amount of yield forage. These populations can be used for areas with a long growth time.

Finally in this study populations were located as five groups based on morphology and phenology traits. Members of each group are similar for mentioned traits and can be recommended for breeding programs. Also, the results indicated no relationship between studied traits and the origin of populations.

Conclusions and implications The findings showed the high variation of morphology and yield traits in different species and populations of vetch. These differences are very important to select the type of companion crops and methods of integration to improve yields of both crops (food and forage) without significant effect of one on the other. Vicia sativa (Kermanshah, Javanrod) and V. villosa (Karaj) were superior in terms of fresh and dry forage yields. V.michauxii var.stenophylla (Qom), V. monantha (Kermanshah), V. sativa (Gilan, Astara), and V. villosa (Karaj), are recommended by having tall plant and big pods. However, more comprehensive studies and additional experiments are required to complete information for breeding programs.

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Acknowledgments

The authors thank Dr. Jalilian for identification of plants and the director of Gene Bank for providing seeds and making the lab facilities available for our study and RIFR in Iran for financial support. Literature cited: 1.

Mozaffarian V. A dictionary of plant names. 1st ed. Farhang moaser publication, Tehran, Iran. 2006.

2.

Rebole A, Alzueta C, Ortiz LT, Barrol C, Rodriguez ML, Caballero R. Yields and chemical composition of different parts of the common vetch at flowering and at two seed filling stages. Span J Agric Res 2004;2(4):550-557.

3.

Gurmani ZA, Shafiq ZM, Bashir M. Performance of Vetch, Vicia sativa cultivars for fodder production under rain fed condition of Pothwar region. J Agric Res 2006;44(4): 291-299.

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Kebede G. Correlation and cluster analysis for quantitative and qualitative traits of accessions of vetch species in the central highlands of Ethiopia. G J Adv Res 2016; 3(7):56-72.

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Duc G, Bao SY, Baum M, Redden B, Sadiki M. Suso MJ. Diversity maintenance and use of Vicia faba L. genetic resource. Field Crops Res 2010;115:270-278.

6.

Büyükkartal HN, Çölgeçen H, Pınar NM. Seed coat ultrastructure of hard-seeded and softseeded varieties of Vicia sativa. Turk J Botany 2013;37:270–275.

7.

Smýkal P, Coyne CJ, Ambrose MJ, Maxted N, Schaefer H, Blair MW, et al. Legume crops phylogeny and genetic diversity for science and breeding. Crit Rev Plant Sci 2015;34:43104.

8.

Rahmati T, Azarfar A, Mahdavi A, Khademi K, Fatahnia F, Shaikhahmadi H, Darabighane B. Chemical composition and forage yield of three Vicia varieties (Vicia spp.) at full blooming stage. Ital J Anim Sci 2012;1(3):308-311.

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9.

Dong R, Jahufer MZZ, Dong DK, Wang YR, Liu ZP. Characterisation of the morphological variation for seed traits among 537 germplasm accessions of common vetch (Vicia sativa L.) using digital image analysis. N Z J Agric Res 2016;59(4):422–435.

10. Demirkan AK, Nizam I, Orak A, Şen C, Serkan Tenikecier H, Güler N, Ersoy H. Determination of some morphological characters and forage yield of Vetch (Vicia sp.) genotypes collected from thrace region of Turkey. Int J Adv Res 2018;(11):276-283. 11. Kebede G. Morpho‑agronomic performance of vetch species and their accessions grown under nitosol and vertisol conditions in the central highlands of Ethiopia. Agric Food Secur 2018;7(90):1-14. 12. Mikić A, Mihailović V, Karagić D, Milošević B, Milić D, Vasiljević S, Katanski S, Zivanov D. Common vetch (Vicia sativa) multi-podded mutants for enhanced commercial seed production. Proc Appl Bot Genet Breed 2019;180(1):78-81. 13. Sanchez-Gutierrez RA, Figueroa-Gonzáles JJ, Rivera-Vázquez JS, Reveles-Hernández M, Gutiérrez-Bañuelos H, Espinoza-Canales A. Yield and nutritional value of common vetch (Vicia sativa l.) during fall-winter in Zacatecas, Rev Mex Cienc Pecu 2020;11(1):294-303. 14. Grela ER, Samolinska W, Rybinski W, Kiczorowska B. Nutritional and anti-nutritional factors in Vicia sativa l. Seeds and the variability of phenotypic and morphological characteristics of some vetch accessions cultivated in european countries. Animals 2021; 11(44):1-15. 15. Saberi A. Investigation of yield and morphological traits of some new forage products and forgotten forage plants in Golestan province. Appl Res Pl Ecophysi 2019;6(2):45-57. 16. Abbasi MR, Vaezi1 S, Baghaie N. Genetic diversity of bitter vetch (Vicia ervilia) collection of the National Plant Gene Bank of Iran based on agro-morphological traits. IR J Rangelands and Forests Pl Breeding and Gene Res 2007;15(2):113-128. 17. Arzani H, Ahmadi Z, Azarnivand H, Bihamta MR. Forage quality of three life forms of rangeland species in semi-arid and semi-humid regions in different phenological stages Desert 2010;15:71-74. 18. SAS. SAS/STAT User´s Guide. Statistical Analysis System. Inc. Cary, NC. Versión 9.1. 2011.

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19. Julia CC, Waters DLE, Wood RH, Rose TJ. Morphological characterization of Australian ex situ wild rice accessions and potential for identifying novel sources of tolerance to phosphorus deficiency. Genet Resour Crop Evol 2016;63:327-337. 20. Peratoner G, Seling S, Klotz Florian C, Figl U, Schmitt AO. Variation of agronomic and qualitative traits and local adaptation of mountain landraces of winter rye (Secale cereale L.) from Val Venosta/Vinschgau (South Tyrol). Genet Resour Crop Evol 2016;63:61-273. 21. Loumerem M, Alercia A. Descriptors for jute (Corchorus olitorius L.). Genet Resour Crop Evol 2016;63(7):1103-1111. 22. Shen G, Girdthai T, Liu ZY, Fu YH, Meng QY, Liu FZ. Principal component and morphological diversity analysis of Job’s-tears (Coix lacryma-jobi L.). Chil J Agric Res 2019;79:131-143. 23. Ebrahimi M, Bihamta MR, Hoseinzade AH, Golbashy M, Khialparast F. A study of agronomy and morphologic traits of white bean genotypes using multivariate analysis. J Crop Breed 2009;1(3):1-13. 24. Berhanu T, Abera M. Adaptation and forage yield of vetches (Vicia spp.) in the southern highlands of Ethiopia. Agric Sci Pract 2017;4(1):46-49. 25. Ghafoor A, Ahmad Z, Qureshi AS, Bashir M. Genetic relationship in Vigna mungo (L.) Hepper and V. radiate (L.) R. Wilczek based on morphological traits and SDSPAGE. Euphytica 2002;123:367–378. 26. Chebouti A, Meziani N, Bessedik F, Laib M, Amrani S. Variation in morphological traits and yield evaluation among natural populations of Medicago truncatula and Medicago laciniata. Asian J Biol Sci 2019;12:596-603. 27. Getnet A, Tekleyohanes B, Lemma G, Mesfin D, Diriba G. Major herbaceous forage legumes: Some achievements in species and varietal evaluation in Ethiopia. In: Kemal A, et al, editors. Food and forage legumes of Ethiopia: progress and prospects. Proc Workshop Food Forage Legumes 22–26 September 2003. Addis Ababa, Ethiopia. 28. Goghari M, Dashti H, Madah Hosseini Sh, Dehghan E. Evaluation of genetic diversity of lentil germplasm using morphological traits in Bardsir. IR J Field Crop Sci 2014; 45(4):541-551.

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29. Almayehu N, Becker H. Enotypic diversity and patterns of variation in a germplasm material of Ethiopian mustard (Brassica carinata A. Braun). Genet Resour Crop Evol 2002;49(6):573-582.

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https://doi.org/10.22319/rmcp.v13i4.5963 Article

Effect of soil cover on the growth and productivity of buffel grass (Cenchrus ciliaris L.) in degraded soils of arid zones

Ernesto Herssaín Pedroza-Parga a Aurelio Pedroza-Sandoval a* Miguel Agustín Velásquez-Valle b Ignacio Sánchez-Cohen c RicardoTrejo-Calzada a José Alfredo Samaniego-Gaxiola d

a

Universidad Autónoma Chapingo. Unidad Regional Universitaria de Zonas Áridas. Bermejillo, Durango, México. Km. 40 Carr. Gómez Palacio – Chihuahua. Bermejillo, 35230, Durango, México. b

Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias (INIFAP). Campo Experimental Saltillo. Departamento de Manejo Integrado de Cuencas. Saltillo, Coahuila, México. c

INIFAP. Centro Nacional de Investigación Disciplinaria en Relaciones Agua Suelo Planta Atmósfera. Gómez Palacio, Durango, México. d

INIFAP. Departamento de Fitopatología del Centro de Investigación Regional Norte Centro. Matamoros, Coahuila, México.

* Corresponding author: apedroza@chapingo.uruza.edu.mx

Abstract: The objective of this study was to evaluate the use of corn crop residues as mulch and its impact on soil moisture content and the establishment, development and productivity of

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buffel grass (Cenchrus ciliaris L). A randomized block design with three replications was used. The treatments were: sowing of 10 kg ha-1 of buffel grass seed (Bs); vegetation cover on soil with 10 t ha-1 of corn crop residues (Vc); Bs + Vc combination; and control (no grass sowing and no vegetation cover). The Bs + Vc treatment maintained a higher soil moisture content (P≤0.05), with 13.8 % vs 10.6 % of the control. Consequently, the number of grass plants m-2, buffel grass cover, plant height, chlorophyll index and dry biomass production had a tendency to respond better, with values of 518.5 plants m-2, 51.23 %, 31.8 cm, 162 and 167.8 g m-2, respectively, and they exhibited a tendency toward a statistically similar response as to this treatment when applied separately (Vc and Bs). Photosynthesis (µmol s-2s-1), stomatal conductance, transpiration (mmol H2O m-2 s-1), and water use efficiency were not affected by any of the treatments in this study, their response being equivalent to that of the control. Key words: Plant stress, Soil moisture, Pasture, Extensive livestock farming.

Received: 16/03/2021 Accepted: 02/06/2022

Introduction Every year, the productive capacity of 10 million hectares of agricultural land is lost due to soil degradation caused by a series of natural and anthropogenic factors (1,2). Water erosion is one of the main causes of soil degradation in arid areas, where rainfall is erratic and torrential, producing high volumes of water runoff in a short period with a strong erosive impact (3). Among the soil properties that determine water erosion are those related to infiltration and sediment stability, such as texture, organic matter content, and type of particle aggregates(4). The vegetation cover over the soil reduces particle shedding by intercepting raindrops and reducing their erosive energy. Vegetation and surface plant debris reduce the velocity of water flow over the soil and promote sediment settling (5). The impact is greater in these regions due to the lack of adequate vegetation cover, low organic matter content, and low soil moisture retention capacity, among other factors (6). In order to mitigate soil degradation, agronomic practices are carried out according to the type of agricultural production system and the specific conditions of each region (7,8).

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The construction of curbs on contour lines, the construction of masonry to reduce the velocity of rainwater, on-site rainwater harvesting systems based on micro-watersheds, the replanting of native grasses with conventional tillage methods, the establishment of different species of native or introduced plants with forage potential, and the use of different types of soil moisture retainers(9) are some of the technologies applied to mitigate the problem of erosion. Most of these techniques are aimed at retaining soil moisture in the face of high potential evaporation rates, which can be up to ten times higher than precipitation in semi-arid areas. Livestock production systems in semi-arid zones are vulnerable due to recurrent droughts, the presence of soils with low vegetation cover, and low organic matter content, which generate a process of natural resource degradation that results in low productive potential(10). In addition, overgrazing is one of the most recurrent problems that reduce the productivity of pasture areas with deficient precipitation(11). All this makes it necessary to strengthen the lines of research and generate strategies to improve the use and management of water, soil, plant and animal resources in livestock areas based on native grazing vegetation and the regular presence of pastureland, so as to greater sustainability from the productive, economic, social and environmental points of view(12). One factor that improves physical soil conditions to increase and conserve moisture after rainfall is the use of soil cover(13). If the use of vegetation cover is complemented with the replanting of native grasses of the region, there is a greater possibility of mitigating the degradation of pasture land. Buffel grass (Cenchrus ciliaris L.) is an introduced species in Mexico that has shown adaptation to critical environmental conditions in semi-arid zones, which to a large extent sustain their economy through extensive cattle raising on pastureland(13,14). Even though this grass species has a high potential for adaptation and development in degraded soils of semiarid areas(9,15), the establishment of this forage species in marginal environmental conditions requires an adequate management of natural resources to guarantee its germination, growth and productivity according to its development potential(16,17). From this perspective, vegetative soil covers and other soil moisture retainers, among other practices, are proving to be an effective strategy in the sustainable development of pasture-based livestock areas in degraded soils of arid zones(6,18,19). The objective of this study was to evaluate the use of corn crop residues as soil cover, and its impact on soil moisture content and the establishment, development and productivity of buffel grass in degraded soils of arid zones in northern Mexico.

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Material and methods Geographic location

The study was carried out in an area with microphyllous and rosette scrub vegetation and small areas of grassland in the municipality of Mapimí in the north of the State of Durango, Mexico. The area is located at 25° 52' 23.65" N and 103° 43' 41.74" W and at an altitude of 1,176 m, with an average annual rainfall of 304 mm, a maximum temperature of 44 °C and a minimum of 10.2 °C(20).

Description of the experimental site

According to physical-chemical soil analysis, the experimental site presents a sandy loam soil with 56, 28, and 16 % sand, silt and clay respectively; a permanent wilting point (PWP) of 9.6 %, and a field capacity (FC) of 19.7 %. These soils are low in macro and microelements, although they have good levels of potassium (68.4 mg·kg-1) and calcium (33.7 meq·L-1), the latter making them alkaline soils with a pH of 8.3 and a slope of 1 % (Figure 1)(21). Figure 1: Geographic location of the study area in the Municipality of Mapimí, State of Durango, Mexico

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Experimental and treatment design

A randomized block experimental design was used with three replications and four treatments: sowing of 10 kg ha-1 with buffel grass seed (Bs); no sowing of grass and only application of 10 t ha-1 of corn stubble as mulch on the soil (Vc); the combination of the treatments Bs + Vc, plus the control (no sowing of grass or application of vegetative cover). Each experimental unit had a dimension of 5x5 m. The study was carried out in the summer-autumn of 2017, for which soil preparation of the experimental area was performed by using a rake to a depth of 5 cm. In the grass sowing treatments, the seeds were scattered, ensuring their even distribution on the ground, and then covered with a light layer of the same soil by a second pass of the rake, in order to prevent the seeds from being exposed to the wind and dragged by it. The treatments using dry corn stubble as soil cover were applied immediately after planting. The experiment was established on dry soil, and the treatments were exposed to the first rain, which occurred in July with a rainfall volume of 64.8 mm, whereby the grass seed was allowed to germinate. Rainfall in the area of experimental influence during the study period was measured using a La Crosse TechnologyTM Heavy Weather Pro WS 2800 microclimatic station (USA).

Variables measured

The soil moisture content (%) was quantified using a Soil TesterTM Model HB-2 digital tensiometer (Ontario, Canada); while plant variables such as the number of grass plants m-2 were measured with a 20x20 cm quadrant, counting the number of plants within the quadrant; grass height (cm); grass cover (%) estimated in one m2 using a 20x20 cm quadrant and using a scale of 0 to 100 to estimate the % of ground cover by grass per unit area. All these variables were measured at six different dates: 36, 52, 67, 87, 107, and 127 d after sowing (DAS), and three measurements were taken as sampling unit per treatment at each evaluation date. The physiological variables of the grass were: chlorophyll index, measured using a Spectrum Technologies Inc . Fieldscout CM 1000 chlorophyll meter; photosynthesis (µmol CO2 m-2 s-1); stomatal conductance; transpiration (mmol H2O m-2 s-1) —these last three measurements were made with a model LI-6400XT infrared gas flow analyzer, (LI-COR®, Inc. Lincoln, Nebraska, USA)—; water use efficiency, product of the quotient of the amount of CO2 assimilated and the amount of water transpired by the plant. These variables were measured only once at 107 DAS, for which three plants were taken per experimental unit. At the end

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of the experiment (127 DAS), the dry biomass produced from the grass (g m-2) was obtained by cutting and drying the whole plant, except the root, at constant weight.

Data analysis An analysis of variance and a Tukey multiple range test of means (P≤0.05) were performed using the SAS package (Version 9.0) to identify the effect of the treatment.

Results and discussion According to precipitation records in the study area, the precipitation in year 2017 was 277.4 mm, slightly lower than the annual average, which 304 mm. The July-September period had the highest rainfall, with a total of 165.5 mm, representing 59.6 % of the total for the year (Figure 2). Buffel grass thrived adequately under these rainfall conditions, since the optimal range of summer rainfall reported a growth of 150 to 550 mm(22), which coincides with that recorded at the study site. Martin et al(23) reported that, for a period of 3 yr, the growth activity of this species was observed 15 d after a rainfall of 20 mm or more, a condition that occurred in the months of July and September in the present study. In arid grasslands of southern New Mexico, it was found that rainfall of < 20 mm in one day does not contribute to adequately wet the topsoil by 0.1 m(24). Figure 2: Behavior of pluvial precipitation in the study area during the year 2017. Mapimí, Durango, Mexico

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Soil moisture content, grass growth and development The average soil moisture content was significantly higher (P≤0.05) in the treatment with buffel grass sowing + soil cover of corn crop residues (Bs + Vc) than that of the control, exhibiting values of 13.8 vs 10.6 %, respectively; the former (Bs + Vc) showed no statistical difference with respect to the other two treatments (Bs and Vc) applied separately (Table 1). Table 1: Effect on soil growth and development of buffel grass (Cenchrus ciliaris L) with and without the use of a vegetative soil cover consisting of corn crop residues Soil Grass cover (%) Plant height Treatments Number of moisture plants m-2 (cm) (%) Control*

10.6b

172.8b

12.65c

17.1bc

Bs

12.2a

358.0ab

7.11c

6.5c

Vc

13.0ab

481.5ab

25.68b

22.3ab

Bs + Vc

13.8a

518.5a

51.23a

31.8a

* No grass sowing or soil cover was applied, only natural-born grass. Bs= Sowing of 10 Kg ha-1 of Buffel grass seeds without the application of corn crop residues on the soil. Vc= Application of 10 t ha-1 of corn crop residues on the soil as soil cover. Bs + Vc= Combination of the last two treatments mentioned above. ab Figures with the same letters within the same column are statistically equal (P≤0.05).

As a result of this water availability condition in the Bs + Vc treatment, the number of grass plants m-2, grass cover, chlorophyll index, and grass plant height were significantly higher than in the Bs + Vc treatment (P≤0.05), with values of 518.5 plants m-2, 51.23 %, 162 and 31.8 cm, respectively; the control registered the lowest values for these variables, with no statistical difference between the control and the Bs treatment. There was no consistent response to the Bs and Vc treatments when applied separately, since they fluctuated between statistically similar values to those of the Bs + Vc treatment and the control (Table 1). The above results are consistent with those reported by Cruz-Martínez et al(9), who found that buffel grass improved growth, chlorophyll content, and grass cover in the soil when hydrogel was applied at different doses as soil moisture retainers. Alcalá(25), indicates that the development of buffel grass depends largely on the amount of water retained in the soil. On the other hand, soil moisture conservation practices in pasture sites have been reported to increase water infiltration and, therefore, plant productivity(26). In contrast, physical soil

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degradation negatively affects the growth and yield of agricultural crops, as a consequence of limited root depth, low soil moisture reserves, and low availability of nutrient content, which negatively affects soil organic carbon, nitrogen, phosphorus and potassium contents, and soil pH(27).

Physiological indicators and grass biomass productivity

The Bs + Vc treatment stood out for its higher chlorophyll index with respect to the control, which would be reflected in an adequate photosynthetic activity(28). Pezeshki(29) and Carter and Knap(30) identified that a degradation of chlorophyll by any stress factor has repercussions in the reduction of the photosynthetic capacity of the leaves, as it limits the photochemical process in the absorption of radiation. With the treatment that combined the sowing of 10 kg ha-1 of pasture and the application of 10 t ha-1 of corn crop residues as soil cover (Bs + Vc), the chlorophyll content and biomass production were significantly higher (P≤0.05) than with the rest of the treatments —with values of 162.0 and 167.8 g m-2, respectively—, compared to the control, which registered values of 18.9 µmol m-2s-1, 105.7 and 54.4 g m-2. This represents an increase of 12.1, 53.2 and 208.4 % between these variables, respectively, which suggests that the sowing of grass needs to be complemented with the incorporation of a soil cover (in this study, corn crop residues) or some other type of soil moisture retainer, as reported by various authors(12,17,28). Stomatic conductance, transpiration, and water use efficiency were not affected by the treatments applied in this study (Table 2).

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Table 2: Physiological indicators and biomass productivity of buffel grass (Cenchrus ciliaris L) in different grass seeding treatments and use of corn crop residues as soil cover Treatm.

Photosynthesis Stomatic (µmol m-2s-1) conductance

ab Control* 18.9

Transpiration Dry WUE Chlorophyll (mmol H2O2 matter index m-2s-1) (g m-2)

0.156ª

2.75a

6.9a

105.7b

54.4c

Bs

14.1b

0.111a

2.16a

7.1a

75.1c

53.3c

Vc

20.1ab

0.176a

2.95a

7.0a

146.4a

102.7b

Bs + Vc

21.2a

0.138a

2.53a

8.4a

162.0a

167.8a

Treatm.= Treatments. WUE= water use efficiency. * No grass sowing or soil cover application: only naturalborn grass. Bs= Sowing of 10 Kg ha-1 of buffel grass seeds; no application of corn crop residues to the soil. Vc= application of 10 t ha-1 of corn crop residues as soil cover. Bs + Vc= combination of the last two treatments mentioned above. abc Figures with the same letters in the same column are equal (P≤0.05).

In a projection of dry biomass production measured in g m-2, the best treatment (Bs + Vc) yielded 1.6 t ha-1, while the control produced 0.54 t ha-1, 208.4 % more of the former with respect to the latter, and an overall average of 0.89 t ha-1 among all the treatments. Therefore, in terms of productivity, this technology also opens up a prospect, given the low bioproductivity of these areas. The results in Tables 1 and 2 show that lower soil moisture corresponded to a significant (P≤0.05) decrease in photosynthetic activity, at least in the Bs + Vc treatment, with respect to the control. This is consistent with the findings of Tezara et al(31), who report that the presence of moisture in the soil favors plant photosynthesis, while water deficit decreases it. The positive result of the chlorophyll index as a function of higher soil moisture content is contrary to that reported by Meléndez et al(32) and Trujillo et al(33), who observed that the chlorophyll content increases in soils with low moisture gradients and decreases in soils with high soil moisture gradients. In contrast, in a study on Opuntia ficus-indica, Aguilar and Peña(34) reported that the chlorophyll concentration decreased significantly in plants under drought, consistently with the findings of this study. The above contrasting results regarding the response to water stress in terms of chlorophyll content may be related to the genetic nature of the plant materials used, such as cactus, and to the ecological conditions in which the different studies were conducted(35). Additionally, Cabrera(36) points out that the physiological activity, such as photosynthesis, conductance, and transpiration of buffel grass, depends on the fluctuations of the weather conditions of each year.

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Conclusions and implications The use of soil cover with corn crop residues in combination with the sowing of buffel grass (Cenchrus ciliaris L.) was the treatment with the best effect on the soil moisture content, which favored the growth and development of the grass plant, with a better number of plants per unit area, a higher plant cover, a higher chlorophyll index, and a higher dry matter production. However, these same treatments applied separately showed inconsistent behavior, with a response similar to that of the combination of both practices, but differentiated from the response of the control. Grass plant physiology in terms of photosynthesis, stomatal conductance, transpiration, and water use efficiency showed no effect of the soil cover practices tested in this study. Literature cited: 1.

David P, Burgess M. Soil erosion threatens. Food production. Agriculture 2013;3(3):443-463.

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Encinas RA, Ibarra J. La degradación del suelo y sus efectos sobre la población. Población y Desarrollo 2003:5-9.

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Bolaños GMA, Paz PF, Cruz G, Carlos O, Argumedo EJA, Romero B, et al. Mapa de erosión de los suelos de México y posibles implicaciones en el almacenamiento de carbono orgánico del suelo. Terra Latinoamericana 2016;34(3):271-288.

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Diaz GM. Alternativas para el control de la erosión mediante el uso de coberturas convencionales, no convencionales y revegetalización. Ingeniería e Investigación 2011;31(3):80-90.

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Distrito de Conservación de Recursos del Condado de Monterrey. Guía de prácticas para el manejo de erosión y escorrentía agrícola en laderas. 2016: https://www.rcdmonterey.org/pdf/rcdmc_hillslope_guide_en_espanol-10-5-16final.pdf.

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Pedroza-Sandoval A, Trejo-Calzada R, Sánchez-Cohen I, Yáñez-Chávez JA, CruzMartínez A, Figueroa-Viramontes U. Water harvesting and soil water retention for forage production in degraded areas in arid lands of Mexico. In: New perspectives in forage crops. Loiola ER, Leilson RB editors. USA : Editorial Intechopen; 2018:3-23.

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7.

Pedroza-Sandoval A, Chávez-Rivero JA, Trejo-Calzada R, Sánchez-Cohen I, RuizTorres J. Captación y aprovechamiento integral del agua de lluvia y manejo de aguas residuales en zonas áridas. En: Tópicos selectos de sustentabilidad: Un reto permanente Volumen IV. Moreno RA, Reyes CJL, editores. México, DF: Editorial CLAVE. 2016:69-90.

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Sánchez-Cohen I, Díaz-Padilla G, Velásquez-Valle M, Slack DC, Heilman P, PedrozaSandoval A. A decision support system for rainfed agricultural areas of Mexico. Computers Electronics Agric 20151;14:178-188.

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Cruz-Martínez A, Pedroza-Sandoval A, Trejo-Calzada R, Sánchez-Cohen I, SamaniegoGaxiola JA, Hernández-Salgado R. Captación de agua de lluvia y retención de humedad edáfica en el establecimiento de buffel (Cenchrus ciliaris). Rev Mex Cienc Pecu 2016;7(2):159-172.

10. Kéfi S, Rietkerk M, Alados CL, Pueyo Y, Papanastasis VP, Elaich A, de Ruiter PC Spatial vegetation patterns and imminent desertification in Mediterranean arid ecosystems. Nature 2007;449(7159):213-227. 11. Velásquez VMA, De Alba AA, Gutiérrez LR, García EG. Prácticas de restauración de suelos para la conservación del agua. Centro Investigación Regional Norte Centro del INIFAP. Campo Experimental de Zacatecas. Folleto Técnico. Núm. 46; 2012. 12. Roco FL, Engler PA, Jara-Rojas R. Factores que influyen en la adopción de tecnologías de conservación de suelos en el secano interior de Chile Central. Rev FCA UNCUYO 2012;44(2):31-45. 13. Sharma BA, Lewi, SD, Gaston A, Darapuneni M, Wang JJ, Sepat S, Bohara H. Winter cover crops effect on soil moisture and soybean growth and yield under different tillage systems. Soil Tillage Res 2019;195. 14. Brorens BAHV, VanEs HM, Verheyden SML, Schindelbeck RR. Soil hydraulic properties as affected by tillage. Final report. Master degree. Department of Soil, Crop and Atmospheric Sciences. Ithaca, NY: Cornell University; 1991. 15. Saucedo TRA. Guía técnica para el establecimiento y utilización de plantaciones de chamizo. Centro de Investigación Regional Norte Centro del INIFAP. Campo Experimental de Zacatecas. Folleto Técnico Núm. 10;2003. 16. Velásquez VMA, Muñoz VJA, Macías RH, Esquivel AG, Rivera GM. Producción de forraje de variedades de zacate buffel [Pennisetum ciliare L. (Link.) Sin. Cenchrus ciliaris L.] en la región árida del Estado de Durango, México. Rev AGROFAZ 2014;14(1):69-76.

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17. Yáñez-Chávez LG, Pedroza-Sandoval A, Sánchez-Cohen I, Velásquez-Valle MA, Trejo-Calzada R. Management practices and bioproductivity in grassland of dry areas. In: Grasses as food and feed. Publisher: Intech Open (Edited by Zerihun Tadele). London, SE19SG-United Kindom 2018;49-65. 18. Yáñez-Chávez LG, Pedroza-Sandoval A, Martínez-Salvador M, Sánchez-Cohen I, Echavarría-Cháirez FG Vásquez-Valle MA, López-Santos A. Uso de retenedores de humedad edáfica en la sobrevivencia y crecimiento de dos especies de pastos Bouteloua curtipendula [Michx.] Torr. y Chloris gayana Kunth. Rev Mex Cienc Pecu 2018;9(4):702-718. 19. Huerta-Olague JJ, Oropeza MJL, Guevara GRD, Ríos BJD, Martínez MMR, Barreto GOA, et al. Efecto de la cobertura vegetal de cuatro cultivos sobre la erosión del suelo. Idesia (Arica) 2018;36:153-162. 20. Medina GG, Díaz PG, López HJ, Ruíz CJA, Marín SM. Estadísticas climatológicas básicas del estado de Durango. (Periodo 1961 – 2003). Libro Técnico № 1. Campo Experimental Valle del Guadiana. CIRNOC-INIFAP; 2005. 21. García I, Martínez JJG. Caracterización de la Reserva de la Biosfera Mapimí Mediante el uso de sistemas de información geográfica. En: Memorias del IV Simposio Internacional sobre la Flora Silvestre en Zonas Áridas. Universidad Autónoma de Chihuahua-Universidad de Sonora; 2004:369-377. 22. Cox JR, Martin RMH, Ibarra FFA, Fourie JH, Rethman NFG, Wilcox DG. The influence of climate and soils on the distribution of four African grasses. J Range Management 1988;41(2):127–139. 23. Martin RM, Cox JR, Ibarra FF. Climatic effects on buffelgrass productivity in the Sonoran Desert. J Range Management 1995;48(1):60–63. 24. Herbel CH, Gibbens RP. Matric potential of clay loam soils on arid rangelands in southern New Mexico. J Range Management 1989;42(5):386–392. 25. Alcalá GC. Guía práctica para el establecimiento, manejo y utilización del zacate buffel. Patronato del Centro de Investigaciones Pecuarias del Estado de Sonora, AC. 1995. 26. Beltrán LS, Loredo OC, Núñez T, González ELA. Buffel titán y buffel regio nuevas variedades de pasto para el altiplano de San Luis Potosí. Folleto técnico N° 35. Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias. 2008. 27. Lal R, Singh BR. Effects of soil degradation on crop productivity in East Africa. J Sust Agr 2008;13(1):15-36.

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28. Pedroza-Sandoval A, Yáñez-Chávez LG, Sánchez-Cohen I, Samaniego-Gaxiola JA, Trejo-Calzada R. Hydrogel, biocompost and its effect on photosynthetic activity and production of forage maize (Zea mays L.) plants. Acta Agronómica 2017;66(1):63-68. 29. Pezeshki SR. Wetland plant responses to soil flooding. Environ Exper Botany 2001;46:299-312. 30. Carter GA, Knapp AK. Leaf optical properties in higher plants: linking spectral characteristics to stress and chlorophyll concentration. Am J Botany 2001;88(4):677684. 31. Tezara WM, Driscoll SD, Lawlor DW. Water stress inhibits plant photosynthesis by decreasing coupling factor and ATP. Nature 1999;1401:914-917. 32. Meléndez L, Hernández A, Fernández S. Efecto de la fertilización foliar y edáfica sobre el crecimiento de plantas de maíz sometidas a exceso de humedad en el suelo. Bioagro 2006;18(2):107-114. 33. Trujillo ME, Méndez JR, Hossne AJ, Parra FJ. Efecto de la humedad y compactación de un Ultisol de la sabana del estado Monagas sobre la concentración de clorofila y carotenoides, lavado de electrolitos y contenido relativo de agua en plantas de soya. Acta Universitaria 2010;20(3):18-30. 34. Aguilar BG, Peña VCB. Alteraciones fisiológicas provocadas por sequía en nopal (Opuntia ficus-indica). Rev Fitotec Mex 2006;29(3):231-127. 35. Bacarrillo-López R, Pedroza-Sandoval A, Inzunza-Ibarra MA, Flores-Hernández A, Macías-Rodríguez FJ. Productividad de forraje de variedades de nopal (Opuntia spp.) bajo diferentes regímenes de humedad del suelo. Ecosist Recur Agropecu 2021;8(3): e2878. 36. Cabrera HM. Respuestas ecofisiológicas de plantas en ecosistemas de zonas con clima mediterráneo y ambientes de alta montaña. Rev Chilena Historia Natural 2002;75:625637.

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https://doi.org/10.22319/rmcp.v13i4.6005 Article

Typology of honey consumers with a university education in Mexico

Fidel Ávila Ramos a Lizeth Paula Boyso Mancera a Mercedes Borja Bravo b* Venancio Cuevas Reyes c Blanca Isabel Sánchez Toledano d

a

Universidad de Guanajuato. Departamento de Veterinaria y Zootecnia. Guanajuato, México.

b

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Campo Experimental Pabellón, Km. 32.5 carretera Aguascalientes-Zacatecas, Pabellón de Arteaga, Aguascalientes, México. c

INIFAP. Campo Experimental Valle de México. Estado de México, México.

d

INIFAP. Campo Experimental Zacatecas. Zacatecas, México.

*Corresponding author: borja.mercedes@inifap.gob.mx

Abstract: Mexico is a honey-producing country, paradoxically, its per capita consumption is low compared to European countries. The objective was to make a typology of honey consumers in Mexico with a minimum educational level of bachelor’s degree in ages from 20 to 60 years and to determine their socioeconomic characteristics and aspects that motivate consumption. A questionnaire was applied to a sample of 1,003 honey consumers who met the conditions of age and school level. The information was analyzed using cluster and discriminant analysis. Three types of consumers were identified: 1) educated consumers with average income (34.4 %), they were those who consume honey frequently, have extensive knowledge about beekeeping by-products and honey properties, prefer to buy the product from

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beekeepers; 2) highly educated consumers with high income (25.8 %), most of them have postgraduate degrees and receive income greater than $5,000 per week, they were people of mature age and with moderate consumption of honey, a third of this group only know honey, have knowledge of its properties and qualities, they are indifferent to the place of purchase; and 3) educated consumers with low income (39.8 %), it grouped young consumers who only have a bachelor’s degree, their consumption is moderate, they prefer to buy the product in markets. The groups of consumers formed provide information on a segment of the honey market in Mexico, it is necessary to continue conducting research on issues related to consumption and preference of honey consumers in Mexico. Key words: Honey consumption, Socioeconomic characteristics, Clusters.

Received: 11/06/2021 Accepted: 07/03/2022

Introduction Honey is the main product obtained from beekeeping; it is defined as a sweet substance made by bees from the nectar of flowers, which they collect, combine with specific substances, transform and store to serve as energy food(1). In 2019, Mexico produced 61.9 thousand tonnes of honey and during 2010-2019, the average annual growth rate was 1.2 %(2). In 2019, 43.4 % of production went to Germany and the United States, and Mexico ranked among the first exporting countries(3). Currently, there is a tendency in consumers to purchase food products with general (taste, price, safety, organic and certified) and subjective attributes related to environmental, social and ethical issues; in addition, they should promote health, well-being and reduce the risk of developing diseases(4,5). Honey is a product appreciated for its properties and health benefits, as a sweetener and natural remedy; it contains carbohydrates, water, proteins, vitamins, minerals and phenolic compounds. Consequently, its intake is associated with a better antioxidant capacity, modulation of the immune system, antimicrobial activities, influence on lipid values, regulation of glycemic responses, among others(5). This has contributed to the growing trend in world consumption, which, during 2008 to 2018, increased 5.3 % and in 2018, consumption was 2.55 million tonnes(6).

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In contrast, in Mexico honey consumption has decreased; during 2017-2019, an apparent national consumption of 22.3 thousand tonnes was recorded(2,3). From 2010, the trend in consumption was downward, with an average annual growth rate of -2.8 %, until 2019. Although the country is one of the main world producers, the Mexican population does not show a culture of honey consumption and it is reflected in the per capita consumption of 170 g, well below some European countries, which exceed 1,000 g per person per year(6). There are studies that have determined the factors that influence honey consumption, among them sociodemographic factors such as age, occupation and education(7,8,9). Other influencing factors were color, taste, variety and price(9,10). In another study, it was mentioned that consumption is influenced by the income level of households and the purchase decision is determined by consumers’ knowledge of the value of honey(11). Attributes such as therapeutic properties have become important in the purchase decision and the product is valued as traditional, healthy and for its use in alternative medicine(5,12). Studies conducted in Croatia, Romania, Italy, Serbia and Brazil(13-16) indicate that the educational level of the honey consumer is relevant and influences the purchase decision, because the person may have greater knowledge about the qualities of the product. This aspect should be considered for Mexico, where the studies conducted deal with the production chain, commercialization(17,18) and consumer preferences at the regional level(19). However, information on the identification of consumer profiles and types for market segments is limited, even though this type of information contributes to the understanding of how consumption decisions are made, reveals information for agri-food chains and provides elements to producers and industrialists for value addition(16,20). Knowing the types of consumers supports the design of market strategies that position the product in the market and motivate its consumption. Therefore, the objective of this work was to make a typology of honey consumers in Mexico with a minimum educational level of bachelor’s degree in ages from 20 to 60 years and to determine their socioeconomic characteristics and aspects that motivate consumption.

Material and methods Sample size

The type of research was exploratory, and the information was obtained through a structured survey. The sampling was directed to the Mexican honey-consuming population with

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university education, between 20 and 60 years of age. The sample size was obtained using the simple random sampling formula for finite populations(21,22): 𝑍 2 𝑁 𝑝𝑞 𝑛= (𝑁 − 1)𝑒 2 − 𝑍 2 𝑝𝑞 Where n was the sample size; N represents the population, equal to 57.34 million inhabitants, population between 20 and 60 years of age according to the Census of Population and Housing (INEGI)(23); Z was the 90 % confidence level; e was the error of 4.1 %; P was the 50 % probability that the sample is representative, and q was the probability that the sample is not representative (q=1-p). The estimated sample size was 990 surveys, but in practice 1,003 were conducted.

Instrument used and sources of information

The information was collected through a questionnaire of 15 questions on age, gender, schooling, size of the city where they lived, weekly income, monthly consumption of honey, habits in the consumption of honey, place of purchase, consumer knowledge of properties and uses and by-products of honey. The questions were closed with dichotomous, multiple and scale answers(24). The design of the survey was made on the Google Apps server through Drive®, where the name of the survey was first established and each of the questions raised with their respective answers was described. Subsequently, the link that indicates the abbreviation of the URL was generated. Prior to the application, pilot tests were conducted to ensure the clarity of the questions and minimize errors (n= 10). Once validated, the survey was applied via the internet, sharing the link in social networks. With the information obtained, a database was created in Excel 2016 spreadsheets.

Statistical analysis

The typology of honey consumers was made using multivariate techniques, first a hierarchical cluster (CA) and K-mean analysis was applied. The hierarchical CA was based on Ward’s grouping method and was used to identify the number of groups graphically and by means of Mojena’s criterion (𝛼̃ + 𝑘𝑠𝛼 ); where 𝛼̃ is the mean of the Euclidean distances, sα is the standard deviation of the distances and k is a constant(25). Subsequently, the analysis was complemented with that of K-means for a better identification of the groups.

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To verify and validate the results obtained in the CA of K-means, the classification and assignment of each individual to the group formed was evaluated with a discriminant analysis (DA)(22,26); where the independent variables that most discriminated against the groups were determined and it was verified that the conformation of groups of the CA was robust. In the DA, the stepwise variable selection method was used. To select the variables, the Wilk’s statistic Lambda was used, which, for its interpretation, considers that, if its value is close to zero, the total variability will be due to the differences between groups and, therefore, the corresponding set of variables will discriminate against the groups. If its value is close to 1, the groups will be mixed and the set of independent variables will not be suitable for constructing the discriminant functions(27,28). The statistical analysis of the data was performed with the SPSS 27.0 software for Windows(29) and Minitab 18.1.

Results Statistical results

The hierarchical CA allowed identifying graphically three types of honey consumers (Figure 1), likewise this result was corroborated by estimating Mojena’s Criterion, where 𝛼̃= 2.68, 𝑘= 1.25 and 𝑠𝛼 =0.54, which resulted in 3.35. The number of clusters identified in the hierarchical CA was used for the CA of K-means. Figure 1: Dendrogram of honey consumers with university education in Mexico

The groups of honey consumers formed were analyzed by a discriminant analysis to verify the goodness of the classification. With the analysis, it was determined that 97.5 % of the respondents were classified correctly and, therefore, the classification in three clusters was

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valid. Similarly, the Wilk’s statistic Lamda of 0.115, a value close to zero, means that the groups formed were statistically different (Table 1).

Statistic

Value

Wilk’s Lambda

0.115

Table 1: Multivariate statistics Fisher Degrees of Degrees of distribution freedom of freedom of value the the numerator denominator 255.14 12 1990

Probability greater than F calculated <0.000

According to the values obtained, Wilk’s Lamda and the F statistic, six of the nine variables (weekly income, age, monthly consumption, motivation to consume, by-products and place of purchase) contributed to the discrimination of groups by their level of P>0.05 and F value greater than 3.8. The variables that did not contribute to the separation of groups were gender, form of consumption and size of the city (Table 2). Table 2: Mean test between the differentiated groups Variable Gender

Wilk’s Lambda 0.994

F 3.242

Significance 0.059

Age

0.695

219.515

0.000

Weekly income

0.321

1058.353

0.000

Size of the city

0.999

0.610

0.543

Monthly consumption

0.716

198.139

0.000

Form of consumption

0.991

4.471

0.062

Place of purchase

0.908

50.893

0.000

By-products

0.920

43.663

0.000

Motivation to consume

0.791

132.168

0.000

Characterization of the types of honey consumers

Once the types of consumers were defined, they were characterized based on the variables included in the analysis (Tables 3 and 4) and the particularities of each one were determined. The name assigned to each group was considering the educational level and weekly income.

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Group 1: Educated consumers with average income

This group consisted of 345 consumers (34.4 %), of which 64.6 % were women and the rest were men. Most of the people in this group were women aged 26 to 40 and adults aged 41 to 60, and just over half have postgraduate studies. With respect to income, the population was concentrated in the middle categories (more than $3,000 per week) and they live in large cities (Table 3). It was identified that they were the most frequent consumers of honey for sweetener or home remedy. They acquire honey directly from the beekeeper, they know more about the derivatives of the hive and their reasons for purchase are related to the natural properties of honey (Table 4). Table 3: Socioeconomic and demographic characteristics of the types of honey consumers (%) Variables Gender Men Women Age From 20 to 25 yr old From 26 to 40 yr old From 41 to 60 yr old Schooling Bachelor’s degree Postgraduate degree Weekly income Less than 1,500 From 1,500 to 3,000 From 3,000 to 5,000 More than 5,000 Size of the city More than 100,000 From 30,000 to100,000 From 10,000 to 30,000 Less than 10,000

Group 1 (n=345)

Group 2 (n=259)

Group 3 (n=399)

35.4 64.6

40.2 59.8

30.6 69.4

4.9 47.8 47.2

6.6 59.1 34.4

56.4 33.8 9.8

46.4 53.6

37.8 62.2

81.2 18.8

3.2 26.1 32.2 38.6

0.0 0.0 31.7 68.3

54.9 44.6 0.5 0.0

59.4 20.3 9.6 10.7

59.1 20.1 10.4 10.4

55.4 22.3 10.0 12.3

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Table 4: Characteristics in honey consumption by type of consumer (%) Group 1 (n=345) Monthly consumption 10 g 0.9 50 g 11.9 100 g 39.1 500 g 48.1 How do you consume honey? Sugar substitute 71.0 Home remedy 25.5 In cosmetics 3.5 Where do you buy it? Market 20.0 Self-service store 12.2 Beekeeper 67.8 By-products of beekeeping you know Honey 8.1 Honey, pollen, royal jelly 23.5 Honey, pollen, royal jelly, apitoxin 68.4 Why do you consume honey? Because of its properties 63.8 It is a natural product 25.8 It is a healthy product 9.9 Because of family custom 0.6

Group 2 (n=259)

Group 3 (n=399)

31.7 32.0 29.7 6.6

27.6 33.8 27.3 11.3

65.6 32.0 2.3

61.7 32.1 6.2

44.4 23.6 32.0

52.6 8.3 39.1

31.3 29.3 39.4

26.6 30.3 43.1

14.7 32.4 35.5 17.4

23.6 34.6 24.3 17.5

Group 2: Highly educated consumers with high income

The second group was made up of 259 consumers, 25.8 % of the respondents. This group is composed mostly of mature consumers between 26 and 40 yr of age, located in large cities. This group was characterized by having the highest school degree and high income (Table 3). They showed a low consumption of honey, and they are indifferent to where to buy it, the motivation they have to acquire it is associated with the idea of consuming a natural and healthy product, but they also do it because it is a family custom.

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Group 3: Educated consumers with lower incomes

Group three consisted of 399 consumers, which corresponded to 39.8 % of the sample. The members were young people with a bachelor’s degree and weekly income of less than $3,000. They showed a low consumption of honey and they used it as a sugar substitute. This type of consumers had a preference to buy the product in markets and directly from beekeepers, they have knowledge of the products derived from the hive and their purchase motivations are determined by the fact that it is a natural, healthy product with properties, and by family custom.

Discussion The results obtained in the characterization were similar to those found in a comparative analysis on honey consumption in Romania, Italy and Serbia, where it was mentioned that the educational level and the amount of income participate in the behavior of honey consumers in those countries(13). In several studies on honey, it is mentioned that the sociodemographic factors that positively influence consumption were the age, gender, educational level and income of people(30,31). This same condition was reflected in this analysis, where the main variable that segmented the population studied by type of consumers was income. In other European countries, honey is considered an expensive product compared to other sweeteners, so its acquisition is conditioned to the income of the consumer(5,9,14), this explanation describes the condition of Mexican consumers. A second variable that influenced the differentiation of the groups was age; although a sample in ages between 20 and 60 years was considered, the difference between the groups by age ranges was noticeable; in the first group, no predominant range was observed; however, group 2 was made up of mature people and group 3 was made up of the youngest. It is assumed that older generations consume honey more frequently than younger consumers(30,32,33); these characteristics of consumption coincided with the Mexican consumers interviewed, since the adult population of group 1 were the ones who consume the most and the young people of group 3 were the ones who consume the least. On the other hand, a greater trend of honey consumption in women has been identified in other parts of the world(9) and that this consumption tends to increase when it comes to health care, both in prevention and treatment of diseases(34,35,36). In this regard, it was found that most of the interviewed population were also women, and they consume honey.

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In addition to the above, the consumption of honey of groups 1 and 3 is directly related to the age, educational level, gender and income of consumers. However, consumers in group 2 do not meet these conditions, as they are highly educated people, with high income, of middle aged and low consumption. This behavior can be due to several factors, for example, in Slovakia and Romania(34), family size and frequency of honey consumption during childhood are determinants in the consumer profile. With regard to the motivation to consume honey, it was observed that, in the three groups, the properties of the product and its natural origin are appreciated and they conceive it as a healthy product, these results were similar to those reported in studies carried out in European countries(5,9), where they mention that the perception that consumers have about honey is usually more important in the purchase decision than the price it can have in the market. The perception of honey has developed in recent years and was the product of a greater knowledge of consumers about its properties and contributions to human health, so it is now recognized as a natural sweetener, healthy food and there is information on the numerous therapeutic properties it has(5). In addition to the above, it is assumed that the educational level of the sample influenced the perception of the consumers surveyed, since, as Lucchese and Gerber(16) mentioned, at a higher school level, the discourse of the benefits of honey is oriented to the nutritional aspect, associated with the advantages of consuming vitamins, nutrients and medicinal qualities that contribute to having good health and better quality of life. A difference that was distinguished between groups was consumption due to family tradition, mainly in groups 2 and 3. In a study conducted on young Poles(37), it was mentioned that this type of population consumes honey due to family tradition and the eating habits learned from their families; this same situation occurs with young Mexican consumers, who preserve their eating habits until their adulthood. The most frequent consumers, who were those of Group 1, showed greater knowledge of the by-products of the hive and they buy honey directly from beekeepers, this result coincided with the behavior of consumers in Croatia, where 75 % of them buy honey directly from producers(15). However, the place of purchase of honey provides important information about the consumer and the commercialization of the product. Acquiring it directly from the beekeeper indicates that consumers link foods to a concept of natural goods or services produced by companies in rural areas, with an established socioeconomic identity that they tend to prefer(38). On the other hand, the predominance of beekeepers as the main points of sale is confirmed, who maintain an important market share in frequent consumers, in addition to pointing out that honey is marketed without a brand and label, which are extrinsic aspects of quality and are not very relevant for consumers. In this regard, Arvanitoyannis and Kristallis(14) pointed out that these consumers are traditional and they acquire quality through criteria based on experience and a personal relationship between consumer and beekeeper. 888


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On the other hand, group 3 showed a greater tendency to buy honey in markets and in a smaller percentage from beekeepers; whereas, for Group 2, a preferred place to make the purchase was not observed, which denotes that this type of consumer does not base its decision criteria on this aspect. The classification made in this study considered only one segment of the honey market, represented by consumers with university education between 20 and 60 yr of age. These particularities of the study were considered relevant because, in the case of Mexico, there are no studies focused on specific market segments, in addition to the fact that, when conducting the survey on line, the level of participation of this segment of the population has been observed to be higher, as indicated by studies carried out in Romania(14) and Croatia(15), which highlight the greater participation of consumers with a high educational level in the answering of online surveys. Although numerous studies on profiles and types of honey consumers have been conducted in other countries(13,15,30), in Mexico this has been a little explored topic. The importance of this type of studies is highlighted by the way in which it allows producers to target their product and promote a better commercialization of it. One of the limitations of this study was that variables about tastes and preferences, consumer perception of quality, types of honey and extrinsic characteristics that are appreciated in other countries were not included(39). The results obtained represent a first approach to the types of honey consumers for the case of Mexico. Likewise, it is important to conduct this type of analysis for other market segments that allows identifying opportunities for the increase in national honey consumption.

Conclusions and implications The typology obtained showed the differences that exist between honey consumers with university education in an age range of 20-60 yr in Mexico. This type of consumers is grouped into three groups, the first consists of educated consumers with an average income and they differed from others because they consume honey frequently, have extensive knowledge of the by-products of beekeeping and properties, prefer to buy the product directly from beekeepers. A second group is the one made up of highly educated consumers, having mostly postgraduate degrees and receiving high incomes, these are people of mature age and with a moderate consumption of honey, even when they have knowledge of the properties and qualities of the product. A third of this group only know honey and no other by-product and they are indifferent to the place of purchase. Group 3, which consists of educated consumers with low incomes, groups young consumers who only have a bachelor’s degree, their consumption is moderate, and they prefer to buy the product in markets. Those in group

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1 were the most frequent and receptive consumers of honey and, therefore, potential consumers. Therefore, it is necessary to define strategies for promoting the product to inform the positive and healing aspects of honey and thus reinforce their knowledge and purchase decision. The strategy for consumers of groups 2 and 3 should focus on publicizing beekeeping as a sustainable activity, showing the different products derived from honey and the benefits of each by-product. Local honey producers should be aware that the reactivation of the beekeeping sector in Mexico could be achieved through the promotion of domestic consumption. Although the results obtained in this study are not definitive, the findings could have repercussions on producers and marketers, in order to potentiate the consumption of honey in Mexico through effective marketing strategies for each consumer profile. It is recommended to study other market segments, deepen in the analysis of consumption preferences and the influence of motivational and subjective aspects on the consumption of honey in Mexico. Literature cited: 1. Crane E. A book of honey. USA:Oxford Univ Press; 1980. 2. SIAP. Servicio de Información Agroalimentaria y Pesquera. Cierre de la producción pecuaria (1980-2019). https://nube.siap.gob.mx/cierre_pecuario/. Consultado 10 Nov, 2020. 3. SE. Secretaría de Economía. Sistema de Información Arancelaria Vía Internet. http://www.economia-snci.gob.mx/. Consultado 12 Nov, 2020. 4. Annunziata A, Scarpato D. Factors affecting consumer attitudes towards food products with sustainable attributes. Agric Econ 2014;60(8):353-363. 5. Testa R, Asciuto A, Schifani G, Schimmenti E, Migliore G. Quality determinants and effect of therapeutic properties in honey consumption. An exploratory study on Italian consumers. Agriculture 2019;9(174):1-12. 6. FAO. Organización de las Naciones Unidas para la Alimentación y la Agricultura. FAOSTAT: datos. http://www.fao.org/faostat/es/#home. Consultado 15 Nov, 2020. 7. Pocol CB, Bolboacă SD. Perceptions and trends related to the consumption of honey: A case study of North-West Romania. Int J Consum Stud 2013;37(6):642-649. 8. Gyau A, Akalakou C, Degrande A, Biloso A. Determinants of consumer preferences for honey in the democratic republic of Congo. J Food Prod Mark 2014;20(5):476-490. 9. Kowalczuk I, Jezewska-Zychowicz M, Trafiałek J. Conditions of honey consumption in selected regions of Poland. Acta Sci Pol Technol Aliment 2017;16(1):101-112.

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10. Šánová P, Svobodová J, Hrubcová B, Šeráková P. Segmentation of honey buyers’ behaviour by conjoint analysis. Sci Agric Bohem 2017;48(1):55-62. 11. Roman A, Popiela-Pleban E, Kozar M. Factors influencing consumer behavior relating to the purchasing of honey. Part 1. The buying process and the level of consumption. J Apic Sci 2013;57(2):159-172. 12. Badolato M, Carullo G, Cione E, Aiello F, Caroleo MC. From the hive: Honey, a novel weapon against cancer. Eur J Med Chem 2017;142:290-299. 13. Ignjatijević SD, Prodanović RV, Bošković JZ, Puvača NM, Tomaš SMJ, Peulić TA, Đuragić OM. Comparative analysis of honey consumption in Romania, Italy and Serbia. Food Feed Res 2019;46(1):125-136. 14. Arvanitoyannis I, Krystallis A. An empirical examination of the determinants of honey consumption in Romania. Int J Food Sci Technol 2006;41:1164-1176. 15. Brščić K, Šugar T, Poljuha D. An empirical examination of consumer preferences for honey in Croatia. Applied Economics 2017;49(58):5877-5889. 16. Lucchese CT, Gerber RM. Consumo de mel de abelhas: análise dos comportamentos de comensais do Estado de Santa Catarina. Informações Econômicas 2009;39(10):22-31. 17. Magaña MMA, Moguel OYB, Sangines GJR, Leyva MCE. Estructura e importancia de la cadena productiva y comercial de la miel en México. Rev Mex Cienc Pecu 2012;3(1):49-64. 18. González RFJ, Rebollar RS, Hernández MJ, Guzmán SE. La comercialización de la miel en el sur del Estado de México. Rev Mex Agronegocios 2014;34(18):806-815. 19. Tapia CE, Castañeda SMC, Ramírez AJP, Macías MJO, Barajas PJS, Tapia GJS, et al. Caracterización fisicoquímica, contenido fenólico y preferencias de los consumidores de miel de Apis mellífera honey en el Sur de Jalisco, México. Interciencia 2017;24(9):603-609. 20. da Silva SCM, Oliveira AA, Ramos SR, Ibiapina A, Dos Santos AL, De Souza MGA. Tipologia do consumidor de frutos do cerrado. Revista Desafíos 2019;6(Especial):134139. 21. Téllez-Delgado R, Mora-Flores JS, Martínez-Damián MA, García-Mata R, GarcíaSalazar JA. Caracterización del consumidor de carne bovina en la zona metropolitana del Valle de México. Agrociencia 2012;46(1):75-86.

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22. Borja BM, Vélez IA, Ramos GJL. Tipología y diferenciación de productores de guayaba (Psidium guajava l.) en Calvillo, Aguascalientes, México. Región y Sociedad 2018;30(71):1-22. 23. INEGI. Instituto Nacional de Estadística y Geografía. Población total por entidad federativa y grupo quinquenal de edad según sexo, 1990 a 2010. https://www.inegi.org.mx/app/tabulados/interactivos/?px=Poblacion_01&bd=Poblacio n. Consultado 12 Feb, 2020. 24. Schnettler MB, Mora GM, Millis QN, Miranda VH; Sepúlveda MJ, Denegri CM, et al. Tipologías de consumidores según el estilo de vida en relación a la alimentación: un estudio exploratorio en el sur de Chile. Rev Chil Nutr 2012;39(4):165-172. 25. Martín MMT, Cabero MQ, Santana YRP. Tratamiento estadístico de datos con SPSS. Madrid, España: Paraninfo; 2007. 26. Díaz de Rada IV. Diseño de tipologías de consumidores mediante la utilización conjunta del análisis clúster y otras técnicas multivariantes. Economía Agraria 1998;(182):75104. 27. Vivanco AM, Martínez CFJ, Taddei BIC. Análisis de la competitividad de cuatro sistemas producto estatales de tilapia en México. Estud Soc 2010;18(35):167-207. 28. Ferrán–Aranaz, M. SPSS para Windows. Análisis estadístico. México: McGraw–Hill; 2001. 29. IBM Corporation. SPSS software. https://www.ibm.com/mx-es/analytics/spss-statisticssoftware. 30. Pocol CB, Teselios CM. Socio-economic determinants of honey consumption in Romania. J Food Agric Environ 2012;10:18–21. 31. Pocol, CB. Modelling the honey consumption behaviour in Romania by using sociodemographic determinants. Afr J Agric Res 2011;6:4069–4080. 32. Guziy S, Šedík P, Horská E. Comparative study of honey consumption in Slovakia Russia. Potravin Slovak J Food Sci 2017;11(1):472–479.

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33. Ismaiel S, Kahtani S, Adgaba N, Al-Ghamdi A, Zulail A. Factors that affect consumption patterns and market demands for honey in the Kingdom of Saudi Arabia. Food Nutr Sci 2014;5:1725-1737. 34. Šedík P, Pocol CB, Horská E, Fiore M. Honey: ¿food or medicine? A comparative study between Slovakia and Romania. British Food J 2019;121(6):1281-1297.

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35. Zhang S, Lu Z, Chunling T, Zhang Q, Liu L, Meng G, Yao Z, et al. Associations between honey consumption and prehypertension in adults aged 40 years and older. Clinical Exper Hyper 2020;42(5):420-427. 36. Münstedt K, Männle H, Riepen T. Survey of reasons why women utilize honey therapeutically, and reasons for not utilizing honey. Heliyon 2020;6(10):1-5. 37. Żak, N. Honey market in the opinion of young consumers. Handel Wewnętrzny 2017;366(1):424-438. 38. Wilkins JL, Bowdish E, Sobal J. Consumer perceptions of seasonal and local foods: A study in a US community. Ecology Food Nutr 2002;41(5):415-439. 39. Cosmina M, Gallenti G, Marangon F, Troiano S. Attitudes towards honey among Italian consumers: A choice experiment approach. Appetite 2016;99:52–58.

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https://doi.org/10.22319/rmcp.v13i4.5839 Article

Vertical and spatial price transmission in the Mexican and international cattle and beef market

José Luis Jaramillo Villanueva a*

a

Colegio de Postgraduados. Boulevard Forjadores de Puebla # 205, Santiago Momoxpan. 72760, Mpio. San Pedro Cholula, Puebla, México.

*Corresponding author: jaramillo@colpos.mx

Abstract: From 2000 to 2019, the Mexican beef subsector has undergone significant structural changes; the most important was the concentration of both production and marketing stages. In 2019, the Mexican Federal Commission of Competence revealed that, Mexican households’ income diminished between 16 and 31 % due to a lack of market efficiency. In the case of meat, the reduction may be up to 98 %. In this context, the objective of this study was to examine the degree of spatial price transmission between national and international live cattle prices and the vertical transmission between live cattle prices and carcass meat prices to evaluate market efficiency. The econometric approach consists of the estimation of a vector error correction model, using monthly beef real prices, for the period 1990-2019. Findings from this research provide information for decision-makers and stakeholders in this industry: these comprehend unidirectional transmission of international beef prices t o domestic beef prices and from farm price to processor price. Also point to the existence of asymmetric price transmission, which is related to whether cattle and beef prices are increasing or decreasing. Results indicate that a long-run single cointegration relationship exists between international and farmer prices, and between processor and farm price. The direction of price transmission tends to go from producers to processors and from international price to farmer price. When the international price increases, the speed of adjustment tends to be significantly slower, in contrast to when the international price decreases, resulting in a significantly faster rate of adjustment. Key words: Asymmetric price transmission, Beef prices, Vector error, Correction model. 894


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Received: 06/11/2020 Accepted: 01/06/2022

Introduction The livestock and beef industry in Mexico

The Mexican beef cattle sub-sector has substantial economic and social relevance. Production of beef represents almost half the value of Mexican gross animal product (1). Mexico occupies the eighth position in the world ranking for beef production, with 1.91 million t, 3.35 % of world beef production(2). The growth average rate (GAR) of cattle production in Mexico was 1.92 % in 2000-2018 (SIAP(3)). From a social perspective, it is the main economic activity carried out by small family farms, accounting for 1.06 million cattle production units in 2018 that generated 1.2 million direct jobs, and three million related jobs(4). In Mexico, from 1990 to 2019, the beef cattle and beef industry have undergone significant consolidation, as since 2000, both cattle and beef packing have rapidly reorganized into fewer and larger plants. The outcome of these processes is a highly concentrated cattle and beef industry. Thus, Sukarne® is the largest beef processing company in Mexico and has dominated beef export growth from 2010 to 2019. SuKarne® ranks as the sixth-largest beef packing company in North America, it accounts for 74 % of total Mexican beef exports(5). However, the Mexican Federal Commission of Competence(6) revealed that Mexican households lose between 16 and 31 % of their income. The rapid concentration of beef and cattle industry generates pressure on small processing firms, government, and consumers because retail beef prices grew at a GAR of 4.99 % from 2009 to 2018. However, farmer prices increased at a GAR of 2.43 %, calculated with data from USDA(2) and INEGI(7). Market concentration in the beef cattle industry is associated with non-competitive behavior that may result in economic inefficiency and a decline in consumer welfare(8). Some research(9) pointed out that market concentration is among the major causes of asymmetric price transmission in agricultural market chains. Nonetheless, in Mexico, up to now, there are few studies about beef cattle and beef price transmission and then, no consensus exist on this issue.

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Spatial price transmission

Spatial price transmission refers to the transmission of price shocks across different areas and commodities(10). The critical underlying theoretical explanation of spatial price transmission is revealed in the spatial arbitrage relationship, known as the Law of One Price (LOP). It implies that the difference between prices at different market locations will never exceed transaction costs; otherwise, arbitrageurs would exploit these profit opportunities(10). Suppose that PA and PB represent the prices for a homogeneous commodity in two spatially separated markets in t, and r A B represents transfer costs to move one unity of merchandise from B to A, the LOP asserts that: PB-PA ≤ r AB. The price difference in t for a commodity at two spatially separated markets should not differ by more than transfer costs. If the spatial price difference exceeds that of transfer costs, economic agents make spatial arbitrages. Following the adjustment process, a new equilibrium is reached, and the LOP is once again maintained. As literature(11) asserted, trade between two markets implies they are integrated.

Vertical price transmission

Vertical price transmission analysis is useful for assessing the efficiency of integrating different economic actors into a market. The extent and speed at which price changes are transmitted from one level to another in the market have important policy implications for welfare distribution and competitiveness. In a competitive market, price shocks at one level of the market chain should be reflected by similar changes at other levels, as market efficiency hypothesize a relationship of mutual price equilibrium(12). From 1990 to 2010, extensive studies examined market links between farm, processor, and retail markets(13). The extent of adjustment and speed with which shocks transmit between farmer, processor and retailer market prices is an essential factor that reflects market participants’ actions at varying market levels. The nature, speed and extent of adjustment to market shocks may also have important implications for marketing margins, spreads, and mark-up pricing practices(9,12). The objective of this study was to estimate the speed of price transmission between the price of the Mexican beef cattle processor (carcass) and cattle farmer (calf) (vertical transmission) and between the price of Mexican and international cattle price (calf) (spatial price transmission) in order to know on possible asymmetric price transmission and related

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economic consequences on producer and consumers. The hypothesis was that domestic and international beef price transmission is asymmetric.

Material and methods The study method consisted of econometric tests using time series of monthly spot real price data, deflated using the consumer price index, from 1990 to 2019. Farmer price was represented by calf prices, intermediate (processor) price was represented by carcass price, and international and import price by calf prices. The last one in dollars, but converted to Mexican real pesos using the bilateral exchange rate. International beef price (calf) comes from the U.S. Department of Agriculture (USDA). Additional information on other indicators originates from official statistical sites in Mexico, among which are the Sistema de Información Agropecuaria y Pesquera (SIAP), the National Institute of Statistics, Geography and Informatics (INEGI by its acronym in Spanish), the National Confederation of Livestock Organizations (CNG by its acronym in Spanish), and from the ANETIF (National Association of Federal Inspection Type) Foundation. Verification of each series´ integration order was conducted, using the Augmented DickeyFuller and Phillips-Perron (PP) tests(14,15). These tests were followed by an estimation of the long-run relationship, using the Engle-Granger two-step cointegration and the Johansen test(16). Finally, asymmetric Vector Error Correction Model (VECM) was performed; a test to select the lag order for an asymmetric VECM and a F-test on the coefficient of ECT+ and ECT- (positive and negative changes in the error term respectively) in order to test the null hypothesis of symmetry: H0: B+i = B-i.

Test for cointegration; long-run relationship

Once a unit´s root existence is proved, cointegration between variables in the series is necessary for a long-term equilibrium relationship. A variable vector with a unit root is cointegrated if a linear combination of these variables is stationary(17). The Engle-Granger twostep cointegration test(17) and the Johansen test(16) were applied to test for a long-run relationship. The first approach consists of estimating the cointegration regression, equation (1), by Ordinary Least Squares (OLS) method:

ptout = a + b1 ptin + mt

(1)

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where

ptout is a firm output price in period t, ptin is the input price in t and Ut is the error term.

The estimation of equation (1) generated the residual ût, to which was applied a unit root test for ût. As the coefficient of Ut-1 was less than unity, a cointegration relationship exists. Subsequently, a regression of equation (2) was performed.

Dmt = a + b1mt-1 + b2 Dmt-1

(2)

A negative coefficient of the error term (between -2 and zero) confirms a long run relationship between prices. In contrast, the Johansen test derived the distribution of two test statistics for the null of no cointegration; the Trace and Eigenvalue test(16). Once cointegration between prices was verified, a two-step Error Correction Model (ECM) was applied to capture the short- and long-term effects of which

ptin on ptout , and the speed of adjustment at

ptout restores equilibrium after a change in ptin . Two econometric models were

estimated: the spatial asymmetric model and the vertical asymmetric model.

Spatial asymmetric price transmission

Considering that farm and international prices have a unit root and were cointegrated, symmetric and asymmetric VECM were estimated to investigate possible price interdependence. Following an econometric approach(18), the ECM for spatial price transmission is depicted in equation (3). farm int Dptfarm = a + b1Dptint + b2 ECTt-1 + b3 (L)Dpt-1 + b4 (L)Dpt-1

(3)

In equation 3, the contemporaneous response term was also segmented(18). It leads to equation (4), in which contemporaneous and short run response to departures from the cointegrating relationship are asymmetric if 1  1 and 2  2 , respectively. int Dptfarm = a + b1+ Dptint + b1- Dptint + b2+ ECTt-1+ + b2- ECTt-1- + b3(L)Dpt-1farm + b4 (L)Dpt-1

An F-test was used to test for the null hypothesis of symmetry.

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Vertical asymmetric price transmission

In the literature, an approach based on cointegration theory was proposed(17) to test for possible asymmetries in the beef value chain. It indicates that two non-stationary time series may be long-term co-integrated if both series, from the same order, are integrated. In contrast, using an asymmetric VECM, Cramon-Taubadel(19) tested for Asymmetric Price Transmission (APT) in the presence of non-stationary series, by applying the two-step Engel and Granger approach. For this approach, the authors proposed splitting the error-correctionterm into positive and negative components to identify whether prices are transmitted differently, depending on whether they increase or decrease. Following the approach for testing vertical asymmetric price transmission(19), it was estimated equation (5): ret DPt ret = b0 + b1DPt farm + b2 ECTt-1 + B3 (L)DPt-1 + B4 (L)Pt-1farm + e t

(5)

ret where: ECTt-1 = Pt-1 - a 0 - a1 Pt-1farm is the error correction term, and b3 (L), b4 (L) are

polynomial lags. Furthermore, splitting the ECT into positive and negative components (i.e. positive and negative deviations from the long-term equilibrium – ECT+ and ECT-) reveals whether the speed of prices´ transmission differs, depending on it increases or decreases. Furthermore, it enables testing for Asymmetric Price Transmission (APT)(20). Then, we estimated equation (6): + ret DPt ret = b0 + b1DPt farm + b2+ ECTt-1 + b2- ECTt-1 + B3 (L)DPt-1 + B4 (L)Pt-1farm + e t

.

(6)

To test for asymmetry, an F-test was used to test the null hypothesis of symmetry, whether an asymmetric price response exists,

b2+ ¹ b2- .

Results and discussion Results from the ADF and PP(14,15) unit root tests cannot negate the null of non-stationarity of price series; T-statistic values do not corroborate rejection of the null hypothesis of a unit root with 95% confidence (Table 1).

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Table 1: Results of the ADF and PP test on beef price series Price series ADF test 5% critical PP test 5% critical value value International price -2.456 -3.426 -11.886 -21.378 Farm price -2.096 -3.426 -13.455 -21.378 Processor price -3.489 -3.426 -22.733 -21.378 Import price -7.396 -3.426 -86.416 -21.378 Source: own calculations.

These results enabled to use the cointegration technique to calculate the relationship between international and domestic Mexican beef prices and between a processor and farm beef prices. These results concur with previous studies concerning the non-stationarity of beef prices(21,22,23).

Long run cointegration

The estimation of equation (1), for the spatial model (equation 4), shows an R2 of 0.78, a tstatistic of 34.78 and an F statistic of 1209.7, which indicate long-run cointegration. The ADF test on the error term shows a test statistic of -2.57 vs a 5 % critical value of -2.87, which indicates a failure to reject the null of non-stationarity. Different authors(21,22,23) reported similar results for beef prices. For the vertical model (equation 6), it was found an R2 of 0.68, a t-statistic of 27.47, and an F statistic of 742.4. On the error-term, the ADF shows a test statistic of -2.696 vs a 5 % critical value of -2.87, indicating that cannot reject the null of nonstationarity. For the two models, it was estimated equation (2). The results showed a negative coefficient of the error term, which confirms the long run relationship between beef prices (Table 2).

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Table 2: Engle-Granger two-step cointegration test Farmer-Int. price Coefficient Standard error t-value -.148 .032 -4.53* m t-1

Dmt-1 Constant F-test R-squared Farmer-processor

-.240

.052

-4.59*

.000 30.280 0.1501

.004

0.020

mt-1

-.014

.016

-1.291

Dmt-1

-.234

.053

-4.461*

Constant F-test R-squared

.002 11.65 0.164

.002

Source; own estimation. *denote 95% significance.

Results from Johansen’s test (Table 3) provided strong evidence to reject the null hypothesis of non-cointegration between domestic farmer price and international price and between farmer price and processor beef prices, suggesting the existence of a long run single cointegration relationship. Table 3: Johansen cointegration test for price cointegration Farmer-International price

Rank

Eigenvalue . 0.035 0.004

Variable Farmer price International price Constant Farmer-processor

0 1 2 Coeff. 1 -.826 .528 Rank

. 0.035 0.004

Variable Farmer price Processor Price Constant

0 1 2 Coeff. 1 -0.678 5.375

--Eigenvalue

Trace statistic 30.208 1.744*

5% critical value 15.411 3.761

SE -.083 -Trace statistic 14.182* 1.537

Z --9.902 -5% critical value 15.41 3.761

SE

Z

0.1697

-3.998*

Source: Own estimation. Coeff.= Coefficient; SE= standard error. *denote 95% significance.

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Studies on beef and cattle prices, applying the Johansen test, reported cointegration between domestic farm prices and international prices. Long-run cointegration between farmers and processors/retail beef prices was also reported(24). The results suggest that their historical innovations profoundly influence prices in the international beef market. The international beef price has a consistently strong impact on Mexican price movements in the long-run. Given a unit change in the international price, farmer price of live cattle changes by 82 %, which implies a large effect, but different from unity. The remaining of the explanation given by other market fundamentals. For the case of farm-processor relationship, an increase of 1 % of the processor price, induces a 0.68 % increase in farm price. Since the above results confirmed the cointegration of international and domestic farm prices and between farmer and processor beef prices, it was estimated a symmetric and an asymmetric VECM.

Spatial vector error correction model

For the spatial model, it was estimated a VECM to investigate the possible interdependence of domestic and international beef prices considering that farm and international prices have a unit root, and are co-integrated. Results from the VECM show that both farm and international beef prices respond to disequilibria because coefficients are significant at the 5 % level. There is limited correction of price disequilibria, and coefficients are of the correct sign. In a similar study conducted in Europe(25), using the asymmetric VECM, it was found that price movement in global beef markets transmitted to domestic markets, but a lesser extent. Table 4 shows that contemporaneous change coefficients are significantly less than one in both equations. It means that within a single month, farm prices do not react entirely to global price changes. This fact shows that monthly data is adequate for revealing the process of beef price transmission(18).

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Table 4: Results of the VECM; symmetric and asymmetric spatial model Symmetric spatial model Asymmetric spatial model Independent Std. variable Coef. Std. Err. t-value P-value Coef. t-value P-value Err. Pprodt-1 0.27 0.054 4.99* 0.000 0.273 0.061 4.49* 0.000 Pprodt-2 0.017 0.056 0.31 0.768 0.017 0.056 0.31 0.768 Pprodt-3 0.043 0.056 0.77 0.441 0.042 0.056 0.75 0.453 Pprodt-4 0.04 0.055 0.65 0.514 0.034 0.055 0.62 0.535 Pint t-1 -.028 0.022 -1.26 0.210 -0.059 0.024 -2.44* 0.015 Pint t-2 -0.048 0.022 -2.14* 0.033 -0.025 0.023 -2.09* 0.037 Pint t-3 -0.008 0.022 -0.37 0.715 -0.005 0.022 -0.23 0.820 Pint t-4 0.022 0.019 1.17 0.243 0.023 0.018 1.27 0.215 ECTt-1 -0.049 0.015 -3.26* 0.000 ------+ ECT t-1 -------0.049 0.021 -2.38* 0.018 ECT- t-1 -------0.059 0.029 -2.05* 0.042 Constant 0.0011 0.002 0.62 0.552 0.001 0.002 0.58 0.556 Norm. test (Prob>z)=0.000 (Prob>z)=0.000 LM test (Prob>chi2)=0.291 (Prob>chi2)=0.524 DW test 0.299 0.532 R-squared 0.320 0.353

H0 : b1+ = b1-

---

F(1,330)= 0.822

H0 : b2+ = b2-

---

F(1,330)= 12.084 Source: Own estimation.*denote 95% significance.

The t- statistics for ECT+ and ECT- indicate that farm prices respond strongly to negative shocks, but positive shocks in the margin are allowed to persist. The induces a significantly greater change in farm price than the ECT+. A similar result, reported in economic literature(26), showed that VECM indicated that most of the market´s disequilibrium was corrected within a month. Prices correct a small percentage of disequilibria in the markets, mostly by external forces. An F-test of the null hypothesis of symmetry ( b 2

+

= b2- ) leads to

rejection at the 5 % level of significance (F= 12.08). This result implies that when price fall, the transmission is faster than when price rise. Price increases reach producers with a delay, with respect to a fall in international prices, which are transmitted faster. This result is consistent with the fact that international prices react more rapidly when the margin is squeezed than when it is stretched(27). A possible explanation for the price asymmetry is the insufficient access by livestock producers to price information and infrastructure(9). The spatial market integration of livestock and beef prices between international and

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Mexican market is an issue of major importance because is deficit country, and therefore efficient trade has important food security policy implications. From the policy point of view, this should help in the design of agricultural support programs, and risk management tools for the beef industry. The finding of strong transmission effects between international and Mexican prices corroborates the view that participants in the Mexican supply chain need to consider the highly volatile nature of international prices in their decision-making process.

Vertical vector error correction model Because cointegration exists between processor and farm beef prices, a VECM was estimated, following Cramon-Taubadel’s approach (equation 5). The output of the symmetric and asymmetric VECM in Table 5 indicates that both the coefficient for ECT and the shortterm parameter are significant at the 5 % level. Table 5: Results of VECM; vertical symmetric and asymmetric model Symmetric vertical model Asymmetric vertical model Independent Std. tvariable Coef. Std. Err. t-value P-value Coef. P-value Err. value Pprodt-1 0.077 0.036 2.120* 0.035 0.094 0.031 2.944* 0.004 Pprodt-2 -0.079 0.056 -1.411 0.158 -0.075 0.056 -1.321 0.187 Pprodt-3 0.026 0.056 0.47 0.645 0.023 0.056 0.41 0.675 Pprodt-4 0.067 0.053 1.242 0.214 0.068 0.054 1.263 0.21 Pint t-1 0.111 0.032 3.440* 0.001 0.104 0.033 3.090* 0.002 Pint t-2 0.065 0.032 2.000* 0.046 0.063 0.032 1.93 0.054 Pint t-3 0.002 0.032 0.075 0.948 0.003 0.032 0.121 0.908 Pint t-4 -0.04 0.031 -1.28 0.202 -0.042 0.031 -1.33 0.184 ECTt-1 -0.029 0.008 -3.690* 0.000 ------ECT+ t-1 -------0.033 0.0118 2.811* 0.006 ECT- t-1 -------0.042 0.0117 3.560* 0.000 Constant 0.001 0.002 0.88 0.375 0.001 0.0015 0.89 0.375 Normality test (Prob>z)=0.000 (Prob>z)=0.000 LM test (Prob>chi2)=0.336 (Prob>chi2)=0.605 DW test 0.344 0.612 R-squared 0.341 0.391 Test:

b2+ ¹ b2-

---

F(1,330)= 14.371 Source: Own estimation. *denote 95% significance

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This result suggests that processor and farmer’s prices share a relationship of long-term equilibrium. A change in farmer’s prices has a significant effect on processor prices during the subsequent period. The ECT-induces a significantly greater change in the processor price than ECT+. These results corroborate the hypothesis that price changes are not transmitted efficiently from one level to another(28). It also supports the hypothesis(9) that beef processors may have some market power. The asymmetric VECM results reveal that the transmission of beef prices is asymmetrical for the speed of adjustment. The t- statistics for ECT+ and indicate that retail prices respond strongly to negative shocks, indicating that when producer prices decrease, the speed of adjustment tends to be significantly faster. Moreover, when prices increase, there are statistically significant changes in the speed of adjustment. An F-test of the null hypothesis of symmetry ( b 2+ = b 2- ) leads to rejection at the 5 % level of significance (F= 14.37). It suggests that farm prices react more rapidly when the margin is squeezed than when it is stretched. In a study of the US beef market(29), it was pointed out a price transmission asymmetry that is much more critical for wholesale-retail than for farm-wholesale. Likewise, positive price shocks are transmitted with higher intensity than negative ones. Focusing on adjustments of retail prices to restore equilibrium, estimates of the adjustment coefficients indicate that, within a month, retail prices adjust so as to eliminate approximately 4.2 % of a unit negative change in the deviation from the equilibrium relationship created by changes in producer prices. On the other hand, retail prices adjust by 3 % of a positive change in deviation from the equilibrium created by changes in producer prices. Because beef and carcasses are nonstorable commodities subject to production lags with inelastic supply in the short run, producers are unable to adjust production in response to transitory price changes. By contrast, beef processor can immediately respond to changes in producer prices by adjusting their prices. Furthermore, processor, unlike feedlots, face significant fixed costs. In the short run, margins may thus be reduced in an attempt to keep a plant operating near full capacity. Therefore, as a result of competition between different processor, farm prices may be bid down more quickly than they are bid up. Given that asymmetric price transmission implies a certain degree of market power and / or market inefficiency, more research is needed to delve into the possible causes of asymmetric price transmission of livestock and beef, not only at the national level but also regional.

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Conclusions and implications This research provides for Mexico that the transmission of beef prices is asymmetric in the domestic and international markets. A long-run cointegration relationship exists between international and Mexican beef farm prices and between farm and domestic processor price. For the spatial analysis, both farm and international prices show a significant response to price disequilibria and asymmetric price transmission. Price movements in international markets are transmitted asymmetrically to the M exican market, indicating that a decrease in international prices tends to be transmitted faster to farmers than an increase in international prices. Considering the vertical price transmission model during the following period, a change in producer prices has a significant effect on processor prices. The speed at which prices tend to converge to ent i rel y correct for deviation is moderately slow, but when producer prices decrease, the speed of adjustment tends to be significantly faster. Asymmetric price transmission in the Mexican beef market have policy implications. The role of government intervention in the market via various price support programs may have welfare and income redistribution effects. For example, bovine livestock support programs in Mexico may be benefiting more to processors than farmers (feedlots). Findings from this research can provide valuable contributions to the policy debate, revealing a unidirectional transmission of beef prices from producers to processors, and that the transmission of beef prices is asymmetrical, depending on whether prices are increasing or decreasing.

Acknowledgments and conflict of interest

I am grateful for the financial support of the Colegio de Postgraduados-Campus Puebla for the development of the database and to Ms. Leticia Portilla Durán for her collaboration in the development of the database. I state that there is no conflict of interest of any kind between the funding institution and the published data and results.

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Literature cited: 1. SIAP. Servicio de Información Agroalimentaria y Pesquera. Indicadores Económicos. https://www.gob.mx/siap 2018. Consultado 9 Dic, 2019. 2.

USDA. United States Department of Agriculture. Livestock and poultry. https://apps.fas.usda.gov/psdonline/circulars/livestock_poultry.pdf. 2019. Accessed Jan 11, 2020.

3. SIAP. Servicio de Información Agroalimentaria y Pesquera. Anuario estadístico de la producción ganadera. https://nube.siap.gob.mx/cierre_pecuario/ 2019. Consultado 16 Oct, 2019. 4. Consejo Mexicano de la Carne. Compendio estadístico 2018. https://comecarne.org/ 2018. Consultado 9 Ene, 2019. 5. SUKARNE 2020. Página oficial. Consultado 16 Abr,2021. www.grupovix.mx, y http://www.sukuero.com.mx/celebrando-45-anos-de-sukarne/ 6. COFECE. Comisión Federal de Competencia Económica. Poder de mercado y bienestar social. www.cofece.mx 2019, Consultado Ene 14, 2019. 7. INEGI. Instituto Nacional de Estadística y Geografía. Índice Nacional de Precios al Consumidor y al Productor, 2018. http://www.inegi.org.mx. Consultado Ene 14, 2019. 8. Xia T, Li X. Consumption inertia and asymmetric price transmission. J Agric Resour Econ 2010;35(2):209–227. 9. Meyer J, Cramon TSV. Asymmetric price transmission: a survey. J Agric Econ 2004;55(3):581-611. 10. Fackler PL, Goodwin BK. Spatial price analysis. In Gardner B, Rausser G, editors. Handbook of agricultural economics. Amsterdam: Elsevier 2001;1(2):971-1024. 11. Ganneval S. Spatial price transmission on agricultural commodity markets under different volatility regimes. Econ Model 2016;52(A):173-185. 12. Serra T, Goodwin BK. Price transmission and asymmetric adjustment in the Spanish dairy sector. Appl Econ 2003;35(18):1889-1899. 13. Miller DJ, Hayenga ML. Price cycles and asymmetric price transmission in the U.S. Pork market. Am J Agr Econ 2001;83(3):551-562. 14. Dickey DA, Fuller WA. Likelihood ratio statistics for autoregressive time series with a unit root. Econometrica 1981;49(4):1057-1072.

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15. Phillips PCB, Perron P. Testing for unit root in time series regression. Biometrika 1988;75(2):335-346. 16. Johansen S. Estimation and hypothesis testing of cointegration vectors in Gaussian vector autoregressive models. Econometrica 1991;59(6):1551-1580. 17. Engle RF, Granger CWJ. Cointegration and error correction: Representation, estimation and testing. Econometrica 1987;55(2):251-276. 18. Cramon-Taubadel SV, Loy JP. Price asymmetry in the international wheat market: comment. Can J Agric Econ-Rev Can Agroecon 1996;44(3):311-317. 19. Cramon-Taubadel SV. Estimating asymmetric price transmission with error correction representation. Application to the German Pork market. Eur Rev Agric Econ 1998;25(1):1-18. 20. Granger CWJ, Lee TH. Investigation of production, sales and inventory relationships using multicointegration and non-symmetric error correction models. J Appl Econom 1989;4(S1):S145-S159. 21. Chung C, Rushin J, Surathkal P. Impact of the livestock mandatory reporting act on the vertical price transmission within the beef supply chain. Agribusiness 2018;34(3):562578. 22. Goodwin BK. Spatial and vertical price transmission in meat markets. Workshop of market integration and vertical and spatial price transmission in agricultural markets. University of Kentucky Lexington 2006. 23. Barahona JF, Trejos B, Lee JW, Chulaphan W, Jatuporn C. Asymmetric price transmission in the livestock industry of Thailand. APCBEE procedia 2014;8:141-145. 24. Guillen J, Franquesa R. Testing for market power in the Spanish meat market: Price transmission elasticity and asymmetry using econometric models. IJCEE 2010;1(3/4):294-308. 25. Ihle R, Brümmer B, Thompson SR. Structural change in European calf markets: Decoupling and the blue tongue disease. Eur Rev Agric Econ 2012;39(1):157-180. 26. Bekele S, Alemu D. Analysis of beef cattle market integration in the case of Wolaita Zone, Southern Ethiopia. J Market Consumer Res 2016;30:9-15. 27. Darbandi E, Saghaian SH. Vertical price transmission in the U.S. beef markets with a focus on the great recession. J Agribus 2016;34(2):99-120.

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28. Lloyd T. Forty years of price transmission research in the food industry: Insights, challenges and prospects. J Agr Econ 2017;68(1):3-21. 29. Emmanouilides CJ, Fousekis P. Vertical price dependence structures: copula-based evidence from the Beef supply chain in the USA. Eur Rev Agric Econ 2015;42(1):7797.

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https://doi.org/10.22319/rmcp.v13i4.6138 Article

Exploring bovine fecal bacterial microbiota in the Mapimi Biosphere Reserve, Northern Mexico

Irene Pacheco-Torres a Cristina García-De la Peña b* César Alberto Meza-Herrera a Felipe Vaca-Paniagua c,d,e Clara Estela Díaz-Velásquez c Claudia Fabiola Méndez-Catalá c Luis Antonio Tarango-Arámbula f Luis Manuel Valenzuela-Núñez b Jesús Vásquez-Arroyo g

a

Universidad Autónoma Chapingo. Unidad Regional Universitaria de Zonas Áridas, Bermejillo, Durango, México. b

Universidad Juárez del Estado de Durango. Facultad de Ciencias Biológicas, Av. Universidad s/n Fracc. Filadelfia, 35010 Gómez Palacio, Durango, México. c

Facultad de Estudios Superiores Iztacala. Laboratorio Nacional en Salud, Diagnóstico Molecular y Efecto Ambiental en Enfermedades Crónico-Degenerativas. Tlalnepantla, Estado de México. d

Instituto Nacional de Cancerología. Ciudad de México, México.

e

Universidad Nacional Autónoma de México. Facultad de Estudios Superiores Iztacala Unidad de Biomedicina, Tlalnepantla, Estado de México, México. f

Colegio de Postgraduados, Campus San Luis Potosí, Salinas de Hidalgo, San Luis Potosí, México.

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Universidad Juárez del Estado de Durango. Facultad de Ciencias Químicas. Gómez Palacio, Durango, México.

* Corresponding author: cristina.garcia@ujed.mx

Abstract: In Mexico, information on the bovine fecal microbiota (Bos taurus) is scarce. The present study describes the diversity and abundance of bacteria in fecal samples from rangeland bovines, collected in the Mapimi Biosphere Reserve in the central part of the Chihuahuan desert. Fecal samples were analysed using high-throughput next generation massive sequencing using V3-V4 16S rRNA on Illumina Miseq. A total of 17 phyla, 24 classes, 33 orders, 50 families, 281 genera, and 297 species were identified. Firmicutes and Verrucomicrobia were the most abundant phyla. The most abundant genera were Sporobacter, PAC000748_g (genera into the Ruminococcaceae family) and Eubacterium_g23. Three genera (Clostridium, Corynebacterium and Fusobacterium) and one species (Campylobacter fetus) potentially pathogenic bovine bacteria were registered. This information represents a bacteriological baseline for monitoring the grazing bovine intestinal health status, and to trace possible interactions with the fecal microbiota of native roaming wildlife in the area. Key words: Bos taurus; Campylobacter fetus; Bacterial diversity; 16S rRNA gene; Massive sequencing.

Received: 13/01/2022 Accepted: 06/04/2022

Introduction The microbial community of the gastrointestinal system of cattle remains understudied. Due to its influence on nutrient absorption, productivity, potential reservoir of human and animal pathogens, as well as overall animal health, there is a need to better understand bovine gut microbial communities(1). Recently, high-throughput sequencing using 16S rRNA amplicons has provided deeper information on the fecal bovine microbiota composition, and the results obtained to date indicate a high diversity(2).

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The central part of the Chihuahuan Desert in Mexico has a high diversity of wildlife(3,4). The bovine (Bos taurus) has been raised as grazing livestock since its introduction at the end of the 16th century, being the most important economic activity in this area(5). However, this activity is the main reason of ecological deterioration which affects wildlife; for example, this ruminant species competes for forage resources with endemic animal species (i.e., Gopherus flavomarginatus, Bolson tortoise)(3). Cattle grazing also exerts strong pressure on plant populations, modifying their cover; this may increase soil erosion susceptibility in this desert(3,6). The cattle gut microbiome has many microbial species that play an important role in health and productivity(7,8). These microbes are essential for the fermentation of consumed plant matter that is converted into energy for the host(9). However, bovines asymptomatically transport bacterial species that are potential pathogens to wildlife as Escherichia coli, Campylobacter spp., Salmonella spp. and Listeria spp.(6,10). In recent years, the extensive use of land for agriculture has increased the densities of cattle populations creating positive correlations with pathogenic infections by fecal bacteria(11). Though, knowledge about bovine fecal bacterial diversity under grazing management systems is relatively scarce(12). This study aimed to explore for the first time the diversity and abundance of fecal bacteria from bovines under grazing-marginal conditions in the Mapimi Biosphere Reserve, center of the Chihuahuan desert, using next-generation sequencing (16S rRNA).

Material and methods All the methods and activities of this study were in strict accordance with accepted guidelines for ethical use, care and welfare of animals in research at international(13) and national(14) levels, with institutional approval reference number UJED-FCB-2018-07.

Study area The study was developed in the Mohovano de las Lilas locality, northeast of the Mapimi Biosphere Reserve in Mexico (26°00’ and 26°10’N, 104°10’ and 103°20’W) in the center of the Chihuahuan desert. This area has warm, very arid climate, with an average annual temperature of 25.5 ° C, and an average annual precipitation of 264 mm. The predominant vegetation is rosette and microphile scrub, as well as halophyte, and gypsophila plants(15).

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Field work In July 2018, three fresh fecal samples were collected from three healthy male bovines. From each fecal sample, 0.25 g was collected from the center of the sample and deposited it in BashingBead™ cell lysis tubes (Zymo Research Corp.) adding 750 μL of lysing/stabilizing solution. Each tube was processed in a TerraLyzer™ cellular disruptor (Zymo Research Corp.) during 20 sec according to the equipment specifications.

Laboratory work DNA was extracted from the samples using the Xpedition™ Soil/Fecal DNA MiniPrep kit (Zymo Research Corp.) in a laminar UV flow hood in sterile conditions. The amount of DNA obtained was measured in a Qubit™ fluorometer (Invitrogen). Then, the V3-V4 region of the 16S rRNA gene was amplified using the following primers(16): S-D-Bact-0341-b-S-17, 5´CCTACGGGNGGCWGCAG-3´ and S-D-Bact-0785-a-A-21, 5´GACTACHVGGGTATCTAATCC-3´. The step after sequencing was realized using a Illumina protocol(17,18) and thereafter, the samples was sequenced in MiSeq of 2 × 250 paired final. The complete sequencing process is available in García-De la Peña et al(19).

Data availability The files used in this study were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: PRJNA614584).

Bioinformatic analysis The DNA sequences were analyzed using Quantitative Insights into Microbial Ecology bioinformatics software (QIIME)(20). Both forward and reverse sequences were assembled using the PEAR program(21) considering Q30 the quality criterion (one false base for every 1,000 bases). Chimeric sequences were discarded with USEARCH(22). Then, the operational taxonomic units (OTUs) were selected with the UCLUST method(22) at 97 % similarity; a representative sequence for each OTU was obtained, and the taxonomy was assigned using EzBioCloud database as reference(23). A simple random rarefaction process was performed(24) in order to obtain a standardized file for all samples. The relative abundance for the phylum and family levels were represented as stacked bar plots using R, and genus level was visualized as a heatmap using Morpheus software (Morpheus,

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https://software.broadinstitute.org/morpheus); hierarchical clustering (average linkage method with Euclidean distance) was used to visualize samples dendrogram(25).

Results and discussion In this study, the average number of sequences assembled was 155,915. A mean ± sd of 109,814 ± 16,686 bacterial sequences were obtained after taxonomic designation. The average number of OTUs with a 97 % of similarity was 6,661 ± 431 (Table 1). Table 1: Fecal sequences information of Bos taurus at the Mohovano de las Lilas locality, Mapimi Biosphere Reserve, Mexico Sample

Total

Assembled

Discarded

BS

BSS

OTUs

1

322,428

138,862

183,566

131,275

98,084

6,293

2

223,470

145,379

78,091

136,807

102,441

6,556

3

305,380

183,506

121,874

173,177

128,916

7,135

Mean

283,759

155,915

127,843

147 086

109 814

6,661

BS= bacteria sequences after taxonomical designation, BSS= bacteria sequences after singletons removal; OTUs= operational taxonomic units.

A total of 17 phyla, 24 classes, 33 orders, 50 families, 281 genera, and 297 species were determined. The most abundant phyla (Figure 1) were Firmicutes (𝑥̅ = 88.9 %) and Verrucomicrobia (𝑥̅ = 6.4 %). The same phyla were reported in grazing Mongolian cattle in Hulunbuir grassland and Alxa Desert in China(26). These phyla are considered normal components in the basic fecal microbiota of domestic herbivores(27,28) and other species of ruminants(29,30). Firmicutes has been reported as the most frequent phylum in fecal samples of cattle, horses(2,31,32), and red deer(33). This abundance is related to high fiber intake(34). Verrucomicrobia was the second abundant phylum in the cattle samples in this study. Aricha et al(26) determined that this phylum was very abundant in the intestinal tract of the grazing Mongolian cattle in the Alxa Desert, and argue that this may be related to the extremely strong disease resistance of this breed of cattle. It is important to analyze later if this phylum confers resistance to cattle diseases in the Mapimi reserve, which would represent an advantage for the bovine’s health in this area. Also, Bacteroidetes was reported in previous studies Mongolian(26), and Holstein Friesian(36) as the second most abundant phylum in other cattle species such as grazing and feedlot Angus Beef(35). However, Bacteroidetes was found in a minimum proportion (0.001%) in the cattle samples of the Mapimi reserve. This disparity 914


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can be related to the type of diet(37), geographical differences(26), and the environment in which they are distributed(38). Nevertheless, this information can only be confirmed by developing specific studies in this respect. Figure 1: Relative abundance (%) of fecal bacteria taxa (phylum level) from three samples of Bos taurus at the Mohovano de las Lilas locality. Only the first 10 more abundant phyla are shown

At family level, Ruminococcaceae (𝑥̅ = 68.9 %) and Lachnospiraceae (𝑥̅ = 10.9 %) were abundant in the fecal samples collected (Figure 2); both families are found in the mammalian gut environment and have been associated with good health(39). Some genera of the Ruminococcaceae family are part of the normal intestinal microbiota of cattle, sheep, and goats metabolizing cellulose, and colonizing the rumen(40); these bacteria taxa are important for the degradation and fermentation of polysaccharides in the diet of ruminants(41). In addition, it has been reported that members of the Lachnospiraceae family exhibit pectin hydrolysis activities in the cattle´s rumen(42) associated to the butyric acid production and providing energy for the growth of intestinal epithelial cells(43). The high abundance of Lachnospiraceae in cattle protects the intestine and acts as a barrier that favors the adaptation of the host to its environment; it also promotes a decrease in the incidence of intestinal diseases(26).

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Figure 2: Relative abundance (%) of fecal bacteria taxa (family level) from three samples of Bos taurus at the Mohovano de las Lilas locality. Only the first 10 more abundant families are shown

From 281 classified genera found in this study, 36.6 % have a taxonomic name; this percentage is higher than the reported by Kim and Wells(44) in feces of cattle where only 110 genera were classified, and about 41 % of the total sequences couldn’t be assigned to a known genus (Figure 3). Nevertheless, the results showed here increase the number of genera of the B. taurus fecal microbiota previously reported (12,45,46), who confirmed that the fecal bacterial microbiota is extremely diverse in cattle, and has not yet fully described. Sporobacter was the most abundant genus found in the fecal samples of cattle in this study. This genus was reported in alpaca(47), deer sika(28), horse(48), donkey(49), and the Bolson tortoises Gopherus flavomarginatus(19). This genus is related to digestion of plant ligno-cellulosic matter; however relatively little is known about the role of this bacteria in the degradation process(50). Durso et al(51) reported Faecalibacterium, Ruminococcus, Roseburia, and Clostridium as important components of the fecal bovine microbiota. These genera were also determined in the present study. According to some studies(52,53) these bacteria constitute 50 to 70 % of the total number of microorganisms in the digestive system of ruminants. These animals have specific gut microbial taxa as they are dependent on these bacteria to extract energy and nutrients from food(54), besides having specialized anatomical and physiological adaptations to the cellulolytic fermentation of low nutrition - high fiber vegetal material(55). The presence of other bacterial genera reported in this study could be the result of environmental and genetic factors, age, breed, diet, phylogeny, among others(56,57,58). Recently(56,59,60), was demonstrated that herbivorous animals have the most diverse microbiota since they depend on microbial metabolic pathways to maximize energy and nutrient extraction from feeding(61). 916


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Figure 3: Heatmap of Bos taurus fecal bacteria sample at genus level at the Mohovano de las Lilas locality. Only the first 40 more abundant genera are shown

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Although the gut microbiome usually remains stable over time assisting as a defense system against pathogens and other disease-causing agents in the host, the disturbance of this community can lead to animal disease(62,63). In the present study, the samples collected were obtained from apparently healthy bovines. However, bacteria considered of veterinary importance were found in these animals; this could be a potential health risk because they are carriers of these microorganisms. For example, Campylobacter, Clostridium, Corynebacterium and Fusobacterium were found in the fecal samples. These genera have been associated with cattle disease. Campylobacter has been reported as a cause of infertility and abortion in ruminants(64,65); also represents a critical threat to public health, because it can be transmitted from cattle to humans(66,67,68). Clostridium has been reported causing diseases and death in ruminants, especially in cattle; examples are respiratory diseases(69), botulism(70) and the blackleg(71). Corynebacterium has been reported in beef and dairy cattle associated with renal disease(72), mastitis(73,74), and tuberculosis(75,76,77); also, it is considered as an important emergent pathogen for humans(78). Finally, Fusobacterium was reported by others(79-82), causing abscesses in cattle. It is important to develop other studies that provide information on the pathogenicity and dynamics of these potential pathogens in the bovines of the Mapimi reserve. At species level, Pseudobacteroides cellulosolvens and Campylobacter fetus were registered in the present study. Pseudobacteroides cellulosolvens is anaerobic bacteria that degrade plant cell wall polysaccharides and cellulosic, being capable of using cellulose or cellobiose as a sole carbon source(83). Campylobacter fetus is a relevant species; the main reservoirs of this bacteria are both the intestinal and the genital tracts in cattle and sheep(64,65). This species causes spontaneous abortion and infertility in cattle, while it is also an opportunistic pathogen to humans(84). Due to the free-grazing management in the Mapimi Reserve, the bovine feces remain over the soil until natural processes degrade them. Consequently, native fauna can be in contact with these feces, increasing the probability of interspecific transmission of some bacteria(85). Although it has been previously reported that there are no evidence of cross-parasite infection between cattle and mule deer in the Mapimi Biosphere Reserve(86), it is important to clarify whether this same scenario occurs for bacteria. McAllister and Topp(87) estimate that about 77 % of the pathogens that usually infect livestock can also affect wildlife. However, also wildlife is considered an important source of microorganisms that could cause infectious diseases to domestic animals and humans(88,89). For these reasons, it is important to develop studies focused on risk management at the interface of domestic species and native fauna, considering the implications for the transmission of microorganisms with pathogenic potential(88,89). This information could lead to establish microbiological control strategies for wild fauna populations and livestock within the area.

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Conclusions and implications Information about the bovine fecal microbiota under extensive grazing conditions is scarce. From economic, ecological and health perspectives, it is crucial to determine the bacterial diversity -from phyla to species-, in the intestine of domestic ruminants. The present study is the first insight into the fecal bacterial composition of bovines in the Mapimi Biosphere Reserve in Mexico using next generation sequencing. This information significantly expands the knowledge about the composition and abundance of bacteria that are part of the microbiological community of the bovine intestine. In this case, the approach was through the analysis of feces in free grazing cattle. Although a large number of bacterial taxa were reported from the collected samples, it was not possible to determine the genus or species of some bacteria, so it is still necessary to go further into the taxonomy using specific molecular markers. However, the results obtained in the present study could be used as a bacteriological baseline for monitoring the grazing bovine intestinal health status, and to trace possible interactions with the fecal microbiota of native roaming wildlife in the area. Finally, it is important to emphasize that the next generation massive sequencing is a very effective technique that simplifies the analysis of complete bacterial communities; therefore, complementary studies on the microbiota in this and other bovine populations in Mexico are warranted.

Acknowledgments

To S.I. Barraza-Guerrero, D. Acosta-Astorga and R. Zapata-Fernández for their support in the fieldwork. The owners of the Mohovano de las Lilas locality gave their authorization to take bovine fecal samples.

Conflict of interest

The authors declare that they have no conflict of interest.

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https://doi.org/10.22319/rmcp.v13i4.6042 Article

Phytochemical profile, antimicrobial and antioxidant activity of extracts of Gnaphalium oxyphyllum and Euphorbia maculata native to Sonora, Mexico

Priscilia Yazmín Heredia-Castro a Claudia Vanessa García-Baldenegro a Alejandro Santos-Espinosa a Iván de Jesús Tolano-Villaverde a Carmen Guadalupe Manzanarez-Quin b Ramón Dolores Valdez-Domínguez c Cristina Ibarra-Zazueta c Reyna Fabiola Osuna-Chávez c Edgar Omar Rueda-Puente c Carlos Gabriel Hernández-Moreno c Susana Marlene Barrales-Heredia c Jesús Sosa-Castañeda c*

a

Universidad Estatal de Sonora (UES). Ingeniería en Horticultura. Sonora, México.

b

Centro de Investigación en Alimentación y Desarrollo A. C. (CIAD, A. C.) Tecnología de Alimentos de Origen Animal. Sonora, México. c

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

* Corresponding autor: jesus.sosa@unison.mx

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Abstract: The use of synthetic chemical compounds to preserve foods or treat diseases of bacterial origin is limited because they can cause health damage. Therefore, the food and livestock industries seek natural strategies to preserve foods and preserve the health of animals intended for human consumption. In this sense, some extracts of plant from Sonora, Mexico could be an alternative due to the great diversity of plants and the fact that some of them are traditionally used to treat diseases. On the other hand, there are few studies that support the biological activity of ethanolic extracts of Gnaphalium oxyphyllum (E1) and Euphorbia maculata (E2). In this study, phytochemical content was determined by spectrophotometry, antimicrobial activity was determined by agar diffusion and antioxidant activity was evaluated by ABTS, DPPH and FRAP. The results showed that the E1 and E2 extracts had total phenols, total flavonoids, flavones and flavonols, total flavanones and dihydroflavonols, as well as total tannins, total chlorogenic acid and total polysaccharides. In addition, both extracts showed higher antimicrobial activity against Listeria monocytogenes ATCC 19115, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella enterica serovar Typhimurium ATCC 14028 when 1 mg ml-1 was used (P<0.05). In addition, they presented antioxidant activity by the methods of ABTS, DPPH and FRAP. Therefore, the antimicrobial and antioxidant potential of these plants represents a natural alternative to control some Gram-positive and Gram-negative bacteria in the livestock industry, as well as for food preservation. Key words: Gnaphalium oxyphyllum, Euphorbia maculata, Antimicrobial activity, Antioxidant, Natural alternative, Food industry.

Received: 16/08/2021 Accepted: 05/05/2022

Introduction Consumer interest in avoiding foods with synthetic chemical compounds has increased due to their potential harm to health. In the scientific community, there is a growing interest in the search for natural strategies for food preservation; as well as in livestock production to prevent recurrent diseases of domestic animals(1). Some of the natural alternatives that have been considered in the food industry and in veterinary medicine include the use of probiotics, bacteriocins, antioxidants and chemical compounds derived from plants(1,2). Considering the above, plant extracts have advantages, since, in some of them, their antioxidant and antimicrobial potential has been shown(3). In this context, Mexico is one of the countries with great plant biodiversity worldwide, ranks fourth with approximately 31,000 different species of plants. Of these, it is estimated that more than 3,350 are used in the preparation of traditional medicine treatments(4), and in some of

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these plants, it has been seen that they have the same active ingredient that is used in the preparation of commercial drugs(5). However, the studies carried out with plants native to Mexico are incipient since phytochemical compounds and their biological activity lack scientific evidence of their activity. In addition, there are few scientific studies that have characterized the antimicrobial activity of plants native to Sonora, Mexico(6-9) and those that evaluate their antioxidant activity are very few. Particularly, Gnaphalium oxyphyllum is a plant known as “Gordolobo” in Sonora and is endemic to northwestern Mexico. It is traditionally used in the treatment of some conditions, such as flu, asthma, cough, fever, bronchitis, swelling, stomach diseases, wounds, low back pain, in the prevention of malaria and urinary tract problems derived from prostatitis and neuritis. As well as for angina pain, antipyretic and to lower blood pressure(10,11). In addition, its ability to inhibit the growth of some pathogenic bacteria and fungi has been demonstrated(11,12). However, the antimicrobial and antioxidant activity of the ethanolic extracts of this plant has not been evaluated(11). On the other hand, Euphorbia maculata is a plant native to northwestern Mexico, locally known as “Golondrina”. It is traditionally used to treat stomach upsets and eye problems, in addition, in Chinese medicine it is used in blood disorders such as hematuria, hemoptysis, epistaxis and hemafecia, for the treatment of anthrax and some wounds. However, the antimicrobial, antifungal and antioxidant activity has been poorly documented, and there are no studies that evaluate its antimicrobial potential in ethanolic extracts(13,14). The evidence indicates that these plants are of high biological value, but they have been little studied and have not been harvested in Sonora, Mexico, so the biological activity of the plants can be compromised, because their phytochemical profile can vary depending on factors such as altitude, cultivation site, agronomic and environmental conditions in which they grow(15). Therefore, and considering that plants can also be used as a food supplement(16), it is interesting to evaluate the nutritional value, antimicrobial, antioxidant activity and phytochemical profile of these plants grown in Sonora, Mexico.

Material and methods Preparation of ethanolic extracts

The extracts were obtained from Gnaphalium oxyphyllum (E1) and Euphorbia maculata (E2), the plants were harvested at the Department of Agriculture and Livestock (DAG, for its acronym in Spanish) of the University of Sonora (DAG-UNISON). The stems and leaves of each plant were dehydrated at 34 °C in a hot air oven (Thelco, Precision Science, model 28, USA). The dehydrated plant material was then pulverized in a mill (Pulvex Mini 100, Mx) to a particle size of 100 microns. Subsequently, 100 g of the pulverized plant material was mixed with 100 ml of 99 % purity ethanol (Sigma-Aldrich, St. Louis MO) in an amber glass bottle and stored for 5 d(17). Finally, the extracts were filtered with

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Whatman No. 41 filter paper and the remaining alcohol in the plant material was evaporated. The yield was calculated by difference in weight of the plant material, and finally, the ethanolic extracts were stored at 4 °C in the dark.

Phytochemical profile of ethanolic extracts

The contents of total phenols and total flavonoids were quantified by the methodologies used by Al-Rifai et al(18) and the data were expressed as milligrams of gallic acid equivalent per gram of extract (mg GAEq. g-1) for total phenols, while for total flavonoids, the data were expressed as milligrams of quercetin equivalent per gram of extract (mg QEq. g-1). The content of flavones and flavonols, as well as the content of total flavanones and dihydroflavonols were determined following the methodologies proposed by Popova et al(19) and the results were expressed as milligrams of hesperetin equivalent per gram of extract (mg HEq. g-1). The total tannin content was determined by the methodology reported by Price and Butler(20) and the results were expressed in milligrams of catechin equivalent per gram of extract (mg CEq. g-1), while the chlorogenic acid content was quantified following the methodology reported by Griffiths et al(21), where the results were expressed as milligrams of chlorogenic acid per gram of extract (mg CA g-1). Finally, the total polysaccharide content was determined by the methodology reported by DuBois et al(22) and the data were expressed as milligrams of glucose equivalent per gram of extract (mg GEq. g-1). Calibration curves were used in all determinations and absorbances were read on a spectrophotometer (Spectro Max MD, EU).

Antimicrobial activity of ethanolic extracts

The Gram-positive bacteria Listeria monocytogenes ATCC 19115 and Staphylococcus aureus ATCC 25923, and the Gram-negative bacteria Escherichia coli ATCC 25922 and Salmonella enterica serovar Typhimurium ATCC 14028, from the Laboratory of Microbiology of the Department of Chemical-Biological Sciences of the University of Sonora, were used. The bacteria were reactivated in BHI (brain-heart infusion, BD Difco, Sparks, MD) broth culture medium, and two plates with BHI (brain-heart infusion, BD Difco, Sparks, MD) agar were used for each bacterium. Four sterile discs of Whatman No. 41 filter paper of 6 mm in diameter were then placed on each plate and 20 μL of ethanolic extract was added to each disc. Subsequently, the plates were incubated at 37 °C for 24 h and antimicrobial activity was measured in inhibition halos, where halos greater than 3 mm were considered as inhibition(23).

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Physicochemical analysis of plants

The analytical methods of the AOAC(24) were used. Total solids were determined by the oven-drying method (990.19); ashes by the gravimetric method (945.46); crude fat by the ether extraction method (920.39); crude protein by the micro-Kjeldahl method (991.20) and moisture by numerical difference. The data were expressed in grams per 100 grams of dry matter (g 100 g-1). Additionally, the pH was measured with an electronic potentiometer (Hanna Instruments pH 211, Cluj, Romania).

Determination of minerals in plants

The amount of calcium (Ca), magnesium (Mg), sodium (Na) and potassium (K) from each plant was determined on a model 5000 flame atomic absorption spectrophotometer (PerkinElmer®, CT, USA)(25), while the phosphorus concentration (P) was determined by a colorimetric method of ammonium molybdovanadate in a model 3030 spectrophotometer (PerkinElmer®, CT, USA)(26). The results were expressed in grams per 100 grams of dry matter (g 100 g-1).

2,2-diphenyl-1-picrylhydrazyl (DPPH) radical inhibition method

The concentrations of each extract were adjusted to 0.1, 0.5, 1.0 and 2.0 mg ml -1, then 1 ml of each extract was mixed with 2 ml of a methanolic solution prepared with the 2,2diphenyl-1-picrylhidrazyl (DPPH●) radical at a concentration of 1 x 10-4 M. The mixture was left to react for 16 min in the dark at room temperature. Finally, the absorbance was measured in a spectrophotometer (Spectro Max MD, EU) at a wavelength of 517 nm and the DPPH● solution was used as control(27).

2,2'-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) radical inhibition method

A mixture in 1:1 ratio (v/v) of the 2,2'-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS●+) radical (7 mM) and potassium persulfate (4.95 mM) was prepared and kept in the dark for 16 h at room temperature. The mixture was then diluted with methanol until an absorbance of 1 to 1.5 was obtained. Next, 0.1 mL of each extract was mixed at different concentrations (0.1, 0.5, 1.0 and 2.0 mg mL-1) with 3.9 ml of the ABTS●+

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solution. Finally, absorbance values were measured on a spectrophotometer (Spectro Max MD, EU) at a wavelength of 734 nm. The ABTS●+ solution was used as control(27).

Ferric-reducing antioxidant power (FRAP) method

The FRAP reagent was prepared by mixing 10 parts of sodium acetate buffer solution (300 mM) at a pH of 3.6 with one part of TPTZ (10 mM) (2,4,6-tri (2-pyridyl)-s-triazine) and one part of FeCl3 hexahydrate (20 mM). Then, 0.2 ml of extract was mixed with 3.8 ml of FRAP reagent and the mixture was left to react for 30 min at 37 °C. Finally, absorbance was measured on a spectrophotometer (Spectro Max MD, EU) at a wavelength of 593 nm(27).

Statistical analysis

A completely randomized one-way experimental design was used at 95 % confidence with three repeats per treatment. The mean comparison test was performed by TukeyKramer at a significance level of 0.05 and the Pearson correlation coefficient was performed with 95 % confidence. The statistical software used was NCSS version 11.

Results and discussion The results of the proximate analysis of the plants showed in E2 higher moisture, less total solids and ashes with respect to the E1 plant (P<0.05) (Table 1), while no differences were found in the amount of fat and protein of both plants (P>0.05). The results in the amount of moisture, total solids and ashes of this study are similar to those found in wild edible plants from Bangladesh and in plants consumed by native tribes of India(28,29). The variability in the results can be attributed to biological, environmental factors or the age of the plants(30). In addition, the moisture content of plants could depend on the humidity and temperature of the environment, as well as on the harvest time of the plant, while the ash content refers to the inorganic part of the plant, which includes salts (phosphates, sulfates, chlorides) and some minerals (sodium, potassium, calcium, magnesium, iron and manganese), and their amount may depend on the mineral content of the soil where the plant is established(31). Likewise, plant lipids are mainly found in the form of triacylglycerols, phospholipids, galactolipids and sphingolipids, and their amount is usually very low in plants(30,32,33), which coincides with what is found in the E1 and E2 plants, and with what was reported in plants from Bangladesh and India(28,29,30). Although this study found no difference in the amount of lipids between E1 and E2 plants (P>0.05),

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it has been reported that the variation in lipid content may depend on the species and the environmental conditions in which the plant is found(30,34).

Plant E1 E2

Table 1: Proximate analysis of E1 and E2 plants Moisture Total solids Ashes Fat a a 61.53 ± 2.24ª 38.47 ± 2.23 5.74 ± 0.63 2.12 ± 0.12a 68.22 ± 1.22b 31.78 ± 1.13b 4.56 ± 0.73b 2.05 ± 0.15a

Protein 11.98 ± 0.85a 11.17 ± 0.73a

E1= Gnaphalium oxyphyllum; E2= Euphorbia maculata; data expressed in g 100 g-1 of dry matter. ab Different literal indicates difference between the data in the same column (P<0.05).

Likewise, the protein content of E1 and E2 plants was similar to that found in green leafy vegetable plants(35), and it has been reported that the amount of protein in plants may depend on the physiological state, age, environmental conditions and nutrients present in the soil(36). On the other hand, the content of P, Na and K was higher in plant E1 with respect to plant E2 (P<0.05), while the content of Mg was higher in plant E2 (P<0.05), and no differences were found in the content of Ca between both plants (P>0.05) (Table 2). These results are similar to those found in plants from Iran and India(30,37), and it has been reported that the variability in the mineral content of the plants could be related to the mineral composition of the soil, as well as to the geographical area where the plants are established(38).

Plant E1 E2

Table 2: Mineral content of the E1 and E2 plants Ca P Mg Na a b a 1.12 ± 0.13 0.33 ± 0.05 0.21 ± 0.03 1.63 ± 0.03b 1.15 ± 0.14a 0.25 ± 0.03a 0.55 ± 0.02b 1.22 ± 0.33a

K 1.23 ± 0.05b 1.07 ± 0.33a

E1= Gnaphalium oxyphyllum; E2= Euphorbia maculata; data expressed in g 100 g-1 of dry matter. ab Different literal indicates difference between the data in the same column (P<0.05).

The results of the antimicrobial activity showed that the E1 and E2 extracts inhibited the growth of the four evaluated pathogens (P<0.05) and the greatest inhibition occurred when the pathogens were exposed to 1 mg ml-1 of each extract (Table 3). On the other hand, the E1 extract was more efficient in inhibiting S. aureus and L. monocytogenes with respect to the E2 extract (P<0.05), while both extracts did not show differences in inhibition against E. coli and S. enterica serovar Typhimurium. Similar results were reported in hexane extract from Gnaphalium oxyphyllum flowers, which was able to inhibit the growth of S. aureus, B. cereus, E. coli and S. enteric serovar Typhimurium, in addition, the methanolic extract of these flowers inhibited the growth of S. aureus and B. cereus, while the hexane extract of the leaves of Gnaphalium oxyphyllum had antimicrobial activity against S. aureus, B. cereus and E. coli(10). Another study showed that hexane and chloroform extracts from the aerial part of Gnaphalium oxyphyllum inhibited the growth of S. aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, E. coli and Candida albicans(12). In addition, it has been reported that the hydroethanolic extract from leaves of Euphorbia maculata showed antimicrobial activity against S. aureus(39), while methanolic extracts from other plants of the genus

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Euphorbia showed antimicrobial activity against S. aureus, Bacillus megaterium, Proteus vulgaris, Klebsiella pneumoniae, E. coli, Pseudomonas aeruginosa and Candida albicans, Candida glabrata, Epidermophyton spp. and Trichophyton spp.(40), which is similar to what was found in this study. The antimicrobial activity of the extracts is associated with cell wall damage and decrease in cytoplasmic pH in Gram-positive and Gram-negative pathogenic bacteria, in addition, the antimicrobial activity of plants is attributed to a wide variety of secondary metabolites, such as tannins, alkaloids, phenolic compounds, flavonoids, xanthones and hyperforin(41,42). In this context, the results showed that the content of total phenols, total flavonoids, flavones and flavonols, total chlorogenic acid and total polysaccharides was higher in the E1 extract with respect to E2 (P<0.05) (Table 4), while no difference was found in the content of total flavanones and dihydroflavonols, and total tannins between the extract E1 and E2 (P>0.05). On the other hand, the E1 extract had a pH of 4.18 and the E2 extract had a pH of 5.26, while the yield of the extracts of these plants varied from 12.24 to 15.68 %, respectively. Similar results were reported by Rojas et al(12), who reported yields of 1.76 % and 5.64 % in hexane and chloroform extracts from the Gnaphalium oxyphyllum plant. The differences in the yield of this plant may be due to the polarity of the type of solvent that was used for the extraction of phytochemical compounds. In addition, the variation in the pH of plant extracts may be due to the acidic nature of the compounds present, such as flavonoids, tannins, benzoic acid, oleic acid, stearic acid, lignoceric acid, among others(43). In this sense, it has been reported that, in Gnaphalium oxyphyllum and other species of the genus Gnaphalium, the presence of diterpenoids, flavonoids, acetylene compounds and carotenoids was found(10,44), while in Euphorbia maculata, the presence of polyphenols and flavonoids has been reported(45,46,47), and in other species of Euphorbia, the presence of sesquiterpenes, diterpenes, sterols, flavonoids and other polyphenols has been reported(14). Table 4: Phytochemical profile, pH and yield of E1 and E2 extracts Phytochemicals Extracts E1 E2 a 181.62 ± 0.04 173.22 ± 0.06b Total phenols, mg GAEq. g-1 114.30 ± 0.05a 103.42 ± 0.04b Total flavonoids, mg QEq. g-1 Flavones and flavonols, mg HEq. g-1 110.15±2.35a 98.33±2.44b Total flavanones and dihydroflavonols, mg 23.68±1.89a 21.58±2.16a HEq. g-1 Total tannins, mg CEq. g-1 8.21±0.16ª 7.92±0.67a Total chlorogenic acid, mg CA g-1

33.14±1.01a

28.78±1.11b

Total polysaccharides, mg GEq. g-1 pH of the extract Extract yield, %

257.92±2.19a 5.26 12.24

236.59±2.16b 4.18 15.68

ab

E1= Gnaphalium oxyphyllum; E2= Euphorbia maculata. Different literal indicates difference between the data in the same column (P<0.05).

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The antioxidant activity of plants is associated with the presence of vitamins, phenolic compounds, carotenoids, among others. Particularly, in this study, it was found that the E1 and E2 extracts showed greater antioxidant activity by the DPPH and FRAP methods when they were evaluated at a concentration of 1 mg ml-1 (P<0.05) (Table 5), while in the ABTS method, the greater antioxidant activity of the E1 and E2 extracts was observed when they were evaluated at a concentration of 0.5 mg ml-1 (P<0.05). To date, there is no universal method to measure the antioxidant activity of plants because the chemical reagents used by these methods do not react the same with the different types of antioxidants present in plants. For example, ABTS● reacts with lipophilic and hydrophilic antioxidants, which allows it to be applicable in aqueous and lipid systems, while DPPH● can only be dissolved in an organic medium so it reacts well with low polar or non-polar compounds, and both methods are based on the ability of antioxidants to neutralize reference free radicals (ABTS● and DPPH●). Therefore, in the E1 and E2 extracts, there could be more phenolic compounds of a hydrophobic nature than of a hydrophilic nature. Likewise, the FRAP method is based on the ability of antioxidants to reduce the ferric ion to the ferrous state and measures the total antioxidant capacity of the sample, which shows the presence of phenolic compounds in the E1 and E2 extracts(48). These results of antioxidant activity are similar to those reported by Luyen et al(49), who observed high antioxidant power in methanolic extracts, ethyl acetate and aqueous extracts of Euphorbia maculata using the ORAC method, while other studies have shown the antioxidant activity of plants of the genus Euphorbia, where Basma et al(50) evaluated the antioxidant activity of leaves, stems, flowers and roots of Euphorbia hirta using DPPH and FRAP techniques. In addition, Upadhyay et al(51) found antioxidant activity in Euphorbia hirta leaves by the DPPH and FRAP methods, while Zhang et al(52) reported antioxidant activity in Euphorbia lathyris stems, roots, seed and seed cover using the DPPH and FRAP methods. Table 5: Antioxidant activity of E1 and E2 extracts Extract

DPPH (mg QEq. g-1)

(mg ml-1) E1

E2

ABTS (mg QEq. g-1)

FRAP (mg FeSO4Eq. g-1)

E1

E1

E2

E2

0.1

0.028±0.001a 0.025±0.003a 0.008±0.0001a 0.005±0.0002a 0.062±0.002ª 0.054±0.004a

0.5

0.127±0.004b 0.128±0.006b 0.035±0.0002b 0.032±0.0002b 0.084±0.004b 0.072±0.006b

1

0.146±0.004c 0.140±0.004c 0.037±0.0002b 0.035±0.0003b 0.099±0.003c 0.095±0.005c

2

-

ab

-

-

-

-

-

E1= Gnaphalium oxyphyllum; E2= Euphorbia maculata; (-)= not quantifiable. Different literal indicates significant difference between the data of the same column and between the treatments of the same method (P<0.05).

Finally, the ABTS, DPPH and FRAP methods are commonly used to measure the antioxidant activity of phenolic compounds because of the high correlation that can be found between them. Therefore, it has been suggested that it is not necessary to apply more than one method to measure the antioxidant activity; however, it has been reported that this is not always the case, due to the nature of the phytochemicals present in plants(27). In this study, a high correlation coefficient (R2) was found between the DPPH, 936


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ABTS and FRAP methods (DPPH vs ABTS= 0.99; DPPH vs FRAP= 0.93; ABTS vs FRAP= 0.88), which confirms the presence of antioxidant phenolic compounds found in E1 and E2 extracts and shows the accuracy of the methods used.

Conclusions and implications The extracts of Gnaphalium oxyphyllum and Euphorbia maculata showed the presence of the phytochemicals: total phenols, total flavonoids, flavones and flavonols, total flavanones and dihydroflavonols, total tannins, total chlorogenic acid and total polysaccharides. In addition, both extracts had antimicrobial activity against Grampositive and negative pathogenic bacteria, as well as antioxidant activity by the DPPH, ABTS and FRAP methods. Therefore, the extracts from plants native to Sonora, Mexico, Gnaphalium oxyphyllum and Euphorbia maculata, represent a natural alternative in the food and livestock industry to reduce the use of synthetic chemical compounds.

Acknowledgements

To the University of Sonora and the State University of Sonora for the support in the use of materials and facilities, as well as to Lic. Gerardo Reyna Cañez for his technical support. This research work was conducted in collaboration with the UES-PII-20-UAHIH-02 project. Literature cited: 1. Aguilar CN, Ruiz HA, Rubio RA, Chávez-González M, Sepúlveda L, Rodríguez-Jasso RM, et al. Emerging strategies for the development of food industries. Bioengineered 2019;10(1):522-537. 2. Lillehoj H, Liu Y, Calsamiglia S, Fernandez-Miyakawa ME, Chi F, Cravens RL, et al. Phytochemicals as antibiotic alternatives to promote growth and enhance host health. Vet Res 2018;49(1):1-18. 3. Mateos-Maces L, Chávez-Servia JL, Vera-Guzmán AM, Aquino-Bolaños EN, AlbaJiménez JE, Villagómez-González BB. Edible leafy plants from Mexico as sources of antioxidant compounds, and their nutritional, nutraceutical and antimicrobial potential: A review. Antioxidants 2020;9(6):541. 4. Mata R, Figueroa M, Navarrete A, Rivero-Cruz I. Chemistry and biology of selected Mexican medicinal plants. Prog Chem Org Nat Prod 2019;108:1-142.

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41. Egamberdieva D, Wirth S, Behrendt U, Ahmad P, Berg G. Antimicrobial activity of medicinal plants correlates with the proportion of antagonistic endophytes. Front Microbiol 2017;9:199. 42. Manandhar S, Luitel S, Dahal RK. In vitro antimicrobial activity of some medicinal plants against human pathogenic bacteria. J Trop Med 2019;2019:1-5. 43. Ochoa PA, Marin MJ, Rivero BD, Saborít A. Caracterización física, físico-química y química de extractos totales de hojas frescas de Petiveria alliacea L. con acción antimicrobiana. Rev Mex Cienc Farm 2013;44(1):52-59. 44. Zheng X, Wang W, Piao H, Xu W, Shi H, Zhao C. The genus Gnaphalium L.(Compositae): phytochemical and pharmacological characteristics. Molecules 2013;18(7):8298-8318. 45. Agata I, Hatano T, Nakaya Y, Sugaya T, Nishibe S, Yoshida T, et al. Tannins and Related Polyphenols of Euphorbiaceous Plants. VIII. Eumaculin A and Eusupinin A, and Accompanying Polyphenols from Euphorbia maculata L. and E. Supina RAFIN. Chem Pharm Bull 1991;39(4):881-883. 46. Liu S, Jiang DS, Li L. Research on the total flavonoids' content and antioxidant activity of Euphorbia maculata and Euphorbia humifusae. J Hunan Agric Univ 2007;33(3):287. 47. Elmore CD, Paul RN. Phenolic deposits and kranz syndrome in leaf tissues of spotted (Euphorbia maculata) and prostrate (Euphorbia supina) spurge. Weed Sci 1983;31:131-136. 48. León MG, Osorio FMDR, Torrenegra ME, Gil GJ. Extracción, caracterización y actividad antioxidante del aceite esencial de Plectranthus amboinicus L. Rev Cubana Farm 2015;49(4):708-718. 49. Luyen BTT, Tai BH, Thao NP, Lee SH, Jang HD, Lee YM, et al. Evaluation of the anti-osteoporosis and antioxidant activities of phenolic compounds from Euphorbia maculata. J Korean Soc Appl Bi 2014;57(5):573-579. 50. Basma AA, Zakaria Z, Latha YL, Sasidharan S. Antioxidant activity and phytochemical screening of the methanol extracts of Euphorbia hirta L. Asian Pac J Trop Med 2011;4(5):386-390. 51. Upadhyay A, Chattopadhyay P, Goyary D, Mazumder PM, Veer V. Euphorbia hirta accelerates fibroblast proliferation and Smad-mediated collagen production in rat excision wound. Pharmacogn Mag 2014;10(39):534-542. 52. Zhang L, Wang C, Meng Q, Tian Q, Niu Y, Niu W. Phytochemicals of Euphorbia lathyris L. and their antioxidant activities. Molecules 2017;22(8):1335.

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CONC 0.1 0.5 1 2 3

Table 3: Antimicrobial activity of E1 and E2 extracts against Gram-positive and negative bacteria Gram positive Gram negative S. aureus L. monocytogenes E. coli S. typhimurium E1 E2 E1 E2 E1 E2 E1 E2 D Ca C Ba A Aa A 8.43±0.42 ª 6.10±0.52 7.50±0.20 ª 4.32±0.31 3.00±0.70 ª 2.30±0.21 2.50±0.15 ª 2.50±0.12Aª 12.10±0.61Eb 10.13±0.42Db 9.50±0.36CDb 8.11±0.43Cb 5.50±0.70Bb 4.20±0.32ABb 3.50±0.20Ab 3.50±0.14Ab 16.00±0.32Fc 14.00±0.36Ec 13.24±0.43Dc 10.34±0.41Cc 8.50±0.70Bc 8.10±0.34Bc 5.52±0.40Ac 6.52±0.22Ac 16.22±0.28Ec 14.10±0.51Dc 13.53±0.38Dc 10.40±0.36Cc 8.55±0.70Bc 8.30±0.41Bc 5.54±0.32Ac 6.54±0.32Ac 16.31±0.53Ec 14.23±0.39Dc 13.56±0.41Dc 10.48±0.38Cc 8.57±0.70Bc 8.35±0.42Bc 5.56±0.45Ac 6.55±0.31Ac

CONC= concentration of extracts (mg mL-1); E1= Gnaphalium oxyphyllum; E2= Euphorbia maculata; data expressed in mm of inhibition halo. Different uppercase literal indicates significant difference between data in the same row and different lowercase literal indicates significant difference between data in the same column (P<0.05).

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https://doi.org/10.22319/rmcp.v13i4.5958 Article

Effects of acid whey on the fermentative chemical quality and aerobic stability of rehydrated corn grain silage

Ediane Zanin a* Egon Henrique Horst b Caio Abércio Da Silva a Valter Harry Bumbieris Junior a

a

State University of Londrina. Department of Animal Science, Londrina, Paraná, Brazil, 86057-970. b

Midwestern Paraná State University. Department of Veterinary Medicine, Guarapuava, Paraná, Brazil.

* Corresponding author: ediane.z@hotmail.com

Abstract: The objective was to evaluate the fermentative, chemical characteristics and aerobic stability of corn grain silages rehydrated with whey fluid (WF) or whey powder (WP) and water, with or without the addition of inoculant (I). The corn grain was ground and hydrated adding water without chlorine and/or whey to reach 35 % humidity and stored in silos of 4.36 kg. After 45 d of fermentation, samples of the silages were submitted to chemical-fermentative analyses in opening of silos and 240 h of exposure to air. The aerobic stability of the silages was evaluating during 240 h has considered to the loss when the temperature of the ensiled mass exceeded the ambient temperature by 2 °C. A reduction in the acid detergent fiber (ADF) and lignin content of the silages was observed with the use of WF and WP. The levels of ammoniacal nitrogen (NH3-N) were the lowest for WF and WP (0.7 and 0.9 g/kg TN) and pH was 4.31 for WF after 240 h of aerobic exposure. The use of inoculants provided higher levels of Ash, ether extract (EE), and low buffering capacity (BC), in addition to reductions in ADF levels. The inoculated silages showed higher levels of NH3-N and pH after 240 h. The silage of corn grains rehydrated with WF provided ideal pH values, low NH3-N content, reduced levels of ADF and lignin, and improved aerobic stability. In addition to being a sustainable

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alternative, the use of fluid whey to rehydrate corn grains adds nutritional value and improves silage fermentation. Key words: Acid whey, Byproduct, Corn grain, Inoculant, Silage, Sustainability.

Received: 04/03/2021 Accepted: 13/05/2022

Introduction Corn grain is one of the most used energy ingredients in animal feed; furthermore, it can also be subjected to rehydration to be stored as silage. Rehydrated corn grain silage is a strategy used to guarantee the availability of feeds throughout the year(1) decrease logistical costs(2), and minimize the effects of fluctuations in the price of this commodity(3). The process of milling the dry corn grain and its subsequent rehydration also aims to increase its digestibility, reflecting positively on animal performance(4). In particular, these resources are appropriate since the corn used in most countries is characterized as flint, which has lower digestibility. Associated with rehydration, the fermentation of grain corn is an interesting process; dry grain is not suitable for ensiling due to its low moisture and sugar content, which results in limited production of total acids(5). Thus, rehydration, commonly conducted with water and aimed at reaching final levels between 35 to 37 % humidity(6,7), is a practical application. The use of a liquid source with low added value or one that has polluting but non-toxic characteristics can also be used for the rehydration of dry grain corn. Byproducts such as acid whey fluid, which have considerable concentrations of lactic acid bacteria and lactose(8) as well as recognized nutritional value, constitute a suitable example for this purpose, with advantages given to the supply of more nutrients to the silage(9) and an appropriate final destination for this byproduct. In order to improve fermentation, reduce nutrient losses, and inhibit the growth of undesirable microorganisms(5,9), microbial inoculants composed of homofermentative and heterofermentative bacteria are also incorporated into the ensiled mass, which can prolong the aerobic stability of moist and rehydrated corn grain silages(7,10). Given this context, our hypothesis was that the composition of whey fluid and its microbiological

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capacity promotes the improvement of silage quality, reduces the use of water for rehydration of grains, and contributes to an appropriate destination for this byproduct. For whey powder, in addition to considering the nutrient load of its composition as a liquid source for rehydration, it is available on a commercial scale for purchase. Finally, the addition of inoculants may help these two liquid sources to improve the fermentative quality and aerobic stability of the silage. Thus, the objective of the present study was to evaluate the effects of rehydration of corn grains with acid whey or whey powder and water, with or without the addition of an inoculant, on the chemical, fermentative characteristics and aerobic stability of the silages.

Material and methods Corn grain and preparation of silage

The corn grains were obtained from the storage silos of Cooperativa Agropecuária Cocamar®, Londrina, Paraná, Brazil, and their genetic identity is not known. These grains were initially processed in a hammer mill to reach an average particle size of 1.5 mm, and submitted to moisture content evaluation according to the methodology described in AOAC(11), with an average value of 117 g/kg dry matter (DM). The acid whey was obtained from the dairy company Volpato®, in the city of Arapongas, Paraná, Brazil, during the processing of milk for the production of derivatives and was used shortly thereafter in natura to rehydrate the grains. The powdered whey used was purchased from Cooperativa Cativa®, in the city of Londrina, Paraná, Brazil. The corn grain was ground and subjected to hydration according to each treatment by adding water without chlorine and/or whey to reach 35 % humidity, with or without the addition of an inoculant defining five products, which were incorporated into the dry corn grain corresponding to the experimental treatments: Corn grain silage rehydrated with water (CON); Corn grain silage rehydrated with whey fluid (SWF); Corn grain silage rehydrated with fluid whey, plus inoculant (SWF + I); Corn grain silage rehydrated with water-reconstituted whey powder (SWP); and Corn grain silage rehydrated with powdered whey reconstituted with water, plus inoculant (SWP + I). The microbial inoculant added to the mass to be ensiled was previously diluted to 2.5 mL of the product to 7 L of water without chlorine and/or whey for each 20 kg of ground corn and manually homogenized. The inoculant used was Biotrato SLO® (SLO Biotecnologia & Agropecuária, Cambé, Paraná, Brazil) which consists of Propionibacterium acidipropionici, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus acidilactici, Enterococcus faecium, Lactobacillus buchneri, and Lactbacillus curvatus at a concentration of 70×109 UFC/g and 8 % of cellulolytic enzymes.

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Once hydrated, the mass of each of the five products was stored in six polyethylene silos with a capacity of 4 L each, determining units with an initial average weight of 4.36 ± 0.17 kg. Compaction was carried out manually, with an average specific density of 1,020 ± 0.04 kg natural matter (NM)/m3. All silos were sealed with a lid and appropriate plastic tape and stored in a dry and ventilated place for 45 d until the opening date, when they reached a final weight of 4.28 ± 0.20 kg. The experimental design was completely randomized with five treatments and six replications corresponding to each silo.

Chemical analysis

Samples of corn grain prior to ensiling (883 g DM/kg in natural matter (NM), 92.7 g crude protein (CP)/kg DM, 11.5 g Ash/kg DM, 31.8 g ether extract (EE)/kg DM, 126.2 g NDF/kg DM, 25.8 g ADF/kg DM, and 11.3 g lignin/kg DM) and the ensiled mass after opening the silos (Table 1) were evaluated according methodologies to AOAC(11), and neutral detergent fiber (NDF) assayed with a heat stable alpha amylase and sodium sulphite (aNDF), acid detergent fiber (ADF) and lignin (lignin (sa)) using sulphuric acid and corrected for ash were evaluated according to the methodology described by Van Soest et al(12). The values of total digestible nutrients (TDN) were calculated according to Sniffen et al(13), total carbohydrates (TCHO) according to the equation proposed by Chandler(14), and non-fibrous carbohydrates (NFC) according to Hall(15). The composition of fluid whey before silage was: 60 g DM/kg NM, 865 g CP/kg DM, 3.40 g Ash/kg DM, 3.50 g EE/kg DM, pH 6.30, and acidity of 0.13 for lactic acid. The whey powder showed the following characteristics: 970 g DM/kg NM, 110 g CP/kg DM, 60 g Ash/kg DM, 15 g EE/kg DM, pH 6.30-6.80, and acidity of 0.13 for lactic acid. These analyses followed the procedures described by Zenebon et al(16). Samples of the silages for each treatment were collected when the silos were opened to determine the chemicalfermentative composition using a near-infrared spectroscopy system (NIRS DS2500; Foss, Denmark) (Table 2) from the 3rlab® laboratory (Chapecó, Santa Catarina, Brazil). The analysis results shown in Table 2 are only exploratory and descriptive, since it is a characterization of silages without the application of statistical analysis.

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Table 2: Chemical-fermentative composition of rehydrated corn grain silages determined by the NIRS system Treatment 1 Variables 2 (g/kg DM)

SWF Without 609.4 390.6 98.5 504.8 97.9 00.6 02.2 06.2 28.6 74.9 69.3 04.9 64.9 50.5

With 655.0 344.0 100.1 520.6 99.2 00.9 02.9 09.1 31.4 87.9 82.1 05.1 58.5 49.0

SWP Without 641.5 358.5 100.7 504.0 100.1 00.7 02.8 06.6 26.0 85.5 78.7 04.6 53.7 50.4

With 621.7 378.3 95.1 509.5 94.4 00.7 02.3 07.5 27.4 76.6 69.4 04.7 61.8 48.3

Starch 698.8 Starch, g/kg NFC 919.7 Digestibility of rumen starch, g/kg 466.4 starch 0h

703.9 905.1 394.1

688.9 905.0 417.6

704.7 916.4 354.0

699.9 904.4 458.1

Digestibility of rumen starch, g/kg 701.9 starch 7h

709.2

701.6

686.4

740.7

Lipids Ash Calcium Phosphorus Potassium Magnesium Sulfur Lactic acid Acetic acid Protein equivalent of NH3-N NH3-N, g/kg of CP pH Kd of starch (using 3.7 h) %h

33.1 17.9 00.5 02.0 03.7 00.8 00.9 24.8 05.0 03.6 37.0 3.89 17.19

35.3 18.3 00.5 02.0 03.9 00.9 00.9 26.3 03.6 03.1 31.3 4.13 16.96

29.7 17.8 00.5 02.2 04.0 00.9 00.9 22.2 03.0 02.9 28.8 4.23 16.29

38.1 18.6 00.5 02.2 03.8 00.9 00.9 24.8 04.2 03.8 40.1 4.15 18.86

DM, g/kg NM Moisture CP Protein soluble g/kg CP Protein available ADIP NDIP ADIP g/kg CP ADF NDF aFDNmo Lignin Lignin, g/kg NDF Sugars (carbohydrates soluble in water)

CON 652.9 347.1 97.2 496.0 96.2 01.0 02.8 10.0 31.3 91.0 85.4 05.1 56.3 44.5

36.2 18.5 00.5 02.2 03.9 00.9 00.9 21.5 03.2 03.3 33.6 4.78 17.00

1

Treatment: CON= corn grain silage rehydrated with water; SWF= corn grain silage rehydrated with whey fluid; SWF + I= corn grain silage rehydrated with fluid whey, plus inoculant; SWP= corn grain silage rehydrated with water-reconstituted whey powder; SWP + I= corn grain silage rehydrated with powdered whey reconstituted with water, plus inoculant.

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Variables: DM= Dry matter (g/kg of natural matter); ADIP= acid detergent insoluble protein; NDIP= Neutral detergent insoluble protein; ADF= acid detergent fiber; NDF= neutral detergent fiber; aNDFmo= neutral detergent fiber with amylase and expressed excluding residual ash; NH3-N= ammoniacal nitrogen. Kd= fractional rate of degradation. Analyses determined by the laboratory 3rLab.

Fermentative analysis and aerobic stability

To evaluate the fermentation profile of the silages, buffering capacity (BC) and ammoniacal nitrogen (NH3-N) were determined according to the methodology of Playne and McDonald(17) at the opening of the silos and after 240 h of exposure to air (Table 3). The aerobic stability of the silages was determined using 3 kg of the ensiled mass, homogenized and deposited in the silos according to each treatment, which remained in a closed room exposed on a bench at ambient temperature for 240 h. The ambient (25.43 ± 2.38 °C) and silage temperatures were obtained every 12 h using a digital thermometer (TP101 Xtrad 145 mm; Shenzhen Handsome Techn., Guangdong City, China). The thermometer rod was inserted 10 cm deep in the center of the mass for this measurement. The loss aerobic stability was considered when the temperature of the ensiled mass exceeded the ambient temperature by 2 °C(18). The pH of the ensiled mass during aerobic exposure was measured using a potentiometer (AZ Temp Meter; AZ Instrument Corp., Taichung City, Taiwan) according to the methodology of Phillip and Fellner(19). These analyses were delineated in subdivided plots, where the main portion was the treatment and the subplot was the time of aerobic exposure.

Statistical analysis

The data were analyzed according to a completely randomized design using the General Linear Model (PROC GLM) procedure of SAS (see 9.2; SAS Inst. Inc., Cary, NC, USA). Contrasts were used to verify scientific hypotheses using the CONTRAST command, making it possible to compare the impacts of using fluid whey and powdered whey on the investigated variables, as well as comparing the effects of using inoculants with these reconstituting agents. The proposed model was as follows: 𝐘𝐢𝐣𝐤𝐥= µ + α𝑖 + β𝑗 + δ𝑘 + (αβ)𝑖𝑗 + (αδ)𝑖𝑘 + (βδ)𝑗𝑘 + ξ𝑖𝑗𝑘𝑙

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where: Yijkl = observed value regarding level i of factor A, combined with level j of factor B and level k of factor C, in repetition l; µ = overall average; αi = level effect i of factor A; βj = level effect j of factor B; δk = level effect k of factor C; (αβ)ij = interaction effect of A with B; (αδ)ik = interaction effect of A with C; (βδ)jk = interaction effect of B with C; ξijkl = experimental error associated with Yijkl and considered independent and identically distributed, with distribution N(0, σ2). The (αβδ)ijk interaction was initially tested, but due to its low magnitude, it was removed from the statistical model. The results are presented as means ± standard deviation, as well as the corresponding standard error. Significance was declared at P<0.01 and P <0.05, and trends were discussed when P<0.10. For the aerobic stability pH data, regression analysis (α= 0.05) was performed to split the time interaction per treatment in the RStudio statistical program (v. 3.6.0; 2019).

Results Quality of chemical composition

The ADF contents of the corn grain silage rehydrated with SWP differed significantly (P<0.01) between treatments (Table 1), with a lower observed ADF value of 14.7 g/kg DM for the silage without inoculation, followed by SWP + I with 21.0 g/kg of DM. There was a significant interaction (P<0.01) between the liquid sources used, in which the SWP silages had the lowest ADF values. The lignin content differed significantly (P<0.10) between the SWF silages and other treatments, with the highest lignin content observed for SWF + I at 7.8 g/kg DM, in addition to presenting a significant interaction (P<0.05) between SWF and SWP, with a lower observed lignin content for SWF silage (Table 1). The EE value differed (P<0.05) for the silages where SWF was used as a liquid source to rehydrate corn kernels, with a higher content of this nutrient for SWF + I. There was a significant interaction (P<0.01) between the liquid sources of SWF and SWP used to rehydrate the grains (Table 1), in which the SWF source showed an increase in EE compared to that in the silages for the control and SWP treatments.

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For levels of Ash a significance difference was observed for the sources of treatments (P<0.05), addition of inoculants (P<0.01) and interaction between the liquid sources (P<0.05). The corn grain silages that were rehydrated with SWP had the highest levels of Ash compared to those in the other sources of rehydration, and the addition of inoculant in the SWP and SWF treatments provided the highest levels of this component (Table 1). The DM, CP, NDF, TCHO, and NFC values of the silages were not influenced (P>0.05).by the liquid sources used or by the addition of inoculants.

Fermentation profile

The NH3-N of the silages after 240 h of exposure to air from the ensiled mass were significantly different between silages (Table 3), with and without inoculation (P<0.05), for treatments with the liquid sources SWP and SWF (P<0.01), and there was significant interaction between the sources (P<0.01). The silages with the addition of inoculant and the control treatment showed the highest levels of NH3-N at 240 h. The pH values evaluated in the silages showed a significant difference both in the opening of the silos and after 240 h of exposure to air (Table 3). For the pH values during the opening of the silos, a significant difference was observed between the silages with the liquid sources of SWF (P<0.05) and SWP (P<0.01) without the addition of inoculant, with a lower pH value of 4.26 for SWF. After 240 h of exposure, the pH values differed between the SWF and SWP treatments (P<0.01; 0.10, respectively), with a lower observed value of 4.31 for the WF and a tendency towards a lower pH value for the silage with SWP when compared to those in the control (Table 3). For the addition of inoculant, corn grain silages rehydrated with SWP showed a higher pH value compared to that in the control and SWF treatments (P<0.01). In addition, a significant interaction (P<0.01) was observed between the silages with SWP and SWF, in which the lowest pH values were observed for SWF, regardless of the addition of inoculant. The inoculated silages (P<0.05) that were independent of the liquid sources used showed the lowest BC values compared to those of the SWF and control sources.

Aerobic stability

The loss of aerobic stability differed significantly (P<0.05) between the corn grain silages rehydrated with water and SWF + I and those from the other silages, which broke the stability after 84 h of oxygen exposure, showing greater stability after opening the silos (Table 4). The time required for the ensiled mass of the treatments with water and SWF

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+ I to increase the temperature of the ensiled mass by 2 °C above the ambient temperature was significantly shorter (76 and 75 h, respectively) than that in the other treatments (P<0.05), a behavior that characterizes the loss of aerobic stability and onset deterioration of silage. The time to reach the maximum temperature of the ensiled mass, except for the silage made with corn grain rehydrated with SWF (P<0.05), was longer than 200 h, with a significant difference between treatments in the final pH of the silages exposed to air for 240 h (Table 4, Figure 1). Table 4: Aerobic stability parameters of corn grain silages submitted to rehydration Treatments Parameters

CON

SWP

SWF

SWP+I

SWF+I

Aerobic stability, hour Time to maximum temperature, hour pH-240 h

76b

84a

84a

84a

75b

Pvalue 0.0007

234a

234a

104b

204a

201a

0.0001

5.50±1.2ac 7.35±1.2b 4.40±0.3c 6.71±1.6ab 6.88±1.4ab 0.0006

CON= corn grain silage rehydrated with water; SWF= corn grain silage rehydrated with whey fluid; SWF + I= corn grain silage rehydrated with fluid whey, plus inoculant; SWP= corn grain silage rehydrated with water-reconstituted whey powder; SWP + I= corn grain silage rehydrated with powdered whey reconstituted with water, plus inoculant.

Figure 1: pH values after aerobic exposure of rehydrated corn grain silages

CON: 6.06±1.18, Ŷ=4.23+0.01x+0.0002x2-0.000001x3, R²=0.72, P=0.0011, CV=9.54; SWP: 5.38±1.18, Ŷ=4.52-0.0042x+0.0001x², R²=0.88, P=0.028, CV=21.88; SWF: 4.31±0.27, P=0.123, CV=6.26; SWP+I: 6.37±1.60, Ŷ=4.51-0.024x+0.0006x²-0.000002x³, R²=0.92, P=0.001, CV=25.17; SWP+I: 5.11±1.40, Ŷ=4.57-0.011+0.0001x², R²=0.89, P=0.005, CV=27.35.

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Discussion Quality of chemical composition

It was observed that the inoculant contributed to ADF reduction when compared to that in the control treatment. These lower ADF values may be related to the dilution of the fiber content evaluated(20) and to the cellulolytic enzymes present in the inoculants, which degrade the fiber and alter the three-dimensional structure of the grain cell wall(9,21), determining positive results for the digestibility of this food. The ADF and NDF results for the silages that used SWF were in agreement with Rezende et al(9), who also observed small reductions in the ADF levels in treatments with acid whey associated with inoculants, and in general observed lower NDF content for the treatments with rehydration of the corn grain with acid whey, without adding inoculant. For these variables and the liquid fluid whey source, it is still unclear how reductions in the levels of these nutrients occur. However, the whey acidity contributes to potentiating fermentation, which, together with the acidic hydrolysis of hemicellulose, can reduce the levels of these fibers. The levels of lignin in all silages regardless of treatment could be considered low, since the content obtained in the corn kernels before rehydration was 11.3 g/kg DM. This reduction can occur after ensiling, due to the acid hydrolysis process of fibers and acidification that will weaken the complex lignin molecules(20) and thus obtain lower values of this component. However, the action of fluid and whey sources on reductions in lignin content in rehydrated corn grain silages requires more specific studies in terms of fiber structures, since this nutrient, as well as NDF and ADF, are directly related to feed digestibility. The lignin levels determined in the present study for the control treatment differed from those obtained by Oliveira et al(1), who found values for lignin between 13.7 and 14 g/kg DM in corn grain silages rehydrated with water plus enzymatic additive. One factor that may explain this variation is that the levels of lignin present in rehydrated grain silages can vary widely due to the diversity of available corn cultivars, phase, and agronomic management. The addition of the inoculant may have contributed to a higher value of content of EE for SWF + I, as this increase was also observed by Tres et al(22) and Arcari et al(3) in rehydrated corn grain silages inoculated with L. buchneri. The SWF source increased in EE compared to that in the silages for the control and SWP treatments. This increase can be explained by the microbiological potential and availability of nutrients that the SWF presents as a fresh product(8) at the time of rehydration, since there was an adjustment in

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the DM base of the tested liquid sources, for homogeneous distribution of the presented nutrient load. The EE levels determined in the present study for the control treatment were similar to the results obtained by Oliveira et al(1), Mombach et al(23), and Tres et al(22) who identified values of 53.1, 45.2, and 39.6 g EE/kg DM, respectively, in samples of grain silages rehydrated with water without adding inoculants. The use of the SWP liquid source may have contributed to higher observed levels of Ash, due to its composition having 60 g Ash/kg DM, while SWF had 3.40 g Ash/kg DM. The DM, CP, NDF, TCHO, and NFC values of the silages were similar to the results observed by Rezende et al(9) in corn grain silages rehydrated with whey, and by Da Silva et al(10) and Oliveira et al(1), who evaluated corn grain silages rehydrated with water, plus inoculants, and their effects on nutrients.

Fermentation profile

The corn grain silage rehydrated with SWF showed superior fermentation quality compared to that of the silage with SWP and water, with the lowest value of NH3-N at 0.7 g/kg TN after exposure to air for 240 h. According to McDonald et al(20), as the pH rapidly decreases in the silage, the protein fraction is preserved and the concentrations of NH3-N will be lower, thus characterizing an adequate fermentation. An increase in NH3N concentrations in silages may be related to the proteolytic activity of microorganisms from the epiphyte and/or inoculated population during silage, which will affect the decomposition of the prolamine of the cereals, as well as the digestibility of starch, and as a consequence there will be higher levels of protein available for NH3-N production(21). In the present study, the use of fluid whey may have contributed to a rapid reduction in pH and preservation of the protein fraction during storage and exposure to air due to the low proteolytic activity of lactic acid bacteria(9,24) present in the microbiological profile of whey(8). Conversely, the inclusion of the inoculant may have altered the profile of the bacterial population of the silo(10,24) and consequently reduced the prolamine content of the inoculated silages during exposure to air, as higher concentrations of soluble protein and NH3-N were observed at the opening of the silos (Table 2) and after 240 h of exposure to air (Table 3), respectively, in the silages in which the inoculants were incorporated, which may indicate greater proteolysis(5,21). This hypothesis was supported by the results obtained by others(10), who, when evaluating the addition of L. buchneri in moist and reconstituted corn grain silages, found that the bacterial profile of the silage was modified, increasing the concentration of lactic acid bacteria as the silage storage period increased, with a higher concentration of NH3-N after 90 d of storage in both types of silages. The lower pH value of 4.26 for SWF in opening silos reinforced the hypothesis that the microbial profile of fluid whey preserved the protein in corn kernels due to its low proteolytic activity of lactic acid bacteria, in addition to contributing to a rapid drop in 953


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pH after ensiling. It is known that the starch-protein matrix of corn grains presents greater degradation due to microbial activity than from simple solubilization of final fermentation products, such as acids(21). The values of pH observed after 240 of exposure to air showed the action of source WF in silages and inoculants. The modification of the bacterial profile in silos can be due to the addition of inoculants with L. buchneri, that can creating ecological niches and to benefit proteolytic bacteria in convert lactic acid into acetic acid, consequently increasing the pH during the fermentation process in this type of environmental(21,24). This fact observed in the inoculated silages, which presented higher pH values after 240 h of exposure to air (Table 3). The pH of an ensiled sample is a measure of its acidity, which corresponds to the sum of the concentrations of acids present in the ensiled mass, whose main acids were acetic, propionic, and lactic acid. Lactic acid is produced by lactic acid bacteria and has a higher concentration, contributing more to the decline in pH during fermentation(25). Some authors(12,20) consider pH values from 3.8 to 4.2 as ideal; however, the pH itself is not able to inhibit the action of undesirable microorganisms, which are also dependent on the speed of pH reduction, observed through the BC of the silages. The concentration of lactic acid in SWF and SWF + I were more expressive in samples obtained for the silages at the opening of the silos (Table 2), which presented the lowest pH values after 240 h of exposure to air. The corn grain silage rehydrated with SWF maintained the pH at the opening and after the exposure to air, which was close to the values considered ideal for fermentative quality of the silage. This fact may also be due to the composition of the serum, which included sugars that were used as substrates by the lactic acid bacteria in the fermentation process, contributing to a rapid drop in pH. Considerable values were also observed for SWP, unlike water, which presented higher pH values at the opening of the silos. The results obtained for the control treatment were in accordance with those from Oliveira et al(1), who found pH values of 4.25 at the opening of the silo and 6.50 on the fifth day of exposure to air in corn silage rehydrated with water. The inoculated silages that were independent of the liquid sources used showed the lowest BC values compared to those of the SWF and control sources (Table 3), which could be characterized as an important action of the inoculant in terms of fermentation right after ensiling, with a direct effect of inoculation on the speed of pH reduction and consequent improvement in silage BC. According to Jobim et al(26), this measurement depends on the composition of the plant regarding the contents of CP, inorganic ions, and the combination of organic acids and their salts, in addition to providing information on the speed of pH reduction, which must be low to facilitate this acidification during fermentation, culminating in improved conservation and silage quality. In the present study, these characteristics (Table 2) could be considered for a positive BC of rehydrated corn grain silages. 954


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Aerobic stability

The loss of aerobic stability in corn grain silages rehydrated with water and whey occurred after 55 h(9); however, in this work it was obtained greater aerobic stability with values of 75 to 84 h of exposure without losing stability, which characterizes a positive effect of rehydration. One of the factors that can influence the deterioration of the silages is the humidity of the ensiled mass, as there is a favoring of the medium so that undesirable microorganisms develop when the moisture content and acetic acid concentration are high(9). In the present work, the moisture content was between the recommended intervals of 35 to 37 for a high quality of rehydrated grain silage (6,7), and the concentrations of acetic acid were similar between silages. In addition, the use of mandatory heterofermentative bacteria in inoculants, such as L. buchneri, increases the aerobic stability of moist and rehydrated corn grain silages(5,7,27), which justifies the results achieved for the loss of stability of the silages inoculated at 84 h of exposure to air. Regarding the variable time to reach the maximum temperature of the ensiled mass, as the heating rate was obtained through the maximum temperature records divided by the time to reach the maximum temperature, it was observed that the maximum temperature was reached from the eighth to the ninth day of exposure to air, except for the SWF, which reached the maximum temperature on the fourth day, although the maximum temperature values reached for this silage were low. In general, the rehydrated corn grain silages, regardless of the liquid source used for rehydrating the grains, were efficient in the time to reach the maximum temperature in this process. This justification may be related to the fermentative profile of these silages and the increased effect of the bacterial inoculant used, which improves the aerobic stability of the silages. Despite SWF reaching the maximum temperature before the other silages, with a final pH value close to the ideal (4.40) in terms of quality, after 240 h of exposure to air, the silage was still superior in qualitative terms and fermentations to those in the other silages. These results were similar to those obtained by Rezende et al(9), who observed that after 40 h of aerobic exposure, rehydrated grain silages increased the temperature, regardless of the liquid used for rehydration. Although the loss of aerobic stability for the corn grain silage rehydrated with SWF occurred at 84 h of exposure, and it was observed that these silages, when challenged with the time of exposure to air, also presented average pH values lower than those of the silages made with the other sources of rehydration (Figure1). This characteristic of maintaining stability after opening the silos can be explained by the microbial profile of the liquid source used for rehydration, which has low proteolytic activity of lactic acid bacteria(24) and acceleration of grain fermentation by a rapid fall in pH after ensiling due to the presence of lactic acid (Table 2).

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The treatments that received an increase in microbial inoculant also showed a final pH value, in aerobic exposure, lower than that of the SWP, but not lower than that of the SWF without the inoculant and the control silage (Table 4). This shows that although the inoculation was efficient at prolonging the time to reach the heating rate of the silages, the pH values could still be considered above the ideal values, such as the value obtained for SWF. A significant increase in pH was also observed for the treatments with water, SWP, and SWP + I after 60 h of exposure (Figure 1). The pH values of the silages increased with exposure to air, due to the action of yeasts that can use lactate as carbon and energy sources, favoring an environment for the growth of molds and aerobic bacteria, which are responsible for the deterioration of silage(20). The pH values for the corn grain silage rehydrated with SWF remained more stable during the 240 h of exposure and were close to the values at the opening of the silo, when compared with those of the other treatments. This behavior can explain the significant difference in the final pH value in the aerobic exposure of this treatment. However, corn grain silage rehydrated with water, which also had a lower pH value at the end of the exposure, did not show stable behavior during the exposure. Conversely, the corn grain silage rehydrated with SWP showed a constant pH increase during the exposure and reached a higher final pH value (7.35). These results are similar to those observed by Oliveira et al(1), who found that the pH behavior of corn grain silages rehydrated with water showed a constant increase from 48 h to 120 h of exposure to air. The final pH of the silages is influenced by several factors, but according to Kung Junior et al(25), is more related to the concentration of lactic acid and TC in ensiled food, as shown in Table 2, in which the highest concentrations of lactic acid at the opening of the silos were for SWF, followed by the inoculated silages. A previous study(10) showed that increased aerobic stability with increased storage period for rehydrated and moist corn grain silages inoculated with L. buchneri are due to the accumulation of fermentation products such as acetic and propionic acid, which have antifungal properties. The corn grain silages rehydrated with SWF were superior according to the quality parameters evaluated in the present study. In addition, the use of fluid whey in the rehydration of the grain can be considered as an alternative liquid source to water that provides conservation of ensiled food and an appropriate destination for this byproduct for preservation of the environment. Although whey powder showed improvements in the chemical and fermentative quality compared to those of the commonly used liquid source, it may not be an appropriate alternative option for rehydrating grains, as it requires water to dilute the powder before rehydrating the grain and distances itself from the production of food in a sustainable way. Another fact that may disadvantage the use of this source is the process becoming more expensive, since the product is acquired through purchase and the production of whey powder involves several processes that add value to the product to be sold.

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Conclusions and implications The use of fluid whey presents itself as a suitable alternative to the use of water for rehydration of corn grains for silage, because in addition to being a sustainable alternative and that to preserve the environment, it also justifies its use by adding chemical, fermentative improvements and aerobic stability to the silage. In addition, these data suggest that more specific research is needed regarding the action of whey on the reduction of rehydrated corn grain silage fibers and the microbiological potential of the product to be used as a possible biological additive in the conservation of these silages.

Acknowledgments

The authors would like to thank the postgraduate program in Animal Science of the State University of Londrina (UEL, Londrina, Brazil) and the Coordination of Improvement of Higher Education Personnel (CAPES, Brasília, Brazil) for granting by scholarship.

Conflicts of interest

The authors declare no conflict of interest. Literature cited: 1. Oliveira ER, Takiya SC, Del Valle AT, et al. Effects of exogenous amylolytic enzymes on fermentation, nutritive value, and in vivo digestibility of rehydrated corn silage. Anim Feed Sci Technol 2019;251:86-95. 2. Ferraretto LF, Fredin SM, Shaver RD. Influence of ensiling, exogenous protease addition, and bacterial inoculation on fermentation profile, nitrogen fractions, and ruminal in vitro starch digestibility in rehydrated and high-moisture corn. J Dairy Sci 2015;98:7318-7327. 3. Arcari MA, Martins CMMR, Tomazi T, Gonçalves JL, Santos MV. Effect of substituting dry corn with rehydrated ensiled corn on dairy cow milk yield and nutrient digestibility. Anim Feed Sci Technol 2016;221:167-173. 4. Ferraretto LF, Crump PM, Shaver RD. Effect of cereal grain type and corn grain harvesting and processing methods on intake, digestion, and milk production by dairy cows through a meta-analysis. J Dairy Sci 2013;96:533-550.

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5. Kung Junior L, Schmidt RJ, Ebling TE, Hu W. The effect of Lactobacillus buchneri 40788 on the fermentation and aerobic stability of ground and whole high moisture corn. J Dairy Sci 2007;90:2309-2314. 6. Silva CM, Amaral PNC, Baggio RA, et al. Estabilidade de silagens de grãos úmidos de milho e milho reidratado. Rev Bras Saúde Prod Anim 2016;17:331-343. 7. Da Silva NC, Nascimento CF, Nascimento FA, Resende FD, Daniel JLP, Siqueira GR. Fermentation and aerobic stability of rehydrated corn grain silage treated with different doses of Lactobacillus buchneri or a combination of Lactobacillus plantarum and Pediococcus acidilactici. J Dairy Sci 2018;101:4158-4167. 8. Rektor A, Vatai G. Membrane filtration of Mozzarella whey. Desalination 2004; 162:279-286. 9. Rezende AV, Rabelo SHC, Veiga MR, et al. Rehydration of corn grain with acid whey improves the silage quality. Anim Feed Sci Technol 2014;197:213-221. 10. Da Silva NC, Nascimento FC, Campos AMV, et al. Influence of storage length and inoculation with Lactobacillus buchneri on the fermentation, aerobic stability, and ruminal degradability of high-moisture corn and rehydrated corn grain silage. Anim Feed Sci Technol 2019;251:124–133. 11. AOAC. Association of Official Analytical Chemists. Official methods of analysis. (17th ed.) AOAC Internacional, Arlington, VA: AOAC International, 2000. 12. Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci 1991;4:3583-3597. 13. Sniffen CJ, O'Connor JD, Van Soest PJ, Fox DG, Russell JB. A net carbohydrate and protein system for evaluating cattle diets: II. carbohydrate and protein availability. J Anim Sci 1992;70:3562-3577. 14. Chandler P. Energy prediction of feeds by forage testing explorer. Feedstuffs 1990; 62:1-12. 15. Hall MB. Neutral detergent-soluble carbohydrates nutritional relevance and analysis. A laboratory manual. Gainesville, Florida, EUA, 2000. 16. Zenebon O, Pascuet NS, Tiglea P. Métodos físico-químicos para análise de alimentos, Instituto Adolfo Lutz, São Paulo. 2008;819-877. 17. Playne MJ, McDonald P. The buffering constituents of herbage and of silage. J Sci Food Agric 1966;17:264-268. 18. Taylor CC, Kung Junior L. The effect of Lactobacillus buchneri on fermentation and aerobic stability of high moisture corn in laboratory silos. J Dairy Sci 2002;85:1261532. 958


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19. Phillip LE, Fellner V. Effects of bacterial inoculation of high-moisture ear corn on its aerobic stability, digestion, and utilization for growth by beef steers. J Anim Sci 1992;70:3178-3187. 20. McDonald P, Henderson AR, Heron SJE. The biochemistry of silage. 2 ed. Marlow, UK, 1991. 21. Junges D, Morais G, Spoto MHF et al. Short communication: Influence of various proteolytic sources during fermentation of reconstituted corn grain silages. J Dairy Sci 2017;100:9048-9051. 22. Tres TT, Bueno AVI, Jobim CC, Daniel JLP, Gritti VC. Effect of okara levels on corn grain silage. Rev Bras Zootec 2020;49:e20190184. 23. Mombach MA, Pereira DH, Pina DS, Bolson D, Pedreira BC. Silage of rehydrated corn grain. Arq Bras Med Vet Zootec 2019;71:959-966. 24. Oude Elferink SJWH, Krooneman J, Gottschal JC, Spoelstra SF, Faber F, Driehuis F. Anaerobic conversion of lactic acid to acetic acid and 1,2-propanediol by Lactobacillus buchneri. Appl Environ Microbiol 2001;67:125-132. 25. Kung Junior L, Shaver RD, Grant RJ, Schmidt RJ. Silage review: Interpretation of chemical, microbial, and organoleptic components of silages. J Dairy Sci 2018;101:4020-4033. 26. Jobim CC, Nussio LG, Reis RA, Schmidt P. Avanços metodológicos na avaliação da qualidade da forragem conservada. Rev Bras Zootec 2007;36:101-119. 27. Basso FC, Bernardes TF, Roth APTP, Rabelo CHS, Ruggieri AC, Reis RA. Fermentation and aerobic stability of high-moisture corn silages inoculated with different levels of Lactobacillus buchneri. Rev Bras Zootec 2012;41:2369-2373.

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Table 1: Chemical quality (g/kg DM) of grain corn silages rehydrated with water, powdered whey and fluid whey Treatment 1 SWF Variables 2

Without

With

SEM 3

SWF

SWP

I

SWF × SWP 4

SWP

CON Without

With

DM

633.4 ± 0.38 628.3 ± 0.53 637.0 ± 0.34

629.8 ± 0.68

625.7 ± 1.84

0.97

ns

ns

ns

ns

Ash

11.3 ± 0.25

12.0 ± 0.07

12.7 ± 0.06

0.06

ns

**

*

**

CP

103.0 ± 0.19 104.7 ± 0.42 103.2 ± 0.27

101.3 ± 0.17

104.7 ± 1.74

0.31

ns

ns

ns

ns

EE

37.0 ± 0.55

35.4 ± 0.30

43.6 ± 0.30

0.40

**

ns

ns

*

NDF

122.1 ± 2.07 112.8 ± 0.77 128.6 ± 1.42

137.4 ± 2.21

101.4 ± 0.96

1.44

ns

ns

ns

ns

ADF

23.0 ± 0.46

20.2 ± 0.23

22.1 ± 1.00

14.7 ± 0.75

21.0 ± 0.21

0.27

ns

*

**

*

Lignin

3.4 ± 0.57

3.3 ± 0.27

7.8 ± 0.14

3.4 ± 0.23

4.2 ± 0.31

0.24

***

ns

ns

**

TCHO

847.7 ± 0.77 847.0 ± 0.46 843.2 ± 0.22

851.3 ± 0.36

845.9 ± 2.01

1.04

ns

ns

ns

ns

TDN

812.9 ± 0.02 813.1 ± 0.01 812.8 ± 0.05

813.3 ± 0.04

813.0 ± 0.01

0.02

ns

***

***

**

NFC

718.8 ± 2.64 734.2 ± 1.01 714.6 ± 1.29

714.0 ± 2.39

744.5 ± 2.49

2.12

ns

ns

ns

ns

11.1 ± 0.06 37.3 ± 0.35

11.8 ± 0.05 41.8 ± 0.47

1

Treatment: CON= corn grain silage rehydrated with water; SWF= corn grain silage rehydrated with whey fluid; SWF + I= corn grain silage rehydrated with fluid whey, plus inoculant; SWP= corn grain silage rehydrated with water-reconstituted whey powder; SWP + I= corn grain silage rehydrated with powdered whey reconstituted with water, plus inoculant. 2 DM= Dry matter (g/kg of natural matter); Ash= mineral matter; CP= crude protein; EE= ether extract; NDF= neutral detergent fiber; ADF= acid detergent fiber; TCHO= carbohydrates total; TDN= Total digestible nutrients; NFC= non-fibrous carbohydrates. 3 SEM= standard error of the mean. 4 Interaction “SWF x SWP”. *P<0.01; ** P<0.05; *** P<0.10; ns= no significant.

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Table 3: Fermentative quality (g/kg DM) of grain corn silages rehydrated with water, powdered whey and fluid whey Treatment 1

NH3-N-0 h

0.6 ± 0.04

0.5 ± 0.02

0.4 ± 0.01

0.5 ± 0.009

0.4 ± 0.03

0.02

ns

ns

ns

SWF × SWP 4 ns

NH3-N-240 h

1.9 ± 0.08

0.7 ± 0.01

1.3 ± 0.04

0.9 ± 0.03

2,20 ± 0.08

0.05

*

*

**

*

pH-0 h

4.53 ± 0.07

4.26 ± 0.09

4.43 ± 0.03

4.38 ± 0.14

4.35 ± 0.12

0.10

**

*

ns

ns

pH-240 h

6.13 ± 0.08

4.31 ± 0.11

5.14 ± 0.70

5.44 ± 0.41

6.47 ± 0.18

0.33

*

***

*

*

BC

259.9 ± 1.78 276.8 ± 5.27

266.3 ± 1.73

231.6 ± 3.03

243.4 ± 1.67

3.11

ns

ns

**

ns

Variables

1

CON

2

SWF Without

SWP With

Without

With

SEM

3

SWF

SWP

I

Treatment: CON= corn grain silage rehydrated with water); SWF= corn grain silage rehydrated with whey fluid; SWF + I= corn grain silage rehydrated with fluid whey, plus inoculant; SWP= corn grain silage rehydrated with water-reconstituted whey powder; SWP + I= corn grain silage rehydrated with powdered whey reconstituted with water, plus inoculant. 2 BC= buffering capacity (e.mg/100g DM); NH3-N= ammoniacal nitrogen g/kg of total nitrogen) and pH in opening (0h) and exposure to air (240 h); 3 SEM= standard error of the mean; 4 Interaction “SWF x SWP”. * P<0.01; ** P<0.05; *** P<0.10; ns= no significant.

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https://doi.org/10.22319/rmcp.v13i4.6023 Article

Growth performance and carcass classification of pure Pelibuey and crossbred lambs raised under an intensive production system in a warmhumid climate

Miriam Rosas-Rodríguez a Ricardo Serna-Lagunes b Josafhat Salinas-Ruiz a Julio Miguel Ayala-Rodríguez c Benjamín Alfredo Piña Cárdenas d Juan Salazar-Ortiz a*

a

Colegio de Postgraduados. Campus Córdoba. Carretera Federal Córdoba-Veracruz km 348, Congregación Manuel León, 94946, Amatlán de los Reyes, Veracruz, México. b

Universidad Veracruzana. Facultad de Ciencias Biológicas y Agropecuarias, región Orizaba-Córdoba, Veracruz, México. c

Colegio de Postgraduados. Campus Montecillo-Ganadería. Estado de México, México.

d

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Campo Experimental La Posta. Veracruz, México.

*Corresponding author: salazar@colpos.mx

Abstract: The effect of breed on growth, characteristics, and carcass classification was investigated using 11 Charollais x Pelibuey (ChP) lambs, 10 Dorper x Pelibuey (DP) lambs, and 18 Pelibuey (P) lambs in an intensive production system in a warm-humid climate. A significant effect of genotype (𝑃<0.05) was observed on birth weight (BW), weaning weight (WW), and

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daily weight gain (DWG), all of which were higher in the ChP genotype. ChP and DP lambs reached commercial weight 35 and 23 d earlier, respectively than P lambs. Genotype has a marked influence on the carcass characteristics, affect conformation and classification of carcass. The probability of obtaining a carcass with good conformation and MEX 1 classification (good: MEX 1) is 72 % higher for the ChP genotype than for the P genotype. The loin and leg yields of the ChP genotype were higher than those of the other genotypes. The pH, temperature, and instrumental color of the carcass, meat, and subcutaneous fat were affected by genotype. ChP lambs showed better growth, characteristics, and carcass classification than lambs of the DP and P genotypes. Key words: Lamb, Hair sheep, Carcass, Commercial cuts, Meat.

Received: 15/07/2021 Accepted: 31/01/2022

Introduction In Mexico, sheep production is important due to the high demand for and insufficient domestic production of meat(1). In warm-humid regions, local forage production is favorable, and sheep meat production could be improved year-round to meet domestic demand. In local sheep production systems, including those in warm-humid regions, meat production can be improved by the use of specific technologies and management strategies(2). One of these strategies is the crossing of local breeds such as the Pelibuey breed of sheep with large wool breeds to produce crossbred lambs, since the animals resulting from these crosses present greater daily weight gain(3). The Pelibuey is a medium-sized hair breed and is distributed throughout a large portion of the Mexican territory(4). It is a breed that is used for meat production because it possesses hardiness characteristics that allow it to adapt to different climates(5,6). It has low reproductive seasonality, high prolificacy, and resistance to parasites, although fattening animals have lower growth rates than the traditional wool breeds(7). Due to their specific characteristics and under the conditions of the production systems in use in Mexico, Pelibuey sheep can be used as a maternal breed for crossing with other breeds specialized for meat production(6) to obtain lambs that develop carcasses with better meat conformation. However, the productive performance and carcass characteristics of the Pelibuey breed have been less satisfactory than those of other breeds with better meat conformation(4), and sometimes these carcass

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characteristics are not improved by crossing with certain specialized wool breeds(5). Due to the recent introduction into Mexico of new breeds of sheep with greater specialization for meat production (for example, the Charollais and Dorper breeds) and the possibility that these breeds may be of potential use in crossing with the Pelibuey breed, it is necessary to evaluate the productive performance (birth weight, weaning weight, daily weight gain, and fattening days) and the carcass meat characteristics of lambs that result from crossing with the Pelibuey breed. Standards for the evaluation of the quality of sheep carcass meat vary worldwide. To guide and strengthen the chain of production, processing, marketing, and consumption of sheep meat and to define the quality characteristics of sheep carcasses for national commercialization in Mexico, the Mexican standard for the classification of sheep carcass meat, NMX-FF-106-SCFI-2006, was used. However, there are few reports of its application in the evaluation of sheep carcass meat. Likewise, there is little information on the growth and characteristics of either purebred Pelibuey lambs or lambs obtained by crossing this breed with breeds such as Dorper and Charollais. To meet the current and future demand of the domestic market, it is important to determine the effect of breed on the growth of lambs, their age at slaughter, and the quality of the carcass meat for breeds currently in use in Mexico and thereby to generate information that contributes to the commercialization of quality carcass meat. The objective of the present study was to evaluate the effect of breed on the growth, characteristics, and carcass classification of purebred Pelibuey lambs and lambs obtained by crossing the Pelibuey breed with the Dorper and Charollais breeds.

Material and methods The research was conducted from 2015 to 2017 at the facilities of the Ovine Experimental Area of the Córdoba Campus Colegio de Postgraduados, located on the Córdoba-Veracruz federal highway at km 348, Amatlán de los Reyes, Veracruz, Mexico. The geographic location is 18° 51' 20" N and 96° 51' 37" W, at an altitude of 650 m asl. The climate is warmhumid with abundant rains in summer, the average annual temperature is 22 °C, and the annual rainfall is 2,000 mm(8). The experiment was conducted according to the criteria set forth in the Official Mexican Standard on technical specifications for the production and sanitary meat processing (NOM-009-ZOO-1994), technical specifications for the production and humane treatment in the mobilization of animals (NOM-051-ZOO-1995), use of laboratory animals (NOM-062-ZOO-1999), and methods for killing domestic and wild animals (NOM-033-SAG/ZOO-2014), in accordance with the Regulations for the Use and Care of Animals Intended for Research of the Colegio de Postgraduados.

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Experimental animals and diet

Thirty-nine (39) male lambs from an experimental herd: Charollais x Pelibuey (n= 11), Dorper x Pelibuey (n= 10), and Pelibuey (n= 18), were reared by their mothers. In the first week of lactation, the lambs stayed with their mothers; after this period, the ewes went out to pasture from 1000 to 1400 h, returning to nurse their young and to stay all night. The lambs were provided with a commercial diet (Agribrands Purina Mexico® creep feeding) from birth to weaning in feeders with restricted access for mothers. After weaning, the lambs were fed a diet of mechanically minced sugarcane forage with an approximate particle size of 3.0 cm and a commercial feed concentrate (Agribrands Purina Mexico®) containing 15% crude protein (CP) and consisting mainly of ground cereals, a combination of oilseed pastes, cereal by-products, molasses, coconut paste, and vegetable oil. This food was offered freely to the lambs only once daily (0700 to 0800 h). Water was available ad libitum in cup drinkers. In the central region of Veracruz, Mexico, where the present study was developed, sheep producers manage the diet tested in this research during the fattening period. In this sense, the management conditions that sheep producers currently apply are being evaluated, so the diet of the sheep studied was not modified, in order to adopt and transfer the results of this research to the producer’s local sheep.

Lamb growth performance

The weight of the lambs was measured at birth (within the first 24 h of life, BW) and every 15 d thereafter until they reached slaughter weight (approximately 45 kg). The lambs’ weaning weights (WW) were also recorded (approximately 75 d). The fattening days (FD) were determined as the number of days between weaning and slaughter. The daily weight gain (DWG) was determined from the difference in the slaughter weight and the WW divided by the FD. The lambs were slaughtered at similar average live weights.

Lamb carcass yield

The slaughter of the animals was conducted in the municipal slaughterhouse of Orizaba, Veracruz, Mexico, at 18 km from the facilities of the Colegio de Postgraduados under the specifications established in standard NOM-033-SAG/ZOO-2014. Each lamb was individually carried, and the animals were transported at a population density of 0.2 m2/lamb to minimize the likelihood of injury. Prior to slaughter (1200 h), food availability to the lambs 965


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was reduced, and they were fasted for 4 h and transported to the slaughterhouse on the day of the slaughter. The live weight (LW) of the lambs was recorded at the slaughterhouse. They were then stunned using a captive bolt pistol, and exsanguination was performed through a cut in the carotid artery and the jugular vein. The animals were then skinned and eviscerated. Non-carcass components such as the head and hooves were removed and weighed separately. The weights of the blood, skin, full and empty green viscera, red viscera, and bile were also recorded. The hot carcass weight (HCW) was recorded immediately to determine the hot carcass yield (HCW/LW*100). When the carcasses reached room temperature, they were stored in a cold room at 4 °C for 24 h, where they were hung by both Achilles tendons. Subsequently, the following measurements were made: cold carcass weight (CCW) for determining the cold carcass yield (CCW/LW), weight loss (HCW−CCW), dorsal fat thickness, and rib eye perimeter. To determine the fat thickness and the area of the rib eye of the longissimus dorsi, a cut was made between the 12th and 13th ribs, the thickness of the dorsal fat was measured with a digital caliper, and the perimeter of the rib eye was drawn on acetate paper. The rib eye area was estimated from the perimeter using an LI 3100 leaf area meter (LICOR®, Lincoln, NE, USA).

Lamb carcass conformation-classification and commercial meat cuts

The carcasses were evaluated by five trained evaluators. The training of the evaluators consisted of several training sessions in ovine carcass quality. The photographic standards used for the evaluation of the carcasses obtained in this experiment are shown in Figure 1. The evaluation of the carcasses was based on the criteria of the Mexican standard for the classification of lamb carcasses (NMX-FF-106-SCFI-2006)(9). This standard describes three carcass conformation categories (excellent, good, and deficient) and four quality grade categories for the complete carcass; in order of decreasing quality, the better are Mexico Extra (MEX EXT), Mexico 1 (MEX 1), Mexico 2 (MEX 2), and Out of Classification (O/C). The criteria for classification include age of the animal, slaughter weight, carcass conformation, and dorsal fat thickness in the longissimus dorsi muscle at the height of the 12th rib (fat/conformation ratio).

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Figure 1. Lamb carcasses classified according to the Mexican standard NMX-FF-106SCFI-2006

(A) Charollais x Pelibuey (Good conformation, MEX 1 quality grade); (B) Dorper x Pelibuey (Good conformation, MEX 1 quality grade); and (C) Pelibuey (Deficient conformation, MEX 2 quality grade).

The carcasses were divided longitudinally along the dorsal spine. The right half was divided into six commercial sections in a modification of the procedure described: cuello (neck, 1st to 5th cervical vertebrae); hombro (shoulder, bone base: scapula and humerus including the first 5 ribs in a perpendicular section located under this); brazuelo (foreshank and breast, including the radius, from the 2nd to the 11th rib in a perpendicular section with the flank); costillar (ribs, 5th to 12th thoracic vertebrae); lomo (loin, longissimus lumborum from the 13th thoracic vertebra to the 7th lumbar vertebra); and pierna (leg, the section between the last lumbar vertebra and the first sacral vertebra)(10). The sections were weighed individually, and the yield (%) was determined with respect to the weight of the right half of the carcass(11).

Measurements of carcass color, temperature, and pH

Instrumental color, temperature, and pH of the carcasses were measured at 30 min and 24 h after slaughter. The instrumental color was measured according to the CIE L*a*b* scale. For the color30min of the carcass, the reading was made of the rectus abdominis muscle(11); for the color24h, the reading was made of the longissimus dorsi, and for the fat color, the reading was made of the fat coverage of the leg. A portable colorimeter was used to measure this variable (Mod CR-300/410, Minolta, Tokyo, Japan). Illuminant D65 was used as an observation standard at a visual angle of 10º and an 8 mm of aperture. The temperature of the hot carcass (HC) and that of the cold carcass (CC) were measured by inserting a food-grade punch

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thermometer into the muscle mass (leg). The pH30min was measured using a potentiometer equipped with a puncture electrode (pH meter Mod HI 99163, Hanna, TX, USA) after calibration of the equipment using buffer solutions at pH 4.0 and 7.0, choosing the same point for all the carcasses. The pH24h of the meat (longissimus dorsi) was measured according using a potentiometer (Mod pH 1100, Oaklon, Eutech Instruments, Singapore) previously calibrated with pH 4.0, 7.0, and 10.0 buffer solutions. All measurements were performed in triplicate.

Statistical analysis

The data were analyzed using the GLIMMIX procedure in SAS 9.3 (SAS Institute Inc., Cary, NC, USA). The type of breed was considered as the main effect in the model. For the DWG and FD variables, the following covariance model was used: yij = μ + breedi + (β + δi )X ij + animalj + εij ; where 𝒊 = 1,2,3; 𝒋 = 1, ⋯ ,39, 𝒚𝒊𝒋 are the FD of breed i in animal 𝑗, 𝝁 is the general mean, 𝒃𝒓𝒆𝒆𝒅𝑖 is the fixed effect due to genotype 𝑖, (𝜷 + 𝜹𝒊 )𝑿𝒊𝒋 , 𝛽 is the intercept of the covariate 𝑋𝑖𝑗 weaning weight, 𝜹𝒊 is the slope of the genotype, 2 ), 𝒂𝒏𝒊𝒎𝒂𝒍𝒋 is the random effect due to the animal, assuming 𝑎𝑛𝑖𝑚𝑎𝑙𝑗 ~𝐼𝐼𝐷𝑁(0, 𝜎𝑎𝑛𝑖𝑚𝑎𝑙 2 ). 𝝐𝒊𝒋 is the experimental error with 𝜀𝑖𝑗𝑘 ~𝐼𝐼𝐷𝑁(0, 𝜎 To analyze the variables of productive development, carcass characteristics, commercial cuts, and carcass and meat quality, the following mixed model was used: yij = μ + breedi + animalj + εij ; where 𝒊 = 1,2,3; 𝒋 = 1, ⋯ ,39, 𝒚𝒊𝒋 is the variable of the response of the type of cross 𝑖, in animal 𝑗, 𝝁 is the general mean, 𝒃𝒓𝒆𝒆𝒅𝑖 is the fixed effect due to breed, 𝑎𝑛𝑖𝑚𝑎𝑙𝑗 is the random effect due to the animal, 2 ), assuming 𝑎𝑛𝑖𝑚𝑎𝑙𝑗 ~𝐼𝐼𝐷𝑁(0, 𝜎𝑎𝑛𝑖𝑚𝑎𝑙 𝝐𝒊𝒋 is the experimental error, assuming, 𝜀𝑖𝑗𝑘 ~𝐼𝐼𝐷𝑁(0, 𝜎 2 ).

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Fisher's LSD method and Satterthwaite's degrees of freedom correction method were used to compare means. The cumulative logit model was used to compare the carcass conformation and the quality grade of the genotypes. The linear predictor is 𝜂𝑐𝜄 = 𝜂𝑐 + 𝜏𝜄 , where 𝜂𝑐𝜄 is the linear predictor in category 𝑐th (𝑐 = 0, 1) for the 𝑖th genotype (𝑖 = 1,2,3), 𝜂𝑐 is the intercept for the 𝑐th category, and 𝜏𝜄 is the 𝑖th fixed effect of the genotype. The Mexican standard for the classification of lamb carcasses establishes three categories for the conformation of the carcass (excellent, good, and deficient) and four categories for the quality grade of the whole carcass (in decreasing order of quality, these categories are MEX EXT, MEX 1, MEX 2, and O/C). In this study, only two categories for conformation (good and deficient) and two categories for quality grade (MEX 1 and MEX 2) were obtained and considered in the model for the analysis.

Results and discussion Lamb growth performance

The productive performance of the lambs according to genotype is shown in Table 1. The analysis of variance showed that there is a highly significant effect (𝑃= 0.0001) of genotype on the variables BW, WW, and DWG. The average BW and WW were significantly greater in the Charollais x Pelibuey (ChP) genotype than in the Dorper x Pelibuey (DP) genotype (by 0.47 kg and 2.94 kg, respectively) and in the Pelibuey (P) genotype (by 0.69 kg and 4.05 kg, respectively). There was a significant effect of genotype (𝑃=0.0020) and covariate weaning weight (𝑃=0.0073) on the number of FD. The number of FD required for the lambs to reach commercial weight was not significantly different in ChP and DP lambs, but FD in those groups differed significantly from that in P lambs. ChP and DP lambs reached commercial weight 35 and 23 d earlier than P lambs, respectively. The average daily weight gain (DWG) from weaning to slaughter differed significantly in the three groups; ChP and DP lambs showed higher DWG than P lambs (Figure 2). DWG was greater in the ChP genotype than in the DP genotype.

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Table 1. Productive behavior of Charollais x Pelibuey (ChP), Dorper x Pelibuey (DP), and Pelibuey (P) lambs ChP DP P Variable (n= 11) (n= 10) (n= 18) Birth weight, kg

3.93 ± 0.24a

3.46 ± 0.24ab

3.24 ± 0.16b

Weaning weight, kg

19.39 ± 0.91a

16.45 ± 0.92b

15.34 ± 0.60b

FD (weaning to slaughter)

106.87 ± 7.07b

118.01 ± 6.76b

141.35 ± 5.36a

DWG, kg/d (weaning to slaughter)

0.278 ± 0.01a

0.235 ± 0.01b

0.207 ± 0.01c

FD= fattening days; DWG= daily weight gain; The data are reported as the mean ± the standard error. abc Means within the same row marked with different letters are significantly different (𝑃<0.05).

The present study showed that crossing of Pelibuey ewes with Charollais rams results in better productive development (BW, WW, FD and DWG) of lambs raised in conditions of high temperature and humidity, that is, in the study area where the present investigation was developed, the temperature varies from 12 to 32 °C (minimum of 9 °C and maximum of 37 °C) and it rains 25.1 d on a month with at least 1 mm of precipitation. The lambs from ewes with meat suitability (Katahdin) and rams of four breeds (Charollais, Dorper, Suffolk, and Textel) under better environmental conditions for the production of sheep (dry temperate climate at 1,962 m asl), were that the best productive behavior was found in lambs from the Katahdin x Charollais crossing(12). The production values cited in that study are similar to the values found in the present study. The BW values found in this study are superior to those reported in other studies(13) in lambs from Black Belly x Pelibuey ewes and rams of three different breeds (Dorset, Hampshire, and Suffolk) and lambs from Pelibuey ewes and hair breed rams (Pelibuey, Katahdin, and Dorper), with average reported values of 3.18 ± 0.34 and 2.9 ± 0.09 kg, respectively. The DWG (kg/d) in the present study for the three genotypes was higher than the values in P (0.181 ± 0.02), Pelibuey x Suffolk (0.206 ± 0.03), DP (0.222 ± 0.03), F1 x Dorset (0.217 ± 0.05), F1 x Hampshire (0.219 ± 0.05), and F1 x Suffolk (0.222 ± 0.04) lambs(4,13). These differences may be due to the breeds used in the crossbreeding and to the management of the lambs during fattening. It is important to mention that no BW, WW, FD, or DWG values have previously been reported for the Charollais x Pelibuey (ChP) crossing. For the first time, this study showed that the performance of ChP lambs with respect to the variables is better than that of other crosses, even under stressful climatic conditions of high temperature and humidity of study area. Thus, this crossing is a good alternative to produce sheep in the tropics. Figure 2 shows the change in live weight from birth to 5.5 mo according to genotype. The graph shows that at the age of 5.5 mo, lambs of the ChP and DP genotypes showed a higher

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growth rate, with average monthly weight gains of 6.71 and 5.42 kg, respectively, than lambs of the P genotype (5.02 kg), despite the fact that the initial weights of the lambs of the three genotypes were very similar. ChP lambs reached the commercial weight for slaughter 35 and 12 d before lambs of the P and DP genotypes, respectively. The Dorper breed has been recommended for the production of lambs with meat suitability in crossbreeding(14). However, the results obtained in this investigation show that crossing of Charollais rams with Pelibuey ewes results in lambs that are more suitable to produce meat due to their better growth rate. Figure 2. Change in live weight from birth to 5.5 months of age of Charollais x Pelibuey (ChP, closed circles), Dorper x Pelibuey (DP, open boxes), and Pelibuey (P, closed boxes) lambs. The regression lines are presented for each breed from birth to 5.5 mo of age 45.0

ChP y = 5.42x + 2.86 R² = 0.9964

40.0 35.0

DP y = 6.71x + 3.80 R² = 0.9952 P y = 5.02x + 2.55 R² = 0.9985

Live weight (kg)

30.0 25.0 20.0 15.0 10.0 5.0 0.0 0

1

2 3 Age (months)

4

5

Lamb carcass yield

The analysis of variance showed a highly significant effect of genotype on carcass weight loss (𝑃=0.0072) and on the weight of empty green viscera (𝑃=0.0001). This means that the Charollais x Pelibuey breed presented less loss of empty green viscera, followed by the Dorper x Pelibuey breed, while the pure Pelibuey breed had a lower loss of this characteristic. There was no significant effect of genotype on the remaining variables (Table 2), such as weight loss, dorsal fat thickness, area of the rib, and red viscera, so the behavior of the characteristics of the carcasses was similar between races and crosses of sheep evaluated. 971


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Weight loss between hot and cold carcasses was lower in the ChP genotype; the weight of empty green viscera was significantly different among the three breeds and was higher in the hair breeds (P and DP). Lambs of the three genotypes showed similar average cold carcass yield (CCY) values because the weight of the animals at slaughter was standardized. Table 2. Characteristics of the carcasses of Charollais x Pelibuey (ChP), Dorper x Pelibuey (DP), and Pelibuey (P) lambs ChP DP P Variable (n = 11) (n = 10) (n = 18) Empty live weight, kg 44.85 ± 0.85 42.96 ± 0.89 43.17 ± 0.66 Hot carcass weight, kg

22.21 ± 0.47

21.45 ± 0.49

21.40 ± 0.36

Cold carcass weight, kg

21.86 ± 0.46

21.03 ± 0.49

20.91 ± 0.36

Hot carcass yield, %

49.53 ± 0.44

49.89 ± 0.46

49.53 ± 0.34

Cold carcass yield, %

48.74 ± 0.43

48.90 ± 0.45

48.46 ± 0.33

b

ab

0.50 ± 0.02a

Loss of weight, kg

0.35 ± 0.03

Dorsal fat thickness, mm

1.51 ± 0.17

1.63 ± 0.18

1.56 ± 0.13

Area of the rib, cm2

14.66 ± 0.69

15.43 ± 0.81

15.83 ± 1.33

Red viscera, kg

1.98 ± 0.39

1.98 ± 0.41

2.39 ± 0.30

Empty green viscera, kg

3.84 ± 0.13c

4.31 ± 0.13b

4.87 ± 0.10a

abc

0.42 ± 0.03

The data are reported as the mean ± the standard error (SE). Means within a row marked with different letters are significantly different (𝑃<0.05).

In this study, genotype was not found to affect the CCY and lambs with standardized weights at slaughter and from hair sheep ewes and rams of the Dorset, Hampshire, Suffolk, Pelibuey, and Rambouillet breeds(5,13). Those authors showed that there were no significant differences in CCY among the genotypes studied, but the values reported for CCY were lower than those obtained in this investigation. Carcass yield can be affected by factors such as the age of the lamb, wool growth, nutrition, and breed(15). Results in this research showed no effect of breed on the CCY. In this sense, state that with the standardization of the weight at slaughter, the carcass yield is not affected(5). In this study, ChP lamb carcasses lost less weight at 24 h after slaughter (0.15 kg) than P and DP carcasses. In contrast, in the crossing of Pelibuey with Rambouillet and Suffolk breeds, no differences were found in this variable(5). Another important characteristic of the carcass is dorsal fat thickness. Low values of this parameter are an indicator of lean meat, which is preferred in the Mexican market(5). The average dorsal fat thickness values in this study were very low (ChP= 1.51 ± 0.17 mm, DP= 1.63 ± 0.18 mm, P= 1.56 ± 0.13 mm) despite the high slaughter weight of the animals. Similar values have been reported for Pelibuey (1.2), Pelibuey x Kathadin (1.8 mm), DP (1.8 mm),

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Pelibuey x Rambouillet (1.7 mm), and Pelibuey x Suffolk (1.4 mm) lambs(16,17). On the other hand, higher values of dorsal fat thickness (6.33 ± 1.22 mm) have been reported in lambs obtained by crossing Kathadin ewes with Charollais rams(16). This may be because the Katahdin and Charollais breeds undergo rapid growth and accumulate dorsal fat at a young age compared to the Pelibuey breed, which is slower-growing and tends to accumulate more visceral fat than dorsal fat(2). The rib eye area is an indicator of the muscle conformation of the carcass(18); the greater the rib eye area, the better is the muscle conformation. In this study, genotype did not influence the rib eye area or the area of the longissimus dorsi muscle (ChP= 14.66 ± 0.69 cm2, DP= 15.43 ± 0.81 cm2, and P= 15.83 ± 1.33 cm2). Lower values have been reported in DP (11.01 cm2), Pelibuey, Pelibuey x Rambouillet and Pelibuey x Suffolk (5.16 ± 0.13 cm2) and Black Belly (10.89 cm2) lambs(5,16,17,19). The highest rib eye area values (19.8 ± 0.5 cm2) were found in Kathadhhin x Charollais and Katahdin x Dorper lambs treated with β-adrenergic agonists during fattening(20). Breed, diet, and hormonal treatment affect muscle development(21).

Classification, conformation of the carcass, and commercial cuts

The estimated probabilities of carcass conformation and quality grade according to genotype are shown in Figure 3. The f analysis showed a statistically significant effect among genotypes (𝑃 < 0.0382) on the carcass conformation and quality grade. Of the carcasses evaluated according to the NMX-FF-106-SCFI-2006 standard, the ChP genotype showed better carcass conformation and quality grade than the DP and P genotypes. The lambs obtained from the crossing of Pelibuey ewes with the Charollais breed showed better carcass conformation and quality grade than the other animals, as shown by the fact that the probability of obtaining good conformation and MEX 1 quality grade in the ChP genotype is 0.10 and 0.72 units higher, respectively, than that for the DP and P genotypes. The P genotype showed the highest percentage (72 %) of carcasses with deficient conformation and MEX 2 quality grade, whereas 10 % of the carcasses of the DP genotype and none of the carcasses of the ChP genotype displayed deficient quality and conformation. In general, crossbreeding DP and ChP resulted in better classification, better carcass conformation, and better-quality grade than was obtained with genotype P.

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Figure 3. Estimated probabilities of achieving specific carcass conformation categories and quality grades for Charollais x Pelibuey (ChP), Dorper x Pelibuey (DP), and Pelibuey (P) lambs using a cumulative logit model Good/MEX 1

1 1

Deficient/MEX 2 0.9

Probability

0.8

0.722

0.6 0.4

0.278

0.2

0.1

0 0 ChP

DP Genotype

P

Figure 1 shows the photographic standards used to evaluate the carcasses obtained in this experiment. The crossing of Pelibuey ewes with rams from the Charollais and Dorper breeds conferred better carcass conformation and quality grade because the latter two breeds present better meat conformation(1) than the pure Pelibuey breed. In this study, the crossing of Katahdin x Charollais was shown to yield carcasses with excellent conformation and MEX EXT quality grade; in that case, both of the breeds used in the cross are suitable for meat production(16). The average weight of the half carcass and the weight and yield of commercial cuts according to genotype are shown in Table 3. The analysis of variance showed a highly significant effect of genotype on the average weight of the neck (𝑃=0.0036), loin (𝑃=0.0339), and leg (𝑃= 0.0001), but no significant effect of genotype was observed for the other commercial cuts or for the average weight of the half carcass. In the yield of the commercial cuts, there was a significant effect of genotype on the neck (𝑃=0.0060), the foreshank+breast (𝑃=0.0289), the loin (𝑃=0.0484), and the leg (𝑃=0.0088). The P genotype presented higher neck weight and yield than the ChP and DP genotypes, whereas only the P genotype presented greater loin weight than the DP genotype. It was observed that of the three genotypes ChP presented greater leg weight and yield and greater foreshank+breast yield than the other two genotypes.

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Table 3. The average weight of the half carcass and the weight and yield of the commercial cuts of Charollais x Pelibuey (ChP), Dorper x Pelibuey (DP), and Pelibuey (P) lambs Variable

ChP (n = 11)

DP (n = 10)

P (n = 18)

Half-carcass weight 10.45 ± 0.26 Commercial cuts (kg) Neck 0.58 ± 0.03b Shoulder 1.57 ± 0.05 Foreshank+breast 2.43 ± 0.12 Ribs 1.18 ± 0.07 Loin 1.58 ± 0.08ab Leg 3.44 ± 0.09a Yield of commercial cuts (%) Neck 5.68 ± 0.37b Shoulder 15.52 ± 0.44 Foreshank+breast 24.28 ± 1.05a Ribs 10.51 ± 0.35 Loin 15.31 ± 0.71ab Leg 32.81 ± 0.58a

10.17 ± 0.27

10.36 ± 0.20

0.56 ± 0.03b 1.57 ± 0.05 2.23 ± 0.13 1.14 ± 0.07 1.46 ± 0.08b 3.13 ± 0.09b

0.71 ± 0.02a 1.54 ± 0.04 2.13 ± 0.09 1.14 ± 0.06 1.72 ± 0.06a 3.15 ± 0.08b

5.64 ± 0.35b 15.51 ± 0.42 21.91 ± 0.99ab 11.22 ± 0.33 14.39 ± 0.68b 31.35 ± 0.55ab

6.96 ± 0.28a 14.83 ± 0.33 20.59 ± 0.79b 10.47 ± 0.26 16.57 ± 0.53a 30.41 ± 0.43b

ab

The data are reported as the mean ± the standard error (SE). Means within the same row marked with different letters are significantly different (𝑃<0.05).

The leg and loin are cuts of great commercial value and represent 43.3 % of the yield of the carcass(10). In this study, the yield obtained for both cuts was greater than the reported value in all three genotypes: ChP (48.12 %), DP (45.74 %), and P (46.98 %). In this study, the crossing of Pelibuey with Charollais resulted in greater weight of the leg, which is a cut of high commercial value(5). However, 1 to 4 % differences among the genotypes were observed in the weights of the neck, loin, leg, and foreshank+breast cuts minimal differences in the weights of the majority of commercial cuts in the evaluation of 15 wool breeds specialized for the production of wool or meat(22). In crossbred lambs of hair breeds (DP) and hair x wool breeds and reported differences in cut yield of approximately 1%, similar to the differences found in this study(23).

pH, temperature, and instrumental color of the carcass and meat

Table 4 presents the average values of pH, temperature, and instrumental color of the rectus abdominis muscle, meat, and subcutaneous fat according to genotype. Of the variables measured in the carcass, T30min (𝑃=0.0658), L* (𝑃=0.0001), and a* (𝑃=0.0107) were affected by genotype. The analysis of variance also showed significant differences in the variables 975


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pH24h (𝑃=0.0607), L* (𝑃=0.0001), a* (𝑃=0.0001), and b* (𝑃=0.0006), measured in the meat (longissimus dorsi) and in L* (𝑃=0.0001) of the subcutaneous fat; no differences were observed in the remaining variables. The ChP genotype presented a higher carcass temperature than the P genotype; however, the 24-h post mortem temperature was similar among the three genotypes (T24h, 𝑃=0.2643) because the carcasses were maintained under the same storage conditions (24 h at 4°C). The average value of pH24h was greater in the ChP genotype than in the P genotype. Table 4. The pH, temperature, and instrumental color of the rectus abdominis muscle and meat of Charollais x Pelibuey (ChP), Dorper x Pelibuey (DP), and Pelibuey (P) lambs ChP DP P Variable (n = 11) (n = 10) (n = 18) pH30min

6.64 ± 0.06

6.72 ± 0.06

6.75 ± 0.04

pH24h

5.66 ± 0.02a

5.61 ± 0.02ab

5.59 ± 0.02b

T30min (°C)

39.88 ± 0.23a

39.37 ± 0.24ab

39.19 ± 0.18b

T24h (°C)

4.64 ± 0.19

4.20 ± 0.20

4.33 ± 0.15

Rectus abdominis Longissimus dorsi24h Subcutaneous fat

ab

L* a* b* L* a* b* L* a* b*

b

41.35 ± 0.78 12.78 ± 1.40b -0.81 ± 0.33 33.69 ± 0.66b 14.73 ± 0.42b 4.29 ± 0.23a 68.41 ± 1.38b 3.32 ± 0.45 4.29 ± 0.46

c

38.20 ± 0.82 13.30 ± 1.46b -0.94 ± 0.35 32.61 ± 0.69b 13.84 ± 0.45b 3.33 ± 0.24b 67.98 ± 1.45b 3.6 ± 0.48 4.31 ± 0.49

44.32 ± 0.61a 17.80 ± 1.09a -1.22 ± 0.26 37.31 ± 0.51a 17.24 ± 0.33a 4.66 ± 0.18a 73.15 ± 1.08a 3.14 ± 0.35 4.93 ± 0.36

The data are reported as the mean ± the standard error (SE). Means within the same row marked with different letters are significantly different (𝑃<0.05).

The pH and color of meat are important indicators of quality and influence the visual appearance of the meat(24). The difference in pH at 24 h post mortem among the genotypes in this study was probably because the T30min of the carcass tends to be lower in hair breeds than in wool breeds(25), and it has been shown that the meat of fast-growing lambs tends to have a higher pH(26). However, the pH24h values for the three genotypes in this study fell within the preferred range for this parameter(27). The color of the rectus abdominis muscle (30 min post mortem) was significantly affected by genotype in this study. The values of a* were similar to those reported in Rasa Aragonesa lamb carcasses for different thicknesses of dorsal fat with a slaughter weight of 50-60 kg(28). Values higher reported in the present study for L* (51.12) and a* (11.64) in Rasa Aragonesa lambs, with a slaughter weight of 24 kg(29). 976


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In the color of meat (longissimus dorsi), the ChP and DP genotypes presented indices of L* (luminosity) and a* (red) lower than those presented by the P genotype. During storage, the ferric metmyoglobin (MetMb) accumulation rate on the surface of the meat is governed by intrinsic factors (age of the animal, breed, sex, diet, pH, and metabolic type of the muscle) and extrinsic factors (temperature, oxygen availability, lighting, growth of surface microbes, and type of packaging) or a combination of these factors(30,31). In this study, it was observed that genotype affected the color of the meat; meat color was probably also affected by the exposure time of the carcasses prior to cold-room storage(26). Found an effect of genotype on the a* and b* indices of meat color in hair breed lambs and lambs obtained by crossing hair and wool breeds(32). With respect to subcutaneous fat, there were no significant differences in the indices a* (𝑃= 0.7484) or b* (𝑃=0.4617) among the three genotypes. The b* (yellow) index of subcutaneous fat was similar in the three genotypes because the lambs underwent the same management during fattening and remained stabled; grazing lambs tend to have higher b* index values(26) due to the presence of high levels of carotenoids in the fat(32), resulting in a yellow color that is unattractive to consumers.

Conclusions and implications Breed had a significant effect on the growth, characteristics, and classification of lamb carcasses. The crossing of Pelibuey sheep with the Charollais breed (ChP) resulted in higher DWG. ChP and DP lambs reached commercial weight one month earlier than P lambs. With the ChP crossing, there is a high probability (0.72) of obtaining carcasses that show good conformation and good quality grade (MEX 1). Therefore, the ChP crossing can be an option for the commercial breeding and fattening of lambs to produce quality meat in hot, humid climates. In this study it was found that the genotype of the evaluated sheep presented different conditions in the composition of the meat, such as an increase in pH, temperature variations, changes in the instrumental color of the carcass, amount of meat and subcutaneous fat, however, these parameters are within the acceptable ranges of meat quality for each breed. In this sense, it is feasible for sheep producers in the Center of Veracruz, México that they can use the crosses of the breeds evaluated in this study to maintain or increase the productivity of their herds.

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Acknowledgments and declaration of conflict of interest

We thank the Liaison Branch of the Colegio de Postgraduados Campus Córdoba for the support provided for this work in the Microregion de Atención Prioritaria. This research was funded and was supported by a student scholarship from Consejo Nacional de Ciencia y Tecnología (CONACyT) Mexico. The authors declare no conflicts of interest. Literature cited: 1. Partida JA, Vázquez E, Rubio MS, Méndez D. Effect of breed of sire on carcass traits and meat quality of Katahdin lambs. J Food Res 2012;1(4):141-149. 2. Muñoz-Osorio GA, Aguilar-Caballero AJ, Sarmiento-Franco LA, Wurzinger M, CámaraSarmiento R. Technologies and strategies for improving hair lamb fattening systems in tropical regions: A review. Ecosist Recur Agropec 2016;3(8):267-277. 3. Pineda J, Palma JM, Haenlein GFW, Galina MA. Fattening of Pelibuey hair sheep and crossbreds (Rambouillet-Dorset×Pelibuey) in the Mexican tropics. Small Ruminant Res 1998;27(3):263-266. 4. Partida de la Peña JA, Braña VD, Martínez RL. Desempeño productivo y propiedades de la canal en ovinos Pelibuey y sus cruzas con Suffolk o Dorset. Téc Pecu Méx 2009;47(3): 313-322. 5. Gutiérrez J, Rubio MS, Méndez RD. Effects of crossbreeding Mexican Pelibuey sheep with Rambouillet and Suffolk on carcass traits. Meat Sci 2005;70(1):1-5. 6. Partida de la Peña JA, Braña VD, Jiménez SH, Ríos RFG, Buendía RG. Producción de carne ovina. Libro técnico. 2013;5:6-18. 7. Wildeus S. Hair sheep genetic resources and their contribution to diversified small ruminant production in the United States. J Anim Sci 1997;75:630-640. 8. García E. Modificación al sistema de clasificación climática de Koppen. 5a ed. México, Distrito Federal. Universidad Nacional Autónoma de México. 2005. 9. Secretaría de Economía. Productos pecuarios-carne de ovino en canal–clasificación. 1ra ed. Distrito Federal, México. Secretaría de Economía. 2006. 10. Martínez DE, Núñez GFA, Rodríguez AFA. Manual para la evaluación de corderos en pie y en canal. Chihuahua, México. Universidad Autónoma de Chihuahua. 2007.

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11. Colomer F, Morand FP, Kirton AH, Delfa R, Sierra I. Métodos normalizados para el estudio de los caracteres cuantitativos y cualitativos de las canales caprinas y ovinas. Madrid, España, Cuadernos del INIA. 1987. 12. Hinojosa-Cuéllar JA, Oliva-Hernández J, Torres-Hernández G, Segura-Correa JC. Productive performance of F1 Pelibuey × Blackbelly lambs and crosses with Dorper and Katahdin in a production system in the humid tropic of Tabasco, México. Archiv Med Vet 2013;45(2):135-143. 13. Bores QRF, Velázquez MPA, Heredia A. Evaluación de razas terminales en esquemas de cruza comercial con ovejas de pelo F1. Téc Pecu Méx 2002;40:71-79. 14. Vázquez-Soria ET, Partida de la Peña JA, Rubio-Lozano M, Méndez-Medina D. Comportamiento productivo y características de la canal en corderos provenientes de la cruza de ovejas Katahdin con machos de cuatro razas cárnicas especializadas. Rev Mex Cienc Pecu 2011;2:247-258. 15. Gardner, GE, Williams A, Ball AJ, Jacob RH, Refshauge G, Hocking EJ, Behrendt R, Pethick DW. Carcass weight and dressing percentage are increased using Australian Sheep Breeding Values for increased weight and muscling and reduced fat depth. Meat Sci 2015;99:89-98. 16. Estrada A, Dávila H, Herrera RS, Robles JC, La OO, Castro BI, Portillo JJ, Ríos FG, Contreras G. Carcass characteristics and yield of the primary cuts of lambs fed broom millet (Sorghum bicolor var. Technicum, jav). Cuban J Agr Sci 2012;46:145-150. 17. Ríos FG, Gómez-Vázquez A, Pinos-Rodríguez JM, García-López JC, Estrada-Angulo A, Hernández-Bautista J, Portillo JJ. Effect of breed on performance and carcass characteristics of Mexican hair sheep. South African J Anim Sci 2011;41:275-279. 18. Hopkins DL, Wotton JSA, Gamble DJ, Atkinson WR. Lamb carcass characteristics. 2. Estimation of the percentage of saleable cuts for carcasses prepared as ’trim’ and traditional cuts using carcass weight, fat depth, eye muscle area, sex, and conformation score. Australian J Exp Agr 1995;35(2):161-169. 19. Salinas-Ríos T, Sánchez-Torres-Esqueda MT, Hernández-Bautista J, Díaz-Cruz A, NavaCuellar C, et al. Carcass characteristics, physicochemical changes and oxidative stress indicators of meat from sheep fed diets with coffee pulp. Arq Brasileiro Med Vet Zoot 2014;66:1901-1908. 20. Partida de la Peña JA, Cesaya RTA, Rubio LMS, Méndez MRD. Efecto del clorhidrato de zilpaterol sobre las características de la canal en cruzas terminales de corderos Katahdin. Vet Méx OA. 2015;2:1-13.

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21. Ferreira OGL, Rossi FD, Coelho RAT, Fucilini VF, Benedetti M. Measurement of ribeye area by the method of digital images. Rev Brasileira Zoot 2012;41:811-814. 22. Kirton AH, Carter AH, Clarke JN, Sinclair DP, Mercer GJK, Duganzich DM. A comparison of 15 ram breeds for export lamb production 2. Proportions of export cuts and carcass class. NZ J Agr Res 1996;39:333-340. 23. Santos CV, Ezequiel JMB, Morgado ES, de Sousa JSC. Carcass and meat traits of lambs fed by-products from the processing of oil seeds. Acta Scient Anim Sci 2013;35:387394. 24. Hopkins DL, Fogarty NM. Diverse lamb genotypes-2. Meat pH, colour and tenderness. Meat Sci 1998;49:477-488. 25. Hernández-Cruz L, Ramírez-Bribiesca JE, Guerrero-Legarreta MI, Hernández-Mendo O, Crosby-Galvan MM, Hernández-Calva LM. Effects of crossbreeding on carcass and meat quality of Mexican lambs. Arq Brasileiro Med Vet Zoot 2009;61:475-483. 26. Priolo A, Micol D, Agabriel J, Prache S, Dransfield E. Effect of grass or concentrate feeding systems on lamb carcass and meat quality. Meat Sci 2002;62:179-185. 27. Sañudo C. Calidad de la canal y de la carne en los ovinos: factores que la determinan. Rev Argentina Prod Anim 2006;26:155-167. 28. Sañudo C, Alfonso M, Sánchez A, Delfa R, Teixeira A. Carcass and meat quality in light lambs from different fat classes in the EU carcass classification system. Meat Sci 2000;56:89-94. 29. Ripoll G, González-Calvo L, Molino F, Calvo JH, Joy M. Effects of finishing period length with vitamin E supplementation and alfalfa grazing on carcass color and the evolution of meat color and the lipid oxidation of light lambs. Meat Sci 2013;93:906913. 30. Jeong JY, Hur SJ, Yang HS, Moon SH, Hwang YH, Park GB, Joo ST. Discoloration characteristics of 3 major muscles from cattle during cold storage. J Food Sci 2009;74:C1-C5. 31. Renerre M. Factors involved in the discoloration of beef meat. Int J Food Sci Technol. 1990;25:613-630. 32. Burke JM, Apple JK, Roberts WJ, Boger CB, Kegley EB. Effect of breed-type on performance and carcass traits of intensively managed hair sheep. Meat Sci 2003;63:309-315.

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https://doi.org/10.22319/rmcp.v13i4.6065 Article

Effect of weight and body condition score from pregnant cows on the carcass characteristics of their progeny: Meta-analysis

Sander Martinho Adams a* John Lenon Klein a Diego Soares Machado b Dari Celestino Alves Filho a Ivan Luiz Brondani a Luiz Angelo Damian Pizzuti a

a

Universidade Federal de Santa Maria. Laboratório de Bovinocultura de Corte. Av. Roraima nº 1000 Cidade Universitária, Camobi, Santa Maria - RS, Brazil. b

Instituto Federal de Educação Ciência e Tecnologia Farroupilha – Alegrete. Brazil.

*Corresponding author: sander.adams@hotmail.com

Abstract: The objective of the meta-analysis was to evaluate the effects of beef cows weight variation during the 2nd and / or 3rd trimester pregnancy on some parameters of the progeny carcass. The cow weight gain during this gestational period was calculated to standardize the treatments: moderate loss (ML= cows that lost 0 to 5 % of weight) and moderate gain (MG= cows that gained 0 to 5 % of weight). The effect size for all parameters was calculated as medium difference (MD) with a 95% confidence interval and heterogeneity determined using the Q test and the I2 statistic. A random effects metaanalysis was performed for each indicator separately as the medium control and experimental groups. The cow’s weight variation during the studied time variation did not influence the progeny carcass characteristics (P>0.05). Although, a trend towards greater hot carcass weight (P=0.15) and thickness of subcutaneous fat (P=0.10) was observed in calves from MG cows in relation to calves from ML cows. However, the meta-analysis demonstrated that small variations in cow weight during the final half of pregnancy do not affect progeny carcass characteristics. 981


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Key words: Beef cows, Marbling. Steers. Subcutaneous fat.

Received: 16/09/2021 Accepted: 30/05/2022

Introduction Among factors that can influence the postnatal performance of beef cattle, it can be highlight the cow nutritional insults during pregnancy, uterine changes, also known as fetal programming. The prenatal development of cattle influences productive performance throughout the postnatal period(1). The authors add that the number of muscle and fat cells an animal will have throughout its life is determined in the fetal phase and is influenced by pregnant cow nutrition, because the myogenesis and adipogenesis processes are exclusive from the fetal period(2). Thus, Du et al(3), conclude that calves of cows kept under restricted supply of nutrients during pregnancy have a compromised meat production potential. According to Reynolds et al(4), the structural and functional changes in organs and tissues caused by the supply of nutrients during pregnancy serve to allow a rapid adaptation of the developing fetus to the pressure of uterine environmental selection. These changes can be related to the health and productive potential of the progeny in adulthood. However, the nutritional challenge during fetal formation can form a phenotype with greater adaptability when nutritional conditions were more challenging in the postnatal period(5). Thus, the effects of fetal programming are more noticeable in the first months from progeny's life(6). The authors state that the real effects of fetal programming in beef cattle are still contradictory and need further clarification, since there are many divergences between researches, such variability of the studied nutrients, gestational period and intensity of nutritional restriction, as well the progeny characteristics evaluated. Therefore, the objective of this meta-analysis was to evaluate the effects of cow weight variation during pregnancy at progeny carcass characteristics.

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Material and methods Literature search

Literature searches were performed using specific search databases on the platforms: Scientific Electronic Library Online (https://scielo.org), Portal de Periódicos Capes (https://www.periodicos.capes.gov.br), ScienceDirect (https://www.sciencedirect.com) and Google Scholar (http://scholar.google.com). The searches were based on the keywords: “fetal programming in beef cows and the performance of steers progeny” or “fetal programming in beef cattle and progeny performance.” The literature searches included publications from the last ten years (2009 - 2019).” This meta-analysis was performed using combined data from 10 studies (9 peer-reviewed articles, 1 doctoral thesis), with a total record of 1053 calves during termination and after slaughter phases. When possible, the same study was inserted two or more times in the meta-analysis database. The reviewed studies evaluated the effects of maternal nutrition during gestation on the postnatal performance of progeny, as described in Table 1. Table 1: Description of the studies included in the database for conducting the metaanalysis Study Year

Cow Local Sex Breed

Initial Comparations BW1

534 14 534 All 14 498 Male 15 408 Male 54

7

2013

USA

AxC

7

2013

USA

AxC

8

2009

USA

AxS

9

2019

ARG

A

10

2015a

USA

AxS

Male

440 28

11

2015b USA

AxS

All

463 3

12

2013

USA

A

Male

13

2015

USA

AxS

All

575 9 600 Male 7

± Supl. x No Supl. ± High ECC x Low ECC ± Supl. x No Supl. ± High CP x Low CP Positive energy ± status x Negative Positive energy ± status x Negative ± Supl. x No Supl. ± Supl. x No Supl.

983

Number of observations HCW SFT

M

LDA

228

228

228 228

228

228

228 228

24

24

24

24

24

24

24

24

11

11

11

11

101

101

101 101

40

40

40

40

71

71

71

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14

2016

USA

AxS

15

2019

BRA

CxN

Total

-

-

684 ± 100% TDN x 86 7 125% TDN 413 ± Weight Gain x Male 240 8 Moderate Loss Male

-

-

1053

86

86

86

240

-

240

1053 813 1053

Cow breed, A= Aberdeen Angus; S= Simental; C= Charolês; N= Nelore. 1 Initial body weight (kg) of cows and ± SEM. Variables, HCW= Hot carcass weight; SFT= Subcutaneous fat thickness; M= Marbling; LDA= Longissimus dorsi area. CP= crude protein; TDN= total digestible nutrients.

Inclusion and exclusion criteria

In total, 21 studies published between 2009 and 2020 were identified from the preestablished search. The criteria established for inclusion of studies in database were: 1) possibility of calculating cow average daily weight gain during gestation and adequacy to treatments; 2) provide carcass progeny variables; 3) the period of nutritional insult occurs in second or third pregnancy trimester (cow greatest demand); and 4) report information on sample size and variability measurements of interest (i.e. deviation or standard error). In case of studies that reported the standard error of mean (S.E.M.), the standard deviation (σ) was it obtained through the equation: 𝜎 = 𝑆. 𝐸. 𝑀 ∗ √𝑛 A total of 11 studies obtained by the search terms were excluded from this meta-analysis because they did not answer the criteria mentioned above: criterion 1) 6 studies excluded; criterion 2) 2 studies excluded; criterion 3) 3 studies excluded. A large number of studies were excluded from this research for not meeting the inclusion criteria. In addition, this is justified by the wide variation between studies, especially concerning the intensity of food restriction and distance between treatments, period of food restriction, as well as the great diversity of variables evaluated, as reported by Klein et al(6) in a literature review on the subject.

Data selection and group formation

Four carcass traits of the progeny selected as response variables, including males and females. For this meta-analysis, the measurements of hot carcass weight (HCW), obtained prior to entering the cold chamber, subcutaneous fat thickness (SFT), marbling and Longissimus dorsi area (LDA) used. These last three measurements obtained in the Longissimus dorsi muscle between the 12th and 13th ribs. The average daily weight gain (ADG) evaluated during the breastfeeding period, and the post-weaning ADG considered as the daily weight gain of calves during the rearing phase. 984


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The weight variation of cows during gestation was used to standardization the tested effects (treatments), according to the equation below: 𝑊𝑉 =

(𝐼𝑊 − 𝐹𝑊) 𝑋 100 𝐼𝑊

where WV represents the variation in weight of the cow between the beginning of the experimental period and calving; IW represents the weight of the cow at the beginning of experiment; FW represents the weight of cow at calving. This standardization was necessary due to great variability of treatments of the researches included in the database. Thus, the meta-analysis consists of two groups according to weight variation classes: moderate loss (ML= cows that lost 0 to 5 % of weight during gestation) and moderate gain (MG= cows that gained 0 to 5 % of weight during gestation). In this meta-analysis, the moderate loss (ML) is used as a control group. The data for each study, such as number of replicates, means and standard deviations, organized in Microsoft® Office Excel® spreadsheets for further analysis.

Meta-analytical procedure

Statistical analyses were performed using the software R version 4.0.2(16), through the ‘meta’ package, ‘metacont’ function(17). Egger’s linear regression asymmetry was used to examine the presence of publication bias(18), with a significant bias value when P<0.05, through the 'metabias' function. In addition, funnel plots were used to evaluate publication bias in meta-analysis through the 'funnel' function. The funnel plot graphically shows the precision of the estimated intervention effect, where smaller studies had a wider variance and larger ones had less spread of variability. In the absence of bias, the funnel plot should be approximately symmetrical. The effect size was calculated as the mean difference (MD), which is the difference between the control and experimental groups (subgroups weight gain and severe loss WG and SL from cow’ body weight). The MD requires that all studies have the same unit of measurement but allows for the interpretation of effect size in the original units(19). The effects of variation in the weight of the cow during gestation were expressed in forest plot graphs, constructed from the 'forest' function, using the estimated MD. The meta package provides a forest plot with the effect size and weighted contribution to each study from fixed and random effect models(17). The consistency of results between the experiments was quantified using the measures of heterogeneity of the Chi-square test (Q) and I2 statistics(20), which quantifies the impact of heterogeneity on the meta-analysis, whit a mathematical criterion independent of the number of studies and the metric effect of each treatment. Although the Q test is helpful in identifying heterogeneity, the measure I2 was used to measure heterogeneity(20). The I2 statistic is given by: 985


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𝑄 − (𝑘 − 1) 𝑋 100 𝑄 where Q is the χ2 heterogeneity statistic and k is the number of trials. The I2 statistic describes the percentage of variation across studies due to heterogeneity. Negative values of I2 are set equal to zero; consequently, I2 lies between 0 and 100%(21). Its value might not be important if it falls within the range 0–40 %. However, a value of 30–60 % often indicates moderate heterogeneity, 50–90 % might represent substantial heterogeneity, and value in the range of 75–100 % represents considerable heterogeneity(22). 𝐼 2 (%) =

Results The funnel plots for cow weight variation effect, during pregnancy at progeny carcass characteristics are expressed in Figure 1, and no substantial asymmetry was observed in most characteristics analyzed(22). The variation from cow weight (ML and MG), the number of studies, the mean gross difference and the size of the effect of each variable, P values and heterogeneity, are demonstrated in Table 2. Egger’s test showed that the variables evaluated don’t have publication bias (P>0.05). In general, the meta-analysis did not identify major effects of cow weight variation during pregnancy at progeny carcass characteristics (P>0.05). Despite the low studies number published in this research line, the hot carcass weight showed a favorable trend for the progeny of MG cows (P=0.15), which produced 3.25 kg more carcass at progeny from ML cows (Figure 2). Likewise, animals from MG cows at gestation end showed tendency (P=0.10) for greater thickness of subcutaneous fat compared to animals from ML cows (Figure 3). The average difference was 0.05 cm between the groups studied.

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Figure 1: Funnel plot for cow weight variation during gestation at progeny performance

a) Hot carcass weight; b) Subcutaneous fat thickness; c) Marbling; d) Longissimus dorsi area. Each point represents an individual randomized trial. The y-axis is the standard error of the trials and the x-axis is the effect size. The Larger studies appear toward the top of the plot and cluster around of effect size (mean) and smaller studies appear toward the bottom of the plot. When publication bias has occurred one expects an asymmetry in the scatter of small studies with more studies showing a positive result than those showing a negative result.

Table 2: Effect size and heterogeneity for weight variation in beef cows during pregnancy on progeny performance Number 95% PPI2 PA Item of MD confidence Q B C value value (%) valueD studies intervals HCW, 10 3.23 -1.25, 7.72 0.1580** 2.54 0.9797 0 0.7841 kg SFT, 10 0.05 -0.01, 0.10 0.1030** 8.07 0.5275 0 0.0825 cm M, 9 -12.96, 12.63 0.9802NS 9.61 0.2932 17 points 0.16 LDA, 10 1.13 -0.55, 2.82 0.1881NS 15.20 0.0857 41 0.9031 cm² A

Item, HCW= Hot carcass weight; SFT= Subcutaneous fat thickness; M= Marbling; LDA = Longissimus dorsi area. B P-value for MD, *Significant at 5% probability; ** Tendency; NS Not significant. C P-value for Q statistics; 2 I , Statistics of the estimated heterogeneity. D P-value for Egger’s test; - Number of studies (k<10) too small to test for small study effects (18).

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Figure 2: Forest plot for hot carcass weight (HCW, kg) of the progeny from cows with different weight variations during gestation

The solid line of the x-axis is the no-effect line and dotted lines represent the estimated difference of the random model; therefore, the points to the left of the line represent a reduction in the trait, while the points to the right of the line indicate an increase. Each square relative weight of the study of the overall estimate of effect size with the larger squares representing a larger weight. The upper and lower bound of the squared line represents the upper and lower confidence intervals of 95% for the size of the effect. The diamond at the bottom represents the 95% confidence interval for the global estimate.

Figure 3: Forest plot for subcutaneous fat thickness (SFT, cm) of the progeny from cows with different weight variations during gestation

The solid line of the x-axis is the no-effect line and dotted lines represent the estimated difference of the random model; therefore, the points to the left of the line represent a reduction in the trait, while the points to the right of the line indicate an increase. Each square relative weight of the study of the overall estimate of effect size with the larger squares representing a larger weight. The upper and lower bound of the squared line represents the upper and lower confidence intervals of 95% for the size of the effect. The diamond at the bottom represents the 95% confidence interval for the global estimate.

The progeny of ML and MG cows didn’t show differences (P= 0.9802) in meat marbling content (Figure 4), with average value of 438 points, equivalent to small marbling content according the classification used. Likewise, the Longissimus dorsi area was not influenced (P= 0.1881) by weight variation from pregnant cows (Figure 5).

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Figure 4: Forest plot for Marbling (points*) of the progeny from cows with different weight variations during gestation

The solid line of the x-axis is the no-effect line and dotted lines represent the estimated difference of the random model; therefore, the points to the left of the line represent a reduction in the trait, while the points to the right of the line indicate an increase. Each square relative weight of the study of the overall estimate of effect size with the larger squares representing a larger weight. The upper and lower bound of the squared line represents the upper and lower confidence intervals of 95% for the size of the effect. The diamond at the bottom represents the 95% confidence interval for the global estimate. * 100 = Practically Devoid; 200 = Traces; 300 = Slight; 400 = Small; 500 = Modest.

Figure 5: Forest plot for Longissimus dorsi area (LDA, cm2) of the progeny from cows with different weight variations during gestation

The solid line of the x-axis is the no-effect line and dotted lines represent the estimated difference of the random model; therefore, the points to the left of the line represent a reduction in the trait, while the points to the right of the line indicate an increase. Each square relative weight of the study of the overall estimate of effect size with the larger squares representing a larger weight. The upper and lower bound of the squared line represents the upper and lower confidence intervals of 95% for the size of the effect. The diamond at the bottom represents the 95% confidence interval for the global estimate.

Discussion Among factors that can modify the uterine environment(23), maternal nutrition during pregnancy stands out, which according to the authors can modify developing fetus

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metabolism and physiology. Several studies have demonstrated interferences from nutrition of the pregnant cow and the consequent variation in cow's weight and body score, but with many divergences, on the progeny performance in adulthood. In the metaanalysis, it was found some influences of cow’s weight variation at final gestation period on the steer’s carcass characteristics. The trends for higher hot carcass weight and subcutaneous fat thickness for the progeny of MG cows presented in Figures 2 and 3, respectively. The results corroborate the theories described by Du et al(2), who state that improving nutrition during the final stage of pregnancy favors the processes of myogenesis and adipogenesis of progeny, and consequently improves muscle mass and fat in the carcass. In a similar study, Rodrigues et al(24) obtained higher HCW in cows that gained up to 10 % of their body weight during pregnancy compared to cows that lost 0 to 10 % and 10 to 20% of weight during that period. The authors did not observe changes in SFT in that study. Body growth depends on the processes of hyperplasia and hypertrophy of preformed muscle fibers during pregnancy(2), and the nutritional restriction in this period impairs these processes due to the lower nutritional priority compared to other fetal tissues and organs(25). Unlike subcutaneous fat, the weight variation of the pregnant cow did not alter the intramuscular fat deposition, known as marbling fat (Figure 4). Like the body fat deposition, the formation of adipocytes during pregnancy follows a chronological sequence. In a scheme presented by Du et al(26), there is sequential and overlapping deposition of visceral, subcutaneous, intermuscular and intramuscular fat. Du et al(3) conclude that the formation of intramuscular adipocytes, the last to be formed, can extend over the first months of an individual’s life (approximately 250 d). Thus, postnatal life nutrition could have more effect than fetal programming intramuscular adipogenesis(27), according to the results obtained in the present meta-analysis, since adipocytes, despite being scarce, can increase their size as nutritional leftovers occur. In general, the similarity in adipose tissue deposition may be a consequence of the small variation in weight among cows that lost or gained weight during pregnancy, with an average of less than 5%. The cow weight gain during late pregnancy did not improve the Longissimus dorsi area (Figure 5), corroborating the findings of Rodrigues et al(24). This result may be a consequence of environmental adaptation of cow calves after birth. Webb et al(5) describe that malnutrition or food restriction during pregnancy ends up producing a phenotype that has greater adaptive skills when exposed to unfavorable environments in adulthood. Ramírez et al(27) conclude that the severe nutrient restriction during pregnancy can also compensate for the individual’s growth after birth, when it is exposed to restricted environments also after birth. Bell et al(28) also add that there may be a plasticity of postnatal rearing systems in regulation of muscle hypertrophy capable of overcoming the negative effects at pregnancy nutritional restriction.

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In addition to the greater adaptation capacity from progeny in postnatal life, the fetal programming effects and the nutrients supply of the fetus can be dependent on metabolic adaptation capacity of pregnant cows. Bauman et al(29) describe that nutrient partition of cows through hemorrhagic and homeostatic mechanisms, where the fetus has nutritional body priorities. These mechanisms may explain the mobilization of body reserves and cow weight loss during pregnancy to maintain an adequate supply of nutrients to the fetus under moderate conditions of nutritional restrictions(5). Thus, a small reduction in body weight of the pregnant cow, within 0 to ± 5%, can be accepted in production systems as it does not interfere with the progeny carcass characteristics. Thus, these results corroborate those of Klein et al(6), who found through the literature review that effects of fetal programming, or pregnant cow nutrition, are more noticeable in the early months of the progeny 's life, with lesser effects with the advancing age of these animals. Brameld et al(30) complement that, with enough time during postnatal life, the animal is able to overcome or compensate for most of these initial differences, resulting in only small (if any) residual effects on body composition in later growth stages. In general, the absence of effects on pregnant cow nutrition at carcass characteristics verified in this study can be attributed to the low weight variation or challenge to pregnant cows. The intensity of nutritional insult is an important factor to be considered in evaluation of the effects from fetal programming on offspring quality. Therefore, the adoption of a nutritional system that provides weight gain to pregnant cows not only depend on progeny performance evaluation, but also on a economic analysis of the entire calf production cycle according the desired goals.

Conclusions and implications The results obtained in this meta-analysis indicate that small cow's weight variations effects during the second and / or third trimester of pregnancy are difficult to be found in adulthood and post-slaughter carcass characteristics of the progeny. Literature cited: 1. Zago D, Canozzi MEA, Barcellos JOJ. Pregnant cow nutrition and its effects on fetal weight – a meta-analysis. J Agric Sci 2019;157(1):1-13. 2. Du M, Tong J, Zhao J, Underwood KR, Zhu MJ, Ford SP. Nathanielsz PW. Fetal programming of skeletal muscle development in ruminant animals. J Anim Sci 2010;88(1):51-60. 3. Du M, Wang B, Fu X, Yang Q, Zhu MJ. Fetal programming in meat production. Meat Sci 2015;109(1):40-47.

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4. Reynolds LP, Borowicz PP, Caton JS, Crouse MS, Dahlen CR, Ward AK. Developmental programming of fetal growth and development. Vet Clin North Am Food Anim Pract 2019;35(1):229-247. 5. Webb MJ, Block JJ, Funston RN, Underwood KR, Legako JF, Harty AA, et al. Influence of maternal protein restriction in primiparous heifers during mid and/or late-gestation on meat quality and fatty acid profile of progeny. Meat Sci 2019;152(1):31-37. 6. Klein, JL, Soares DSM, Adams SM, Alves Filho DC, Brondani IL. Efeitos da nutrição materna na gestação sobre a qualidade da progênie – uma revisão. Res Soc Dev 2021;10(2):1-10. 7. Bohnert DW, Stalker LA, Nyman A, Falck SJ, Cooke RF. Late gestation supplementation of beef cows differing in body condition score: Effects on cow and calf performance. J Anim Sci 2013;91(1):5485-5491. 8. Larson DM, Martin JL, Adams DC, Funston RN. Winter grazing system and supplementation during late gestation influence performance of beef cows and steer progeny. J Anim Sci 2009;87(1):1147-1155. 9. Maresca S, López Valiente S, Rodriguez AM, Testa LM, Long NM, Quintans GI, Pavan E. The influence of protein restriction during mid- to late gestation on beef offspring growth, carcass characteristic and meat quality. Meat Sci 2019;153(1):103108. 10. Mohrhauser DA, Taylor AR, Gonda MG, Underwood KR, Pritchard RH, Wertz-Lutz AE, Blair AD. The influence of maternal energy status during mid-gestation on beef offspring tenderness, muscle characteristics, and gene expression. Meat Sci 2015;1101:201-211. 11. Mohrhauser DA, Taylor AR, Underwood KR, Pritchard RH, Wertz-Lutz AE, Blair AD. The influence of maternal energy status during midgestation on beef offspring carcass characteristics and meat quality. J Anim Sci 2015;93(1):786-793. 12. Mulliniks JT, Mathis CP, Cox SH, Petersen MK. Supplementation strategy during late gestation alters steer progeny health in the feedlot without affecting cow performance. Anim Feed Sci Technol 2013;185(1):126-132. 13. Wilson TB, Schroeder AR, Ireland FA, Faulkner DB, Schike DW. Effects of late gestation distiller’s grains supplementation on fall-calving beef cow performance and steer calf growth and carcass characteristics. J Anim Sci 2015;93(1):4843-4851. 14. Wilson TB, Faulkner DB, Shike DW. Influence of prepartum dietary on beef cow performance and calf growth and carcass characteristics. Livest Sci 2016;184(1):2127.

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15. Rodrigues LS. Nutrição no terço final da gestação: eficiência produtiva da vaca e desempenho da progênie até os doze meses de idade. [doctoral thesis]. Brazil, RS: Universidade Federal de Santa Maria; 2019. 16. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria, 2020. 17. Schwarzer G. Meta: General package for meta-analysis. 2016. https://cran.rproject.org/web/packages/meta/index.html, Accessed 15 July, 2020. 18. Egger M, Smith GD, Schneider M, Minder C. Bias in meta-analysis detected by a simple, graphical test. BMJ 1997;315(1):629-634. 19. Appuhamy JADRN, Strathe AB, Jayasundara S, Dijkstra J, France J, Kebreab E. Antimethanogenic effects of monensin in dairy and beef cattle: A meta-analysis. J Dairy Sci 2013;96(8):5161-5173. 20. Lean IJ, Rabiee AR, Duffield TF, Dohoo IR. Invited review: Use of meta-analysis in animal health and reproduction: Methods and applications. J Dairy Sci 2009;92(8):3545-3565. 21. Lean IJ, Thompson JM, Dunshea FR. A meta-analysis of zilpaterol and ractopamine effects on feedlot performance, carcass traits and shear strength of meat in cattle. PLoS One 2014;9(12):1-28. 22. Higgins JPT, Thompson SG, Deeks JJ, Altman DG. Measuring inconsistency in metaanalyses. BMJ 2003;327:557-560. 23. Tsuneda PP, Hatamoto ZLK, Duarte Júnior MF, Silva LES, Delbem RA, Motheo TF. Efeitos da nutrição materna sobre o desenvolvimento e performance reprodutiva da prole de ruminantes. Invest 2017;16(1):56-61. 24. Rodrigues LS, Moura AF, Alves Filho DC, Brondani IL, Klein JL, Adams SM, Cocco JM, Pereira LB. Análise dos componentes principais da variação de peso da vaca durante a gestação na programação fetal em fêmeas. Res Soc Dev 2021;10(2):1-14. 25. Zhu MJ, Ford SP, Means WJ, Hess BW, Nathanielsz PW, Du M. Maternal nutrient restriction affects properties of skeletal muscle in offspring. J Physiol 2006;575(1):241-250. 26. Du M, Huang Y, Das AK, Duarte MS, Dodson MV, Zhu MJ. Manipulating mesenchymal progenitor cell differentiation to optimize performance and carcass value of beef cattle. J Anim Sci 2013;91(1):1419-1427. 27. Ramírez M, Testa LM, Valiente SL, La Torre E, Long NM, Rodriguez AM, Pavan H, Maresca S. Maternal energy status during late gestation: Effects on growth performance, carcass characteristics and meat quality of steers progeny. Meat Sci 2020;164(1):1-7.

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28. Bell AW, Greenwood PL. Prenatal origins of postnatal variation in growth, development and productivity of ruminants. Anim Prod Sci 2016;56(8):1217–1232. 29. Bauman DE, Currie B. Partitioning of nutrients during pregnancy and lactation: a review of mechanisms involving homeostasis e homeorhesis. J Dairy Sci 1980;63(9):1514-1529. 30. Brameld JM, Greenwood PL, Bell AW. Biological mechanisms of fetal development relating to postnatal growth, efficiency and carcass characteristics in ruminants, in: Greenwood PL, et al, editors. Managing the prenatal environment to enhance livestock productivity. Dordrecht: Springer Science and Business Media 2010;93120.

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https://doi.org/10.22319/rmcp.v13i4.6006 Article

Risk factors associated with lentivirus seroprevalence in sheep and goat herds from northeastern Mexico

Rogelio Ledezma Torres a José C. Segura Correa b Jesús Francisco Chávez Sánchez a Alejandro José Rodríguez García a Sibilina Cedillo Rosales a Gustavo Moreno Degollado a Ramiro Avalos Ramírez a*

a

Universidad Autónoma de Nuevo León, Nuevo León. Facultad de Medicina Veterinaria y Zootecnia. Campus de Ciencias Agropecuarias, calle Francisco Villa s/n colonia ExHacienda El Canadá, 66050. General Escobedo, Nuevo León, México. b

Universidad Autónoma de Yucatán. Facultad de Medicina Veterinaria y Zootecnia. Campus de Ciencias Biológicas y Agropecuarias. Mérida, Yucatán, México.

*Corresponding author: ramiro.avalosrm@uanl.edu.mx

Abstract: A cross-sectional study was conducted with the purpose of determinate the risk factors associated with the serological frequency of small ruminant lentivirus (SRLV) in sheep and goats from northeastern Mexico. From 128 herds, 71 of goats, 32 of sheep and 25 mixed herds (goats + sheep), 768 individual sera were collected from animals ≥1 yr old. From each herd, 4 to 5 serum samples were mixed and analyzed by ELISA to identify antibodies against SRLV glycoprotein 135. Samples were obtained from randomly selected animals in 2019 and 2020. A questionnaire was applied to the producers and the data were analyzed to

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determine the risk factors associated with herd seropositivity by logistic regression. The proportion of seropositive herds, overall, was estimated at 50.6 %. According to the type of herd, seropositivity in goat herds was 62.0 %, in sheep herds 25.4 % and 50.2 % in mixed herds. The risk factors associated with the presence of antibodies against SRLV were the presence of animals with arthritis, veterinary care, reuse of needles, nerve alterations, low pregnancy rate, type of herd and mastitis. Serological frequency indicates a high endemicity of SRLV in small ruminant herds from northeastern Mexico. Key words: Retrovirus epidemiology, Small ruminants, Arthritis, Veterinary care, Biosecurity on farms.

Received: 11/06/2021 Accepted: 07/04/2022

Introduction In northeastern Mexico, sheep and goats are the two species of ruminants with the greatest territorial dispersion and form one of the main economic livelihoods for the rural population of this area(1,2). In most of the herds from this area, a semi-extensive management system is practiced, in which the animals graze during the day, and before the spend the night in artisanal pens made of plant material from the region. Usually, no protein supplement, vitamins are offered, or adequate sanitary management is applied. Reports associated with health, reproduction and productivity disorders are common in herds(3,4,5). Obviously, the lack of technical assistance, training, absence or lack of biosecurity, among others, contribute to these health problems(2,6). Of the viral agents that affect sheep and goats, the infection caused by the small ruminant Lentivirus (SRLV) has become relevant in recent years(7,8). SRLV is a non-zoonotic virus of the genus Lentivirus, subfamily Orthoretrovirinae and family Retroviridae, highly contagious and infectious among goats and sheep(9). Initially, SRLV was named caprine arthritis encephalomyelitis virus or ovine progressive pneumonia virus (also called MaediVisna virus) since it was considered as two different pathogens specific to goats and sheep, respectively. However, it has been recognized that this virus can cross the species barrier and infect both ruminants(10,11). In addition, molecular genetic studies have shown that, genetically, SRLV is the same virus, so it is currently recognized as a single virus with viral variants adapted to goats and sheep(12). SRLV infections are lifelong and are characterized

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by causing a chronic multisystem inflammatory disease, with slow and progressive development, that may or may not manifest itself clinically in the life of the animal(13). They are characterized by gradual emaciation that leads to poor body condition and shortness of breath associated with interstitial pneumonia; alterations in the central nervous system, multiple arthritis and indurative mastitis in both species(9). The clinical manifestation depends on the genetic characteristics of the infecting SRLV strain, its tissue tropism, the affected animal species and its genetics(9,14). SRLV infections have a worldwide distribution(7) and are associated with significant economic losses(15,16). In Mexico, the serological presence of SRLV in goats from Mexico was reported in 1985(17) and the isolation of the virus in 1999(18). The serological presence of SRLV in goat herds from northeastern Mexico was reported in 1994(19). Initially, a higher seroprevalence of SRLV was estimated in goat herds under intensive management in milk production and newly imported from the United States of America(17,19). In Mexico, serological detection(20,21,22) and pathological damage associated with SRLV infection in goats and sheep(23) have been reported. Until 2012, Mexico was considered free of SRLV infection in sheep, but this infection is currently considered endemic and is within group 3 of diseases and pests in the national territory(24). Serological and molecular evidence of SRLV infection in Mexican Pelibuey sheep was demonstrated in herds of Jalisco, Veracruz and Chiapas(25), State of Mexico and Querétaro(22). However, studies of the presence, effects and impact of SRLVs on the health and productivity of goats and sheep from Mexico are scarce. The coexistence and multiple interrelationships between small ruminant populations in Mexico, particularly in the northeast of the country, usually increases the risk of acquisition and spread of SRLV and other pathogens(6,22). It is known that sheep and goats can harbor multi-species infectious agents with the potential to affect these and other animal species and even humans(26,27). In fact, SRLV can be considered within this category, so associated infections could trigger disease outbreaks and mortality. Given these conditions, a high serological frequency of SRLV at the herd level is considered and can be potentiated by at least one risk factor. Therefore, the objective of this study was to estimate the seroprevalence and determine the risk factors associated with Small Ruminant Lentivirus infection in sheep and goat herds in northeastern Mexico.

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Material and methods Location and characteristics of herds

A cross-sectional study was conducted, selecting 128 herds located in northeastern Mexico, in the states of Coahuila, Nuevo León and Tamaulipas. The management system of the herds was mostly semi-extensive. In general, the animals showed nutritional, reproductive and health complications.

Number of herds and animals sampled

A total of 768 animals were sampled from 71 goat herds, 32 sheep herds and 25 mixed herds (n=128 herds). For the state of Nuevo León, the sampling considered the total number of ranches registered in the 2017 list of beneficiaries of the Sustainable Livestock Production and Livestock and Beekeeping Management Program obtained by the Secretariat of Agriculture, Livestock, Rural Development, Fisheries and Food of Mexico. For the states of Coahuila (Laguna Region) and Tamaulipas, the animals sampled were herds of farmers who, in a direct interview, expressed their desire to cooperate. The sample size of 128 herds and 768 animals was calculated using the computer program EpiMuestra(28). Because there was no information on SRLV infections for goats and sheep in northeastern Mexico, the following was considered: an expected prevalence of 50 %, a confidence level of 95 % and absolute accuracy of 5 %. The sampling was in two stages, first selecting the herds and then the animals within each herd, arbitrarily considering a design effect of 2(28). The sampling unit for analysis was the herd. Serological analyses were analyzed by groups that corresponded to a homogeneous mixture of 4 to 5 sera per herd(29) at a rate of 200 μL per animal. The herd whose serum mixture was positive in the commercial ELISA test was considered positive.

Serum samples and their handling

Serum samples were obtained between the autumn of 2019 and late spring of 2020. Blood was obtained by puncture of the jugular vein and vacuum tubes with coagulation activator gel (Becton Dickinson, www.bd.com). The samples were identified and transported to the 998


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laboratory under refrigeration conditions at 7 °C (±3) in a polystyrene container. In the laboratory, the sera were separated from the clot after centrifuging the tubes at 2,500 rpm for 5 min. Each serum sample was deposited in new sterile plastic tubes of 2 ml. Each tube was labeled with its individual code, date and origin. All samples were stored at -20 °C in the serum bank of the Laboratory of Virology of FMVZ-UANL, until their use in the ELISA test.

Field information collection

The identification of possible risk factors was determinated based on the responses of the farmers in an individually applied survey. The survey consisted of 30 questions, and they included aspects of the type of farm, health and animal health aspects, as well as the identification and location of the herd.

Detection of anti-SRLV antibodies

The detection of anti-SRLV antibodies was performed by competitive ELISA with the commercial kit Small Ruminant Lentivirus Antibody Test Kit, cELISA, (WMRD Inc., Pullman, WA, USA). This test detects antibodies directed against highly conserved antigenic sites of glycoprotein 135 of caprine arthritis encephalomyelitis virus. The sensitivity and specificity reported for the test was 100 % and 96.4 %, respectively(30). The reading of the reaction in each well was made at an optical density of 650 nm in the ELISA reader (ELx800, Bio-Tek®) and using the computer package KC Junior software (www.biotek.com). The presence of antibodies was derived from the calculation of the percentage of inhibition according to what was recommended by the manufacturer, using the formula: I = 100 {1 – (OD of the sample/ OD of the NC)} Where: I is the percentage of inhibition; OD is the optical density detected; NC is the negative control. For the validation of the test, an average of the OD of the NC ≥ 0.300 was considered. If the I value of the sample was ≥ 35 %, it was considered as positive, while an I <35 % as negative(30).

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Statistical analysis

From the positive reactions in the ELISA test of each herd, the actual seroprevalence of SRLV was estimated by means of the online tool WinEpi version 2.0(31). For the estimation of proportions and 95 % confidence intervals (CI95%), the sensitivity and specificity reported by the manufacturer of the ELISA kit was included(30). To determine the association between risk factors and SRLV seropositivity, initially, possible risk factors were identified in a univariate analysis by the Chi-square test or Fisher’s exact test (PROC FREQ). Those factors with a (P>0.20) were subjected to a multivariate logistic regression analysis (Table 1) by means of the LOGISTIC procedure. Factors significant to Fisher’s exact test with fewer than 5 observations were not included in the logistic regression analysis. All analyses were performed using the statistical package SAS of 2010.

Results and discussion Herd seroprevalence

The herd seroprevalence value against SRLV of 50.6 %, obtained in the present study (Table 1), is consistent with those obtained in other parts of the world(30,31,32) but contrasts with previous studies conducted in Mexico(21,33,34,35). Recently, Martínez-Herrera et al(33), using an indirect ELISA test, reported a lower herd-level seroprevalence in Creole goats from Veracruz, Mexico, with 6.4 %. Also, Torres-Acosta et al(21), using agar gel immunodiffusion (AGID), reported in 2003 an apparent seroprevalence of 3.6 % in goat herds, mostly Creole from the state of Yucatan, Mexico. Previously, in 1984 Adams et al(34), using AGID, reported at the individual level in goats from the State of Mexico and Guanajuato serological frequencies of 22.1 % and 6.3 %, respectively. These same researchers mentioned not finding antibodies in native Creole goat herds(34). Santiago et al(35), using the same ELISA test as the present study, found a herd-level seropositivity of 41.3 % in samples of goats from the state of Guanajuato, Mexico. According to the above, the design of the study, the management, type and purpose of the herds, as well as the lack of biosecurity measures against SRLV probably influenced the seropositivity parameters of the present and each of the previous studies. In 1984 and 1985, a high seropositivity in goats of dairy breeds imported into Mexico and absence of seropositivity in native Creole goats were reported(17,34). These observations and data from the present study suggest that SRLV entered native Creole herds from northeastern Mexico perhaps through contact with imported breed goats for the purpose of improving productivity. In the present study, no significant difference was found(36) between

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the seroprevalence of the types of herds, of goats (63.0 %), of sheep (25.4 %) or mixed (50.2 %). These data contrast with those obtained in similar herds from other countries(30,31,32) in which the seropositivity indices are relatively low compared to those of the present study. However, this coincides with what has been reported in previous studies for the management of a single species, either sheep or goats(32,36,37) and when they are managed under mixed conditions(36). The type of serological test used, the management and characteristics of the environment could explain the differences found. Table 1: Prevalence of antibodies against small ruminant lentivirus (SRLV) in sheep and goat herds in northeastern Mexico Herd Goats Sheep Mixed (goats + sheep) Overall

n 71 32 25 128

(+) 45 9 13 67

ActP 62.0 25.4 50.2 50.6

CI95% 50.7 – 73.3 10.3 – 40.5 30.6 – 69.8 41.9 – 59.2

ActP = actual prevalence, *Sensitivity (100%) and specificity (96.4%) of the ELISA test (30), 95% confidence level(31).

Risk factors associated with serology at the herd level

After analysis in contingency tables, a total of 21 factors out of 30 were selected to evaluate their association with SRLV seropositivity in goat and sheep herds. The risk factors that contributed significantly to the explanation of SRLV seropositivity were: type of herd, veterinary care, multiple use of needles, low pregnancy rate, presence of animals with arthritis, report of nerve alterations and animals with mastitis; these last three alterations associated with chronic inflammatory processes (Table 2). The logistic regression analysis showed a significant effect of the same factors as in Fisher’s exact test or Ji-square test; but the following factors were not included: herd size, introduction of animals, quarantine, biosecurity and mixed herds because each of them had ≤5 observations. Several reports(32,33,36) have indicated these last variables as risk factors so they were included in the discussion. Table 2 shows the risk factors associated with SRLV seropositivity in goat and sheep herds from northeastern Mexico. In northeastern Mexico, it is relatively common to find mixed goat-sheep herds. The coexistence of both species could facilitate the transmission of SRLV not only through direct contact but also through the intake of colostrum or milk(37,38) and through other management practices such as the use of needles in several animals during the application of medicines, vaccines or identification earrings(6,37,39).

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Table 2: Herd seroprevalence and risk factors for SRLV seropositivity in goat and sheep herds from northeastern Mexico Variable

n

Seroprevalence

P

Odds Ratio (IC95%)

ART

Yes 63 No 65

82.5 23.1

<0.0001

31.3 (6.7-142.8) 1

CARE

Yes 19 No 109

94.7 44.9

<0.0001

11.9 (1.0-142.9) 1

NEED

Yes 69 No 59

63.8 38.9

0.005

9.6 (2.1-43.5) 1

NERV

Yes 47 No 81

78.7 37.0

<0.001

7.4 (1.9-28.6) 1

PREG

Yes 50 No 78

74.0 38.5

<0.0001

6.8 (1.8-25.6) 1

Caprino 71 Mixto 25 Ovino 32

63.4 52.0 28.1

0.0041

5.4 (1.0-28.2) 2.2 (0.4-12.3) 1

Yes 66 No 62

71.2 32.5

<0.0001

4.9 (1.3-17.9) 1

HER

MAST

CI = class intervals, ART = presence of arthritis, CARE = veterinary care, NEED = repeated use of needles, NERV = presence of nerve alterations, PREG = low pregnancy rate, HER = type of herd, MAST = presence of mastitis.

A strong association was found between the type of herd, of goats (OR 5.4; CI95%= 1.0-28.2) or mixed (goats + sheep) (OR 2.1; CI95%= 0.3-12.3), with SRLV seropositivity. Similar observations have reported that the presence of goats is a risk factor that contributes to the SRLV seropositivity in sheep(39,40). Herd size has been reported as another important factor that influences SRLV seropositivity, because one of the routes of transmission of this virus is through direct contact between infected animals(36,39,40). However, this variable was excluded from the logistic regression analysis due to the low number of observations and to meet the data quality criteria for analysis. In the present work, a strong association was found between the presence of animals with arthritis (OR 31.2; CI95%= 6.7-142.8) and with mastitis (OR 4.8; CI95%= 1.3-17.8) in seropositive herds. SRLV infections are characterized by being strongly related to these clinical-pathological conditions(40,41,42). For sheep, a high association has been reported between the occurrence of mastitis and SRLV infection in endemic

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herds(9,39,40). In a recent study, it was determined that when the goat farmer recognizes the presence of arthritis, SRLV infection is widespread in the herd(42). It has also been proposed that the development of arthritis depends on the genetic characteristics of the infecting SRLV strain(43). What was observed in the present study indicates that animals with chronic arthritis are present with high frequency in seropositive herds regardless of the animal species and the type of management. An association was found between reproductive problems and SRLV seropositivity. Herds in which the producer recorded a low pregnancy rate (≤50 %) were more likely to be seropositive than those with pregnancy rates ≥50 %. Few studies have focused on knowing the impact of SRLV on reproduction in small ruminants. Recently, the ability of this virus to induce intrauterine infections in small ruminants and be transmitted via semen either with artificial insemination or natural mounting was reported(44). However, no association was found between the presence of abortions or animals with low birth weight in the herd, which contrasts with previous studies in which the delayed development of newborn kids was associated with the seropositivity of the mother(37,39). Probably, the differences between the two observations are explained due to the nature of both studies or the bias in the responses given by the producers in the present research. Interestingly, an association (P<0.0001) was found between SRLV seropositivity and veterinary care (OR=11.9; CI95%= 1.0-142.8). An important form in the horizontal transmission of SRLV is contact with humans, particularly the movement of veterinarians and workers between and within herds(39). It is possible to consider that veterinary care would play an important role in controlling SRLV infection; the increase in seropositivity in herds of small ruminants with vectorization by the veterinarian from one herd to another during their visits has been reported(39). The data obtained in the present work probably reflect that the same veterinarian cares for different herds. This was not included in the surveys, allowing the horizontal transfer of the virus in the region studied due to ignorance of the presence of the virus in the region, its consequences and forms of dispersion. One study indicates that the presence of humans, as well as the number of employees and years of experience in management within herds are related to the presence and circulation of the virus(39). On the other hand, the absence of biosecurity, hygiene and disinfection measures greatly increases the presence of SRLV infection(42). Therefore, it is necessary to make producers aware of this agent, as well as to let them know the biosecurity guidelines to prevent the circulation of the virus in their herds. Based on the above, the epidemiological observations indicated for more than 25 yr for SRLV in goats from Mexico are confirmed and expanded(17,19,34). In addition to this, the present work is the first serological report of SRLV infection in sheep from northeastern Mexico. SRLV is considered as a single virus with genetic variants adapted to goats or sheep(8,9). The possibility of cross-infections(10,11) and the isolation of recombinants among the genetic 1003


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variants of the virus have also been reported(41). Given the above, it is interesting to determine if the serological response found in small ruminants is directed towards genetic variants of SRLV adapted to goats or sheep(22). Likewise, to be able to specify if recombinant SRLVLs(8,10) that have managed to adapt to both sheep and goats in the area(11) circulate in the northeast of Mexico.

Conclusions and implications SRLV seropositivity in sheep and goats from northeastern Mexico is relatively high. This is the first serological report of SRLV infection in sheep from northeastern Mexico. The estimated seroprevalence and risk factors detected in seropositive herds should be considered in the design of biosecurity programs and public policy applied to the health and productivity of goat and sheep herds in Mexico.

Acknowledgements

To the participating goat and sheep producers from northeastern Mexico, without whose willingness the present work would not have been possible. The study was financially supported through the 2019 Research, Science and Technology Support Program (PAICyT, for its acronym in Spanish) of the Autonomous University of Nuevo León. Literature cited: 1. Escareño-Sánchez LM, Wurzinger M, Pastor-López F, Salinas H, Sölkner J, Iñiguez L. La cabra y los sistemas de producción caprina de los pequeños productores de la Comarca Lagunera, en el norte de México. Rev Chapingo Serie Cienc Forest Amb 2011;17(Esp):235-246. https://doi.org/10.5154/r.rchscfa.2010.10.087. Consultado 5 Oct, 2021. 2. Alva-Pérez J, López-Corona LE, Zapata-Campos CC, Vázquez-Villanueva J, BarriosGarcía HB. Condiciones productivas y zoosanitarias de la producción caprina en el altiplano de Tamaulipas, México. Interciencia 2019;44(3):154-160. 3. Mellado M, Valdez R, Lara LM, Garcıa JE. Risk factors involved in conception, abortion, and kidding rates of goats under extensive conditions. Small Ruminant Res 2004;55:191-198.

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4. Avalos-Ramírez R, Cedillo-Rosales S, Salinas-Meléndez JA, Morales-Loredo A, Cervantes-Vega R, Domínguez-Díaz D, et al. Parasitosis y enfermedades comunes de caprinos en majadas de Nuevo León: Prevalencia y descripción. 1ª ed. México: Consorcio Técnico del Noreste de México, AC. 2010. 5. Salinas-González H, Valle Moysen ED, de Santiago-Miramontes MDLA, Veliz-Deras FG, Maldonado-Jáquez JA, Vélez-Monroy LI, et al. Análisis descriptivo de unidades caprinas en el suroeste de la región lagunera, Coahuila, México. Interciencia 2016;41:763-768. 6. Avalos-Ramírez R, Cedillo-Rosales S, Salinas-Meléndez JA, Morales-Loredo A, Cervantes-Vega R, Domínguez-Díaz D, et al. Bioseguridad en hatos caprinos: Protocolos aplicados en majadas de Nuevo León. 1ª ed. México: Consorcio Técnico del Noreste de México, AC. 2010. 7. Gomez-Lucia E, Barquero N, Domenech A. Maedi-Visna virus: current perspectives. Vet Med (Auckl) 2018;9:11-21. 8. Ramírez H, Reina R, Amorena B, de Andrés D, Martínez HA. Small Ruminant Res Lentiviruses: genetic variability, tropism and diagnosis. Viruses 2013;5(4):1175-1207. 9. Minguijón E, Reina R, Pérez M, Polledo L, Villoria M, Ramírez H, et al. Small ruminant research Lentivirus infections and diseases. Vet Microbiol 2015;181(1-2):75-89. 10. Da Cruz JC, Sigh DK, Lamara A, Chebloune Y. Small ruminant lentiviruses (SRLVs) break the species barrier to acquire new host range. Viruses 2013;5(7):1867-1884. 11. Leroux C, Chastang J, Greenland T, Mornex JF. Genomic heterogeneity of small ruminant lentiviruses: existence of heterogeneous populations in sheep and of the same lentiviral genotypes in sheep and goats. Arch Virol 1997;142(6):1125-1137. 12. Reina R, Berriatua E, Lujan L, Juste R, Sánchez A, Andres D, et al., Prevention strategies against small ruminant lentiviruses: an update. Vet J 2009;182(1):31-37. 13. Blacklaws BA. Small ruminant lentiviruses: Immunopathogenesis of visna-maedi and caprine arthritis and encephalitis virus. Comp Immunol Microbiol Infect Dis 2012;35(3):259-269. 14. Callado AKC, Castro RS, Teixeira MFS. Lentivírus de pequenos ruminantes (CAEV e Maedi-Visna): revisão e perspectivas. Pes Vet Bras 2001;21(3):87-97. 15. Azevedo DAA, Santos VWS, Sousa, ALM, Peixoto RM, Pinheiro RR, Andrioli A, et al., Small ruminant lentiviruses: economic and productive losses, consequences of the disease. Arqui Inst Biol 2017;84:1-10.

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16. Martínez-Navalón B, Peris C, Gómez EA, Peris B, Roche ML, Caballero C, et al., Quantitative estimation of the impact of caprine arthritis encephalitis virus infection on milk production by dairy goats. Vet J 2013;197(2):311-317. 17. Nazara SJ, Trigo FJ, Suberbie E, Madrigal V. Estudio serológico de la artritis-encefalitis caprina en México. Tec Pecu Mex 1985;48:98-101. 18. Daltabuit Test M, de la Concha-Bermejillo A, Espinosa LE, Loza Rubio E, Aguilar Setién A. Isolation of caprine arthritis encephalitis virus from goats in Mexico. Can J Vet Res 1999;63(3):212-215. 19. Villarreal-Cavazos DA. Prevalencia serológica del virus de la artritis encefalomielitis caprina (VAEC) en algunos hatos caprinos del Noreste de México [tesis licenciatura]. México, NL: Universidad Autónoma de Nuevo León; 1994. 20. Molina RM, Trigo FJ, Cutlip RC. Estudio serológico de la neumonía progresiva ovina en México. Vet Méx 1986;17:269-273. 21. Torres-Acosta JF, Gutiérrez RE, Butler V, Schmidt A, Evans J, Babington J, et al., Serological survey of caprine arthritis-encephalitis virus in 83 goat’s herds of Yucatán, México. Small Ruminant Res 2003;49:207-211. 22. Loeza CJG. Detección serológica y molecular de lentivirus de pequeños rumiantes que circulan de forma natural en ovinos de dos estados del altiplano mexicano. [tesis especialidad]. México, Estado de México: Universidad Autónoma del Estado de México; 2017. 23. Eguiluz C, Aluja A. Neumonía intersticial progresiva (Maedi) y adenomatosis pulmonar en vísceras de óvidos decomisadas. Vet Méx 1981;12:235-237. 24. SADER. Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación. DOF. Acuerdo mediante el cual se dan a conocer en los Estados Unidos Mexicanos las enfermedades y plagas exóticas y endémicas de notificación obligatoria de los animales terrestres y acuáticos. México. 2018. 25. Sánchez JH, Martínez HA, García MM, Garrido G, Gómez L, Aguilar JA, et al., The presence of small ruminant lentiviruses in Mexican Pelibuey sheep. Theriogenology 2016;86(1):1953–1957. 26. Ganter M. Zoonotic risks from small ruminants. Vet Microbiol 2015;181(1-2):53-65. 27. Villagra-Blanco R, Barrantes-Granados O, Montero-Caballero D, Romero-Zúñiga JJ, Dolz G. Seroprevalence of Toxoplasma gondii and Neospora caninum infections and associated factors in sheep from Costa Rica. Parasite Epidemiol Control 2019;4:e00085. https://doi.org/10.1016/j.parepi.2019.e00085 . 1006


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28. Segura-Correa JC, Honhold N. Métodos de muestreo para la producción y salud animal. Universidad Autónoma de Yucatán. Mérida, Yucatán, México. 2000. 29. Hernandez-Medrano JH, Espinosa-Castillo LF, Rodriguez AD. et al. Use of pooled serum samples to assess herd disease status using commercially available ELISAs. Trop Anim Health Prod 2021;53:507. https://doi.org/10.1007/s11250-021-02939-1. 30. Herrmann LM, Cheevers WP, McGuire TC, Adams DS, Hutton MM, Gavin WG, et al. Competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to caprine arthritis-encephalitis virus: diagnostic tool for successful eradication. Clin Diagn Lab Immunol 2003;10(2):267-71. 31. de Blas I, Ruiz-Zarzuela I, Vallejo A. WinEpi: Working in epidemiology. An online epidemiological tool. ISVEE 11: Proc 11th Sympe Int Soc Vet Epidemiol Econom, Cairns (Australia), August 6-11 2006. Theme 4 - Tools & training for epidemiologists: Poster session. 2006;800. 32. Pérez M, Biescas E, de Andrés X, Leginagoikoa I, Salazar E, Berriatua E, et al. Visna/maedi virus serology in sheep: survey, risk factors and implementation of a successful control programme in Aragón (Spain). Vet J 2010;186(2):221-225. 33. Martínez–Herrera DI, Villagómez-Cortes JA, Hernández-Ruiz SG, Peniche-Cerdeña AEJ, Pardío-Sedas VT, Torres-Acosta F, et al. Seroprevalence and risk factors for caprine arthritis-encephalitis in the state of Veracruz, Mexico. Agrociencia 2020;54(1):15-29. https://agrocienciacolpos.mx/index.php/agrociencia/article/view/1879/1876. Consultado 24 nov. 2021. 34. Adams DS, Oliver RE, Ameghino E, DeMartini JC, Verwoerd DW, Houwers DJ, et al. Global survey of serological evidence of caprine arthritis-encephalitis virus infection. Vet Rec 1984;115:493-495. 35. Santiago BCI, Gutierrez HJL, Herrera LE, Palomares REG, Díaz AE. Diagnóstico serológico de Lentivirus de Pequeños Rumiantes (LvPR) en rebaños caprinos del estado de Guanajuato. Quehacer Científico en Chiapas 2017; 12(1):15-19. https://dgip.unach.mx/images/pdf-REVISTA-QUEHACERCIENTIFICO/2017-enerjun/1.Diagnostico_serologico_de_Lentivirus.pdf. Consultado 30 nov 2021. 36. Michiels R, Van Mael E, Quinet C, Welby S, Cay AB, De Regge N. Seroprevalence and risk factors related to small ruminant lentivirus infections in Belgian sheep and goats. Prev Vet Med 2018;151:13-20. 37. Norouzi B, Taghavi RA, Azizzadeh M, Mayameei A, Najar NMV. Serological study of small ruminant lentiviruses in sheep population of Khorasan-e-Razavi province in Iran. Vet Res Forum 2015;6(3):245-249.

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38. Gjerset B, Jonassen CM, Rimstad E. Natural transmission and comparative analysis of small ruminant lentiviruses in the Norwegian sheep and goat populations. Virus Res 2007;125(2):153-161. 39. Junkuszew A, Dudko P, Bojar W, Olech M, Osiński Z, Gruszecki TM, et al. Risk factors associated with small ruminant lentivirus infection in eastern Poland sheep flocks. Prev Vet Med 2016;127:44-49. 40. Kalogianni AI, Bossis I, Ekateriniadou LV, Gelasakis AI. Etiology, Epizootiology and Control of Maedi-Visna in Dairy Sheep: A Review. Animals (Basel). 2020;10(4):616. 41. Gayo E, Cuteri V, Polledo L, Rossi G, García MJF, Preziuso S. Genetic characterization and phylogenetic analysis of small ruminant lentiviruses detected in Spanish Assaf sheep with different mammary lesions. Viruses 2018;10(6):315. 42. Czopowicz M, Szaluś-Jordanow O, Mickiewicz M, Moroz A, Witkowski L, Bereznowski A, et al., Relationship between the dissemination of small ruminant lentivirus infection in goat herds and opinion of farmers on the occurrence of arthritis. PLoS One. 2018;13(9):e0204134. 43. Pérez M, Biescas E, Reina R, Glaria I, Marin B, Marquina A. et al. Small ruminant lentivirus-induced arthritis: clinicopathologic findings in sheep infected by a highly replicative SRLV B2 genotype. Vet Pathol 2015;52(1):132-139. 44. Furtado-Araújo J, Andrioli A, Pinheiro RR, Sider LH, deSousa ALM, de Azevedo DAA, et al. Vertical transmissibility of small ruminant lentivirus. PLoS One. 2020;15(11):e0239916.

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https://doi.org/10.22319/rmcp.v13i4.6100 Article

Family sheep production systems in the Mixteca region of Oaxaca, Mexico

Jorge Hernández Bautista a Héctor Maximino Rodríguez Magadán a Teódulo Salinas Rios a Magaly Aquino Cleto a Araceli Mariscal Méndez a*

a

Universidad Autónoma Benito Juárez de Oaxaca. Facultad de Medicina Veterinaria y Zootecnia, Oaxaca, México

*Corresponding author: mariscalma@hotmail.com

Abstract: Family sheep production is common in rural Mexico. It is an important element of subsistence systems in these areas but is generally rustic. Better understanding of rustic sheep production is a first step in developing strategies and programs to support family producers. Family sheep production units in two municipalities in the Mixteca region of Oaxaca, Mexico, were characterized in terms of production system, market access and land use. A mixed methodology was applied, employing a structured questionnaire addressing socioeconomic and productive variables, and participatory observation in 29 family sheep producers. All the surveyed producers see sheep farming as their main income source. Most (86 %) use a subsistence system, and all use family labor. The main feeding strategy was grazing of communal land, and production was largely intended for sale of live animals to intermediaries or in local markets for eventual processing for meat, and/or for self-use. Most (83 %) of the production units included a pen built from regional materials, and these pens were most frequently on the family property. Implementation of management plans and animal health and safety measures were minimal. Analysis of these productive systems 1009


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identified how producers manage sheep production. Management strategies respond to the environmental services available on communal lands, and involve family-type production which fulfills economic, social, environmental and cultural functions, but provides low productivity. Unit productivity and producer livelihood could be improved by implementing measures such as pasture rotation and adopting technological innovations. Broadening producer access to government programs and creating public policy that promotes development in marginal rural areas could greatly improve productivity and consequently reduce poverty and food insecurity. Key words: Family production, Sheep Production, Small producers, Mixteca.

Received: 24/11/2021 Accepted: 08/04/2022

Introduction Mexico has a sheep population of 8.9 million(1). Sheep farming occurs in different regions throughout the country and production systems respond to local resource availability and market conditions. Production unit scale is influenced by socioeconomic conditions, land access, and input and technology availability. Extensive, subsistence family production units (FPUs) are the most common but are largely limited to valleys, hills and mountains in rural areas(2). Improving management practices in FPUs in marginal agricultural zones can provide environmental, socioeconomic and/or nutritional benefits(3). Though considered production systems, FPUs are also a way of life, a structure of social relationships and an element of identity in peasant cultures(4). Most (63.4 %) subsistence UPFs are in the states of Mexico, Oaxaca, Guerrero, Puebla, Chiapas, Veracruz, Hidalgo and Michoacán; 52.0 % of these are in highly marginalized areas and 16.4 % in extremely marginalized areas(5). Of the thirty-two states in Mexico, Oaxaca is among the five poorest, with 61.7 % of its population living below the poverty line(6). It is also has the sixth largest sheep population in the country, most of which are produced in subsistence FPUs in the Mixteca and Central Valleys regions(7). A majority (78 %) of the state’s highly and extremely marginalized municipalities are in the Mixteca region, and 77.4 % of the population in this region lives in rural, small and dispersed localities; the main economic activities are seasonal agriculture and small ruminant production(8). Small livestock production based on extensive grazing and communal work has been present in the

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region since 1530, and is still widely used(9). Small ruminants are an integral element in regional culinary tradition, and a vital contribition to the peasant economy since their production is low-cost and they provide multiple benefits(10,11). Traditional production systems of this kind help to mitigate poverty by promoting food sovereignty and security, and generating employment in agriculture, in addition to contributing to environmental, climate and cultural sustainability in rural areas(12). However, FPUs face myriad challenges such as technological, social, economic, environmental and political changes (e.g., globalization). Given their precarious economic situation, small rural producers can be acutely affected and experience technological regression in production systems(13). The large livestock population in Oaxaca’s Mixteca region provides very low production value; for example, the current $59.74 kg average regional price for sheep is much lower than the $76.34 kg national average(7,14). Rural production systems in Mexico are extremely heterogeneous. They must adapt to varying availabilities of different natural, human and financial resources, and inconsistent and unequal access to institutions and markets. Strategies intended to promote and strengthen small family livestock production must encompass this heterogeneity to generate policies differentiated by producer type that are not overly generalized in scope(13). Tailoring policy design to meet the needs of specific production systems requires identification of their particular characteristics, including their scale, management practices and territory. Strategies can then be designed and implemented based on PU type and resources, which also allow for their analysis, promote organization and social actor participation, and result in differentiated policies that help to develop marginalized rural areas. The present study objective was to analyze sheep FPUs in the municipalities of Suchixtlahuaca and Coixtlahuaca in the Mixteca region of Oaxaca, Mexico.

Material and methods The study was carried out in the municipalities of Coixtlahuaca (17°38’ and 17°49’ N, 97° 09’ and 97° 25’ W; altitude 2,000 to 2,900 m asl) and Suchixtlahuaca (17°43’ and 17°72’ N, 97°22’ and 97°36’; altitude 2,000 to 2,900 m asl) in the Mixtec region of Oaxaca (Figure 1). Both municipalities have a temperate sub-humid climate with summer rains, a 15.6 °C average annual temperature and 500 to 1,000 mm annual rainfall(15,16).

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Figure 1: Study area. San Juan Bautista Coixtlahuaca and San Cristóbal Suchixtlahuaca, Mixteca region, Oaxaca, Mexico

Scale 1:250000. Political boundaries as of 2012(17).

Vegetation cover is limited in both municipalities, and includes oak, juniper, savin and cacti, as well as shrubs used as sheep forage such as Leucaena leucocephala, Vachellia farnesiana, Prosopis laevigata and Morus spp. This is partially due to the region’s low rainfall and consequent semi-arid hydrogeography. Just north of the town of Suchixtlahuaca is the Rio Grande and to the south is the Rio de la Cruz, both of which are seasonal(15). La Culebra river, the main drainage in Coixtlahuaca, is predominantly seasonal(16). The population of Coixtlahuaca largely considers itself indigenous (64.72 %) and is highly marginalized in socioeconomic terms. In Suchixtlahuaca, most (65.59 %) of the population considers itself indigenous and experiences moderate marginalization(17). From August 2017 to February 2018, a mixed methodology was used to study sheep producers using a FPU system in these populations. Since the number of production units and their locations were not known, a non-probabilistic (a.k.a. snowball) sampling method was applied(18), resulting in a sample of 29 producers. Data on population socioeconomic parameters and sheep producer systems was collected using a questionnaire structured in two sections:

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1) Socioeconomic data. Items addressed the variables of gender, age, education level, years of experience in the activity, main source of herd management technical knowledge, nature of production unit, land tenure, production system type and main economic activity. 2) Production system data. Items addressed the variables of livestock inventory, breed, zootechnical purpose, production purpose, food strategy, infrastructure, health management, access to government programs and production diversification. Questionnaire data was supported and triangulated through participatory observation from the participating sheep producers. Family sheep production systems were classified into three categories based on market access and production system(19,20): a) Subsistence. Sheep diet is based on grazing grasses and legumes in rangeland. During the dry season, animals are fed stubble and straw harvested in the rainy season, in addition to sporadic supplements of salt and minerals; health and safety management is commonly poor. These systems have a holding pen. The animals represent savings for the producer, and are occasionally sold. The subsistence system also encompasses sheep production units (SPUs) in which animals remain in a holding pen all day and are fed stubble and poor quality straw. b) Transition. Sheep are fed by grazing in extensively managed paddocks with supplements. Preventive health management is used and producers have market access, although their market articulation is hindered by intermediaries. c) Consolidated. This intensive system involves two management strategies. One involves stabling animals and feeding with silage, hay, balanced feed and integrated rations. The feeding strategy is adjusted according to animal physiological stage. In the other, animals are intensively grazed in fenced areas on improved forages, commonly supplemented with concentrates. Both strategies employ an animal health calendar and a record system. Producers have access to sufficient feed within their production units and access to local markets. However, this system depends heavily on government support and other income sources for livestock development. Data analysis was done by descriptive statistics and an analysis of variance (ANOVA) run with the Infostat statistical package. Participant observation data served to triangulate and contextualize the statistical analysis.

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Results and discussion The total analyzed sample of sheep producers was 29, nineteen (66 %) of which were in Coixtlahuaca and ten (34 %) in Suchixtlahuaca. Most (86 %, n= 25) production systems were subsistence and of these fifteen (60 %) were in Coixtlahuaca and ten (40 %) in Suchixtlahuaca. The remaining four systems (14 % of total) were transition systems located in Coixtlahuaca (Table 1). All the analyzed SPUs used family labor. Table 1: Sheep production systems by municipality Suchixtlahuaca Coixtlahuaca Production system n % n % Subsistence 10 100 15 79 Transition 0 0 4 21 Total 10 34 19 66

n 25 4 29

Total % 86 14 100

n= number of production units; %= proportion of total.

Socioeconomic data

Average producer age was 55.5 yr. Most (62 %, n= 18) were aged 20 to 59 years and the remainder (38 %, n= 11) were 60 yr or older. This coincides with the average age (52.6 yr) of heads of household reported for small SPUs(19). Although production unit owners were older in age, their children did participate in production activities. However, family participation does not ensure intergenerational continuity. Just because producers’ children have learned how to raise sheep and goats is no guarantee that they will continue in the activity once they inherit the production units. This raises the question of how to manage intergenerational turnover in production systems in a manner that maintains them as culturally relevant agroecosystems. Attaining this transition will require livestock breeds that provide economic value, are marketable in the region and beyond, meet local subsistence requirements and contribute to natural resources conservation and/or resilience. A majority of producers (97 %, n= 28) were men, with just one woman (3 %) in the sample. There are reports of the frequent participation of women in sheep production, particularly in subsistence systems. However, cultural perceptions in the two studied municipalities consider management of land and food production as work too strenuous for women and children; nonetheless, women and children do engage in these activities when men are otherwise occupied.

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Education level varied between the subsistence and transition systems evaluated in the present study. Of the producers involved in a subsistence system, 12 % (n= 3) had no formal education, 48 % (n= 12) had an elementary level education, 32 % (n= 8) had a middle school education, and 8 % (n= 2) a high school education. These data are consistent with previous reports of family-managed subsistence-level sheep production systems(17,18). Among the producers using transition systems, 50 % (n= 2) had a middle school education and 25 % (n= 1) a high school education and professional training. Average education level among the subsistence level producers (6.8 yr) was clearly lower than among the transition system producers (11.7 yr). This supports previous reports that producers with a higher education level tend to employ greater technification in their production systems(20). It also coincides with observations that agricultural activity in rural areas in Mexico is largely managed by peasants with low education and specialization levels(10). Indeed, the studied municipalities are highly to moderately marginalized and their populations suffer social deficiencies such as low education levels. Limited access to education prevents rural populations from specializing or acquiring training. This is coupled with their greater dependence on agricultural activities, and the fact that knowledge of productive activities is transferred between family members. No formal education is required since, through social reproduction, they acquire knowledge and understanding of their territory from social interaction and use of tangible and intangible assets. The average number of years dedicated by producers to sheep farming was 28.1 yr, highlighting the deep-rooted tradition of sheep production in these communities. Among the producers using a subsistence system, average years of experience was 29.5 yr (± 2.18), while among those in a transition system it was 19.5 yr (± 5.45). One common aspect among all the producers was that they had entered the activity because a relative had already begun it; in other words, they continued their predecessors’ efforts, essentially preserving a tradition. They continue the tradition even though production can be hampered by challenges such as health problems and low market prices, among other factors. Agricultural activities (agriculture and livestock) were the sole economic activity for most (65.5 %, n= 19) of the producers, and sheep production was the principal income source. Slightly more than half (52 %, n= 13) the subsistence producers identified themselves as peasants living off agriculture and livestock, while 20 % (n= 5) perceived themselves as only ranchers, and 24 % (n= 6) as vendors. Among the transition producers, only one (25 %) stated agriculture and livestock to be their main economic activity, while another two (50 %) perceived themselves as vendors (Table 2). This agrees with previous studies observing that small agricultural producers tend to diversify their income strategies, mostly pursuing agriculture, sheep production and sales(10,21).

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Table 2: Main economic activities of sheep producers by production system Sheep production system Main occupation Subsistence Transition n % n % Farmer/Rancher

13

52

1

25

Rancher

5

20

0

0

Construction

1

14

0

0

Vendor

6

24

2

50

Teacher

0

0

1

25

n= number of producers; %= proportion of total.

Among the subsistence producers, most (88 %, n= 22) used communal lands and 12 % (n= 3) owned small properties. All (100 %, n= 4) the transition producers used communal lands. These results contradict reports stating that use of small private properties is more widespread in transition and commercial systems(20). In the present study, this discrepancy may exist because in the historically agricultural Mixteca region communal land tenure is the primary form of tenure(22). This highlights the fact that the Mixteca region is a socially-constructed, rather than a merely geographic, space within which communal access is allowed on some land resources under certain rules. Therefore, sheep producers can graze their animals in the same area year round, regardless of a pasture’s carrying capacity.

Production system data

Total sheep population in all the studied SPUs was 1,222, and average herd size was 42 heads (SD, s= 43). At the municipality level, average herd size was 23.58 heads (± 11.16) in Coixtlahuaca and 68.50 (± 12.54) in Suchixtlahuaca. In the subsistence SPUs, average animal inventory was 50.08 heads (± 8.10) and in the transition SPUs it was 15.50 heads (± 19.83). An SPU’s animal inventory is linked to the production system and the feasibility of implementing technological improvements. Making technological improvements is particularly difficult for small producers since they are generally rudimentary, have only limited infrastructure, experience difficulty in accessing credit and are managed by producers with low education levels. In the present results, the subsistence SPUs had larger inventories, perhaps because they are based on grazing resources available in their natural surroundings, which keeps costs low. In contrast, transition SPUs employ more technology, consequently raising herd management costs.

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The SPUs studied here produced sheep for live sale and eventual processing for meat. In the subsistence SPUs, animals were sold in bulk at a price imposed by an intermediary. Animal weight was not considered; perhaps for this reason weight is not recorded in this type of system. In the transition SPUs, average final animal weight for sale was 35 kg. Most (55.1 %, n= 16) of the SPUs used Creole breeds or Creole x commercial breed crosses as their main breed. The subsistence SPUs mostly used Creole breeds or crosses (56 %, n= 14), although many (40 %, n= 10) did use Pelibuey or Pelibuey crosses. Half (50 %, n= 2) the transition SPUs employed Creole animals or Creole crosses, although Pelibuey and Dorper animals were present at one unit each (i.e., 25 %) (Figure 2). Overall, Creole breeds continue to dominate among the studied SPUs, although commercial breeds are increasingly used. Local or Creole breeds may remain popular in the study area because they are adapted to local conditions and therefore conserved by small producers. They form an integral part of sustainable use strategies in which sheep can feed on crops and/or wild vegetation, then provide food and other resources to people(23). Figure 2: Sheep genotypes in subsistence and transition production systems in Coixtlahuaca and Suchixtlahuaca, Oaxaca, Mexico

25

Breed

Pelibuey crossbreed Pelibuey

0

Dorper croossbreed

0

28

12 4 25

Dorper

0 25

Creole crossbreed

48

25

Creole

8

0

10

20

Transition

30 40 Percentage

50

60

Subsistence

All (100 %, n= 29) the SPUs had holding pens and basic livestock infrastructure. Most (83 %, n= 24) of the infrastructure was made from regionally available materials (mesquite and oak wood), and the rest (17 %, n= 5) were made with metal; 40 % (n= 2) of those with metal structures were transition SPUs. Most (60 %) of the producers with metal infrastructure had accessed it via social programs, while the others had repurposed metal elements as a way of keeping down costs. Feeders and drinkers in pens were used in all the transition SPUs, whereas none of the subsistence SPUs had feeders and 76 % (n= 19) had drinkers inside pens.

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The lack of drinkers in some of the subsistence SPUs, and feeders in all of them, may be because they are extensive grazing systems in which the animals only spend the night in a pen; producers may feel a feeder or drinker is unnecessary. These results coincide with previous reports of the use of regionally available materials in family-managed SPUs(24), which allows producers to exploit natural assets and/or ecosystem services. Most (88 %, n= 22) of the subsistence producers used grazing as the sole feed source. This occurs from 0800 to 1600 h every day, with the producers leading animals to pasture in the morning and penning them in the afternoon (Table 3). The remaining producers (12 %, n= 3) based feeding on a combination of forage and grain. These same producers have small properties and, lacking extensive grazing land, must feed in pens. All four (100 %) transition SPUs fed in pens. Two (50 %) fed with forage, one (25 %) fed an integrated diet, and another (25 %) used a combination of grazing and an integrated diet. These producers may have more knowledge and technical training as well as economic resources to invest in their production system. The subsistence producers are taking advantage of the surrounding natural environment to lower production costs, but it is also an element of their participation in a regional socio-cultural agroforestry system. Grazing is done on communal lands, which provides substantial cost savings on the main production input, but also requires active participation in the community’s social structures. Regional tree-shrub forages such as L. leucocephala, V. farnesiana, P. laevigata and Morus spp. are quite palatable to sheep, but also provide high crude protein content(25). When managed rationally using strategies such as rotation, this renewable natural resource can be extremely productive and environmentally stable. The 19 (66 %) subsistence SPUs implemented pasture rotation. However, they did not use technical-productive strategies to manage this rotation, rather they based it on how the animals use the land and the paths the animals graze along; animal load was not considered. Year round grazing of an area or region allows for no prolonged rest period for vegetation renewal and regeneration, decreasing potential animal productivity. Grazing natural vegetation on communal lands can be sustainable and economically viable if ecosystem services are efficiently exploited by applying a grazing plan. This needs to consider the amount of forage provided by the vegetation, the most appropriate animal load to effectively exploit available forage, the most efficient grazing time, the target number of animals to be sold yearly, and herd productive and reproductive events.

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Table 3: General characteristics of subsistence and transition sheep production units Subsistence n % 22 88

Category Regional materials Metal and other materials Feeder Pen infrastructure Drinker Sale Production Self-use purpose Sale/self-use Intermediary Market Local market Not sold Integrated diet Forage Forage/grain Feeding strategy Grazing Grazing/integrated diet Grazing/forage/grain Pen

Pasture rotation Land tenure Vaccination Deparasitization Livestock production diversification

Transition n % 2 50

3

12

2

50

0 19 21 1 3 20 4 1 0 0 3 10

0 76 84 4 12 80 16 4 0 0 12 40

4 4 3 0 1 0 4 0 1 2 0 0

100 100 75 0 25 0 100 0 25 50 0 0

0

0

1

25

12

48

0

0

Yes

19

76

0

0

No

6

24

4

100

Communal

22

88

4

100

Small property

3

12

0

0

Yes

0

0

3

75

No

25

100

1

25

Yes

24

96

4

100

No

1

4

0

0

Yes

22

88

1

25

No

3

12

3

75

n= number of units, %= proportion of total.

Planned grazing provides ecosystem benefits such as manure, the growth of smaller-sized plant species, and reduction of dry plant material, a potential fuel for fires(26). If implemented based on a well-designed plan, exploitation of natural resources by sheep producers can be seen as effective usufruct of communal lands that interweaves their livelihoods. If no plan properly guides this use, it can lead to degradation of vegetation, increased soil erosion, deteriorated soil fertility and structure, and a consequent reduction in forage availability and thus animal productivity. 1019


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Among the subsistence SPUs, production was aimed at sale of live animals in most cases (84 %, n= 21), while fewer units (12 %, n= 3) used it for sale of live animals and self-use, and just one (4 %) was only for self-use. Of the four transition SPUs, three (75 %) used production for sale of live animals and one (25 %) for sale of live animals and self-use. These results support previous research indicating that in Mexico small ruminants are widely used for sale and self-use, a dynamic adapted to the rural and culinary culture which includes traditional dishes such as roasted lamb in the Mixteca region of Oaxaca(11). Family production units like those in the present study fulfill tangible and intangible functions (4). Tangible functions include cash generation from sale of animals, food for self-use and in some cases manure which serves as the fertilizer that completes the productive cycle. Intangibles include the roasted sheep which is an integral element in regional food culture since it is prepared for family celebrations and religious festivities, is transmitted and enriched intergenerationally, and forms a part of regional sociocultural dynamics. The family nature of the studied SPUs also has the intangible function of perpetrating a cultural element since grazing occurs on communal land, the traditional production methods are transferred from parents to children and the herd itself represents both the family’s livelihood and its continued participation in cultural traditions(2). It is essentially a lifeway contained within a territory with its own landscape, natural environment and customs aimed at reproducing the sheep production system and with it regional culture. Market access differed between the production systems (P<0.05). In the subsistence SPUs, most (80 %, n= 20) sold their animals to intermediaries, while only four (16 %) sold them in local markets. All (100 %, n= 4) the transition SPUs sold in local markets (Table 3), where the animals were acquired by intermediaries, roasters, finishers or other producers. The dependence of the subsistence SPUs on intermediaries may be linked to the generally low education level among producers, and their lack of resources, organization and information. This translates into marked inequity in market access, lower prices imposed by intermediaries and low added value. In contrast, the transition SPUs sell directly in the markets and are thus able to command better prices. Vaccinations were only applied in three (75 %) of the transitions SPUs (Table 3), although infrequently, and the producers were unclear as to what vaccine had been used. Internal deworming was done by all the transition SPUs (100 %, n= 4) and most of the subsistence SPUs (96 %, n= 24). Very few of the producers took additional health care measures such as administering vitamins and minerals. These results coincide with a previous study indicating that family livestock production systems largely lack health and safety measures and receive minimal management. Productivity is consequently low, as is the income generated for producers(24). Only two (6.8 %) of the studied producers (both transition SPUs) had accessed government programs. That all the subsistence SPUs had no access to these programs suggests that low 1020


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producer education level may play a role, although other factors also surely come into play, such as insufficient program information and promotion, and a lack of specific programs, differentiated policies, technical assistance, research and targeted financing focused on subsistence producers. The absence of institutional benefits in these two studied municipalities highlights their marginalization. Diversification of agricultural production was present in most (79 %, n= 23) of the producers. Only one (25 %) of the transition SPUs diversified its production, whereas 22 (88 %) of the subsistence SPUs did so. Poultry for self-use (eggs and/or meat) was used in all (100 %) the diversified SPUs, but pigs (meat), cows (meat, milk, work animals) and horses (work animals) were also raised for self-use (Table 3 and Figure 2).

Conclusions and implications Sheep farming in the studied municipalities is a largely subsistence activity following a peasant approach in that exploitation focuses on goods and services provided by the land, such as grazing areas and zoogenetic resources. Sheep farming is a livelihood as well as a traditional activity that fulfills socioeconomic, environmental and cultural functions in the region. Limiting factors in the studied production units include advanced age of the producers, their low educational level, their ignorance of and/or minimal participation in government programs, lack of organization and access to efficient marketing channels. In conjunction, these factors substantially lower productivity and profitability of sheep production. Only a small proportion of the production units were in transition, emphasizing the need to promote public policies for development in marginalized rural areas. The guiding axis of these policies needs to be organization and association of small family producers to work towards innovation, technology transfer, and access to financing and local markets. Improving sheep production practices in regions that depend heavily on this activity can be a very effective way of addressing the ongoing issues of poverty and food insecurity.

Conflict of interest

The authors declare no conflict of interest related to the research reported herein or its publication.

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Literature cited: 1. SADER. Secretaría de Agricultura y Desarrollo Rural. Atlas agroalimentario 2012-2018. 2019. https://nube.siap.gob.mx/gobmx_publicaciones_siap/pag/2018/AtlasAgroalimentario-2018. Colsultado 20 jul, 2019. 2. Partida de la Peña J, Braña V, Jiménez S, Ríos R, Buendía R. Producción de carne ovina. México: Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. 2013. 3. Moreno DC, Grajales HA. Caracterización de los sitemas de producción ovinos de trópico alto en Colombia: Manejo e indicadores productivos y reproductivos. Rev Med Vet Zoot 2017;64(3):36-51. 4. Samper M. Sistemas territoriales de agricultura familiar. IICA, Ed. San José, Costa Rica. 2016. 5. SAGARPA, & FAO. Agricultura familiar con potencial productivo en México. http://www.sagarpa.gob.mx/programas2/evaluacionesExternas/Lists/Otros%20Estudio s/Attachments/42/Agricultura%20Familiar_Final.pdf . Consultado 2 feb, 2022. 6. CONEVAL. Consejo Nacional de Evaluación de la Política de Desarrollo Social. Medición de la pobreza en entidades federarivas. https://www.coneval.org.mx/coordinacion/entidades/Oaxaca/Paginas/principal.aspx. Consultado 2 feb, 2022. 7. SIACON NG. Sistema de Información Agroalimentaria de Consulta. Pecuario Estatal 2020. México: Servicio de Información Agroalimentaria y Pesquera, SADER. https://www.gob.mx/siap/documentos/siacon-ng-161430. Consultado 20 mar, 2022. 8. UTM. Universidad Tecnológica de la Mixteca. Diagnóstico Regional de la Mixteca. Oaxaca: Comité de Planeación para el Desarrollo del Estado. 2017. 9. Romero F. Economía y vida de los españoles en la Mixteca Alta: 1519-1720. México, DF: Instituto Nacional de Antropología e Historia. 1990. 10. Desauguste M, Lerdon J, Moreira L, Alomar C. Caracterización de la producción ovina en la agricultura familiar de la comuna de Paillaco, Región de los ríos, Chile. Agro Sur, 2011;39(2):88-94. 11. Vásquez G. Ganado menor y enfoque de género. Aportes teóricos y metodológicos. Agricultura, Sociedad y Desarrollo 2015;12(4):515-530. 12. López S, Carrión A. Geografía, economía y territorios rurales en América Latina: presentación del dossier. Eutopía U. 2018;(4):7-22.

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13. CEPAL. Comisión Económica para América Latina y el Caribe. Efectos sociales de la globalización sobre la economía campesina: reflexiones a partir de experiencias en México, Honduras y Nicaragua. México CEPAL. 1999. 14. SEDAPA. Secretaria de Desarrollo, Agropecuario, Pesca y Acuacultura/SNIDRUS. Atlas agroalimentario del Estado de Oaxaca. 2017. 15. Hernández B. Plan Municipal de Desarrollo 2008-2010. H. Ayuntamiento Constitucional San Cristóbal Suchixtlahuaca, Oax. 2008 https://www.finanzasoaxaca.gob.mx/pdf/inversion_publica/pmds/08_10/129.pdf. 16. Juárez F. Plan Municipal de Desarrollo. H. Ayuntamiento Constitucional de San Juan Bautista Coixtlahuaca, Oaxaca. 2008 . https://finanzasoaxaca.gob.mx/pdf/inversion_publica/pmds/08_10/176.pdf. 17. INEGI. Instituto Nacional de Estadísticas Geografía e Informática. Encuesta inter censal. Oaxaca: Dirección General de Población de Oaxaca. 2015 https://www.inegi.org.mx/contenido/productos/prod_serv/contenidos/espanol/bvinegi/ productos/nueva_estruc/inter_censal/panorama/702825082307.pdf Consultado 2 feb, 2022. 18. Hernández SR, Fernández CC, Baptista LP. Metodología de la investigación 6a. ed. México DF: McGraw-Hill; 2014. 19. Hernández-Bautista J, Salinas-Rios T, Rodríguez-Magadán HM, Aquino-Cleto M, Mariscal-Méndez A, Ortiz-Muñoz IY. Características que determinan el sistema de producción ovina en el estado de Oaxaca, México. Rev Mex Agroecosist 2017;4(1):3847. 20. Pérez H, Vilaboa A, Chalete M, Bernardino M, Díaz R, López O. Análisis descriptivo de los sistemas de producción con ovinos en el estado de Veracruz, México. Rev Cientifica 2011:XXI(4):327-334. 21. Robles B. La pequeña agricultura campesina y familiar: construyendo una propuesta desde la sociedad. Entre Diversidades 2016;(7):46-83. 22. Lazos ChE. Conocimientos, poder y alimentación en la mixteca Oaxaqueña: Tareas para la “gobernanza ambiental”. CLACSO. 2012 . 23. Díaz T, Valencia P. Lineamientos para el Fortalecimiento de la Producción Pecuaria Familiar en América Latina y el Caribe. En: Salcedo S, Guzmán L. Agricultura familiar en América Latina y el Caribe: recomendaciones de política. Santiago, Chile 2014:165174.

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24. Cuéllar OJA, Tórtora PJ, Trejo GA, Román RP. La producción ovina mexicana, particularidades y complejidades. S./FES-UNAM Ed. Distirto Federal, México: Ariadna; 2012. 25. Hernández HJ. Valoración de la caprinocultura en la Mixteca Poblana: socioeconomía y recursos arbóreo-arbustivos [tesis doctoral]. Cuba: Universidad de Camagüey; 2006. 26. Lasanta T. Pastoreo en áreas de montaña: Estrategias e impactos en el territorio. Rev Estudios Geográficos 2010:LXX1(268):203-233.

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https://doi.org/10.22319/rmcp.v13i4.5277 Review

Hypocalcemia in the dairy cow. Review

Carlos Fernando Arechiga-Flores a* Zimri Cortés-Vidauri a Pedro Hernández-Briano a Renato Raúl Lozano-Domínguez a Marco Antonio López-Carlos a Ulises Macías-Cruz b Leonel Avendaño-Reyes b

a

Universidad Autónoma de Zacatecas. Unidad Académica de Medicina Veterinaria y Zootecnia. El Cordovel, Enrique Estrada, Zacatecas, México. b

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

*Corresponding author: arechiga.uaz@gmail.com

Abstract: Calcium (Ca) levels decrease in blood and cytosol at the time of calving, altering nerve impulse transmission, muscle contraction, and immune cell activity. In the nervous system, Ca participates in the conduction of stimuli. In the muscular system, it decreases contractions, causing alterations in smooth muscle, uterus and mammary gland. In the uterus, there is retention and storage of uterine fluids and waste, with bacterial complications. In the immune system, the function of neutrophils is important, and it manifests itself with a decrease in cells engaged in phagocytosis, predisposing to mastitis and metritis. In bovine hypocalcemia, two manifestations are distinguished: clinical and subclinical. In the clinical one (Ca values less than 5.5 mg/dl), homeostasis alters, with loss of appetite, decubitus and lethargy.

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Subclinical hypocalcemia is more common (Ca between 8.0 and 5.5 mg/dl), and homeostasis does not alter, but muscle contraction and immune function decrease. The treatment is based on the application of calcium orally in standing cows, and intravenously in prostrate cows. Prevention depends on the inclusion of rations that contain anionic salts, which favors the stimulus to maintain blood Ca levels to control the level of cations and anions. In addition, Ca can be administered orally. Calcium homeostasis in lactation is regulated by the serotonin hormone, which stimulates the parathyroid hormone and bone resorption in osteoclasts. Key words: Hypocalcemia, Dairy cow, Homeostasis, Calcium, Serotonin, Metritis.

Received: 22/02/2019 Accepted: 12/03/2020

Introduction The transition period of the dairy cow comprises 3 wk before and 3 wk after calving(1) and several physiological changes occur in the obtaining of nutrients for the calving process, expulsion of fetal membranes and production of colostrum and milk. Therefore, circulating levels of calcium (Ca) decrease in blood and cytosol(2,3). Homeostasis or self-regulation of Ca normally uses the following feedback mechanism: it decreases the concentration of ionized calcium (iCa2+), stimulating the parathyroid gland to secrete the parathyroid hormone (PTH). PTH binds to its hormone receptors in kidneys and bone tissue. In the kidneys, PTH increases renal reabsorption of Ca as well as the increase in production of 1,25dihydroxyvitamin D, the active form of vitamin D(4). Vitamin D stimulates the epithelial cells of the intestine to increase the active transport of Ca(5). If the calcium in the diet is insufficient to generate homeostasis, the mechanism is directed to bone tissue(4). Dairy cows slowly begin the reabsorption of Ca from bone tissue, but the accelerated demand of the mammary gland induces clinical hypocalcemia(2). The parathyroid gland (PTH) participates in the homeostasis of Ca, but there is also the function of the parathyroid hormone-related protein (PTHrP), secreted in the mammary gland(6). The serotonin hormone is responsible for stimulating the production of the PTHrP protein(7). Serum Ca is present in three forms: ionic (iCa2+) or free calcium (50 % of total calcium), bound to proteins (approximately 40 %) and in the form of complexes with anions (10 %). iCa2+ is the only biologically active calcium. Calcium participates in nerve, muscle and immune functions(8-10). At the nervous level, it participates in the conduction of stimuli. At

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the muscular level, in muscle contraction, and in the immune part, with the function of immune cells. Therefore, cows with hypocalcemia alter these functions depending on the severity in the decrease in calcium. There are two types of hypocalcemia: 1) clinical and 2) subclinical. The purpose of this review is to succinctly evaluate the incidence of hypocalcemia, as well as its consequences on immune function, metritis and mastitis(11-13).

Hypocalcemia Hypocalcemia is a metabolic-nutritional disease, caused by the decrease in blood Ca. It usually occurs after calving, its manifestation can be clinical and subclinical.

Clinical hypocalcemia

Clinical hypocalcemia, also known as milk fever or puerperal paresis, is characterized by a momentary imbalance in the regulation of the concentration of calcium (Ca) in the blood between 48-72 h postpartum. Serum Ca levels decrease to 5.5 mg/dL, with the subsequent alteration in homeostasis(14-15). This disease causes great economic losses in dairy production units, mainly due to the cost of treatments, secondary complications and the deaths it causes(13). Among the risk factors for hypocalcemia, the following are considered: 1) The age of the cow, 2) The high demand for Ca to produce colostrum and milk, 3) The diet consumed during the transition period. Animals recovered from puerperal hypocalcemia produce 5 to 15 % less milk in that lactation(14-15). That is, the homeostasis of Ca(16) alters, mainly affecting highly producing cows, showing loss of appetite, decubitus and lethargy. Its incidence varies from 5 to 7 %(14-21) and increases as lactations progress. Calcium is related to muscle contraction. During milk fever, muscle contraction and tone in the gastrointestinal and cardiovascular systems are not maintained, and it can cause the death of the animal. Immune function decreases(2,22), and the risk of postpartum diseases such as mastitis, fetal membrane retention (FMR), metritis and abomasum displacement increases(11-14,23-26). The clinical signs of hypocalcemia are divided into three phases(11). In phase I, the cow does not show paresis, it may even go unnoticed, its signs are tenuous and transient; it is hypersensitive, nervous, excitable, with muscle tremors, anorexia, ataxia and general weakness. Some cows lose weight quickly and drag their hind limbs. The animal avoids walking or moving, does not feed, body temperature can be normal, and it can remain several hours in that state. Some cows show clinical signs of hypocalcemia similar to those described, but without having calved. This alteration usually occurs after periods of stress or decrease in dry matter consumption. This condition is more common in cows in estrus or heat, with severe digestive

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disorders or severe toxic mastitis. Transient hypocalcemia may occur in cows with anorexia and low intestinal motility(17). Phase II, (prodromal), it exhibits moderate to severe depression, partial paralysis and the characteristic sign of lying down with the neck bent and with the head directed towards the flank. The tetany observed in the first phase progresses to impossibility to get up, paresis and prolonged decubitus, cold extremities, dry muzzle and temperature higher than normal (36.5 to 38 °C); weak arterial pulse, heart sounds, barely audible, and moderate heart rate (80/min). Absence of rumen movements is detected, which can lead to states of secondary exhaustion. Phase III is the most severe. The animal exhibits complete lateral decubitus. Severe cardiac depression, irregular pulse (almost imperceptible), decreased shallow breathing. Animals without an established therapy die in a few hours, with the manifestation of a state of shock. The diagnosis of milk fever is based on the history of the animal, age of the mother and serum calcium concentration. The decrease in serum magnesium and phosphorus levels may be associated with eosinopenia and lymphopenia (adrenal hyperactivity), but the latter are not specific. It is necessary to make the differential diagnosis with hepatic steatosis, septic endometritis, mastitis and acute rumen acidosis.

Subclinical hypocalcemia

It occurs when blood Ca decreases to levels less than 2.00 to 1.38 nM, but homeostasis continues(14). The normal concentration of Ca is 8.5 to 10 mg/dL (2.1 - 2.5 nM). It can start 12-24 h after calving, when the lowest concentration of Ca is recorded, and increases with a greater number of lactations, affecting up to 50 % of cows(2,14,20,27-28). That is why subclinical hypocalcemia is more expensive(29-30). The decrease in blood Ca is related to the transmission of nerve impulses that lead to less muscle contraction; with lower rumen and abomasal motility, with the subsequent displacement of the abomasum and lower food consumption(14,31). For example, with the reduction of the blood level of Ca to 7.5 and 5 mg/dl, the abomasal motility of cows decreases by 30 to 70 %, respectively(32). Its effects on muscle contraction also prevent the effective closure of the mammary nipple duct (teat), which contributes to the occurrence of mastitis, and its biological and economic consequences(13-14). In addition to the relationship of Ca with muscle contraction, Ca also affects immune function and insulin secretion(12). The function of neutrophils decreases, as the cytosolic concentration of ionized calcium (iCa2+) in peripheral blood mononuclear cells decreases(2,33). Therefore, the severity of the problem will manifest itself with secondary disorders related to production and reproduction, such as retention of fetal membranes and metritis(12,30-33). iCa2+ corresponds to approximately 50 % of total calcium, the rest is bound

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to proteins and is biologically inactive. Cows with subclinical hypocalcemia decrease insulin secretion and increase blood glucose concentration(3,33-35). Because the entry of glucose into peripheral tissues reduces, as happens in the period of insulin resistance during the postpartum period of the dairy cow(36). The cytosol of pancreatic cells requires iCa2+, which decreases during hypocalcemia, for the release of insulin(35-37). The decrease in insulin allows the release of the lipase hormone, responsible for participating in lipolysis. This increases the plasma concentration of non-esterified fatty acids (NEFAs)(12,20,23,27,37-39) with its corresponding risk of ketosis(23-25) due to the increase of ketone bodies: acetone, βhydroxybutyrate and acetoacetic acid in the bloodstream.

Immune function

The immune system contains cells and molecules with the ability to recognize and eliminate invading or foreign microorganisms; it is regulated by the cytosolic concentration of ionic calcium(8-10). The [Antigen-Receptor] binding of the immune cell triggers a series of events characterized by the increase of iCa2+ in the cytosol and depletion of iCa2+ reserves in the endoplasmic reticulum; this continues with the obtaining of additional iCa2+ from the extracellular space(40). Hypocalcemia reduces the cytosolic concentration of iCa2+ in the mononuclear cells of the blood, also reducing immune function(2). The ATPase pumps for iCa2+ of the sarcoplasmic and endoplasmic reticula regulate the entry and replacement of iCa2+ in the endoplasmic reticulum(41). The cytosolic increase of iCa2+ is needed for the adhesion of neutrophils to endothelial cells, their transmigration into tissues, chemotaxis and phagocytosis(42). This could be altered in cases of the decrease in extracellular iCa2+. In addition, control in the magnitude, amplitude and duration of the destination of iCa2+ in the immune cell is also required for the functions of immune cells(43). Immunity can be innate and specific.

Innate immunity

Innate immunity is activated quickly and constitutes the first immune defense when the infection begins. It depends on phagocytes such as polymorphonuclear neutrophils, macrophages and mammary epithelial cells. Macrophages identify and recognize foreign pathogens, produce cytokines (interleukin-1γ, interleukin-6 and tumor necrosis factor-α) to begin the immune response, they also recruit polymorphonuclear neutrophils. In addition, they phagocytize and eliminate invading pathogens and constitute a bridge between the innate response and the specific response through the major histocompatibility complex class

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II, to prepare T cells(44). After the start of the inflammatory response, the predominant cells are polymorphonuclear neutrophils, which, through blood circulation, are directed by chemotaxis to locate the site of invasion(45). Polymorphonuclear neutrophils, as well as macrophages, engulf and eliminate foreign microorganisms. In activated phagocytes, oxidative burst (respiratory burst) is triggered by the activation of the nicotinamide-adeninedinucleotide phosphate (NADPH) enzyme that catalyzes the reduction of oxygen to the superoxide anion, extremely toxic to foreign microorganisms. Finally, foreign microorganisms are eliminated by exocytosis. In hypocalcemia(12,22), the function of neutrophils reduces, the percentage of neutrophils engaged in phagocytosis decreases(3,12,22,3334) , the mononuclear cellular response to the antigen-activated stimulus weakens(2) and the oxidative burst response reduces after incubation with pathogenic bacteria(3). In neutrophils of cows with subclinical hypocalcemia, it has been observed that the cytosolic level of iCa2+ decreases more rapidly than in normocalcemic cows, therefore the influx of calcium is not sufficient to maintain and use the cytosolic iCa2+, or replenish endoplasmic reticulum deposits, or both. This leads to a decrease in their ability to phagocytize and eliminate pathogenic bacteria(3). The reduction of the immune response leads to the manifestation of other infections of bacterial origin such as mastitis(23-24) and metritis(12,46-48).

Specific immunity

It depends on antibodies, macrophages and T and B lymphocytes that recognize specific microorganisms(47). This immunity is activated if the infection persists. T cells are subdivided into helper T lymphocytes and cytotoxic T lymphocytes. Helper cells produce cytokines, such as interleukin (IL-2) and interferon gamma (IFN-γ), crucial in the immune response. Cytotoxic T cells recognize and eliminate cells infected with an antigen, as well as predecessor immune cells or damaged cells, which, when present, increase the susceptibility of infection. B lymphocytes differentiate into plasma cells that produce antibodies or immunoglobulins (Igs): IgG1, IgG2 and IgM, or memory cells(47).

Metritis Metritis (puerperal metritis) is a postpartum bacterial complication that can be caused by less contraction of the uterine muscle (myometrium), facilitating the entry and proliferation of bacteria in the uterus, or by less activity of immune cells. Both factors caused by hypocalcemia. This infection can lead to negative consequences on reproductive function during the postpartum period(48-49). In the first 65 d postpartum, the percentage of gestation

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at first service has been found in 39.4 % in cows diagnosed with metritis, 38.7 % in cows with clinical endometritis and 51.4 % in cows without uterine infection(50). Immune function is compromised before metritis. Circulating neutrophils in these cows have shown a decrease in glycogen at the time of calving, and monocytes stimulated by the bacterium Escherichia coli have reduced the expression of tumor necrosis factor-α(51). Metritis is characterized by an increase in uterine size, dark red and foul-smelling aqueous uterine discharges, associated with decay, loss of appetite, high heart rate, fever and decrease in milk production(52-54). There are predisposing factors such as fetal membrane retention (FMR)(55), fetal maceration and dystocia(53-58). The incidence of metritis ranges from 2.2 % to 37.3 %(59). At the herd level, the factors of greatest risk for the occurrence of metritis are the size of the herd (greater in large herds), time of year (greater in November and April), number of calving (greater in animals of three calvings or less), dystocia and placental retention(60-61). The process of infection is as follows: after calving, the cervix and cervical canal remain open for a few days for the expulsion of fluids and waste from the uterus, through the contraction of the uterine muscles(62-65). This process is more efficient in normocalcemic vs hypocalcemic cows(60,66-69). Hypocalcemic cows are more prone to retention and stagnation of uterine fluids and waste, and therefore to a greater risk of bacterial complications(60,66-69). Stagnation of fluids and waste is an excellent medium for bacterial multiplication(70-73). The opening of the cervix allows bacteria to enter the uterus, although their presence will not necessarily develop the infection. Bacteria have been isolated in most cows after calving(60,74-75), but it is controlled by the action of neutrophils and other leukocytes(57-60,67,76-79). They migrate to the uterine lumen in response to the presence of bacteria and are generally able to control bacterial populations until the infection is eliminated. The cow remains healthy and has a normal postpartum period: milk production, and a new conception and gestation. The above, however, does not always happen. In some cows with subclinical hypocalcemia(12), neutrophils do not stop the infection, bacterial populations grow, and females have purulent and fetid discharges, characteristic of metritis(80-81). In the diagnosis of gestation by rectal palpation, the uterus presents an increase in size and its inflammation suppresses postpartum follicular growth and development(79-82). The cows have a fever and remain depressed and inappetent. The lack of adequate food consumption predisposes to the presence of other disorders such as abomasum displacement and the fatty liver complex/ketosis. If the inflammation continues, it usually progresses to endometritis, which greatly compromises the cow’s fertility. Cows with subclinical hypocalcemia had a lower gestation rate and a longer interval from calving to conception compared to normocalcemic cows; the risk of metritis decreases with high levels of Ca in the blood(12).

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Mastitis Two models of mastitis transmission are recognized: environmental mastitis and contagious mastitis(83).

Environmental mastitis

Some normal microorganisms in the environment such as Escherichia coli, Klebsiella spp., Enterobacter spp., Serratia spp., Pseudomona spp., Proteus spp., and some gram-positive bacteria such as Streptococcus uberis and Streptococcus dysgalactiae, are the ones involved in causing environmental mastitis(84-87). The cow uses innate immunity to combat environmental mastitis, with physical barriers such as the teat sphincter; chemical barriers such as keratin and lactoferrin, and immune system components such as macrophages, dendritic cells, mast cells, neutrophils, eosinophils and natural killer cells (NKCs)(88-93). Hypocalcemia affects the teat canal and neutrophils. It can influence the immune system through the secretion of cortisol during calving. The teat canal is the first line of defense against mastitis because it is the pathway by which pathogens can enter the mammary gland. The canal is sealed between milkings and during the dry period by a keratin plug derived from the lining of the stratified epithelium of the canal. Probably, the main function of this waxy plug is to establish a physical barrier to prevent bacterial penetration. The teat has muscles in its sphincter that keep it closed between one milking and another. After milking, it takes two hours for the contraction of the sphincter and closure of the teat canal(94). Calcium decreases at the time of calving, both in the blood circulation and in the internal deposits of blood cells(2). Normally, the calcium recovers within a few days. In cows with hypocalcemia, this decrease is accentuated, which leads to other alterations linked to Ca. In cows with subclinical hypocalcemia, probably the teat sphincter remains distended for longer due to inefficient muscle contraction caused by Ca deficiency. In addition, when starting lactation, cows remain prostrate for long periods, compared to normocalcemic cows. This facilitates the entry of environmental pathogens through the teat canal, which reach the cistern of the mammary gland, where they proliferate and consequently induce mastitis(95). Lactoferrin is a protein that exerts different functions related to innate immunity, is synthesized in neutrophils(96), and has a high affinity for iron (Fe; chelating activity), so it binds to free iron and reduces it. Microorganisms require Fe for their growth(97-99), its bacteriostatic effect prevents bacterial proliferation(100-101), although lactoferrin can also act as a bactericide(102). In hypocalcemia, the function of neutrophils reduces(3,22), and the activity of lactoferrin decreases, generating a higher incidence of mastitis in hypocalcemic cows.

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Hypocalcemia can reduce immune function through cortisol at the time of calving. The fetus starts the calving in the cow, stimulating the hypothalamic-pituitary-adrenal axis and increasing the secretion of cortisol. Cortisol changes the steroidogenic pathway, instead of directing it towards the synthesis of progesterone (P4), it directs it towards the synthesis of estradiol (E2). As a result, the synthesis of progesterone reduces and the synthesis of estradiol increases, inducing calving. Cortisol secretion increases considerably in cows with hypocalcemia. Cortisol secretion is higher in hypocalcemic cows than in normocalcemic cows(103). In addition, cortisol is considered a very potent immunosuppressive agent and probably increases the immunosuppression observed in the cow during peripartum(104), with the subsequent risk of occurrence of mastitis.

Contagious mastitis

The microorganisms involved in contagious mastitis are: Staphylococcus aureus, Streptococcus agalactiae, Arcanobacterium pyogenes, Mycoplasma spp.(105-106). The spread of the bacterium responsible for the infection occurs during milking, due to practices such as the shared use of towels to wash and dry udders, through contaminated hands of milkers and use of teatcups of mechanical milking without disinfecting between cow and cow. The use of individual gloves or towels, as well as the milking separately and milking of infected cows to the end, with prior disinfection of the milking units, helps to prevent infection(107-109).

Treatment Treatment should be applied immediately. The best option is to apply calcium orally to cows that are still standing. The blood calcium level increases over the course of 30 min after administration(110) and remains elevated for 4 to 6 h(110-111). Intravenous treatment rapidly increases blood calcium levels, but this increase can be extreme and potentially dangerous, and can cause fatal cardiac complications, so it is not advisable to administer it in cows that are still standing(112). After intravenous treatment, the level of blood Ca decreases again to lower than normal concentrations; consequently, the cow again shows hypocalcemia in a period of 12 to 18 h(112-113). Even the dosage of Ca intravenously suspends the animal’s ability to mobilize the necessary Ca and meet the requirements at critical times(111-113). Experimentally, atropine-induced arrhythmia has been reversed by alternating states of hypercalcemia and hypocalcemia in dairy cows(114). For cows in phases II and III of clinical disease, 500 ml of 23 % calcium gluconate solution should be administered immediately slowly intravenously. This provides 10.8 g of elemental calcium, which is enough to correct

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the total calcium deficiency in the cow (4-6 g). The administration of Ca intravenously is little recommended(115). In cows that respond favorably to treatment, it is important to reinforce it with oral administration 12 h after recovery, to avoid relapses(111).

Prevention Hypocalcemia is prevented by manipulating the diet and administering calcium orally(113-119).

Diet manipulation

Low-calcium diets (LCDs) are administered, and the ration is adjusted to meet nutritional needs considering the dietary cation-anion difference (DCAD). Feeding with LCDs leads to transient hypocalcemia, with subsequent reabsorption from bone tissue and increase in absorption from the small intestine and increases in calcium availability(120). Rations with 8 to 10 g of calcium per day produce favorable effects for the aforementioned purpose(120). The use of anionic salts to reduce hypocalcemia is based on their acidogenic nature, which causes digestive and metabolic acidity, and generates optimal conditions for the circulation of Ca in the body(121-122). Another dietary strategy to reduce the occurrence of hypocalcemia consists of providing a ration deficient in Ca before calving. This causes a negative Ca balance in the cow before calving and stimulates the secretion of parathyroid hormone (PTH) and 1,25dihydroxyvitamin D, promoting Ca homeostasis at calving. In the field, it is recommended to provide prepartum rations with reduced levels of Ca (approximately 0.5 % of Ca)(123-124). The acidification of the pH in the rumen and intestine leads to the increase of the solubilization of Ca; acidosis promotes the activation of parathormone (PTH), and this in turn participates in the absorption of intestinal Ca(124-125). Acidity increases the function of osteoclasts, responsible for bone resorption, transferring iCa2+ from the bones to the blood circulation and increasing the excretion of Ca in the urine(125-127). Cows fed a negative DCAD diet in the prepartum increase the blood concentration of iCa2+(127). Under normal conditions, the body maintains a pH between 7.35 and 7.45(128), through various physical-chemical regulatory mechanisms, such as: the buffer systems of the plasma (bicarbonates and proteins) and bone tissue. There are other physiological regulators such as the elimination of CO2 by respiratory route to the detriment of bicarbonates, the elimination of acids through the kidneys and the reabsorption of bicarbonates. The concentration of ions (milliequivalents) establishes an equality in the different media, the sum of anions (negatively charged ions with acid nature) is equal to the sum of cations (positively charged ions with basic nature). Based on the fact that Na+, K+ and Cl- ions (bioavailable ions that cannot be metabolized in

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simpler forms) determine the acid-base balance of the plasma medium and the acidogenic function of sulfates (SO=; they directly acidify biological fluids)(129), these four ions are taken into account for the calculation of the cation-anion balance of the raw material and the formulation of diets for dry cows(117-118,125). By calculating the dietary cation-anion difference (DCAD), the diet can be formulated on the ionic balance and pH of the plasma medium(117118) . DCAD is defined as the difference between cations and anions. Feeding cows with a negative DCAD diet to dry cows at the end of gestation increases anions (SO4= and Cl-), so the blood buffer capacity alters, and the blood acidifies(125). In response, the body releases cations (H+) to neutralize anions and maintain electroneutrality. This causes the pH to decrease and generates acidity in the urine and greater excretion of Ca; the level of iCa 2+ in the blood circulation reduces and stimulates the secretion of PTH and this in turn participates in the active formation of 1,25 dihydroxyvitamin D3 (1,25(OH)2 D3) and in the mobilization of bone Ca(4,130) with the increase in the concentration of iCa2+ in the blood(123-125,130). In addition, cows fed negative DCAD diets increase their concentrations of serotonin(127), which is important for the function of the mammary gland during lactation. Based on the above, the use of DCAD reduces the incidence of hypocalcemia(34,116-118,124,131-132), with the subsequent increase in leukocyte function and reproductive health(34). A problem with anionic salts is their low palatability, as they reduce feed consumption and predispose to other eating disorders such as low energy intake in the transition period. Fortunately, the new DCAD are more palatable and avoid this situation. Another disadvantage is their cost, but the costbenefit of their use must be analyzed(133-137).

Oral administration of calcium

The favorable effect of oral calcium for the prevention of hypocalcemia in dairy cows has been demonstrated, even having access to rations with anionic salts or in herds with low incidence of milk fever cases(119). When the animal consumes less Ca than required, the absorption of Ca increases. On the contrary, when the animal consumes more Ca than required, the absorption of Ca decreases(138). Events that cause changes in efficient absorption of Ca begin with changes in plasma Ca but depend on the control of the active metabolite of vitamin D3, known as 1-25 dihydroxyvitamin D3 [1-25-(OH)2 D3]. Although there is evidence of pre-duodenal absorption of Ca, the greatest absorption of Ca occurs in the duodenum or upper part of the small intestine(139). The transfer of Ca through the intestinal villi occurs by facilitated transport and is initiated by 1,25-(OH)2 D3, which enters the enterocyte by means of cell diffusion and binds to its receptor in the cellular cytoplasm (140). The 1,25-(OH)2 D3 receptor complex moves to the chromatin fraction in the cell nucleus and this hormone-receptor complex synthesizes more messenger RNA and specific proteins that regulate Ca transport.

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There are several components that limit the bioavailability and absorption of Ca. Oxalates, which could reduce the amount of Ca in hays and alfalfas, or low levels of phosphorus (P) in the diet, as well as high levels of magnesium fluoride, concentration of lipids in the diet, or by nucleic acids produced by bacteria or bacterial cell walls(138). There are compounds such as calcium chloride (CaCl3) that have the ability to maintain the concentration of blood calcium(110-111), this is due to its bioavailability and its ability to stimulate the acid response in the cow, which increases its own mobilization of calcium(110). Good absorption is obtained with 50 g of elemental calcium dissolved in 250 ml of water. However, care should be taken with the dosage of calcium. There is a risk of inhalation, and it is very caustic for the tissues of the upper airways(110). Calcium propionate is absorbed slowly, probably because it does not increase acidity. The administration of 75 to 125 g dissolved in water and propylene glycol offers good results(110-111). Calcium carbonate dissolved in water is another presentation that has been evaluated, without satisfactory results since it does not increase the level of blood calcium(110), probably due to its low bioavailability. In addition, calcium carbonate produces an alkalogenic response, which acts in the opposite way to anionic salts and prevents the mobilization of bone calcium. To facilitate the dosage of calcium, the use of boluses with calcium chloride and sulfate has been studied. The bolus is administered immediately after calving and 12 h after. With this treatment, the ionic concentration of plasma calcium has been increased(134-136). This bolus has the advantage of being palatable, Ca is not wasted, there are no risks of inhalation, and the release of calcium is slower and more effective. The application of calcium subcutaneously is not recommended because it causes irritation and necrosis in the tissues(11).

Serotonin

Serotonin regulates the physiology of the mammary gland during lactation. It is synthesized in various tissues of the body from the L-tryptophan amino acid, by the action of the tryptophan hydroxylase (TPH) enzyme to transform it into 5-hydroxytryptophan (5-HTP). Decarboxylase converts 5-HTP into serotonin(137-140). In rodents, serotonin is synthesized in the intestine and other tissues(141-144), travels through the bloodstream and acts on the mammary gland. The mammary gland has receptors for serotonin(145-148). In addition, it expresses the TPH enzyme(147) and synthesizes serotonin during lactation(148-151). Serotonin stimulates the synthesis and secretion of parathyroid hormone-related protein (PTHrP) in the mammary gland(145-146,151-157) and participates in the expression of calcium-sensitive receptors (CaSRs) during lactation(158-160). PTHrP is secreted into the maternal circulation and acts on bone cells to stimulate bone resorption in osteoclasts, releasing calcium into the systemic circulation(142), destined for the mammary gland(160). The addition of 5-HTP, the precursor of serotonin, to the feed ration, during the gestation-lactation transition period in

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rodents, increases the circulating concentration of serotonin, PTHrP and Ca, as well as the content of Ca in milk(151). PTHrP, unlike PTH, acts as a paracrine regulator and is located in the circulation during lactation or humoral hypercalcemia(160). Therefore, like PTH, PTHrP acts as a hormonal regulator of Ca and is important for the homeostasis and mobilization of Ca during calving and lactation(161-162). In addition, PTHrP is detected in blood only during lactation. It decreases its production, plasma concentration and the preservation of bone mass(159). The zero serum concentration of PTHrP during lactation is restored with the application of 5-HTP over the course of 1 h(146). This demonstrates the importance of 5-HTP in the production of PTHrP during lactation and in the increase of blood Ca(152-153). CaSR identifies variations of extracellular free calcium, its ions bind, and the link for cellular response is established in various organs(163-167). During lactation, the production of PTHrP is inhibited and the transport of calcium to milk is stimulated, it is activated with the increase of calcium in the circulation(158,168). Therefore, the mammary gland acts as a calciumsensitive organ during lactation, which responds to changes in its extracellular concentration, mainly through the calcium-sensitive receptor. Which identifies the concentration of Ca in the blood circulation and together with the PTHrP regulates its level in the blood(159). Serotonin helps maintain calcium homeostasis in lactation. PTHr releases calcium from bone tissue into the circulation, and activates CaSRs, which have negative feedback on PTHrP. Consequently, the stimulation on the osteoclasts is suspended, that is, their release from bone tissue is decreased. CaSR also promotes the transport of blood calcium to milk. Consequently, it reduces Ca in the blood and causes greater secretion of PTHrP in the mammary epithelial cells, increasing calcium reabsorption. Therefore, the mammary gland regulates its own calcium requirements under a negative feedback system(159) that allows it to maintain its calcium requirements during milk production. In dairy cattle, a process similar to that of rodents may occur. There is expression of serotonin receptors in the mammary epithelium(144,169). The circulating concentration of serotonin on the first day of lactation has been positively correlated with the circulating level of Ca in dairy cows(153-156), as well as throughout most of lactation(170). The intravenous application of 5-HTP, the precursor of serotonin, administered at the end of lactation in non-pregnant Holstein cows(157) and at the end of gestation in pregnant cows(148-151) has increased the systemic level of serotonin and calcium, and has decreased the elimination of calcium in the urine, increasing the concentration of calcium in milk and colostrum. The effect of serotonin is independent of parathyroid hormone(151). In addition, PTHrP has been previously identified in the cow’s circulatory system(170-172).

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Conclusions Hypocalcemia can occur in the peripartum due to alterations in calcium homeostasis when the blood and cytosolic concentration of Ca decreases. Hypocalcemia can occur clinically and subclinically. The reduction of calcium leads to a decrease in immune function and in smooth muscle contractions, increasing the risk of metritis and mastitis, among other alterations. Clinical hypocalcemia is treated with intravenous calcium and subclinical hypocalcemia with oral calcium. Prevention requires the addition of anionic salts in the ration and the addition of calcium orally. In addition to inducing mild prepartum hypocalcemia to stimulate the secretion of parathyroid hormone (PTH) and of 1,25-dihydroxyvitamin D and thus induce Ca homeostasis after calving. Efficient absorption of Ca depends on the plasma Ca level and on the active metabolite of vitamin D3, called 1,25-dihydroxyvitamin D3 [1-25(OH)2 D3]. During lactation, serotonin participates in maintaining calcium homeostasis through the synthesis and secretion of parathyroid hormone-related protein (PTHrP), and this effect is independent of the action of parathyroid hormone. Literature cited: 1. Drackey JK. Biology of dairy cows during the transition period the final frontier? J Dairy Sci 1999;82:2259-2273. 2. Kimura K, Reinhardt TA, Goff JP. Parturition and hypocalcemia blunts calcium signals in immune cells of dairy cattle. J Dairy Sci 2006;89:2588-2595. 3. Martinez N, Sinedino LDP, Bisinotto RS, Ribeiro ES, Gomes GC, Lima FS, et al. Effect of induced subclinical hypocalcemia on physiological responses and neutrophil function in dairy cows. J Dairy Sci 2014;97:874-887. 4. Horst RL, Reinhardt TA. Vitamin D metabolism in ruminants and its relevance to the periparturient cow. J Dairy Sci 1983;66:661-678. 5. Goff JP. Calcium and magnesium disorders. Vet Clin North Am Food Anim Pract 2014;30:359-381. 6. Moseley JM, Kubota M, Diefenbach-Jagger H, Wetthenhall RE, Kemp BE, Suva LJ, et al. Parathyroid hormone-related protein purified from a human lung cancer cell line. Proc Natl Acad Sci USA 1987;84:5048-5052. 7. Horseman ND, Hernandez LL. New concepts of breast cell communication to bone. Trends Endocrinol Metab 2014;25:34-41.

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99. Legrand D, Elass E, Pierce A, Mazurier L. Lactoferrin and host defence: an overview of its immune-modulating and anti-inflammatory properties. Biometals 2004;17:225-229. 100. Smith KL, Schanbacher FL. Lactoferrin as a factor of resistance to infection of the bovine mammary gland. J Am Vet Med Assoc 1977;170:1224-1227. 101. Rainard P. Bacterioestatic activity of bovine milk lactoferrin against mastitic bacteria. Vet Microbiol 1986;11:387-392. 102. Orsi N. The antimicrobial activity of lactoferrin: current status and prespectives. Biometals 2004;17:189-196. 103. Horst RL, Jorgensen NA. Elevated plasma cortisol during induced and spontaneous hypocalcemia in ruminants. J Dairy Sci 1982;65(12):2332-2337. 104. Kehril ME Jr, Nonnecke BJ, Roth JA. Alterations in bovine lymphocyte function during periparturient period. Am J Vet Res 1989;50:215-220. 105. Bradley A. Bovine mastitis: an evolving disease. Vet J 2002;164:116-128. 106. White LJ, Lam TJGM, Schukken YH, Green LE, Medley GF, Chappell MJ. The transmission and control of mastitis in dairy cows: a theoretical approach. Prev Vet Med 2006;74(1):67-83. 107. Halasa T, Huijps K, Østerås O, Hogeveen H. Economic effects of bovine mastitis and mastitis management: a review. Vet Quart 2007;29(1):18-31. 108. Venjakob PL, Borchardt S, Heuwieser W. Hypocalcemia-Cow-level prevalence and preventive strategies in German dairy herds. J Dairy Sci 2017;100(11):9258-9266. doi: 10.3168/jds.2016-12494. 109. Contreras GA, Rodriguez JM. Mastitis: comparative etiology and epidemiology. J Mamm Gl Biol Neoplasia 2011;16:339-356. 110. Goff JP, Horst RL. Oral administration of calcium salts for treatment of hypocalcemia in cattle. J Dairy Sci 1993;76:101-108. 111. Goff JP, Horst RL. Calcium salts for treating hypocalcemia: carrier effects, acid-base balance, and oral versus rectal administration. J Dairy Sci 1994;77:1451-1456. 112. Thilsing-Hansen T, Jorgensen RJ, Ostergaard S. Milk fever control and principles: a review. Acta Vet Scand 2002;43:1-19. 113. Curtis RA, Cote JF, McLennan MC, Smart JF, Rowe RC. Relationship of methods of treatment of relapse rate and serum levels of calcium and phosphorus in parturient hypocalcemia. Can Vet J 1978;19:155-158. 1046


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114. Littledike ET, Glazier D, Cook HM. Electrocardiographic changes after induced hypercalcemia and hypocalcemia in cattle: reversal of the induced arrhythmia with atropine. Am J Vet Res 1976;37(4):383-388. 115. Doze JG, Donders R, van der Kolk JH. Effects of intravenous administration of two volumes of calcium solution on plasma ionized calcium concentration and recovery from naturally occurring hypocalcemia in lactating dairy cows. Am J Vet Res 2008;69:1346-1350. 116. Oetzel GR, Olson JO, Cartis CR, Fettmen M. Ammonium chloride and ammonium sulfate for prevention of parturient paresis in dairy cows. J Dairy Sci 1988;71:33023309. 117. Tucker WB, Houge JF, Waterman DF, Swenson TS, Xing Z. Role of sulfur and chloride in the dietary cation-anion balance equation for lactating dairy cattle. J Anim Sci 1991;69:1205-1213. 118. Moore SJ, Vandehaar MG, Sharma BK. Effects of altering dietary cation-anion difference on calcium and energy metabolism in peripartal cows. J Dairy Sci 2000;83:2095-2104. 119. Oetzel GR, Miller BE. Effect of oral calcium bolus supplementation on early lactation health and milk yield in commercial dairy herds. J Dairy Sci 2012;95:7051-7065. 120. Horst RL, Goff JP, Reinhardt TA, Buxton DR. Strategies for preventing milk fever in dairy cattle. J Dairy Sci 1997;80:1269-1280. 121. Block E. Manipulation of dietary cation-anion difference on nutritionally related production diseases, productivity, and metabolic responses of dairy cows. J Dairy Sci 1994;77:1437-1450. 122. Block E. Manipulating dietary cations and anions for prepartum dairy cows to reduce incidence of milk fever. J Dairy Sci 1984;67:2939-2948. 123. Goff JP. The monitoring, prevention, and treatment of milk fever and subclinical hypocalcemia in dairy cows. The Vet J 2008;176(1):50-57. 124. Goff JP, Koszewski NJ. Comparison of 0.46% calcium diets with and without added anions with a 0.7% calcium anionic diet as a means to reduce periparturient hypocalcemia. J Dairy Sci 2018;101(6):5033-5045. 125. Muhammad SH, Muhammad AS, Mahr-Un-Nisa, Muhammad S. Dietary cation anion difference: impact on reproductive and productive performance in animal agriculture. Afr J Biotechnol 2010;9:7976-7988.

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126. Goff JP, Horst RL. Role of acid-bas physiology on the pathogenesis of parturient hypocalcemina (milk fever)-the DECAD theory in principal and practice. Acta Vet Scand 2003;97:51-56. 127. Rodney RM, Martinez N, Block E, Hernandez LL, Celi P, Nelson CD, Santos EP, Lean IJ. Effects of prepartum dietary cation-anion difference and source of vitamin D in dairy cows: vitamin D, mineral, and bone metabolism. J Dairy Sci 2018;101:2519-2543. 128. Nagy O, Kovác G, Seidel H, Weissová T. The effect of arterial blood sampling sites on blood gases and acid-base balance parameters in calves. Acta Vet Hung 2001;49:331340. 129. Whiting SJ, Drapper HH. Effect of a chronic acid load as sulfate or sulfur amino acid on bone metabolism in adult rats. J Nutr 1981;111:1721-1726. 130. Horst RL, Littledike ET, Patridge TA. Plasma concentrations of 1,25-dihydroxivitamin D, 1,24R,25-trihydroxivitamin D3 and 1,25,26-trihydroxivitamin D3 after administration to dairy cows. J Dairy Sci 1983;66:1455-1460. 131. Gaynor PJ, Mueller FJ, Miller JK, Ramsey N, Goff JF, Horst RL. Parturient hypocalcemia in Jersey cows fed alfalfa haylage based-based diets with different cation to anion ratios. J Dairy Sci 1989;72:2525-2531. 132. Goff JP, Horst RL, Mueller FJ, Miller JK, Kiess GA, Dowlen HH. Addition of chloride to a prepartal diet high in cations increases 1,25-dihidroxivitamin response to hypocalcemina preventing milk fever. J Dairy Sci 1991;74:3863-3871. 133. Lean IJ, DeGaris PJ, McNeil DM, Block E. Hypocalcemia in dairy cows: meta-analysis and dietary cation anion difference theory revisited. J Dairy Sci 2006;89(2):669-684. 134. Lean IJ, Santos JEP, Block E, Golder HM Effects of prepartum dietary cation-anion difference intake on production and health of dairy cows: A meta-analysis. J Dairy Sci 2019;102(3):2013-2133. 135. Santos JEP, Lean IJ, Golder H, Block E. Meta-analysis of the effects of prepartum dietary cation-anion difference on performance and health of dairy cows. J Dairy Sci 2019;102(3):2134-2154. 136. Charbonneau E, Pellerin D, Oetzel GR. Impact of lowering dietary cation-anion difference in nonlactating dairy cows: A meta-analysis. J Dairy Sci 2006;89(2):537-548. 137. Goff JP. The monitoring, prevention, and treatment of milk fever and subclinical hypocalcemia in dairy cows. The Vet J 2008;176(1):50-57.

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138. Horst RL. Regulation of calcium and phosphorus homeostasis in the dairy cow. J Dairy Sci 1986;69(2):604-616. 139. Braithwaite GD. Calcium and phosphorus metabolism in ruminants with special reference to parturient paresis. J Dairy Res 1976;43(3):501-520. 140. Wasserman RH, Fullmer CS. Vitamin D and intestinal calcium transport: facts, speculations and hypotheses. The Journal of Nutrition 1995;125(suppl 7):1971S-1979S. 141. Sampson JD, Spain JN, Jones C, Castensen L. Effects of calcium chloride and calcium sulfate in an oral bolus given as a supplement to postpartum dairy cows. Vet Ther 2009;10:131-139. 142. Wang L, Erlandsen H, Haavik J, Knappskog PM, Stevens RC. Three-dimensional structure of human tryptophan hydroxylase and its implications for the biosynthesis of the neurotransmitters serotonin and melatonin. Biochem 2002;41:12569-12574. 143. Ormsbee HS, Fondacaro JD. Action of serotonin on the gastrointestinal tract. Proc Exp Biol Med 1985;178:333-338. 144. Berger M, Gray JA, Roth BL. The expanded biology of serotonin. Annu Rev Med 2009;60:1514-1529. 145. Wysolmerski JJ. Parathyroid hormone-related protein: an uptake. J Clin Endocrinol Metab 2012;97:2947-2952. 146. Wyler SC, Lord CC, Lee S, Elmqist JK. Liu C. Serotonergic control of metabolic homeostasis. Front Cell Neurosci 2017;11:277. 147. Hernandez LL, Limesand SW, Collier JL, Horseman ND, Collier RJ. The bovine mammary gland expressed multiple functional isoforms of serotonin receptors. J Endocrinol 2009;203:123-131. 148. Hernandez LL, Gregerson KA, Horseman ND. Mammary gland serotonin regulates parathyroid hormone-related protein and other bone-related signals. Am J Physiol Endocrinol Metab 2012;302:E1009-1015. 149. Matsuda M, Imaoka T, Vomachka AJ, Gudelsky GA, Hou Z, Mistry M, Bailey JP, Nieport KM, Walter DJ, Bader M, Horseman ND. Serotonin regulated mammary gland development via an autocrine- paracrine loop. Dev Cell 2004;6:193-203. 150. Weaver SR, Prichard AP, Endres EL, Newhouse SA, Peters PL, Crump PM, Akins MS, Crenshau TD, Bruckmaier RM, Hernandez-Castelam LL. Elevates of circulating serotonin improves calcium dynamics in the prepartum dairy cows. J Endocrinol 2016;230:105-123.

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151. Weaver SR, Jury NJ, Gregerson KA, Horseman ND, Hernandez L. Characterization of mammary-specific disruptions for Tph1 and Lrp5 during murine lactation. Scientific Reports 2017;7:151-155. 152. Hernández-Castellano LE, Hernandez LL, Sauerwein H, Bruckmaier RM. Endocrine and metabolic changes in transition dairy cows are affected by prepartum infusion of a serotonin precursor. J Dairy Sci 2017;100(6):5050-5057. 153. Hernández-Castellano LE, Hernandez LL, Weaver S, Bruckmaier RM. Increased serum serotonin improves parturient calcium homeostasis in dairy cows. J Dairy Sci 2017;100:1580-1587. 154. Laporta J, Peters TL, Weaver SR, Merriman KE, Hernandez LL. Feeding 5-hydorxi-ltryptophan during transition from pregnancy to lactation increases calcium mobilization from the bone in rats. Domest Anim Endocrinol 2013;44:176-184. 155. Laporta J, Moore SAE, Peters NW, Peters TL, Hernandez LL. Short communication: circulating serotonin (5-HT) concentrations on day 1 of lactation as a potential predictor of transition-related disorders. J Dairy Sci 2013;96:5146-5150. 156. Laporta J, Keil KP, Vezina CM, Hernandez LL. Peripheral serotonin regulates maternal calcium trafficking in mammary epithelial cells during lactation in mice. PloS One 2014a 9:E110190. 157. Laporta J, Keil KP, Weaver SR, Cronick CM, Prichard AP, Crenshaw TD, et al. Serotonin regulates calcium homeostasis in lactation by epigenetic activation of hedgehog signaling. Mol Endocrinol 2014;11:1866-1874. 158. Laporta J, Gross JJ, Creshaw TD, Bruckmaier RM, Hernandez LL. Short communication: Timing of first milking affects serotonin concentrations. J Dairy Sci 2014;97:2944-2948. 159. Laporta J, More SAE, Weaver SR, Cronick CM, Olsen M, Pichard AP, et al. Increasing serotonin (5-HT) alters calcium and energy metabolism in late-lactation dairy cows. J Endocrinol 2015;226:43-55. 160. VanHoulten JN, Dann P, McGeoch G, Browen EM. Calcium sensing receptor regulates mammary gland parathyroid hormone-related protein production and calcium transport. J Clin Invest 2004;113:598-608. 161. VanHoulten JN. Calcium sensing by the mammary gland. J Mammary Gland Biol Neoplasia 2005;10:129-139. 162. Wysolmerski JJ. Interactions between breast, bone, and brain regulate mineral and skeletal metabolism during lactation. Ann NY Acad Sci 2010;1192:161-169. 1050


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163. Rakoupoulos M, Vargas SJ, Guillespie MT, Ho PW, Diefenbach-Jagger H, Leaver DD, et al. Production of parathyroid hormone-related protein by the rat mammary gland in pregnancy and lactation. Am J Physiol 1992;263:E1077-E1085. 164. Ratcliffe WA, Thompson GE, Care AD, Peaker M. Production of parathyroid hormone related protein by the mammary gland of the goat. J Endocrinol 1992;133:87-93. 165. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Litton J, Hebert SC. Cloning and characterization of an extracellular Ca(2+)sensing receptor form bovine parathyroid. Nature 1993;366:575-580. 166. Hoffer AM, Brown EM. Extracellular calcium sensing and signaling. Nat Rev Mol Cell Biol 2003;4:530-538. 167. Brown EM, Conigrave AD. Regulation of cellular signal transduction pathway by extracellular calcium-sensing receptor. Curr Pharm Biol 2009;10:270-281. 168. Quinn SJ, Kifor O, Kifor I, Butters RR Jr. Role of the cytoskeleton in extracellular calcium-regulated PTH release. Biochem Biophys Res Comm 2007;354:8-13. 169. Brennan SC, Thiem U, Roth S, Agarwal A, Fetahu ISh, Tennakon S, et al. Calcium sensing receptor signaling in physiology and cancer. Biochem Biophys Acta 2013;1833:1732-1744. 170. Ardeshirpour L, Dann P, Polland M, Wysolmerski J, VanHulten J. The calcium-sensing receptor regulates PTHrP production and calcium transport in the lactating mammary gland. Bone 2006;38:787-793. 171. Suárez-Trujillo A, Argüello A, Rivero MA, Capote J, Castro N. Differences in distribution of serotonin receptor subtypes in the mammary gland of sheep, goats, and cows during lactation and involution. J Dairy Sci 2019;102(3):2703-2707. 172. More SA, Laporta J, Crenshaw TD, Hernandez LL. Plasma of circulating serotonin and related metabolites in multiparous dairy cows in the peripartum period. J Dairy Sci 2015;98:3754-3765. 173. Corbellini CN. Etiopatogenia e controle da hipocalcemia e hipomagnesemia em vacas leiteiras. Seminário internacional sobre deficiências minerais em ruminantes, 1998;28. 174. Beede DK, Wang C, Donovan GA, Archbald LF, Sanchez WK. Dietary cation-anion difference (electrolyte balance) in late pregnancy. In: Florida Dairy Production Conference Proc 1991:1-6.

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175. Hernández EGS, Bouda J, Cecilio AA, Doubek J, Forero FHV. Efecto de la aplicación de prostaglandina F2α en las primeras horas posparto sobre las concentraciones séricas de calcio en vacas lecheras. Vet Méx 2014;1(2):1-13. 176. Albornoz L, Albornoz JP, Cruz JC, Fidalgo LE, Espino L, Morales, et al. Estudio comparativo de los niveles de calcio, fósforo y magnesio durante el periparto en vacas lecheras en diferentes sistemas de producción en Uruguay y España. Veterinaria (Montevideo), 2017;53(205):1-11. 177. Wittwer F, Heuer G, Contreras PA, Böhmwald TM. Valores bioquímicos clínicos sanguíneos de vacas cursando con decúbito en el sur de Chile. Arch Med Vet 1993;15:83-88. 178. Wagemann C, Wittwer F, Chihuailaf R, Noro M. Estudio retrospectivo de la prevalencia de desbalances minerales en grupos de vacas lecheras en el sur de Chile: a retrospective study. Arch Med Vet 2014;46(3):363-373. 179. Sánchez JM, Saborío-Montero A. Hipocalcemia e hipomagnesemia en un hato de vacas Holstein, Jersey y Guernsey en pastoreo. Agronom Costarric 2014;38(2):55-65. 180. Ceballos-Márquez A, Villa NA, Betancourth TE, Roncancio DV. Determinación de la concentración de calcio, fósforo y magnesio en el periparto de vacas lecheras en Manizales, Colombia. Rev Colomb Cienc Pec 2004;17(2):125-133. 181. Sánchez JM, Saborío-Montero A. Prevalencia de hipocalcemia en cuatro hatos Jersey en pastoreo en Costa Rica. Agronomía Costarricense 2014;38(2):33-41 182. Reinhardt TA, Lippolis JD, McCluskey BJ, Goff JP, Horst RL. Prevalence of subclinical hypocalcemia in dairy herds. The Vet J 2011;188(1):122-124. 183. Reyes C, Mellado M. Ocurrencia de desórdenes derivados del parto y mastitis en vacas Holstein, en función del número de partos y meses del año. Vet Méx 1994;25(2):133135.

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Table 1: Blood levels of calcium (Ca) in dairy cows exposed to different treatments and conditions of the production system Country

Treatment

No. of animals

Ca (mg/dl)

Argentina

Control Treatment3

240 280

7.11 9.32

United States of America Mexico

Control4 Treatment5

Spain and Uruguay

10 10

System: Intensive

256

Silvopastoral

354

Costa Rica

Colombia

80 % (8/10) 40 % (4/10)

10.96 + 0.06 9.35 + 0.09 2.0-2.6 mmol/L

76 Prepartum: 1986-20026

471

2003-2011

270

Postpartum: 1986-2002

1041

2003-2011

766

Breed: Holstein Jersey Guernsey

49 62 41

Dairy production: Low

FMR1 (%)

Fallen cows (%) 12.5 0.6

Ref2

173

82 % 30 %

174

0 % (0/10) 0 % (0/10)

175

176

51 %

26

177 178

2.37 0.14 2.29 0.18

+

2.35 0.14 2.27 0.12

+

+

+

7.85 7.49 8.06

27 (55 %) 31 (50 %) 18 (44 %)

2 (4 %) 8 (13 %) 0 (0 %)

Stage:

179

180

Prepartum Postpartum

High

Clinical hypocalcemia

9.4 6.0

3.8 4.3

Control Treatment

Chile

Subclinical hypocalcemia

Prepartum Postpartum

2.14 0.10 2.39 0.10 2.42 0.11 2.40 0.12

+ + + +

1

FMR= Fetal membrane retention (%). 2 Ref= Bibliographic references. 3 Treatment: mixture of mineral salts (150g Cl2Ca, 150g NHSO4, 29g MgO2). 4 Control= Diet with +50 meq/kg; 5 Diet with -250meq/kg. 6 Periods (years).

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Table 2: Comparison of incidence of cases of subclinical hypocalcemia and clinical hypocalcemia in dairy herds in different countries. Country

Breed

No. of animals (n=)

Plasma levels of calcium (mg/dL)

Subclinical hypocalcemia (%)

Clinical hypocalcemia (%)

Ref.1

Costa Rica (Grazing)

Holstein Jersey Guernsey

49 62 41

7.85 7.49 8.06

27 (55) 31 (50) 18 (44)

2 (4) 8 (13) 0 (0)

180

Costa Rica

:

No. of calvings

Jersey

Clinical hypocalcemia (%) 1 4 6 10 8 13

181

(Grazing)

Subclinical hypocalcemia (%) 25 41 49 51 54 42

United States

Holstein

Subclinical hypocalcemia (%) 53 42 78 44 47 63

Clinical hypocalcemia (%) 6 13 2 29 29 25

182

Risk indices FMR2

Hypocalcemia

183

0.68 1.33 1.57 1.65 1.12

0.31 0.32 5.80 3.37 2.14

No. of animals 454 447 291 166 72 32

1 2 3 4 5 6 No. of calvings

(Housed)

Mexico

1 2 3 4 5 6

Holstein

No. of calvings 1-2 3-4 5-6 7-8 >9

(Housed)

1

Ref= Bibliographic reference. FMR=fetal membrane retention.

2

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https://doi.org/10.22319/rmcp.v13i4.5217 Thecnical note

Productive performance and nutritional value of Pennisetum purpureum cv. Cuba CT-115 grass at different regrowth ages

Gloria Esperanza de Dios-León a Jesús Alberto Ramos-Juárez a* Francisco Izquierdo-Reyes a Bertín Maurilio Joaquín-Torres a† Francisco Meléndez-Nava b

a

Colegio de Postgraduados, Campus Tabasco. Periférico Carlos A. Molina. Km 3.5 carretera Cárdenas-Huimanguillo, 86500, H. Cárdenas, Tabasco, México. b

Universidad Popular de la Chontalpa. Departamento de Zootecnia. Cárdenas, Tabasco.

*Corresponding author: ramosj@colpos.mx

Abstract: Grass species forage yield and nutritional value directly affect livestock production performance. They also vary in response to regional climate and soil conditions. Forage yield and nutritional value in Pennisetum purpureum cv. Cuba CT-115 were evaluated at five regrowth ages (30, 45, 60, 75 and 90 d) in three seasons (dry, rainy and northwinds). A completely randomized block design with repeated measures was used, with four replicates per treatment. In all three seasons, maximum height was reached at 75 d: 127.1 cm in the dry season, 151.6 cm in the rainy season and 137.0 cm the northwind season. Forage yield was highest (27.0 t DM ha-1) at 90 d in the rainy season, with a growth rate of 300.2 kg DM ha -1 d-1, 7.3% crude protein and 37.0% in situ digestibility of dry matter. The leaf:stem ratio was highest at 30 d in all seasons, with a 1.65 average value. Crude protein content was highest in the northwind season at 30 and 45 d, with a 15.6 % average value. In all three seasons, digestibility was highest at 30 (mean= 49.3 %), 45 (51.8 %) and 60 d (48.2 %). Based on forage yield, use of P. purpureum cv. Cuba CT-115 grass for open grazing is recommended 1055


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for cutting at 90 days’ regrowth and based on its nutritional quality is recommended for grazing at 60 days’ regrowth, both during the rainy season. Key words: Pennisetum purpureum, Forage yield, Growth rate, Quality.

Received: 14/01/2019 Accepted: 30/08/2021

In the humid tropics of Mexico, forages are the main feed source for cattle. Forage availability and its nutritional value varies between seasons. Production is highest during the rainy season and declines during the northwind and dry seasons(1). Meat and milk production in grazing cattle respond directly to forage production, with decreased yields as forage production drops. Consequently, there is an ongoing search for forage species that meet animal nutritional requirements while maintaining constant, year-round production(2). The grass Pennisetum purpureum cv. Cuba CT-115 is part of this search. Created from a clone of King grass generated through tissue culture, this cultivar was originally introduced in Cuba in the 1990s. Its short internodes and low height make apt for direct grazing. In addition, beginning at four to six months of age it has high biomass production (15 t MS-1 ha) and higher nutritional value than the Cameroon, Dwarf and Taiwan cultivars of King grass(2,3). Understanding a forage species’ growth and production performance, and consequent forage availability, in a specific region is vital to designing management strategies that maximize animal production(4). Seasonal variations in growth rate, leaf biomass, leaf area index and plant height are used as criteria for guiding optimal and sustainable pasture management(5,6). Cutting frequency also influences forage yield(7). Seasonal and annual forage growth and yield are a direct function of weather conditions, soil fertility and management practices (8). To obtain maximum forage yield each forage species requires specific seasonal management(9). It is therefore important to understand a forage species’ productive behavior and optimal harvest time since these parameters directly affect forage yield and pasture persistence(10). No data has yet been published on the productive behavior of P. purpureum cv. Cuba CT115 under the climatic and soil conditions of the state of Tabasco, Mexico. The present study objective was to evaluate the forage yield and nutritional value of P. purpureum cv. Cuba CT-115 at different regrowth ages during the dry, rainy and northwind seasons in Cambisol soil in the Chontalpa region of Tabasco.

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The experiment was carried out under seasonal conditions from April 2011 to April 2012, at the Experimental Field of the Colegio de Postgraduados, in the state of Tabasco, Mexico, (17°59'15.6" N, 93°35'06.9" W; 12 m asl). Regional climate is Am, warm humid, with summer rains, 2,251 mm average annual rainfall and 26 °C average annual temperature(11). Rainfall during the experimental period was 2,576 mm, with 6. 9% falling in the dry season, 70.3 % in the rainy season and 22.8 % in the northwind season. The highest rainfall (723 mm) during the experimental period was recorded in October. Average temperature during the experimental period was 24.4 °C, with seasonal averages of 25.6 °C in the dry season, 25.7 °C in the rainy season and 21.8 °C in the northwind season. Maximum temperature during the experimental period was 35.3 °C in April and the minimum was 16.0 °C in December (Figure 1). Soil in the experimental field is Cambisol, with a clay loam texture, pH 5.5, 1.9 % organic matter (OM), 0.14 % nitrogen (N) and 21.4 mg kg-1 phosphorous (P). Figure 1: Average monthly temperature and rainfall during experimental period at Experimental Field at Cárdenas, Tabasco

The P. purpureum cv. Cuba CT-115 pasture used in the present study was planted in 2009, in furrows spaced at 0.80 m and 1 m between plants. Since then, it has been grazed with cattle. A uniform manual cut was done on April 1, 2011, at an approximate height of 10 cm above ground surface. After this initial cut the field was fertilized with 100 kg nitrogen (urea) in three 33.3 kg applications: one in April, July and October. Weed control was done manually at the beginning of the experimental period. Experimental design was a completely random block design involving five regrowth ages (30, 45, 60, 75 and 90 d) and three seasons (dry, March-May; rainy, June-October; and northwind, November-February). Four replicates were done of each treatment (e.g., regrowth age), using season as a repeated measurement(12). Samples were collected after reaching each successive regrowth age. Each of the twenty experimental plots consisted of four rows (4 m 1057


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long, 0.80 m apart). The plots measured 4 x 3.2 m, with a total area of 12.8 m 2, and an effective area of 4.8 m2 consisting of the central furrows. Measurements were taken of plant height and these used to calculate forage yield, and growth rate (GR). The leaf:stem ratio was quantified using plant samples. Analyses were done of dry matter (DM), crude protein (CP), in vitro digestibility of dry matter (IDDM), neutral detergent fiber (NDF) and acid detergent fiber (ADF). Plant height, from soil surface to the top of the flag leaf(13), was measured immediately prior to cutting. Forage yield was estimated by harvesting plants inside the 4.8 m2 effective area from ground level. A representative 3 kg subsample was taken from the harvested forage, washed and dried at 65 °C for 72 h in a forced air oven. Calculation of dry matter yield (DM) was done using the formula: DM = FM x % DM/100, where FM= fresh matter(13). The leaf:stem ratio was calculated using a 2 kg subsample of the harvested forage. Its leaf and stem components were separated, weighed and dried at the temperature and time indicated above. Growth rate (GR) was calculated with the formula: GR= HF/t, where GR= growth rate (kg DM ha-1d-1), HF= harvested forage (kg DM ha-1) and t= days elapsed between forage harvests(14). Both DM and CP content were measured following the applicable AOAC techniques(15). Established protocols were used to quantify IDDM at 24 h(16), and NDF and ADF(17). All analyses were run at the Animal Science Laboratory of the Colegio de Posgraduados, Tabasco.

Results were evaluated with an analysis of variance (ANOVA) to identify statistical differences between the studied factors: treatments, seasons and the treatments x season interaction. A Tukey test (α=0.05) was applied for a multiple comparison of means for treatments, seasons and the treatments x season interaction. Significant differences were analyzed following the general guide A factor (treatments) effects in each B factor (season) level(18). All analyses were run with the Proc Mixed procedure and Slice instruction in the SAS ver. 9.4 software(19). The ANOVA identified differences (P<0.05) in the treatments x seasons interaction in all the evaluated variables. Plant height increased as regrowth age increased, the highest value (165.1 cm) being recorded at 90 d in the rainy season; this is 10.8 % higher than in the northwind season and 15.4 % higher than in the dry season. Forage yield also increased as regrowth age increased, the highest yield (27.0 t DM ha-1) also being observed at 90 d in the rainy season. In all three seasons, leaf:stem ratio values decreased as regrowth age increased, the highest value (1.79) being recorded at 30 d in the rainy season. Growth rate (GR) was highest (300.2 kg DM ha-1d-1) at 90 d in the rainy season. Average GR in the rainy season was 237.3 kg DM ha-1d-1, which is 105 % higher than in the northwind season and 148 % higher than in the dry season (Table 1).

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Table 1: Plant height, forage yield, leaf:stem ratio and growth rate in Pennisetum purpureum cv. Cuba CT-115 at different regrowth ages in three seasons Regrowth age (days) Seasons 30 45 60 75 90 Plant height (cm) Dry 59.4c 91.5b 96.0b 127.1a 147.2a Rainy 64.4c 103.8b 123.3b 151.6a 165.1a Northwind 45.0c 64.7c 98.5b 137.0a 138.9a Forage yield (t DM ha-1) Dry 4.0b 4.2b 3.9b 5.0b 10.7a Rainy 5.9c 6.6c 16.3b 20.3b 27.0a Northwind 3.1c 2.9b 7.8ab 11.8a 11.3a Leaf:stem ratio Dry 1.73a 1.69a 0.79b 0.82b 0.76b Rainy 1.79a 1.15b 0.88bc 0.72c 0.56c Northwind 1.43a 1.26ab 0.85bc 0.94bc 0.75c Growth rate (kg DM ha-1 d-1) Dry 134.8a 92.9a 65.4a 66.8a 119.3a Rainy 196.4bc 147.7c 272.0ab 270.9ab 300.2a Northwind 103.4ab 65.3b 129.2ab 156.9a 125.5ab abc

Different letter superscripts in the same row indicate statistical difference (Tukey, P<0.05).

In all three seasons, DM increased as regrowth age increased. The highest average values were recorded at 90 d: 23.7 % in the dry season, 19.4 % in the rainy season and 15.4 % in the northwind season. At all five regrowth ages, DM values were higher in the dry season, with a 19.7 % average. This average is 16.2 % higher than in the rainy season and 35 % higher than in the northwind season. Crude protein (CP) values decreased as regrowth age increased. The highest values were all recorded at 30 d: 15.7 % in the northwinds season, 12.5 % in the dry season and 10.4 % in the rainy season. At all five regrowth ages, average CP values were highest (13.1 %) in the northwind season. This value is 50.6 % higher than in the rainy season and 57.8 % higher than in the dry season. In all three seasons, IDDM remained unchanged up to 60 d regrowth and decreased after 75 d regrowth. The lowest NDF values were observed at 45 d in the dry and rainy seasons. No differences were found at any of the regrowth ages in the northwind season. In contrast, ADF content increased as regrowth age increased, the highest value (47.1 %) being observed at 90 d in the rainy season, which had 43.2 % average ADF. An increase in plant height at greater regrowth ages is normal behavior in upright growth grasses(20). The highest height was recorded in the rainy season and can be attributed to the higher rainfall (1,812 mm) and temperature (25.7 °C) values recorded in that season. In P. 1059


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purpureum, both higher rainfall and temperature favor photosynthesis, and consequently growth. As expected, the lower precipitation (586 mm) and temperature (21.8 °C) of the dry season, in addition to its shorter days, high winds and greater cloud cover, negatively affected plant photosynthesis capacity, slowing growth. Lower heights have been reported previously for P. purpureum cv. Cuba CT-115. For example, during the dry season heights of 31 cm at 75 d regrowth and 53 cm at 90 d have been reported(21). In a study evaluating P. purpureum clones, height was 68 cm at 60 d regrowth during the rainy season and 64 cm at 90 d in the dry season(22). In an evaluation of 12 P. purpureum species, heights during the rainy season were 56.4 cm at 60 d regrowth and 66.3 cm at 90 d. In the present study, regardless of season, average plant height at 60 d regrowth was 105.9 cm. Since maximum height for direct grazing of this grass cultivar is 100 cm(23), 60 d regrowth is apparently the optimal time of use for this grass under the present study conditions. Forage yield was highest (27.0 t DM ha-1) in the rainy season at 90 days’ regrowth; this yield was 139 % higher than in the northwind season and 151 % higher than in the dry season. There is a positive correlation between plant age and yield, and rainfall and yield, as observed elsewhere(24). The forage yields observed in the present study are notably higher than previously reported for the studied cultivar. For example, one study found a 3.8 t DM ha-1 yield during the rainy seasons and a 1.2 t DM ha-1 yield during the dry season(22). In eight P. purpureum clones, yields of 2.5 t DM ha-1 were observed in the rainy season and 0.47 t DM ha-1 in the dry season(25). Such broad discrepancies in results may result from variations in climate conditions, crop management practices and soil fertility. Forage yield distribution in the present study was 5.6 t DM ha-1 in the dry season, 7.4 t DM ha-1 in the northwinds season and 15.2 t DM ha-1 in the rainy season. The lower yield observed during the dry season can be attributed to the substantially lower rainfall (178 mm) during this season, which negatively affects the biochemical process of plant photosynthesis(24). In the northwind season, the lower yield is more probably due to its lower temperatures rather than the relatively lower precipitation. In all three seasons the leaf:stem ratio was highest at 30 d regrowth, which can be attributed to the higher number of leaves present at early ages in this grass species. This parameter decreased from 1.65 at 30 d to 0.69 at 90 d, analogous to the decrease from 1.33 at 33 d to 0.77 at 90 d reported elsewhere for P. purpureum(26). The high GR observed in the present study during the rainy season can be attributed to the higher rainfall and temperatures (1,812 mm and 25.7 °C, respectively) occurring during this season. These favor plant metabolic activity, increasing the amount of photosynthates and, consequently, DM production. In contrast, the lack of rainfall in the dry season clearly limits plant growth. In all three seasons GR increased at 60, 75 and 90 d regrowth, indicating an increase in DM yield with age. The same response has been reported previously in P. purpureum cv. Cuba CT-115(27,28), as well as in P. purpureum cv. King(29). 1060


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The above growth response also accounts for the greater fiber accumulation with age observed in the present results, which is normal in tropical grasses(26). It occurs because the proportion of cell wall in a plant, directly associated with DM content, increases with age as the leaf:stem ratio tips in favor of stems, more vascular bundles appear, cell content decreases and water is lost(28,29). In the present results, DM content was highest (23.7 %) in the dry season, perhaps due to water stress caused by greater leaf maturation and senescence, and consequent DM accumulation. Compared to stems, leaves have a higher senescence rate because their surface is more sensitive, causing them to lose more water(29). The lower CP content with greater regrowth age observed in the present results can be attributed to the lower leaf:stem ratio with plant age. The higher leaf:stem ratio at younger ages results in higher CP content since protein occurs in greater quantities in leaves. In addition, synthesis of structural components such as cellulose, hemicellulose and lignin increases as plants mature, lowering forage quality in grasses. The highest CP content in the present results were lower than reported elsewhere for this cultivar during the rainy season (14.5 % at 28 d, 12.0 % at 56 d and 11.0 % 84 d regrowth)(28). However, CP contents at all the regrowth times and in all three seasons in the present study were above the 7 % minimum CP level required for proper rumen functioning(30). The lower IDDM values with greater regrowth age observed in the present study were due to the higher leaf:stem ratio at 30, 40 and 60 days’ regrowth than at 75 and 90 days’ regrowth (Table 2). Older forage plants have higher DM percentages and more NDF and ADF content because, as they mature, the proportion of stems increases and that of leaves decreases (i.e., the leaf:stem ratio drops), increasing the amount of structural carbohydrates and lignin, which directly influence forage digestibility and use efficiency(31). The average IDDM in the present results (46.2 %) is considered low, although it is only slightly lower than the 50.1 % reported for this cultivar at 56 days’ regrowth and 24 h incubation(32). The average rainy season NDF (75.5 %) and ADF (43.2 %) values in the present results are similar to the 72.2 % NDF and 44.1 % ADF reported for another Pennisetum species(33). In the present study, both NDF and ADF contents were highest in the rainy season at 60, 75 and 90 days’ regrowth. This is probably due to the higher rainfall and temperature values in this season, which would generate more growth, more stem production and, consequently, more DM, cellulose, hemicellulose and lignin accumulation(17). The present results coincide with the literature, which indicates that tropical grasses grow and mature quickly, causing changes in their chemical composition and decreased forage nutritional quality.

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Table 2: Nutritional value of Pennisetum purpureum cv Cuba CT-115, at different age of regrowth in three seasons Regrowth age (days) Seasons 30 45 60 75 90 Dry matter (%) Dry

17.4b

18.4b

19.3b

19.6b

23.7a

Rainy

14.4b

14.7b

16.2ab

17.9ab

19.4a

Northwind

10.6b

11.2ab

12.2ab

14.4ab

15.4a

Crude protein (%) Dry

12.5a

7.8b

7.1b

7.0b

7.4b

Rainy

10.4a

10.9a

7.9b

7.0b

7.3b

Northwind

15.7a

15.5a

13.0b

11.2bc

10.3c

In situ degradation of dry matter (%) Dry

48.1ab

54.3a

49.4ab

46.7b

42.9b

Rainy

51.3a

49.8a

45.1ab

40.2b

37.0c

Northwind

48.6a

51.3a

50.1a

39.0b

39.3b

Neutral detergent fiber (%) Dry

60.7c

62.9bc

67.4ab

70.5a

64.4bc

Rainy

70.1b

70.0b

77.0a

79.7a

80.6a

Northwind

65.4a

68.0a

67.1a

70.8a

68.2a

Acid detergent fiber (%) Dry

31.6c

33.6bc

39.3ab

34.3bc

43.6a

Rainy

38.8b

40.3b

44.9ab

45.0ab

47.1a

Northwind

33.8b

34.9ab

35.9ab

36.9ab

40.8a

abc

Different letter superscripts in the same row indicate statistical difference (Tukey, P<0.05).

Forage yield of the grass Pennisetum purpureum cv. Cuba CT-115 studied here increased with regrowth age, providing a yield distribution of 53.9 % in the rainy season, 26.2 % in the northwind season and 19.9 % in the dry season. Total annual production under the experimental conditions was 28.2 t DM ha-1. Forage nutritional value in terms of CP, IDDM, and NDF and ADF content decreased with regrowth age. From a forage yield and quality perspective, Pennisetum purpureum cv. Cuba CT-115 grass is best at 60 days’ regrowth for direct grazing and 90 days’ for cutting.

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Literature cited: 1. Sosa REE, Cabrera TE, Pérez RD, Ortega RL. Producción estacional de materia seca de gramíneas y leguminosas forrajeras con cortes en el Estado de Quintana Roo. Téc Pecu Méx 2008;46(4):413-426. 2. Araya MM, Boschini C. Producción de forraje y calidad nutricional de variedades de Pennisetum purpureum en la Meseta Central de Costa Rica. Agron Mesoamericana 2005;16(1):37-43. 3. Cruz R, Torres V, Herrera RS, Martínez RO. Cultivo de tejido y fitotecnia de las mutaciones de pastos tropicales. Pennisetum purpureum: otro ejemplo para la obtención de nuevos clones. Rev Cubana Cienc Agríc 1996;30(1):1-10. 4. Hernández GA, Hodgson JG, Matthew C. Effect of spring grazing management on perennial ryegrass and ryegrass-white clover pastures. 1. Tissue turnover and herbage accumulation. N Z J Agric Res 1997;40(1):37-50. 5. Da Silva SC, Hernández GA. Manejo del pastoreo en praderas tropicales. En: Velasco ZME et al, editores. Los Forrajes y su impacto en el trópico. 1ra. ed. Universidad Autónoma de Chiapas. Chiapas, México; 2010:63-95. 6. Velasco ZME, Hernández GA, González HVA, Pérez PJ, Vaquera HH. Curvas estacionales de crecimiento del ballico perenne. Rev Fitotec Mex 2002;25(1):97-106. 7. Herrera RS, Martínez RO, Cruz R, Tuero R, García M, Guisado I et al. Producción de biomasa con hierba elefante (Pennisetum purpureum) y caña de azúcar (Saccharum officinarum) para la ganadería tropical. II. Carbohidratos solubles y estructurales. Rev Cubana Cienc Agric 1995;29(2):245-252. 8. McKenzie BA, Kemp PD, Moot DJ, Matthew C, Lucas RJ. Enviromental effects on plant growth and development. In: White J, Hodgson J editors. New Zealand pasture crop science. Oxford: University Press; 1999:29-44. 9. Zaragoza EJA, Hernández GA, Pérez PJ, Herrera HJG, Osnaya GF, Martínez HP et al. Análisis de crecimiento estacional de una pradera asociada alfalfa-pasto ovillo. Téc Pecu Méx 2009;47(2):173-188.

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10. Santana PAA, Pérez LA, Figueredo AME. Efectos del estado de madurez en el valor nutritivo y momento óptimo de corte del forraje napier (Pennisetum purpureum Schum.) en época lluviosa. Rev Mex Cienc Pecu 2010;1(3):277-286. 11. García E. Modificaciones al sistema de clasificación climática de Köppen. Quinta edición. Serie Libros No. 6. Anexo. Instituto de Geografía, Universidad Nacional Autónoma de México, México; 2004. 12. Gumpertz ML, Brownie C. Repeated measures in randomized blocks and split experiments. Institute of Statistics Mimeograph. Series No. 2202. NCSU, NC, USA; 1991. 13. Toledo JM, Schulze-Kraft R. Metodología para la evaluación agronómica de pastos tropicales. En: Toledo JM editor. Manual para la evaluación. Red Internacional de Evaluación de Pastos Tropicales (RIEPT). Centro Internacional de Agricultura Tropical (CIAT), Cali Colombia; 1982:91-110. 14. Garduño VS, Hernández GA, Herrera HJG, Martínez HPA, Joaquín TBM. Rendimiento y dinámica de crecimiento estacional de Ballico perenne, pastoreado con ovinos a diferentes frecuencias e intensidades. Téc Pecu Méx 2009;47(2):189-202. 15. AOAC. Official Methods of Analysis. 20th Edition. Maryland, USA: Association of Official Analytical Chemists. 2016. 16. Orskov ER, Howell DeBFD, Mould F. The use of the nylon bag technique for the evaluation of feedstuffs. Trop Anim Prod 1980;5(3):195-213. 17. Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci 1991;74(10):3583-3597. 18. Maxwell SE, Delaney HD. Designing experiments and analysing data: A model comparison perspective. Brooks/Cole Publishing Company, Pacific Grove, CA.USA;1990. 19. SAS. SAS User’s Guide: Statistics (version 9.4). Cary NC, USA: SAS Institute Inc. 2013.

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20. Crespo G, Álvarez J. Comparison of biomass production of Penissetum purpureum clones N fertilized. Cuban J Agric Sci 2014;48(3):287-291. 21. Casanovas E, Figueredo Y, Soto R, Novoa R, Valera R. Efecto de la frecuencia de corte en el comportamiento fenológico y productivo de Pennisetum purpureum vc. Cuba CT115 en el periodo poco lluvioso. Rev Cubana Cienc Agríc 2006;40(4):465-470. 22. Herrera RS, García M, Cruz AM, Romero A. Assessmet of Pennisetum purpureum clones obtained by in vitro tissue culture. Cuban J Agric Sci 2014;46(4):427-433. 23. Tarazona AM, Ceballos MC, Naranjo JF, Cuartas CA. Factores que afectan el comportamiento de consumo y selectividad de forrajes en rumiantes. Rev Colomb Cienc Pecu 2012;25(3):473-487. 24. Sanderson MA, Stair DW, Hussey MA. Physiological and morphological responses of perennial forages to stress. Adv Agron 1997;59:171-224. 25. Herrera RS. Clones of Pennisetum purpureum for different ecosystems and productive purposes. Cuban J Agric Sci 2015;49(4):515-519. 26. Luna MR, Chacón ME, Ramírez RJ, Álvarez PG, Álvarez PP, Plúa PK et al. Rendimiento y calidad de dos especies del género Pennisetum en Ecuador. Rev Electrón Vet 2015;16(8):1-10. 27. Fortes D, Herrera RS, García M, Cruz AM, Romero A. Growth analysis of the Pennisetum purpureum cv. Cuba CT-115 in the biomas bank technology. Cuban J Agric Sci 2014;48(2):167-172. 28. Valenciaga D, Chongo B, Herrera RS, Torres V, Oramas A, Cairo JG et al. Efecto de la edad de rebrote en la composición química de Pennisetum purpureum cv. Cuba CT-115. Rev Cubana Cienc Agríc 2009;43(1):73-79. 29. Chacón HPA, Vargas RCF. Digestibilidad y calidad del Pennisetum Purpureum cv. King grass a tres edades de rebrote. Agron Mesoamericana 2009;20(2):399-408. 30. Van Soest PJ. Nutritional ecology of the ruminant. Second ed. Ithaca NY, USA: Cornell University Press; 1994.

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31. Vieira RMA, Fernández AM. Importancia de los estudios cuantitativos asociados a la fibra para la nutrición y alimentación de los rumiantes. 43 Reunión de la Sociedad Brasilera de Zootecnia. Joao Pessoa, Brasil: Sociedad Brasileira de Zootecnia; 2006. 32. Valenciaga D, Chongo B, Lao O. Caracterización del clon Pennisetum CUBA CT-115. Composición química y degradabilidad ruminal de la materia seca. Rev Cubana Cienc Agríc 2001;35(4):349-354. 33. Valles MB, Castillo GE, Bernal BH. Rendimiento y degradabilidad ruminal de materia seca y energía de diez pastos tropicales cosechados a cuatro edades. Rev Mex Cienc Pecu 2016;7(2):141-158.

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https://doi.org/10.22319/rmcp.v13i4.5959 Technical note

Bacterial evaluation of Zacazonapan artisanal cheese matured under noncontrolled conditions in two production periods

Jair Jesús Sánchez-Valdés a Vianey Colín-Navarro a Felipe López-González a Francisca Avilés-Nova b Octavio Alonso Castelán-Ortega c Julieta Gertrudis Estrada Flores a*

a

Universidad Autónoma del Estado de México. Instituto de Ciencias Agropecuarias y Rurales. Campus UAEM El Cerrillo, El Cerrillo Piedras Blancas. 50295, Toluca, Estado de México, México. b

Universidad Autónoma del Estado de México. Centro Universitario UAEM Temascaltepec. Temascaltepec, Estado de México, México. c

Universidad Autónoma del Estado de México. Facultad de Medicina Veterinaria y Zootecnia. Campus UAEM El Cerrillo, Toluca, Estado de México, México.

* Corresponding author: jgestradaf@uaemex.mx

Abstract: Traditional Zacazonapan cheeses have unique organoleptic characteristics and are characterized by being linked to the territory of origin. In the maturation process, there are many interactive variables that are responsible for physical, chemical, biological and structural changes. In order to evaluate the bacteriological evolution of artisanal cheeses during their maturation under non-controlled conditions in two production periods, samples of raw milk and cheese were collected at 0, 30, 60, 120 and 150 d of maturation. The

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presence of molds and yeasts (MaY), mesophilic aerobic bacteria (MAB), Staphylococcus spp. (Staph), total coliforms (TC), fecal coliforms (FC), Salmonella spp. (Salm) and Listeria spp. (List) was determined. The average microbial load was 9.68, 9.38, 8.55 and 8.10 log10 CFU/g of cheese for MaY, MAB, Staph and TC respectively, as well as 2.68 log10 MPN/g of cheese for FC. Salm was not detected but List was. The microbiological evolution of Zacazonapan matured cheese had counts that exceed the maximum levels of the Official Mexican Standard 243 SSA1 2010. Key words: Environmental maturation, Aging, Microbiological evolution, Raw milk.

Received: 08/03/2021 Accepted: 07/04/2022

Traditional cheeses are produced from a complex system that gives rise to unique organoleptic characteristics and are characterized by strong links with their territory of origin(1). In the process, there are several interactive variables that are responsible for physical, chemical, biological and structural changes. Their quality depends on environmental factors, and on interactions between inoculated microorganisms and curd substrates that result from variations in the quality of raw milk and processing conditions(2). Lactic microflora is of particular interest because the biochemical activities of these organisms are involved in cheese making and may play a role in the development of organoleptic characteristics during maturation(3); however, due to their process of preparation and use of raw milk, they can generate outbreaks of food poisoning(4). Zacazonapan cheese is handmade with raw milk from Creole cattle from the southern region of the State of Mexico in the rainy season (July-November). In this season, the feeding of the cattle is based on grazing and the cheeses are consumed after four months of maturation at environmental temperature and relative humidity. This cheese has also been described as Zacazonapan aged cheese(5). Knowing the evolution of the main microbial groups during the maturation of this cheese and the final microbiological quality can suggest modifications to the maturation process that improve the quality without losing any of its characteristics. Therefore, the objective of the study was to evaluate the bacteriological evolution of artisanal cheeses during their maturation under non-controlled conditions in two production periods.

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The work was carried out in seven cheese factories in the municipality of Zacazonapan (19° 07’ 27” N, 100° 02’ 57” W and 1,470 m asl). Its average annual temperature and precipitation is 23.0 °C and 1,041.8 mm respectively. The study was carried out at the end of the cheese production season (from November 2010 to April 2011), which was called a batch of “dry season cheese” due to the environmental conditions in which it is matured. In the middle of the following year’s production season (September 2011 to February 2012), the batch was called “rainy season cheese”. Before the cheese was made, a milk sample was taken from each production site according to the Official Mexican Standard (NOM, for its acronym in Spanish) 109 (handling and collection of samples)(6). The samples were placed in closed sterile containers and transported at 4 °C for their analysis in the microbiology laboratory the next day. Two pieces of fresh cheese weighing approximately 2.0 kg were acquired in both sampling seasons and taken to a cheese factory in the area, where they were left to mature for 150 d under normal conditions of temperature and relative humidity. Using a cork borer, two samples of 50.0 g were collected from each piece of cheese at 0, 30, 60, 120 and 150 d of maturation. Campeche wax was used to seal the holes in the place where the sample was taken. From each sample of cheese, the presence of molds and yeasts (MaY), mesophilic aerobic bacteria (MAB), Staphylococcus spp. (Staph), total coliforms (TC), fecal coliforms (FC), Salmonella spp. (Salm) and Listeria spp. (List) was determined. To determine MaY, 10 g of the central part of the cheese + 10 ml of milk from each sample obtained were homogenized in 90 ml of 0.1 % peptone water (in duplicate) and decimal dilutions were prepared from this first dilution, obtaining dilutions 10-2 to 10-7. Subsequently, they were incubated for 24 h at a temperature of 25 °C(7). To determine MAB, Staph, TC, FC, Salm and List, 25 g from the central part of the cheese + 25 ml of milk from each sample were collected and homogenized in 225 ml of 0.1 % peptone water (in duplicate). Decimal dilutions up to 10-7 were made from this solution, then incubated for 24 h at a temperature of 35 °C(7). According to NOMs, the presence of MaY was determined by plate counting with potato dextrose agar after incubation at 25 °C for 48 h(8). For MAB, tryptone-yeast extract agar was used, incubating at 35 °C for 48 h(9). TCs were determined in plate by violet red bile agar after incubation at 35 °C for 24 h(10). The presence of FC was determined by the most probable number technique, using lauryl sulfate tryptose broth, after incubating at 35 °C for 24 h(11). The determination of Staph with Baird-Parker agar and the addition of egg yolk tellurite, incubated at 37 °C for 48 h after growth(12). Analysis for Salm in SalmonellaShigella agar incubated for 48 h at 35 °C(13) and List incubated at 35 °C for 48 h in Oxford agar(14). The data obtained were normalized by log10, an experimental design of completely randomized blocks was used and analyzed with the command of the general linear model of

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minitab V.14(15). When significant differences were observed (P<0.05), the Tukey test was applied. The evolution of temperature and precipitation in the study region is shown in Figure 1. The maturation temperature of dry season cheeses starts at 24.8 °C in November and ends with 31.8 °C in April; rainfall is almost zero (<12 mm/mo), typical characteristics of the dry season (Figure 1a). In rainy season cheeses, the maturation temperature begins with 22.1 °C in September and ends with 22.7 °C in February, rainfall was 240 mm at the beginning of maturation and 36 mm at the end (Figure 1b). Figure 1: Precipitation and temperature during the environmental maturation of Zacazonapan cheese

a) Dry season cheese

b) Rainy season cheese

Relative humidity, temperature and time are important factors during cheese maturation (16). The maturation temperatures observed in this work favored the development of microorganisms and rainfall affected the final texture of the cheeses; the absence of rains produced drier cheeses, which showed cracked crusts, and the presence of rains produced softer cheeses with wet crusts, which prevented the cheeses from bursting due to the production of gas. However, these cheeses had holes and putrefactive areas inside, a phenomenon known as late swelling(17).

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The counts made on the milk used for cheese making (Table 1) were 7.03, 7.02 and 4.9 (log10 CFU/ml of milk) for MaY, MAB and TC. The use of poor microbiological quality milk is a common practice during the production of artisanal raw milk cheeses and is attributed to poor milking practices. Similarly, the absence of a cold chain and poor transport conditions leads to the counts of MaY, MAB and TC(18). Table 1: Microbiological quality of milk and Zacazonapan cheese during maturation Milk / Cheese MaY 1 Milk 7.03 Cheese maturation days 0 9.250b 30 9.632b 60 9.628b 120 9.908ab 150 10.102a Average 9.704 Cheese production season Dry 9.722 Rainy 9.648 Average 9.685 SEM 0.21

MAB 1 7.02

Staph 1 ND

TC 1 4.9

FC 2 ND

9.499 a 9.882 a 9.403 a 8.858 b 8.900 b 9.308

9.090ab 9.827a 7.133b 8.438b 7.797b 8.457

9.468 a 9.854 a 7.276 b 6.680 b 7.147 b 8.085

3.040 a 3.040 a 3.040 a 2.340 b 1.767 c 2.645

8.912 b 9.865 a 9.388 0.26

7.992 b 9.123 a 8.558 0.529

8.189 8.022 8.106 0.947

3.040 a 2.195 b 2.618 0.264

MaY= molds and yeasts; MAB= mesophilic aerobic bacteria; Staph= Staphylococcus spp. TC= total coliforms; FC= fecal coliforms. 1= log10 colony-forming units / g of cheese or ml of milk. 2= log10 most probable number / g of cheese. ac= different letters within a column indicate differences (P<0.05). SEM= standard error of the mean. ND: Not determined

In both batches of cheese, it is observed that, in the first days of maturation, there is an increase in microbial counts (d 30), which decrease throughout the maturation process due to the biochemical and microbiological processes that occur inside the cheese, such as the reduction of water content, the concentration of solids, the increase in acidity and a reduction in pH caused by the action of lactic bacteria. This causes a microbial competition for nutrients(19). The group of MaY (Table 1) had significant differences (P<0.05) between days of maturation. This is due to the physical retention of microorganisms in rennet and microbial multiplication during milk curdling and whey draining(18). The reduction of MaY from day

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60 is attributed to the fact that, as the maturation progresses, the center of the cheese becomes compressed, and the oxygen needed to microbial reproduction reduces(20). The MaY counts observed in this study are consistent with those found in similar cheeses. For example, the Tepeque aerated cheese had counts of 7.6 log10 CFU/g of cheese in the dry season and 7.7 log10 CFU/g of cheese in the rainy season; and when the Tepeque cheese was taken to maturity, it had 6.2 log10 CFU/g in the dry season and 6.4 log10 CFU/g in cheese matured in the rainy season(21). During the maturation of Kurdish cheese, fresh cheese starts with 5.6 log10 CFU/g of cheese, after 20 d it presents 5.95 log10 CFU/g of cheese, until reaching 9.28 log10 CFU/g of cheese at 40 d, then the load begins to decrease, reaching 9.06 log10 CFU/g of cheese on d 60(22). The same population dynamics were observed in the present study and are related to the depletion of lactose content due to the simultaneous use of lactic acid bacteria(18,20,22). NOM 243 SSA1, which indicates the sanitary provisions and specifications for milk and milk products(23), does not specify the determination of MAB as quality indicator microorganisms in cheeses, because this group includes lactic acid bacteria, which are desirable microorganisms in cheese maturation(24), which, due to their long-term proteolytic and lipolytic activities, contribute to the development of flavor and aroma (18,20,22), providing the typical characteristics to the product of a region. In this study, MAB had significant differences (P<0.05) between days of maturation and between production periods. In this regard, the counts of the dry season cheese were 8.91 log10 CFU/g of cheese and that of the rainy season was 9.86 log10 CFU/g of cheese (Table 1). Microbiological counts increased in the first 30 d of maturation due to the presence of inherent lactic acid bacteria in the milk or rennet used to make the cheese, obtained from young calves from the region. The decrease in MAB counts from d 60 is attributed to the fact that the growth of these organisms during cheese maturation is controlled by some physicochemical factors such as water activity, salt concentration, pH, organic acids, temperature during maturation, oxidation-reduction potential and presence of nitrates(25). The population dynamics of MAB in this study are consistent with those of other cheeses. In artisanal pore cheese three days after being made, MAB were present with a concentration of 6.77 log10 CFU/g of cheese and after 12 d of being made, they increased to 7.44 log10 CFU/g of cheese(16). In the Tepeque aerated cheese, concentrations of 7.9 log10 CFU/g of cheese were reported in the dry season and 7.6 log10 CFU/g of cheese in the rainy season. In Tepeque cheese taken to maturity, the concentration in both the cheese produced in the dry season and in the rainy season was 6.1 log10 CFU/g of cheese(21). In the study 1072


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carried out by Hernández et al(26) on the Zacazonapan aged cheese, the cheeses were matured at a temperature of 24 °C and at a relative humidity of 65 % and at 27 d of maturation, MAB were present at a concentration of 1.5 to 7.8 log10 CFU/g of cheese. The group of Staph (Table 1) had differences (P<0.05) between days of maturation and between production periods. The average of the counts made to the dry season cheese was 7.99 log10 CFU/g of cheese and that of the rainy season was 9.12 log10 CFU/g of cheese. The presence of staphylococci and the appearance of enterotoxins in foods are important parameters in the evaluation of food safety(27). Staphylococcus aureus is often found in raw milk and in the environment of cheese plants (equipment and staff), is salt tolerant and can grow under a wide range of conditions; low acid production can allow staphylococci to grow and produce enterotoxins(16,27). In artisanal pore cheese, it was found that S. aureus was present with an average concentration of 5.91 log10 CFU/g of cheese in three-day old cheese and 6.29 log10 CFU/g of cheese with 12 d of maturation(16). In Tepeque aerated cheese, the microbial load was 7.9 and 7.7 log10 CFU/g of cheese in cheese from the dry season and the rainy season, respectively(21). In Kurdish cheese, the concentration was 3.06 log10 CFU/g of cheese in fresh cheese and 1.12 log10 CFU/g of cheese at 40 d of maturation(22). Results similar to those found in this study indicate serious failures in the hygienic-sanitary conditions of cheese factories(28) and, consequently, the consumption of this cheese can cause staphylococcal poisoning, representing a danger to the consumer(29). Significant differences (P<0.05) were observed in the TC content between days of maturation, decreasing throughout the process. The average of both batches of cheese was 8.10 log10 CFU/g of cheese (Table 1). TCs are a good indicator of hygienic quality, their presence is undesirable because it causes structural defects in the cheese and are an indicator of fecal contamination and reflect lack of hygiene during the preparation or handling of the product and warn of the possible presence of other pathogens (30). During salting, there is a slow process of natural dehydration of cheeses, favoring the survival of these bacteria for longer(16). The bacterial load of TC in cheeses at day zero (Table 1) was similar to that reported in fresh cheeses(28,29). The minimum load observed in this study was 6.68 log10 CFU/g of cheese at 120 d, similar to matured Tepeque cheese(21). However, it exceeds the counts reported in aerated or controlled maturation cheeses, such as artisanal pore cheese(16), Zacazonapan aged cheese(26) and Corrientes cheese(31), which makes clear the importance of controlling the maturation conditions of this type of cheese.

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The counts for FC (Table 1) showed significant differences (P<0.05) between production seasons, being higher in dry season cheeses (3.04 log10 MPN/g of cheese). The results of this study were lower than those found in Mexican tropical cream cheese made with unpasteurized milk from the region of Tonalá, Chiapas(24) and their presence in cheese indicates contamination by feces(32). The decrease in counts after 120 d of maturation in rainy season cheeses is due to the action of lactic acid bacteria(27). The results of the analyses carried out for Salm and List during the maturation of the cheeses (Table 2) indicate that Salm was only detected in the cheeses of d 0 (fresh cheeses) and List was present throughout the maturation. Table 2: Salmonella spp. and Listeria spp. during the maturation of Zacazonapan cheese Days of maturation Microbial group Batch 0 30 60 120 150 Dry season P Salm Rainy season P Dry season P P P P P List Rainy season P P P P P Salm= Salmonella spp.; List= Listeria spp.; P= Present in 25 g of sample.

The presence of Salm in cheeses is related to the preparation of products with unpasteurized milk and it has been detected in fresh cheeses(29), in the first days of maturation(22,27,32). In cheeses with 7 d of maturation, Salm was not detected, and this is attributed to the accumulation of lactic acid and the decrease in pH (< 4.7)(27). The presence of List in cheeses (Table 2) suggests contamination when using milk contaminated by cows that suffer from subclinical mastitis(33). Bacteria of the genus List have been detected in equipment used in the manufacture of cheeses(34), in aerated and matured cheeses(21,28). Although the species was not determined, the risk to consumer health that List represents must be considered, since it can produce important alterations to the central nervous system and even death(34). The results obtained in this research; the bacterial count of the studied groups exceeds what is allowed by NOM 243 SSA1(23). However, there is a worldwide trend of consuming artisanal products that are sought after for their taste and quality linked to the place of origin. Microbial diversity as well as interactions between populations are the main factors that contribute to the taste of traditional cheeses(16,20).

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It is concluded that the maturation for 150 d of both batches of cheeses, as is traditionally done, is insufficient to inhibit the development of pathogenic microorganisms, although the rainy season cheese had a lower microbial load. The presence of bacteria of the genus List and Salm indicated a greater health risk, and it is not suitable for consumption. It is necessary to carry out actions throughout the cheese process, and it is suggested to implement good manufacturing practices for the production of cheese, and that a space of the cheese factory be adapted as a maturation chamber. Acknowledgements and conflicts of interests Work funded by the projects FE016/2009 (COMECYT) and 3101/2011 (Autonomous University of the State of Mexico). The authors thank the National Council for Science and Technology (CONACYT, for its acronym in Spanish) for the grant to the first author in his postgraduate studies and the cheese producers of the municipality of Zacazonapan for the support provided for carrying out this research. The authors of this paper declare that there is no conflict of interest of any kind. Literature cited: 1. Cardoso VM, Dias RS, Soares BM, Clementino LA, Araújo CP, Rosa CA. The influence of ripening period length and season on the microbiological parameters of a traditional Brazilian cheese. Braz J Microbiol 2013;44:743-749. 2. Sicard M, Perrot N, Leclercq-Perlat MN, Baudrit C, Corrieu G. Toward the integration of expert knowledge and instrumental data to control food processes: Application to Camembert-type cheese ripening. J Dairy Sci 2011;94:1-13. 3. Licitra G, Carpino S. The microfloras and sensory profiles of selected protected designation of origin Italian cheeses. Microbiol Spectr 2014;2(1):1-12. 4. Soto VZ, Pérez LL, Estrada AD. Bacterias causantes de enfermedades transmitidas por alimentos: una mirada en Colombia. Salud Uninorte Barranq 2016;32(1):105-122. 5. Hernández MC, Hernández MA, Villegas de Gante AZ, Aguirre ME. El proceso sociotécnico de producción de Queso Añejo de Zacazonapan, Estado de México. Rev Mex Cienc Pecu 2011;2:161-176. 6. Norma Oficial Mexicana NOM-109-SSA1-1994. Bienes y servicios. Procedimientos para la toma, manejo y transporte de muestras de alimentos para su análisis microbiológico. México: Diario Oficial de la Federación, 4 de noviembre de 1994.

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7. Norma Oficial Mexicana NOM-110-SSA1-1994. Bienes y servicios. Para la preparación y dilución de muestras de alimentos para su análisis microbiológico. México: Diario Oficial de la Federación, 28 de abril de 1994. 8. Norma Oficial Mexicana NOM-111-SSA1-1994. Bienes y servicios. Método para la cuenta de mohos y levaduras en alimentos. México: Diario Oficial de la Federación, 28 de abril de 1994. 9. Norma Oficial Mexicana NOM-092-SSA1-1994. Bienes y servicios. Método para la cuenta de bacterias aerobias en placa. México: Diario Oficial de la Federación, 23 de marzo de 1994. 10. Norma Oficial Mexicana NOM-113-SSA1-1994. Bienes y servicios. Método para la cuenta de microorganismos coliformes totales en placa. México: Diario Oficial de la Federación, 28 de abril de 1994. 11. Norma Oficial Mexicana NOM-112-SSA1-1994. Bienes y servicios. Determinación de bacterias coliformes fecales. Técnica del número más probable (NMP). México: Diario Oficial de la Federación, 10 de mayo de 1995. 12. Norma Oficial Mexicana NOM-115-SSA1-1994. Bienes y servicios. Método para la determinación de Staphylococcus aureus en alimentos. México: Diario Oficial de la Federación, 20 de febrero de 1995. 13. Norma Oficial Mexicana NOM-114-SSA1-1994. Bienes y servicios. Método para la determinación de Salmonella en alimentos. México: Diario Oficial de la Federación, 28 de abril de 1994. 14. Norma Oficial Mexicana NOM-143-SSA1-1995. Bienes y servicios. Método de prueba microbiológico para alimentos. Determinación de Listeria monocytogenes. México: Diario Oficial de la Federación, 19 de noviembre de 1997. 15. Minitab V.14. Statistical software. User´s guide II: Data analysis and quality tools, graphics, and Macros 2003; USA. 16. Alejo-Martínez K, Ortiz-Hernández M, Recino-Metelín BR, González-Cortés N, Jiménez-Vera R. Tiempo de maduración y perfil microbiológico del queso de poro artesanal. Rev Iberoamericana Cienc 2015;2 (5):15-24. 17. Vázquez-Fontes C, Sánchez-Vera E, Castelán-Ortega O, Espinoza-Ortega A. Microbiological quality of artisan-made Mexican Botanero cheese in the Central Highlands. J Food Saf 2010;30:40-50.

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18. Volken De SCF, Dalla RT, Zachia AA. Changes in the microbiological and physicochemical characteristics of Serrano cheese during manufacture and ripening. Braz J Microbiol 2003;34:260-266. 19. De Dea LJ, Bernini V, De Lorentiis A, Pecorari A, Neviani E, Gatti M. Parmigiano Reggiano cheese: evolution of cultivable and total lactic microflora and peptidase activities during manufacture and ripening. Dairy Sci Technol 2008;88:511–523. 20. Marino M, Maifreni M, Rondinini G. Microbiological characterization of artisanal Montaisa cheese: analysis of its indigenous lactic acid bacteria. FEMS Microbiol Letón 2003;229:133–140. 21. Solís MAD, Martínez LR, Solorio SJ, Estrada FJG, Avilés NF, Gutiérrez IAT, Castelán OOA. Características del queso Tepeque de la tierra caliente de Michoacán: Un queso producido en un sistema silvopastoril intensivo. Trop Subtrop Agroecosystems 2013;16:201-214. 22. Milani E, Shahidi F, Mortazavi SA, Reza-Vakili SA, Ghoddusi HB. Microbiological, biochemical and rheological changes throughout ripening of Kurdish cheese. J Food Saf 2014;34:168-175. 23. Norma Oficial Mexicana NOM-243-SSA1-2010. Productos y servicios. Leche, fórmula láctea, producto lácteo combinado y derivados lácteos. Disposiciones y especificaciones sanitarias. Métodos de prueba. México: Diario Oficial de la Federación, 27 de septiembre de 2010. 24. Romero-Castillo PA, Leyva-Ruelas G, Cruz-Castillo JG, Santos-Moreno A. Evaluación de la calidad sanitaria de queso crema tropical mexicano de la región de Tonalá, Chiapas. Rev Mex Ing Quim 2009;8:111-119. 25. Beresford TP, Fitzsimons NA, Brennan NL, Cogan TM. Recent advances in cheese microbiology. Int Dairy J 2001;11:259-274. 26. Hernández MC, Hernández MA, Aguirre ME, Villegas de Gante AZ. Physicochemical, microbiological, textural and sensory characterization of Mexican Añejo cheese. Int J Dairy Technol 2010;63(4):552-560. 27. Amran AM, Abbas AA. Microbiological changes and determination of some chemical characteristics for local Yemeni cheese. Jordan J Biol Sci 2011;4 (2):93-100. 28. Castro-Castillo G, Martínez-Castañeda FE, Martínez-Campos AR, Espinoza-Ortega A. Caracterización de la microbiota nativa del queso Oaxaca tradicional en tres fases de elaboración. Rev Soc Ven Microbiol 2013;33(2):105-109. 1077


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29. González-Montiel L, Franco-Fernández MJ. Perfil microbiológico del queso de aro consumido en la Cañada Oaxaqueña. Brazilian J Food Technol 2015;18(3):250-257. 30. Sengul M, Ertugay MF. Microbiological and chemical properties of cheese Helva produced in Turkey. Int J Food Prop 2006;9:185-193. 31. Vasek OM, Mazza SM, Giori GS. Physicochemical and microbiological evaluation of corrientes artisanal cheese during ripening. Food Sci Technol 2013;33(1):151-160. 32. Martins JM, Galinari E, Pimentel-Filho NJ, Ribeiro JJI, Furtado MM, Ferreira CLLF. Determining the minimum ripening time of artisanal Minas cheese, a traditional Brazilian cheese. Braz J Microbiol 2015;46(1):219-230. 33. Meyer-Broseta S, Diot A, Bastian S, Rivière J, Cerf O. Estimation of low bacteria concentration: Listeria monocytogenes in raw milk. Int J Food Microbiol 2003;80:115. 34. Arguello P, Lucero O, Castillo G, Escobar S, Albuja A, Gallegos J, Carrascal A. Calidad microbiológica de los quesos artesanales elaborados en zonas rurales de Riobamba (Ecuador). Perspectiva 2015;16 (18):65-74.

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https://doi.org/10.22319/rmcp.v13i4.5976 Technical note

Antigen production and standardization of an in-house indirect ELISA for detection of antibodies against Anaplasma marginale

Elizabeth Salinas Estrella a* María Guadalupe Ortega Hernández b Erika Flores Pérez b Natividad Montenegro Cristino b Jesús Francisco Preciado de la Torre a Mayra Elizeth Cobaxin Cárdenas a Sergio D. Rodríguez a

a

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad (CENID-SAI), Carretera Cuernavaca-Cuautla no. 8534, Col. Progreso, 62574, Jiutepec, Morelos, México. b

Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria (SENASICA), Jiutepec, Morelos, México.

*Corresponding author: mvz.elisalinest@gmail.com, salinas.elizabeth@inifap.gob.mx

Abstract: Serologic tests are important for the detection of specific antibodies against infectious agents. Commercial indirect ELISA are costly and usually as effective as in-house ELISAs. In the present work, it was prepared a batch of Anaplasma marginale crude antigen from infected blood, and tested it against official positive and negative serum controls and compared with an old batch of antigen. The new antigen batch showed an efficiency similar to the old batch. The sensitivity of the test was comparable between the new and old batches. Both, new and

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old antigen lots are being used at an excess. The new antigen lot is large enough to run thousands of tests at a more affordable price than commercial kits. Key words: Bovine anaplasmosis, Indirect ELISA, Serologic diagnostics.

Received: 06/04/2021 Accepted: 08/03/2022

Anaplasmosis, caused by the Gram-negative bacterium Anaplasma marginale (Rickettsiales; Anaplasmataceae), is an infectious disease that affects cattle and wild ruminants(1). The disease has a high prevalence in tropical, subtropical and even temperate zones and causes jaundice, anemia, production losses of meat and milk, reproductive inefficiency, mortality and related therapeutic costs, as well as trade restrictions for the movement of positive reactors(2). Infection with A. marginale induces the production of antibodies that can be detected by laboratory tests(3,4,5). Many serologic tests have been developed for the detection of specific anti-A. marginale antibodies, including the card-agglutination test(6), complement fixation test(7), indirect fluorescent antibody(8,9) and enzyme linked immunosorbent assay (ELISA)(4,5,10). Serologic tests do not discern between infected and non-infected animals but the presence of specific antibodies can be used for the elimination of positive reactors when purchasing or introducing cattle within an anaplasmosis-free herd or area. The efficiency of the test is of considerable relevance, and serologic data can also be valuable in evaluation of vaccine effectivity(4,11). The Terrestrial Manual, Chapter 3.4.1 of the World Organization for Animal Health (OIE)(12), recommends commercial ELISA kits for the detection of A. marginale antibodies. These include a competence-ELISA (cELISA) based on a recombinant MSP5 protein(5,13,14) and a conventional indirect ELISA (iELISA) kit also based on rMSP5 (15). The Terrestrial Manual also recommends the use of in-house iELISA and provides a protocol for preparation of the antigen and the assay(12). In-house ELISA’s have been developed for serological diagnosis of many pathogens(16-20). At INIFAP, an in-house iELISA kit was developed for the detection of antibodies against A. marginale in cattle serum. The assay is based on the use of initial bodies extracted from infected erythrocytes. Although the production of this antigen requires an initial substantial investment, the quantity and quality and longevity of the antigen allows for the performance of thousands of tests over a long period. This assay was standardized more than 20 yr ago(11) and the batch of antigen prepared then, is still in use. The procedure of A. marginale antigen

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production was carried out again to ascertain the validity of the procedure and the test. The results in the present study are consistent for the serological diagnosis of anaplasmosis in experimental and field samples and at different antigen concentrations. For the present work it was produced an antigen lot following a protocol originally developed for the preparation of antigen for the card agglutination-test (CAT-Ag), and Complement fixation test and adapted for ELISA(9,21,22). The antigen was also tested for antigenicity, against control and field samples. Black Aberdeen Angus calves from the Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria (SENASICA) stock were used for replication of the microorganism. At the beginning of the procedure all animals were negative at end-point PCR for A. marginale(23), Babesia bovis and B. bigemina(24,25) as well for antibodies against all three pathogens. During the experimental period, animals were fed a balanced diet according to weight; water was provided ad libitum. Animals were maintained in isolation and handled under conditions that provided safety for both cattle and operators. Surgery, post-surgical treatment and care of cattle were performed according to the protocol approved by the Internal Committee for the Care and Use of Experimental Animals (CICUAE) of the CENID-SAI of INIFAP(27), based on the surgical technique described by Alexander(28) by certified veterinary personnel. MEX-31-096-01 Anaplasma marginale Mexican strain Tizimín(11,26), was used as the source of the antigen. This strain is cryopreserved in liquid nitrogen as 50% packed cell volume in 10% PVP-40 at 17% infected erythrocytes. For monitoring, blood samples were drawn from the coccygeal vein using evacuated tubes with heparin as anticoagulant. Rectal temperature (RT), packed cell volume (PCV) estimated by the microhematocrit technique and percentage of infected erythrocytes (PIE, quantitated by observation of blood smears at the microscope) were recorded for analysis with each sample collection. Calf 1 was inoculated intramuscularly (IM) with 4 ml of just thawed Tizimín strain infected blood. Monitoring was carried three times a week until appearance of infected erythrocytes in Giemsa-stained blood smears at which time, monitoring was performed daily. The contents of one CPD-blood-bag was drawn. Buffy coat and plasma were removed by centrifugation and aspiration (Hermle Labortechnik, Model: Z 400 K, Series: 50095021); sedimented cells were suspended at 50% in physiological saline (0.85% NaCl), and IM inoculated to the second calf. The same procedure was used for the inoculation of the third calf. At PIE peak, blood from the third calf was drawn by puncture of the jugular vein in commercial CPD-blood bags fitted with 16G needles, (450 ml + 10 % CPDA). Xylazine at an appropriate dose was applied as a sedative to avoid suffering and the animal was physically restrained using ropes.

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The blood was filtered through sterile gauze and erythrocytes sedimented by centrifugation in 500 ml volumes at 2,500 rpm at 4°C for 20 min (Hermle Labortechnik, Z 400 K, Series: 50095021). The buffy coat and plasma were removed by aspiration. Erythrocytes (red blood cells or RBCs) were suspended at 50% in cold PBS pH 7.2 and washed by centrifugation a total of three times; in the last wash, the RBCs were suspended in PBS-antibiotics (PBS-Ab, penicillin 1,000 U/streptomycin 1 mg/ml). The erythrocyte suspension was disrupted in a microfluidizer (Microfluidics, Hc-8000, Series: 99100) at 7,400 PSI) The lysate was centrifuged at 10,000 xg (Hitachi, RPR 9 12 818 rotor) at 4 °C for 30 min; the supernatant was discarded and the sediment suspended with 30 ml of PBS-Ab and homogenized by sonication (Omni Sonic Ruptor 400) for three cycles of 2 min at 50 % power in an ice bath with 1 min breaks to avoid overheating. The homogenate was processed in a high-pressure homogenizer at 1,200 PSI to release all the initial bodies that remained in the non-lysed RBCs and centrifuged at 16,300 xg, for 30 min at 4 °C. Giemsa-stained smears were made from the sediment of every step to monitor for white cell nuclei and RBC membranes. The Ag was adjusted to 4 % w/v in acetate buffer and homogenized by agitation with a magnetic stirrer for 30 min at room temperature. The protein in the Ag was quantified by the micro-Bradford (BioRad®) method using bovine serum albumin as the standard. The antigenicity of this new batch of A. marginale antigen (Ag-2018) was verified by comparing it with a batch in use (Ag-2012). Enough antigen of each lot was mixed with an equal volume of 0.1% SDS in H2O(29) and incubated for 30 min at room temperature (20 to 25 °C). The antigen-SDS mixtures were diluted in carbonate-bicarbonate buffer pH 9.6 to a final volume of 25 mL. Ag-2012 was routinely used at 1.07 µg/200 µL of protein/well. Both antigens were adjusted at the aforementioned concentration. For the purpose of this study, wells in microtiter plates received 200 µL volumes per well of reagents and washing solutions at each step of the procedure. Plates received 1.07 µg of protein/well (1X concentration) and incubated overnight at 4 °C, plates were then washed three times with PBS pH 7.2-Tween20 at 0.05 % (PBS-T20) and blocked with 5% skim milk in PBS-T20 pH 7.2 for 30 min. After three washes with PBS-T20 control serum samples diluted 1:100 in PBS-T20 were run either in duplicates or triplicates. The plates were incubated for 1 h at 37 °C, and washed three times with PBS-T20. Rabbit anti-bovine IgG-alkaline phosphatase conjugate (Jackson ImmunoResearch Laboratories, Lot: 112108) diluted 1:10,000 in PBS-T20 were placed in each well; plates were incubated for 60 min at 37 °C and washed as described; Pnitrophenylphosphate 0.075% in Tris pH 9.5 buffer were added and allowed to incubate for 30 min at 37 °C. The plates were read at 405 nm in a microplate absorbance reader (BioRad, iMark™) and the optical density (OD) values were recorded. Two wells containing all components, except for serum, were used as blanks. The mean absorbance of these blank wells was subtracted from the absorbance values of every other well in the plate. The mean and standard deviation of replicates and, positive and negative sera controls was also calculated.

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The new antigen was subjected to three assays to: a) verify its antigenicity in comparison to Ag-2012, against known control sera, b) define the limit of antigenicity and c) verify its sensitivity against field serum samples. To verify antigenicity, 12 positive and 10 negative control sera routinely used to carry out the iELISA and officially used in the Hemoparasite Laboratory of the National Center for Animal Health Verification Services (CENAPA), were ran in duplicates against each, Ag2012 and Ag-2018 at the same protein concentration (at 1.07 µg/200 µL, 1X). The mean, standard deviation and error were calculated for each set of replicates to determine that the assay had no errors. To test for the antigenicity limit, four positive and four negative control sera were run in duplicate against each antigen at 2X (2.14 µg), 1X (1.07 µg), ½ X (0.535 µg) and ¼ X (0.268 µg) concentrations. The remainder of the assay was carried as described. To verify the antigenicity against field samples, twenty (20) unknown problem sera were tested against both antigen batches at 1X concentration; all samples were run in duplicates. The cutoff point (CP) was calculated as described. Results are expressed as the OD value of the sample minus the value of the blank reading. The positivity index (PI) for each serum sample was calculated as the quotient of the mean absorbance divided by the CP value, where 1= positive; <1= negative. Results were subjected to analysis of variance (ANOVA) by applying the Student t-test using Social Science Statistics available at https://www.socscistatistics.com/tests/anova/Default2.aspx. For the purposes of this report, the percentage that represents the value of the standard deviation (SD) from the mean of each pair of repetitions was used as error, in order to verify the performance of the operator. Whenever the error was  20, the result was discarded and the sample repeated. Means of field samples were subjected to χ2 analysis with the on-line tools VassarStats, (http://vassarstats.net), and Diagnostic test evaluation calculator (https://www.medcalc.org/calc/diagnostic_test.php). Using the procedure described, a maximal 65.7 PEI was reached in the last calf on d 7 with a 26 % PCV. Fourteen 500 ml blood-bags of infected blood were drawn from this last animal. Blood was processed and the antigen produced had a total yield of 144.26 mg of protein in a final volume of 210 ml, equivalent to 0.687 µg/µL of antigen. Ag-2012 which is routinely used at 1/200 dilution, was re-quantified as it was used as reference; this batch had 0.494 µg/µL. To verify antigenicity, 12 positive and 10 negative control sera were run in duplicates against each, Ag-2012 and Ag-2018 at the same protein concentration (1.07 µg/well). The mean,

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standard deviation and error were calculated for each pair to determine that the assay had no errors. Analysis of variance and Student t test of mean values of each serum showed no significant differences (P≥0.5) with an F value of 0.99826 between positive control samples with either antigen. Comparison of negative sera showed that the OD readings were slightly higher when tested against Ag-2012 (Figure 1); analysis of variance of negative OD means showed a significant difference (P>0.01) between the results for Ag-2018 vs Ag-2012 with an F value of 33.54294. From this first comparison the calculated cut-off point for the negative sera tested with Ag2018 was 0.40, while for the same sera tested with the Ag-2012 it was 0.570. The mean positivity index for all positive sera tested with Ag-2018 was 3.44, whereas for the same sera tested with Ag-2012 this was 2.73 (Figure 2). Figure 1: OD values of control sera. Scatter distribution of 12 positive and 10 negative control sera tested against Ag-2012 and Ag-2018 C ONTR OL S ER A DIS TR IBUT IO N positives 2012

positives 2018

negatives 2012

negatives 2018

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0

2

4

6

8

10

12

14

All serum samples were diluted 1:100 and tested in duplicates. Antigens were used at 1.07 g / well.

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Figure 2: Positivity index (PI) scatter distribution. PI values 1= positive (red line); <1= negative

The mean PI value for all positive sera tested with Ag-2018 was 3.44 (dotted line); mean PI for the same sera tested with Ag-2012 was 2.73 (dashed line).

PI values for all positive sera (Figure 2) were calculated for their respective CP. The positivity indexes were 2.69 and 2.093 with Ag-2018 and Ag-2012, respectively. If is divided the mean PI (2.69) for Ag-2018 by Ag-2012 PI (2.093), it gives a quotient of 1.28, this value indicates that Ag-2018 is 1.28 times better at discriminating positive sera than Ag-2012. Determination of the antigenicity limit. The two antigens were tested at four protein concentrations against four positive and four negative control sera as described. As observed, positive and negative sera showed similar distributions at decreasing concentrations against both Ag-2012 (upper panel) and Ag-2018 (lower panel, Figure 3). Analysis of variance within each antigen and between antigens was carried out taking the OD reading of each replica as an independent value. No significant differences were observed when comparing the results between antigens or by concentration against the positive sera; in the case of Ag2018, when the values of the positive sera are taken, F test was not significant is observed, f = 0.56757 and a value of P= 0.639377, at P>0.05. Likewise, when the positive sera are tested against different Ag-2012 concentrations, no significant difference is observed either, f = 1.66871 and a value of P= 0.187871, which is not significant at P>0.05.

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Figure 3: Antigenicity limit for AG-2012 and AG-2018

Four positive and four negative control sera were tested against (left to right) 0.25 X, 0.5 X, 1.0 X and 2.0 X antigen concentrations. Upper panel shows the distribution of positive and negative control relation among the concentrations used for Ag-2012, while lower panel shows the respective for Ag-2018.

When applying ANOVA to the values of each positive sera for each concentration between antigens, there were significant differences. For example, when the results of the sera were compared at the two-fold concentration (2.14 µg/well), a value of F= 18.56523 was obtained; the P value was 0.000342 which was significant for P>0.05. Finally, in order to verify the efficiency of the antigens at discriminating field samples, 20 sera from field cases were tested against both antigens in parallel at 1.07 µg of protein per well. The sera were run in duplicates. Additionally, three negative and three positive control sera were run in the same plate. The cutoff point for each antigen was 0.446 and 0.354 for Ag-2012 and Ag-2018 respectively. 13/20 samples were positive and 7/20 negative against Ag-2012; in contrast to 12/20 positives and 8/20 negatives when tested against Ag-2018.

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Additionally, two samples positive for Ag-2012 were negative for Ag-2018; on the other hand, one negative sample for Ag-2012 was positive for Ag-2018, thus, there was not 100 % coincidence between the two antigens (Table 1). However, when the results were analyzed by χ2 test, the Fisher exact test statistic value was 0.2049; thus, there was no significant difference at P>0.05 between the two antigens at discriminating positives from negatives. According to the diagnostic test evaluation calculator (Table 2), there was 100.00 % sensitivity, 87.5 % specificity, a positive predictive value of 92.31 % and a negative predictive value of 100.00% for the two tests (Table 2). Table 1: Antigenicity verification against field samples. Both antigens were used at a protein of 1.07 µg/well Ag 2012 Ag 2018 Sera

Mean

PI

Mean

PI

1

0.514

1.15

0.365

1.03

2

0.449

1.01

0.386

1.09

3

0.466

1.05

0.386

1.09

4

1.123

2.52

1.167

3.30

5

0.350

0.79

0.269

0.76

6

0.397

0.89

0.310

0.87

7

0.491

1.10

0.356

1.00

8

0.464

1.04

0.328

0.93

9

0.967

2.17

0.937

2.65

10

0.462

1.04

0.335

0.95

11

0.353

0.79

0.279

0.79

12

0.509

1.14

0.435

1.23

13

0.424

0.95

0.376

1.06

14

0.368

0.83

0.316

0.89

15

0.414

0.93

0.319

0.90

16

0.466

1.04

0.431

1.22

17

0.999

2.24

0.973

2.75

18

0.691

1.55

0.583

1.65

19

0.497

1.12

0.444

1.26

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20

Negative control

0.370

0.306

0.427

0.334

0.385

0.299

0.400

Positive control

0.83

0.404

0.291

1.114

1.051

1.125

1.023

1.132

1.124

1.123

Mean of negatives

0.404

0.308

SD

0.021

0.023

PC

0.446

0.354

0.86

0.308

1.066

Serum samples were diluted 1/100 and ran in duplicates. The test conditions were the same as those used for routine examination of unknown samples. Samples in yellow background are those with a positivity index ≥ 1.

Table 2: MedCalc Diagnostic test evaluation. The 2 statistic is 7.2. The P-value is 0.00729 Statistic Value 95% CI (%) Sensitivity 100.00 73.54 to 100.00 % Specificity

87.50

47.35 to 99.68 %

Positive likelihood ratio

8

1.08 to 43.43

Negative likelihood ratio

0

1.28 to 50.04

Disease prevalence (*)

60.00

36.05 to 80.88 %

Positive predictive value (*)

92.31

65.73 to 98.69 %

Negative predictive value (*)

100.00

Accuracy (*)

95.00

75.13 to 99.87 %

* This result is significant at P<0.01. The Yates-corrected 2 statistic is 5. The P-value is .025347. Not significant at P<0.01.

Bovine anaplasmosis is an infectious disease that occurs mainly in tropical and subtropical regions, it is of global importance and, in Mexico, it is distributed throughout the national territory(30,31) causing considerable losses in cattle(2). Serologic diagnosis of this disease is a useful tool for prophylaxis, treatment and control strategies at the individual and herd levels(4). Several techniques have been adapted for the detection of antibodies against A. marginale but immunoassays are preferred because of their scalability, suitability for 1088


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automation, objectivity, and often higher sensitivity and specificity for the identification of asymptomatic bovines(3). When an indirect ELISA test for bovine anaplasmosis diagnosis was developed and compared with the complement fixation test (CFT)(8,32), the iELISA showed several advantages over the CFT, such as objective interpretation and the ability to use hemoglobin tainted serum (not ideal but very common when working with field samples). Moreover, the iELISA is based on antibody-antigen affinity and not antibody concentration. However, several adaptations to the technique have been made since. As the presence of both A. marginale and A. centrale is common in other countries the use of recombinant antigens in iELISA for the detection of antibodies against both agents have been incorporated(5). Absence of A. centrale in Mexico allows for the continued use of iELISA with crude antigen for the routine serological diagnosis of A. marginale. In addition, reports of a positive rate for an ELISA based on whole-bacteria that was significantly greater than the seropositivity rate for an ELISA were recombinant antigens are used(16,33,34) led to the hypothesis that crude native antigens allow for the detection of a wider range of antibodies as they present more than one epitope of the pathogen. In the present work, a batch of crude A. marginale antigen was produced starting from blood of an infected splenectomized steer. The production of crude antigens involves great effort in terms of man-hours and costly disease-free animals, furthermore, it requires surgeries in the experimental animals and the postoperative care during recovery. Through this procedure though, it was possible to reach 65 % infected erythrocytes in the last bovine, from which approximately 14 L of blood were exsanguinated. After a laborious extraction and washing process, 210 mL of antigen were obtained with a final protein concentration of 144.26 mg, equivalent to 0.687 µg/µL. This concentration is higher compared with recombinant protein concentrations(5) where the yield was 40 mg/L and 60 mg/L of culture for each recombinant protein used in that work. This supports the use of crude native proteins as the coating antigen for iELISA tests as its yield implies economic savings and more affordable prices for producers. To test to for antigenicity of the new antigen, 12 positive and 10 negative control sera were run against both antigens. Mean OD readings of positive sera ran against Ag-2012 and Ag2018 were not significantly different; in contrast, the OD values of the negative sera were higher when tested against Ag-2012 than with Ag-2018 (Figure 1). The mean of all negative sera was 0.314 for Ag-2018, while it was 0.450 when tested with Ag-2012. When the mean for positives was divided by the mean for negatives tested with Ag-2012, the quotient was 2.93 whereas that value was 3.79 for Ag-2018. Dividing 3.79 by 2.93 it gets a 1.28 value. This indicates that Ag-2018 is 1.28 times better at discriminating positives from negatives.

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For the antigenicity limit test, antigens were assayed at 2X, 1X, ½X and ¼X protein concentration against a lot of four positive and four negative control sera. While there was a gradual decline in OD readings corresponding to antigen concentration, this decline was not statistically significant, indicating that both antigens were being used at an excess. The variations observed between concentrations of Ag-2018 were minimal, whereas the values of the same sera at the same concentrations of Ag-2012 were more heterogeneous even when they were not significantly different. It was not known if these variations are due to ageing or presence of debris in the antigen lot yet reading with the new lot were more consistent. In the third test, the efficiency of the new antigen was tested against 20 field sera. There were 13 and 12 positives for Ag-2012 and Ag-2018 respectively. While there was no 100 % coincidence (Table 1), the samples that did not coincide were different only by hundredths of a unit (PI), i.e., when they were positive for one and negative for the other, they were right above the PI and vice versa, when they were negative they were right below the PI. This gives an 80 % correlation between antigens. Yet, statistical analysis showed 100 % sensitivity (a remarkable improvement over previously reported for a similar antigen)(32), 87.5 % specificity, 92.31 % positive predictive value, 100 % negative predictive value and 95 % accuracy (Table 2). These results in terms of sensitivity and specificity are comparable to more recently developed ELISA’s(3,5,17). Thus, these results show that both antigen lots are equally reliable to routinely run the in-house iELISA. The present work provides evidence of the reproducibility in the production from one lot of antigen to another, even many years after being prepared, as long as the protocol is followed. The only difference in the preparation of these two antigen batches was the use of newer technology for the disruption of the infected erythrocytes i.e., a microfluidizer, a device that facilitates the process in terms of larger volumes being disrupted and apparently less debris in the final preparation. These two batches of antigen are now in use at the Laboratorio del Departamento de Helmintos y Hemoparásitos of the SENASICA for the diagnosis of bovine anaplasmosis in the cattle of Mexican producers. It was concluded that the new batch of antigen (Ag-2018) is as good as the old batch. Both batches need to be titrated to reduce the concentration in order to optimize the use of each batch, which will in turn reduce the cost of the test making it more affordable for producers. Antigens produced by a governmental laboratory have a high quality standard and are reliable as they are regulated by international and international agencies. The need for commercially available ELISA kits is obviated when an in-house iELISA is available reducing the costs and importing periods thus making testing more efficient and expeditious.

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Acknowledgments The results are part of the activities of the fiscal projects "Conservation of germplasm of the rickettsia Anaplasma marginale" (INIFAP 1162734713) and “Establishment of in vitro culture of Mexican strains of Anaplasma marginale in tick cells” (INIFAP 13341734501). Thanks to all facilities provided by SENASICA for the realization of this work. Literature cited: 1.

Aubry P, Geale DW. A review of bovine anaplasmosis. Transbound Emerg Dis 2011;58(1):1-30.

2.

Rodríguez SD, García-Ortiz MA, Jiménez-Ocampo R, Vega y Murguía CA. Molecular epidemiology of bovine anaplasmosis with a particular focus in Mexico. Infect Genet Evo 2009;9(6):1092-1101. doi: 10.1016/j.meegid.2009.09.007.

3.

Primo ME, Thompson CS, Valentini BS, Sarli M, Novoa MB, et al. Development of a novel fusion protein with Anaplasma marginale and A. centrale MSP5 improved performance of Anaplasma antibody detection by cELISA in infected and vaccinated cattle. PLoS One 2019;14(1):e0211149. DOI: 10.1371/journal.pone.0211149

4.

Rodríguez PJL, Forlano RMD, Meléndez MRD. Dinámica de anticuerpos e infección activa por Anaplasma marginale en becerras. Rev Med Vet 2020;(40):35-44. https://doi.org/10.19052/mv.vol1.iss40.4

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Sarli M, Thompson CS, Novoa MB, Valentini BS, Mastropaolo M, et al. Development and evaluation of a double-antigen sandwich ELISA to identify Anaplasma marginale– infected and A. centrale–vaccinated cattle. J Vet Diagn Invest 2020;32(1):70-76. doi: 10.1177/1040638719892953.

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Löhr KF, Ross JP, Meyer H. Studies on homologous and heterologous antibody responses to infections with Anaplasma marginale and A. centrale using the indirect fluorescent antibody and capillary tube agglutination tests. Z Tropenmed Parasitol 1973;24(1):86-95.

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Price KE, Brock WE, Miller JG. An evaluation of the complement-fixation test for anaplasmosis. Am J Vet Res 1954;15(57):511-516.

8.

Gonzalez E, Long R, Todorovic R. Comparisons of the complement-fixation, indirect fluorescent antibody, and card agglutinationtests for the diagnosis of bovine anaplasmosis. Am J Vet Res 1978;39:1538–1541.

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9.

González BC, Obregón D, Alemán Y, Alfonso P, Vega E, Díaz A, Martínez S. Tendencias en el diagnóstico de la anaplasmosis bovina. Rev Salud Anim 2014;36(2):73-79.

10. Barry DN, Parker RJ, De Vos AJ, Dunster P, Rodwell BJ. A microplate enzyme-linked immunosorbent assay for measuring antibody to Anaplasma marginale in cattle serum. Aust Vet J 1986;63(3):76-79. 11. Rodríguez CSD, García OMA, Cantó AGJ, Hernández SG, Santos CN, et al. Ensayo de un inmunógeno experimental inactivado contra Anaplasma marginale. Tec Pecu Mex1999;37(1):1-12. 12. OIE. Organización Mundial de Sanidad Animal. Manual de las pruebas de diagnóstico y de las vacunas para los animales terrestres (mamíferos, aves y abejas). 2004, Section 3.4.; Chapter 3.4. Bovine Anaplasmosis. https://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.04.01_BOVINE_A NAPLASMOSIS.pdf. 13. Knowles D, Torioni de Echaide S, Palmer G, McGuire T, et al. Antibody against an Anaplasma marginale MSP5 epitope common to tick and erythrocyte stages identifies persistently infected cattle. J Clin Microbiol 1996;34(9):2225-2230. doi: 10.1128/jcm.34.9.2225-2230.1996. 14. Chung C, Wilson C, Bandaranayaka-Mudiyanselage CB, Kang E, Adams DS, et al. Improved diagnostic performance of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5glutathione S-transferase fusion protein as antigen. J Vet Diagn Invest 2014;26(1):6171. doi: 10.1177/1040638713511813. 15. Morzaria SP, Katende J, Musoke A, Nene V, Skilton R, et al. Development of serodiagnostic and molecular tools for the control of important tick-borne pathogens of cattle in Africa. Parassitologia 1999;41 Suppl 1:73-80. 16. Ortona E, Riganoa R, Margutti P, Notargiacomo S, Ioppolo S, et al. Native and recombinant antigens in the immunodiagnosis of human cystic echinococcosis. Parasite Immunol 2000;22(11):553-539. doi: 10.1046/j.1365-3024.2000.00336.x. 17. Schweiger A, Grimm F, Tanner I, Müllhaupt B, Bertogg K, et al. Serological diagnosis of echinococcosis: the diagnostic potential of native antigens. Infection 2012;40(2):139152. doi: 10.1007/s15010-011-0205-6. 18. Galo SS, González K, Téllez Y, García N, Pérez L, et al. Development of in-house serological methods for diagnosis and surveillance of chikungunya. Rev Panam Salud Publica 2017;41:e56. doi: 10.26633/RPSP.2017.56.

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19. Alandijany TA, El-Kafrawy SA, Tolah AM, Sohrab SS, Faizo AA, et al. Development and optimization of in-house ELISA for detection of human IgG antibody to SARSCoV-2 full length spike protein. Pathogens 2020;9(10):803. doi: 10.3390/pathogens9100803. 20. Sil BK, Jahan N, Haq MA, Oishee MJ, Ali T, et al. Development and performance evaluation of a rapid in-house ELISA for retrospective serosurveillance of SARS-CoV2. PLoS One 2021;16(2):e0246346. doi: 10.1371/journal.pone.0246346. 21. Rogers TE, Hidalgo RJ, Dimopoullos GT. Immunology and serology of Anaplasma marginale. I. Fractionation of the complement-fixing antigen. J Bacteriol 1964;88(1):81-86. doi: 10.1128/jb.88.1.81-86.1964. 22. Amerault TE, Roby TO. A rapid card agglutination test for bovine anaplasmosis. J Am Vet Med Assoc 1968;153(12):1828-1834. 23. Torioni de Echaide S, Knowles DP, McGuire TC, Palmer GH, Suarez CE, et al. Detection of cattle naturally infected with Anaplasma marginale in a region of endemicity by nested PCR and a competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5. J Clin Microbiol 1998;36(3):777-782. doi: 10.1128/JCM.36.3.777-782.1998. 24. Figueroa JV, Chieves LP, Johnson GS, Buening GM. Detection of Babesia bigeminainfected carriers by polymerase reaction amplification. J Clin Microbiol 1992;30:25762582. doi: 10.1128/JCM.30.10.2576-2582.1992. 25. Figueroa JV, Chieves LP, Johnson GS, Buening GM. Multiplex polymerase chain reaction-based assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood. Vet Parasitol 1993;50;69–81. doi: 10.1016/0304-4017(93)90008-B. 26. Martínez-Ocampo F, Quiroz-Castañeda RE, Amaro-Estrada I, Cobaxin Cárdenas M, Dantán-González E, et al. Draft genome sequences of Anaplasma marginale strains MEX-15-099-01 and MEX-31-096-01, two Mexican isolates with different degrees of virulence. Microbiol Resour Announc 2019;8(45):e01184-19. doi: 10.1128/MRA.01184-19. 27. Salinas EE, Preciado TJF, Rodríguez CSD, Cobaxin CME, Amaro EI, Quiroz CRE. Esplenectomía experimental en bovinos como apoyo en el estudio de Anaplasma marginale. INIFAP. 2019. Libro Técnico No. 20, ISBN: 978-607-37-1152-4 28. Alexander A. Técnica quirúrgica en animales y temas de terapéutica quirúrgica, 4th ed. México: Interamericana. 1979:170–172.

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29. Winkler GC, Brown GM, Lutz H. Detection of antibodies to Anaplasma marginale by an improved enzyme-linked immunosorbent assay with sodium dodecyl sulfatedisrupted antigen. J Clin Microbiol 1987;25(4):633-636. doi:10.1128/jcm.25.4.633636.1987. 30. Fragoso Sánchez H. 2do Seminario Internacional de Parasitología Animal. Memorias. 1991:153-160. 31. Preciado TJF, García-Ortiz MA, Hernández-Ortiz R, Rodríguez-Camarillo SD. Anaplasmosis bovina en las cuencas lecheras de México. En: Milian SF, Hernández AL, Hernández OR. Situación epidemiológica de la ganadería lechera en México, CENID Microbiología animal, INIFAP. 2015:51-73. 32. Tello RM, Álvarez MJA, Ramos AJA, Aboytes TR, Cantó AGJ. La prueba de ELISA en el diagnóstico de la anaplasmosis. Téc Pecu Méx 1986;52:45-50. 33. Ito A, Xiao N, Liance M, Sato MO, Sako Y, et al. Evaluation of an enzyme-linked immunosorbent assay (ELISA) with affinity-purified Em18 and an ELISA with recombinant Em18 for differential diagnosis of alveolar echinococcosis: results of a blind test. J Clin Microbiol 2002;40:4161-4165. doi:10.1128/JCM.40.11.41614165.2002. 34. Magnarelli LA, Bushmich SL, Sherman BA, Fikrig E. A comparison of serologic tests for the detection of serum antibodies to whole-cell and recombinant Borrelia burgdorferi antigens in cattle. Can Vet J 2004;45(8):667-673.

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Revista Mexicana de Ciencias Pecuarias

Edición Bilingüe Bilingual Edition

Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 4, pp. 846-1094, OCTUBRE-DICIEMBRE-2022

ISSN: 2448-6698

ARTÍCULOS / ARTICLES

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Evaluation of morphological and yield traits in the populations of Vicia spp.

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Efecto de la cobertura del suelo sobre el crecimiento y productividad del zacate buffel (Cenchrus ciliaris L.) en suelos degradados de zonas áridas

Effect of soil cover on the growth and productivity of buffel grass (Cenchrus ciliaris L.) in degraded soils of arid zones Ernesto Herssaín Pedroza-Parga, Aurelio Pedroza-Sandoval, Miguel Agus�n Velásquez-Valle, Ignacio Sánchez-Cohen, RicardoTrejo-Calzada, José Alfredo Samaniego-Gaxiola………………………....................866

Tipología de consumidores de miel con educación universitaria en México

Typology of honey consumers with a university education in Mexico Fidel Ávila Ramos, Lizeth Paula Boyso Mancera, Mercedes Borja Bravo, Venancio Cuevas Reyes, Blanca Isabel Sánchez Toledano......……..…….....…….....……................……..…..........................................…….879

Vertical and spatial price transmission in the Mexican and international cattle and beef market

Transmisión vertical y espacial de precios en el mercado mexicano e internacional de ganado vacuno José Luis Jaramillo Villanueva……………………………………………….......……...................…….....……......…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....……..........…….....…….....……...........894

Exploring bovine fecal bacterial microbiota in the Mapimi Biosphere Reserve, Northern Mexico

Irene Pacheco-Torres Cristina García-De la Peña, César Alberto Meza-Herrera, Felipe Vaca-Paniagua, Clara Estela Díaz-Velásquez, Claudia Fabiola Méndez-Catalá, Luis Antonio Tarango- Arámbula, Luis Manuel Valenzuela-Núñez, Jesús Vásquez-Arroyo…….................…..….…............….…............….…............…..........……..910

Perfil fitoquímico, actividad antimicrobiana y antioxidante de extractos de Gnaphalium oxyphyllum y Euphorbia maculata nativas de Sonora, México

Phytochemical profile, antimicrobial and antioxidant activity of extracts of Gnaphalium oxyphyllum and Euphorbia maculata native to Sonora, Mexico Priscilia Yazmín Heredia-Castro, Claudia Vanessa García-Baldenegro, Alejandro Santos-Espinosa, Iván de Jesús Tolano-Villaverde, Carmen Guadalupe Manzanarez-Quin, Ramón Dolores Valdez-Domínguez, Cris�na Ibarra-Zazueta, Reyna Fabiola Osuna-Chávez, Edgar Omar Rueda-Puente, Carlos Gabriel Hernández-Moreno, Susana Marlene Barrales-Heredia, Jesús Sosa-Castañeda………………...........……..............…….....…….....…….....…….....…….....…….....……...…….....……...…….....……...…….....……...…….....…….....……..........….............928

Effects of acid whey on the fermentative chemical quality and aerobic stability of rehydrated corn grain silage

Efectos del suero ácido sobre la calidad química fermentativa y la estabilidad aeróbica del ensilado de grano de maíz rehidratado Ediane Zanin, Egon Henrique Horst, Caio Abércio Da Silva, Valter Harry Bumbieris Junior.........……………….............………….………….………….………….………….………….………….………….…………........................…...... 943

Growth performance and carcass classification of pure Pelibuey and crossbred lambs raised under an intensive production system in a warm-humid climate

Rendimiento productivo y clasificación de canales de corderos Pelibuey puros y cruzados criados bajo un sistema de producción intensivo en un clima cálido-húmedo Miriam Rosas-Rodríguez, Ricardo Serna-Lagunes, Josa�at Salinas-Ruiz, Julio Miguel Ayala- Rodríguez, Benjamín Alfredo Piña Cárdenas, Juan Salazar-Or�z.…….....……..….....…….....…....….....……...……..…..962

Effect of weight and body condition score from pregnant cows on the carcass characteristics of their progeny: Meta-analysis

Efecto del peso y la puntuación de la condición corporal de vacas gestantes en las características de la canal de su progenie: Meta análisis Sander Mar�nho Adams, John Lenon Klein, Diego Soares Machado, Dari Celes�no Alves Filho, Ivan Luiz Brondani, Luiz Angelo Damian Pizzu�……………………….…….......….…………..............................……….981

Factores de riesgo asociados a la seroprevalencia de lentivirus en rebaños ovinos y caprinos del noreste de México

Risk factors associated with lentivirus seroprevalence in sheep and goat herds from northeastern Mexico Rogelio Ledezma Torres, José C. Segura Correa, Jesús Francisco Chávez Sánchez, Alejandro José Rodríguez García, Sibilina Cedillo Rosales, Gustavo Moreno Degollado, Ramiro Avalos Ramírez……………………………………………….....……..........…….....…….....…….....…….....…….......…….....……...........…......995

Caracterización de los sistemas de producción familiar ovina en la Mixteca Oaxaqueña, México

Family sheep production systems in the Mixteca region of Oaxaca, Mexico Jorge Hernández Bau�sta, Héctor Maximino Rodríguez Magadán Teódulo Salinas Rios, Magaly Aquino Cleto, Araceli Mariscal Méndez………………………....…….......….....…….......….....….....….....…….....……...1009

REVISIONES DE LITERATURA / REVIEWS La hipocalcemia en la vaca lechera. Revisión

Hypocalcemia in the dairy cow. Review Carlos Fernando Arechiga-Flores, Zimri Cortés-Vidauri, Pedro Hernández-Briano, Renato Raúl Lozano-Domínguez, Marco Antonio López-Carlos, Ulises Macías-Cruz, Leonel Avendaño-Reyes…............…1025

NOTAS DE INVESTIGACIÓN / TECHNICAL NOTES Comportamiento productivo y valor nutricional del pasto Pennisetum purpureum cv Cuba CT-115, a diferente edad de rebrote

Productive performance and nutritional value of Pennisetum purpureum cv. Cuba CT-115 grass at different regrowth ages Gloria Esperanza de Dios-León, Jesús Alberto Ramos-Juárez, Francisco Izquierdo-Reyes, Ber�n Maurilio Joaquín-Torres, Francisco Meléndez-Nava……………………………….........………....……....……....…….......1055

Evaluación bacteriana de queso artesanal Zacazonapan madurado bajo condiciones no controladas en dos épocas de producción

Bacterial evaluation of Zacazonapan artisanal cheese matured under non-controlled conditions in two production periods Jair Jesús Sánchez-Valdés, Vianey Colín-Navarro, Felipe López-González, Francisca Avilés-Nova, Octavio Alonso Castelán-Ortega, Julieta Gertrudis Estrada Flores......……..………………..………...……....…..……1067

Antigen production and standardization of an in-house indirect ELISA for detection of antibodies against Anaplasma marginale

Producción de antígenos y estandarización de un ELISA casero indirecto para la detección de anticuerpos contra Anaplasma marginale Elizabeth Salinas Estrella, María Guadalupe Ortega Hernández, Erika Flores Pérez, Na�vidad Montenegro Cris�no, Jesús Francisco Preciado de la Torre, Mayra Elizeth Cobaxin Cárdenas, Sergio D. Rodríguez…….........................………........………........………........………........………........………........………........………........………........………........………...............…………..…..1079

Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 4, pp. 846-1094, OCTUBRE-DICIEMBRE-2022

CONTENIDO CONTENTS

Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 4, pp. 846-1094, OCTUBRE-DICIEMBRE-2022