RMCP Vol. 10, Num. 3 (2019): July-September [english version] by Revista Mexicana de Ciencias Pecuarias

Revista Mexicana de Ciencias Pecuarias

Edición Bilingüe Bilingual Edition

Rev. Mex. Cienc. Pecu. Vol. 10 Núm. 3, pp. 522-800, JULIO-SEPTIEMBRE-2019

ISSN: 2448-6698

CONTENIDO CONTENTS Efecto de la inclusión de granos secos de destilería con solubles (DDGS) en la calidad de la canal y de la carne de conejos en crecimiento Effect of dietary inclusion of distiller’s dried grains with solubles (DDGS) on carcass and meat quality in growing rabbits Ysnagmy Vázquez Pedroso, Hugo Bernal Barragán, Manuel Isidoro Valdivié Navarro, Erasmo Gu�érrez Ornelas, Luis Marino Mora Castellanos, Ernesto Sánchez Alejo, Carlos Alberto Hernández Mar�nez....522 Survival of classic swine fever virus in hams made from the meat of pigs vaccinated with the PAV-250 strain and unvaccinated pigs

Supervivencia del virus de la fiebre porcina clásica en jamones elaborados a partir de la carne procedente de cerdos vacunados con la cepa PAV-250 y de cerdos no vacunados Heidi Amezcua Hempel, María Salud Rubio Lozano, Eliseo Manuel Hernández Baumgarten, Pablo Correa Girón, Oscar Torres Ángeles, María Antonia Coba Ayala, Jose Abel Ciprián Carrasco, Susana Elisa Mendoza Elvira …………………………………………………………………………………………………………………………………….......…………………………………......…..…536

Dietary supplementation of inulin or flavomycin and type of cut of rabbit meat: changes on fatty acid profile and sensorial characteristics

Suplementación dietética de inulina o flavomicina y tipo de corte de carne de conejo: cambios del perfil de ácidos grasos y características sensoriales María Eugenia Juárez-Silva, Mario Cuchillo-Hilario, Enrique Villarreal-Delgado……………………………………………………………………………………………………………………………….......552

Effects of injecting increased doses of vitamins C and E on reproductive parameters of Holstein dairy cattle

Efectos de la inyección de dosis aumentadas de vitaminas C y E en los parámetros reproductivos del ganado lechero Holstein Juan González-Maldonado, Raymundo Rangel-Santos, Raymundo Rodríguez-de Lara, Gustavo Ramírez-Valverde, J. Efrén Ramírez Bribiesca, J. Manuel Vigil-Vigil, M. Fernando García-Espinosa…………...571

Improved farrowing rate using intrauterine insemination in sows Mejoramiento del porcentaje de parición mediante el uso de inseminación artificial en cerdas Fernando Cane, Norma Pereyra, Valen�na Cane, Patricia Marini, Juan Manuel Teijeiro…………………………………………………………………………………………………………………………..583 Economic evaluation of post-weaning and finishing cattle supplemented on pasture Evaluación económica de ganado post-destete y finalizado suplementado en pastoreo de Brachiaria brizantha Aroldo Brandão de Oliveira, Robério Rodigues Silva, Fabiano Ferreira da Silva, Gleidson Giordano Pinto de Carvalho, Ana Paula Gomes da Silva, João Wilian Dias da Silva, Daniele Soares Barroso, Grabriel Dallapicola da Costa……………………………………………………………………………………….......………595

Key factors influencing the sale of bulls in livestock auctions

Factores clave que influyen en la venta de toros en subastas de ganado Giovana Tagliari Evangelista, Jusecléia Ferreira Lopes, Giordano Bruno Fornari, Ricardo Pedroso Oaigen, Thaís Lopes Gonçalves, Tamara Esteves de Oliveira, Luís Kluwe de Aguiar, Júlio Otávio Jardim Barcellos..610

Vertical and spatial price transmission in the Mexican and international milk market

Transmisión de precios vertical y espacial en el mercado mexicano e internacional de leche José Luis Jaramillo-Villanueva, Adriana Palacios-Orozco………………………………………………………………………………………………………………………………………………………..…623

Genetic variability in a Holstein population using SNP markers and their use for monitoring mating strategies Variabilidad genética en una población de vacas Holstein utilizando marcadores SNP y su uso para monitorear estrategias de apareamiento Kathy Scienski, Angelo Ialacci, Alessandro Bagnato, Davide Reginelli, Marina Durán-Aguilar, Maria Giuseppina Strillacci……………………………………………………………………………………….643 Definición de curvas de crecimiento con modelos no lineales en borregas de siete razas con registro de pureza en México Defining growth curves with nonlinear models in seven sheep breeds in Mexico Joel Domínguez-Viveros, Edwin Canul-Santos, Felipe Alonso Rodríguez-Almeida, María Eduviges Burrola-Barraza, Juan Ángel Ortega-Gu�érrez, Francisco Cas�llo-Rangel………………………………….664 Factores de riesgo a nivel de establo asociados con el desempeño reproductivo en el sistema de producción de leche a pequeña escala en México Farm-level risk factors associated with reproductive performance in small-scale dairy farms in Mexico Luis Javier Mon�el-Olguín, Eliab Estrada-Cortés, Mario Alfredo Espinosa-Mar�nez, Miguel Mellado, Josafath Omar Hernández-Vélez, Guillermina Mar�nez-Trejo, Laura Hérnández-Andrade, Rubén Hernández-Or�z, Arcelia Alvarado-Islas, Felipe J Ruiz-López, Héctor Raymundo Vera-Avila……………………………………………………………………………...…676

Actividad acaricida de extractos etanólicos de tres genotipos de Leucaena spp. sobre Rhipicephalus microplus en condiciones in vitro In vitro acaricide activity of extracts from three Leucaena spp. genotypes versus Rhipicephalus microplus Guadalupe González-López, Melina Maribel Ojeda-Chi, Fernando Casanova-Lugo, Iván Oros-Ortega, Luis Ignacio Hernández-Chávez, Ángel Trinidad Piñeiro-Vázquez, Roger Iván Rodríguez-Vivas………..692 REVISIONES DE LITERATURA Bases del sistema inmune de la abeja melífera (Apis mellifera). Revisión Fundaments of the honey bee (Apis mellifera) immune system: Review Alejandra Larsen, Francisco José Reynaldi, Ernesto Guzmán-Novoa……………………………………………………………………………………………………………………………………………..705 Anatomy, physiology, manipulation and veterinary applications of the reticular groove. Review Anatomía, fisiología, manipulación y aplicaciones veterinarias del surco reticular. Revisión María-José Mar�n-Alonso, Luis G. Cal-Pereyra, Maximino Fernández-Caso, José-Ramiro González-Montaña………………………………………………………………………………………………….729 NOTAS DE INVESTIGACIÓN Morfología de nopal forrajero cv Miúda (Nopalea cochenillifera Salm Dyck) en sistemas de cultivo del agreste de Pernambuco, Brasil Organic matter fertilization improves morphological variables in Nopalea cochenillifera Salm Dyck cv. Miúda grown as forage in Pernambuco, Brazil Paulina Vazquez Mendoza, Toni Carvalho de Sousa, Mercia Virginia Ferreira Dos Santos, Oscar Vicente Vazquez Mendoza, Jose Carlos Ba�sta Dubeux Junior, Mario de Andrade Lira……………………..756 Development and evaluation of equations to predict body weight of Pelibuey ewes using heart girth Desarrollo y evaluación de ecuaciones para predecir el peso corporal de ovejas Pelibuey mediante la circunferencia torácica Alfonso J. Chay-Canul, Ricardo A. García-Herrera, Rosario Salazar-Cuytún, Nadia F. Ojeda-Robertos, Aldenamar Cruz-Hernández, Mozart A. Fonseca, Jorge R. Canul-Solís…………………………………767 Eficacia del humo de frutos de Guazuma ulmifolia (Sterculiaceae) y vapores de timol para el control de Varroa destructor infestando abejas africanizadas Effectiveness of the smoke of fruits of Guazuma ulmifolia (Sterculiaceae) and vapors of Thymol for control of Varroa destructor infesting Africanized bees William de Jesús May-Itzá, Luis Abdelmir Medina Medina………………………………………………………………………………………………………………………………………………………778 Rendimiento, parámetros agronómicos y calidad nutricional de la Tithonia diversifolia con base en diferentes niveles de fertilización Yield, agronomic parameters and nutritional quality of Tithonia diversifolia in response to different fertilization levels Julián Mauricio Botero Londoño, Arnulfo Gómez Carabalí, Mónica Andrea Botero Londoño…………………………………………………………………………………………………………………..789

Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 10 Núm. 3, pp. 522-800, JULIO-SEPTIEMBRE-2019

Pags.

Rev. Mex. Cienc. Pecu. Vol. 10 Núm. 3, pp. 522-800, JULIO-SEPTIEMBRE-2019

REVISTA MEXICANA DE CIENCIAS PECUARIAS Volumen 10 Número 3, JulioSeptiembre, 2019. 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 04-2016-060913393200-203. 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 julio de 2019. Ganado Charolais. Fotografía tomada por: Rafael Jiménez Ocampo. Concurso de fotografía INIFAP 2010.

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

EDITOR EN JEFE Arturo García Fraustro

EDITORES POR DISCIPLINA Dra. Yolanda Beatriz Moguel Ordóñez, INIFAP, México Dr. Ramón Molina Barrios, Instituto Tecnológico de Sonora, México Dra. Maria Cristina Schneider, PAHO, Estados Unidos Dra. Elisa Margarita Rubí Chávez, UNAM, 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. Sergio D. Rodríguez Camarillo, INIFAP, México Dr. Martin Talavera Rojas, Universidad Autónoma del Estado de México, 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 Dra. Silvia Elena Buntinx Dios, Facultad de Medicina Veterinaria y Zootecnia, UNAM, México Dr. José Armando Partida de la Peña, INIFAP, México Dr. José Luis Romano Muñoz, INIFAP, México. Dr. Alejandro Plascencia Jorquera, Universidad Autónoma de Baja California, México Dr. Juan Ku Vera, Universidad Autónoma de Yucatán, México Dr. Ricardo Basurto Gutiérrez, INIFAP, México. Dr. Luis Corona Gochi, Facultad de Medicina Veterinaria y Zootecnia, UNAM, México Dr. Juan Manuel Pinos Rodríguez, Facultad de Medicina Veterinaria y Zootecnia, Universidad Veracruzana, México Dr. Carlos López Coello, Facultad de Medicina Veterinaria y Zootecnia, UNAM, México Dr. Arturo Francisco Castellanos Ruelas, Facultad de Química. UADY Dra. Guillermina Ávila Ramírez, UNAM, México. Dr. Emmanuel Camuus, CIRAD, Francia. Dr. Héctor Jiménez Severiano, INIFAP., México Dr. Juan Hebert Hernández Medrano, UNAM, México. Dr. Adrian Guzmán Sánchez, Universidad Autónoma Metropolitana-Xochimilco, México Dr. Eugenio Villagómez Amezcua Manjarrez, INIFAP, CENID Salud Animal e Inocuidad, México Dr. Fernando Cervantes Escoto, Universidad Autónoma Chapingo, México Dr. Adolfo Guadalupe Álvarez Macías, Universidad Autónoma Metropolitana Xochimilco, México Dr. Alfredo Cesín Vargas, UNAM, México.

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REVISTA MEXICANA DE CIENCIAS PECUARIAS REV. MEX. CIENC. PECU.

VOL. 10 No. 3

JULIO-SEPTIEMBRE-2019

CONTENIDO ARTÍCULOS

Pág. Efecto de la inclusión de granos secos de destilería con solubles (DDGS) en la calidad de la canal y de la carne de conejos en crecimiento Effect of dietary inclusion of distiller’s dried grains with solubles (DDGS) on carcass and meat quality in growing rabbits Ysnagmy Vázquez Pedroso, Hugo Bernal Barragán, Manuel Isidoro Valdivié Navarro, Erasmo Gutiérrez Ornelas, Luis Marino Mora Castellanos, Ernesto Sánchez Alejo, Carlos Alberto Hernández Martínez............................................................................................................................. ..................522

Survival of classic swine fever virus in hams made from the meat of pigs vaccinated with the PAV-250 strain and unvaccinated pigs Supervivencia del virus de la fiebre porcina clásica en jamones elaborados a partir de la carne procedente de cerdos vacunados con la cepa PAV-250 y de cerdos no vacunados Heidi Amezcua Hempel, María Salud Rubio Lozano, Eliseo Manuel Hernández Baumgarten, Pablo Correa Girón, Oscar Torres Ángeles, María Antonia Coba Ayala, Jose Abel Ciprián Carrasco, Susana Elisa Mendoza Elvira ……………………………………………………………………..………………………………………………………..…536

Dietary supplementation of inulin or flavomycin and type of cut of rabbit meat: changes on fatty acid profile and sensorial characteristics Suplementación dietética de inulina o flavomicina y tipo de corte de carne de conejo: cambios del perfil de ácidos grasos y características sensoriales María Eugenia Juárez-Silva, Mario Cuchillo-Hilario, Enrique Villarreal-Delgado............................. ..552

Effects of injecting increased doses of vitamins C and E on reproductive parameters of Holstein dairy cattle Efectos de la inyección de dosis aumentadas de vitaminas C y E en los parámetros reproductivos del ganado lechero Holstein Juan González-Maldonado, Raymundo Rangel-Santos, Raymundo Rodríguez-de Lara, Gustavo Ramírez-Valverde, J. Efrén Ramírez Bribiesca, J. Manuel Vigil-Vigil, M. Fernando García-Espinosa 571

III

Economic evaluation of post-weaning and finishing cattle supplemented on pasture Evaluación económica de ganado post-destete y finalizado suplementado en pastoreo de

Brachiaria brizantha

Aroldo Brandão de Oliveira, Robério Rodigues Silva, Fabiano Ferreira da Silva, Gleidson Giordano Pinto de Carvalho, Ana Paula Gomes da Silva, João Wilian Dias da Silva, Daniele Soares Barroso, Grabriel Dallapicola da Costa ........................................................................................................... 595

Key factors influencing the sale of bulls in livestock auctions Factores clave que influyen en la venta de toros en subastas de ganado Giovana Tagliari Evangelista, Jusecléia Ferreira Lopes, Giordano Bruno Fornari, Ricardo Pedroso Oaigen, Thaís Lopes Gonçalves, Tamara Esteves de Oliveira, Luís Kluwe de Aguiar, Júlio Otávio Jardim Barcellos ........................................................................................................................................... 610

Vertical and spatial price transmission in the Mexican and international milk market Transmisión de precios vertical y espacial en el mercado mexicano e internacional de leche José Luis Jaramillo-Villanueva, Adriana Palacios-Orozco ................................................................. 623

Definición de curvas de crecimiento con modelos no lineales en borregas de siete razas con registro de pureza en México Defining growth curves with nonlinear models in seven sheep breeds in Mexico Joel Domínguez-Viveros, Edwin Canul-Santos, Felipe Alonso Rodríguez-Almeida, María Eduviges Burrola-Barraza, Juan Ángel Ortega-Gutiérrez, Francisco Castillo-Rangel ...................................... 664

Factores de riesgo a nivel de establo asociados con el desempeño reproductivo en el sistema de producción de leche a pequeña escala en México Farm-level risk factors associated with reproductive performance in small-scale dairy farms in Mexico Luis Javier Montiel-Olguín, Eliab Estrada-Cortés, Mario Alfredo Espinosa-Martínez, Miguel Mellado, Josafath Omar Hernández-Vélez, Guillermina Martínez-Trejo, Laura Hérnández-Andrade, Rubén Hernández-Ortíz, Arcelia Alvarado-Islas, Felipe J Ruiz-López, Héctor Raymundo Vera-Avila ......... 676

Rhipicephalus microplus

Guadalupe González-López, Melina Maribel Ojeda-Chi, Fernando Casanova-Lugo, Iván Oros-Ortega, Luis Ignacio Hernández-Chávez, Ángel Trinidad Piñeiro-Vázquez, Roger Iván Rodríguez-Vivas ..... 692

REVISIONES DE LITERATURA

Bases del sistema inmune de la abeja melífera (Apis mellifera). Revisión Fundaments of the honey bee (Apis mellifera) immune system: Review Alejandra Larsen, Francisco José Reynaldi, Ernesto Guzmán-Novoa ............................................... 705

Anatomy, physiology, manipulation and veterinary applications of the reticular groove. Review Anatomía, fisiología, manipulación y aplicaciones veterinarias del surco reticular. Revisión María-José Martín-Alonso, Luis G. Cal-Pereyra, Maximino Fernández-Caso, José-Ramiro GonzálezMontaña ............................................................................................................................................ 729

NOTAS DE INVESTIGACIÓN

Morfología de nopal forrajero cv Miúda (Nopalea cochenillifera Salm Dyck) en sistemas de cultivo del agreste de Pernambuco, Brasil Organic matter fertilization improves morphological variables in Nopalea cochenillifera Salm Dyck cv. Miúda grown as forage in Pernambuco, Brazil Paulina Vazquez Mendoza, Toni Carvalho de Sousa, Mercia Virginia Ferreira Dos Santos, Oscar Vicente Vazquez Mendoza, Jose Carlos Batista Dubeux Junior, Mario de Andrade Lira ................................ 756

Development and evaluation of equations to predict body weight of Pelibuey ewes using heart girth Desarrollo y evaluación de ecuaciones para predecir el peso corporal de ovejas Pelibuey mediante la circunferencia torácica Alfonso J. Chay-Canul, Ricardo A. García-Herrera, Rosario Salazar-Cuytún, Nadia F. Ojeda-Robertos, Aldenamar Cruz-Hernández, Mozart A. Fonseca, Jorge R. Canul-Solís .......................................................................................................................................................... 767

Eficacia del humo de frutos de Guazuma ulmifolia (Sterculiaceae) y vapores de timol para el control de Varroa destructor infestando abejas africanizadas Effectiveness of the smoke of fruits of Guazuma ulmifolia (Sterculiaceae) and vapors of Thymol for control of Varroa destructor infesting Africanized bees William de Jesús May-Itzá, Luis Abdelmir Medina Medina ............................................................... 778

Rendimiento, parámetros agronómicos y calidad nutricional de la Tithonia diversifolia con base en diferentes niveles de fertilización Yield, agronomic parameters and nutritional quality of Tithonia diversifolia in response to different fertilization levels Julián Mauricio Botero Londoño, Arnulfo Gómez Carabalí, Mónica Andrea Botero Londoño ............ 789

Actualización: abril, 2018 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.

indican, empezando cada uno de ellos en página aparte. Página del título Resumen en español Resumen en inglés Texto Agradecimientosy conflicto de interés Literatura citada Cuadros y gráficas

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.

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 formato de derechos patrimoniales disponibles en el propio sitio oficial de la revista.

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

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

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

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

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

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). 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.

Introducción Materiales y Métodos Resultados Discusión Conclusiones e implicaciones

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

VII

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.

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.

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

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.”). I)

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

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.

Sólo número sin indicar volumen. II) Stephano HA, Gay GM, Ramírez TC. Encephalomielitis, reproductive failure and corneal opacity (blue eye) in

VIII

pigs associated with a paramyxovirus infection. Vet Rec 1988;(122):6-10.

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.

No se indica el autor. IV) Cancer in South Africa [editorial]. S Afr Med J 1994;84:15.

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

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

Libros y otras monografías

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.

Autor de capítulo. IX)

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)

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

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

Organización como autor. 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. Oficial 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.

Publicaciones electrónicas 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. 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. 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. 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.

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

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 gráficas y figuras se deberán elaborar en Word, Power Point, Corel Draw y enviadas en archivo aparte (nunca insertarlas como imágenes en el 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. 18. Abreviaturas de uso frecuente: cal cm °C DL50 g ha h i.m. i.v. J kg

caloría (s) centímetro (s) grado centígrado (s) dosis letal 50% gramo (s) hectárea (s) hora (s) intramuscular (mente) intravenosa (mente) joule (s) kilogramo (s)

versus

gravedades

Cualquier otra abreviatura se pondrá entre paréntesis inmediatamente después de la(s) palabra(s) completa(s). 19. Los nombres científicos y otras locuciones latinas se deben escribir en cursivas.

Updated: April, 2018 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.

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 â&#x20AC;&#x153;Guide for submit articles in the Web site of the Revista Mexicana de Ciencias Pecuariasâ&#x20AC;?. Manuscripts should be prepared, typed in a 12 points font at double space (including the abstract and tables). At the time of submission, the application form, must be filled out, as well as a letter of originality and no duplication and patrimonial rights format, available on the official website of the journal.

References Tables and Graphics 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).

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

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

a) Research Articles. They should originate in primary

works and may show partial or final results of research. The text of the article must include the following parts: Introduction Materials and Methods Results Discussion Conclusions and implications

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.

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.

Research articles will not exceed 20 double spaced pages, without including Title page and Tables and Figures (8 maximum). 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.

b) Technical Notes. They should be brief and be evidence for technical changes, reports of clinical cases of special interest, complete description of a limited investigation, or research results which should be published as a note in the opinion of the editors. The text will contain the same information presented in the sections of the research article but without section titles.

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.

c) Reviews. The purpose of these papers is to

Title page Abstract Text Acknowledgments

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. References. All references should be quoted in their original language. They should be numbered consecutively in the order in which they are first mentioned in the text. Text, tables and figure references should be identified by means of Arabic numbers. Avoid, whenever possible, mentioning in the text the name of the authors. Abstain from using abstracts as references. Also, “unpublished observations” and “personal communications” should not be used as references, although they can be inserted in the text (inside brackets).

Journals

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

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

Issue with no volume 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.

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.

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

No author given

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

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

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

c. Accepted articles, even if still not published, can be included in the list of references, as long as the journal is specified and followed by “in press” (in brackets).

Organization, as author

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 names(s), the number of the edition, the country, the printing house and the year.

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

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.

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

XII

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

Books and other monographs

Author(s)

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

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

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.

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.

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.

Conference paper X)

XI)

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.

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.

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 incorporated all the corrections and modifications asked for by the editors. Graphs and figures should be submitted separately in Microsoft Word, MS Power Point, or Corel Draw. Figures must not be inserted as images within the text. In Tables do not use internal horizontal or vertical lines.

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

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.

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

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.

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.

17. List of abbreviations:

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

cal cm °C

XIII

calorie (s) centimeter (s) degree Celsius

DL50 g ha h i.m. i.v. J kg km L log Mcal MJ m Âľl Âľm mg ml mm min ng

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) mega joule (s) meter (s) micro liter (s) micro meter (s) milligram (s) milliliter (s) millimeter (s) minute (s) nanogram (s)

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

versus

gravidity

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

XIV

https://doi.org/10.22319/rmcp.v10i3.4356 Article

Effect of dietary inclusion of distiller’s dried grains with solubles (DDGS) on carcass and meat quality in growing rabbits Ysnagmy Vázquez Pedroso a Hugo Bernal Barragán b* Manuel Isidoro Valdivié Navarro a Erasmo Gutiérrez Ornelas b Luis Marino Mora Castellanos a Ernesto Sánchez Alejo b Carlos Alberto Hernández Martínez b

Instituto de Ciencia Animal. Carretera Central, Km. 47½. San José de las Lajas, Mayabeque, Cuba. b

Universidad Autónoma de Nuevo León. Facultad de Agronomía. Campus de Ciencias Agropecuarias. Gral. Escobedo N.L. México.

*Corresponding author: hugo.bernalbr@uanl.edu.mx; hubernal05@yahoo.com.mx Abstract: Distiller’s dried grains with solubles (DDGS) are widely in livestock diets to replace costly ingredients. An evaluation was done of the effect of dietary inclusion of different levels (0, 10, 20 and 30 %) of DDGS on carcass and meat quality in rabbits. At 96 d, after the growth period, a sample of 56 rabbits (Negro Azteca x Chinchilla) were slaughtered. Carcass characteristics were measured and calculated using twenty rabbits (5 per treatment): carcass proportions of anterior limbs, posterior limbs, ribs and loins; weight of meat, bone and loin fat; and the meat:bone ratio. A sensory evaluation of rabbit meat acceptance was done with a panel of 46 untrained evaluators who expressed their perceptions of meat aroma, color, flavor and texture. Color of the Longissimus dorsi muscle was quantified with the CIELAB system, and texture measured via shear force. Carcass and meat quality results were analyzed with an ANOVA. Sensory evaluation results were assessed with non-parametric statistics. No differences (P>0.05) were present in the carcass, organoleptic and meat texture results. The b* chromatic parameter was higher (P<0.05) in the treatments containing 10, 20 and 30 % DDGS (11.77, 12.17 and 12.22, respectively) than in the control diet (9.68). Sensory evaluation showed that 522

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rabbit meat with or without DDGS was perceived as having an agreeable aroma and taste, pale color and soft texture. Dietary inclusion of DDGS at up to 30 % had no effect on carcass or meat characteristics in rabbits. Key words: DDGS, Rabbits, Carcass quality, Meat quality.

Received: 24/01/2017 Accepted: 07/05/2018

Introduction

Ethanol production has grown notably worldwide. From 16.6 million liters in 2001 production it has boomed to 83.4 million in 2011(1), and will continue expanding in response to global demand for biofuels(2,3). The raw materials used to produce ethanol vary by region and country. In general, the European Union produces ethanol from different grains, while Brazil generates it from sugar cane(4,5) and the United States from corn. The largest ethanol producer in the world is the United States, which reached a total production of 60 billion liters in 2014. Distillerâ&#x20AC;&#x2122;s dried grains with solubles (DDGS) are a biofuels industry byproduct, the nutritional value, availability and costs of which provide an opportunity for their use in animal feeds(6). Ethanol production in Mexico is based on sugarcane and sweet sorghum, neither of which produce DDGS(7). However livestock producers in Mexico have found DDGS to be a valuable resource that can replace grains such as maize and sorghum, as well as soy flour, in animal diets. Their financial and sustainability advantages have led to heavy consumption and consequent importation of DDGS from the U.S.(8). Imported, competitively-priced DDGS represent a source of protein, amino acids, fat, energy and minerals that can replace conventional ingredients, many of which are also used for human food. In the United States, DDGS is mostly used in ruminants (66 % beef cattle and 14 % dairy cattle), but pig production has reached 12 % of total DDGS consumption, while poultry production uses about 8 % of available DDGS(9). Use of DDGS in animal feed is expected to increase in coming years due positive results when included in poultry feed(10,11). Very little research has been done on DDGS in diets for rabbits. Studies have been done on productive performance(12-15), nutrient digestibility(16,17), morphometry and other 523

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carcass traits(18,19,20). Even if DDGS can replace grains and oilseed byproducts in rabbit diets, with corresponding benefits for producers, evaluations are still needed on the effect dietary inclusion of DDGS may have on marketable portions of the carcass and rabbit meat sensory characteristics. The present study objective was to evaluate the effects of dietary inclusion of DDGS on carcass and meat quality in growing rabbits.

Materials and methods

The research was done at the rabbit production facilities of the La Ascension Unit of the Faculty of Agronomy of the Autonomous University of Nuevo Leon (Universidad Autonoma de Nuevo León – UANL) in Aramberri, Nuevo Leon, Mexico. Some analyses were done at the Sensory Evaluation Laboratory of the Food Industries Research and Development Center of the UANL. Animals were 56 hybrid rabbits (Negro Azteca x Chinchilla) weaned at 40 days of age, with an average live weight of 752 ± 39 g. Management and feeding conditions were similar for all animals, with free access to water and feed. All animals were housed at a density of two rabbits per cage in galvanized wire cages (840 x 330 x 400 mm) provided with a feeder and water bottle. Each cage was treated as an experimental unit. Four DDGS inclusion levels (0, 10, 20 and 30 %) were tested, and each level was considered a treatment (n = 7 cages per treatment). Addition of DDGS was mostly compensated for by reducing contents of sorghum, soy flour and monocalcium phosphate (Table 1), based on the control diet (0% DDGS)(15).

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Table 1: Diet composition and nutrient contribution as fed basis Ingredients Alfalfa meal Sorghum grain Soybeanmeal DDGS Molasses Monocalcium phosphate Salt Vit+trace min premix1 DL-Methionine L-Lysine Soya oil Analyzed contribution: Crude protein, % Crude fat, % NDF, % ADF, % Gross energy, kcal/kg Calculated composition: Crude fiber, % Digest energy, kcal/kg Total phosphorous, % Calcium, % Lysine, % Methonine + Cysteine, % 1

DDGS Treatments (%) 10 20 49.05 53.88 26.94 17.20 9.60 4.60 10.00 20.00 3.00 3.00 0.54 0.46 0.50 0.50 0.20 0.20 0.14 0.14 0.00 0.02 0.00 0.00

0 50.38 30.00 13.70 0.00 3.00 0.68 0.50 0.20 0.14 0.00 1.40

30 55.28 10.40 0.00 30.00 3.00 0.36 0.50 0.20 0.14 0.12 0.00

17.05 3.23 18.89 15.32 3006

16.73 2.82 22.30 17.85 3106

16.94 3.57 24.82 18.87 3239

17.42 4.99 27.94 20.95 3286

17.43 2814 0.45 0.88 0.77 0.60

17.57 2714 0.45 0.85 0.71 0.60

19.46 2635 0.45 0.90 0.65 0.60

20.36 2583 0.45 0.91 0.65 0.60

Vit + trace mineral premix provided (per kilo premix): Vit. A: 12,000,000 UI; Vit. D3: 1,500,000 UI; Vit. E: 60,000 UI; Vit. K3: 2 g; thiamin (B1): 2 g; riboflavin: 6 g; pyridoxin (B6): 3.5 g; B12: 20 mg; biotin: 150 mg; folic acid: 520 mg; niacin: 60 g; pantothenic acid: 15 g; and choline chloride: 500 g. Minerals: manganese 40 g; zinc: 100 g; iron: 90 g; copper: 10 g, iodine: 480 mg; selenium: 240 mg.

At the end of the finishing trial (96 days), at an average commercial weight of 1.955 Âą 86 g, twenty rabbits were randomly selected (five per treatment) and slaughtered without previous fasting. The animals were slaughtered with a single blow to the base of the skull, on the upper portion of the neck, in the occipital region, and death confirmed by circulation ceasing(21). These animals were used to provide meat for the sensory tests. Weight was measured of the anterior and posterior limbs, rib section and loin. Each portion was then boned and weight measured for total meat and bone, and loin fat. These figures were used to calculate the carcass meat:bone ratio following an established methodology(22). The Longissimus dorsi muscle (LD) to the 5th lumbar vertebra was extracted to evaluate meat color and tenderness. Carcasses were butchered according to a common methodology(23). After 24 h refrigeration, meat color was quantified with a colorimeter 525

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(CR-400, Konica, Minolta, Japan) by measuring the color parameters used in the CIELAB color space(24): luminosity (L*); index (a*), greens (negative values) to reds (positive values); and index (b*) blues (negative values) to yellows (positive values). Meat tenderness was evaluated with a texturometer (TA-XT Plus, Texture Analyzer, Stable Micro Systems, Godalming, UK), equipped with a triangular cut Warner-Bratzler blade(25) to measure shear force. Meat sensory evaluation was done by using affective analysis with the participation of potential or current consumers, who express their preferences among several products offered for evaluation(26). The panel of 46 evaluators (age range = 17 to 56 yr) were prepared following a method developed for pork(27). In an effort to offer a meat product at least somewhat familiar to the panelists, for each independent treatment the meat samples were prepared as meatballs (fried) without added spices, except salt. Each panelist was offered four samples (one from each treatment) on a tray along with a glass of water. Samples were randomly identified with a code. The organoleptic characteristics of aroma, color, flavor and texture were measured with a 1-to-5 sensory scale(28). For aroma the scale corresponded to very disagreeable (1), disagreeable (2), neither agreeable nor disagreeable (3), agreeable (4), and very agreeable (5). The color scale was very strong (1), strong (2), neither pale nor strong (3), pale (4), and very pale (5). The five flavor values were strongly dislike (1), dislike (2) neither like nor dislike (3), like (4) and like very much (5). For texture they corresponded to very firm (1), firm (2), neither soft nor firm (3), soft (4), and very soft (5). Statistical analysis of the variables for cold carcass weight, carcass quality and meat quality (expressed as a percentage cold carcass weight) was done with the StatSoft program(29). The theoretical assumptions of the analysis of variance were tested with the Levene variance homogeneity analysis(30), and the Shapiro-Wilk error normality test(31). The data met these assumptions and therefore required no transformation. An analysis of variance was then run following a completely randomized design with four treatments and five replicates per treatment. Differences between treatments were identified with a Duncan Test(33) using a P<0.05 significance level. Analyses were done with the INFOSTAT ver. 2012 statistical package(32). Meat sensory evaluation results were examined with a non-parametric statistics (Ď&#x2021;2) analysis of response frequency to identify differences between treatments for each indicator. This analysis was run with the SPSS ver. 24 package.

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Results and discussion

No differences (P>0.05) were observed for any of the carcass weight and edible cut variables (Table 2). Inclusion of up to 30% DDGS in the diet of growing rabbits had no effect on carcass characteristics. Even though the question arises if this absence of effect on carcass composition may be due to the number of replicates employed (n= 5 per treatment). The present results agree with those of previous studies involving inclusion levels of up to 20% DDGS(19,20), and up to 28% DDGS(34) in diets for rabbits without alterations in carcass yield.

Table 2: Carcass weight and commercial cut proportion in rabbits fed diets containing different levels of DDGS Indicators Cold carcass weight, g Posterior limbs, % Loin, % Ribs, % Anterior limbs, %

0 1057.1 34.15 26.39 21.15 18.31

DDGS (%) 10 20 957.7 1004.8 31.84 31.66 32.41 31.89 19.37 20.86 16.39 15.59

30 956.9 31.18 29.40 23.00 16.42

SE (Âą)

35.31 1.47 1.84 1.34 0.72

0.1851 0.5070 0.1213 0.3294 0.0913

As in the present study up to 30% DDGS inclusion in the diet caused no negative effects in carcass composition, this highlights the benefit of DDGS inclusion in diets for growing rabbits since up to 65 % of sorghum grain and 100 % of soybean were replaced in the diet. In a previous study(34), DDGS was used in diets for growing rabbits to replace 65 % alfalfa hay and 100 % soybean meal in the reference diet. In contrast, the goal of our research group is to evaluate the use of more fodder and agro-industrial byproducts in livestock diets without negatively affecting, or ideally improving, productive performance. This coincides with a study in which up to 65 % of grains and 95 % of soybean meal were substituted with up to 30 % DDGS in diets for growing rabbits with good results in growth indicators(14). The studied DDGS inclusion levels did not affect (P>0.05) the carcass meat, bone or fat proportions (Table 3). The meat proportion averaged 65 Âą 1.24 %, which corresponded to 2.2 times the bone proportion. Fat content was less than 2.5 % in all treatments. These results are similar to those of a study in which no differences were observed in the rabbit leg meat:bone ratio and in visceral fat content in response to DDGS inclusion levels ranging from 0 to 28 % in diets for growing rabbits(34).

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Table 3: Carcass meat, bone and fat proportions and the meat:bone ratio in rabbits fed diets containing different levels of DDGS Indicators

DDGS (%) 10 20 64.95 65.72 30.36 30.98 1.59 1.04 2.15 2.15

0 65.52 28.46 2.30 2.32

Meat, % Bone, % Loin fat, % Meat:Bone

30 64.35 29.95 2.28 2.18

SE (Âą)

1.24 1.29 0.36 0.13

0.8615 0.5704 0.0743 0.7319

In the sensory evaluation differences (P<0.05) were observed in response frequency for each of the five categories in each evaluated rabbit meat parameter: aroma, color, flavor and texture (Table 4). However, panelist opinions did not differ between treatments (P>0.367).

Table 4: Sensory evaluation of meat from rabbits fed diets containing different levels of DDGS (n= 46 panelists) Responses

0 No.

Aroma: Very agreeable Agreeable Neither agreeable nor disagreeable Disagreeable Very disagreeable Color: Very pale Pale Neither pale nor strong Strong Very strong Flavor: Like very much Like Neither like nor dislike Dislike Dislike very much

DDGS (%) 10 No. %

20 No.

30 No.

14 24 8

30.4ab 52.2 a 17.4 b

13 22 10

28.3 a 47.8 a 21.7 a

9 27 10

19.6 b 58.7 a 21.7 b

10 26 10

21.7 b 56.5 a 21.7 b

0 0

0.0 c 0.0 c

1 0

2.2 b 0.0 c

0 0

0.0 c 0.0 c

0 0

0.0 c 0.0 c

6 18 18

13.0 b 39.1 a 39.1 a

3 18 18

6.5 b 39.1 a 39.1 a

5 17 16

10.9 b 37.0 a 34.8 a

6 16 17

13.0 b 34.8 a 37.0 a

4 0

8.7 b 0.0 c

7 0

15.2 b 0.0 c

8 0

17.4 b 0.0 c

5 2

10.9 b 4.3 b

7 21 13

15.2 b 45.7 a 28.3ab

11 25 6

23.9ab 54.3 a 13.0 b

8 26 11

17.4 b 56.5 a 23.9 b

10 22 10

21.7 b 47.8 a 21.7 b

4 1

8.7 b 2.2 b

3 1

6.5 b 2.2 b

0 1

0.0 c 2.2 c

2 2

4.3 c 4.3 c

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Texture: Very soft Soft Neither soft nor firm Firm Very firm a,b,c

6 20 11 9 0

13.0 b 43.5 a 23.9 a 19.6 b 0.0 c

6 15 7 16 2

13.0ab 32.6 a 15.2ab 34.8 a 4.3 b

2 22 8 14 0

4.3 b 47.8 a 17.4ab 30.4 a 0.0 c

4 18 11 9 4

8.7 b 39.1 a 23.9ab 19.6ab 8.7 b

Different letter superscripts in the same column indicate differences in panelist response frequencies (P<0.05). 1 Number of panelists.

Between 35 (75 %) and 38 (82 %) of the 46 panelists (P=0.687) stated that the aroma of the rabbit meat was agreeable to very agreeable, with no differences between treatments. In contrast between 8 (17 %) and 10 (21 %) felt the aroma to be neutral (neither agreeable nor disagreeable), with no differences between treatments (P=0.957). Meat color was perceived as neutral (i.e. neither pale nor strong) by 16 to 18 of the evaluators in each treatment (P=0.984). Fewer (P<0.05; Table 4) felt it to be very pale (3 to 6 panelists for treatment; P=0.753), or strong (4 to 8 responses per treatment; P=0.644). From 21 (45 %) to 26 (56 %) of the panelists said they liked the flavor of the rabbit meat, while 8 (17 %) to 11 (24 %) said they very much liked the meat from the treatments with 10 to 30 % DDGS (P=0.774). As far as the meat from the control treatment, 21 (45 %) said they liked it, and seven said they very much liked it, with no differences between treatments (P=0.774; Table 4). Very few (<11%) panelists stated they did not like the meat, with no differences among treatments (P=0.717). Most of the panelists expressed their approval of the rabbit meat both with and without DDGS. This represents a potential market niche worth exploring in more detail, especially since consumption of rabbit meat is notcommon in the region where the sensory evaluation was done. Between 45 and 56 % of the panelists perceived the meat to be soft or very soft, with no differences among treatments (P>0.05). From 15 to 24 % (Table 4) were of the opinion that it was medium texture (neither soft nor firm), again with no differences (P=0.711). The meat was stated to be of firm texture by 19 to 35 % of the panelists with no differences among treatments (P=0.367). Less than 9 % described its texture as very firm, with no differences (P=0.414). Overall, the panelists expressed varying opinions of their degree of acceptance of the rabbit meat in the different evaluated sensory quality categories. The sensory analysis method applied here is known as an affective test(26). The main objective of this kind of test is for a group of consumers or potential consumers to express their personal responses when evaluating a product using a given set of response options. In the present case it was focused on characterizing the sensory perceptions of a panel of potential rabbit meat consumers regarding meat from rabbits fed diets containing different levels of DDGS.

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The panel consisted of a broad sample of 46 untrained panelists. Another possibility would have been to use eight to ten trained evaluators to evaluate meat quality. These follow specific methodologies and use specialized equipment. However, in a study including sensory evaluation of rabbit meat, trained evaluators were unable to detect differences (P>0.900) between meat samples(35). No previous studies exist of sensory analysis of meat from rabbits fed diets containing DDGS. However, the present results agree with a study done using pigs fed diets containing DDGS in which no negative effects were found on the sensory attributes of pork from these pigs(36). Color is an important quality factor in meats. In the present results the L* and a* chromatic coordinates of Longissimus dorsi muscle did not differ between treatments (P>0.05) (Table 5). But there were differences (P<0.05) in the b* coordinate between the treatments containing DDGS levels (10, 20 and 30 %) and the control. This indicates that the Longissimus dorsi from rabbits in the DDGS treatments exhibited a more intense yellow color. This may be due to the carotenoid pigments in the DDGS, the source of the yellow color of corn grains, which would have occurred in higher concentrations in the DDGS treatments than in the control(37). These results agree in general with those from a study of the color of the carcass and Longissimus dorsi muscle of rabbits fed diets containing DGGS from different sources (barley, wheat, corn) at three concentrations (0, 20 and 40 %)(20). Carcass color did not differ among treatments, which was also true of the Longissimus dorsi except for a higher a* value (reds) in the treatment with 20 % DDGS from wheat. Luminosity values (L*) did not differ between the treatments in the present study, indicating the analyzed rabbit meats had similar levels of clarity. The levels observed here (L* = 59.42 to 62.23) are within the ranges reported in the literature for rabbit meat, which are generally high (L*>50)(38). They also indicate that the analyzed rabbit meat should be considered pale, since L* values greater than 52 in rabbit meat are indicative of pale meat(39). The sensory evaluation (Table 4) generally supports these results in that panelists largely perceived the rabbit meat to be pale or neither pale nor strong in color. Numerical values for shear force were higher in the meat from rabbits fed diets containing higher DDGS proportions (Table 5), although the differences were not significant (P>0.05). The present shear force values are slightly higher than reported values (2.9 to 3.5 kg/cm2)(22), indicating the meat evaluated here was firmer. This discrepancy may be due to slaughter age since the animals in the present study were slaughtered at 96 d of age while those in the previous study were slaughtered at 63 d of age(22). Older animals are known to produce firmer meat than younger animals mainly due to the increase in connective tissue and its characteristics(40). The texture evaluation results coincided with those of the sensory evaluation, and in both cases differences in meat texture among treatments were not significant. No previous studies have included texture analyses of meat from rabbits fed diets containing DDGS. Studies of pork from finishing pigs fed diets containing up to 20 % DDGS found no negative effects on meat quality determined by shear force in cooked loin chops(41). In another study inclusion of 10 or 20 % DDGS 530

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in the diets of growing-finishing pigs had no effect on the shear force nor the overall palatability of bacon and pork chops(42). This agree in general with the present results.

Table 5: Chromatic coordinate (L*, a* and b*) and shear force values for Longissimus dorsi muscle from rabbits fed diets containing different levels of DDGS Indicators L* a* b*

0 60.37 8.79 9.68a

Shear force, kg/cm2

3.25

a,b

DDGS (%) 10 20 30 60.51 59.42 62.23 8.58 6.75 8.22 b b 11.77 12.17 12.22b 3.50

3.65

3.88

SE (Âą) 1.44 0.82 0.56

P 0.5904 0.3237 0.0157

0.35

0.637

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

Conclusions and implications

Inclusion of up to 30 % DDGS in rabbit diets had no effect on carcass or meat characteristics. This widely available agricultural byproduct is an interesting alternative for replacing costlier ingredients such as soybean and sorghum in diets for growing rabbits. Sensory evaluation showed the rabbit meat from the DDGS treatments to have favorable organoleptic characteristics for human consumption, although further promotion would be needed for consumers in the study area to accept its taste.

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26. Deliza R, Abreu Glória MB. Sensory perception. In: Nollet LML and Toldrá F editors. Sensory analysis of food of animal origin. USA: CRC Press; 2011:61-86. 27. Mariezcurrena-Berasain MA, Braña-Varela D, Mariezcurrena-Berasain MD, Domínguez-Vara IA, Méndez-Medina D, Rubio-Lozano MS. Características químicas y sensoriales de la carne de cerdo, en función del consumo de dietas con ractopamina y diferentes concentraciones de lisina. Rev Mex Cienc Pecu 2012;3(4):427-437. 28. Nute GR. Sensory descriptors. In: Nollet LML, Toldrá F editors. Sensory analysis of food of animal origin. USA: CRC Press; 2011:49-60. 29. StatSoft, Inc. STATISTICA (data analysis software system), versión 7. 2003. 30. Levene H. Robust tests for the equality of variance. Contributions to probability and statistics. USA: Stanford University Press; 1960. 31. Shapiro S, Wilk B. An analysis of variance test for normality (complete samples). Biometrika 1965;52(3-4):591-611. 32. Di Rienzo JA, Casanoves F, Balzarini MG, Gonzalez L, Tablada M, Robledo CW. InfoStat, Software Estadístico. Manual de Usuario. Versión 2012, Universidad Nacional de Córdoba, Argentina; 2012. 33. Duncan B. Multiple range and multiple F tests. Biometrics 1955;11:1-42. 34. Lima PJDO, Watanabe PH, Candido RC, Ferreira ACS, Vieira AV, Rodrigues BBV, Nascimento GAJ, Freitas ER. Dried brewers grains in growing rabbits: nutritional value and effects on performance. World Rabbit Sci 2017;25:251-260. 35. Martínez AM, Hernández P. Evaluation of the sensory attributes along rabbit loin by a trained panel. World Rabbit Sci 2018;26:43-48. 36. McClelland KM, Rentfrow G, Cromwell GL, Lindemann MD, Azain MJ. Effects of corn distillers dried grains with solubles on quality traits of pork. J Anim Sci 2012;90(11):4148-4156 37. Salinas MY, Saavedra AS, Espinosa TE. Physicochemical characteristics and carotenoid content in yellow corn (Zea mays L.) grown in the state of Mexico. Agric Téc Méx 2008;34(3):357-364. 38. Pla M, Hernández P, Blasco A. The colour of rabbit carcasses and meat. Meat Focus International 1995;4(5):181-183. 39. Hulot F, Ouhayoun J. Muscular pH and related traits in rabbits: A review. World Rabbit Sci 1999;7(1):15-36. 40. Bailey AJ, Light ND. The role of connective tissue in determining the textural quality of meat. In: Connective tissue in meat and meat products. London, UK: Elsevier Applied Science; 1989. 534

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41. Whitney MH, Shurson GC, Guedes RC. Effect of including distillers dried grains with solubles in the diet, with or without antimicrobial regimen, on the ability of growing pigs to resist a Lawsonia intracellularis challenge. J Anim Sci 2006;84(7):1870-1879. 42. Widmer MR, McGinnis L.M, Wulf DM, Stein HH. Effects of feeding distillers dried grains with soluble, high-protein distillers dried grains, and corn germ to growingfinishing pigs on pig performance, carcass quality, and the palatability of pork. J Anim Sci 2008;86(8):1819-1831.

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https://doi.org/10.22319/rmcp.v10i3.4528 Article

Survival of classic swine fever virus in hams made from the meat of pigs vaccinated with the PAV-250 strain and unvaccinated pigs

Heidi Amezcua Hempel a María Salud Rubio Lozano b Eliseo Manuel Hernández Baumgarten a Pablo Correa Girón c† Oscar Torres Ángeles d María Antonia Coba Ayala c Jose Abel Ciprián Carrasco a Susana Elisa Mendoza Elvira a*

Universidad Nacional Autónoma de México, FES-Cuautitlán, Laboratorio de Virología. Av. Primero de Mayo S/N, Campo I, Col. Santa María las Torres, Cuautitlán Izcalli. Estado de México. México. b

Universidad Nacional Autónoma de México, Facultad de Medicina Veterinaria y Zootecnia, Centro de Enseñanza Práctica en Salud y Producción Animal. México. c

Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias, CENIDMicrobiología, Ciudad de México. México. d

Universidad Autónoma de Morelos, Facultad de Farmacia. Cuernavaca, Morelos, México.

*Corresponding author: seme_6@yahoo.com.mx

Abstract: The study was to determine the presence of Classical Swine Fever virus (CSFv), in the meat of vaccinated pigs with the PAV-250 strain and then challenged using the same strain. Five treatment groups were established (each with four pigs). Group A: Pigs that 536

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were fed with processed hams from negative animals; Group B: Pigs that were fed with processed hams from commercial pigs inoculated with the ALD (reference strain) (titre of 104.0/ml); Group C: Pigs fed with processed hams from pigs infected with the virulent ALD strain (titre of 102.5/ml); Group D: Pigs fed with processed hams from pigs vaccinated with the PAV-250 strain and challenged with the ALD strain (titre of 101.1/ml); and Group E: Pigs fed with processed hams from pigs vaccinated with two doses of the PAV-250 strain and challenged with the ALD strain (negative). Blood samples were taken at d 1, 5, 10, 15 and 20 for biometric analysis. Groups B, C and D manifested clinical signs of CSFv: 40 Â°C temperature, anorexia, paralysis, vomiting, diarrhea, tremor, hirsute hair and cyanosis. Pigs were slaughtered and necropsies performed to identify lesions in tissues. Results of direct immunofluorescence testing of tissues were positive and the virus was recovered. Under these study conditions, it was found that CSFv resisted the cooking method at 68 Â°C for 40 min in hams from unvaccinated pigs, and that the virus was able to transmit the disease to healthy unvaccinated pigs, whereas the hams from the vaccinated animals did not transmit the virus. Key words: Classic swine fever, PAV-250 vaccine, Hams, Mexico.

Received: 13/06/2017 Accepted: 30/08/2018

Introduction

The Classic swine fever virus (CSFv) belongs to the Flaviviridae family, genus Pestivirus and is closely associated to the viruses that cause bovine viral diarrhea/mucosal disease and border disease, which can also infect pigs(1,2). The CSF is a highly contagious disease, whose acute form affects the nervous system, the vascular endothelium, and the reticuloendothelial cells(2). Contagion occurs primarily through contact with different types of secretions and nasal, lacrimal, urinary, and faecal excretions, but it can also be mechanically transmitted by mosquitoes, birds, utensils, contaminated food waste, infected or contaminated pork meat(3-7). The virus can remain infective in pigs and pork by-products for months, constituting an epizootic factor of considerable importance. Additionally, the virus can survive in infected pigs or swine by-products for months or even years, when the meat is stored in frozen or refrigerated conditions(8-10).

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In Mexico, backyard and subsistence pig-raising represent a production and commercialization system characterized by a continuous purchase and sale cycle of animals after short fattening periods; although in some cases, the producers raise their own pigs by keeping one male and as many as five breeding females. However, the facilities are rustic, located near homes, and the workers are usually family members(11). Traditionally, the feed for pigs raised under these conditions consists of scraps and waste (escamocha) from household food preparation and consumption or from restaurants, hotels, hospitals, markets, food distribution centres and agricultural industries, among others. These materials often include pork products or by-products. For these reasons, feeding backyard pigs with scraps and waste constitute an important source for CSFv, susceptible pigs can acquire the disease when fed with inadequately heat-treated kitchen waste or food scraps, as it has happened in Mexico for many years. It has been proven that feeding pigs with food scraps and waste is one of the main causes of CSF outbreaks, because the virus can resist in bone marrow for long periods, especially if cooking temperatures are not sufficiently high to inactivate the virus(12). McKerker et al(13) found that the persistence of African Swine Fever virus in the serrano and Iberian hams (112 d post-processing) was up to 5-to-6 mo, which was supported by the findings from Botija(14). Effective vaccines against CSF are available and their use can reduce costs and limit the spread of this disease. However, the efficacy of vaccines depends on their ability to induce a strong immune response, which can be obtained using the modified live vaccine, which was very successful to control CSF in countries where it was endemic. Serial transmission studies in pigs of the live-attenuated CSF vaccine have demonstrated that reversion to virulence does not occur(15). Around 2005, Mexico was engaged in the swine fever control and eradication program, where the country was divided into three regions: Region 1 had an intensive vaccination program; Region 2, where the eradication of the virus was obtained through vaccination; and Region 3, which corresponded to a disease-free phase. Because phases 1 and 2 require intensive vaccination, the restricted use of the vaccine prompted the research team -along with the Lelystad Institute- to perform a study for determining the antigenic composition of the currently use of vaccine and compare it to others that were applied in the past. The Mexican field strains included in this study revealed heterologous reactions in their secondary epitopes, which were distantly related to the conserved neutralization sites. This variation was restricted to the secondary neutralization sites of CSFv. Only the PAV-250 vaccine has been used in phases 1 and 2 of Mexicoâ&#x20AC;&#x2122;s CSFv eradication campaign. Pigs vaccinated with PAV-250 and challenged on the 14th post-vaccination day with the virulent ALD-CSFv did not transmit the challenge virus to susceptible pigs. However, a low releasing level of the virulent virus from vaccinated pigs was detected following this challenge; therefore, the virus remained at levels below the infectious dose. The PAV-250 vaccine proved to be superior to other swine vaccines available in Mexico(16,17). The aim of this study was to determine the presence of CSFv in the meat of pigs vaccinated with the PAV-250 strain and then challenged. Additionally, to ascertain the virus status in hams prepared from meats belonging to those animals. 538

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Material and methods

Meat from experimentally vaccinated and challenged pigs

Meat used in this experiment came from a previous one that had five treatments: Group I, meat from negative control pigs; Group II, meat from commercial pig legs inoculated with the ALD strain with a titre of 106.0/ml; Group III, meat from pigs infected with the virulent ALD strain with a titre of 104.7/ml; Group IV, meat from pigs vaccinated with the PAV-250 strain and challenged with the ALD strain with a titre of 103.1/ml; and Group V, meat from pigs vaccinated with two doses of the PAV-250 strain and challenged with the ALD strain with negative titre(18-21).

Preparation of the hams

The legs from each experimental group were selected and processed independently. The average weight of the legs before processing was 2.2 kg. Bone was removed from the legs, and then the meat was injected with 20 % of brine and kept for 18 h at 4 째C before cooking. The brine contained the following ingredients: 20 g of NaCl, 0.24 g of sodium nitrite, 0.24 g of sodium nitrate, 6.0 g of food grade phosphate, 0.66 g of sodium ascorbate, 3.6 g of refined sugar, 0.18 g of monosodium glutamate and 0.11 g of hydrolyzed vegetable proteins. Hams were wrapped with a cotton mesh and placed in molds(22) and then cooked to an internal temperature of 68 째C for 40 min. A thermometer was inserted at the piece core to measure the temperature, as specified by the NMX-F123-S-1982(23). The molds containing the hams were refrigerated for 24 h and then hams were washed with water at 28 째C. Finally, the hams were stuffed into plastic casings and stored at 4 째C until its use to feed the animals.

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Animals

A total of 20 commercial Yorkshire ď&#x201A;´ Landrace cross pigs obtained from a commercial farm and with a weight of 16 to 18 kg were used. The pigs were serologically negative for CSF, PRRS and Aujeszky virus according to the ELISA technique(24-26).

Ham from experimentally vaccinated and challenged pigs

Five treatment groups were sorted with four pigs each. Group A corresponded to pigs that were fed with processed hams from negative animals; Group B belonged to pigs that were fed with processed hams from commercial pig legs inoculated with the ALD strain (with a titre of 104.0/ml); Group C corresponded to pigs fed with processed hams from pig legs infected with the virulent ALD strain (with a titre of 102.5/ml); Group D was formed with pigs fed small pieces of the ham processed hams from pig legs vaccinated with the PAV250 strain and challenged with the ALD strain (with a titre of 101.1/ml); and Group E belonged to pigs fed with processed hams from pig legs vaccinated with two doses of the PAV-250 strain and challenged with the negative ALD strain. All the pigs of the four groups were fed with shredded ham and were given approximately 200 g for each pig in a single occasion.

Clinical signs

All pigs in each group had their rectal temperature monitored and clinical signs evaluated on a daily basis, during a period of 7 to 21 d.

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Blood biometrics

Blood samples were taken from all animals. A baseline sample was drawn at the beginning of the experiment; the following samples were then taken every 5th day for a total of 5 (d 1, 5, 10, 15 and 20). At the end of the experiment (i.e. d 21), the pigs were slaughtered(27).

Slaughter and pathology evaluation

Necropsies were performed on all animals that died during the experiment and also on the slaughtered pigs. Euthanizing was carried out by sedation with 3 mg/kg of azaperone and deep anaesthesia with 0.3 ml/kg of a mixture of xylazin and tiletamin with zolazepam, followed by exsanguination(28). The study design was approved by the Ethics Committee for Animal Experiments of the Veterinary Medicine Faculty at UNAM (Universidad Nacional Autónoma de México); the experiment was conducted in compliance with the Mexican Regulations for Animal Care and Maintenance(29).

Immunofluorescence test

The tonsils, ganglia and spleens of the experimental pigs were collected. The conjugate for CSF diagnosing was added to previously fixed tissue sections, which were incubated immediately for 30 min at 37 °C in a humid chamber. The lamellae were washed and mounted with glycerol/PBS 1/1 and observed under immunofluorescence microscopy(24-26).

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Viral isolation and titration

The virus was isolated in the PK-15 cell line from a suspension that included tissues from the lymph nodes, spleens and tonsils, as well from the bone marrow of the femur. Viral titration was performed using direct immunofluorescence testing(20-21).

Statistical analysis

A factorial design with random data from the CSFv-infected groups was analysed using the SAS software by means of a +/- standard deviation (SD) or standard error above the mean (SEM) with an ANOVA.

Results

Preparation of the hams

The legs from the above-mentioned pig groups were selected and processed by the cooking method, in order to obtain the hams. Under these study conditions, CSFv was found after cooking (68 Â°C for 40 min) in hams from unvaccinated pigs, as the viral titer decreased.

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Viral isolation and titration

Virus isolation and titration revealed the following: CSFv was not isolated from Group A hams. For Group B commercial meat that had an inoculum of the ALD strain of the CSF virus at 104.0/ml was used. The ham of Group C had a titre of 102.5/ml; whereas the ham of Group D had a titre of 101.1/ml. Finally, the meat belonging to Group E had a negative titre.

Temperature and clinical signs

After feeding the pigs with ham pieces made from the legs of (1) PAV-250 vaccinated animals, (2) non-vaccinated but challenged pigs with the ALD reference strain and (3) pigs inoculated directly with the ALD strain, the findings in the infected pigs revealed a variety of clinical signs characteristic of CSFv (Table 1).

Table 1: Clinical signs: Expressed as percentages Groups (n=4 ) Temperature 40 Â°C Anorexia Paralysis Vomiting Diarrhea Tremor Hirsute hair Cyanosis

Group A 0 0 0 0 0 0 0 0

Group B 100 100 100 25 25 0 75 100

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Group C 100 100 100 100 100 100 100 100

Group D 100 100 50 75 75 50 25 50

Group E 0 0 0 0 0 0 0 0

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Hematic biometry

The blood values for Group C were markedly different from the ones of those pigs that were fed with free CSFv hams, as their red blood cell count was 10x6 l. However, there were no statistical differences in terms of the percentages of cellular packet, haemoglobin (g/dl), monocytes and eosinophils (103 l) with P<0.05. Table 2 shows the cell values that were affected after the ingestion of hams infected with CSFv in Groups B and C. Those animals showed leukopenia, which is characteristic of this disease and other highly significant values, such as decreased leucocytes, indicating that Group A behaved quite similarly to Group E. These findings indicate that the hams fed to the vaccinated and challenged animals had no viral particles capable of causing CSF infections.

Necropsies

The animals were slaughtered for the evaluation of the macroscopic tissue lesions in the different experimental groups (Tables 2 and 3).

Table 2: Blood values observed in the experimental groups Groups (n=4)

Group A

Group B 17.31a

Average: DE: 2.67 SEM: 0.94

Group C 10.77 b

Average: DE: 4.02 SEM: 1.42

Group D

Group E

Leucocytes x 103 l

Average: DE: 4.02 SEM: 1.42

Lymphocytes x 103 l

Average: 3.68 a Average: 1.18 b DE: 1.14 DE: 0.22 SEM: 0.43 SEM: 0.07

Average: 2.68 b Average: 1.38 b Average: 3.38 a DE: 1.24 DE: 1.24 DE: 1.24 SEM: 0.43 SEM: 0.43 SEM: 0.43

Segmented neutrophils x 103 l

Average: 7.50 a Average: 3.50 b DE: 2.18 DE: 2.18 SEM: 0.77 SEM: 0.77

Average: 4.60 b Average: 3.30 b Average: 7.80 a DE: 2.18 DE: 2.18 DE: 2.18 SEM: 0.77 SEM: 0.77 SEM: 0.77

abcMeans

11.97 b

11.56 b

Average: DE: 3.02 SEM: 1.42

with different superscript in the same row are different significantly (P<0.05).

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Average: 16.88 a DE: 4.02 SEM: 1.42

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Table 3: Lesions observed in the experimental groups Groups (n=4) Group A Group B Group C Group D Group E Haemorrhagic and oedematous 0 100 100 25 0 lymphonodus Kidneys and/or bladder with 0 50 75 25 0 petechial haemorrhaging Cyanotic skin 0 0 75 50 0 Infarcted spleen 0 100 75 50 0 Ileocecal valve and intestine with 0 100 75 50 0 ulceration Pulmonary lesions 0 100 75 25 0

CSFv identification by IFD in organs and tissues and viral isolation

The fluorescence assay performed in all groups was positive, except for Group A (i.e. negative control). Viral isolation was performed using a mix of lymph nodes, spleens and tonsils (named as the suspension), and bone marrow from each animalâ&#x20AC;&#x2122;s femora. The found titres varied widely, ranging from 102 to 104 (Table 4).

Table 4: CSFv identification by direct immunofluorescence testing and viral isolation Groups (n= 4) Group A Group B Group C Group D Group E Organs and tissues positives by IFD, expressed in percentages Tonsils 0 100 100 75 0 Lymphonodus 0 100 100 75 0 Spleen 0 75 50 50 0 Viral isolation Mix of lymphonodus; spleens and tonsils Negative Titre: 104.3 Titre: 103.9 Titre: 102.2 Negative suspension

Discussion

The main result is that the PAV-250 vaccine reduced the infection titres in the vaccinated and infected animals, which means that cooking does not eliminates the virus. 545

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The contribution of this study is that it was shown that the PAV-250 vaccine, in addition to protecting the animals, contributed to maintain low infection titers after the challenge. For several years, Mexico maintained a control and eradication campaign of the Classic Swine Fever, until the country was declared free of this disease in 2012(30), whereas the CSF was eradicated in the USA during the year of 1978(31). However, the outbreak danger of this disease in high swine-density areas in Central America and south-eastern Mexico persists. It is important to keep in mind that pork meat plays a key role in food safety. Although Classic Swine Fever does not affect humans, previous studies show that it can be transmitted in poorly processed food products that may be used to feed pigs, which increases the risk of disease re-emergence(32). Although many swine-raising operations are large-scale in modern production systems, there are also small-scale domestic or â&#x20AC;&#x2DC;backyardâ&#x20AC;&#x2122; pig raising practices, where the animals are fed with scraps and waste. Therefore, these practices represent a high risk for large-scale production units, as well. A study by Mebus et al(33) that utilized meat from animals inoculated with CSFv demonstrated the presence of the virus in different types of meats and sausages after preparation, revealing a high-risk scenario. The meat from those animals contained large amounts of the virus in certain products and by-products. The Italian and USA meat industries determined a period of 189 d for deactivating the CSFv virus in hams produced by the Prosciutto di Parma technique. In other dried or pickled products, the CSFv survived for 70 d in bone marrow and 90 d in fat and muscle; these results agreed with those from other studies(34-36). However, the virus persisted for long periods in the lymph nodes, bone marrow and fat from the products used in the study. The test used to determine the presence of CSFv showed that inoculated animals were sensitive and substantial amounts of viral particles were detected. These results agree with the present research, since it was isolated the virus in the PK15 cell line, as well as in viral titres. In the study by Mebus et al(33), the inoculated pigs developed CSF during in vivo tests using Iberian ham. Of the three-studied viruses, the CSFv persisted for a longer period in the tested products. This was proven by observation of classic clinical signs of CSF in the experimental pigs fed with those hams(35). The analysis of haemograms showed the presence of leukopenia and normal counts, while those levels fell below 9,000 and reached a low of 3,000 (between d 4-7) in the CSF pigs. It is important to consider that healthy pigs under 5 wk of age normally have lower leucocyte counts. Upon disease onset, the animals showed a low white blood cell count, weakening of the immune system and multiple internal haemorrhages that occurred when the bacteria invade the animals(27). The pigsâ&#x20AC;&#x2122; organs presented macroscopic lesions caused by the CSFv found in the experimental hams. Moreover, this was confirmed by direct immunofluorescence testing of the tissue sections obtained from the experimental pigs. These results showed that the cooking treatment reduced the viral titer by 2 logarithms in the legs inoculated directly with the ALD reference strain (Group B) and those from pigs infected with this same strain (Group C). Another way to prove the presence of this virus, involved viral isolation using a suspension prepared with a mixture of lymphatic ganglia, spleens and tonsils, and bone

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marrow from the femora. The titres obtained using this method ranged from 102 to 104. Based on these tests and animal observation throughout the experiment, it was possible to prove that the virus was indeed present in the hams made from the legs of infected pigs. As has been shown, the CSFv persists for different periods in the tissues of exposed pigs: 14 d in blood, 21 d in ganglia, and 94 d in intestinal â&#x20AC;&#x2DC;buttonâ&#x20AC;&#x2122; ulcers. In addition, it has been detected in the spleen of approximately 1.2 % healthy animals from exposed groups sent to slaughter and in waste materials from restaurants, which contain meat and bone remains from sick pigs that were killed at the slaughterhouse. In ham, hotdogs and similar products, the virus can survive up to 85 d, whereas in the bone marrow of salted meat, it can persist for 73 d and 60 d in salami. Furthermore, it can also resist refrigeration and freezing for 5-10 yr. Feeding pigs with inadequately cooked scraps was the main factor responsible for the 18 and 22 % of outbreaks that occurred in the USA between 1972 and 1973, respectively(8). On the other hand, the virus can survive for 25 d in bacon. It has been proven that feeding pigs with scraps is one of the main outbreak causes of CSF, because the virus survives for long periods in bone marrow, especially when cooking temperatures are too low for effectively inactivate any virus particles that may be present. Often, pigs infected with cholera are sent to the slaughterhouse and the meat enters the market, where it is consumed in restaurants. The food scraps that contain incompletely cooked bones are generally sold as food for swine, constituting another way in which the infection cycle can be perpetuated and cause an infection. CSFv also survives for 2 d in open corrals and from 2-to-4 d in manure. In fact, this virus survived for several weeks in experimentally infected manure; while in another experiment, the CSFv was detected along 1,600 m of an open sewage canal that ran from laboratories that produce vaccinations against CSF(12). The vaccination strain PAV-250 produces good immunity in pigs and does not allow the spreading of the virus in the field among herds on the same farm. These data were confirmed by the experiment, since the pigs from Group E did not show any clinical signs or pathological lesions. Additionally, they had negative results on the DIF tests and a zero recovery of the virus from lymphoid tissues and bone marrow. In Mexico, the use of the vaccination strain PAV-250 during the Control and Eradication CSF Program, succeeded through the implementation of a vaccination strategy based on zones, which not only controlled the CSF but also achieved a total eradication of the disease(9,14,27,29). The eradication of FPC in Mexico was carried out only with the use of vaccination, it was a very exceptional process and it is not mentioned in any other publication. Yes, Mexico is already free of CSFv was due in large part to the benefits of the PAV-250 vaccine, while in other Latin American countries continue to have this big problem, basically due to the use of several vaccine strains of FPC and continue to feed them with food waste(15,30,37). The use of this vaccine for the process of obtaining ham, is not referred to in any other publication. Although Mexico is already free of CSFv, which is largely due to the benefits

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of the PAV-250 vaccine, however, in other Latin American countries they still have this great problem, due to the use of several vaccine strains and to feed with squid.

Conclusions and implications

The PAV-250 vaccine reduced the infection titers in the vaccinated and infected animals, which means that cooking does not eliminate the virus. Under these study conditions, it was found that CSFv resisted the cooking method at 68 Â°C for 40 min in hams from unvaccinated pigs, and that the virus was able to transmit the disease to healthy unvaccinated pigs, whereas the hams from the vaccinated animals did not transmit the virus. The eradication of FPC in Mexico was carried out only with the use of vaccination. The contribution of this study is that it was shown that the PAV-250 vaccine, in addition to protecting the animals, contributed to maintain low infection titres after the challenge.

Acknowledgments

Thanks to the support of the Grants: PAPIIT IN228516 PIAPI1827.

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Dunne HW. Diseases of swine: Hog cholera. 4th ed. Ames, Iowa: Iowa State University Press; 1975.

Mengeling WL, Drake L. Replication of hog cholera virus in cell culture. Am J Vet Res 1969;30:1817-1823.

10. Ramírez NR, Pijoan AC. Diagnóstico de las enfermedades del cerdo. México DF Ramírez NR-Pijoan AC; 1982. 11. Suárez B, Barkin D. Porcicultura, producción de traspatio, otra alternativa. Centro de Desarrollo. Tríptico de la UAM-Xochimilco. México DF; 1990. 12. Helwing DM, Keast JC. Viability of virulent swine fever virus in cooked and uncooked ham and sausage casings. Austr Vet J 1966;42:131-135. 13. McKerker PD, Yedloutschning RJ, Callis JJ, Murpgy R, Panina GF, Civardi A et al. Survival of viruses in “Prociuto di Parma” (Parma Ham). Can Inst Sci Technol 1987;20:267-272. 14. Botija C. African Swine Fever: new developments. Rev Sci Tech Int Epiz 1982;1:1065-1094. 15. Van Oirschot J. Vaccinology of classical swine fever: from lab to field. Vet Microbiol 2003;96:367–384. 16. Correa-Girón P. La fiebre porcina clásica en las Américas: Características más importantes de la vacuna PAV-250 y de las vacunas contra la fiebre porcina clásica (CSF) usadas en México. Primer Symposium International 1998. Morilla GA editor. INIFAP, SAGARPA, IICA, FUPPUE; 2000. 17. González C, Pijoan C, Ciprián A, Correa P, Mendoza S. Airborne transmission of Hog Cholera virus in susceptible pigs and the effect of vaccination with the PAV250 HCV strain on the airborne transmission of HVC. J Vet Med Sci 2001;63:991996. 18. Coba A, Martínez A, Mendoza ES, Ciprián CA, Correa P. Antibody development in swine against a Hog Cholera lethal strain. J Anim Vet Advances 2008; (1):94-99.

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19. Mendoza A, Correa-Giron P, Aguilera EA, Colmenares G, Torres O, Cruz T, et al. Antigenic differentiation of classical swine fever vaccine strain PAV-250 from other strains, including field strains from Mexico. Vaccine 2007;25:7120–7124. 20. Mendoza ES, Alvarado NJL, Hernández-Baumgarten E, Ciprián A. Manual de diagnóstico virológico. Primera ed. Universidad Nacional Autónoma de México. 2008. 21. Ramírez H, Valero G, Fraire M. Diagnóstico veterinario. Requisitos, proceso, interpretación, ventajas y desventajas de técnicas diagnósticas: Diagnóstico serológico de enfermedades virales. Primera ed. SARH, CENID-MicrobiologíaINIFAP, PAIEPEME, SMPV. 1993. 22. Dott Gaetano. Elaboración de productos cárnicos. México: Trillas; 1983. 23. NMX-F-123-S-1982. Alimentos. Jamón cocido. Especificaciones. Norma Mexicana. Dirección General De Normas. 1982. 24. Correa GP. Diagnóstico de Fiebre Porcina Clásica (CSF) por la técnica directa de inmunofluorescencia. Secretaria de Agricultura y Recursos Hidráulicos, México. 1993. 25. Dinfer. Diagnostic Virology, a review of methods at the National Veterinary Institute: Inmunofluorescence (IF) test. Swedish International Developing Authority; 1989. 26. Mendoza ES, Aguilera CE, Torres AO Correa GP, Hernandez-Baumgarten E, Ciprian CA. OIE Symposium on Classical Swine Fever (Hog Cholera): Classical swine fever; recent findings and perspectives regarding differential serological diagnosis between street and vaccine viruses. Birmingham, England. 1998. 27. Calderón ANL, García ERM, Paasch, MLH. Estudios hematológicos de la fiebre porcina clásica aguda. Aportaciones a la patogénesis de la diátesis hemorrágica. Vet Méx 1997;28:21-24. 28. Meyns T, Maes D, Calus D, Ribbens S, Dewulf J, Chiers K, et al. Interactions of highly and low virulent Mycoplasma hyopneumoniae isolates with the respiratory tract of pigs. Vet Microbiol 2007;120:87-95. 29. Norma Oficial Mexicana. Regulación Mexicana para el cuidado y mantenimiento de los animales, NMX-F-062-200-1999. 30. Diario oficial. Acuerdo del 14 de agosto del 2012, la República Mexicana Libre de Fiebre Porcina Clásica. 2012. 31. Terpstra C. Infectious diseases of livestock: Hog cholera. 2nd ed. South Africa: Oxford University Press; 1994. 32. Organización Mundial de Sanidad Animal. http:/www.oie.int/doc/ged/D13957.PDF. 550

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33. Mebus CA, House C, Ruiz-Gonzalvo F, Pineda JM, Tapiador J, Pire JJ, et al. Survival of foot and mouth disease, African swine fever and Hog Cholera virus in Spanish Serrano cured hams, shoulders, and loins. Food Microbol 1993;10:133-144. 34. Savi P, Torlone V, Tilili F. Sulla sopravivenza del virus de lla peste suina in alcuni prodotto di salumeria. Vet Ital 1964;15:760-769. 35. Cottral GE, Cox BF, Baldwin DE. The survival of foot and mouth disease virus in cured and uncured meat. Am J Vet Res 1960;21:288-297. 36. Van Oirshot JT. Persistent and unapparent infections with swine fever virus of low virulence their effects on the immune system [thesis], State University of Utrecht; 1980. 37. Correa-Girón P. Enfermedades virales de los animales domésticos (mono-gástricos): Cólera porcino. 4ª. ed. Coordinación y Producción. Arte e Impresos BJ. México; 1981.

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https://doi.org/10.22319/rmcp.v10i3.4714 Article

Dietary supplementation of inulin or flavomycin and type of cut of rabbit meat: changes on fatty acid profile and sensorial characteristics

María Eugenia Juárez-Silva a* Mario Cuchillo-Hilario a Enrique Villarreal-Delgado a

Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ). Departmento de Nutrición Animal Fernando Pérez-Gil Romo, Ciudad de México, México.

*Corresponding author: eugenia.juarezs@incmnsz.mx

Abstract: The demand for meat from animals raised with the minimum use of antibiotics is growing. Also, the use of prebiotics, antibiotics, type of cut of meat to modify the fatty acid profile and the effects on consumer preferences are still not clear. The present study investigated the fatty acid profile, the health, and risk fatty acid indices and the consumer sensory evaluation of rabbit’s meat fed inulin and flavomycin as additives. Forty-eight (48) New Zealand rabbits were randomly arranged into 4 treatments of 12 animals each. The control group did not receive antibiotic or inulin supplementation. The second group was supplemented with inulin (2.5 g of inulin/kg of feed) while the third group received flavomycin as supplement (0.1 g of flavomycin/kg of feed). The fourth group received both inulin and flavomycin. Inulin addition in rabbit’s diet increases beneficial fatty acids (CLA, P=0.0001; and n3-PUFA, P=0.0001) and enables a better health-promoting index (P=0.0004) while reducing the atherogenic (P=0.001) and thrombogenic indices (P=0.042) of meat. The type of cut of meat (loin, fore legs and hind legs) had a minor impact on changing the fatty acid profile. In contrast, inulin or flavomycin addition showed larger modifications than type of cut of meat on this respect. Flavomycin reduced hedonic properties of meat (taste, P=0.0001; color, P=0.01; and aroma, P=0.0001). Loin tended to be the most preferred cut of meat (P=0.01). Inulin is a good alternative to avoid the utilization of antibiotics in rabbit’s feeding. 552

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Key words: Fatty acids, Growth promoter, Prebiotic, CLA, Aroma, Meat rabbit.

Received: 04/12/2017 Accepted: 16/05/2018

Introduction

In recent years, increasing interest in the lipid composition of meat and in the impact of fatty acids on human health has occurred. Rabbit’s meat is highly valued for its nutritional properties due to the high content of protein with excellent biological value(1,2), low fat content with low amounts of cholesterol(2). There is considerable interest in increasing the levels of polyunsaturated fatty acid (PUFA), especially n-3 fatty acids in animal products to enhance the functional properties of foods while promoting human health(3,4). Diet is a crucial way to modify the fatty acid profile of rabbit’s meat(5). The addition of functional substances to animal’s diet can generate favorable response in the meat quality increasing the content of polyunsaturated n-3 fatty acids(6). Fatty acids n-3 consumption may help to balance the n-6/n-3 ratio, which might impact on the prevention of cardiovascular diseases, hypertension, diabetes, arthritis, osteoporosis, among other diseases(2,4). Moreover, the close relationship between diet and health has modified consumer’s habits, with an increasing demand for products that not only meet nutritional needs but also healthy food choices(7). Food research linked to human health has included cholesterol and fatty acid profile to represent foods as promoters or threats to health(8-11). Rabbit’s meat is a good source of n-6 fatty acids and is a limited source of n-3 fatty acids as well as eicosapentaenoic fatty acid (EPA) and docosahexaenoic fatty acid (DHA)(12). Due to this low n-3 fatty acids content, the n-6/n-3 ratio in rabbit meat is high with values ranging from 7 to 11(12,13). Therefore, lowering the n-6/n-3 ratio in rabbit’s meat is highly desirable. Flavomycin is an antibiotic frequently used in pig, poultry, and cattle farming(14,15). However, the use of antibiotics as growth promoters in animal production has been associated with bacterial resistance in humans. The European Union banned the use of antibiotics as additives in 2006. These circumstances have stimulated the study of alternative products, such as inulin, a prebiotic fiber composed of a chain of fructose units with a terminal of glucose(16). Many studies have shown that fructooligosaccharides supplementation have advantageous effects in humans and animals. Supplementation

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promotes the maturation of the gastrointestinal tract of suckling rabbits, i.e. gastric pH gradually reach lower values if young rabbits are supplemented with prebiotics. Lower pH (around 2.0) promotes enzyme activity (amylase and proteases) in comparison to higher gastric pH values (around 4.0). Further, complementary effects of supplementation comprise better immune response and enhancement of the growth (number) and permanency of beneficial microbiota including Bifidobacterium and Lactobacillus(17) in the gut while reducing the risk of pathogenic infections(18). Inulin added to rabbit and poultry diets reduces the total deposition of fat in the body, lowers abdominal fat and modifies the cholesterol, lowers triglyceride values and lowers lipoprotein concentrations(19). Therefore, the meat of rabbits fed inulin may be a good alternative for human consumption, because inulin adjusts microbiota metabolism, induces the synthesis of favorable compounds, and effectively increases desirable nutrients to maintain human wellbeing status. In contrast, the utilization of flavomycin in emerging economies as in Mexico is not prohibited despite consumers from such regions of the globe are increasing their preference for products free of antibiotics. Sensory evaluation and preference of consumers can be modified by the addition of ingredients in the animal diets that change the hedonic properties of animal products. Also, different cuts of meat may drive the consumer preferences, owing to variations on physical and specific sensory properties of animal muscles. Therefore, consumer preferences can shift the markets according to specific demands. Therefore, it was tested inulin and flavomycin and their combination to investigate the potential benefits on nutritional aspects and the consumer preferences of non-antibiotic versus antibiotic agentsâ&#x20AC;&#x2122; utilization. Though flavomycin has an impact on disease prophylaxis, the present study did not focus on those effects. The objectives of this study were to evaluate the dietary supplementation of inulin or flavomycin as well as the type of cut of meat (loin, fore legs and hind legs) on the profile of long fatty acids and on the sensorial characteristics and consumer preference of rabbitâ&#x20AC;&#x2122;s meat.

Material and methods

Experimental setup

Forty-eight (48) New Zealand rabbits (female= 24/male= 24) 40 d old (790 Âą 150 g) were randomly arranged into four treatments of 12 animals each. Rabbits were obtained from 554

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the experimental farm “Granja Veracruz” at the Universidad Nacional Autónoma de México (UNAM). The study lasted 57 d, the first 15 d of the trial were used for adaptation to the animal management, the housing and the experimental diets. The ingredients and composition of the diets for each treatment are displayed in Table 1. The diets covered the necessary nutritional needs indicated for the species. Each treatment was prepared by mixing the individual ingredients. The control group (CG) did not receive antibiotic (Flaveco40 ECO-Animal Health) or inulin (IPS Raftifeed, Megafarma-Orafti) supplementation. The second group (I+) was supplemented with 2.5 g of inulin/kg of feed. The third group (F+) received 0.1 g of flavomycin/kg of feed. The fourth group (IF) received both inulin and flavomycin doses as previously discussed (2.5 g of inulin/kg and 0.1 g of flavomycin/kg of feed). Rabbits were housed individually in stainless steel cages. Food and water were provided ad libitum throughout the research period. Protocols for animal housing, management and sampling were approved by the Institutional Animal Care and Research Advisory Committee (Comité de Investigación en Animales-CINVA) at the INCMNSZ under the registration number NAN-059-09-10-1. Rabbits were slaughtered following the guidelines of the Official Mexican Standard of methods for slaughter domestic and wild animals (NOM-033-SAG/ZOO-2014).

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Table: Ingredients and chemical composition of the diet (g/kg) Ingredients

Control

Inuline (I)

Flavomycin (F)

116 251 129 31 438 10

Antioxidant BHTb

0.01

Potassium sorbate fungicide

0.01

Inulin Flaveco 40ÂŽc Coccidiostatd Binderd Sodium chloride Chemical composition Crude protein, g/kg NDF, g/kg ADF, g/kg Ether extract, g/kg Gross energy, MJ/kg

0 0.0 0.5 20 5

2.5 0.0 0.5 20 5

0 0.1 0.5 20 5

2.5 0.1 0.5 20 5

169 525 225 39 10.8

169 530 202 30 13.5

168 517 193 29 11.7

171 514 250 42 12.8

Corn Wheat bran Soybean meal Soybean oil Alfalfa Calcium phosphate Vitamin and mineral Premixa

Mixture of vitamins and minerals content in grams per kilogram: vit A 32 000 UI, vit D3 4000 UI, vit E 100 g, vit K3 4g, vit B1 8.0 g, vit B2 8.0 g, vit B6 8.0 g, vit B12 40 g, Biotin 200 mg, Panthotenic acid 40 g, Iron 4 g, Copper 6 g, Cobalt 1 g, Zinc 60 g, Manganese 43 g, Iodine 32 mg and Selenium 8 mg. (BASF, Mexico). b BHT, hidroxitolueno butilado; c Flaveco 40 contains 40 g of flavophospholipol per kg. dCoccidiostat

robenidine hydrochloride at 33 ppm (Alpharma AS). eCarboxymetil

cellulose.

Collection and preparation of samples

The rabbits were slaughtered by cervical dislocation, following the recommendation of the NOM-062-ZOO-1999 (Mexican Official Norm - NOM, 2001). The hot carcasses were placed in a ventilated area for 1 h before being cut to get the three meat cuts (loin, fore legs and hind legs) per single rabbit on each of the four treatments according to the guidelines of Blasco and Ouhayoun(20). Cuts of meat were individually packed into hermetically sealed plastic bags and stored at -18 Â°C until evaluation. For fatty acid evaluation, the experimental unit was the cut of meat (loin, fore legs and hind legs) of six 556

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animals within the treatments. The rest of cuts of meat from the animals of each treatment (six rabbits) were used for sensorial evaluation. The experimental unit was the pool of the same cuts of meat of two animals to obtain enough material for 10 panelists.

Chemical analysis of diets

Crude protein was measured by Kjeldahl nitrogen analysis AOAC cod 976.05(21). The neutral detergent fiber (NFD) and acid detergent fiber (ADF) contents were determined according to the Ankom method, following the protocol of the manufacturer, using model F-57 filter bags (Ankom Technology, NY, USA). The ether extract was extracted by anhydrous diethyl ether using a Soxhlet apparatus AOAC 920.15 and 963.39(21). Energy was calculated for each diet in a Parr calorimeter model 1241 (Parr Instrument Company, IL, USA), following the protocol of the manufacturer(22).

Fatty acid extraction and determination of methyl-esters fatty acids (FAME) and conjugated linoleic acid (CLA)

Fatty acids were extracted from the meat using a 2:1 chloroform-methanol mixture(23) and a gravimetric calculation according to the official method 696.33, AOAC(21). Fatty acid methyl esters (FAME) were quantified by gas chromatography-GC (Varian, Inc., Palo Alto, CA, USA) using a CP-3380 chromatograph equipped with a split injector, FID, and an autosampler CP 8400, in a DB 23 column (30 m×0.25 mm inner diameter; Varian, Inc., Palo Alto, CA, USA) with a film thickness of 0.25 μm. Nitrogen was used as the carrier gas at a flow rate of 30 ml/min. The column temperature was held for 1 min at 120 °C, then increased at a rate of 10 °C/min to 200 °C, and finally at 5 °C/min to 230 °C. The injector and FID temperatures were 250 °C and 300 °C, respectively. The volume of injection was 1 μL. Integration of each fatty acid was performed with a Varian Star Chromatography Workstation Software version 4.51. Identification of the peaks was made on the basis of retention times of standard methyl esters of each individual fatty acid (FAME mix C4-C24 no. 18919-1 AMP; Sigma-Aldrich Inc., St. Louis, MO, USA). Conjugated linoleic acid (CLA) in particular was identified using a CLA methyl ester standard with a mixture of cis- and trans-9,11-, and -10,12- octadecadienoic acids (Cat. 557

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no. O5632. Sigma-Aldrich Co., USA). Myristoleic acid (C: 14 9-tetradecenoic acid; Cat. no. M3525 Sigma-Aldrich Co., USA) was added to the methylated meat fat samples prior to GC analysis and using an internal standard. We only evaluated the long chain fatty acids because of their high relevance on human health while volatile fatty acids were not considered in the present study. Results were reported as the percentage of long chain fatty acids (g/100 g).

Health and risk indices (AI, TI and HPI)

Atherogenic (AI) and thrombogenic (TI) indices, were calculated according to Ulbricht and Southgate(9) using the following formulas: AI = C12:0 + (4 x C14:0) + C16:0 / n-6 PUFA + n-3 PUFA + MUFA. TI = C14:0 + C16:0 + C18:0 / (0.5 MUFA) + (0.5 n-6 PUFA) + (3 n-3 PUFA) + (n-3 PUFA/ n-6 PUFA). Health promoting index (HPI) was calculated according to the recommendation of Chen et al(10) : HPI= n-6 PUFA + n-3 PUFA + MUFA / C12:0 + (4x C14:0) + C16:0.

Consumer sensory panel evaluation

Twelve hours before the sensorial evaluation’s day, cuts of meat were defrosted at a refrigeration temperature (4 °C). Further, cuts of meat were chopped (dices, 2 x 1 x 1 cm) and later were pooled within each treatment. The cuts of meat of the four treatments were cooked using water (1:1 w/v) at an intern temperature of 71 °C using conventional pots with lids (diameter 25 cm and volume of 4 L) for 60 min. The four pots were heated simultaneously (100 °C) on a gas stove (IEM). No additive was added during the cooking process. The cooked samples (2 cm3/12 g and pH 5) were offered on disposables plastic white plates. The evaluation was performed at the sensorial evaluation laboratory of the INCMNSZ. A mild-white light was used at the individual evaluation rooms(24). A total of 30 untrained panelist (male and women 15:15) generated 1,080 results (30 panelist x 4 treatments x 3 types of cut of meat x 3 meat sensory characteristics). Double-blind evaluation was carried out. To score the preference of taste, color and aroma, a scale from 1 to 4 was used (4= like it very much; 3= like it; 2= do not like; 1= dislike). Plain water and a slice of white bread was offered to clean and eliminate residuals between samples. 558

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Statistical analysis

The data was processed by ANOVA using the Proc GLM(25). The model utilized was: Yij = μ + AAi + PMj + AAi x PMj + eij; Where: Y is the target variable; μ is the mean; AAi = Additive agent i [Inulin (I+), Flavomycin (F+) and Inulin plus flavomycin (IF)]; PMj = Piece of meat j (loin, fore legs and hind legs); e = experimental error. Differences were stablished with Tukey test (α= 0.05).

Results

Table 1 shows the chemical composition of the different diets. No differences were observed between diets in any of the analized variables. Total protein content of the four diets was on average 169 ± 1 g/kg, whereas NDF and ADF were 522 ± 7 and 218 ± 26 g/kg, respectively. The content of ether extract did not differed significantly among treatments. Also, inulin did not modify the gross energy of the diets in which this ingredient was added (I+ and IF). Fatty acid concentration of rabbit’s meat was influenced by the addition of inulin or flavomycin as well as the type of cut of meat (Table 2). However, feeding treatment showed to have more impact on fatty acid concentration than type of cut of meat on this respect and there were few interactions of these two main factors. For example, no significant effect was observed for myristic acid (C14:0) because of the type of cut of meat; however, I+ and IF treatments were lower in the values of this fatty acid, due to the diet effect (P=0.0001). Palmitic (C16:0) acid was numerically higher in CG, moreover was only different from the fore legs when rabbits were fed F+ and IF (P=0.004). Differences in heptadecanoic acid (C17:1) in all feeding treatments and cut of meat were not detected, except for loin (F+) and fore legs (IF) with an effect type of cut of meat (P=0.04). Stearic acid (C18:0) increased when rabbits were fed with F+ and the same results were observed when flavomycin was combined with inulin. Flavomicine (F+) 559

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influenced the increase in stearic acid (C18:0) content in the three types of cut of meat (loin, fore legs and hind legs), however, the hind legs in F+ and IF showed the maximum value while the lowest was for CG. Oleic acid (C18:1) concentration was highly influenced by feeding treatment, type of cut of meat, and their interaction (P=0.02; P=0.01, P=0.001). The concentration of Îą-linolenic acid (C18:3) was highest when rabbits were fed I+, in contrast, IF reported the lowest value of this fatty acid. Eicosanoic acid (C20:0) in fore legs from rabbits fed F+ was higher than the rest of types of cut of meat in all treatments (P=0.007). Arachidonic acid (C20:4 n-6) was eleven times (3.4 %) higher in hind legs from IF group than fore legs from CG (0.3 %). This outcome was affected by the two main factors and their interactions (P=0.001; P=0.0001; P=0.0003).

Table 2: Percentage of long chain fatty acids (g/100 g) of cooked rabbitâ&#x20AC;&#x2122;s meat influenced by the adittion of inulin and flavomycin and type of cut of meat (n=6) Control

Inulin (I) Loin

Flavomycin (F)

Fore legs Hind legs

Loin

Fore legs Hind legs

Loin

P value

Fore legs Hind legs SEM

Diet

Cut of meat

Diet x Cut of meat

Loin

Fore legs

Hind legs

C 12:0

0.07

0.08

0.14

0.11

0.17

0.09

0.12

0.1

0.23

0.16

0.13

0.08

0.01

0.47

0.74

0.11

C14:0

1.53a

1.6a

1.7a

1.34ab

1.2ab

0.8b

1.4a

1.6a

1.5a

1.4a

1.3ab

1.2ab

0.04

<0.0001

0.27

0.08

C 16:0

21.35a

20.5a

21.7a

20.23ab

20.2abc

19.2abc

17.2bc

19.2abc

19.5abc

17.08c

19.5abc

0.2

<0.0001

0.004

0.49

C 16.1 n-9

1.98

1.5

1.7

1.42

1.9

2.03

1.4

1.3

1.5

1.2

1.3

1.4

0.07

0.001

0.43

0.14

C 17:1

0.54ab

0.5ab

0.6ab

0.62ab

0.5ab

0.6ab

0.5b

0.7ab

0.6ab

0.8a

0.6ab

0.02

0.10

0.04

0.27

C 18:0

6.72cd

6.8bcd

6.5d

6.9bcd

7.07abcd

7.6abcd

8.12ab

8.1ab

8.3a

7.5abcd

7.9abc

8.3a

0.11

<0.0001

0.16

0.50

C:18.1 n-9

33.62ab

35.2a

36.3a

37.4a

36.5a

34.3ab

35.9a

33.6ab

36.8a

33.5ab

30.3b

0.41

0.02

0.01

0.001

C18:2 n-6

19.65c

20.8abc

20.9abc

20.7abc

20.3bc

20.9abc

21.33abc

23.02a

22.7ab

21.2abc

20.3abc

20.5abc

0.23

0.0003

0.37

0.25

C 18:3 n-6

0.03

0.02

0.07

0.11

0.03

0.05

0.006

0.1

0.03

0.02

0.03

0.02

0.01

0.27

0.98

0.03

C18:3 n-3

3.45ab

3.6ab

4.03ab

3.8ab

4.2a

3.8ab

3.5ab

3.6ab

3.4b

3.3b

2.5c

0.06

<0.0001

0.07

0.0003

C 20:0

0.51b

0.6b

0.72b

0.7b

0.5b

1.2a

0.7b

0.6b

0.7b

0.6b

0.03

0.09

0.007

0.0005

C 20:4 n-6

1.93abc

0.3d

1.3cd

0.83cd

1.1cd

2.9ab

1.4bcd

0.5cd

1.4bcd

1.2cd

1.8 abcd

3.4a

0.13

0.001

<0.0001

0.0003

C 20:5 n-3

0.14ab

0.06abc

0.07abc

0.12ab

0.1abc

0.12abc

0.06bc

0.2a

0.14ab

0.01

<0.0001

0.36

0.24

C 22:6 n-3

0.07ab

0.04ab

0.03ab

0.04ab

0.05ab

0.07ab

0.02ab

0.05ab

0.1a

0.09a

0.01

0.0001

0.46

0.20

SFA

30.18

29.7

30.6

29.34

29.2

29.3

28.1

29.9

29.07

27.04

29.7

0.33

0.12

0.06

0.77

MUFA

36.14ab

37.3a

38.5a

39.4a

39.03a

36.9ab

36.12ab

37.9a

35.7ab

38.7a

35.5ab

32.3b

0.42

0.008

0.03

0.004

PUFA

25.36

24.9

26.4

25.5

25.7

27.8

26.2

27.2

27.5

26.01

25.8

26.7

0.34

0.18

0.04

0.90

n -3 PUFA

3.7abc

4.14ab

3.9abc

4.3a

3.9abc

3.5bcd

3.6abc

3.4cd

3.7abc

3.6abc

2.7d

0.06

<0.0001

0.055

0.0005

n-6/n-3

5.9bcd

5.8cd

5.4cd

5.5cd

4.9d

6.14bcd

6.6bc

6.5bc

7.2b

6.2 bcd

6.3bcd

8.9a

0.12

<0.0001

SFA/ PUFA

1.21

1.2

1.1

1.05

1.121

1.03

1.09

1.12

1.073

1.11

0.02

0.03

0.40

0.80

n-6 PUFA

21.6

21.2

22.2

21.6

21.4

23.9

22.7

23.6

24.2

22.4

22.2

23.9

0.31

0.03

0.008

0.84

CLA

5.3abc

3.9abc

4.09abc

8.3a

6.04abc

7.8ab

2.6c

1.9c

3.1c

2.9c

4.05abc

3.9bc

0.37

<0.0001

0.40

0.60

SFA= saturated fatty acids; PUFA= polyunsaturated fatty acids; MUFA= monounsatured fatty acids; CLA= conjugated linoleic acid isomers (cis-9, trans-11; trans-9, cis-11; trans-10, cis-12; mg g-1 of fat); SEM= standard error of the mean; ND= not detected. a,b,c,d Means

with different letters within the same row are significantly different (P<0.05).

In the analysis of SFA and PUFA the results indicated that they were not modified by inulin, flavomycin or the type of cut. MUFA content was affected by feeding and type of cut of meat and showed significant interaction (P= 0.008; P= 0.03 and P= 0.004). Moreover, the n3-PUFA and monounsaturated fatty acids (MUFA) content increased 560

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with the inulin adittion and in CG. The ratio n6/n3 decreased in the CG and I+ with respect to F+ and IF, but only hind legs from IF was different from the rest (P= 0.0001). This is more evident when we analized the effects by cuts of meat; i.e., inulin decreased the n6/n-3 ratio in fore legs (4.9) while IF increased this value in hind legs (8.9 %). CLA content (8.3 %) of loin from animals fed with inulin was three times higher than the average of the three cuts of meat (2.5 %) from rabbits fed with flavor. Atherogenic and health promoting indices were influenced by feeding while thrombogenic, was influenced by feeding and type of cut of meat factors (Figure 1).

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Figure 1: Atherogenic, thrombogenic and health promoting indices of rabbitâ&#x20AC;&#x2122;s meat influenced by the adittion of inulin or flavomycin and type of cut of meat (n= 30)

AI = atherogenic index [C12:0 + (4 x C14:0) + C16:0 / n-6 PUFA + n-3 PUFA + MUFA]. TI = thrombogenic index [C14:0 + C16:0 + C18:0 / (0.5 MUFA) + (0.5 n-6 PUFA) + (3 n-3 PUFA) + (n3 PUFA/ n-6 PUFA)]. HPI = health promoting index [n-6 PUFA + n-3 PUFA + MUFA / C12:0 + (4 x C14:0) + C16:0]. a,b,c

Means with different letters are significantly different (P<0.05).

CG = Control group; I+= 2.5 g of inulin/kg; F+= 0.1 g of flavomycin/kg; IF= inulin and flavomicyn.

Fore legs from control group was the most preferred cut of meat (taste= 3.4, color= 3.2 and aroma= 3.3) within all analyzed treatments. In contrast, hind legs from F+ and IF (taste= 2.47 and 2.47; color= 2.90 and 2.50; aroma= 2.53 and 2.57, respectively) were the least preferred cut of meat of all samples evaluated.

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Discussion

Inulin addition to animal diet did not change the gross energy of the diets in which this ingredient was added (I+ and IF). In another study where inulin was used, the protein and fat digestibility increased while the gross energy of diet remained unchanged(26). Though, inulin is a source of carbohydrates, the percentage of inclusion of inulin in both studies was not enough to modify the gross energy values of diets. In the present study, although ADF content slightly decreased while NDF increased in I+ treatment, no statistical differences were detected. Feeding treatment modified the fatty acid profile as in other studies(27,28). The fatty acid profile in monogastric animals is almost a direct reflection of their dietary fatty acids. In the present study, inulin (I+) which is a linear polymer and oligomer of fructose with a terminal of glucose, favored the increase of fatty acids such as oleic (C18:1), Îą-linolenic (C18:3) and DHA (C22:6) while reducing the content of myristic acid (C14:0). It has been shown that cecal fermentation of rabbits can be modified with the use of inulin, by shifting the precursor of n-6 fatty acids (linoleic acid, C18:2) to Îą-linolenic acid (C18:3), the n-3 precursor, stimulating the production of healthier unsaturated fatty acids (27). In contrast, the antibiotic flavomycin inhibited the viability and growth of not only pathogen microbiota but also the beneficial microbiota along the gut and caecum(29). According to Bovera et al(30), endogenous production of saturated fatty acids as palmitic (C16:0) and stearic (C18:0) made it impossible to lower the values with the addition of inulin. Flavomycin alone and inulin plus flavomycin supplemented together, decreased the concentration of C16:0 when these ingredients were added. In contrast, flavomycin alone and Inulin plus flavomycin supplemented together, increased the concentration of C18:0. Divergent effects on particular fatty acids are possible, because gut and cecal microorganisms are capable of hydrogenating unsaturated fatty acids into more saturated ones, or vice versa through elongation (addition of two-carbon units to the carboxyl ends) and desaturation (introduction of double bonds into the long-chain acyl CoAs) of palmitic acid as discussed by other authors(29,30). This explains why in the present study, F+ and IF had lower value of palmitic acid (C16:0) than I+ and CG. In contrast to palmitic acid (C16:0), stearic acid (C18:0) increased in F+ and IF and decreased in I+ and CG. The possible explanation is that microbial population is altered, yielding distinct microbiota composition, further, modifying the metabolites produced. Likely, palmitic acid is elongated to stearic acid more rapidly in F+ and IF than in I+ and CG as shown in Table 2.

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Coprophagia of soft feces performed by rabbits mainly at diurnal cycles, can increase PUFA concentration in meat(31). However, in the present study, no differences were observed in the total PUFA content. Moreover, I+ increased CLA (C18:2 cis-9, trans-11), total PUFA-n3 and MUFA as palmitoleic acid (16:1), which were able to positively affect the HPI while reducing the atherogenic and thrombogenic indices. Consequently, the n6/n-3 ratio decreased in the CG and I+ with respect to F+ and IF. In contrast, CG showed the highest TI and AI indices and a reduced HPI, possibly due to the high content of SFA as myristic (C14:0), palmitic (C16:0) and the reduced content of PUFA. These results are in accordance to those found by Bovera et al(30), when feeding rabbits a prebiotic mannanoligosccharide additive (at 0.5, 1.0 and 1.5 of g/kg of diet), which resulted in a reduction in the TI but no differences were detected in the AI. In the present study, inulin inclusion positively affected AI and TI indices in comparison to CG, but the addition of inulin did not decrease the AI and TI indices in relation to F+ and IF. Therefore, it would be prudent to boost the concentration of desirable fatty acids such as conjugated linoleic acid isomers (CLA), Îą-linolenic, EPA and DHA to intensify this trend. The inclusion of rich sources of n-3 fatty acids in the rabbit diets e.g. oils and meals from oleaginous, would be good option for this purpose(13,28). The occurrence of these metabolites in rabbit meat may contribute to its shelf life and improved human health as was reported in other animal products(11,32-34). Daily intake recommendation for eicosapentanoic (C20:5) and docosahexanoic (C22:6) acids, which belong to the family n3, is 500 mg to maintain a healthy cardiovascular system in humans. The intake of rabbit meat fed from CG, I+, F+ and IF contribute to the n-3 fatty acids daily ingestion of about 145, 165, 121 and 110 mg/100 g of meat, corresponding to 29, 33, 24 and 22 % of the recommended daily allowance, respectively(35). Analyzing the contribution of n-3 fatty acids by cuts of meat; loin, fore legs and hind legs would contribute to 135, 144 and 126 mg/100g of meat, corresponding to 27, 28 and 25 % of the recommended daily allowance, respectively. The n-3-PUFA and MUFA content slightly increased with the inulin addition and the health promoting index while reducing the atherogenic and thrombogenic indices, thus indicating smaller feasibility to cause an atherogenic and a thrombogenic event. This may be due to the caecotrophes ingestion by rabbits that increase the recycle of nutrients, i.e., rabbits eating soft faeces, increased the availability of unsaturated fatty acid in the diet(29). In that process, non-utilized lipids, which escape from intestinal digestion, are hydrogenated/dehydrogenated in the ceacum by the microbial population and then liberated to the lower part of the hindgut, further, the rabbits ingest them through the caecotrophes. Likely, inulin may exert the metabolic pathway to produce unsaturated fatty acids. This explains why CG showed the highest thrombogenic and atherogenic indices and a reduced health-promoting index. Also, the utilization of inulin by some bacteria as a food source, produced short chain fatty acids, which might favor the acidification of the gut and ceacum environment, facilitating the colonization of beneficial lactic acid bacteria and hampering the presence and activity of potential pathongens(15,29).

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In respect to cuts of meat influence on fatty acid profile, no clear difference was found among cuts for SFA, MUFA and PUFA. Petracci et al(6) found that the content of n-3 PUFA in rabbit’s fed 3%, 6% and 9% linseed diets, increased more in leg (2.4, 6.0, 8.5 11.0 % of the total fatty acids) than in loin (2.1, 4.6, 6.8 and 8.85 % of the total fatty acids). Leg pieces tended to increase the PUFA amount than loin. These results are higher than those found in the present study, where n-3 PUFA averaged 3.7 % for loin, fore legs and hind legs. The authors concluded that the large percentage of linseed (up to 9 %), which is a rich source of PUFA, probably increased the α-linolenic acid (18:3) and further the n-3 fatty acids content. The lower concentration of α-linolenic acid (18:3) in the present study compared to the higher content in the study reported by Petracci(6), helps to explain these differences. Moreover, their results are similar to the present study findings, because hind legs showed the highest concentration of PUFA compared to loin and fore legs. Currently, no studies have assessed consumer’s sensory evaluation of rabbit meat fed a functional supplement versus an antibiotic as growth promoter. However, it is feared that over use of antibiotics employed as growth promoters in animal feeding may cause bacteria and microorganism resistance. In the current investigation, flavomycin reduced the preferences of panelists in terms of taste, color, and aroma of meat. Because of flavomycin modifying the thickness of intestinal wall, constrained intestinal bacteria growth by inhibition of peptidoglycan biosyntesis, and increase the absorption of nutrients(15,16), these might substantially modify nutrient transport to animal blood stream and to animal tissues. Therefore, nutrient availability and lipid deposition changes triggered by flavomicyn might help to explain at some extent those differences(19). Diet is a major driver for sensorial modifications of animal products(28,32). Inulin may favor the hedonic traits as shown by Mendez-Zamora et al(36) who evaluated the inclusion of 15% (dry weight basis) of inulin as a flavor additive in sausages. In that study, inulin improved the color and the overall acceptance of sausages by consumers. These results suggest that inulin added to the diet would have similar effects in the rabbit meat apart from the prebiotic benefit by increasing hedonic properties. However, this effect was not evident when inulin was mixed with flavomycin.

Table 3: Sensorial evaluation and consumer preference of rabbit’s meat influenced by the addition of inulin or flavomycin and type of cut of meat (n = 30) Control Loin

Fore legs

Inulin (I) Hind legs

Loin

Fore legs

Flavomycin (F) Hind legs

Loin

Fore legs

Hind legs

IF Loin

Fore legs

SEM Hind legs

P value Diet

Cut of meat

Diet x Cut of meat

Taste

3.3abc 3.46a ±0.52 ±0.57

2.97abcd 3.03abcd 2.73cd ±0.80 ±0.41 ±0.63

2.90abcd 3.03abcd 2.87bcd ±0.54 ±0.66 ±0.73

2.47d 3.43abc 2.80cd ±0.68 ±0.67 ±0.92

2.47d 0.007 <0.0001 ±0.73

0.01

0.0003

Color

3.10ab 3.23a ±0.60 ±0.56

2.90ab ±0.75

3.0ab ±0.45

2.83ab ±0.74

3.03ab ±0.71

3.0ab ±0.78

2.77ab ±0.72

2.90ab ±0.71

3.30a ±0.65

2.83ab ±0.91

2.50b 0.008 ±0.90

0.01

0.18

3.13ab

3.06abc

3.03abcd

2.77bcd

3.0abcd

3.13ab

2.80abcd

2.53d

3.10ab

2.83abcd

2.57cd

0.33

0.03

Aroma

3.30a

±0.43 ±0.70

a,b,c,d Means

±0.73

±0.49

±0.56

±0.45

±0.50

±0.71

±0.73

±0.54

±0.74

±0.67

0.01

0.006 <0.0001

with different letters within the same row are significantly different (P<0.05). SEM= standard error of the mean.

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When main effects (feeding and cuts of meat) were tested, loin tended to be the most preferred cut in all sensorial traits evaluated, but when specific effects of feeding treatment by single cut of meat was evaluated; fore legs from CG was the most preferred piece among all cuts evaluated. Though we did not evaluate rheological properties, they may play an important role for this result, where texture, hardness, tenderness, springiness and chewiness between pieces of meat are determinants for consumer’s preferences(2).

Conclusions and implications

Inulin addition in rabbit’s diet increases beneficial fatty acids (CLA and n3-PUFA) and enables a better health promoting index while reducing the atherogenic and thrombogenic indices of meat. The cuts of meat had a smaller impact on changing the fatty acid profile than inulin or flavomycin addition. Flavomycin reduced the score of the preferences among panelists in terms of taste, color, and aroma. Rheological properties of meat should be take into account to determine their influence on consumers’ preference. Inulin is a good alternative to avoid the utilization of antibiotics in the rabbits feeding. Complementary research on animal performance and economic benefits should be done to support the use of inulin as prebiotic.

Acknowledgements

Special thanks to Irene Torres Acosta for the assistance during the experimental phase with the animals and to Silvia Carrillo Dominguez for the statistical analysis recommendations. All authors read and approved the final version of the manuscript. Compliance with ethical standards Protocols for animal housing, management, slaughter and sampling were approved by the Institutional Animal Care and Research Advisory Committee (Comité de Investigación en Animales-CINVA) at the INCMNSZ under the registration number NAN-059-09-10-1. All procedures were in accordance with the Mexican Official Norm on Principles of Laboratory Animal Care (NOM 062-ZOO1999).

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Conflict of interest

The authors declare that they have no conflict of interest.

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22. Parr, Instrument Company. Oxygen bomb calorimetric and combustion methods. : Parr Instrument Company (Technical manual No. 130). ed.; Moline, llinois. USA. 1966. 23. Folch JM, Less M, Sloane-Stanley G. A simple method of the isolation and purification of total lipids. J Biol Chem 1957;226497-504. 24. Juárez S, Carlin S, Rebollo F, Verdugo R, Martínez G. Sensory evaluation of cooked pork meat (M. bícepsfemoris) fed with and without ractopamine hydrochloride associated to age but not gender of the non-trained panelist. J Anim Plant Sci 2016;26(1):40-45. 25. SAS SAS/STAT® 9.2. User's Guide. Second edition, 1; SAS. Institute Inc: Cary, North Carolina, USA. 2009. 26. Alzueta C, Rodríguez ML, Ortiz LT, Rebolé A, Treviño J. Effects of inulin on growth performance, nutrient digestibility and metabolisable energy in broiler chickens. Br Poul Sci 2010;51(3):393-398. 27. Dal Bosco A, Mugnai C, Roscini V, Paci G, Castellini C. Effet of genotype on estimated indexes of fatty acid metabolism in rabbits. World Rabbit Sci 2014;22(2):21-28. 28. Jiya E, Ijaiya A, Ayanwale BA, Olorunsanya AO. Fatty acid composition of meat from the hind leg cut of rabbits (Oryctolagus cunniculus) fed doets containing graded levels of processed tallow (Detarium Microcarpum) seed meal. Biotech Anim Husb 2015;31(2):283-290. 29. Bovera F, Lestingi A, Iannaccone F, Tateo A, Nizza A. Use of dietary mannanoligosaccharides during rabbit fattening period: Effects on growth performance, feed nutrient digestibility, carcass traits, and meat quality. J Anim Sci 2012;90(11):3858-3866. 30. Bovera F, Lestingi A, Piccolo G, Iannaccone F, Attia YA, Tateo A. Effects of water restriction on growth performance, feed nutrient digestibility, carcass and meat traits of rabbits. Animal 2013;7(10):1600-1606. 31. Leiber F, Meier JS, Burger B, Wettstein H-R, Kreuzer M, Hatt J-M, et al. Significance of coprophagy for the fatty acid profile in body tissues of rabbits fed different diets. Lipids 2008;43(9):853-865. 32. Cuchillo HM, Puga DC, Navarro OA, Pérez-Gíl RF. Antioxidant activity, bioactive polyphenols in Mexican goats' milk cheeses on summer grazing. J Dairy Res 2010;77(1):20-26. 33. Galina MA, Osnaya F, Cuchillo HM, Haenlein GFW. Cheese quality from milk of grazing or indoor fed Zebu cows and Alpine crossbred goats. Small Rumin Res 2007;71(1-3):264-272. 569

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34. Delgadillo PC, Sánchez MB, Nahed TJ, Cuchillo HM, Díaz MM, Solis ZR, et al. Fatty acid content, health and risk indices, physicochemical composition, and somatic cell counts of milk from organic and conventional farming systems in tropical south-eastern Mexico. Trop Anim Health Prod 2014;46(5):883-888. 35. EFSA. Labelling reference intake values for n-3 and n-6 polyunsaturated fatty acids. ESFA. 2009;7(7):1-11. 36. Méndez-Zamora G, García-Macías JA, Santellano-Estrada E, Chávez-Martínez A, Durán-Meléndez LA, Silva-Vázquez R, et al. Fat reduction in the formulation of frankfurter sausages using inulin and pectin. Food Sci Technol 2015;35(1):25-31.

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https://doi.org/10.22319/rmcp.v10i3.4481 Article

Effects of injecting increased doses of vitamins C and E on reproductive parameters of Holstein dairy cattle

Juan González-Maldonadoa Raymundo Rangel-Santosa* Raymundo Rodríguez-de Laraa Gustavo Ramírez-Valverde b J. Efrén Ramírez Bribiescac J. Manuel Vigil-Vigild M. Fernando García-Espinosad

Universidad Autónoma Chapingo. Posgrado en Producción Animal, Departamento de Zootecnia, Estado de México, 56230, México. Tel: +52-595-9521621.

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

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

Universidad Autónoma Chapingo. Departamento de Zootecnia, Estado de México, México.

*Corresponding author: rangelsr@outlook.com

Abstract: Vitamins C and E have been supplemented separately to improve fertility in cattle. The objective of this study was to evaluate the effect of combined injections of increased doses of vitamins C and E on reproductive parameters in dairy cattle. Lactating Holstein cows (n= 44) were randomly assigned to one of three treatments: 1) Control: n= 15, cows were not injected with vitamins; 2) VCE3: n= 15, cows received a single intramuscular injection of 3,000 IU of vitamin E before estrus and multiple subcutaneous injections of vitamin C with a total dose of 3,000 mg before and after estrus; 3) VCE6: n= 14, cows were treated as in VCE3, but doses of vitamins C and E were increased to 6,000 mg and 571

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6,000 IU. The reproductive indicators measured were diameter of the preovulatory follicle, time to estrus, area of the corpus luteum, pregnancy rate 35 and 45 d after AI and plasma concentrations of estradiol and progesterone. There was no effect of treatment on any of the evaluated reproductive parameters (P˃0.05), except that the lowest dose of vitamins sustained similar pregnancy rates among treatments, even though they had lower progesterone concentrations (P≤0.05) (19.4 ± 2.66 vs 10.1 ± 2.55 vs 19.2 ± 0.44 ng mL-1 for Control, VCE3 and VCE6, respectively). In conclusion, the supplementation with the highest amount of vitamin C and E (6,000 mg and 6,000 IU versus 3,000 mg and 3,000 IU) does not significantly increase the reproductive parameters measured. Key words: Antioxidants, Bovines, Fertility.

Received: 08/05/2017 Accepted: 11/07/2018

Introduction

Studies have suggested a physiological role of vitamins C and E in cattle reproduction(1,2). An improvement in cattle fertility after vitamin E supplementation has been reported(3,4). This vitamin may improve fertility by a direct antioxidant effect on follicle and embryo development(5) or by influencing follicular cell apoptosis and proliferation(6). Vitamin C is necessary to re-activate antioxidant activity of vitamin E(7,8). The effect of vitamin C on reproductive function is mediated by its participation in collagen synthesis, hormone secretion and its antioxidant properties(9). It has been suggested that several injections of vitamin C before and after estrus can improve fertility in repeat breeder cows(10). Unfortunately, there is little research that evaluates the effect of this vitamin on dairy cattle reproductive performance. Recent studies analyzing the impacts of vitamin C on fertility are lacking, researchers may have lost interest in evaluating reproductive responses of cattle to this vitamin because it is thought that bovines do not need vitamin C supplementation(11). It is known that pregnancy rate in cows is improved when 3,000 mg of vitamin C and 3,000 IU of vitamin E are injected at the same time on the expected day of preovulatory follicle emergence, combined with subsequent injections of vitamin C at estrus detection and 2 d after artificial insemination (AI)(12). The first injection of these vitamins aimed to affect follicle development(6,13) and possibly oocyte quality. The second injection of 572

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vitamin C was administered to emulate the natural rise of this vitamin during estrus in cattle(14). The third dose of vitamin C was injected to influence corpus luteum functionality(15,16). Thus, based on previous experience, the hypothesis tested in this study was that cows injected with 6,000 mg of vitamin C and 6,000 IU of vitamin E before and after synchronized estrus will have a higher pregnancy rate than cows injected with 3,000 mg of vitamin C and 3,000 IU of vitamin E.

Material and methods

All the technical and animal management procedures in this study were performed following the guidelines of the Canadian Council on Animal Care(17).

Animals, treatments and experimental design

The experiment was performed at the dairy cattle research farm of the Universidad Autónoma Chapingo, México. Lactating 4.6 ± 0.35-yr-old Holstein dairy cows (n= 44) with an average of 163.4 ± 20.0 d in milk and in a herd with historical record of 22 L d-1 cow-1 were assigned randomly to one of three treatments: 1) Control: n= 15, cows were not injected with vitamins; 2) VCE3: n= 15, cows received a single i.m. injection of 3,000 IU of vitamin E ((±)α-tocopherol®, Sigma-Aldrich) on d-5 (d 0 is the day of intravaginal device removal) and s.c. injections of 3,000 mg of vitamin C (ascorbic acid®, Q.P., Reasol) on d-5, immediately after estrus detection and 2 d after artificial insemination; 3) VCE6: n= 14, cows were treated as in VCE3, but the doses of vitamins E and C were increased to 6,000 IU and 6,000 mg, respectively. The experimental design was completely random, and the experimental unit was one cow.

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Estrus synchronization and breeding

The follicular wave of the cows was synchronized with an intravaginal device containing 1.0 g of progesterone (Sincrogest®, Ourofino Agronegocio, Sao Paulo, Brazil), inserted intravaginally for 8 d, and an i.m. injection of 250 µg of GnRH analogue (GnRH®, Sanfer) at intravaginal device insertion. Corpus luteum regression was induced by i.m. injection of 500 µg of cloprostenol (Celosil®, MSD Animal Health) at intravaginal device removal. Once the intravaginal device was removed, the animals were monitored constantly (at least every 2 h) by direct observation for signs of standing estrus (a cow was considered in estrus when accept mounting by another cow). The cows were artificially inseminated 12 h after estrus detection with a single dose (approximately 20 x 106 spermatozoa) of semen from a single bull of proven fertility.

Nutrition and feeding

The animals received a diet providing 1,117 IU of vitamin E (51.5 kg d-1 cow-1 of a total mixed ratio: fresh alfalfa (21.9 kg), corn silage (21.9 kg) and commercial concentrate (7.7 kg), named Ganadero 18, Productos Agropecuarios Tepexpan, S.A. de CV with protein 18%, fat 4%, fiber 12%. Vitamin E content in diet was determined by high performance liquid chromatography(18).

Reproductive parameters

The measured reproductive parameters were diameter of the preovulatory follicle, time to estrus after intravaginal device removal, area of the corpus luteum (CL), pregnancy rate and blood concentrations of estradiol and progesterone. The diameter of the preovulatory follicle and area of CL were measured by real time ultrasonography (Aloka Prosund 2, equipped with 7.5 MHz linear-array transducer, Hitachi Aloka Medical, Ltd Japan) performed by the same technician. Diameter of the preovulatory follicle was 574

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calculated by averaging horizontal and vertical measurements immediately after estrus detection, while the area of CL was calculated directly in the ultrasound 9 d after AI. The pregnancy test was performed by ultrasonography 30 and 45 d after AI. Blood samples were collected from the coccygeal vein, using tubes containing sodium heparin as anticoagulant (BD Vacutainer®), immediately after estrus detection and 9 d after AI. The blood samples were centrifuged at 3,000 rpm for 10 min and plasma was separated and stored at -20 °C until the day of analysis for estradiol and progesterone concentrations determination by ELISA (Estradiol- and Progesterone-Elisa, DRG Instruments, GmbH, Germany).

Statistical analysis

The statistical analysis was performed on variables collected only from cows that exhibited estrus. The number of cows that exhibited estrus for each of the treatments were Control=14, VCE3=13 and VCE6=14. A normality test was run in PROC CAPABILITY on the residuals of the final model in each variable. When residuals did not satisfy the normality test, the data was subjected to logarithmic transformation. The statistical model included the fixed effect of treatment. In addition, days in milk and age of the cow were included in the final model only when statistical significance was found. The results are presented as mean ± standard error (SE). In all cases, P≤0.05 was considered significant. The data were analysed by PROC GLM, except for pregnancy rate, and the means were compared by the Tukey Test. The pregnancy rate at 30 and 45 d were analysed by PROC GLIMMIX considering a binary distribution and using the link function logit. The SAS statistical package was used for all analyses.

Results

The impacts of injecting increased doses of vitamins C and E on ovarian structure development and hormone concentrations in dairy cattle are shown in Table 1. Overall, cows supplemented with the highest doses of vitamins C and E tended to have a smaller (P=0.06) preovulatory follicle, but blood concentrations of estradiol were not affected by vitamin injections (P˃0.05). The size of the corpus luteum was not different among 575

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treatments. However, cows receiving the lowest dose of vitamins had less (P≤0.05) blood progesterone concentrations than those of the control group and those that received the highest dose of vitamins. Moreover, pregnancy rate 30 and 45 d after AI of cows in the control group showed no differences compared to the groups of cows supplemented with vitamins (Figure 1).

Table 1: Effect of supplementation (mean±SE) with 3,000 mg and 3,000 IU, 6,000 mg and 6,000 IU of vitamins C and E, on ovarian structure size, estrus presentation and hormone concentrations in Holstein dairy cows Treatment Variable Time to estrus, h Diameter of the preovulatory follicle, mm Plasma estradiol concentrations, pg mL-1 Area of the corpus luteum, cm2 Plasma progesterone concentrations, ng mL-1

Control

VCE3

VCE6

48.1±5.17

55.2±5.36

62.1±5.10

0.17

18.9±0.71

17.1±0.73

16.5±0.69

0.06

37.8±4.19

40.1±4.00

38.8±3.85

0.92

6.7±0.52

7.3±0.54

6.0±0.52

0.25

10.1±2.55* 19.2±2.44

0.02

19.4±2.66

* Significantly different from other groups (P≤0.05). VCE3 group supplemented with 3,000 mg of vitamin C and 3,000 IU of vitamin E. VCE6 group supplemented with 6,000 mg of vitamin C and 6,000 IU of vitamin E.

Figure 1: Pregnancy rate 30 and 45 days after AI of Holstein cows in control (white bars) VCE3 (black bars) and VEC6 (hatched bars) groups 120 Pregnancy (%)

100 80 60 40 20 0 30

45 Days after AI

VCE3 group supplemented with 3,000 mg of vitamin C and 3,000 IU of vitamin E; VCE6 group supplemented with 6,000 mg of vitamin C and 6,000 IU of vitamin E.

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Discussion

A relationship between size of preovulatory follicle and probability of a cow diagnosed as pregnant after a fixed-time AI has been reported in dairy cattle(19). Cows with preovulatory follicles between 13.5 to 17.5 mm are more likely to become pregnant after a fixed-time AI(20). A possible explanation for the effect of preovulatory follicle size on pregnancy rate could rely on the degree of oocyte competence. According to results of an in vitro study(21), as follicle increases in size from 3 to 15 mm, the oocyte diameter also increases, and larger oocytes have been reported to have greater developmental competence(22). Another possibility is that young corpora lutea from larger follicles produce more progesterone than those from small preovulatory follicles(23). Supporting the above findings, donor cows with preovulatory follicles larger than 12.5 mm had a higher probability of yielding good quality embryos(24), but those with preovulatory follicles larger than 20 mm in size are at risk of pregnancy loss(25). The cows injected with vitamins C and E had preovulatory follicles falling under the threshold at which the probability of pregnancy after AI increases(20). Since cows injected with the highest dose of vitamins tend to have a small preovulatory follicles, a similar tendency was expected in estradiol concentrations. However, concentrations of this hormone and pregnancy rate among experimental groups was not different. Results from in vitro studies indicate that vitamin C does not affect follicular estradiol production, but it does affect follicle structure(13), and vitamin E improves granulosa cell survival(6). Results of the present study agree with those obtained from in vitro studies regarding estradiol production. In addition, previous research found that estradiol concentrations and pregnancy rate are not influenced by preovulatory follicle size in cows showing estrus and ovulating spontaneously(26), as in the case of the present study. The progesterone produced by the corpus luteum after AI is responsible for pregnancy maintenance. Dairy cows with good genetic merit for fertility traits had a larger corpus luteum and produce more progesterone than those with poor genetic merit(27). Thus, increasing corpus luteum size and progesterone production might be targeted to improve fertility in dairy cattle. Based on its physiological relevance, vitamin C may be an important asset to influence corpus luteum development. It has been reported that ascorbic acid supports collagen biosynthesis during tissue formation and maturation of corpus luteum(15), reaching the highest concentration at mid luteal phase(28). In addition, vitamin C concentrations correlate positively with corpus luteum size and progesterone level(16). However, corpus luteum size was not affected by vitamin supplementation in this study and progesterone concentrations were minor in cows injected with the lowest doses of vitamins C and E. The corpus luteum was observed and measured by real time ultrasound, and a positive correlation between its size and functionality is assumed(29). However, results from this 577

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and other studies are in disagreement. The cows injected with the reduced dose of vitamins regardless of having a similar corpus luteum size, produced less progesterone than the other groups. Similarly previous research did not find a correlation between size of the CL in regression phase and progesterone concentrations in cows(30). In addition, others found that after d 8 of the oestrus cycle, the size of the corpus luteum does not determine progesterone concentrations(31). This finding supports the results of the present work, as corpus luteum was measured on d 9 of the oestrus cycle. Discrepancies among studies are not known, but three points should be considered when CL measurements and progesterone concentrations are to be analysed at the same time. First, from field experience, sometimes ultrasound practitioners fail to find the transducer position that gives the largest view of the CL; this can produce confounding effects when a relationship with progesterone concentrations is sought. Second, the corpus luteum is a dynamic ovarian structure, which is more easily identified and measured during the mid-luteal stage of the oestrus cycle, but measuring this structure at a very early stage (d 2 to 3 after estrus) of development requires a great deal of experience. Third, when diagnosing the status of the corpus luteum, not only its size but also its echographic appearance should be taken into consideration(32). It was not found studies that attempt to evaluate the effect of increasing doses of vitamin E and C on dairy cattle fertility. Other studies have demonstrated a positive effect on pregnancy rate when supplementing vitamins C(14) and E(33) separately. The effect is mediated by enhancing follicle cell survival(6), oocyte competence, corpus luteum functionality(15,16,34) or embryo survival(35,36). Despite previous experiences showing an improvement in pregnancy rate in cows injected with vitamins(12), the results of the present study do not support such findings. However, it is worth noting that cows injected with the lowest dose of vitamins, despite having lower concentrations of progesterone, were capable of sustaining similar pregnancy rates 30 and 45 d after AI compared with the other evaluated groups. Progesterone stimulates changes within the uterine environment allowing embryo receptivity and survival(37). The concentrations of progesterone required to increase the probability of pregnancy occurrence is not well established. One may argue that higher, rather than lower, concentrations of progesterone are better for getting a cow pregnant. However, researches have suggested a range of milk progesterone concentrations within which embryo survival was maximal(38). The existence of a range of progesterone concentrations suitable for pregnancy success is acceptable because a large concentration of progesterone could affect fertility by creating an asynchrony between the uterine environment and the embryo(39); while a uterine environment with low progesterone concentrations will fail to induce the changes necessary for hosting the embryo(40). Besides progesterone concentration, it is well known that embryo quality affects probability of pregnancy, and good quality embryos are better at achieving not only pregnancy, but also live birth in a uterine environment with variable progesterone concentrations, than those embryos of lower quality(41). Therefore, it is possible that cows injected with the lowest dose of vitamins may have had good quality embryos(36) capable

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of surviving and establishing pregnancy in a uterine environment with low progesterone concentrations.

Conclusions and implications

Supplementation with vitamins C and E did not affect preovulatory follicle and corpus luteum size, estradiol production on the day of estrus, or pregnancy rate 30 and 45 d after AI. The supplementation with the highest amount of vitamin C and E did not significantly increased the reproductive parameters measured.

Literature cited: 1.

Allison RD, Laven RA. Effect of vitamin E supplementation on the health and fertility of dairy cows: a review. Vet Record 2000;147:703-708.

Ranjan R, Ranjan A, Dhaliwal GS, Patra RC. L-Ascorbic acid (vitamin C) supplementation to optimize health and reproduction in cattle. Vet Q 2012;32:145150.

Baldi A, Savoini G, Pinotti L, Monfardini E, Cheli F, Orto VD. Effects of vitamin E and different energy sources on vitamin E status, milk quality and reproduction in transition cows. J Vet Med Asoc 2000;47:599-608.

Horn M, Gunn P, Van Emon M, Lemenager R, Burgess J, Pyatt NA, Lake SL. Effects of natural (RRR α-tocopherol acetate) or synthetic (all-rac α-tocopherol acetate) vitamin E supplementation on reproductive efficiency in beef cows. J Anim Sci 2010;88:3121-3127.

Al-Enazi MM. Influence of α-tocopherol on heat stress-induced changes in the reproductive function of Swiss Albino mice. Saudi J Biol Sci 2007;14:61-67.

Ren SQ, Wang JW, Chen HY, Xu MQ, Jiang H, Gao Y, et al. Effect of vitamin E on bovine granulosa cells apoptosis and proliferation through cx43. China Anim Husb Vet Med 2016;43:615-621.

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Dalvit G, Llanes SP, Descalzo A, Insani M, Beconi M, Cetica P. Effect of alphatocopherol and ascorbic acid on bovine oocyte in vitro maturation. Reprod Domest Anim 2005;40:93-97.

Chauhan SS, Celi P, Ponnampalam EN, Leury BJ, Liu F, Dunshea FR. Antioxidant dynamics in the live animal and implications for ruminant health and product (meat/milk) quality: role of vitamin E and selenium. Anim Reprod Sci 2015;54:1525-1536.

Luck MR, Jeyaseelan I, Scholes RA. Ascorbic acid and fertility. Biol Reprod 1995;52:262-266.

10. McIntosh RA. Ascorbic acid (vitamin C) for the treatment of impotency in bulls and sterility in cows. Can J Comp Med 1941;5:267-268. 11. NRC. National Research Council. Nutrient requirements of dairy cattle. 7th ed. Washington, DC, USA: National Academic Press; 2001. 12. GonzĂĄlez-Maldonado J, Santos RR, De Lara RR, Ramirez GV. Impacts of vitamin C and E injections on ovarian structures and fertility in Holstein cows under heat stress conditions. Turk J Vet Anim Sci 2017;41:345-350. 13. Thomas FH, Leask R, Srsen V, Riley SC, Spears N, Telfer EE. Effect of ascorbic acid on health and morphology of bovine preantral follicles during long-term culture. Reproduction 2001;122:487-495. 14. Phillips PH, Lardy HA, Boyer PD, Werner GM. The relationship of ascorbic acid to reproduction in the cow. J Dairy Sci 1941;24:153-158. 15. Luck MR, Zhao Y. Identification and measurement of collagen in the bovine corpus luteum and its relationship with ascorbic acid and tissue development. J Reprod Fertil 1993;99:647-652. 16. Serpek B, Baspinar N, Haliloglu S, Erdem H. The relationship between ascorbic acid, oestradiol 17Î˛ and progesterone in plasma and in ovaries during the sexual cycle in cattle. Rev Med Vet 2001;152:253-260. 17. Canadian Council on Animal Care in Science. CCAC guidelines on: The care and use of farm animals in research, teaching and testing. Ottawa, CA: Canadian Council on Animal Care; 2009. 18. Arnaud J, Fortis I, Blachier S, Kia D, Favier A. Simultaneous determination of retinol, alpha-tocopherol and beta-carotene in serum by isocratic high-performance liquid chromatography. J Chromatogr 1991:572:103-116. 19. Lopes AS, Butler ST, Gilbert RO, Butler WR. Relationship of pre-ovulatory follicle size, estradiol concentrations and season to pregnancy outcome in dairy cows. Anim Reprod Sci 2007;99:34-43. 580

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20. Keskin A, Mecitoğlu G, Bilen E, Güner B, Orman A, Okut H, Gümen A. The effect of ovulatory follicle size at the time of insemination on pregnancy rate in lactating dairy cows. Turkish J Vet Anim Sci 2016;40:68-74. 21. Arlotto T, Schwartz JL, First NL, Leibfried-Rutledge ML. Aspects of follicle and oocyte stage that affect in vitro maturation and development of bovine oocytes. Theriogenology 1996;45:943-956. 22. Otoi T, Yamamoto K, Koyama N, Tachikawa S, Suzuki T. Bovine oocyte diameter in relation to developmental competence. Theriogenology 1997;48:769-774. 23. Robinson RS, Hammond AJ, Hunter MG, Mann GE. The induction of a delayed post-ovulatory progesterone rise in dairy cows: a novel model. Domest Anim Endocrinol 2005;28:285-295. 24. Atkins JA, Smith MF, MacNeil MD, Jinks EM, Abreu FM, Alexander LJ, et al. Pregnancy establishment and maintenance in cattle. J Anim Sci 2013;91:722-733. 25. Colazo MG, Behrouzi A, Ambrose DJ, Mapletoft RJ. Diameter of the ovulatory follicle at timed artificial insemination as a predictor of pregnancy status in lactating dairy cows subjected to GnRH-based protocols. Theriogenology 2015;84:377-383. 26. Perry GA, Smith MF, Lucy MC, Green JA, Parks TE, MacNeil MD, et al. Relationship between follicle size at insemination and pregnancy success. PNAS 2005;102:5268-5273. 27. Cummins SB, Lonergan P, Evans AC, Butler ST. Genetic merit for fertility traits in Holstein cows: II. Ovarian follicular and corpus luteum dynamics, reproductive hormones, and estrus behavior. J Dairy Sci 2012;95:3698-3710. 28. Rapoport R, Sklan D, Wolfenson D, Shaham-Albalancy A, Hanukoglu I. Antioxidant capacity is correlated with steroidogenic status of the corpus luteum during the bovine estrous cycle. Biochim Biophys Acta 1998;1380:133-140. 29. Kayacik V, Salmanoúlu MR, Polat B, Ozluer A. Evaluation of the corpus luteum size throughout the cycle by ultrasonography and progesterone assay in cows. Turkish J Vet Anim Sci 2005;29:1311-1316. 30. Assey RJ, Purwantara B, Greve T, Hyttel P, Schmidt MH. Corpus luteum size and plasma progesterone levels in cattle after cloprostenol-induced luteolysis. Theriogenology 1993;39:1321-1330. 31. Mann GE. Corpus luteum size and plasma progesterone concentration in cows. Anim Reprod Sci 2009;115:296-299. 32. Veronesi MC, Gabai G, Battocchio M, Mollo A, Soldano F, Bono G. Ultrasonographic appearance of tissue is a better indicator of CL function than CL diameter measurement in dairy cows. Theriogenology 2002;68:61-68. 581

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33. Richardson MJ, Lemenager RP, Pyatt N, Lake SL. Natural source vitamin E supplementation and reproductive efficiency in beef cows. Western Am Soc Anim Sci 2008;59:339-342. 34. Vierk JE, Murdoch WJ, Austin KJ, Van-Kirk EA, Hansen TR. Antiluteolytic effect of alpha tocopherol in ewes. J Dairy Sci 1998;81:372. 35. Olson SE, Seidel GE Jr. Culture of in vitro-produced bovine embryos with vitamin E improves development in vitro and after transfer to recipients. Biol Reprod 2000;62:248-252. 36. Sales JN, Dias LM, Viveiros AT, Pereira MN, Souza JC. Embryo production and quality of Holstein heifers and cows with beta-carotene and tocopherol. Anim Reprod Sci 2008;106:77-89. 37. Lonergan P. New insights into the function of progesterone in early pregnancy. Anim Frontiers 2015;5:12-17. 38. Stronge AJ, Sreenan JM, Diskin MG, Mee JF, Kenny DA, Morris DG. Postinsemination milk progesterone concentration and embryo survival in dairy cows. Theriogenology 2005; 64:1212-1224. 39. Randi F, Fernandez-Fuertes B, McDonald M, Forde N, Kelly AK, Bastos-Amorin H, et al. Asynchronous embryo transfer as a tool to understand embryo-uterine interaction in cattle: is a large conceptus a good thing?. Reprod Fertil Develop 2015;28:1999-2006. 40. Kenyon AG, Mendonรงa LG, Lopes G Jr, Lima JR, Santos JE, Chebel RC. Minimal progesterone concentration required for embryo survival after embryo transfer in lactating Holstein cows. Anim Reprod Sci 2013;136:223-230. 41. Yovich JL, Conceicao JL, Stanger JD, Hinchliffe PM, Keane KN. Mid-luteal serum progesterone concentrations govern implantation rates for cryopreserved embryo transfers conducted under hormone replacement. Reproductive Biomed Online 2015;31:180-191.

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https://doi.org/10.22319/rmcp.v10i3.4772 Article

Improved farrowing rate using intrauterine insemination in sows

Fernando Canea Norma Pereyraa Valentina Canea Patricia, Marinib-c Juan Manuel Teijeirob-d*

MEDAX. Sacco Scarafía 365. Chañar Ladeado. Santa Fe. Argentina.

Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva UNR. Argentina. c

Instituto de Biología Molecular y Celular de Rosario-CONICET. Consejo de Investigaciones de la UNR, CIUNR. Argentina. d

Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET. Argentina

* Corresponding author: jteijeiro@fbioyf.unr.edu.ar

Abstract: Intrauterine insemination (IUI), a technique that uses a lower number of spermatozoa than conventional artificial insemination (CAI), could contribute to improve reproductive efficiency of boars. However, since some field trial reports show suboptimal performance for IUI, it is necessary to continue evaluating and standardizing this technique. In this work, the use of fixed reduced sperm amounts and doses volumes for IUI respect to CAI using the same semen samples was assessed. The results show an increase in the farrowing rate using IUI vs CAI (84.80 ± 0.36 vs 71.44 ± 2.63, P<0.05). Parameters such as litter size, live piglets/litter, stillborn or mummified fetuses were analyzed as well and showed non-significant differences between techniques. Statistical positive correlation analyses showed a positive correlation between live piglets/litter and stillborn piglets and between stillborn and total number of piglets, only for CAI. In addition, the economic analysis showed a positive impact on the productivity of the farm, and possibly of the 583

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region, by lowering costs using IUI instead of CAI. In conclusion, the intrauterine insemination had a positive impact on the reproductive performance and on the economic parameters of porcine production. Key words: Porcine production, Intrauterine insemination, Artificial insemination.

Received: 19/02/2018 Accepted: 25/10/2018

Introduction

In the early twentieth century, Ivanow reported the use of the artificial insemination technique (AI) in pig(1,2). However, the commercial application of AI began at the 1980s(3). Its success can be attributed to improvement in the boar:sow ratio, increase of the impact of individual boars in both genetic progress and reproductive efficiency; and limited spread of venereal diseases. Improvement in animal management and quality controls of semen doses and their commercial use have increased the reproductive performance(4). Conventional artificial insemination (CAI) usually employs 2.5 to 4 billion spermatozoa per insemination in a 70 to 100 ml volume of extender, which is deposited through the cervix into the uterus two or three times during the oestrous period(5). Boars used for AI can produce 20 to 40 CAI doses containing 2.5 to 3.0 billion motile sperm in 70 to 100 ml of extender. A reduction in the number of sperm per dose would result in a higher number of doses produced per boar with considerable economic saving, thus new strategies towards lowering the number of spermatozoa per dose in AI are constantly under study(6). Intrauterine insemination (IUI) is an insemination technique that uses a reduced number of sperm per insemination dose respect to CAI(7). However, data reported about the application of this technique show some discrepancy in the number of sperm cells per dose (which is not yet standardized). Moreover, most of the literature comparing treatments does not include groups with similar sperm number per dose, making it difficult to state whether the results are due to sperm number per dose or to the technique itself(4). Boar exposure to sows before insemination is considered to induce myometrial contractions which aid sperm transport; however, there is some uncertainty as to whether boar exposure before IUI catheter insertion is detrimental to the catheter insertion and to whether boar exposure has beneficial effects in CAI. To date, there is not enough

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information to suggest detrimental or beneficial effects for any approach(8). Still other matters of discussion arise when considering the advantages and disadvantages of the application of IUI vs CAI. These are the time consumed to insert the catheters for IUI, which is lower for CAI; backflow due to higher volume in CAI; bleeding at the time of inserting the catheters in IUI; and implementation of fixed-time ovulation induction and insemination. There is information about the use of IUI technology which shows variations not only among countries, but also within each country(9). In spite of considerable variation among countries and farms, AI can be monitored for success using key measures for quality control and reproductive performance(8). Thus, a study was performed comparing the reproductive parameters: farrowing rate, litter size, live piglets/litter, stillborn and mummified fetuses between CAI and IUI. Furthermore, it has been analyzed the economic impact on the studied farm and the possible impact on the region.

Material and methods

Semen collection

Semen samples were collected from adult fertile boars by the glove-hand method in Medax (ChaĂąar Ladeado, Santa Fe, Argentina). Sperm rich fraction was diluted in Vitasem (MagaporÂŽ, Zaragoza, Spain), and conserved at 16 ÂşC until use for no more than 2 d. Viability was measured through eosin exclusion test and the average for the three boars was 92.6 %. Motility was measured subjectively and the average was 91.26 % motile sperm. Morphology was assessed as previously reported(10) and normal sperm in samples were 89 % for boar B, 91 % for boar C and 93 % for boar A.

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Experimental design

The study was conducted at a commercial farm located in Monte Maíz, Córdoba, Argentina (GPS coordinates: -33.206561, -62.600330). Three mature boars, two 415 PIC® (Pig Improvement Company, Pasig City Philippines) (A and B) and one Landrance (C) from Topigs (Topigs Norsvin, Burnsville, USA) were used as semen donors. Boar A showed 91.9 ± 2.13 motile sperm. Boar B showed 91.4 ± 3.2 motile sperm and boar C 90.5 ± 4.0. The inseminations were conducted between February and December of 2015. Five hundred and sixty (560) multiparous housed sows were separated in two groups of 280 sows. The criteria of selection for experimental design were: sows produced by breeding of Large-White female x Landrance boar, age between 190 and 200 d with at least 130 kg and four cycles detected, parity 3.9. A total of 560 inseminations were done. One group was inseminated by conventional artificial insemination (CAI) (280 inseminations) and the other group by intrauterine insemination (IUI) (280 inseminations) using the same boars as donors for each technique. In order to avoid variations on seminal quality due to different factors (individuality of boar, seasonality, physical and sanitary conditions, etc.), distribution of the same ejaculate to practice the inseminations of the two groups was performed. Oestrus detection was performed twice daily by experienced workers. Personnel for insemination procedures were carefully trained and evaluation of return to oestrus before proceeding to new inseminations was done. Sows from the two groups were contemporaneously inseminated by the two techniques. The sows were exposed to the same environment, fed with the same commercial diet and water was provided ad libitum.

Conventional artificial insemination

CAI was performed using spiral catheter (Magapor®, Zaragoza, Spain) and 3 x 109 sperm in 100 ml/dose. All sows were inseminated twice in standing heat in the presence of a boar. CAI was performed with 3x109 spermatozoa in 100 ml.

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Intrauterine insemination

IUI was performed according to Hancock(11) using 1.5 x 109 sperm in 50 ml/dose and in the absence of boars, as recommended for better cannula introduction for IUI (foam catheter M. Magapor®, Zaragoza, Spain). Neither bleeding occurrence nor semen backflow were detected. IUI used 1.5x109 spermatozoa in 50 ml doses.

Analyses of the economic impact

Comparison between the economic parameters obtained with each technique was analyzed by the software “Análisis productivo y económico de granjas porcinas”(12) (Productive and economic analysis of porcine farms, APEC). In this computationally modeled analysis, standard catheter cost, labor cost, labor time, price of kilogram of meat and non-productive days (NPD), were the parameters used. NPD were calculated on the base of a sow productive cycle of 136 d (115 gestational days + 21 lactations days). The techniques and processes were approved for ethics by Servicio de Sanidad y Calidad Agroalimentaria (Agroalimentary sanity and quality service, SENASA, Argentina), resolution 63/2011.

Statistical analysis

Statistical analysis was performed using InfoStat (Universidad Nacional de Córdoba, Córdoba, Argentina). Normality test was performed by Shapiro-Wilks and Wilcoxon signed-rank test for non-parametric statistical hypothesis was applied.

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Results

Analyses of reproductive parameters

When IUI and CAI were compared using the same boar semen samples, statistical data showed an increase in farrowing rate using IUI respect to CAI. The farrowing rate was 71.44 ± 2.63 for CAI, while for IUI it was 84.80 ± 0.36 (Table 1). The other parameters analyzed, live piglets/litter, stillborn or mummified fetuses showed no statistical differences. However, it is to note that a slight non-significant increase in litter size in IUI respect to CAI (14.61 ± 0.06 vs 13.72 ± 0.52) was observed. Since the same boars were used to perform the inseminations and the inseminations of sows were carried out contemporaneously using the two techniques, Pearson correlation coefficient could be applied to give insight into the observed differences. A statistically positive correlation was found between live piglets/litter and stillborn piglets (r= 1; P= 0.0074), and between stillborn and total number of piglets (r= 1; P= 0.0569) only for CAI (Table 2). The analysis for IUI showed no correlation between the studied parameters.

Table 1: Data of farrowing rate, born alive piglets, stillborn piglets, mummified fetuses and litter size obtained using CAI and IUI Number of Boar inseminations

Farrowing rate %

Live Mummified Stillborn piglets/litter fetuses

A B C Mean

74 79 127

Conventional artificial Insemination 72.72 13.13 1.22 0.41 75.5 12.25 0.93 0.15 66.6 11.96 0.84 0.29 71.44 ± 2.63a 12.45 ± 0.61 1.00 ± 0.11 0.28 ± 0.08

A B C Mean

84 70 126

Intrauterine insemination 84.62 12.96 0.88 0.88 84.28 12.97 1.15 0.47 85.5 12.81 1.27 0.42 84.80 ± 0.36b 12.21 ± 0.05 1.10 ± 0.12 0.59 ± 0.15

a,b

Total number of piglets/litter 14.75 13.33 13.08 13.72 ± 0.52

14.71 14.60 14.51 14.61 ± 0.06

Different superscripts indicate significant differences (P<0.05). Means ± standard errors.

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Table 2: Pearson correlation coefficient estimations for the reproductive variables study using IUI and CAI Variable

Variable

Pearson

P-value

Farrowing rate Farrowing rate Farrowing rate Farrowing rate Live piglets/litter Live piglets/litter Live piglets/litter Stillborn Stillborn Mummified fetuses

Conventional artificial insemination Live piglets/litter Stillborn Mummified fetuses Total number of piglets Stillborn Mummified fetuses Total number of piglets Mummified fetuses Total number of piglets Total number of piglets

0.44 0.43 -0.35 0.35 1.00 0.69 0.99 0.70 1.00 0.76

0.7112 0.7186 0.7742 0.7755 0.0074a 0.5146 0.0643 0.5071 0.0569a 0.4502

Farrowing rate Farrowing rate Farrowing rate Farrowing rate Live piglets/litter Live piglets/litter Live piglets/litter Stillborn Stillborn Mummified fetuses

Intrauterine insemination Live piglets/litter Stillborn Mummified fetuses Total number of piglets Stillborn Mummified fetuses Total number of piglets Mummified fetuses Total number of piglets Total number of piglets

-0.98 0.53 -0.34 -0.66 -0.70 0.54 0.80 -0.98 -0.99 0.93

0.1385 0.6465 0.7776 0.5441 0.5080 0.6390 0.4056 0.1311 0.1024 0.2335

Superscripts indicate significant difference (P<0.05)

Analysis of economic impact

The economic impact of using each insemination technique was analyzed, showing an increase on profits using IUI vs CAI in a one-year period, for a farm with 560 sows (Table 3). Based on these data, the potential reproductive performance per sow would be 2.59 farrowing/sow/year, considering 5 d between weaning and further insemination for IUI. This value is above the average for this region of Argentina, which ranges 2.21 to 2.35 farrowing/sow/yr. These results also showed an increase from 2.32 to 2.36 conceptions/sow/yr in favor of IUI. Considering the increase in litter size in 0.89 (14.61 to 13.72) and a mortality percentage of the farm of 6 %, the profit can be calculated as: 589

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0.89 x 2.36 (conceptions/sow/yr) x 280 (sows) = 573.16 â&#x20AC;&#x201C; 6% (mortality) = 538.77. Then, 538.77 x 112 kg (weigh of sale per pig) = 60,342 kg. Contemplating a price of U$S 1.09 per kg of pig, there is a calculated benefit of U$S 65,773 using IUI instead of CAI for the studied farm. Taking into account the prize of the catheter for IUI, which is more expensive than CAI, in the computational modeled economic study, there is a net income of U$S 3,428 in favor of IUI. That is, the difference between the net income for IUI and the net income for CAI is U$S 37,008 â&#x20AC;&#x201C; U$S 33,580. Contemplating an average of 3,000 pig per farm of a similar size in the region, the profit would be of great benefit for the local economy.

Table 3: Economic impact of implementation of IUI and CAI in a farm from the middle region of Argentina Variable Sows Farrowing rate (%) NPD/failure/cycle Farrowing/sow/year pig Kg/sow/year U$S annual net income

CAI

IUI

280 71.44 10 2.32 2,784 33,580

280 84.08 7,38 2.36 2,832 37,008

Dollar values were calculated according to kg of pig prices in Argentina in 2015.

Discussion

Artificial insemination is widely used and there is constant search for new strategies to achieve higher efficiency. Fixed-time AI, use of high genetic merit boars or changing the site of semen deposition are examples of these attempts to improve efficiency. Watson and Behan(7) reported farrowing rates of 91.1, 91.8 and 65.8 % for CAI whereas the IUI technique showed rates of 90.5, 90.5 and 86.9, using 3, 2 and 1 billion spermatozoa in 80 ml, respectively. The mean of the litter sizes with CAI were 12.5, 12.6 and 10.6 and with IUI they were 12.3, 12.3 and 12.1, respectively. They demonstrated that only the 1 billion spermatozoa dose with CAI technique showed a significantly lower farrowing rate and litter size. Rozeboom et al(13), showed that insemination at the beginning of the uterine horn with conventional volumes and spermatozoa numbers (1 billion spermatozoa) produced results similar to insemination in the cervical cavity (4 billion spermatozoa).

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However, litter size and live piglets/litter were lower in IUI than CAI with those amounts of sperm per dose. Moreover, when 0.5 billion spermatozoa were used for IUI, the farrowing rate decreased approximately 10 % in comparison with the CAI group (78 vs 88.2 %, respectively) and, also, the differences in litter size between both techniques favored CAI (9.4 vs 11.6, respectively). Levis(14) reported similar results. Other work(15) established that the farrowing rate did not differ between CAI using 3.5 x 109 spermatozoa in 100 ml and IUI using 2, 1, or 0.5 x 109 spermatozoa in 50 ml. Peltoniemi et al(16) concluded that uterine insemination did not have a significant effect on live piglets/litter and farrowing rate. The results from those studies do not necessarily reflect the data from similar procedures on other farms. In whole, summarized information about pig reproductive technologies with emphasis in field application suggests that high farrowing rates and litter size are becoming common when using standard AI with only 1.5 billion spermatozoa per insemination(17). In the present experimental design, all sows were in the same farm, and were inseminated with the same batches of seminal doses; CAI and IUI were equally distributed in the same day and performed with semen from all the three boars. These controlled parameters make it possible to perform a better comparison between the two techniques. Differences in farrowing rates were found using 3 x 109 spermatozoa in 100 ml for CAI and 1.5 x 109 sperm in 50 ml for IUI, that support the change from CAI to IUI in the studied farm. Moreover, although non-statistically significant, the increase in 0.89 on litter size makes the application of IUI more convenient than CAI. To get higher efficiency, AI must be accomplished reducing the number of spermatozoa, in this work the number of spermatozoa and volume of extender used in each dose were reduced to a half. IUI with reduction in the number of spermatozoa in a fixed volume has already been analyzed(18); however in such work IUI was not contrasted with CAI. They suggested that 0.5 x 109 spermatozoa in 20 ml are sufficient to perform successful IUI, however in this work tested that volume and, the difficulty of handling a 20 ml dose counteracted the possible improvement. The reduction in sperm and doses volume is highly profitable for artificial insemination centers, as the use of superior boars renders twice the doses for IUI than for CAI, thus this feature must be included in future economic impact analyses. In addition, in the presented experiments, a boar was present when developing CAI and absent for IUI. The presence of a boar for IUI is not recommended due to difficulty to introduce de cannula. Instead, it is recommended for CAI to help in standing and to induce myometrial contractions which aid sperm transport, but additional labor for boar movement is required(8). Despite the extra aid for sperm transit due to the presence of the boar, farrowing rate did not improve for CAI. This work agrees with the idea of the additional benefit of IUI on the necessity of less boar handling for its application. Since Steverink et al(19), reported that excessive backflow of semen upon CAI has a negative effect on fertilization results when 1 x 109 spermatozoa in 80 ml are used, it was hypothesized that a reduction in volume would improve the efficiency of the technique by reducing the backflow. In this work, no backflow was observed using 50 ml doses, which seems to be an appropriated volume, easy to handle during the procedure. The main

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obstacle to apply IUI is the complex anatomy of the sowâ&#x20AC;&#x2122;s genital tract composed by cervical folds and the length and coiled nature of the uterine horns. This obstacle was saved by employing well-trained personnel, who were systematically evaluated throughout the experiment. This may be one of the reasons for the success of IUI in this work. The fact that IUI is time-consuming and requires professional trainers must be included in future economic studies. To our knowledge, no statistical correlation analysis was made in the precedent works. Interestingly, a statistically positive correlation between live piglets/litter and stillborn was determined in CAI and such correlation was not observed in IUI (Table 2). The PearsonÂ´s correlation coefficient is a measure of the linear correlation between two quantitative variables, so the results presented here may be interpreted as that more services are necessary to obtain more live piglets/litter when using CAI. It is to note that this correlation could be attributed to boar, sow or environmental effects, but results from IUI, which was performed in the same conditions rules out this possibility. With recent data demonstrating the possibility to perform IUI in primiparous sows(20), it seems that the use of fewer boars in AI centers and production of more AI doses with reduced number of sperm, per boar, is an interesting aspect to consider to improve the efficiency in swine production. Considering that 30 minimal doses could be generated by using one single boar and taking into account health, feed, installations and handling costs, the overall costs could be reduced using IUI instead of CAI. As was demonstrated, the change of CAI to IUI using 1.5 x109 spermatozoa in 50 ml doses would be more profitable.

Conclusions and implications

Using 1.5 x 109 sperm in 50 ml, without the presence of boar and inseminating at heat standing with well-trained personal, IUI had a positive impact on the reproductive performance and on the economic parameters of porcine production.

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Literature cited 1.

Ivanow E. De la fécondation artificielle chez les mamiféres. Arch Sci Biol 1907;(12):377-511.

Ivanow E. On the use of artificial insemination for zootechnical purpose in Rusia. J Agric Sci 1922;(12):244-256.

Reed HCB. Artificial insemination. In: Cole DJA, Foxcroft GR, editors. Control of pig reproduction. London: Butterworth Scientific; 1982:65–90.

Bortolozzo F, Menegat M, Mellagi A, Bernardi M, Wentz I. New artificial insemination technologies for swine. Reprod Domest Anim 2015;(50):80–84.

Roca J, Parrilla I, Bolarin A, Martinez EA, Rodriguez-Martinez H. Will AI in pigs become more efficient? Theriogenology 2016;(86):187-913.

Hernández-Caravaca I, Izquierdo-Rico MJ, Matás C, Carvajal JL. Vieira L, Abril D, Soriano-úbeda C, García-Vázquez FA. Reproductive performance and backflow study in cervical and post-cervical artificial insemination in sows. Anim Reprod Sci 2012;(136):14–22.

Watson P, Behan J. Intrauterine insemination of sows with reduced sperm numbers: Results of a commercially based field trial. Theriogenology 2002;(57):1683–1693.

Knox RV. Artificial insemination in pigs today. Theriogenology 2016;(85):83–93.

Vázquez JM, Roca J, Gil MA, Cuello C, Parrilla I, Vazquez JL, Martínez EA. New developments in low-dose insemination technology. Theriogenology 2008;(70):1216-1224.

10. Teijeiro JM, Cabada M, Marini PE. Sperm binding glycoprotein (SBG) produces calcium and bicarbonate dependent alteration of acrosome morphology and protein tyrosine phosphorylation on boar sperm. J Cell Biochem 2008;(103):1413–1423. 11. Hancock J. Pig insemination technique. Vet Rec 1959;(71):527. 12. Drab S. APEC. Software para análisis productivo-económico en granjas porcinas. Cátedra Producción Porcina. Facultad de Ciencias Veterinarias. Universidad Nacional de Rosario. 2012. 13. Rozeboom K, Reicks D, Wilson M. The reproductive performance and factors affecting on-farm application of low-dose intrauterine deposit of semen in sows. J Anim Sci 2004;(82):2164-2168. 14. Levis D. Liquid boar semen production: current extender technology and where do we go from here. In: Johnson LA, Guthrie HD editors. Boar semen preservation IV. Lawrence, KS, USA: Allen Press Inc; 2000:121–128.

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15. Serret C, Alvarenga M, Cória A, Dias C, Corcini C, Corrêa M, Deschamps J, Bianchi I, Lucia Jr. T. Intrauterine artificial insemination of swine with different sperm concentrations, parities, and methods for prediction of ovulation. Anim Reprod 2005;(2):250-256. 16. Peltoniemi OA, Alm K, Andersson M. Uterine insemination with a standard AI dose in a sow pool system. Reprod Domest Anim 2009;(44):414-841. 17. Martinez EA, Vazquez JM, Roca J, Cuello C, Gil MA, Parrilla I, Vazquez JL. An update on reproductive technologies with potential short-term application in pig production. Reprod Domest Anim 2005;(40):300-309. 18. Mezalira A, Dallanora D, Bernardi MI, Wentz I, Bortolozzo FP. Influence of sperm cell dose and post-insemination backflow on reproductive performance of intrauterine inseminated sows. Reprod Domest Anim 2005;(40):1-5. 19. Steverink D, Soede N, Bouwman E, Kemp B. Semen backflow after insemination and its effect on fertilisation results in sows. Anim Reprod Sci 1998;(54):109-119. 20. Sbardella P, Ulguim R, Fontana D, Ferrari C, Bernardi M, Wentz I, Bortolozzo F. The post-cervical insemination does not impair the reproductive performance of primiparous sows. Reprod Domest Anim 2014;(49):59–64.

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https://doi.org/10.22319/rmcp.v10i3.4896 Article

Economic evaluation of post-weaning and finishing cattle supplemented on pasture

Aroldo Brandão de Oliveiraa* Robério Rodigues Silvaa Fabiano Ferreira da Silvaa Gleidson Giordano Pinto de Carvalhob Ana Paula Gomes da Silvaa João Wilian Dias da Silvaa Daniele Soares Barrosoa Grabriel Dallapicola da Costaa

Universidade Estadual do Sudoeste da Bahia. Programa de Pós-Graduação em Zootecnia. Rod. BR 415, Km 03, Itapetinga, Bahia, Brasil, CEP:45700-000. b

Universidade Federal da Bahia, Escola de Medicina Veterinária e Zootecnia, Departamento de Zootecnia, Salvador, Bahia, Brasil.

* Corresponding author: brandaoitap@hotmail.com

Abstract: The objective of this study was to evaluate the economic viability, through different supplementation strategies, of the post-weaning and finishing stages of cattle supplemented on Brachiaria brizantha cv. Marandu pastures during the rainy and dry seasons. The experimental period was 447 d. The study comprised the post-weaning and finishing stages of 22 intact male crossbred (½ Holstein-Zebu) cattle with an average initial weight of 164.09 ± 12.13 kg and an average age of 7 mo. The animals were distributed in a randomized design with 11 replications per treatment. The following supplementation strategies were tested: strategy 1 (S1): Mineral mix in the 1st and 3rd periods and protein-energy supplementation at 0.2 % of the body weight (BW) in the 2nd period; and strategy 2 (S2): protein-energy supplementation at 0.4 % BW in the 1st and 595

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3rd periods, and protein-energy supplementation at 0.6 % BW in the 2nd period. Strategy 1 resulted in a lower cost per arroba produced and lower cost per hectare, generating a greater net profit per hectare and consequently a higher internal rate of return. When herbage is available, mineral supplementation supplied during the rainy season, associated with low levels of protein-energy supplementation in the dry season (S1), is of greater economic attractiveness for the development of the project, as it leads to higher internal rates of return and net present values in the entire period. Key words: Internal rate of return, Net present value, Supplementation on pasture.

Received: 16/05/2018 Accepted: 30/07/2018

Introduction

The use of supplementation provides greater efficiency to pasture usage, making it an auxiliary tool in pasture management that leads to higher stocking rates and better animal performance and ultimately resulting in a shorter production cycle and increased productivity to the system. However, when a producer chooses to implement supplementation on pasture, forage intake by the animal should be maximized so that it can have better performance, but the viability of the technique must be taken into account at all times. Supplementation is a biologically viable technique(1), because it produces a positive effect on the weight gain of animals or on gain per area. However, the producer must be alert as to the balance between the biological and economic responses, since the economic viability of the system is and will always be a dependent local factor. Whenever dietary supplementation is practiced in grazing-cattle production systems, there will be alterations in the cash flow of the farm, because it will be necessary to invest capital in the purchase of the supplement. In this regard, research involving the use of supplementation for grazing cattle must be subjected to economic analysis, and the obtained information must be quickly passed to farmers, the group with greatest interest in these results. Given the above-stated facts, this study aimed to evaluate the economic viability of rearing beef cattle on pasture under different supplementation strategies during the postweaning and finishing stages.

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Material and methods

The experiment was conducted in Ribeirão do Largo - BA, Brazil (15°26′46″ S, 40°44′24″ W, 800 m asl). The experimental period was 447 d, which were divided into 1st rainy season, 168 d; dry season, 180 d; and 2nd rainy season, 99 d. The study comprised the post-weaning and finishing stages of 22 intact male crossbred (½ Holstein-Zebu) cattle with an average initial weight of 164.09 ± 12.13 kg and an average age of 7 mo. The animals were distributed in a randomized design with 11 replications per treatment. The following supplementation strategies were tested: strategy 1 (S1): mineral mix in the 1st and 3rd periods (1st and 2nd rainy seasons) and protein-energy supplementation at 0.2 % of the BW in the 2nd (dry) period; and strategy 2 (S): protein-energy supplementation at 0.4 % BW in the 1st and 3rd (rainy) periods, and protein-energy supplementation at 0.6 % BW in the 2nd (dry) period. The animals were managed under the intermittent grazing method, in a pasture formed by Brachiaria brizantha cv. Marandu (6.5 ha) that was divided into six paddocks with equal area. Cattle were subjected to the control of ecto- and endo-parasites and vaccinated according to the calendar of the health authority of Bahia State (EBDA) and identified by numbered earrings. Paddocks had a central food court equipped with uncovered plastic troughs (80 cm/animals) with double access and drinkers with an automatic refill system and capacity for 500 L of water. Concentrate and mineral supplements were supplied daily at 1000 h.The animals remained seven days in each paddock, and the groups of animals rotated across the paddocks throughout the grazing cycle, aiming to minimize the paddock (environment) effects. Ingredients used in the supplements provided in both strategies are described in Table 1.

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Table 1: Proportion of ingredients from supplements (as-is basis) Strategy 1 Rainy season

Dry season

Supplement

Ingredient (%)

Mineral (ad libitum) Corn Soybean meal Urea + AS2 Mineral mix3,4

100

Strategy 2 Rainy season

Dry season

Supplement

0.2%1 BW

0.4%1 BW

0.6%1 BW

45.43 45.43 4.99 4.63

BW= body weight; 1Protein-energy 2Urea + ammonium sulfate (9:1) 3Composition: calcium- 235 g; phosphorus- 160 g; magnesium- 16 g; sulfur- 12 g; cobalt- 150 mg; copper- 1.600 mg; iodine- 190 mg; manganese- 1.400 mg; iron- 1.000 mg; selenium- 32 mg; zinc- 6.000 mg; fluorine(maximum) 1.600 mg 4 Composition: calcium- 175 g; phosphorus- 100 g; sodium- 114 g; magnesium- 15 g; zinc- 6.004 mg; manganese- 1.250 mg; copper- 1.875; iodine- 180 mg; cobalt- 125 mg; selenium- 30 mg; fluorine(maximum) - 1.000 mg.

Proposed indexes were used(2) as parameters of economic evaluation of the supplementation strategies; these are described as follows: Number of animals per treatment (n); Experimental period (days); Initial and final body weights - obtained by weighing the animals after a 12-h fasting period, and average body weight in the experimental period (arithmetic mean between initial and final body weights (BW)); Pasture area occupied by each treatment - the total experimental area was divided by the number of elements  6.5 ha/2 = 3.25 ha; Average stocking rate - the average body weight of each animal was multiplied by the number of animals per treatment and divided by the pasture area available per treatment and subsequently divided by 450 (corresponding to one animal unit (AU)) SR = [{(averageBW * 11)/3.25}/450]; Average daily gain of the animals - the weight gain in the experimental period was divided by the number of days in the evaluation period  (finalBW – initialBW)/number of days in each period - ADG during the experiment: S1 0.57 kg/d and S2 0.69 kg/d; Carcass dressing percentage - In the post-weaning phase, a dressing percentage of 50 % was considered, and, at finishing, the animals from S1 obtained 47.39 %, while S2 animals had 50.48 %;

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Average daily intake of concentrate supplement per animal in kg/d - 0.27 kg/d for S1 and 0.57 kg/d for S2 - obtained by the daily supply of chromium oxide together with the supplement, according to methodology proposal(3); Cost per kilogram of concentrate supplement - obtained based on the price of inputs and the respective composition, on a fresh-matter basis, of each concentrate supplement, in which corn: R$ 0.82kg; soybean meal: R$ 1.975 kg; urea: R$1.912 kg; and mineral mix: R$1.36kg Current prices at the commercial fair ofItapetinga-BA, Brazil (November/2015); Price of the arroba (@ = 14.7 kg) of unfinished cattle-mean values referring to the price of the unfinished cattle in the months of June (2014 and 2015) in Bahia State; Price of the @ of the finished cattle in November 2015, according to the Friboi packing plant (JBS Group) in Itapetinga-BA, Brazil; Costs with medications, maintenance of fences and of pastures, and taxes per animal, according to(4) ; Cost with labor, in @ per hectare. Values were obtained according to the data supplied by the owner of the farm where the experiment took place. After the described indices were obtained, it was possible to calculate the production and profitability values of the production system with each of the evaluated supplementation strategies. The variables are detailed as follows: Weight gain per hectare (kg/ha) during the experimental periods  average daily gain multiplied by the number of animals per treatment and by the experimental period, divided by the area occupied by each treatment (ADG * 11 * n of days in the experimental period)/3.25 ha; Meat production per hectare (kg/ha) during the experimental period  weight gain per hectare multiplied by the dressing percentage (DP) considered; Meat production per hectare (@/ha) during the experimental period  meat production in kg/ha divided by 15; Supplement intake per hectare (kg/ha) in the experimental periods  average supplement intake (kg/d) multiplied by the number of animals per treatment and by the experimental period by the area occupied by each treatment: (supplement intake * 11* n of days in the experimental period) / 3.25 ha; Cost with supplement per hectare (R$/ha) in the experimental period  supplement intake per hectare (kg/ha) multiplied by the price of supplement (R$/kg); Cost with supplement per arroba produced (R$/@) in the experimental period  cost with the supplement per hectare (R$/ha) divided by the amount of @ produced per hectare; Cost with labor in R$ per arroba produced (R$/@)  cost with labor per hectare, divided by the number of arrobas produced per hectare;

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Costs with medications, pasture maintenance, and taxes per arroba produced (R$/@) were calculated according to the production cost data (R$/ha) published in ANUALPEC(4), divided by the number of arrobas produced per hectare; Total cost per arroba produced (R$/@)  Sum of costs per arroba (R$/@) with supplement, labor, medications, pasture maintenance, and taxes; Participation of the cost of supplement in the total cost of arroba produced (%) cost with supplement per arroba produced (R$/@), divided by the total cost of arroba produced (R$/@), multiplied by 100; Total cost per animal in the experimental period (R$/animal)  total supplement intake (daily intake * number of days in the experimental period), multiplied by the price of the supplement (R$/kg) plus costs with labor, medication, pasture maintenance, and taxes per animal described in Table 2; Total cost per hectare in the experimental period (R$/ha)  total cost per arroba produced (R$/@) multiplied by the number of arrobas produced per hectare; Net profit per hectare (R$/@), only considering the weight gain in the experimental period with the use of supplementation number of arrobas produced per hectare, multiplied by the price of the arroba of the finished cattle (Table 2); Gross revenue per animal (R$/animal), considering only the weight gain in the experimental period with the use of supplementation  Gross revenue per hectare (R$/ha), multiplied by the pasture area used (3.25ha per treatment), divided by the number of animals per treatment (11 animals); Net revenue, or operating profit, per hectare (R$/ha), considering only the weight gain in the experimental period with the use of supplementation  result of the subtraction of the total cost per hectare from the net revenue per hectare (R$/ha); Total gross revenue per hectare (R$/ha),considering the final body weight of the animals as the sale weight at the price of the @ of the finished cattle (Table 2)  final body weight divided by 30, multiplied by the price of the arroba of finished cattle (R$145.00), multiplied by the number of animals per treatment (11 animals), divided by the pasture area occupied by each treatment (3.25 ha); Cost with the purchase of the unfinished cattle per hectare(R$/ha) initial body weight divided by 30, multiplied by the price of the arroba of unfinished cattle (R$ 145.00 average price of the unfinished cattle in the months of June (2014 and 2015) in Bahia State), multiplied by the number of animals per treatment (11 animals), divided by the pasture area occupied by each treatment (3.25 ha); Capital invested per hectare (R$/ha)  sum of the cost with the purchase of unfinished cattle per hectare (R$/ha) and the total cost per hectare in the experimental period (R$/ha),

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considering the costs with supplement, labor, medications, maintenance of fences and pasture, and taxes per hectare; Reals returned per real invested (R$)  net revenue per hectare divided by the total cost per hectare; Monthly rate of return (%) the net revenue per hectare was divided by the total cost per hectare and multiplied by 100; next, the result was divided by the experimental period and multiplied by 30 d  {(Net revenue ha/Total cost ha) * 100} /n of days of experimental period] * 30; Return on the investment per hectare (R$/ha/n of days of experimental period), considering an investment in the savings account with an average interestrate of 6 % per annum. capital invested in the period per hectare, multiplied by 6%/365, and then multiplied by the experimental period (number of days of experimental period); Percentage of return of the activity (%) net revenue, divided by the invested capital, both in R$/ha, multiplied by 100; Profitability index (%) net revenue (R$/ha), divided by the gross revenue (R$/ha), multiplied by 100. The profitability index indicates the available revenue after the payment of the feed cost (operating cost divided by the gross revenue in R$/ha/period in days multiplied by 100).

Table 2: Performance variables of production of crossbred steers under different supplementation strategies Performance Weight gain, kg/ha Meat production, kg/ha Production, @ of meat/ha Stocking rate, AU/ha

Supplementation strategy S1 S2 865.33 410.41 27.36 2.19

1055.59 533.60 35.57 2.40

CV (%)

P-value

12.39 13.65 13.65 15.87

0.0012 0.0002 0.0002 0.1845

S1= mineral supplementation in the 1st and 3rd periods and protein-energy supplementation at 0.2% BW in the 2nd period; S2= protein-energy supplementation at 0.4% BW in the 1st and 3rd periods and protein-energy supplementation at 0.6% BW in the 2nd period. CV= coefficient of variation; BW = body weight.

Two indices were adapted and used(5) for economic analysis: IRR (internal rate of return) and NPV (net present value). The calculation of the IRR of an investment indicates if it will increase the worth of a company. Therefore, an investment may or may not be made upon analyzing its IRR. For its calculation, it is necessary to project a cash flow that indicates money inputs and outputs stemming from the investments.

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The internal rate of return shows the return on the investment. Therefore, in managerial terms, IRR corresponds to the profitability rate expected from investments in a project. To determine whether the IRR is good or not, a common practice is to compare it with the cost of the invested capital; if the estimated IRR is greater than the cost of capital, then the project is accepted. Otherwise, the project will not be economically viable. In the case of comparison between two or more treatments, the higher the estimated IRR is, the more profitable the treatment will be; i.e., according to the acceptance criteria, the higher the result obtained in the project, the greater the attractiveness for its implementation; also, the investment alternative with the highest IRR will almost always be the preferred one. The calculation of NPV, in turn, represents a mathematical-financial formula that determines the current value of future payments discounted at a proper interest rate, minus the cost of the initial investment. Basically, it is the calculation of how much the future payments added to an initial cost would be worth currently. The concept of money’s worth in time is adopted; e.g., R$ 1,000.00 today will not be worth the same(R$ 1,000.00) in one year, because of the opportunity cost of, for instance, investing this amount in the savings account to earn interest. Thus, the internal rate of return is the ‘R’ value that equates the next expression to zero:

NPV =

CF1+

CF2

CF3 +

(1+R)1

(1 +R)2

(1+ R)3

+ ...+

CFn

CFo + (1 +R)n

in which: CF= net cash flows (0, 1, 2, 3,...,n) and, r= discount rate. The internal rate of return was calculated by projecting the capital inputs and outputs generated by the investment in question. For this purpose, the following variables were considered: Capital invested per hectare in the period (R$/ha/n of days in the experimental period, 447 d)  sum of the cost with the purchase of the unfinished cattle and of the cost with the capital invested per hectare; Daily gross revenue per hectare (R$/ha d)  division of the total gross revenue of each experimental period and total experimental period, per hectare (R$/ha), considering the final body weight of the animals as the sale weight at the price of the arroba of finished cattle, by the number of days in the experimental period. The experimental period was considered as the period of investment. In this way, a capital injection was considered as follows: Total experimental period (447 d): (daily gross revenue * 30 d) *14 mo + (daily gross revenue * 27 d); Rainy season 1 (168 d): (daily gross revenue * 30 d) *5 mo + (daily gross revenue * 18 d); Dry season (180 da: (daily

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gross revenue * 30 d) *6 mo; Rainy season 2 (99 d): (daily gross revenue * 30 d) *3 mo + (daily gross revenue * 9 d). For the other economic index used in the analysis of investments (NPV), three hurdle rates (HR) were considered; these were 5, 10, and 15 % per year, representing 0.41 %, 0.83 %, and 1.25 % per month, respectively. Upon calculating the NPV of the investment in question, the above-described variables were considered. The following mathematical expression represents the calculation of NPV(5): n=i

ĆŠNF/(1+R)t

NPV =

t=o

in which: NPV = net present value; NF = net flow (difference between inputs and outputs); n = number of flows; R = discount rate; t = period of analysis (i = 1, 2, 3...). For the statistical analysis of economic data, each animal was used as an experimental unit. The studied variables were interpreted statistically by analysis of variance and the F test at the 10% probability level.

Results

The weight gains per hectare (kg/ha) and meat production per hectare variables differed (P<0.10) between the two supplementation strategies (Table 2). Total weight gains per hectare and meat production were higher (P<0.10) in strategy S2, increasing from 410.41 kg/ha (27.36@/ha) to 533.60 kg/ha (35.57@/ha), in strategies S1 and S2, respectively. This is corroborated by the difference observed in the ADG of the animals, from 0.57 kg/d in S1 to 0.69 kg/d for S2. The stocking rate observed during the experimental period in the two supplementation strategies did not present differences (P>0.10). The average stocking rate found in this study was 2.29 AU/ha. The animals supplemented with strategy S2 had higher (P<0.10) concentrate intake and costs with supplement when compared with those supplemented with strategy S1, of the orders of 461.68 %, 465.60 %, and 331.35 %, respectively (Table 3).

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Table 3: Operating costs used in the composition of total costs per production of different supplementation strategies for crossbred steers

Variable

Supplementation strategy

Concentrate intake per period, kg/ha Cost with supplement, R$/ha Cost with supplement, R$/@ Cost with labor, R$/@ Cost with medications, R$/@ Cost with pasture maintenance, R$/@ Cost with taxes - IRR,R$/@ Total cost per arroba produced, R$/@ Cost per animal, R$ Participation of supplement in total cost, @ (%)

S1 416.68 604.19 22.66 5.17 2.03 8.76 0.44 39.08 310.05 57.48

S2 2341.16 3417.33 97.78 3.99 1.66 6.75 0.34 110.54 1141.21 88.40

CV (%)

P-value

10.85 10.85 20.26 14.47 12.82 14.47 14.47 18.39 8.89 3.44

<0.0001 <0.0001 <0.0001 0.0004 0.0017 0.0004 0.0004 <0.0001 <0.0001 <0.0001

The costs with labor, medication, pasture maintenance, and taxes differed (P<0.10) between the supplementation strategies adopted. When the supplement cost was added to these costs, the total cost per arroba produced differed (P<0.10) between the two strategies, for which strategy S2 was 2.82 times higher. Also in this context, the cost per animal in strategy S2 was 3.68 times higher than that in S1. The participation of the cost with concentrate in the total cost of arrobas produced represented 57.48 % in strategy S1, whereas in S2 this value was 88.40 % . The cost with the purchase of unfinished cattle in reals in both supplementation strategies did not differ (P>0.10) (Table 4). The difference in weight gain (kg/ha) (P>0.10) between the two strategies, as a result of variations in ADG, led to a difference (P<0.10) in gross revenue per hectare, which considers the final sale price of the animals. The total cost per hectare (R$/ha) between the strategies differed (P<0.10), and the cost of strategy S2 was 3.69 times higher than that of strategy S1, demonstrating an advantage of using S1. The net revenue in the period (R$/ha), observed in each supplementation strategy, was superior in S1 (P<0.10), in which the animals were supplemented with a mineral mix in the rainy periods and with concentrate supplement (0.4% BW) in the dry period of the year.

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Table 4: Economic analysis of different supplementation strategies for crossbred steers Variable Unfinished cattle purchase cost, R$ Gross revenue per animal, R$/animal Gross revenue per hectare, R$/ha Total cost per hectare, R$/ha Net revenue in the period, R$/ha Invested capital, R$/ha R$returned per R$invested Monthly rate of return, % Profitability, % Return from investment at 6% per annum, R$/ha

Supplementation strategy S1 S2 792.22 793.98 1172.16 1524.01 3967.30 5158.20 1046.40 3863.13 2920.90 1295.07 3727.79 6550.46 3.83 1.34 18.99 2.29 73.04 23.76 197.02

197.46

CV (%)

P-value

23.33 13.65 13.65 8.88 31.82 12.91 19.01 31.00 19.61

1.0000 0.0002 0.0002 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

23.33

1.0000

Strategy S1 required greater (P<0.10) capital investment as compared with S2. Thus, S1 allowed a higher (P<0.10) return on the capital invested in the activity. The monthly rates of return and profitability were higher (P<0.10) in strategy S1, which showed to be 73 % more profitable than supplementation strategy S2 (Table 4). Considering the application of the invested capital (6 % return) per hectare in each supplementation strategy in an investment fund (savings account; 6 % per annum), no difference was observed (P>0.10) between the two feeding strategies (Table 4). The internal rate of return did not show differences (P>0.10) between the supplementation strategies (Table 5).

Table 5: Monthly internal rate of return and net present value of different supplementation strategies for crossbred steers Variable Internal rate of return, % Net present value, HR5% Net present value, HR10% Net present value, HR15%

Supplementation strategy S1 0.20 2595.44 2472.84 2355.78

S2 â&#x2C6;&#x2019;0.30 930.62 771.23 619.02

CV (%)

P-value

6.83 35.46 37.00 38.71

0.1521 <0.0001 <0.0001 <0.0001

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The net present value, irrespective of the hurdle rate considered, showed differences (P<0.10) between the supplementation strategies: 172.02 %, 202.26 %, and 241.84 % for the rates of 5, 10, and 15 %, respectively, which were higher for strategy S1.

Discussion

Total weight gain per hectare and meat production were higher (P<0.10) in strategy S2, the greater protein-energy supply from the concentrate supplement(6) would explain the better performance found for the group of animals on S2. Working with supplementation for beef cattle kept(7) on a Tanzania grass pasture and subjected to mineral mixture and concentrate supplementation at 0.2, 0.4, and 0.6 % BW and did not observe increase in the weight gains of animals explained by the high herbage allowance adopted in the experiment (average 12.88 t/ha herbage mass), contrasting the results of the present study. The stocking rate observed during the experiment is higher than the Brazilian national average of 0.5 AU/ha(8). Similarly, when working with cows on a Marandu grass pasture(9) employing two supplementation systems (0.5 and 1.0 % BW) and two energy sources (oat grain and broken corn), found an average stocking rate of 1.68 AU/ha, which is also higher than the national average. Evaluating the effect of different supplementation levels on the performance of purebred Nellore â&#x20AC;&#x2022; a mineral mix and supplementation with concentrate at 0.2, 0.4, and 0.6 % BW(7) â&#x20AC;&#x2022; observed increases in concentrate intake, cost per animal, and total cost, respectively, for the treatments. Higher costs were found for strategy S1, explained by the lower number of arrobas produced, as compared with strategy S2. Comparing feeding strategies (mineral supplementation at 0.2 and 0.3 % BW) in the production of crossbred steers on a Brachiaria brizantha cv. Marandu pasture(10) they found that supplementing animals with concentrate supplement (0.3 % BW) during the rainy and dry seasons led to a 3.11 times higher production cost per animal as compared with the group of animals supplemented only with mineral salt in the rainy seasons and with concentrate supplement (0.2 % BW) in the dry period of the year. This result reinforces the idea that it is important to know the percentage of formation of production costs; in this case, of the arrobas produced. Detailing the costs to produce one arroba allows the producer to seek alternatives that minimize them; one of these alternatives is designing supplementation strategies aimed at satisfactory gains throughout the entire production cycle associated with cost reduction.

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In a supplementation program, a large part of the economic return achieved is a consequence of additional weight gains and anticipated emptying of pastures, which makes them available for other groups of animals or facilitates management practices(11). The cost with the purchase of unfinished cattle in reals in both supplementation strategies did not differed as a function of the initial body weight. Evaluating the economic response of four levels of supplementation (mineral salt 0.3%, 0.6%, and 0.9% BW) in the finishing of Nellore steers on a Brachiaria brizantha pasture in Southeast Bahia State(12) they found, similarly to the present study, higher gross revenues per animal and per hectare for the highest supplementation level. This result was possibly due to the increased amount of arrobas produced and also the differences between the moments when the animals were sold, which represent, in practice, different prices as a function of the month of sale of the lots of animals. Reviewing and discussing protein energy supplementation(13) it was reported an increase in invested capital in their experiment in which they compared mineral salt with concentrate supplementation in the amounts of 0.125, 0.25, 0.50, and 1.0 % BW. The highest performance level, which consequently provided the highest daily revenue, was not the most economically viable; in this way, treatments 0.25 and 0.50 % live weight represented the greatest profitability. This result can be explained by the increased daily cost as the supplementation levels were increased, and these data agree with the results of the present study. In a study conducted with Nellore steers finished on Brachiaria brizantha pastures in Southeast Bahia State, Brazil, tested four levels of concentrate supplementation (control, 0.3, 0.6, and 0.9 % BW)(6) they found higher rates of return and profitability for the lowest supplementation levels, because an increase in costs was observed together with the increase in supplementation levels, which is in line with the results found in the present study. This is due to the lower feed cost observed in treatment S1, which led to a lower total cost of this treatment, as described previously. The positive values of NPV show that both strategies were able to cover the initial investment to purchase the animals, generating additional revenue. Thus, in treatment S1, the NPV were always higher than those in treatment S2, proving its higher profitability. In financial analysis(14) they found economic viability in supplements provided at levels lower than 0.3 % live weight. For the supplementation levels of 0.6 % to 0.9 % LW, however, the authors obtained losses as compared with the supply of mineral mix. In any production system, the economic viability determines the direction to be followed by the many segments of the production chain; among them is the use of forage supplementation, in which case positive and negative aspects should be considered when aiming at an improvement in biological performance despite the increased production cost. To seek balance between biological productivity and financial sustainability is the challenge of modern animal science.

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Conclusions and implications

The mineral or protein-energy supplementation strategies generate benefits to beef cattle farming, with positive effects on performance variables. Mineral supplementation strategies in the rainy season associated with protein-energy supplementation in the amount of 0.2 % of the body weight during the dry season provides the best economic results.

Literature cited 1.

Silva F, Sá J, Schio A, Silva R, Ítavo L, Mateus R. Suplementação a pasto: disponibilidade e qualidade x níveis de suplementação x desempenho. Rev Bras Zootec 2009; 8 (1):371-389.

Almeida V, Silva R, Queiroz A, Oliveira A, Silva F, Abreu Filho G, Lisboa M, Souza S. Economic viability of the use of crude glycerin supplements in diets for grazing cross bred calves. Rev Bras Zootec 2014;43(7):382-389.

Detmann E, Souza MA, Valadares Filho SC, Queiroz AC, Berchielli TT, Saliba EOS, et al. Métodos para análise de alimentos – INCT – Ciência Animal. Instituto Nacional de Ciência Tecnologia de Ciência Animal 2012.

Anualpec - Anuário da Pecuária Brasileira. FNP Consultoria & Comércio 2013;20;1360.

Martin N, Serra R, Oliveira M, Ângelo J, Okawa H. Sistema integrado de custos agropecuários - CUSTAGRI - Informações econômica 1998; 28(1):7-28.

Silva R, Prado I, Carvalho G, Silva F, Santana Júnior H, Souza D, Dias D, Pereira M, Marques J, Paixão M. Novilhos nelore suplementados em pastagens: Consumo, desempenho e digestibilidade. Arch Zoot 2010;59(3):549-560.

Cabral L, Zervoudakis J, Coppedê C. Suplementação de bovinos de corte mantidos em pastagem de Panicummaximum cv. Tanzânia-1 no período das águas. Rev Bras S Prod Anim 2008; (2): 293-302.

Ítavo L, Ítavo C, Dias A, Novais M, Silva F, Mateus R, Schio A. Desempenho produtivo e avaliação econômica de novilhos suplementados no período seco em pastagens diferidas, sob duas taxas de lotação. Rev Bras S Prod Anim 2007;(3):229238.

Agulhon R, Jobim C, Branco A, Calixto JM. Fontes energéticas e níveis de suplementação para vacas em pastagem de capim-marandu 608

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(Brachiariabrizantha Hochst ex. A. Rich. Stapf) no inverno. Rev Bras Zootec 2005;34(1):151-158. 10. Souza, S. Estratégias de suplementação para produção de novilhos mestiços recriados e terminados em pastagens [Dissertação]. Itapetinga, Bahia, Brasil. (Universidade Estadual do Sudoeste da Bahia) 2015. 11. Socreppa L, Moraes E, Moraes K, Oliveira A, Drosghic L, Botini L e Stinguel H. Glicerina bruta para bovinos de corte em pastejo no período das águas: viabilidade produtiva e econômica. Rev Bras S Prod Anim 2015;16(1):232-243. 12. Pinheiro A, Silva R, Junior H, Prado I, Costa L, Lacerda J, Breda A. Avaliação Econômica de Diferentes Níveis de Suplementação na Terminação de Novilhos Nelore em Pastagens de Brachiaria brizantha. Rev Sci Prod Anim 2010;(21):110113. 13. Andrade M, Resende R. Suplementação proteico energética de bovinos de corte sob pastejo no período das águas aspectos econômicos. FAZU em Revista 2013; 72-78. 14. Porto M, Paulino M, Valadares FS, Sales M, Leão M, Couto V. Fontes suplementares de proteína para novilhos mestiços em recria em pastagens de capim-braquiária no período das águas: desempenho produtivo e econômico. Rev Bras Zootec 2009; 38 (8): 553-1560.

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https://doi.org/10.22319/rmcp.v10i3.4609 Article

Key factors influencing the sale of bulls in livestock auctions

Giovana Tagliari Evangelista a Jusecléia Ferreira Lopes a* Giordano Bruno Fornar a Ricardo Pedroso Oaigen b Thaís Lopes Gonçalves b Tamara Esteves de Oliveira a Luís Kluwe de Aguiar c Júlio Otávio Jardim Barcellos a

Universidade Federal do Rio Grande do Sul, Departamento de Zootecnia. Rio Grande do Sul. Brasil. b

Universidade Federal do Pampa. Rio Grande do Sul. Brasil.

Harper Adams University, Department of Food Science. Newport, United Kingdom.

* Corresponding author: jussiferreiralopes@gmail.com

Abstract: This research determines which factors most influence the purchase price of bulls in livestock auctions in the State of Rio Grande do Sul, Southern Brazil. Hence, 760 beef bulls sold in eleven different auctions between August and November 2013 were analysed. The data consists of: breed, muscularity (MUSC), frame (FRAME), body condition score (BCS), scrotal circumference (SC) and body weight (BW). Other data such as the animal entry order and the purchase price of the bulls was collected during the auction. A linear generalized model was used to evaluate the interaction of each variable with the purchase price of the bulls. An ANOVA with Tukey post-hoc was used to compare the differences between the categories that influenced the purchase price of bulls and were realized in the software SPSS 20.0. All breeds presented declining prices 610

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from the first to the second entry order and increasing purchase prices from the order third to forth. Bulls with large frame received higher purchase prices independent of the auction order, except for the second order of entry, in which medium and small animals were more valued. Angus bulls obtained the highest prices in relation to the breeds Brangus and Hereford. The frame and breed constituted the main phenotypic characteristics that influence in price. In addition, the order of entry of bulls in the ring influence the purchase price. Key words: Breed, Bulls, Livestock Auction, Marketing, Price.

Received: 08/09/2017 Accepted: 13/07/2018

Introduction

Brazilian cow-calf systems typically consist of bulls, breeding cows, heifers and calves. In these systems, the number of bulls account for about 3 to 5 % of the animals (1), and approximately 25 % are replaced annually, this constituting a significant component in the production cost(2). This bull for use, it is usually provided by producers of pure breeds, which should know the value of genetic and phenotypic characteristics that affect the selling price(3). Furthermore, as profit margins in cow-calf systems tend to be narrow, to increase profitability farmers must aim at breeding animals which return higher productivity whilst meeting market expectations(4,5). Nevertheless, several variables affect bulls selling price, such as the breed, muscularity, frame, age and scrotal circumference(3,6), but not all livestock buyers are looking for the same characteristics. Often, those features that would best meet the buyerâ&#x20AC;&#x2122;s requirements are overlooked in detriment of others such as animal conformation(7,8), and should be analysed and understand to develop rational marketing strategies. Moreover, since Brazilian bulls are usually traded at auctions, investigating the extent that each factor might influence and bulls selling prices could increase the competitiveness of the sector. However, price formation is complex and influenced by endogenous and exogenous factors such as supply and demand forces, stocks carried over, farmersâ&#x20AC;&#x2122; decision-making and the buyersâ&#x20AC;&#x2122; behaviour during purchase. Therefore, since qualitative and quantitative characteristics influence the sale of the animals(9), this

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research analyses livestock auction arrangements and the most relevant animal features perceived by bull buyers, which affect livestock auction prices.

Material and methods

Data from the sale of 760 bulls was collected from eleven different livestock auctions held in six towns (Alegrete, 1; Dom Pedrito, 3; Esteio, 1; Santa Vitória do Palmar, 1; Santana do Livramento, 3, and Uruguaiana, 2) in the state of Rio Grande do Sul, Brazil, from August to November 2013. The livestock auctions were held annually as part of the traditional bulls’ sale calendar. The auctions were conducted by different auctioneering companies and were typically located at agricultural fairs. However, in two cases the auctions took place at farms (Uruguaiana, 1; Dom Pedrito,1). All bulls were certified by the respective breeder’s associations and were offered between 2 to 3 ys of age. Following established literature on the matter, a standard form was devised to collect relevant information on the animals’ characteristics which could influence buyers’ decision-making. The attributes collected consisted of the breed, muscularity score (MUSC), frame score (FRAME) and body condition score (BCS) of the bulls. The breeds that were analysed in the auction were: Angus, Brangus, Hereford and Braford. The Angus breed originated in Scotland and development in Brazil occurred in different regions, especially the cattle herds in southern Brazil. It is a breed of moderate size, naturally polled and can be black or red in colour(10). The Brangus is formed by Zebu with Angus, and thus has the predominant characteristics in Angus, such as carcass quality, pigmentation, fertility and precocity; with those of Zebu, which are adaptability and rusticity(11). The Hereford breed originates from the County of Hereford in England. The breed is coloured dark red to red-yellow, with a white face, crest, dewlap, and underline. The animals can be polled or with short thick horns that typically curve down at the sides of the head(12). The origin of the Braford breed was by a cross between a Hereford x Brahman or Hereford x Nelore breeds. Currently, the Braford breed has fertility, maternal ability, precocity and meat quality of Hereford with the ability to adapt to the tropics, rusticity and carcass yield of zebu(12). As for FRAME, scores from 1 to 3 were given according to the height measured from animals’ hip as proposed and adapted from the works of the Beef Improvement Federation(13). FRAME 1 represented heights from 104 to 114 cm, typically of smaller biotypes; FRAME 2 varied from 119 to 129 cm, representing biotypes of average height; and FRAME 3 were typical of animals of large biotype.

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Regarding the MUSC, the bulls were classified according the scores 1 – 3, where MUSC 1 represent those of concave muscular profile, narrow width between the hind legs, prominent hip bone and tapered thigh; MUSC 2 was typical of animals of average muscularity, muscular profile less convex, hip bones slightly prominent; and MUSC 3 were those animals of better muscularity, convex muscular profile, large width between the hind legs, well rounded top line and thicker thigh. As for the BCS of 1 to 5 were given based and adapted(14). Those showing a BCS 1 were typically of low muscularity, very lean, and whose ribs were visible; BCS 2 represented lean bulls with low rib fat cover which also presented protruding hip bones; bulls with BCS 3 score had moderate muscular cover whose ribs were practically covered; BCS 4 represented bulls of good muscular cover and which had some fat cover; and BCS 5 were typical of bulls with excess fat cover at the tail fold and ribs. Information available from sales catalogues published by the livestock auctioneers regarding general sale’s conditions (payment terms which could attract a discount for payments paid at sight or in instalments varying from up to 15 or 20 mo); body weight (BW) and scrotal circumference (SC) were used. The bulls were sold following a typical English style auction where the bids were made until the price reached its maximum. To analyse the effect of entry order (ORDER) on the selling price at each auction, the animals were grouped into four stages consisting of 1st quarter (batch 1 to 9), 2nd quarter (10 to 17), 3rd quarter (18 to 29) and 4th quarter (remaining batches). The ORDER was registered against the animals’ initial and selling price and the likely sale terms. For the evaluation of the equivalent fat cattle (EFC), the price of one bull sold in the auction was divided by the price of bullocks’ in the same period (equivalent price obtained in 2013)(15), and with that was determined the number of bullocks’ (EFC= 450 kg) that is equivalent to the price of one bull. Using SPSS software version 20.0, statistical analysis of the data, including frequency, mean, median, maximum and minimum values, standard deviation of price. A linear generalized model was used to evaluate the interaction of each variable with the purchase price of the bulls, and the best model is presented by the Equation 1: P= β+φ+ω+ψ+ϕ+ β*ψ+ φ*ψ+ω*ψ+ϕ*ψ+e in which: P= purchase price; β= breed; φ= frame; ω= muscularity; ψ= entry order; ϕ= body condition score; e= experimental error.

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An ANOVA with Tukey post-hoc was used to compare the differences between the categories that influenced the purchase price of bulls. All analysis considered a 95% level of significance and were realized in the SPSS 20.0.

Results

The average bullsâ&#x20AC;&#x2122; market liquidity in the auctions was approximately 90 %, and the purchase price presented a great price range. The purchase price ranged between $1,645.00 and $14,473.00 with an average price of 4,092.00 dollars (Figure 1). Usually, sellers and buyers referred to the equivalent fat cattle parameter, in this research, the EFC ranged from 2.5 to 6.4 bullocks.

Figure 1: Frequency of bulls sold in auctions in Rio Grande do Sul according to price intervals 20.0 18.0

Percentage (%)

16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0 0.0

Price ($, in dollars)

There was no association between weight and price; in addition, scrotal circumference did not this related to the price. As for the characteristics evaluated subjectively, such as muscularity and body condition score were also not associated with the final price of the bulls at auctions. In this research, no bull presented a BCS below three (average 3.88) and 614

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bulls with light muscularity were to be Angus and Brangus with 63.2 and 31.6 % light muscularity respectively. Only 2.7 % of the animals sampled exhibited a light muscularity whilst 85.5 and 11.6 % of the sample presented a moderate and strong muscularity respectively. Moreover, all the lighter animals were sold during one auction location only, thus contributing to the outliner effect as the selling prices of such an auction might have been higher than in other auction locations. However, other variables also influenced the bulls market(6) as it was seen, in the best model representing the factors influencing the purchase price of bulls in auctions. It was found that the price in 2013 was influenced by the breed and frames of bulls, as well as the entry order of the animals in the auction. The purchase price was also influenced by the interactions of breed and entry order and the interaction of frame and entry order. The price payed for which breed of bull differ between the entry orders. All breeds presented declining prices from the first to the second entry order and increasing purchase prices from the order third to forth. However, from the second to third order Angus and Brangus increased while Hereford and Braford decreased in the purchase price (Figure 2).

Figure 2: Effect of the interactions between breed and entry order over the purchase price of bulls sold in auctions in the state of Rio Grande do Sul, Southern Brazil

Large bulls received higher purchase prices in all orders, except in for the second order of entry, in which medium and small animals were more valued (Figure 3). The purchase price in the fourth entry order was higher than in all the others, and there was also a difference between the first and third entry orders, in which the latter presented the lowest

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prices (Figure 4). The purchase price of smaller bulls was lower than that of mediumsized animals, and the price of the latter did not differ from that of larger bulls (Figure 5).

Figure 3: Effect of the interaction between frame and entry order over the purchase price of bulls sold in auctions in the state of Rio Grande do Sul, Southern Brazil

Figure 4: Effect of the entry order the purchase price of bulls sold in auctions in the state of Rio Grande do Sul, Southern Brazil

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Figure 5: Effect of the frame over the purchase price of bulls sold in auctions in the state of Rio Grande do Sul, Southern Brazil

In the present study it was verified that Angus bulls obtained the highest prices in relation to the breeds Brangus and Hereford, although the supply of Angus animals was only 15 % (Figure 6). However, this may reflect the number of auctions, since one of these is on the market for around 60 yr. Braford and Hereford bulls were marketed in 9 and 10 auctions, respectively. This must have contributed to the fact that they received the lowest prices.

Figure 6. Effect of the breed over the purchase price of bulls sold in auctions in the state of Rio Grande do Sul, Southern Brazil

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Discussion

The high liquidity in the auctions could be attributed to the high demand for bulls in the year of the study (2013). That could also reflect the consolidation in auction houses, which have been marketing for at least 10 yr. Moreover, the reputation of bull breeders historically can exercise some influence in the buyer decisions(3,6,16). In addition, the state of Rio Grande do Sul is a major supplier of bulls of European and synthetic breeds to the other states of Brazil. Moreover, in general, the higher the price, the lower the frequency of animals sold, because bulls with higher phenotypic characteristics tend to require greater care regarding maintenance, increasing their future cost in the farm. This is more concerning since most bulls are sold to typically commercial livestock farmers(17). The equivalent fat cattle (EFC) found in this research indicated that farmers engaged in the breeding bulls attended the needs of different cattle farmers. However, the large majority of bulls sold aimed to meet the demand of commercial herds, as only 10 % of the bulls sold at equivalent fat cattle (EFC) 6.4 or higher. This asymmetric price variation is an indicative of product differentiation, as bull buyers tended to favour animals whose phenotypical characteristics are not directly proportional to the observable parameters(18). Despite the lack of association between weight and price in this research, this has been found in previous researches(19,20,21), possibly because weight influences bullsâ&#x20AC;&#x2122; appearance and could infer a likely weight gain potential, a desirable feature for breeders(17). However, that cannot be assessed only by visual observation, and too much an emphasis on weight at the sale can be detrimental to younger bulls which are lighter, but might be genetically superior. Furthermore, overfeeding also could produce heavier animals that can result in mounting difficulty and low semen quality(22). Therefore, the potential genetic contribution that a heavier bull could have in a herd may be impaired if these animals are not healthy enough by being unable to search for females in heat and reproduce. Since fat bulls with excess fat deposited at the base of the tail need to lose a lot of weight to be used to work in the field. Accordingly, the scrotal circumference, which was not related to price, is another important phenotypical characteristic usually publicised by catalogues, and one of the most important predictor of fertility and precocity in bulls(19,23). However, should SC be obtained during the sales, other environmental effects could be masking the true bullâ&#x20AC;&#x2122;s potential, as overfeeding. Despite being a favourable characteristic in beef herds, muscularity was not related to higher prices, which could be explained by the low representativeness of the sample, since only 2.7 % exhibited light muscularity, whilst 85.5 % and 11.6 % presented moderate and strong muscularity, respectively. Moreover, all the lighter animals were sold during one

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auction, contributing to the outliner effect as the purchase prices of such an auction. Nevertheless, muscularity is an important phenotypic trait as it influences buyer’s evaluation at the purchase, since it is associated to future meat production. Hence, the selection for muscularity should be optimized, not maximized to avoid birth difficulties(24). Body condition score indicates an adequate nutritional and it is an important visual feature for buyers. Hence, since the high nutritional level may exacerbate bulls’ physical qualities, it was expected that buyers favoured bulls with larger BCS, but this feature did not influence the purchase price. The purchase price was higher in the first and last (4th) entries, and the latter presented the highest prices. The reduction in purchase price as the entry order progresses gets to the middle of the auction, second and third quarters, could be attributed to a decreased interest by the buyers, as they could have their needs fulfilled at the beginning of the auction. Nevertheless, the reduction in values according to the entry order can be also explained by a decrease of the relative quality of bulls. Moreover, as the auction progresses the weight of the animals entering the ring diminishes, also reflecting in a lower purchase price. Nevertheless, the highest prices were seen in the last quarter of the auction, mainly for Angus bulls, which also presented higher prices at the end of the auction, as well as Braford and Hereford bulls. The lack of Brangus bulls at the final quarter and of Angus at the third quarter could be a major influence in this analysis. Moreover, small animals received lower purchase prices except in the second quarter, probably because buyers believed that larger bulls could be more efficient in breeding, presenting a lower risk. However, in some breeds, such as Angus, Charolaise, Simmental and Polled Hereford, the frame of bulls increased the purchase price, especially in the USA(25,26), as seen in the calf market(27). Therefore, the removal of extremes (small and large animals) can be helpful to standardize the herd. For breeds, Angus bulls attracted higher prices than Hereford and Brangus bulls, which was expected as it was the results of previews studies in the USA(3,20). Despite the supply of Angus bulls of only 15 %, that did not negatively affect the purchase price. In addition, the small share of Angus sales derived from two auction locations, but one auction event is very traditional and has been taking place in the same site for over 60 yr. The tradition and reputation of the auctions’ events can exercise a positive effect on the price. Moreover, Angus and Brangus were sold only in two traditional events (Uruguaiana and Dom Pedrito). In addition, the different prices between breeds, depended on factors involving buyers’ personal preference, soil and climate conditions, market trends and supply/demand relationships. Despite most Brazilian cattle is of zebu herd, the Southern latitude present overall different environmental characteristics with lower average temperatures and natural pastures (pampa grassland). Hence, the widespread use of European breeds

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(Angus and Hereford) and commercial synthetic breeds (Braford and Brangus) is evidence of how well adapted the cattle is to those conditions. The value of a breed per se reflects the commercial circumstances and the characteristics of the auctions (e.g. time to market). Besides the characteristics of the bulls, there are other factors related to the auction system that influence the formation of bull’s purchase price. However, for more efficient results in the production, actual production weights (birth, weaning, and yearling weight), and production expected progeny differences (EPD’s) (birth, weaning, and yearling) must be considered.

Conclusions and implications

The bulls’ purchase price in livestock auction cannot be determine by only one variable, but the frame and breed constitute the main phenotypic characteristics that influence in price. In addition, the order of entry of bulls in the ring influence the purchase price. These results may be useful to both sellers and buyers of bulls, who need to plan for their purchases and investment. A better understanding of the variables in a bull that would most affect productivity would be a useful instrument to for the purpose of efficient allocation of resources, ensuring liquidity for stocks and possibly a better margin in marketing. In addition, the supply of bulls could be planned according to the expected demand and product carcase specifications.

Literature cited: 1.

Menegassi SRO, Barcellos JOJ, Lampert VDN, Borges JBS, Peripolli V. Bioeconomic impact of bull breeding soundness examination in cow-calf systems. Rev Bras Zootec 2011;40(2):441-447.

Barcellos JOJ. Aspectos práticos e mercadológicos que devem pautar a decisão na comercialização de um touro. In: Barcellos JOJ, et al. editors. Bovinocultura de corte: cadeia produtiva & sistemas de produção. Rio Grande do Sul, Brasil: Agrolivros; 2011:65-69.

Dhuyvetter KC, Schroeder TC, Simms DD, Bolze Jr RP, Geske J. Determinants of purebred beef bull price differentials. J Agr Resource Econ 1996;21(2):396-410.

Barcellos JOJ, Oiagen RP. Cadeia produtiva da carne bovina e os sistemas de produção na bovinocultura de corte. In: Oiagen RP, et al. editors. Gestão na bovinocultura de corte. Rio Grande do Sul, Brasil: Agrolivros; 2014:21-41. 620

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Thomas M, Hersom M. Considerations for selecting a bull. AN218, one of a series of the Animal Sciences Department, UF/IFAS, 2009. https://edis.ifas.ufl.edu/an218. Accessed Aug 5, 2017.

Jones R, Turner T, Dhuyvetter KC, Marsh TL. Estimating the economic value of specific characteristics associated with Angus bulls sold at auction. J Agric Appl Econ 2008;40(1):315-333.

Estigarribia AFL, Ortiz CDP. Producer’s profile upgrade, technological level and criteria for choosing bulls in Presidente Hayes, Paraguai. FAZU 2011;2(8):172-176.

Simms DD, Geske JM, Bolze RP. Commercial cattle producers: bull selection criteria. Agricultural Experiment Station, USA: Kansas State University, 1994;704:56-59.

Christofari LF, Barcellos JOJ, Costa ECD, Oaigen RP, Braccini-Neto J, Grecellé RA. Tendency in the commercialization of calves in Rio Grande do Sul related to your genetic characteristics. Rev Bras Zootec 2008;37(1):171-176.

10. Associação Brasileira de Angus (ABA). Manual do Criador. 2013. http://angus.org.br/wp-content/uploads/2018/04/Manual-do-Criador_WEB.pdf Accessed May 12, 2017. 11. Associação Brasileira de Criadores (ABC). 2018 http://www.newsprime.com.br/abccriadores/Racas.aspx Accessed May 10, 2017. 12. Associação Brasileira de Hereford e Braford (ABHB), http://www.abhb.com.br/Braford/Braford/ Accessed May 12, 2017.

2015.

13. Beef Improvement Federation (BIF). Guidelines for uniform beef improvement programs. 5th ed. Beef Improvement Federation, USA: North Carolina State University. 1986;98. 14. Lowman BG, Scott N, Somerville S. Condition scoring beef cattle. Edingburgh: East of Scotland College of Agriculture; 1976. 15. Agrolink Quotations: cattle. 2013. http://www.agrolink.com.br/cotacoes/historico/rs/boi-gordo-kg-vivo-1kg. Accessed Aug 22, 2016. 16. Commer M, Couvilllon WC, Herndon CW, Brown CJ, Getz WR. The effects of promotion in price determination of beef bulls. Prof Anim Sci 1990;6(1):5-10. 17. Dhuyvetter KC, Turner TK, Marston T, Jones R. Factors influencing the selling prices of purebred Angus bulls. Agr Exp Sta Coop Ext Serv, USA: Kansas State University; 2004;16. 18. Bekkerman A, Brester GW, McDonald TJ. A semiparametric approach to analyzing differentiated agricultural products. J Agr Appl Econ 2013;45(1):79-94. 621

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19. Walburger AM. Estimating the implicit prices of beef cattle attributes: a case from Alberta. Can J Agr Econ 2002;50(2):135–149. 20. Irsik M, House A, Shuffitt M, Shearer J. Factors affecting the sale price of bulls consigned to a graded sale. Bovine Practitioner 2008;42(1):10-16. 21. Marks ML, Parish JA, Smith T, Vann RC, Riley JM. Historical price relationships to performance characteristics and genetic merit of bulls sold in Mississippi Beef Cattle Improvement Association and Hinds Community College Bull Test Sales. Anim and Dairy Sci Ann Rep. Mississippi State University, USA. 2012. 22. Menegassi SRO, Barcellos JOJ, Fornari GB, Canellas LC, Oliveira TE, Soares JCR. Manual de boas práticas para o manejo de touros. 4th ed., Rio Grande do Sul, Brasil. 2015. 23. Menegassi SRO, Pereira GR, Lopes FG, Rocha MK. Exame andrológico. In: Menegassi SRO, Barcellos JOJ editors. Aspectos reprodutivos do touro: teoria e prática. Guaíba, Brasil:Agrolivros; 2015:45-103. 24. Barham B. Bull selection and management guide. Division of Agriculture, USA: University of Arkansas, 2011;39. 25. Atkinson R, Sanders DR, Jones K, Altman IJ. An evaluation of purebred bull pricing: Implications for beef herd management. J Am Soc Farm Man Rural Appr 2010;73:235–243. 26. Mc Hugh N, Fahey AG, Evans RD, Berry DP. Factors associated with selling price of cattle at livestock marts. Animal 2010;4(8):1378–1389. 27. Barham BL, Troxel TR. Factors affecting the selling price of feeder cattle sold at Arkansas Livestock auctions in 2005. J Anim Sci 2007;85:3434–3441.

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https://doi.org/10.22319/rmcp.v10i3.4806 Article

Vertical and spatial price transmission in the Mexican and international milk market José Luis Jaramillo-Villanuevaa * Adriana Palacios-Orozco b

Colegio de Postgraduados, Campus Puebla. Boulevard Forjadores, Núm. 205, Santiago

Momoxpan, 72760 San Pedro Cholula, Puebla. México. b

ISSSTE- Puebla, México.

*Corresponding author: jaramillo@colpos.mx

Abstract: During the last two decades, the Mexican dairy sector experienced important structural changes, especially after the implementation of the NAFTA agreement. In 2016, the Bank of Mexico observed that in milk market, the final prices tend to rise when input prices increase, however; they do not decrease when input prices decrease. In this context, this study examines the degree of spatial and vertical price transmission between farm milk prices and international milk prices as well as between farm milk prices and retail milk prices, in order to assess the efﬁciency level of the Mexican and international dairy market. The ﬁndings of this research provide contributions to decision makers and industry stake-holders: a unidirectional transmission of international milk prices to domestic milk prices and from farm price to retail price along with the existence of asymmetric price transmission which depends on whether milk prices are increasing or decreasing. The results have shown that a long-run single co-integration relationship exists between international and farmer’s prices and between retail and farm price; that the direction of price transmission tends to go from producers to retailers and from international to farmer price and that when international price increase the speed of adjustment tend to be significantly slower, and that when international 623

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price decrease, the speed of adjustment tend to be significantly faster. Key words: Asymmetric price transmission, Milk prices, Vector error correction model. Received: 13/03/2018 Accepted: 14/08/2018

Introduction In the last two decades, the dairy sub-sector in Mexico had undergone a signiﬁcant change. The dairy industry experienced domestic price liberalization; the distribution of milk production among 32 states in México, measured by the Gini index, shows an increase in concentration, from a value of 0.55 in 1990 to 0.63 in 2016. In 1990, six states concentrated 58.71 % of total milk production; in 2008, they contributed with 61.7 %, and in 2016, with 63.5 % (own estimation using data from SIAP-SAGARPA(1). The Bank of Mexico(2) observed that in the Mexican milk market, price to consumers tend to rise when input prices increase; however, they do not decrease when input prices decrease. The concern about the competitiveness of the Mexican dairy market involve several issues; (i) there is a high degree of concentration in the processing stage of milk (a few processing firms) which contrasts with the low concentration in the dairy farmers sector (A large number of farms); (ii) dairy farmers have expressed concerns about the competitiveness of the dairy supply chain, due to the entrance to Mexico of imported milk, at prices below US consumer paid for and even below international prices. The importance of the analysis of price transmission rests on the role of prices as instruments, by which, different levels of the supply chain are linked. Thus, ensuring adequate price signals at the farm gate is fundamental to agricultural productivity(3). A better understanding of the extent to which retailers’ and wholesalers’ prices are efﬁciently transmitted down to producers at the farm gate level is an important issue for the design of policy, that seek not only to reduce the possible causes of market failure to improve competitiveness, but also to increase farm net income. In economic terms, the Mexican agricultural sector accounts for 3.1 % of the total national GDP and contributes with 14.4 % of the employment in the agricultural sector. Cattle production is one of the most important activities of the agricultural sector in Mexico. It 624

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accounts for 28.18 % of the total agricultural GDP and 30 % of employment in the agricultural sector. The cattle inventory in Mexico has grown at an average rate of 2.04 in the last 20 yr, while milk production has an average growth rate of 2.56 % in the same period (own estimation with data from SIAP-SAGARPA(1). Over the last decade, México observed a 6 % increase in the size of its herd, passing from 30.3 million heads in 1996 to 32.2 million in 2016. However, the increase in milk herd was remarkable because it raised 52 % from 1.67 to 2.58 million heads (own estimation with data from SIAP-SAGARPA(1). The number of cattle farms in Mexico fell from 1,129,217 in 2007 to 499,250 in 2016(4,5) with an inventory of 32.2 million heads. There are three main cattle production systems in México: one specialized in milk, a second specialized in beef, and a third that consists of a dual-purpose system producing both milk and beef. The largest part of the cattle production system in Mexico is concentrated in the north of the country and along the Gulf of Mexico. Milk production is an economic activity of social and economic importance in Mexico. This is evidenced by the financial, natural and human resources involved in the productionconsumption supply chain of fluid milk and dairy products, as well as by the income and employment generated by this activity; in Mexico, there are 197 million hectares, of which, livestock in its different modalities occupies 58 %(6); the national population of dairy cattle amounted to 2.5 million heads, producing a total of 11.8 million liters of fluid milk in 2016(1); in value, the milk industry amounts to 106 billion dollars; primary milk production contributed with 46.4 % to milk industry, preparation of milk powder with 22 % and production of dairy products with 31.6 %(7). Six large firms dominate Mexican dairy market (Production, distribution, and processing). These companies, in 2016, traded 60 % of total milk in the country; Liconsa, a state-owned enterprise, contributes with 10.3 %, Grupo LaLa with 21.4 %, Alpura with 10.2 %, Nestle with 7.70 %, Grupo Sigma alimentos with 6.20 % and Grupo Lactalis with 4.10 %(8). Historically, México has been a net importer of milk, however, since 1992, the production deficit began to grow significantly. This fact is explained mainly by the effects of pricing policies on production, which until 1997, were not linked to production costs, because it discouraged investments in technology and genetic material to improve productivity(9). With the adhesion of Mexico to NAFTA, Mexico's dairy industry entered into competition, in prices and quality, with milk industries of United States and Canada. The Annual Average Growth Rate (AAGR) of the national milk production for the period of 1990-2016 was 2.5 % while AAGR of consumption was 2.8 %. The gap between national production and consumption is expected to become wider, and, as a consequence, US milk would play a major role within the Mexican milk market.

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Several authors agree that commercial liberalization of the dairy sector in 1993, the end of the domestic protected market policy and change to a market defined by demand-supply balance, determined a negative impact on the commercial viability of small to medium dairy farms and also affected negatively production of milk, mainly among small to medium farms(4,10). An explanation of the fall of milk production in Mexico is that domestic milk prices were determined by the international price and for internal asymmetries in the Mexican industry, which mean an unbalanced development among types of milk cattle farms, and also an unequitable governmental support among dairy milk farmers. Domestic and international milk prices behavior, in 1995, a year after Mexico entered NAFTA, the producer price follows the international price, and to a lesser extent, the consumer price. The consumer-producer price relationship (Pc/Pp) showed a growing trend, which could imply an asymmetric transmission of prices between different levels of the market. Transmission of market shocks, through stages of the supply chain or through horizontally related markets, is a topic with long tradition in economics. Vertical price transmission analysis can be used to assess how efficiently different actors are integrated in a market. The extent and speed with which price changes are transmitted from one level to the other in the market have important policy implications; for welfare distribution, competitiveness, and sustainability. In a competitive market, price shocks at one level of the market chain should be reflected by similar changes at the other levels, as market efficiency suggests a price equilibrium relationship between them(11). Over the past two decades, extensive studies have been developed to examine market linkages among farm, wholesale, and retail markets(12-15). The main focus of these studies is oriented to assess the nature, extent of adjustment, and speed with which shocks transmit along the different market levels. In these studies, the rate of price response is generally measured through the lag relationship between upstream and downstream price, while the asymmetry of price response is measured as the relative response of downstream prices as upstream prices rise or fall(15). The factors that constrain the complete and symmetric transmission of agricultural commodity prices from one market level to another are classified into: 1) Market power concentration at levels beyond the farm gate; 2) Different adjustment costs when firms change the quantities and/or price of inputs and/or outputs; 3) Government intervention in the pricing of agricultural products; 4) Imperfect information; 5) Different price elasticity at different levels of the market chain; 6) The presence of rapidly perishable goods(12,14,16).

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Spatial price transmission refers to the process based through which markets for a homogeneous commodity, at spatially separated locations, share long-run information(17). Spatial price transmission has been widely analyzed in the context of the â&#x20AC;&#x153;Law of One Price,â&#x20AC;? which hypothesizes that if two markets are linked by trade and they are efficient, the price differential between them is equal to transaction costs(17). Prices are consequently thought of as being connected by a stable long-run equilibrium, with attraction forces of this equilibrium, which result in the correction of temporal deviations that occur due to supply or demand shocks. Therefore, a proportional increase in the international price of an agricultural commodity will lead to an equally proportional increase of its price in domestic markets, at all points in time, assuming markets are integrated(18). In this context, Price transmission analysis measures the extent and speed to which price shocks are transmitted between spatially separated locations(19).

On the other hand, price asymmetry refers to the process in which transmission differs according to whether the prices are increasing or decreasing(16). The literature on spatial price transmission dealt with various factors that constrain the transmission of prices from one market to another. It identifies three groups: transaction costs, trade policies, and market power(20).

The objective was to estimate the degree of price transmission between Mexican milk retail price and farm milk price (vertical transmission) and between Mexican farm milk price and the international one (spatial price transmission) to shed light on the possible asymmetric price transmission and the related consequences for market inefficiency.

Material and methods An econometric analysis was carried out using monthly time series of milk prices from 1990:01 to 2016:12. The Mexican data was downloaded from the website of official statistics from the Agro-Food and Fisheries Information Service(1) of the Ministry of Agriculture, Livestock, Fisheries and Food Service (SAGARPA), The Bank of Mexico (BM), and LACTODATA. The international milk Price was obtained from USDA-AMS(21). Milk prices are monthly spot price. The data was transformed into natural logarithms because the coefficients (Î˛s) of the econometric model are understood as transmission elasticities.

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Verification of the integration order of each series, using the Augmented Dickey-Fuller and Phillips-Perron (PP) tests were performed(22,23). It was followed by estimation of long-run relationship, using the Engle-Granger two-step cointegration and the Johansen tests(24). Finally, Asymmetric Vector Error Correction Model (AVECM) was performed; a test to select the lag order for a AVECM and a F-test on the coefficient of ECT+ and ECT- (positive and negative changes in the error term respectively) to test the null hypothesis of symmetry:

H o :  2   2 .

Test for cointegration; long-run relationship

The cointegration between variables -once the unit root existence has been proved- is a necessary condition for the existence of a long-term equilibrium relationship in the series. A variable vector with unit root is cointegrated if a linear combination of these variables is stationary(25).

To test for long-run relationship, both, the Engle-Granger two-step cointegration test(25) and the Johansen test(24) were used. The first approach consists of estimating the cointegration regression, equation (1), by OLS, obtaining the residual ût and applying a unit root test for ût. Again, ADF and PP test were used. Since the coefficient of Ut-1 is less than unity, a cointegration relationship exists. out is t

Where, p

ptout = a + b1 ptin + mt

(1)

in t

a firm output price in period t, p is the input price in t.

The Johansen test derived the distribution of two test statistics for the null of no cointegration; the Trace and Eigen value tests(24). Once cointegration between prices was verified, a twostep Error Correction Model (ECM) was applied to capture the short- and long-term effects of ptin on ptout , and the speed of adjustment at which ptout returns to equilibrium after a in

change in pt . Two econometric models were estimated; Spatial asymmetric model and Vertical Asymmetric model.

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Spatial Asymmetric Price Transmission

Taking into account that farm and international prices have a unit root and were cointegrated, an Asymmetric Vector Error Correction Model is estimated (AVECM) in order to investigate possible interdependence of prices. Following the approach of Cramon-Taubadel and Loy(26), the ECM for spatial price transmission, takes the following form:

int Dptfarm = a + b1Dptint + b2 ECTt-1 + b3 (L)Dpt-1farm + b4 (L)Dpt-1

(2)

Cramon-Taubadel and Fahlbusch also segment the contemporaneous response term(27). This leads to Equation (3), in which contemporaneous and short run response to departures from the cointegrating relation 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

(3)

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

Vertical Asymmetric Price Transmission

Economic model to analyze vertical price transmission use variations of a model introduced by Wolffram in 1971(28). This model was criticized for being unreliable, since most of the evidence presented to support the assumption that commodity prices were cointegrated was affected by spurious regressions or non-stationary series (29). In order to deal with these econometric shortcomings, Engle and Granger proposed an alternative approach based on cointegration theory, which indicates that two non-stationary time series could be long-term cointegrated if both series are integrated of the same order(25). An initial attempt to use cointegration techniques in testing for asymmetric price transmission was applied by Cramon-Taubadel(30). He used the two-step method approach, based on Engel and Granger, to test for Asymmetric Price Transmission (APT) in the presence of non-stationary series, using an Asymmetric Error Correction Model (AECM). 629

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In this approach, the authors proposed splitting the error correction term into positive and negative components in order to identify if prices are transmitted differently depending on whether they increase or decrease. Following the approach proposed by CramonTaubadel(30) to test for vertical asymmetric price transmission, we estimated equation (4).

DPt ret = b0 + b1DPt farm + b2 ECTt-1 + B3 (L)DPt-1ret + B4 (L)Pt-1farm + e t

(4)

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

polinomial lags. Furthermore, splitting the ECT into positive and negative components (i.e. positive and negative deviations from the long-term equilibrium â&#x20AC;&#x201C; ECT+ and ECT-) allows one to identify if the speed at which prices are transmitted differs depending on whether prices are increasing or decreasing. Furthermore, it makes possible to test for Asymmetric Price Transmission (APT)(31). Then, we estimated equation (5):

DPt ret = b0 + b1DPt farm + b2+ ECTt-1+ + b2- ECTt-1- + B3 (L)DPt-1ret + B4 (L)Pt-1farm + e t

(5)

To test for asymmetry, an F-test was used to test the null hypothesis of symmetry; if b2+ Âš b 2, asymmetric price response exist.

Results and discussion

According to the results of the ADF and PP unit root tests, they cannot reject the null of nonstationarity of price series; T-statistic values do not allow to reject the null hypothesis of a unit root with a 95% confidence interval (Table 1). This result upheld the use of the cointegration technique to calculate the relationship between the international and domestic Mexican milk prices. The above result is in line with previous studies on non-stationarity of milk prices(32).

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Table 1: Results of the ADF and PP test on domestic and international milk price series Price series

ADF test

5% critical value

PP test

5% critical value

International price

-1.864

-3.427

-13.992

-21.358

Retail Price

-1.632

-3.427

-11.84

-21.358

Farm Price

-3.149

-3.427

-18.69

-21.358

Cointegration of spatial model

The estimation of equation (1), show a R2 of 0.59, a t-statistic of 21.84 and a F statistic of 476.94, which indicated a long run cointegration. The ADF test on the error term shows a test statistic of -2.575 vs a 5% critical values of -2.877, which indicates failure of rejection of the null of non-stationarity, then, the following regression was performed:

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

(6)

A negative coefficient of the error term (between -2 and zero) confirmed a long run relationship between milk farm price and international milk price (Table 2). The results of the Johansenâ&#x20AC;&#x2122;s test (Table 3) indicated a strong evidence to reject the null hypothesis of noncointegration between p r i c e s , suggesting the existence of a long run single cointegration relationship. Previous studies on milk prices reported cointegration between domestic farm price and imported milk price (32). Results suggest that price in the international milk market is highly influenced by their own historical innovations, while international milk price has a consistently strong impact on price movements in Mexican milk prices in the long- run. Since the above results confirmed cointegration of international and domestic farm milk price, a VECM was estimated(32,33).

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Cramon-Taubadel & Fahlbusch suggested that in the case of cointegration between nonstationary series, an error correction model (ECM), extended by the incorporation of asymmetric adjustment terms, provides appropriate specification for testing APT(33). An ECM, that relates changes in Pt int to changes in Pt farm for the case of spatial model, as well as the so-called error correction term (ECT), the lagged residuals from the cointegrating equation were estimated. The ECT measures deviations from the long run equilibrium; so, including it in the ECM allows the dependent variable not only to respond to changes in independent variable, but also to â&#x20AC;&#x2DC;correctâ&#x20AC;&#x2122; any deviations from the long run equilibrium that may be left over from previous periods(28,34,35).

Table 2: Engle-Granger two step cointegration test Variable

Coefficient

Std. Err.

t-value

mt-1

-0.1016

0.0186

-5.450

Dmt-1

0.4585

0.0492

9.32

Constant

0.0002

0.0023

0.09

F-test R-squared

50.8 0.3416

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Table 3: Results of the Johansen test for price cointegration

Pfarm - Pint

Cointegrating Equation

Trace

5% critical

Maximun rank

eigenvalue

statistic

value

33.9609

15.41

0.09116

3.1814*

3.76

0.00983

Coefficient

Std. Err.

P-value

0.0488

-8.14

LnPfarm

LnPint

-0.398

Constant

0.741

Spatial Vector Error Correction Model or the spatial model, taking into account that farm and international prices have a unit root and were cointegrated, there was estimated an Asymmetric Vector Error Correction Model (AVECM) in order to investigate possible interdependence of domestic and international milk prices. Following the approach of Cramon-Taubadel and Loy(26), the ECM for spatial price transmission was estimated as in equation (2). The Cramon-Taubadel and Loy approach is the most frequent model to analyze asymmetric price transmission based on an econometric specification that is shown to be inconsistent with cointegration(28). Cramon-Taubadel and Fahlbusch(27), also segmented the contemporaneous response term. Then, we estimated equation (3), in which contemporaneous and short run response to departures from the cointegrating relation are asymmetric if b1+ ยน b1- and b2+ ยน b2respectively. An F-test was used to test the null hypothesis of symmetry. The results of the AECM show that both farm and international milk price respond to disequilibria because coefficients are significant at the 5% level. The correction of price disequilibria is of a small magnitude and coefficients are of the correct sign. In similar studies, using the AECM, several authors found that price swings in global markets are transmitted to domestic markets, but with lower magnitude(35).

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Table 4 shows that contemporaneous change coefficients are significantly less than one in both equations. This means that farm prices do not react completely within one month to international price changes and that monthly data is frequent enough to expose the process of price transmission(26).

Table 4: Results of the VECM; symmetric and asymmetric spatial model Independent Variable

Symmetric Spatial Model Coef. Std. Err. t 0.1173 0.0396 2.96

Asymmetric Spatial Model Coef. Std. Err. t -------

P int t

---

0.3219

0.1159

2.78

 t

---

0.3237

0.1217

2.66

Pfarmt 1

0.5337

0.0555

9.61

0.5186

0.0629

8.24

Pfarmt 2

0.0362

0.0621

0.58

-0.1743

0.0631

-2.76

Pfarmt 3

-0.1796

0.0629

-2.85

0.0386

0.0502

0.77

Pfarmt 4

-0.0106

0.0554

-0.19

0.0246

0.0424

0.58

P int t 1

0.0389

0.0426

0.91

0.0329

0.0622

0.53

P int t 2

-0.0612

0.0421

-1.45

-0.0153

0.0555

-0.28

P int t 3

0.0253

0.0423

0.60

-0.0589

0.0423

-1.39

P int t 4

0.0796

0.0411

1.94

0.0789

0.0412

1.92

ECTt 1

-0.1680

0.0205

-8.19

---

-0.0694

0.0262

-2.65

--0.0003

--0.0020

--0.13

-0.1977 0.0002

0.0329 0.0021

-2.97 0.10

Pint P int

 t 1

ECT

 t 1

ECT

Constant Normality test (Prob>z) LM test (Prob>chi2) DW test R-squared

0.903

0.808

0.5758 1.97 0.4352

0.3989 1.98 0.4378 F(1,306) = 0.85

Test: H0 : b1 = b1

---

Test: H0 : b2 = b2

---

+ +

F(1, 307) = 10.03

Source: Own estimation

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The ECT - induces a significantly greater change in the farm price than the ECT + . Similar results were reported by several studies for spatial price transmission of international milk prices to domestic milk prices(35,36,37). An F-test of the null hypothesis of symmetry ( b 2+ = b 2- ) leads to rejection at the 5% percent level of significance (F = 10.03). Since ECT - indicates that farm milk price is low with respect to the international price, this suggests that milk farm prices react more rapidly when the margin is squeezed than when it is stretched. Therefore, the analysis provides robust statistical evidence for asymmetry in price responses(35).

From the policy point of view, this should help in the design of agricultural support programs, as well as risk management tools for the dairy industry. The finding of strong transmission effects between international and Mexican prices supports the view that trade liberalization in Mexico in the 1990s resulted in greater market orientation. It also shows that participants along the Mexican supply chain need to consider the highly volatile nature of international milk prices in their decision-making process.

Long-run cointegration in the vertical model

In the following, Pt farm is a milk farmerâ&#x20AC;&#x2122;s price in period t and Pt ret is the milk retail price. Hypothesis is that retail price is caused by farm price. Assuming symmetric and linear price transmission, it was estimated equation (1). The results from the cointegrating regression show a R2 of 0.435, a t-statistic on milk farm price of 15.75, and a F statistic of 247.92. The ADF test on error term shows a test statistic of -2.696 vs a 5% critical value of -2.877, which indicates that we cannot reject the null of non-stationarity. Then, it was estimated equation (6). The results show a negative coefficient of the error term, which confirm the long run relationship between prices (cointegration) (Table 5).

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Table 5: Results of the Engle-Granger two step cointegration test Variable

Coefficient

Std. Err.

t-value

mt-1

-0.0523

0.0134

-3.890

Dmt-1

0.3975

0.0510

7.79

Constant

0.0007

0.0031

0.21

F-test

35.35

R-squared

0.2814

Source: Own estimation.

Using Johansen test(24), the null of cointegration cannot be rejected. Because it found that there exists one cointegration relationship between price series (Table 6).

Table 6: Johansen test (1991) for cointegration of Pret and Pfarm

Pretail-

Pfarm

Cointegrating equation LnPret LnPfarm Constant

Maximun rank 0 1 2

eigenvalue . 0.1016 0.01343

Trace statistic 46.0998 3.3528*

Coefficient

Std. Err.

1 -2.175 -2.292

0.2878

-7.56

Source: Own estimation.

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5% critical value 15.41 3.76

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Vertical Vector Error Correction Model

Since cointegration of retail and farm milk prices exist, and following the approach proposed by Cramon-Taubadel(31), it was estimated a VECM (Equation 4). Splitting the ECT into positive and negative components (i.e. positive and negative deviations from the long-term equilibrium – ECT+ and ECT-) makes it possible to test for Asymmetric Price Transmission (APT)(36). Then, was estimated equation (5). To test for asymmetry, if b2+ ¹ b 2- asymmetric price response is present, an F-test was used to test the null hypothesis of symmetry. The output of the symmetric VECM in Table 7, indicates that both, the coefficient of the ECT and the short-term parameter are significant at the 5% level. This result suggests that retail’s and producer’s prices share a long-term equilibrium relationship, and that a change in farmer’s prices does have a significant effect on retailer’s prices in the next period. The

ECT - induces a significantly greater change in the retail price than ECT + . The results support the assumption that price changes are not transmitted efficiently from one level to another(38,39). It also supports the view that Mexican retailers and wholesalers of milk have more market power than milk farmers.

Table 7: Results of the VECM; symmetric and asymmetric vertical model Independent variable

Symmetric Model Coef. Std. Err. t

Asymmetric Model Coef. Std. Err.

Pfarmt

0.327

0.0533

6.13

0.358

0.0536

6.67

Prett 1

0.1273

0.0565

2.25

0.1068

0.0661

1.62

Prett 2

0.0557

0.0570

0.98

0.0575

0.0571

1.01

Prett 3

0.0058

0.0569

0.10

0.0037

0.0570

0.07

Prett 4

-0.0848

0.0571

-1.49

-0.0808

0.0576

-1.40

Pfarmt 1

-0.0593

0.0610

-0.97

-0.0457

0.0652

-0.70

Pfarmt 2

0.0919

0.0601

1.53

-0.0457

0.0604

1.57

Pfarmt 3

-0.1000

0.0615

-1.62

-0.1003

0.0616

-1.63

Pfarmt 4

0.0335

0.0531

0.63

0.0340

0.0532

0.64

ECTt 1

-0.1958

0.0938

-2.09

---

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ECTt 1

---

 t 1

ECT

--Constant 0.0018 Normality test (Prob>z) 0.922 LM test (Prob>chi2) 0.5904 Durbin-Watson (DW) 2.0163 R-squared 0.515   Test H o :  2   2

---

-0.0519

0.0219

-2.37

--0.0020

--0.89

-0.2026 0.0117 0.882 0.5878 2.0171 0.526

0.0546 0.0020

-3.71 0.83

---

F(1,307) =10.36

Source: Own estimation.

This output reveals that the transmission of milk prices is asymmetric with respect to the speed of adjustment, indicating that when producers’ prices decrease, the speed of adjustment tends to be significantly faster, and 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 = 10.36). This suggests that farm prices react more rapidly when the margin is squeezed than when it is stretched. The analysis therefore provides robust statistical evidence for asymmetry in price responses(31). Previous studies(11,40) found, for the US and the Spanish dairy markets, asymmetric price changes between producer and retail stages of the marketing chain. These results suggesting the presence of asymmetric price transmission in the Mexican milk market has important policy implications. First, the role of government intervention in the market via various price support programs could have notable welfare and income redistribution effects. Policy makers have to be very careful in balancing the potential impact of income support programs on producers and its implications for consumer prices in a market where asymmetric price transmission prevails. Also, the existence of imperfect price transmission may also be a warning to policy makers that efforts to further reform and liberalize agricultural markets may not be as beneficial to consumers as expected. Given the limitations of existing models that are primarily price-based, future research that better quantify the impacts of asymmetric price adjustments on producers and consumers are still needed(41).

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Conclusions and implications Long-run cointegration relationship exists between international and Mexican farm milk prices and between farm and domestic retail milk price. For the spatial analysis, both, farm and international prices show significant responses to price disequilibria and asymmetric price transmission. Price movements in international markets are being transmitted asymmetrically to Mexican milk market, indicating that a decrease in international milk prices tend to be transmitted faster to farmers than an increase in international milk prices. For the vertical price transmission model, a change in the producer’s prices do have a significant effect on retailer’s prices in the next period; the speed at which prices tend to converge to fully correct for deviation is moderately slow; and when producers’ prices decrease the speed of adjustment tends to be significantly faster. In this regard, policy makers trying to design mechanisms other than traditional technology transfer approaches to increase small dairy producers’ competitiveness, should pay close attention to measures aimed at increasing the level of price transmission from wholesalers to producers in the marketing chain. The findings of this research provide for the first time important contributions to the policy debate uncovering a couple of issues; a unidirectional transmission of milk prices from producers to retailers, and that the transmission of milk prices is asymmetric depending on whether prices are increasing or decreasing.

Acknowledgments Lic. Leticia Portilla Duran for the collection of secondary information, revision and primary data processing.

Literature cited: 1. SIAP-SAGARPA. Servicio de Información Agroalimentaria y Pesquera. Indicadores económicos. https://www.gob.mx/siap. Consultado 18 Dic, 2016. 2. Banco de México (2017). Indicadores económicos (http://www.baxico.org.mx). Consultado 18 Dic, 2017. 3. Norton RD. Agricultural development policy. Concepts and experiences. Food and Agricultural Organization of the United Nations –FAO. Rome 2004. 4. SAGARPA. Secretaria de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación. https://www.gob.mx/sagarpa. Consultado 18 Dic, 2017.

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5. INEGI. Instituto Nacional de Estadística y Geografía. Índice Nacional de Precios al Consumidor y al Productor, 2017. http://www.inegi.org.mx/ Consultado15 feb, 2017. 6. CONARGEN (2016). Programa Nacional de Recursos Genéticos para la Ganadería. Proyecto del Consejo Nacional de los Recursos Genéticos Pecuarios. Memoria documental. SAGARPA. http://www.sagarpa.gob.mx/ganaderia/Publicaciones/Lists/Otros/Attachments/2/conargen.p df. Consultado15 feb, 2017. 7. LACTODATA. Información sobre el sector http://www.lactodata.info/estadisticas/, Consultado15 feb, 2017.

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8. Fontalvo-Herrera T, De La Hoz-Granadillo E. Morelos-Gómez J. La productividad y sus factores: incidencia en el mejoramiento organizacional. Dimensión Empresarial 2017;15(2):47-60. 9. García HLA, Aguilar VA, Luévano GA, Cabral MA. La globalización productiva y comercial de la leche y sus derivados, articulación de la ganadería intensiva lechera de la Comarca Lagunera. México: Plaza y Valdés editores; 2005. 10. INEGI. Instituto Nacional de Estadística y Geografía. XX http://www.inegi.org.mx/, 2007. Consultado 15 Dic, 2016. 11. Serra T , Goodwin BK. Price transmission and asymmetric adjustment in the Spanish dairy sector. Appl Econ 2003;35(18):1889-1899. 12. Kinnucan HW, Forker OD. Asymmetry in farm-retail price transmission for major dairy products. Am J Agri Economics 1987;69(2):285-292. 13. Schroeder TC, Hayenga ML. Short-term vertical market price interrelationships for beef and pork. North Central J Agr Econ 1987;9(2):171-180. 14. Goodwin BK, Holt MT. Price transmission and asymmetric adjustment in the U.S. beef sector. Amer J Agr Econ 1999;81(3):630- 637. 15. Miller DJ, Hayenga ML. Price cycles and asymmetric price transmission in the U.S. pork market. Amer J Agr Econ 2001;83(3):551-562. 16. Meyer J, Cramon-Taubadel S. Asymmetric price transmission: A survey. J Agr Econ 2004;55(3):581-611. 640

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17. Fackler PL, Goodwin BK. Spatial price analysis. In B. Gardner & G. Rausser (eds), Handbook of agricultural economics. Vol. 1, Amsterdam: Elsevier; 2001. 18. Mundlak Y, Larson DF. On the transmission of world agricultural prices. The World Bank Economic Review 1992;6(3):399-422. 19. Amikuzuno J. Spatial price transmission and market integration in agricultural markets after liberalization in Ghana: Evidence from fresh tomato markets [doctoral thesis]. Goettingen, Germany: University Goettingen; 2009. 20. Rapsomanikis G, Hallam D, Conforti P. Market integration and price transmission in selected food and cash crop markets of developing countries: review and applications. In Commodity Market Review 2003. FAO Rome. 21. USDA-AMS. United States Department http://www.fas.usda.gov/ustrade/. Accessed Dec 8, 2016.

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22. Dickey DA, Fuller WA. Likelihood ratio statistics for autoregressive time series with a unit root. Econometrics 1981;49(4):1057-1072. 23. Phillips PCB, Perron P. Testing for unit root in time series regression. Biometrika 1988;75(2):335-346. 24. Johansen S. Estimation and hypothesis testing of cointegration vectors in Gaussian vector autoregressive models. Econometrics 1991;59(6):1551-1580. 25. Engle RF, Granger CWJ. Co-integration and error correction: Representation, estimation and testing. Econometrics 1987;55(2):251-276. 26. Cramon-Taubadel S, Loy JP. Price asymmetry in the international wheat market: comment. Can J Agric Econ-Rev Can Agroecon 1996;44(3):311-317. 27. Cramon-Taubadel S, Fahlbusch S. Estimating asymmetric price transmission with the error correction representation: An application to the German pork market. Univ Kiel, Dept Agr Econom. 1996. 28. Acquah de-GH, Dadzie KN. An application of the Cramon-Taubadel and Loy error correction models in analyzing asymmetric adjustment between retail and wholesale maize prices in Ghana. J Dev Agric Econ 2010;2(4):100-106.

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29. Ardeni PG. Does the law of one price really hold for commodity prices?. Am J Agr Econ 1989;71(3):66-669. 30. Cramon-Taubadel S. Estimating asymmetric price transmission with the error correction representation. Eur Rev Agric Econ 1998;25(1):1-18. 31. Granger CWJ, Lee TH. Investigation of production, sales and inventory relationships using multicointegration and non-symmetric error correction models. J. Applied Econometrics 1989;4:S145-S159. 32. Jaramillo-Villanueva JL, Sarker R. Los movimientos del tipo de cambio y el comercio de leche en polvo entre MĂŠxico y Estados Unidos. El trimestre econ 2010;77(305):219-246. 33. Cramon-Taubadel S, Fahlbusch S. Identifying asymmetric price transmission with error correction models poster session. EAAE European seminar in reading 1994. 34. Acosta A, Ihle R, Robles M. Spatial price transmission of soaring milk prices from global to domestic markets. Agribusiness 2014;30(1):64-73. 35. Acosta A, ValdĂŠs A. Vertical price transmission of milk prices: Are small dairy producers efficiently integrated into markets?. Agribusiness 2014;30(1):56-63. 36. Popovic R, Radovanov B, Dunn JW. Food scare crisis: the effect on Serbian dairy market. International Food and Agribusiness Management Review 2017;20(1):113-127. 37. Fousekis P, Trachanas E. Price transmission in the international skim milk powder markets. Applied Economics 2016;48(54):5233-5245. 38. Kharin S. Vertical price transmission along the diary supply chain in Russia. Studies in Agricultural Economics 2015;117:80-85. 39. Lloyd T. Presidential address forty years of price transmission research in the food Industry: Insights, challenges and prospects. J Agr Economics 2017;68(1):3-21. 40. Capps O, Sherwell P. Spatial asymmetry in farm-retail price transmission associated with fluid milk products. Agribusiness 2005;23(3):313-331. 41. Awokuse TO, Wang X. Threshold Effects and Asymmetric Price Adjustments in U.S. Dairy Markets. Canadian J Agr Economics 2009;57:269-286.

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https://doi.org/10.22319/rmcp.v10i3.4842 Article

Genetic variability in a Holstein population using SNP markers and their use for monitoring mating strategies

Kathy Scienski a,b,c Angelo Ialacci c Alessandro Bagnato c Davide Reginelli d Marina Durán-Aguilar e Maria Giuseppina Strillacci c*

Texas A&M University, College Station. Interdisciplinary Program in Genetics. Texas, USA. b

Texas A&M University. Department of Animal Science, Texas, USA.

Università degli Studi di Milano. Department of Veterinary Medicine, Via Trentacoste 2, 20134 Milano, Italy. d

Università degli Studi di Milano. Azienda Agraria Didattico Sperimentale Angelo Menozzi, Landriano, Pavia, Italy. e

Universidad Autónoma de Querétaro. Facultad de Ciencias Naturales. Querétaro. México.

* Corresponding author: maria.strillacci@unimi.it

Abstract: As genotyping costs continue to decrease, the demand for genotyping has increased among farmers. In most livestock herds, an important issue is controlling the increase in inbreeding coefficient. While this remains a large motive to genotype, producers are often unaware of the other benefits that genotyping could bring. The aim of this study was to demonstrate that SNP chips could be used as an effective herd management tool by utilizing a population of Italian Holstein-Friesian cattle. After filtering, the total number 643

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of animals and SNPs retained for analyses were 44 and 27,365, respectively. The principal component analyses (PCA) were able to identify a sire and origin-of-sire effect within the herd, while determining that sires do not influence individual genomic selection index values. The inbreeding coefficients calculated from genotypes (FIS) provided a glimpse into the herdâ&#x20AC;&#x2122;s heterozygosity and determined that the genetic variability is being well maintained. On the other hand, inbreeding coefficients on the genomic level were deduced from runs of homozygosity (FROH) and were compared to the inbreeding coefficients based on pedigree (FPED). Furthermore, 1,950 runs of homozygosity (ROH) were identified with the average length of ROH being 4.66 Mb. Genes and QTL within the genomic regions most commonly associated (top 1% and top 5% of SNP) with ROH were characterized. These results indicate that genotyping small herds, albeit at lowdensity, provides insights to the genetic variability within the herd and thus allows producers the ability to manage their stock from a genetic standpoint. Key words: SNP genotypes, Holstein-Friesian, Inbreeding, Runs of homozygosity, Herd management.

Received: 05/04/2018 Accepted: 31/05/2018

Introduction

As the cost of genotyping continues to decrease, it has been met with an increase in demand amongst farmers. Producers recognize that animal genetic resources must be preserved because of their contribution to human livelihood, now and in the future(1). The level of inbreeding within oneâ&#x20AC;&#x2122;s herd has become of particular concern due to selection efforts and has thus been a large motive behind genotyping. An increase in inbreeding leads to a variety of negative effects, such as reduction in phenotypic values for some traits, of genetic variance, and higher frequency of homozygous genotypes(2). Therefore, the desire to manage this coefficient has increased, especially amid small, local herds or dairy stock. Pedigrees may not always be available or sufficient to calculate inbreeding (FPED) within oneâ&#x20AC;&#x2122;s herd, but the coefficient of inbreeding could effectively be determined from genotypes as a means of heterozygosity within the population (FIS) or as a measure of whole genome inbreeding from runs of homozygosity (ROH) (FROH). In fact, several studies in cattle have effectively shown that ROH are suitable to estimate

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genomic inbreeding coefficients in the absence of a pedigree(3-5). Knowledge of runs of homozygosity alone provides not only new possibilities to manage inbreeding in livestock species, but could be used for optimal allocation of resources and maintenance of genetic variation in intensely selected bovine breeds(6). Nevertheless, hesitation still exists among farmers to invest in high-density single nucleotide polymorphism (SNP) chips, so preference has lately been given to the low-density chips, as they are more cost-effective when wanting to genotype a large number of animals. The design of these chips has greatly improved and has led to greater gains in reliability and improved readability among SNP genotypes(7). Another large reason for genotyping among farmers is to obtain the genomic estimated breeding values. In fact, many breeders already embrace this value when purchasing semen(7). Therefore, farmers could already achieve higher annual rates of genetic gain through using genomically tested bulls, but it is becoming increasingly popular to capture extra value from genotyping females(8). This extra value obtained from genotyping oneâ&#x20AC;&#x2122;s herd could be employed as a managerial tool, as genotypes provide considerably more information that just the captured variants at the disease loci featured on the SNP chip. The benefits of this information range from improving the reliability of genomic selection (of both bulls and heifers) to identifying elite females and the best heifers to become herd replacements. As such producers can obtain a better indication of the value of an animalâ&#x20AC;&#x2122;s respect to the solely expected genomic breeding value and use it to avoid or manage inbreeding, and of course prevent genetic defects by avoiding mating that would cause deleterious alleles to surface(8). The objective of this study was to show how SNP chips could be used as an effective management tool in respect of herd genetic variability. We particularly emphasize that the effective level of genomic inbreeding determined from genotypes is comparable to that obtained from an informative pedigree, and that SNP genotyping allows to disclose genomic regions under selection pressure in the herd as identified by genes within runs of homozygosity.

Material and methods

Genotyping

Genotypes were provided by the National Friesian Italian Breeds Association (ANAFI) for a total of 44 Italian Holstein-Friesian cattle aged 12 to 15 mo all coming from a unique

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herd, the University experimental station. All individuals had a marker call rate greater than 98 %. Animals used in this study were part of an experimental population raised at University of Milan’s experimental farm, Azienda Agraria Didattico Sperimentale Angelo Menozzi. Animals were genotyped for 30,125 SNP using the GeneSeek GGP Bovine LD v4 array. After excluding SNPs that were not assigned to a bovine chromosome (BTA) or that were assigned to BTA X or mitochondrial DNA, 27,365 SNP remained.

Principal component analysis (PCA)

Additional information available for each individual included its pedigree, Genomic Productivity, Functionality, Type (GPFT) genomic selection index, and batch of genotyping. The PCA was conducted within SNP with the Variation Suite Golden Helix v8.4 software (SVS) (Golden Helix Inc., Bozeman, MT). The procedure used in SVS considered 20 principal components calculating for each of them a relative eigenvalue; the first principal component (eigenvalue=1.188) was plotted against the second (eigenvalue=0.983).

Inbreeding coefficients

The inbreeding estimate of FIS (or ƒ) was calculated using SVS, defined as 1(HETOBS/HETEXP). This coefficient is equivalent to Wright’s within-subpopulation fixation index with values in the range of -1 to +1. The whole genome inbreeding coefficients (FROH) were calculated according to(9): (LROH)/LAUTO Where: LROH: is the total length of all ROH in the genome of an individual while. LAUTO: refers to the specified length of the autosomal genome covered by SNP (2,505,649,802 bp in the current study). Pedigree-based coefficients of inbreeding (FPED) were derived from Pedigree Viewer(10). The pedigree included records for 2,760 individuals up to 19 generations as calculated by Pedigree Viewer that orders genealogical information in discrete generations. FPED and FROH were compared using linear regression and Pearson’s correlation coefficient to test 646

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their similarity and to highlight differences in the two methods of estimation in a group of animals of the same herd.

Runs of homozygosity

Runs of homozygosity were estimated for each individual using SVS v8.4. This program does not rely on sliding windows to identify ROH, but instead the algorithm works continuously across an entire chromosome by examining every possible run for a match with the specified criteria(11). The following criteria to define ROH were used: 1) Two missing SNP were allowed; 2) One heterozygous genotype was allowed; 3) The minimum number of SNP that constituted the ROH was set to 30; 4) A minimum length of 1 Mb; 5) A maximum gap between consecutive SNP of 1 Mb. Runs of homozygosity were classified into five classes (<2, 2 to 4, 4 to 8, 8 to 16, and >16 Mb) using the same nomenclature as reported by other authors(4,5). The incidence of common runs per SNP was plotted using the Genome Browse tool of Golden Helix, and subsequently aligned to the BTAU 5.0.1 bovine assembly to identify genes consistent with SNP in the top 1% and 5% of runs. The online STRING database classified the network amongst these genes(12), while gene ontology (GO) was performed through the Protein Analysis Through Evolutionary Relationships (PANTHER) classification system (Release 13.1), (http://www.pantherdb.org/pathway/).

Results

Principal component analysis (PCA)

Individuals were grouped based on their batch of genotyping (n= 6), sire (n= 18), sire country of origin (n= 6), and class of GPFT (n= 5). The different batches of genotyping did not cluster whatsoever, indicating that the time of genotyping did not affect individual genotypes (data not shown). On the contrary, there was a strong clustering when 647

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individuals are identified by their sire and their sireâ&#x20AC;&#x2122;s country of origin (Figure 1). Sires 1 and 5 are especially distanced from the others, while those originating from Canada, Italy, and the United States tend to group as well, albeit separately by origin. Individuals were subsequently classified by their GPFT value in increments of 500, and no clustering was observed based upon those assigned categories.

Figure 1: Scatter plot of principal component analysis (PCA) values based on individual SNP genotypes. A) PCA values grouped by sire. B) PCA values grouped by sire origin. C) PCA values grouped by Productivity, Functionality, Type (GPFT) genomic selection index classes

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Inbreeding coefficients

Wrightâ&#x20AC;&#x2122;s inbreeding coefficient estimate of FIS was calculated for each individual. The majority of animals (n= 31) displayed negative FIS, while a smaller proportion (n= 13) possessed positive coefficients (Figure 2). The highest and lowest FIS values were 0.057 and -0.077, respectively. The average FIS existed at -0.018 shoving an increase in heterozygosity due to an outbreeding mating scheme used by the farmer. The majority of individuals possessed FPED coefficients between 0.050 and 0.070 (n= 31); values ranged from 0.044 to 0.116. The whole genome inbreeding coefficient estimate of FROH was higher overall than the inbreeding coefficients calculated from pedigree. The average FPED was 0.064, while the mean FROH >1 Mb equaled 0.083.

Figure 2: Regression of individual inbreeding coefficient. A) Regression of inbreeding calculated by pedigree (FPED) on inbreeding calculated on run of homozigosity (FROH). R2 = 0.205, red line indicates regression line (FROH = 0.042 + 0.621 * FPED). B) Regression of FPED on FIS. R2 = 0.214, red line indicates regression line (Fis= -0.077 + 0.913*FPED)

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Runs of homozygosity

With the ROH criteria set at 1 Mb and minimum of 30 SNP, the SVS software identified a total of 1,950 runs within the population, ranging from 1 to 22 Mb in length (Figure 3). All 44 animals displayed ROH, with the average number of ROH per animal being 44. The largest number of ROH for an individual was 64, while the least number of ROH for an individual existed at 25. The average ROH length was 4.66 Mb, and the greatest amount of ROH existed in the 4-8 Mb range (Table 1). The number of ROH per chromosome was greatest for BTA 10 (122 runs), while lowest for BTA 27 (24 runs). The longest ROH also existed on BTA 10 at over 22 Mb in length, contrary to other results identifying the longest ROH on BTA 8(13-15). The average number of SNPs falling into a ROH was consistent among ROH length category, ranging from 42 (<2 Mb) to 153 (>16 Mb) SNP. ROH length appeared to be relatively proportional to chromosome size, with longer runs appearing on longer chromosomes.

Figure 3: Number of Run of Homozigosity (ROH) according to their class of length

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Table 1: Numbers of ROH per chromosome according to ROH class of length BTA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

<2 Mb 1 1 5 1 7 11 3 5 10 2 2 5 8 8 3 12 2 5 7 2 1 2 4 4 1 1 3 1 7

2-4 Mb 20 31 22 37 36 48 45 32 23 43 36 14 32 50 20 25 17 21 30 24 24 20 16 16 22 21 12 13 18

4-8 Mb 71 75 27 47 40 45 51 56 29 64 41 16 39 21 22 34 21 14 16 43 26 20 12 21 9 11 7 18 20

8-16 Mb 4 10 6 7 15 2 7 10 0 12 6 4 6 0 4 7 3 2 1 7 6 5 2 3 0 2 2 4 3

>16 Mb 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Total 96 117 61 92 98 106 106 103 62 122 85 39 85 79 49 78 43 42 54 76 57 47 34 44 32 35 24 36 48

Total

124

768

916

140

1950

The genomic regions most commonly associated with ROH were identified by selecting the top 1% and 5% of SNPs most frequently observed (Figure 4, Table 2). The incidence of common runs per SNP indicated that the genomic distribution of ROH was nonuniform across chromosomes. Quantitative trait loci (QTL) corresponding with regions of homozygosity housing the top 5% of SNPs were identified using the Animal Quantitative Trait Loci (QTL) Database (AnimalQTLdb) (Table 2)(16).

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Figure 4: Incidence of SNPs in ROH identified by SVS. The red and blue lines indicate the adopted threshold for the top 1% and 5% of observations, respectively

Table 2: Genomic regions of extended homozygosity corresponding with the top 1 and 5% of SNP BTA 1

Number SNP 11

Start (bp)

End (bp)

Genes,

QTL (https://www.animalgenome.org/cgi-bin/QTLdb/BT/index)

49891423

51147114

HSF2BP, ALCAM, CBLB

Milk beta-lactoglobulin percentage QTL (108870); Lean meat yield QTL (37225)

145059132

146790949

PDE9A, PKNOX1, ITGB2, ADARB1, Milk protein percentage QTL (105990); Milk C18 index QTL (32646) POFUT2, ICOSLG, TRPM2, SIK1

15361703

17142680

86178360

88823250

PGAP1, ANKRD44, RFTN2, BOLL, PLCL1, Milk fat yield QTL (122473) SATB2, FBX036

119194847

132528595

ECE1, ALPL, SLC16A14, SP140, SP110, SP140L, CAB39, PSMD1, SPOCD1, FABP3, Fat thickness at the 12th rib QTL (126458); Tick resistance QTL (135875); Milk fat SNRNP40, LAPTM5, HSPG2, USP48, yield (daughter deviation) QTL (25782); Lignoceric acid content QTL (19771, 19709) RAP1GAP

101384522

105043063

55540165

56247946

ANKS1B, AVIL, TSFM, METTL21B, Inhibin level QTL (71509, 71314, 71315, 71316, 71317, 71358, 71359, 71319, 71407, METTL1, CYP27B1, MARCH9, CDK4, 71320); Coat color QTL (37323, 37324, 37325) AGAP2, OS9

62920850

66830677

Fat thickness at the 12th rib QTL (20283); Calving ease QTL (126849); Stillbirth QTL ACTR6, NR1H4, ANO4, SLC5A8, UTP20, (126850); Gestation length QTL (15409, 15410, 15411, 15412); Milk C14 index QTL PARPBP, NUP37, GNPTAB (34847); Lean meat yield QTL (36911)

103006312

104069660

WC1-12, WC1.3, CLSTN3

Shear force QTL (121704)

24733464

26517604

PPP3CA, EMCN, DNAJB14

Clinical mastitis QTL (25244); Body weight (birth) QTL (67220, 67402, 66543); Body weight gain QTL (67403, 67639); Calving ease QTL (106434)

Body weight (yearling) QTL (66889); Udder swelling score QTL (106708); Interval to first estrus after calving QTL (30300)

GPBP1L1, PRDX1, TESK2, ZSWIM5, PTCH2, KIF2C, C3H1orf228, RNF220, ERI3, Body weight (weaning) QTL (24748); Birth index QTL (15168); Calf size QTL (15167, SLC6A9, ST3GAL3, SLC2A1, PPCS, FOXJ3 15169); Calving ease QTL (15170)

73543373

75270817

Curd firmness QTL (95977); Eye area pigmentation QTL (37389); Body weight (birth) QTL (66358); Body weight (weaning) QTL (67229); Body weight (yearling) QTL (67230); Body weight gain QTL (67231); Milk yield (EBV) QTL (16233); Milk fat yield QTL (16234); Milk fat percentage (EBV) QTL (16235); Milk protein percentage QTL KIAA1211, AASDH, SRP72, NOA1, IGFBP7 (16236); Non-return rate (EBV) QTL (16237); Interval from first to last insemination QTL (16238); Milk protein yield (daughter deviation) QTL (26193, 26196); Milk fat yield (daughter deviation) QTL (25834); Milk yield (daughter deviation) QTL (25421); Milk kappa-casein percentage QTL (111104, 112361)

4358132

4953801

CRTC1, CRLF1, ELL, SSBP4, PGPEP1

100624993

NR2F1, FAM172A, KIAA0825, SLF1, Zinc content QTL (24065); Fat thickness at the 12 th rib QTL (24649, 24650); Shear MCTP1, FAM81B, TTC37, PCSK1, ERAP1, force QTL (20767, 31580, 37955); Iron content QTL (23257, 23258); Lean meat yield QTL (37087); Carcass weight QTL (122454); Tenderness score QTL (36406) LNPEP, LIX1

94824183

652

Body weight gain QTL (67930)

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46925080

55060853

SMC5, TRPM3, ABHD17B, C8H9orf85, GDA, Average daily gain QTL (20937); Zinc content QTL (24066); Milk yield QTL (121783); ZFAND5, TMC1, RFK, PRUNE2, GNA14, Milk fat yield QTL (121784); Milk protein yield QTL (121785); Milk protein percentage QTL (121786) VPS13A, GNAQ, CEP78

74227581

77959797

DOCK5, CDCA2, PPP2R2A, PTK2B, GULO, Milk protein yield (daughter deviation) QTL (26264, 26268); Milk fat yield (daughter B4GALT1, NFX1, UBE2R2, KIF24, DNAI1, deviation) QTL (25878); Milk yield (daughter deviation) QTL (25466); Fat thickness at the 12th rib QTL (122442); Interval to first estrus after calving QTL (29993, 30322) CCL19

29967808

FMN1, AVEN, RYR3

Docosapentaenoic acid content QTL (31774); Omega-3 unsaturated fatty acid content QTL (31780); Calving ease (maternal) QTL (44416); Daughter pregnancy rate QTL (44417); Stillbirth (maternal) QTL (44418); Foot angle QTL (44419); Feet and leg conformation QTL (44420); PTA type QTL (44421); Teat placement (front) QTL (44422); Udder attachment QTL (44423); Net merit QTL (44424); Length of productive life QTL (44425); Rear leg placement – rear view QTL (44426); Rear leg placement – side view QTL (44427); Udder height QTL (44428); Rump width QTL (44429); Calving ease QTL (44430); Somatic cell score QTL (44431); Stillbirth QTL (44432); Stature QTL (44433); Udder depth QTL (44434)

39409071

Residual feed intake QTL (23793); Iron content QTL (24060); Calving ease QTL (15185, 44566); Dry matter intake QTL (140483); Body depth QTL (44557, 44572, 44586, 44672); Dairy form QTL (44558, 44573, 44587, 44673); Daughter pregnancy rate QTL (44559); PTA type QTL (44560, 44575, 44589, 44675); Net merit QTL (44561); Length of productive life QTL (44562); Rear leg placement – rear view QTL DPH6, C10H15orf41, MEIS2, RTF1, MGA, (44563, 44578, 44592, 44677); Teat placement – rear QTL (44564, 44579, 44593); MAPKBP1, SPTBN5, PLA2G4E, VPS39, Udder height QTL (44565, 44580, 44594, 44678); Somatic cell score QTL (44567); CAPN3, STARD9, CDAN1, UBR, SCG5, Stillbirth QTL (44568); Stature QTL (44569, 44582, 44596, 44680); Teat length QTL AQR (44570); Udder cleft QTL (44571, 44584, 44598); Conception rate QTL (107126, 107124); Feet and leg conformation QTL (44574, 44588, 44674); Teat placement – front QTL (44576, 44590); Udder attachment QTL (44577, 44591, 44676); Rump width QTL (44581, 44595, 44679); Strength QTL (44583, 44597, 44681); Udder depth QTL (44585, 44599); Shear force QTL (37956, 20778)

49942490

KLHDC2, NEMF, SOS2, CDKL1, MAP4K5, TRIM9, CSNK1G1, FAM96A, DAPK2, HERC1, CA12, APH1B, TLN2, VPS13C, FRMD6, GNG2, NID2, PTGDR, ZNF609, TRIP4, RORA, ICE2

Dairy form QTL (44720); Daughter pregnancy rate QTL (44721); Net merit QTL (44722); Length of productive life QTL (44723); Milk protein percentage QTL (44724, 105566); Calving ease QTL (44725); Somatic cell score QTL (44726); Stillbirth QTL (44727); Milk glycosylated kappa-casein percentage QTL (116745, 111446); Milk fat yield (daughter deviation) QTL (25900); Tick resistance QTL (135843); Dry matter intake QTL (131016, 131002); Body weight (birth) QTL (68188); Body weight gain QTL (68135); Eicosapentaenoic acid content QTL (31775); Omega-3 unsaturated fatty acid content QTL (31781)

59882200

DMXL2, GLDN, TNFAIP8L3, SPPL2A, TRPM7, MYO1E, CCNB2, ADAM10, LIPC, ALDH1A2, POLR2M, MYZAP, CGNL1, TCF12, NEDD4, PRTG, RAB27A, UNC13C, WDR72, FAM214A, MYO5A, GNB5, MAPK6

Tick resistance QTL (135792); Body weight (weaning) QTL (23796); Udder swelling score QTL (106594); Interval to first estrus after calving QTL (28678, 28652); Fat thickness at the 12th rib QTL (122444); Bovine respiratory disease susceptibility QTL (95662); Body weight (weaning) QTL (23795); Conception rate QTL (138570); C22:1 fatty acid content QTL (20512); Calving ease QTL (30516); Shear force QTL (106393)

28781938

30065944

42713194

50086640

60004650

66740333

Calving ease QTL (30516); Shear force QTL (106393); Calving index QTL (30512); Pregnancy rate QTL (65946); Milk zinc content QTL (70034); Conception rate QTL (107130); Body depth QTL (44827, 44850, 44863); Dairy form QTL (44828, 44851, 44864); PTA type QTL (44829, 44853, 44865); Udder attachment QTL (44830, SHC4, CEP152, FBN1, SLC12A1, MYEF2, 44855, 44867); Udder height QTL (44831, 44857, 44869); Rump width QTL (44832, SEMA6D, FERMT2, DDHD1, GABPB1, 44858, 44870); Stature QTL (44833, 44859, 44872); Strength QTL (44834, 44860, 44873); Udder cleft QTL (44835, 44861, 44874); Udder depth QTL (44836, 44862, ATP8B4, FAM227B, GALK2 44875); Feet and leg conformation (44852); Teat placement – front QTL (44854, 44866); Teat placement – rear QTL (44856, 44868); Body weight gain QTL (68136), Somatic cell score QTL (44871); Body weight (weaning) QTL (106643); Gestation length QTL (15473, 15474)

98223275

98398966

Body weight (weaning) QTL (24729)

100918254

101806982

GALC, KCNK10, ZC3H14, EML5

Longissimus muscle area QTL (138414)

4099982

ASTL, CIAO1, KANSL3, LMAN2L, CNNM3, Milk glycosylated kappa-casein percentage QTL (116678) SEMA4C, FAM178B

2254541

36863940

40183335

Milk riboflavin content QTL (64136); Body depth QTL (45332); Calving ease (maternal) QTL (45333); Foot angle QTL (45334); Feet and leg conformation QTL (45335); Milk fat percentage QTL (45336); PTA type QTL (45337); Udder attachment ZAP70, TMEM131, VWA3B, CNGA3, COA5, QTL (45338); Milk fat yield QTL (45339); Milk yield QTL (45340); Net merit QTL MGAT4A, KIAA1211L, ACYP2, SPTBN1, (45341); Milk protein percentage QTL (45342); Milk protein yield (45343); Rear leg EML6, RTN4, CCDC88A, PPP4R3B, placement – rear view QTL (45344); Udder height QTL (45345); Rump width QTL (45346); Calving ease QTL (45347); Stillbirth QTL (45348); Stature QTL (45349); CCDC85A Strength QTL (45350); Udder depth QTL (45351); Shear force QTL (36420, 36421); Udder swelling score (106598); Dry matter intake QTL (121918); Calving interval QTL (121653, 121654)

61014137

63715266

ANGPT4, TBC1D20, HM13, TTLL9, CCM2L, Teat length QTL (47334); Interval to first estrus after calving QTL (28680); Milk iron NOL4L, DNMT3B, SUN5, BPIFB4, SNTA1 content QTL (70225); Scrotal circumference QTL (119789)

4202927

4797465

KCNK9, TRAPPC9, AGO2

Milk fat percentage QTL (33087, 35538, 47482, 61978, 32849, 35540, 14973, 33665, 33581, 35542, 33227, 32960); Milk protein percentage QTL (35774, 113879, 47486, 35776, 113990); Milk protein yield QTL (35306, 35308, 14927); Milk yield QTL (35423, 35425, 14900); Milk casein percentage QTL (108945); Milk riboflavin content QTL (64384); Somatic cell score QTL (64598); Foot angle QTL (47480); Feet and leg conformation QTL (47481); Milk fat yield QTL (47483, 35359, 35360, 31117, 35362); Net merit QTL (47484); Length of productive life QTL (47485); Rear leg placement – rear view QTL (47487); Rear leg placement – side view QTL (47488); Calving ease QTL (47489); Stillbirth QTL (47490); Stature QTL (47491); Strength QTL (47492); Milk oleic acid percentage QTL (32556)

33747933

35216192

C14H8orf34, PREX2, CPA6

Udder swelling score QTL (106736); Age at first calving QTL (140104); Scrotal circumference QTL (138445)

2020646

3805373

Body depth QTL (48079); Calving ease (maternal) QTL (48080); Dairy form QTL PIK3C2B, NFASC, CNTN2, DSTYK, (48081); Daughter pregnancy rate QTL (48082); PTA type QTL (48083); Length of TMCC2, MFSD4A, NUCKS1, PM20D1, productive life QTL (48084); Calving ease QTL (48085); Teat length QTL (48086); Udder cleft QTL (48087); Milk myristic acid percentage QTL (56623); Body weight CTSE, SRGAP2 gain QTL (68840)

5715970

7838108

KCNT2

Milk fat percentage QTL (34602)

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25709923

26613598

21402231

22154025

GPBP1

Body weight gain QTL (69057)

27105033

30747366

HCN1, EMB, PARP8

Calving ease QTL (30553); Average daily gain QTL (131122); Length of productive life QTL (123122, 123133); Milk protein percentage QTL (105307, 105349, 105182, 105395, 105073); Milk protein percentage (daughter deviation) QTL (26866, 26984); Milk yield (daughter deviation) QTL (25661); Body weight QTL (65983)

45232289

47209012

CDH9

-Body depth QTL (50966); Calving ease (maternal) QTL (50967); Daughter pregnancy rate QTL (50968); Stillbirth (maternal) QTL (50969); Foot angle QTL (50970); Feet and leg conformation QTL (50971); PTA type QTL (50972); Teat placement â&#x20AC;&#x201C; front QTL (50973); Udder attachment QTL (50974); Net merit QTL (50975); Length of productive life QTL (50976); Milk protein percentage QTL (50977); Udder height QTL (50978); Rump width QTL (50979); Calving ease QTL (50980); Stature QTL (50981); Strength QTL (50982); Udder depth QTL (50983)

6388853

6900116

CERS3, ADAMTS17

7068248

9209367

Age at puberty QTL (21135, 21136); Sire conception rate QTL (124003); Longissimus MEF2A, LRRC28, TTC23, SYNM, IGF1R, muscle area QTL (22856); Dry matter intake QTL (23894); Body weight QTL (22618); PGPEP1L Interval from first to last insemination QTL (138530); Inseminations per conception QTL (138531)

63970044

65102247

21888915

23828165

TPR1, SUMF1, CNTN4

M. paratuberculosis susceptibility QTL (127097); Body weight (birth) QTL (23910)

50772465

33291018

33669073

LAMA3, ANKRD29, NPC1, TMEM241

Milk protein yield QTL (123993)

50202589

50336324

TSPAN32

Average daily gain QTL (102011, 102012); Growth index QTL (102013)

Discussion

Principal component analyses

The PCA is a useful tool that allows farmers to identify genetically different individuals within their herd based on genotypes. The graphical representation of individuals in fact represent an immediate easy to interpret tool that does not require any specific skill by farmers except the concept that closed points are more similar and distant ones are different. Well-differentiated females can be identified in this herd, mostly due to sire, although a group appearing genetically similar exists belonging to a variety of sires. Sires 1 and 5 are responsible for the out-groups we see, with their derivations being Canada and Italy, respectively. Therefore, as proposed by the farmer involved in this study, this visual is beneficial to determine the a-posteriori analysis of his mating strategies and whether the sires he had chosen are helping to his attempts to limit the reduction in genetic variability of the herd. The PCA is also beneficial in sire selection, as the farmer can ascertain if sires deriving from specific countries have an impact on the genetic distribution in the population. Likewise, it can be visualized how genomic values, in this case GPFT, coincide with different individuals. GPFT of similar value did not cluster, indicating that both sire and sire origin did not impact GPFT in the females of this herd. Sire 1 contributed to individuals with GPFT values ranging from 1,500 to 3,000 and sire 5 to animals with values from 1,000 to 2,500, for example. 654

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Inbreeding coefficients

This result here obtained are concordant with another study performed in Italian Holstein, where the average FPED was 0.044 and the FROH >1 Mb was 0.116(5). Across all 44 animals, the correlation between FPED and FROH was significantly different from 0 with r equal to 0.453 (P<0.01). Intense and accurate artificial selection practiced over many years has resulted in high rates of genetic gain; however, the high rates of gain have been accompanied by large increases in inbreeding(17). Additionally, as population sizes decrease, the probability of mating among relatives increases, especially in small herds or local breeds(13). Producers have largely depended on pedigrees to estimate the coefficient of inbreeding within the herd, but this number can only increase with each generation. Pedigree relatedness gives an expected, not actual, proportion of genomic identity by descent among individuals and it is anticipated that genotype-based estimates provide greater accuracy on relatedness(18). In practice, pedigree information is difficult to obtain, potentially unreliable, and rarely assessed for inbreeding arising from common ancestors(19). When the pedigree-based coefficients of this herd was examined, it was possible that individuals to be up to a tenth inbred. But, in the absence of pedigree data, the extent of a genome under ROH may be used to infer aspects of recent population history, even from relatively few samples(3,9). McQuillan et al.(9) revealed that FROH correlates strongly with the inbreeding coefficient estimated from pedigree (r= 0.86), and it has been found that FROH values are preferable to FPED, as they are thought to be a more realistic reflection of inbreeding level(15,19). However, it is important to note that the setting of the parameters used to derive ROH is crucial to account for the effects of SNP density correctly(20). In this dataset, it is visible a linear relationship between FROH and FPED. The Pearsonâ&#x20AC;&#x2122;s correlation coefficient provides a positive correlation at r= 0.453. The correlation in this work is in concordance with other studies using cattle that were genotyped at similar density(15,20), yet lower than others that were genotyped at medium to high density(3,5). It is possible then that the correlation coefficient may be affected partially by the small number of animals in this study as such as for the use of a lowdensity SNP chip. Additionally, the FROH was larger than the mean FPED in 35 animals, implying that pedigree based inbreeding coefficients could be underestimated. The overall low FIS values are indicative of a low level of inbreeding in the population and of a relatively high number of individuals in a heterozygous state. This may be related to the result of the specific mating strategy implemented by the University herd manager co-author of this work, as he stated his goal was to increase genetic variability and preserve low inbreeding. Therefore, this genomic information is providing unique feedback as to the successful efforts in maintaining inbreeding within this population. Dairy breeds especially have been under more intensive selection and may be related to the recent increase of consanguineous mating resulting from small number of high genetic merit sires used for artificial insemination(5). Because of this, it is important to understand

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the inbreeding occurring at a genomic level, as well as to determine whether or not the genetic variability within oneâ&#x20AC;&#x2122;s herd is being well maintained.

Runs of homozygosity and associated genes The most commonly observed QTL within these regions reported in Table 2 included milk protein percentage, milk fat, calving ease, PTA type, and stature. After aligning the SNP to the BTAU 5.0.1 reference, 260 annotated genes were identified to be consistent with the top 5% of SNP, while 37 genes remained in the top 1%. Genes in the top 1% resided on chromosomes 5 and 10; no annotated genes were identified with the SNP on BTA 16. STRING subsequently identified networks amongst these genes, with 142 genes identified to be involved in some kind of network (Figure 5). These genes also underwent gene ontology in PANTHER. Genes within the top 5% corresponded with 12 biological processes (Figure 6) and 70 pathways (data not shown). The pathway that included the greatest number of genes (n= 12) was the gonadotropin-releasing hormone receptor pathway, while other pathways sharing genes were the 5HT2 type receptor mediated signaling pathway, endothelin signaling pathway, inflammation mediated by chemokine and cytokine signaling pathway, integrin signaling pathway, and Wingless-related integration site (Wnt) signaling pathway. The respective biological process most commonly shared among genes (n= 116) was â&#x20AC;&#x153;cellular processes,â&#x20AC;? specifically cell communication, while another large portion (n= 79) has a role in metabolic processes (Figure 6).

Figure 5: Network of genes corresponding with SNP in the top 5% of incidence of common runs obtained by STRING online database

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Figure 6: Biological processes of genes corresponding with SNP in the top 5% of incidence of common runs 140

120

Number of genes

100

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The practices of intense selection of sires, artificial insemination, and embryo transfer have been featured heavily in some breeds, reducing effective population sizes, genetic diversity and affecting levels of homozygosity(3). The deleterious effects associated with boosted homozygosity arising from inbreeding are predisposed to reduce the genetic gains, implicating in a clear loss of genetic variability(15). ROH give insight to this variability, as they are continuous homozygous segments that are common in individuals and populations. The ability of these segments to give a glimpse into a populationâ&#x20AC;&#x2122;s genetic events makes them a useful tool in providing information about the evolution of the population over time, therefore enabling producers to maintain diversity and fitness within their livestock breed(21). Furthermore, ROH provide useful information about the genetic relatedness among individuals, helping to minimize the inbreeding rate and also helping to expose deleterious variants in the genome(21). Long ROH arise as a result of recent inbreeding, while shorter ROH can indicate more distant ancestral effects such as breed founder effects(3). In point of fact, the presence of segments longer than 10 Mb is

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traceable to inbreeding from recent common ancestors that occurred only five generations ago(22), and 66 % of the animals comprised in this study presented at least one ROH extending over 10 Mb. This signifies that the majority of individuals in this population derived from recent common ancestors and is the product of recent inbreeding, consistent with what we see from the inbreeding coefficients. ROH were also identified as a means to provide insight to the conserved genomic regions within the herd. ROH less than 5 Mb were recently characterized as being short(23). These shorter ROH can be related to a more distant ancestral positive selection effect due to recombination events from repeated meiosis breaking long chromosomes into segments and therefore reducing their size(13,24). Lower SNP density, such as that used in this study, tends to inflate ROH(3). Given that we see primarily short ROH in this herd (average ROH=4.66 Mb), even with the potential inflation, it is possibly to say with confidently that the level of inbreeding within this population has been well maintained, is low, and is recent based upon the presence of long ROH in the majority of individuals yet the overall short ROH throughout the herd. This result identifies with a separate study that established that the Italian Holstein show higher number of ROH segments related to ancient consanguinity(13). Genes within the extended homozygous regions corresponding to SNPs in the top 5% of common runs were found to be largely involved in survival processes. This indicates that basic biological processes, such as cell communication and metabolism, have been maintained in selection efforts. QTL within these regions, however, show that these genomic regions coincide with beneficial production facets such as milk protein percentage, milk fat, and calving ease, although these may be specific to the HolsteinFriesian breed due to stringent selection efforts over time. Additionally, genes in the top 1% (BTA 5, 10, and 16) also correlated with survival and developmental processes. Such processes included cell proliferation (GNG2, ADAM10, ALDH1A2), metabolic function (SCG5, PTGDR, RORA, MYO1E, LIPC, ALDH1A2, MYO5A, GALK2), organ development (ANKS1B, MYO1E, CCNB2, ALDH1A2, PRTG, MAPK6), and immunity (NEDD4). No genes were identified on BTA 16 due to poor annotation, but several genes on BTA 5 and 10 have been well characterized in different phenotypes in cattle. PARP1 binding protein (PARPBP) on BTA 5 has been associated with fat percentage in the third lactation stage in Jersey cattle, while ankyrin repeat and sterile alpha motif domain containing 1B (ANKS1B), also on BTA 5, has been associated with bovine spongiform encephalopathy (BSE) incidence in cattle families(25,26). Meanwhile, genes on BTA 10 were largely found to be involved in fat metabolism and feed intake. Secretogranin V (SCG5) and G protein subunit gamma 2 (GNG2), for example, have been linked to the regulation of hormone metabolic processes involved in feed intake in Holstein(27,28), while transcription factor 12 (TCF12) was found to be associated with lipid and organoleptic traits in European Bos taurus breeds(29). GNG2 was also found to be in a region associated with susceptibility to Mycobacterium avium ssp. Paratuberculosis (Map) infection(30). Lastly, FERM domain containing 6 (FRMD6) was identified to be in a region of positive selection in the Nâ&#x20AC;&#x2122;Dama breed(31). The regions on BTA 10 and 16 harboring the top 1%

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of SNP were also concordant with other studies. Studies involving Holstein were especially similar, with positive selection occurring on BTA 10 at 50-60 Mb and a putative QTL identified at 34.7-56.9 on BTA 10 for somatic cell count and non-return rate at 90 days (paternal effect)(20,32,33). Another study identified a high proportion of ROH on BTA 16, while a separate found selection at regions 24.7-26.7 and 26.5-28.5 on BTA 16(3,20).

Conclusions and implications

Using a population of 44 Italian Holstein-Friesian cattle from the University experimental farm and low-density SNP panels for genotyping, we estimated several genetic variability aspects within the herd population including inbreeding coefficients and runs of homozygosity. The approach was successful to provide a tool to monitor the efficacy of the mating strategies operated in the farm population and to provide insights for the inbreeding management. SNP genotypes can effectively provide inbreeding coefficients in the absence of a pedigree, while runs of homozygosity can further provide information on genomic regions under positive selection and thus indicate the evolving nature of the population. If farmers genotype their young females more extensively, they are able to construct a database with the corresponding information and are therefore enabled to make selection and management decisions as a result. It is to be noticed that the management of the inbreeding is extremely important to reduce the occurrence of mendelian recessive disease and that the genomic approach is providing an innovative and accurate possibility. The farmers are usually genotyping females with low density SNP chip that is shown to be a valuable tool to be used for genomic management purposes. The diffusion of this approach to farmers may lead to a routine genotyping of young females thus providing information for a wide impact of the genomic management of inbreeding.

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Acknowledgments

This work was partially supported by the USDA National Institute of Food and Agriculture under fellowship supplement 2017-38420-26779. The remaining support was received through internal funds from Azienda Agraria Didattico Sperimentale Angelo Menozzi.

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Ouborg NJ, Pertoldi C, Loeschcke V, Bijlsma RK, Hedrick PW. Conservation genetics in transition to conservation genomics. Trends Genet 2010;26(4):177-87.

Purfield DC, Berry DP, McParland S, Bradley DG. Runs of homozygosity and population history in cattle. BMC Genet 2012;13:70.

Ferencakovic M, Hamzic E, Gredler B, Solberg TR, Klemetsdal G, Curik I, et al. Estimates of autozygosity derived from runs of homozygosity: empirical evidence from selected cattle populations. J Anim Breed Genet 2013;130(4):286-93.

Marras G, Gaspa G, Sorbolini S, Dimauro C, Ajmone-Marsan P, Valentini A, et al. Analysis of runs of homozygosity and their relationship with inbreeding in five cattle breeds farmed in Italy. Anim Genet 2015;46(2):110-21.

Schwarzenbacher H. Analysis of genome regions showing strong inbreeding in Brown Swiss and Fleckvieh cattle. Interbull Bulletin 2011;44:130-133.

Schefers JM, Weigel KA. Genomic selection in dairy cattle: Integration of DNA testing into breeding programs. Animal Frontiers 2012;2(1):4-9.

Pryce JE, Hayes BJ, Goddard ME. Genotyping dairy females can improve the reliability of genomic selection for young bulls and heifers and provide farmers with new management tools. In: Proc 38th ICAR Session. Cork 2012. https://www.icar.org/index.php/icar-meetings-news/38th-session-cork-ireland/.

McQuillan R, Leutenegger AL, Abdel-Rahman R, Franklin CS, Pericic M, BaracLauc L, et al. Runs of homozygosity in European populations. Am J Hum Genet 2008;83(3):359-372. 660

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11. Curik I, Ferencakovic M, Solkner J. Inbreeding and runs of homozygosity: A possible solution to an old problem. Livest Sci 2014;166:26-34. 12. Szklarczyk D, Morris JH, Cook H, Kuhn M, Wyder S, Simonovic M, et al. The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res 2017;45(D1):D362-D368. 13. Mastrangelo S, Tolone M, Di Gerlando R, Fontanesi L, Sardina MT, Portolano B. Genomic inbreeding estimation in small populations: evaluation of runs of homozygosity in three local dairy cattle breeds. Animal 2016;10(5):746-54. 14. Kim ES, Cole JB, Huson H, Wiggans GR, Van Tassell CP, Crooker BA, et al. Effect of artificial selection on runs of homozygosity in U.S. Holstein cattle. PLoS One 2013;8(11):e80813. 15. Peripolli E, Stafuzza NB, Munari DP, Lima ALF, Irgang R, Machado MA, et al. Assessment of runs of homozygosity islands and estimates of genomic inbreeding in Gyr (Bos indicus) dairy cattle. BMC Genomics 2018;19(1):34. 16. Hu ZL, Park CA, Reecy JM. Developmental progress and current status of the Animal QTLdb. Nucleic Acids Res 2016;44(D1):D827-33. 17. Rodriguez-Ramilo ST, Fernandez J, Toro MA, Hernandez D, Villanueva B. Genome-wide estimates of coancestry, inbreeding and effective population size in the Spanish Holstein population. PLoS One 2015;10(4):e0124157. 18. Visscher PM, Medland SE, Ferreira MA, Morley KI, Zhu G, Cornes BK, et al. Assumption-free estimation of heritability from genome-wide identity-by-descent sharing between full siblings. Plos Genet 2006;2(3):e41. 19. Keller MC, Visscher PM, Goddard ME. Quantification of inbreeding due to distant ancestors and its detection using dense single nucleotide polymorphism data. Genetics 2011;189(1):237-249. 20. Signer-Hasler H, Burren A, Neuditschko M, Frischknecht M, Garrick D, Stricker C, et al. Population structure and genomic inbreeding in nine Swiss dairy cattle populations. Genet Sel Evol 2017;49(1):83. 21. Peripolli E, Munari DP, Silva M, Lima ALF, Irgang R, Baldi F. Runs of homozygosity: current knowledge and applications in livestock. Anim Genet 2017;48(3):255-271.

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22. Howrigan DP, Simonson MA, Keller MC. Detecting autozygosity through runs of homozygosity: a comparison of three autozygosity detection algorithms. BMC Genomics 2011;12:460. 23. Mastrangelo S, Ciani E, Sardina MT, Sottile G, Pilla F, Portolano B, et al. Runs of homozygosity reveal genome-wide autozygosity in Italian sheep breeds. Anim Genet 2018;49(1):71-81. 24. Kirin M, McQuillan R, Franklin CS, Campbell H, McKeigue PM, Wilson JF. Genomic runs of homozygosity record population history and consanguinity. PLoS One 2010;5(11):e13996. 25. Murdoch BM, Clawson ML, Laegreid WW, Stothard P, Settles M, McKay S, et al. A 2cM genome-wide scan of European Holstein cattle affected by classical BSE. BMC Genet 2010;11:20. 26. Oliveira HRd, Silva FF, Brito LF, Jamrozik J, Lourenco DAL, Schenkel FS. Genome-wide association study for milk, fat and protein yields in different lactation stages in Canadian Holstein and Jersey cattle. World Congress on Genetics Applied to Livestock Production, Auckland, New Zealand. 2018. 27. Xi YM, Yang Z, Wu F, Han ZY, Wang GL. Gene expression profiling of hormonal regulation related to the residual feed intake of Holstein cattle. Biochem Biophys Res Commun 2015;465(1):19-25. 28. Salleh MS, Mazzoni G, Hoglund JK, Olijhoek DW, Lund P, Lovendahl P, et al. RNA-Seq transcriptomics and pathway analyses reveal potential regulatory genes and molecular mechanisms in high- and low-residual feed intake in Nordic dairy cattle. BMC Genomics 2017;18(1):258. 29. Dunner S, Sevane N, Garcia D, Leveziel H, Williams JL, Mangin B, et al. Genes involved in muscle lipid composition in 15 European Bos taurus breeds. Anim Genet 2013;44(5):493-501. 30. Kiser JN, Neupane M, White SN, Neibergs HL. Identification of genes associated with susceptibility to Mycobacterium avium ssp. Paratub erculosis (Map) tissue infection in Holstein cattle using gene set enrichment analysis-SNP. Mamm Genome 2017;1-11. doi: 10.1007/s00335-017-9725-4. 31. Taye M, Lee W, Jeon S, Yoon J, Dessie T, Hanotte O, et al. Exploring evidence of positive selection signatures in cattle breeds selected for different traits. Mamm Genome 2017;28(11-12):528-541. 32. Kuhn C, Bennewitz J, Reinsch N, Xu N, Thomsen H, Looft C, et al. Quantitative trait loci mapping of functional traits in the German Holstein cattle population. J Dairy Sci 2003;86(1):360-368.

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33. Bovine HapMap C, Gibbs RA, Taylor JF, Van Tassell CP, Barendse W, Eversole KA, et al. Genome-wide survey of SNP variation uncovers the genetic structure of cattle breeds. Science 2009;324(5926):528-532.

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https://doi.org/10.22319/rmcp.v10i3.4804 Article

Defining growth curves with nonlinear models in seven sheep breeds in Mexico

Joel Domínguez-Viveros a* Edwin Canul-Santos a Felipe Alonso Rodríguez-Almeida a María Eduviges Burrola-Barraza a Juan Ángel Ortega-Gutiérrez a Francisco Castillo-Rangel a

Universidad Autónoma de Chihuahua. Facultad de Zootecnia y Ecología. Periférico Francisco R. Almada km 1. 31453 Chihuahua, Chih. México.

* Corresponding author: joeldguezviveros@yahoo.com.mx – jodominguez@uach.mx

Abstract: Characterizing growth in livestock is important when making management, marketing and genetic improvement decisions. Nonlinear models were tested to identify those with the best fit for growth curves in seven sheep breeds [Blackbelly (n= 19,084); Pelibuey (n= 39,025); Dorper (n= 35,814); Katahdin (n= 74,154); Suffolk (n= 10,267); Hampshire (n= 7,561); and Rambouillet (n= 7,384)]. Using breed registry databases, live weight was assessed from birth to 230 d of age. The SAS program was applied to test six nonlinear models: Brody, Verhulst, von Bertalanffy, Gompertz, Mitscherlich and logistic. The criteria for selecting the best-fit model were the average prediction error; the prediction error variance; the Durbin-Watson statistic; the coefficient of determination; the root-mean-square error; and the Akaike and Bayesian information criteria. For the Hampshire, Pelibuey and Suffolk breeds the best-fit model was the von Bertalanffy, with a sigmoid curve and an inflection point age between 40 and 57 d. For the Katahdin, Blackbelly, Dorper and Rambouillet breeds the best-fit models were the Brody and Mitscherlich models, with a continuous growth curve, no inflection point and constant growth rate. Marked differences were observed in adult 664

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weight between breeds, with average values (kg) of 44.6 for Blackbelly, 49.2 for Rambouillet, 52.9 for Pelibuey, 55.6 for Hampshire, 60.2 for Katahdin, 64.7 for Suffolk and 65.2 for Dorper; values tended to be highest in the Brody and Mitscherlich models, and lowest in the logistic and Verhulst models. Key words: Growth rate, adult weight, model selection, von Bertalanffy, Brody, Nonlinear regression. Received: 10/03/2018 Accepted: 27/09/2018

Introduction In Mexico the Organization of the Sheep Breeders National Unit (Organismo de la Unidad Nacional de Ovinocultores - UNO) encompasses producers of specialized and pure breed sheep. This organization coordinates genetic improvement plans in sheep breeds based on genealogical records and production controls of the variables included in each breedâ&#x20AC;&#x2122;s selection criteria and objectives. Growth variables such as animal live weight are recorded at five points or ages(1). Live weight data for different ages is used to generate a points distribution over time. This allows analysis and characterization of growth patterns based on nonlinear mathematical models (NLM), which use biological interpretation to summarize variation in live weight over time through a small number of growth parameters and indicators(2,3). Sheep production in Mexico occurs under various technological, agro-ecological and socioeconomic conditions. Organized and truthful documentation of events in the production unit, particularly financial variables, is essential for producers to determine unit profitability. Changes in animal live weight are influenced by genetic and environmental factors, with variable effects through time and during individual development. Each sheep breed has a characteristic growth pattern, requiring the testing of several NLM to identify that with the best fit for each breed. Identifying the NLM with the best fit provides objective and accurate growth pattern data which can be used by producers in decision-making regarding production, management and genetic improvement. The present study objectives were: 1) To identify the best-fit NLM to describe the growth curve in four hair sheep breeds (Blackbelly, Pelibuey, Dorper and Katahdin) and three wool sheep breeds 665

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(Suffolk, Hampshire and Rambouillet); and, 2) To generate growth indicators that can characterize and analyze these growth curves.

Materials and methods The analyzed database includes live weight records for female lambs in seven UNO-registered breeds: Blackbelly (BB), Pelibuey (PE), Dorper (DR), Katahdin (KT), Suffolk (SF), Hampshire (HS) and Rambouillet (RB). Analyzed variables were live weight at birth, 75, 120, 150 and 210 d of age, with measurements taken at intervals of Âą 20 d with respect to the reference age (Table 1). Weight at 75 d corresponds to weaning. Because males are sold beginning at 120 d, only data for females was used in the analyses.

Table 1: Number of records at each age for the seven evaluated sheep breeds Breed Katahdin Pelibuey Dorper Blackbelly Suffolk Hampshire Rambouillet

WB 24,878 14,164 11,487 7,151 3,636 2,597 2,504

W75 21,365 11,796 9,522 5,439 2,836 2,177 1,748

W120 11,500 5,301 5,802 2,475 1,542 1,236 1,189

W150 10,502 4,993 5,510 2,416 1,459 1,056 1,093

W210 5,909 2,771 3,493 1,603 794 495 850

Total 74,154 39,025 35,814 19,084 10,267 7,561 7,384

WB= live weight at birth; W75= live weight in 55 to 95 d interval; W120= live weight in 100 to 140 d interval; W150= live weight in 130 to 170 d interval; and W210= live weight in 190 to 230 d interval.

Data were from flocks mainly distributed in three regions of Mexico. Half (50 %) of the flocks were from the countryâ&#x20AC;&#x2122;s central region and included primarily the SF, HS and RB breeds. The south-southeast region accounted for 22 % of the database, and corresponded to the PE, BB, DR and KT breeds. The north region represented 18 % of the data and included mostly the BB, DR and KT breeds. The remaining 10 % of the data was from flocks in other regions. Production systems in the central region are largely intensive or semi-intensive using stables combined with cultivated pastures. Systems in the north and south-southeast regions are semi-intensive and extensive, combining grazing with corrals. In the north, large arid and semi-arid areas with multispecies pastures and scrub are used, whereas in the south-southeast the tropical climate promotes wide availability of tropical grasses. 666

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Seven NLM were evaluated: Brody (BRO), Verhulst (VER), von Bertalanffy (VBE), Gompertz (GOM), Mitscherlich (MIT) and logistic (LOG). All consisted of three regression coefficients (Î˛1, Î˛2 and Î˛3)(4,5,6). In NLM equations (Table 2), yi represents live weight (kg) measured at time t; Î˛1 is the asymptotic value when t tends to infinity, interpreted as the adult weight parameter (AW); Î˛2 is a fit parameter when y â&#x2030; 0 and t â&#x2030; 0; and Î˛3 is growth rate (GR), expressing weight gain as a proportion of total weight(2,7). The VER, VBE, GOM and LOG models describe growth based on a sigmoid curve, for which inflection point age (IPA) and weight (IPW) were estimated(8,9).

Table 2: Nonlinear models used to describe growth in registered sheep breeds Models Verhulst Logistic Von Bertalanffy Gompertz Brody Mitscherlich

Equation yi = Î˛1*(1 + exp(-Î˛2*t))-Î˛3 + ei yi = Î˛1 / (1 + Î˛2*(exp(-Î˛3*t))) + ei yi = Î˛1*((1 - Î˛2*(exp(-Î˛3*t)))**3) + ei yi = Î˛1*(exp(-Î˛2*(exp(-Î˛3*t)))) + ei yi = Î˛1*(1 - Î˛2*(exp(-Î˛3*t))) + ei yi = Î˛1*(1 - exp(Î˛3*Î˛2 - Î˛3*t)) + ei

yi= live weight in kg measured at time t; Î˛1= asymptotic value; Î˛2= integration constant; Î˛3= curve slope or growth rate.

Analyses were done using the Gauss-Newton method of the NLIN procedure in the SAS statistical program(10). Selection of the model with the best fit was done based on seven criteria(11,12,13): a) the Akaike information criterion [AIC = n*nl(sse/n) + 2k]; b) the Bayesian information criterion [BIC = n*nl(sse/n) + k*nl(n)]; c) the average prediction error [APE= ( â&#x2C6;&#x2018;ni=1 (

lwi â&#x2C6;&#x2019; ewi ewi

) â&#x2C6;&#x2014; 100 )/n]; d)

the prediction error variance [PEV = â&#x2C6;&#x2018;ni=1( ewi â&#x2C6;&#x2019; lwi)2 /n]; e) the Durbin-Watson statistic [DW= 2(1 â&#x20AC;&#x201C; Ď ); đ?&#x153;&#x152; =

2 â&#x2C6;&#x2018;n t=2(et â&#x2C6;&#x2019;etâ&#x2C6;&#x2019;1 ) 2 â&#x2C6;&#x2018;n t=1 et

]; f) the determination coefficient [R2 = (1 â&#x20AC;&#x201C; (sse/tss))]; and g) the đ?&#x2018; đ?&#x2018; đ?&#x2018;&#x2019;

general standard error or model error, from the root-mean-square error (GSE= â&#x2C6;&#x161;đ?&#x2018;&#x203A;â&#x2C6;&#x2019;đ?&#x2018;?â&#x2C6;&#x2019;1. Where: lwi = live weight (kg) at i-th age (d); ewi = estimated live weight (kg) at i-th age (d); n = total number of data; sse = sum-squared error; tss = total sum-squared; k = number of parameters in model; nl = natural logarithm. The APE analyzes the relationship between measured and estimated weight, and, as a function of the symbol, the NLM overestimates (+) or underestimates (-) the predictions. For APE, PEV, GSE, AIC and BIC, the model with the lowest value was considered to have the best fit; for R2 it was the model with the highest value. The DW analyzes for auto correlations in the errors using scenarios: if 2<DW<4, there is a negative auto correlation; if 0<DW<2, there is no auto correlation; and if DW<0, there is a positive auto correlation. 667

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Results and discussion The statistical criteria used for selection of the best-fit model for each breed showed that based on R2 all the NLM explained 94 % or more of the variability in the analyzed data (Table 3). All the NLM also tended to underestimate the predictions (negative APE) without auto correlation in the residuals (0<DW<2). The PEV and APE results did not differ within breeds, but were higher for the LOG model in all breeds. Based on the AIC and BIC, the MIT and BRO model results did not differ within breeds and were the best fit for the KT, BB, DR and RB breeds. For the HS, PE and SF breeds, however, the best-fit model was the VBE, with epi between 40 and 57 d (Table 4), an age within the preweaning period. Based on the NLM, average IPW was 16.4 kg for PE, 20.2 kg for HS and 23.2 kg for SF.

Table 3: Statistics used for selection of best-fit nonlinear models Breedsâ&#x20AC; BB

ModelsÂ§ LOG GOM VBE VER MIT BRO LOG GOM VBE VER MIT BRO LOG GOM VBE VER MIT BRO LOG GOM VBE

*PEV

20.4 19.3 19.1 19.9 18.8 19.0 44.3 41.7 41.1 42.2 40.5 41.0 44.3 42.8 42.6 43.7 42.8 42.8 37.1 35.6 35.3

*APE

-17.8 -10.5 -8.4 -13.5 -5.9 -6.0 -18.4 -10.5 -7.9 -9.8 -5.4 -5.8 -12.4 -7.3 -6.3 -9.9 -5.2 -5.4 -17.0 -9.9 -8.0

*DW

*R2

*GSE

*AIC

*BIC

0.66 0.58 0.56 0.62 0.54 0.56 1.30 1.30 1.32 1.31 1.36 1.39 0.04 0.04 0.04 0.04 0.04 0.04 0.68 0.64 0.64

0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.96 0.95 0.96 0.96 0.95 0.95 0.96 0.95 0.96 0.96 0.95 0.95 0.95

4.3 4.2 4.1 4.2 4.1 4.1 6.4 6.1 6.1 6.2 6.0 6.0 5.7 5.6 5.6 5.6 5.6 5.6 6.0 5.8 5.8

55904 54563 54202 54942 53757 53757 132665 130012 129282 130754 128389 128389 26115 25799 25749 25876 25755 25755 262113 257855 256792

55927 54587 54225 54966 53781 53781 132690 130037 129307 130779 128415 128415 26135 25820 25770 25897 25775 25775 262141 257882 256819

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VER MIT BRO LOG GOM VBE VER MIT BRO LOG GOM VBE VER MIT BRO LOG GOM VBE VER MIT BRO

35.9 35.3 35.4 26.4 25.6 25.5 26.1 25.6 26.2 19.8 18.7 18.5 19.1 18.3 18.4 46.8 45.0 44.8 45.9 44.8 44.9

-9.1 -6.1 -6.1 -15.1 -9.3 -7.0 -8.6 -5.2 -5.9 -5.6 -4.9 -4.2 -5.1 -3.5 -3.7 -9.1 -7.3 -6.1 -9.1 -5.3 -5.3

0.66 0.68 0.67 0.26 0.24 0.24 0.25 0.24 0.26 1.80 1.80 1.80 1.81 1.80 1.82 0.04 0.04 0.06 0.05 0.06 0.07

0.95 0.95 0.95 0.94 0.94 0.94 0.94 0.94 0.94 0.98 0.98 0.98 0.98 0.98 0.98 0.95 0.96 0.96 0.96 0.96 0.96

5.9 5.7 5.7 4.6 4.5 4.5 4.5 4.5 4.5 4.4 4.2 4.1 4.0 4.0 4.0 6.4 6.2 6.2 6.3 6.2 6.2

259020 255755 255755 118402 116815 116583 117161 116745 116745 21873 21119 20914 21355 20629 20629 37846 37354 37276 37467 37277 37277

259048 255782 255782 118428 116841 116608 117187 116771 116771 21894 21139 20935 21376 20650 20650 37867 37376 37298 37489 37299 37299

†

Breeds: BB= Blackbelly; PE= Pelibuey; DR= Dorper; KT= Katahdin; SF= Suffolk; HS= Hampshire; RB= Rambouillet. § Models: VER= Verhulst; LOG= Logistic; VBE= von Bertalanffy; GOM= Gompertz; BRO= Brody; MIT= Mitscherlich. *Statistics for model selection: PEV= prediction error variance; APE= average prediction error; DW= DurbinWatson statistic; R2= determination coefficient; GSE= general standard error; AIC= Akaike information criterion; BIC= Bayesian information criterion.

Table 4: Regression coefficients and growth indicators in evaluated nonlinear models Breeds† BB

Model§ LOG GOM VBE VER MIT BRO LOG GOM VBE

¥β 1

± se

33.1±0.13 36.9±0.21 40.0±0.28 35.2±0.17 61.3±1.22 61.2±1.31 48.8±0.14 54.1±0.22 58.6±0.29

¥β 2

± se

7.37±0.08 2.42±0.01 0.575±0.01 3.35±0.02 -13.11±0.03 0.955±0.02 7.61±0.07 2.48±0.01 0.586±0.01 669

¥β 3

± se

0.0243±0.0001 0.0139±0.0001 0.0103±0.0001 0.0171±0.0002 0.0034±0.0002 0.0034±0.0002 0.0242±0.0001 0.0140±0.0001 0.0105±0.0001

£IPW

£IPA

16.6 13.6 11.9 17.6

82 63 53 86

24.4 19.9 17.4

84 65 54

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VER MIT BRO LOG GOM VBE VER MIT BRO LOG GOM VBE VER MIT BRO LOG GOM VBE VER MIT BRO LOG GOM VBE VER MIT BRO LOG GOM VBE VER MIT BRO

51.8±0.17 88.8±1.23 88.9±1.22 45.4±0.24 50.0±0.37 53.2±0.48 48.0±0.31 68.6±1.36 68.6±1.36 43.5±0.09 48.6±0.15 52.9±0.21 46.3±0.12 84.9±1.01 84.9±0.98 35.9±0.01 40.7±0.18 44.7±0.24 38.7±0.14 78.9±1.40 78.9±1.41 42.7±0.16 45.9±0.23 48.1±0.28 44.5±0.19 56.8±0.62 56.9±0.61 51.7±0.23 57.5±0.36 61.6±0.49 55.1±0.36 84.0±1.61 84.0±1.61

3.43±0.01 -11.52±0.22 0.959±0.0 6.99±0.13 2.31±0.02 0.552±0.03 3.22±0.03 -12.31±0.49 0.935±0.01 7.42±0.04 2.44±0.01 0.581±0.01 3.38±0.01 -12.83±0.17 0.959±0.01 8.48±0.07 2.57±0.01 0.597±0.01 3.55±0.02 -12.01±0.22 0.966±0.01 6.05±0.09 2.14±0.02 0.524±0.02 3.00±0.02 -13.96±0.32 0.915±0.01 7.67±0.13 2.42±0.02 0.571±0.01 3.38±0.02 -12.03±0.35 0.945±0.01

†

0.0171±0.0001 0.0035±0.0001 0.0036±0.0001 0.0285±0.0003 0.0163±0.0002 0.0125±0.0001 0.0201±0.0002 0.0054±0.0002 0.0054±0.0001 0.0241±0.0001 0.0138±0.0001 0.0102±0.0001 0.0171±0.0002 0.0032±0.0001 0.0032±0.0001 0.0256±0.0001 0.0141±0.0001 0.0102±0.0001 0.0174±0.0001 0.0029±0.0001 0.0029±0.0001 0.0259±0.0002 0.0157±0.0001 0.0124±0.0001 0.0191±0.0001 0.0064±0.0001 0.0064±0.0001 0.0276±0.0002 0.0155±0.0001 0.0117±0.0001 0.0191±0.0001 0.0046±0.0001 0.0046±0.0001

25.9

22.7 18.3 15.8 24.0

68 51 40 71

21.8 17.9 15.7 23.2

83 65 54 86

17.9 14.9 13.2 19.4

83 67 57 88

21.4 16.9 14.3 22.3

69 48 36 70

25.8 21.1 18.3 27.6

74 57 46 77

Breeds: BB= Blackbelly; PE= Pelibuey; DR= Dorper; KT= Katahdin; SF= Suffolk; HS= Hampshire; RB= Rambouillet. § Models: VER= Verhulst; LOG= Logistic; VBE= von Bertalanffy; GOM= Gompertz; BRO= Brody; MIT= Mitscherlich. ¥ Regression coefficients in nonlinear models: β1= asymptotic value (kg); β2= fit parameter; β3= growth rate; se= standard error. £ Growth indicators: IPA= inflection point age (d); IPW= inflection point weight (kg).

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The growth curves based on the best-fit models showed the differences in growth pattern by breed (Figures 1 and 2). The growth curve describes and represents the evolution of live weight over time. Analysis of growth curves generates information that can be used in management, feeding and genetic improvement programs. The NLMs express the growth curve according to several components: adult weight, growth rate, degree of maturity, and inflection point age and weight, among others(2,7). Modifying or altering growth therefore requires strategies that improve these components(14,15). The VBE model is characterized by a sigmoid curve (Figure 2), with the inflection point being where the GR transitions from an acceleration process to a deceleration phase. The BRO and MIT models, in contrast, describe a continuous growth curve with no inflection point (Figure 1), and in which the GR as a proportion of the AW is constant over time(3,16).

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Figure 1: Growth curves for Katahdin (KT), Blackbelly (BB), Dorper (DR) and Rambouillet (RB) sheep breeds based on Brody model 60

Live weight, kg

0 1

106

121

136

151

166

181

196

211

226

Age, days

Figure 2: Growth curves for Pelibuey (PE), Suffolk (SF) and Hampshire (HS) sheep breeds based on von Bertalanffy model 60

Live weight, kg

10 PE

0 1

106 121 Age, days

136

151

166

181

196

211

226

Other studies highlight how different NLM provide the best fit for different sheep breeds. Similar studies with the Baluchi(5), Hemsin(17), and West African Dwarf(18) sheep breeds reported that the 672

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BRO model was best fitted to describe growth. In an analysis of growth in Morada Nova sheep(4), the Meloun I and Meloun III models were found to have the best fit, with growth patterns similar to those in the BRO and MIT models used in the present study. However, in the SegureĂąas(9) and Awassi breeds(19) the VBE model has been found to have the best fit. Marked differences were observed in AW in the seven evaluated breeds (Table 4). This parameter tended to be highest in the BRO and MIT models, and lowest in the LOG and VER models. Average values were 44.6 kg in BB, 49.2 kg in RB, 52.9 kg in PE, 55.6 kg in HS, 60.2 kg in KT, 64.7 in SF and 65.2 in DR. Increases in female AW affect maintenance, reproduction and waste value needs. Given that a large percentage of lamb production costs occur in ewes, increasing ewe size can raise production costs; however, asymptotic weight can be kept constant in selection programs while GR is maximized(14,20). Since GR refers to the velocity of growth relative to AW, high GR can result in AW being attained at a younger age. Growth rate (GR) is financially important because it can be used to determine the optimal moment for slaughter, which is usually when the animal has reached maximum GR(13,21). The correlations between AW and GR are essential in strategies aimed at modifying growth curves(15,21). All correlations between AW and GR in the present study were negative and high (-0.70 to -0.99). These negative correlations suggest certain growth curve characteristics: a) older AWs do not derive from high GRs; b) a lower GR may lengthen the time to reach AW; and c) in genetic improvement schemes, GR can be increased without affecting AW(7,15,22).

Conclusions and implications For the Hampshire, Pelibuey and Suffolk breeds, a nonlinear model based on the von Bertalanffy model produced sigmoid type growth curves with an inflection point at 40 to 57 d. For the Katahdin, Blackbelly, Dorper and Rambouillet breeds, a nonlinear model based on the Brody model resulted in growth curves with a continuous growth rate and no inflection point. The differences observed between the breeds as manifested in curve pattern and growth indicators express varying genetic potential, which can be exploited in different production systems.

Acknowledgments The authors thank the Organismo de la Unidad Nacional de Ovinocultores for providing access to its database within the framework of the collaboration agreement between the Universidad 673

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Autónoma de Chihuahua and the Consejo Nacional de los Recursos Genéticos Pecuarios. ECS received a scholarship from the Consejo Nacional de Ciencia y Tecnología to study his Master’s degree.

Literature cited: 1.

CONARGEN. Guía técnica de programas de control de producción y mejoramiento genético en ovinos. Consejo Nacional de los Recursos Genéticos Pecuarios. México, DF. 2010.

Lewis RM, Emmans GC, Dingwall WS, Simm G. A description of the growth of sheep and its genetic analysis. Anim Sci 2002;74:51-62.

Agudelo GDA, Cerón MF, Restrepo LFB. Modelación de las funciones de crecimiento aplicadas a la producción animal. Rev Colomb Cienc Pecu 2008;21:39-58.

de Andrade SL, Souza PLC, Mendes CHM, Fonseca S, Gomes da SF. Traditional and alternative nonlinear models for estimating the growth of Morada Nova sheep. Rev Bras Zootec 2013;42:651-655.

Bahreini BMR, Aslaminejad AA, Sharifi AR, Simianer H. Comparison of mathematical models for describing the growth of Baluchi sheep. J Agr Sci Tech 2014;14:57-68.

Teixeira NMR, da Cruz JF, Neves FH, Santos SE, Souza CPL, Mendes MC. Descrição do crescimento de ovinos Santa Inês utilizando modelos não-lineares seleccionados por análise multivariada. Rev Bras Saude Prod Anim 2016;17:26-36.

Malhado CHM, Carneiro PL, Alfonso PRA, Souza AA, Sarmento. Growth curves in Dorper sheep crossed with the local Brazilian breeds, Morada Nova, Rabo Largo, and Santa Inês. Small Ruminant Res 2009;84:16-21.

Ben HM, Atti N. Comparison of growth curves of lamb fat tail measurements and their relationship with body weight in Babarine sheep. Small Ruminant Res 2013;95:120-127.

Lupi TM, Nogales S, León JM, Barba C, Delgado JV. Characterization of commercial and biological growth curves in the Segureña sheep breed. Animal 2015;9:1341-1348.

10. SAS. SAS/STAT User's Guide (Release 9.0). Cary NC, USA: SAS Inst. Inc. 2005.

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11. Motulsky H, Christopoulos A. Fitting models to biological data using linear and nonlinear regression. A practical guide to curve fitting. Graph Pad Software Inc. 2003. 12. Hossein-Zadeh NG. Modeling the growth curve of Iranian Shall sheep using non-linear growth models. Small Ruminant Res 2015;130:60-66. 13. Hossein-Zadeh NG, Golshani M. Comparison of non-linear models to describe growth of Iranian Guilan sheep. Rev Colomb Cienc Pecu 2016;29:199-209. 14. Owens FN, Dubeski P, Hanson CF. Factors that alter the growth and development of ruminants. J Anim Sci 1993;71:3138-3150. 15. Lupi TM, León JM, Nogales S, Barba C, Delgado JV. Genetic parameters of traits associated with the growth curve in Segureña sheep. Animal 2016;9:729-735. 16. Ribeiro de FA. Curvas de crescimento na produçã animal. Rev Bras Zootec 2005;34:786-795. 17. Kopuzlu S, Sezgin E, Esenbuga E, Bilgin OC. Estimation of growth curve characteristics of Hemsin male and female sheep. J Appl Anim Res 2014;42:228-232. 18. Gbangboche AB, Glele-Kakai R, Salifou S, Albuquerque LG, Leroy PL. Comparison of nonlinear growth models to describe the growth curve in West African Dwarf sheep. Animal 2008;2:1003-1012. 19. Topal M, Ozdemir M, Aksakal V, Yildiz N, Dogru U. Determination of the best nonlinear function in order to estimate growth in Morkaraman and Awassi lambs. Small Ruminant Res 2004;55:229-232. 20. Bathaei SS, Leroy PL. Growth and mature weight of Mehraban Iranian fat-tailed sheep. Small Ruminant Res 1996;22:155-162. 21. Lambe NR, Navajas EA, Simm G, Bünger L. A genetic investigation of various growth models to describe growth of lamb of two contrasting breeds. J Anim Sci 2006;84:2642-2654. 22. Acioli da SLS, Bossi FA, de Lima da SF, Mendes GB, de Oliveira SR, Tonhati H, da Costa BC. Growth curve in Santa Inês sheep. Small Ruminant Res 2012;105:182-185.

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https://doi.org/10.22319/rmcp.v10i3.4825 Article

Farm-level risk factors associated with reproductive performance in small-scale dairy farms in Mexico Luis Javier Montiel-Olguín a,b Eliab Estrada-Cortés c Mario Alfredo Espinosa-Martínez a Miguel Mellado d Josafath Omar Hernández-Vélez e Guillermina Martínez-Trejo f Laura Hérnández-Andrade g Rubén Hernández-Ortíz h Arcelia Alvarado-Islas g Felipe J. Ruiz-López a Héctor Raymundo Vera-Avila a,* a

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). CENID Fisiología y Mejoramiento Animal, km.1 Carretera a Colón, 76280, Ajuchitlán, Colón, Querétaro, México. b

Universidad Autónoma de Querétaro. Facultad de Ciencias Naturales. Querétaro, México.

INIFAP. Campo Experimental Centro Altos de Jalisco. Jalisco, México.

Universidad Autónoma Agraria Antonio Narro. Departamento de Nutrición Animal. Coahuila, México. e

INIFAP. Campo Experimental San Martinito. Puebla, México.

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

INIFAP. CENID Salud Animal e Inocuidad. Ciudad de México, México 676

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INIFAP. CENID Salud Animal e Inocuidad. Morelos, MĂŠxico.

* Corresponding author: hrvera56@gmail.com

Abstract: The profitability of dairy farms is closely linked to reproductive performance. Identifying risk factors that compromise this performance is vital to implementing strategies to improve productivity. An analysis was done of the effects on reproductive performance of artificial insemination (AI) use, herd size and high seroprevalence of reproductive infectious diseases. Data on reproductive events were collected from 52 farms (10-100 cows; 959 lactations) over 18 mo (births 2011-2012). Neosporosis, bovine infectious rhinotracheitis (IBR) and bovine viral diarrhea (BVD) seroprevalences were documented at each farm. Multiple logistic regression analyses were applied to determine the degree of association (odds ratio, OR) between potential risk factors and reproductive variables. Herds of 33 or more cows (OR= 1.5) and high neosporosis seroprevalence (OR= 2.3) were risk factors for assisted calving. High IBR and BVD seroprevalences (OR= 1.3 and 1.9, respectively) were risk factors for days to first service over 70 d in milk (DFS>70). Artificial insemination was a common risk factor for DFS>70 (OR= 2.4) and days open over 110 days in milk (OR= 1.3). Herds of 33 or more cows was a risk factor for nonpregnant cows at first service (OR= 1.7). Artificial insemination, herds of 33 or more cows and high neosporosis, IBR and BVD seroprevalences are factors associated with reproductive performance in small-scale dairy farms in various geographical regions in Mexico. Key words: Artificial insemination, Risk factors, Neosporosis, BVD, IBR. Received: 27/03/2018 Accepted:21/08/2018

Introduction Small-scale milk production systems improve food security and provide income in rural areas worldwide(1). In Mexico, this production system accounts for approximately 23 %of livestock inventory(2), 30 % of national milk production(3), and 73 % of dairy farms(4). Small scale dairy farms in Mexico are characterized by the use of family labor and specialized dairy breeds, the presence of few milking cows and medium-low technology levels(5-7). Improved productive practices at small-scale dairy farms contribute to reducing poverty in rural areas (1), and promoting community development(8,9). 677

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Dairy farm profitability is closely linked to efficient reproductive performance(10,11). Identifying risk factors that compromise reproductive performance is paramount to designing and implementing strategies that improve productivity. Studies of small-scale dairy herds indicate that, compared to natural breeding (NB), artificial insemination (AI) may affect the interval from birth to first postpartum service and the conception rate to first service compared to NB(12,13). In addition, studies of intensive production systems have shown that herd size influences reproductive performance(14-16),as do seroprevalences of reproductive infectious diseases such as neosporosis, bovine infectious rhinotracheitis (IBR) and bovine viral diarrhea (BVD)(17,18). The objective of the present study was to analyze the impact, as potential farm-level risk factors, of AI use, herd size and prevalence of infectious reproductive diseases on reproductive performance in small-scale dairy farms in Mexico. The working hypothesis was that these factors are associated with reproductive performance in dairy cows.

Material and methods Farm selection and data collection An observational prospective cohort study (959 records) was conducted in six states in Mexico with a substantial presence of small-scale dairy production systems. The study included 52 farms distributed among the six states: Jalisco (23); Estado de México (10); Tlaxcala (9); Guanajuato (4); Puebla (3); and Querétaro (3). Selection criteria included: primarily family labor used in production unit; 10 to 100 milking cows; milk production as primary objective of farm; and medium-low technology level. Holstein breed cows accounted for 91.3 % of the animals at the studied farms, and average number of cows per farm was 30.3 ± 2.4. The estimated culling rate was 26.4 %, and milk production per cow was 17.10 ± 0.5 kg/d. The farms included in the study meet the characteristics of smallscale production farms in Mexico(2,7,19). Data collection in the field was done for 18 mo, during which data on reproductive events were recorded: dates of parturition, service types (artificial or natural breeding) and dates, assisted calving or retained fetal membranes, and 50-d postservice gestation diagnosis results.

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Events of interest and classification of potential risk factors linked to reproductive performance

Five events of interest were considered in the analysis: assisted calving (minor and major assistance pooled into the same category); retained fetal membranes (>12 h); days to first service over 70 days in milk (DFS> 70); days open over 110 days in milk (DO>110 ); and non-pregnant cows at first service (NP1S). Based on previous data from smallscale milk production systems, the >70 DFS and >110 DO limit values were established as indicators of reproductive failure(19). Potential risk factors included use of AI, large herd size and high seroprevalence of neosporosis, IBR and BVD. Farms were classified by service type into AI, if at least 75 % of services were AI, and NB, if at least 75 % were NB. Herd size and seroprevalence values were established according to quartile distribution in the study sample(20). Classification of herd size was done based on the average number of producing cows per farm during the field data collection period. The third quartile corresponded to 33 cows (classification <33 or ≥33), and was thus established as the limit to classify farms as high seroprevalence for neosporosis (≥84 %), IBR (≥38 %) and BVD (= 100 %) (Table 1).

Identification of neosporosis, IBR and BVD seropositive animals Blood samples were taken by puncture of the coccygeal vein (vacutainer system) in a randomly selected 10 % of the producing cows in each studied herd. Samples were kept at 4 °C for 24 h, and centrifuged (2,500 xg for 10 min at 4 °C) to separate the serum, which was frozen at -20 °C until analysis. Detection of Neospora caninum antibodies was done with a commercial ELISA test kit (IDEXX Laboratories), following manufacturer instructions. Serum analysis for BVD was run with a commercial ELISA kit for blocking (CIVTEST bovis BVD / Bd P80, Hipra Laboratories), following manufacturer instructions. The IBR analysis was done using the plate neutralization technique, with the MDBK cell line (bovine kidney cells), the IBR758 reference virus, and a 105.6 TCID50% titer at a dilution of 500-1000 infecting doses/ml. Sample positivity was determined by diluting the sera from 1:2 to 1:128, and observing the cytopathic effect produced by the virus(18). Vaccination records were not available for each farm but this practice is common in the studied regions(13,18).

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Statistical analysis All analyzes were done using the SAS 9.3 statistical package (SAS Institute Inc., Cary, NC). Identification of risk factors was done with multiple logistic regression analysis (PROC LOGISTIC), following the methodology implemented by Potter et al(21). Development of these models involved first running simple logistic regression tests between the events of interest and the potential risk factors. Factors with a value of P<0.35 were retained and analyzed later for collinearity(21). Collinearity in multiple models was prevented by generating the correlation coefficients and applying paired χ2 tests of the retained factors using the FREQ procedure with the CHISQ option. When the confidence limit of a factor pair’s correlation coefficient did not include 0 and the P value of χ2 was <0.05, both variables were not included in the same multiple model. Finally, parsimonious multiple models were generated with the BACKWARD option to retain significant variables at a P<0.1(21). The final multiple models included only the main effects, and the odds ratio (OR) was used as a measure of association between the risk factors and the variables of interest.

Results Events of interest and farm-level potential risk factors related to reproductive performance

Prevalence for events of interest were 13.2 % for assistance at delivery, 11.7 % for retained fetal membranes, 64.9 % for DFS>70, 46.4 % for DO>110 and 50.5 % for NP1S. Artificial insemination (AI) was a potential risk factor at 73.9 % of the farms while herd size ≥33 cows was one at 41.3 % of the farms. Seroprevalence for neosporosis, IBR and BVD varied widely among the farms (Table 1). Table 1: Seroprevalences for neosporosis, infectious bovine rhinotracheitis (IBR) and bovine viral diarrhea (BVD) in 52 small-scale dairy farms

Mean ± SE Neosporosis 52.7±4.5 IBR 23.3±1.8 BVD 59.7±3.2

Quartile Quartile Minimum 1 Median 3 Maximum 0 33 50 84 100 0 0 23.5 38 75 0 28 75 100 100

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Risk factors associated with reproductive performance failure The simple logistic regression analyses (Table 2) and multiple models for each event of interest (Table 3) showed herd size ≥33 cows and high neosporosis seroprevalence to be risk factors (P<0.10) for the assisted calving variable (Table 4). No risk factors were identified (OR>1) for retained fetal membranes, although AI and high IBR seroprevalence were significant factors (OR<1, P<0.10; Table 4). For the DFS>70 variable the risk factors were AI and high IBR and BVD seroprevalences (P<0.10; Table 5). The only risk factor identified for DO>110 was AI (P<0.10, Table 5), while the only one identified for NP1S was herd size ≥33 cows (P<0.10).

Table 2: Probability (P) and odds ratio (OR) values for potential risk factors considering different events of interest; simple logistical regression analysis Events of interest Factors AI

AC (P;OR) 0.048; 0.67

RFM (P;OR) DFS>70 (P;OR)

DO (P;OR)

NP1S (P;OR)

0.072; 0.68

<0.001; 2.39

0.054; 1.32

0.162; 0.82

Herd size ≥33 cows 0.090; 1.52

0.821; NC

0.853; NC

0.823; NC

0.001; 1.69

High Neosporosis

<0.001; 2.29

0.099; 0.64

0.414; NC

0.357; NC

0.718; NC

High IBR

0.202; 1.32

0.005; 0.42

0.169; 1.25

0.484; NC

0.916; NC

High BVD

0.010; 0.46

0.793; NC

<0.001; 1.86

0.553; NC

0.049; 0.72

AC=assisted calving; RFM= retained fetal membranes; AI= artificial insemination; DFS>70 = days to first service over 70 d in milk; DO>110 = days open over 110 d in milk; NP1S = non-pregnant cows at first service; NC = OR not calculated due to lack of significance.

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Table 3: Non-collinear potential risk factors for events of interest included in multiple models Events of interest

Model

Potencial risk factors

Assisted calving

AI + High IBR

Herd size ≥33 cows + High Neosporosis

High BVD

AI + High neosporosis

AI + High IBR

High BVD + High IBR

DO>110

NP1S

Herd size ≥33 cows

High BVD

Retained fetal membranes

DFS>70

AI= artificial insemination; DFS>70= days to first service over 70 days in milk; DO>110= days open over 110 days in milk; NP1S= non-pregnant cows at first service; BVD= bovine viral diarrhea; IBR= infectious bovine rhinotracheitis.

Table 4: Effect of study variables on assisted calving and retained fetal membranes in multiple models Variable AC Model 1 Model 2

Model 3 RFM Model 1 Model 2

Effects

CI95%

Service type: NB Service type: AI Herd size: <33 cows Herd size: ≥33 cows Neosporosis: Remainder Neosporosis: High BVD: Remainder BVD: High

Ref. 0.67 Ref. 1.51 Ref. 2.28 Ref. 0.46

N/A 0.45-0.99 N/A 0.93-2.45 N/a 1.52-3.40 N/A 0.26-0.83

N/A 0.048 N/A 0.090 N/A 0.001 N/A 0.010

Service type: NB Service type: AI Service type: NB Service type: AI IBR: Remainder IBR: High

Ref. 0.68 Ref. 0.66 Ref. 0.41

N/A 0.45-1.04 N/A 0.43-1.01 N/A 0.23-0.75

N/A 0.072 N/A 0.055 N/A 0.004

P= probability value; AC= Assisted calving; RFM= Retained fetal membranes NB= natural breeding; AI= artificial insemination; BVD= bovine viral diarrhea; IBR= infectious bovine rhinotracheitis; CI= odds ratio confidence interval; OR= odds ratio; N/A= not applicable. 682

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Table 5: Effect of study variables on days to first service (DFS>70), days open (DO>110) and non-pregnant cows at first service (NP1S) with different multiple models Variable

Effects

CI 95%

Service Type: NB

Ref.

N/A

Service Type: AI

2.42

1.81-3.2

<0.001

IBR: Remainder

Ref.

N/A

IBR: High

1.32

0.95-1.84

0.097

BVD: Remainder

Ref.

N/A

BVD: High

1.86

1.31-2.64

<0.001

Service Type: NB

Ref.

N/A

Service Type: AI

1.32

0.99-1.8

0.054

Herd size: <33

Ref.

N/A

Herd size: â&#x2030;Ľ33

1.69

1.23-2.32

0.001

BVD: Remainder

Ref.

N/A

BVD: High

0.72

0.52-1.00

0.049

DFS>70 Model 1

Model 2

DO>110 Model 1

NP1S Model 1

Model 2

P= probability value; NB= natural breeding; AI= artificial insemination; BVD= bovine viral diarrhea; IBR= infectious bovine rhinotracheitis; CI= odds ratio confidence interval; OR= odds ratio; N/A= not applicable.

Discussion

Small-scale dairy systems in Mexico are highly heterogeneous in terms of productive, reproductive and health status(13,19). The diseases neosporosis, IBR and BVD are associated with reproductive disorders(22,23). In the present data, mean neosporosis seroprevalence values per farm were similar to those previously reported for small-scale dairy systems (51.7 %)(24), but higher than reported for intensive systems in Mexico (~43 %)(18,25). Biosafety measures in small dairy farms may be less stringent, which could increase risk factors associated with the presence of Neospora, such as dogs in production units(26,27). The seroprevalence of IBR in the present study was also similar to previous reports of small dairy 683

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farms in Mexico, with rates near 22 %(24,28), but was notably lower than the 69% seroprevalence reported in other small-scale production systems(18). Average BVD seroprevalence per farm was within reported ranges (52-81%)(18,24). Earlier studies have proposed that these three diseases are endemic and highly prevalent in dairy cattle in intensive, double-purpose and small-scale systems in Mexico(18,29,30). Although these high reported seroprevalences may be due to IBR and BVD vaccine antibodies, their broad distribution and high prevalences in the present results confirm their importance in small-scale dairy farms. It seems this is the first study reporting farm-level risk factors associated with reproductive performance in small-scale dairy farms in Mexico. Farms with high neosporosis seroprevalence were 128 % more probable to require calving assistance, while those with herds of 33 or more cows were 51 % more probable to require it. These results coincide with previous reports of a significant association between assistance in parturition and Neospora-seropositive animals(31). Nonetheless, there are studies in which this association has not been identified(32), highlighting the need to clarify this potential association. A possible reason for assistance in parturition being more prevalent in farms with ≥33 cows may be that at larger farms larger sires (AI or NB) are used, or that problems exist with body condition at birth, which still requires confirmation. Effective management in the peripartum and correct obstetric care are some of the main factors in controlling problems of dystocia(33). Labor at small-scale dairy farms usually consists only of family members, which can´t lead to possible labor shortages at critical moments such as parturition(6). This could be particularly acute at farms with ≥33 cows. At the farms with the highest BVD prevalence and which use AI, risk of the need for assistance was much lower. This result is to be expected since birth weight in calves positive to BVD antigens is 7 kg lighter than in BVD negative calves(34). Moreover, use of AI can also influence the need for calving assistance because there is currently a wide variety of sires in the market offering multiple traits, such as calving ease(35,36). Perhaps the studied farms had been selecting sires with just such a trait (pers. comm., MC Fernando Villaseñor). The farms using AI also exhibited a lower risk of retained fetal membranes (Table 4). Assisted calving is one of the most important risk factors associated with retained fetal membranes at the individual level(37). Use of AI may therefore reduce the need for assistance and consequently placental retention. In the farms with high IBR seroprevalence risk of placental retention was lower, which is apparently counterintuitive since IBR has been associated with abortion and placental retention(38). One possible explanation for the present results is that the high IBR seroprevalences observed here were due to vaccine antibodies(18), which would actually reduce rates of abortion due to IBR, consequently decreasing the risk of placental retention(39). Cows at farms where AI was used were 142 % more likely to have DFS>70. The success of AI rests largely on how efficiently estrus is detected(40). Although not recorded as part of the present study, estrus detection rates are commonly low at both small dairy farms(12), and intensive dairy production systems in Mexico(41). Cows at farms with a high IBR seroprevalence were 32 % more likely to have DFS>70, whereas those at farms with 684

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high BVD seroprevalence were 86 % more likely to have DFS>70; in the case of IBR, this varied widely from a 5 % reduction in probability to an increase of 80 %. The reproductive consequences of BVD infection are well known(42,43), but there are no studies associating high IBR and BVD seroprevalences with days to first service. Indeed, from a pathological point of view, it is unclear how these diseases could increase DFS>70. One possible explanation is that high seroprevalences of these diseases could indirectly impact this indicator(44), or they may be correlated with other risk factors such as overcrowding or mismanagement of biological waste(45). On average, cows at farms where AI was used were 32 % more likely to have DO>110, although this varied from a 1 % reduction to an 80 % increase. AI does allow for greater genetic selection but also has negative impacts on indicators such as days to first service, suggesting that estrus detection techniques are deficient(12,46). Estrus synchronization protocols have been implemented to counteract this tendency in intensive dairy production systems(47). Given the good fertility at first service in the studied system (49.5 %), implementation of fixed-time insemination protocols adapted specifically to this production system could improve reproductive performance(48). Herd size >33 cows made them 69 % more likely to be NP1S. In other production systems, as herd size increases the capacity for effective reproduction management decreases(14-16). Although small-scale dairy production systems clearly have fewer animals to manage than intensive systems, the effect of herd size is still a telling indicator. Presence of BVD has been reported to cause early embryo death and subfertility in dairy cattle(49-51), but the present results showed herds with high BVD seroprevalence to a have lower risk of NP1S. This may seem contradictory, but these high seroprevalences could be due to vaccine antibodies(18). Use of AI was a significant common factor influencing most of the events of interest. This genetic improvement technology reduces post-partum complications without affecting fertility at first service, but increases days at first service and days open (probably due to estrus detection deficiencies). Considering the good fertility rates observed at the studied small-scale dairy farms, one possible strategy to take full advantage of AI would be implementation of fixed-time synchronization protocols for the first service, with sexed semen. However, financial feasibility studies are needed before broad-scale recommendations can be made for this sector. An obvious limitation in the present study is the uncertainty surrounding the source of the antibodies in the serological tests. No reliable vaccination history data were available in the data because the studied small-scale dairy farms did not keep exhaustive vaccination records, even though vaccination is common in the studied regions(13,18). This uncertainty limits the ability to make more accurate inferences based on the present results. However, interpretation of the results has been conservative and can function as a baseline for subsequent epidemiological and pathological studies.

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Conclusions and implications In small-scale dairy farms in Mexico, AI, herd size and high seroprevalences of neosporosis, IBR and/or BVD are factors associated with reproductive performance. The risk factors identified for assisted calvings were herds of 33 or more cows and high neosporosis seroprevalence. Those for DFS>70 were AI and high IBR and BVD seroprevalences, while for DO>110 it was AI. The single risk factor for NP1S was herds of 33 or more cows, and no risk factors were identified for retained fetal membranes. The present study also highlights the need to prevent neosporosis, IBR and BVD, all of which are widely distributed in Mexican dairy farms.

Acknowledgements The research reported here was financed by the Fondo Sectorial de Investigación en Materias Agrícola, Pecuaria, Acuacultura, Agrobiotecnología y Recursos Fitogenéticos (SAGARPACONACYT), proyecto 2010-01-144591.

Conflict of interest The authors declare that they have no financial or personal relationships that could have inappropriately influenced development and reporting of this research.

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Barrera Camacho G, Sánchez Brito C. Caracterización de la cadena agroalimentaria nacional e identificación de sus demandas tecnológicas. Leche. Reporte Final Etapa III. Programa Nacional Estratégico de Necesidades de Investigación y de

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Transferencia de Tecnología Reporte Final Etapa III. Fundación Produce Jalisco. 2003. 4.

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10. Gröhn YT, Rajala-Schultz PJ. Epidemiology of reproductive performance in dairy cows. Anim Reprod Sci 2000;60-61(Suppl C):605-614. 11. Inchaisri C, Jorritsma R, Vos PLAM, van der Weijden GC, Hogeveen H. Economic consequences of reproductive performance in dairy cattle. Theriogenology 2010;74(5):835- 846. 12. Estrada-Cortés E, Espinosa-Martínez MA, de la Torre-Sánchez JF, VillaseñorGonzález F, Vera-Avila HR, Villagomez-Amezcua E, Montiel Olguín LJ. Factores que influyen en la detección del primer estro post-parto en vacas del sistema familiar 687

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23. Mineo TW, Alenius S, Näslund K, Montassier HJ, Björkman C. Distribution of antibodies against Neospora caninum, BVDV and BHV-1 among cows in brazilian dairy herds with reproductive disorders. Rev Bras Parasitol Vet 2006 Oct 1:188-192. 24. Ojeda-Carrasco JJ, Espinosa-Ayala E, Hernández-García PA, Rojas-Martínez C, Álvarez-Martínez JA. Seroprevalencia de enfermedades que afectan la reproducción de bovinos para leche con énfasis en neosporosis. Eco Rec Agro 2016; 3(8):243-249. 25. Garcia-Vazquez Z, Rosario-Cruz R, Ramos-Aragon A, Cruz-Vazquez C, MapesSanchez G. Neospora caninum seropositivity and association with abortions in dairy cows in Mexico. Vet Parasitol 2005;134(1):61-65. 26. Bartels CJM, Wouda W, Schukken YH. Risk factors for Neospora caninumassociated abortion storms in dairy herds in The Netherlands (1995 to 1997) Theriogenology 1999;52(2):247-257. 27. Hobson JC, Duffield TF, Kelton D, Lissemore K, Hietala SK, Leslie KE, et al. Risk factors associated with Neospora caninum abortion in Ontario Holstein dairy herds. Vet Parasitol 2005;127(3):177-188. 28. Magaña-Urbina A, Solorio Rivera JL, Segura-Correa JC. Rinotraqueitis infecciosa bovina en hatos lecheros de la región Cotzio-Téjaro, Michoacán, México. Téc Pecu Méx 2005;43(1):27-37. 29. Solorio Rivera JL. Análisis de riesgo de enfermedades abortivas en el sistema de lechería familiar en la región centro del estado de Michoacán [Tesis doctoral]. México. Universidad Autónoma de Yucatán; 2004. 30. Segura-Correa JC, Solorio-Rivera JL, Sánchez-Gil LG. Seroconversion to bovine viral diarrhoea virus and infectious bovine rhinotracheitis virus in dairy herds of Michoacan, Mexico. Trop Anim Health Prod 2010;42(2):233-238. 31. Pulido-Medellín MO, Díaz-Anaya AM, Andrade-Becerra RJ. Asociación entre variables reproductivas y anticuerpos anti Neospora caninum en bovinos lecheros de un municipio de Colombia. Rev Mex Cienc Pecu 2017;8(2):167-174.

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32. Ansari-Lari M, Rowshan-Ghasrodashti A, Jesmani H, Masoudian M, Badkoobeh M. Association of Neospora caninum with reproductive performance in dairy cows: A prospective study from Iran. Vet Res Forum 2017;8(2):109-114. 33. Mee JF. Prevalence and risk factors for dystocia in dairy cattle: A review. Vet J 2008;176(1):93-101. 34. Kane SE, Holler LD, Braun LJ, Neill JD, Young DB, Ridpath JF, Chase CCL. Bovine viral diarrhea virus outbreak in a beef cow herd in South Dakota. J Am Vet Med Assoc 2015;246(12):1358-1362. 35. Ali TE, Burnside EB, Schaeffer LR. Relationship Between External Body Measurements and Calving Difficulties in Canadian Holstein-Friesian Cattle. J Dairy Sci 1984;67(12):3034-3044. 36. Abo-Ismail MK, Brito LF, Miller SP, Sargolzaei M, Grossi DA, Moore SS, et al. Genome- wide association studies and genomic prediction of breeding values for calving performance and body conformation traits in Holstein cattle. Genet Sel Evol 2017;49(1):82. 37. Kovács L, Kézér FL, Szenci, O. Effect of calving process on the outcomes of delivery and postpartum health of dairy cows with unassisted and assisted calvings. J Dairy Sci 2016;99(9):7568-7573. 38. Sibhat B, Ayelet G, Skjerve E, Gebremedhin EZ, Asmare K. Bovine herpesvirus-1 in three major milk sheds of Ethiopia: Serostatus and association with reproductive disorders in dairy cattle. Prev Vet Med 2018;150:126-132. 39. Gould S, Cooper VL, Reichardt N, O’Connor AM. An evaluation of the prevalence of Bovine herpesvirus 1 abortions based on diagnostic submissions to five U.S.-based veterinary diagnostic laboratories. J Vet Diagnostic Investig 2013;25(2):243-247. 40. Burnett TA, Madureira AML, Silper BF, Fernandes ACC, Cerri RLA. Integrating an automated activity monitor into an artificial insemination program and the associated risk factors affecting reproductive performance of dairy cows. J Dairy Sci 2018;100(6):5005- 5018.

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41. Stevenson JS. Reproductive management of dairy cows in high milk-producing herds. J Dairy Sci 2018;84:E128-143. 42. Grooms DL. Reproductive consequences of infection with bovine viral diarrhea virus. Vet Clin North Am Food Anim Pract 2004;20:5-19. 43. Gonzรกlez-Altamiranda EA, Kaiser GG, Mucci NC, Verna AE, Campero CM, Odeรณn AC. Effect of bovine viral diarrhea virus on the ovarian functionality and in vitro reproductive performance of persistently infected heifers. Vet Microbiol 2013;165:326-332. 44. Szumilas M. Explaining odds ratios. J Can Acad Child Adolesc Psychiatry 2010;19(3):227. 45. De Kruif A. Factors influencing the fertility of a cattle population. J Reprod Fertil 1978;54(2):507-518. 46. Mottram T. Animal board invited review: Precision livestock farming for dairy cows with a focus on oestrus detection. Animal 2016;10(10):1575-1584. 47. Chebel RC, Al-Hassan MJ, Fricke PM, Santos JEP, Lima JR, Martel CA, et al. Supplementation of progesterone via controlled internal drug release inserts during ovulation synchronization protocols in lactating dairy cows. J Dairy Sci 2010;93(3):922-931. 48. Fricke PM, Carvalho PD, Giordano JO, Valenza A, Lopes G, Amundson MC. Expression and detection of estrus in dairy cows: the role of new technologies. Animal 2014;8(s1):134- 143. 49. Virakul P, Fahning ML, Joo HS, Zemjanis R. Fertility of cows challenged with a cytopathic strain of Bovine Viral Diarrhea virus during an outbreak of spontaneous infection with a noncytopathic strain. Theriogenology 2018;29(2):441-449. 50. Houe H, Pedersen KM, Meyling A. The effect of bovine virus diarrhoea virus infection on conception rate. Prev Vet Med 1993;15(2):117-123. 51. McGowan MR, Kirkland PD, Richards SG, Littlejohns IR. Increased reproductive losses in cattle infected with bovine pestivirus around the time of insemination. Vet Rec 1993;133(2):39-43. 691

https://doi.org/10.22319/rmcp.v10i3.4822 Article

In vitro acaricide activity of extracts from three Leucaena spp. genotypes versus Rhipicephalus microplus

Guadalupe González-López a Melina Maribel Ojeda-Chi b Fernando Casanova-Lugo a* Iván Oros-Ortega a Luis Ignacio Hernández-Chávez c Ángel Trinidad Piñeiro-Vázquez d Roger Iván Rodríguez-Vivas b

Tecnológico Nacional de México / I.T. Zona Maya, Quintana Roo, México.

Universidad Autónoma de Yucatán. Facultad de Medicina Veterinaria y Zootecnia. Mérida,

Yucatán, México. c

Tecnológico Nacional de México / I.T.S. Felipe Carrillo Puerto, Quintana Roo, México.

Tecnológico Nacional de México / I.T. Conkal, Yucatán, México.

*Corresponding author: fkzanov@gmail.com

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Abstract: The tick Rhipicephalus microplus is known to develop resistance against some commercial acaracides, driving a search for natural alternatives. An evaluation was done of the acaricide activity against adult and larval R. microplus of ethanol extracts from three Leucaena spp. genotypes: L. leucocephala (Lam.) de Wit (Native); L. leucocephala (Cunningham); and L. Leucocephala x L. padilla (KX2). Larval immersion and adult immersion tests were used to evaluate acaricide activity. Secondary metabolite profiles of the three genotypes were generated using analytical chromatographic plates. Against the larvae, the 50% extract concentration exhibited 91.68% mortality for the Cunningham genotype, 82.00% for the KX2 and 54.06% for the Native. The Native genotype extract was most effective against adults with a 50% mortality at a 20% concentration. Flavonoids and terpenes were identified in all three genotypes and are probably responsible for their acaricide activity. The Leucaena spp. Cunningham and KX2 extracts were effective against R. microplus larvae, but further research is needed to identify the metabolites that provide this acaricide activity, be it individually or synergistically. Key words: Ectoparasites, Secondary metabolites, Vegetal extracts, Subhumid tropics. Received: 23/03/2018 Accepted: 19/06/2018

Introduction The tick Rhipicephalus microplus is among the ectoparasites which cause the greatest losses in livestock. It is also a vector for pathogenic agents such as Anaplasma marginale, Babesia bigemina and B. bovis(1). Worldwide an estimated 80% of cattle are infested with ticks, resulting in annual losses from two to three billion US dollars. Annual losses due to R. microplus infestations in cattle in Mexico were recently estimated to be 573.61 million USD(2).

The most common way of controlling R. microplus is the use of chemical compounds such as pyrethroids (PT), organophosphates (OP), amidines (AM), phenylpyrazolones, tick growth inhibitors and macrocyclic lactones. However, in recent years an increase in ixodicidemultiresistant strains (mainly PT, OP and AM) has been reported in southeastern Mexico(3). There are also now reports of R. microplus resistant to ivermectin(4), and to fipronil(5). This situation is driving a search for alternative methods for controlling R. microplus that also reduce potential damage to the environment and humans(1).

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Plant extracts are promising alternative approaches to arthropod control. They produce secondary metabolites with different action mechanisms such as inhibition of feeding and chitin synthesis; decreased growth, development, and reproduction; and behavioral alterations; this, without adverse effects to non-target species(6). Extracts from plants in the families Lamiaceae, Fabaceae, Asteraceae, Piperaceae, Verbenaceae and Poaceae exhibit the greatest efficacy in tick control(7-11). Secondary metabolites with an acaricide effect versus R. microplus have been identified in plant extracts from Cunila angustifolia, Acacia pennatula, Piscidia piscipula, Leucaena leucocephala, Tagetes minuta, Piper amalago, Lippia graveolens and Milinis minutiflora. These include terpenes, stilbenes, coumarins, acids, alcohols, sulfide compounds, tannins, and aldehydes from essential oils(7,12).

Few studies have been done to date on the effect of Leucaena spp. extracts in tick control. For example, one study assessed the effect of a L. leucocephala extract on R. microplus and found 66.79 % efficacy versus larvae, but none against adults(12). In contrast, a study of defensive proteins produced by L. leucocephala (Lam.) de Wit were found to have an effect against adult R. microplus (56.3 % efficacy)(13).

Three Leucaena genotypes are currently available in southeast Mexico: L. leucocephala (Native); L. leucocephala (Cunningham); and L. leucocephala x L. padilla (KX2). These are widely used as forage shrub plants in tropical silvopastoral systems, and may also contribute to controlling arthropods. The present study objective was to evaluate in vitro the acaricide activity of ethanol extracts from these three Leucaena genotypes against R. microplus adults and larvae.

Materials and methods Experimental site

The study was done at the Maya Zone Technological Institute (Instituto Tecnológico de la Zona Maya - ITZM), Carretera Chetumal–Escárcega Km. 21.5, Ejido Juan Sarabia, Municipality of Othón P. Blanco, in the state of Quintana Roo, Mexico (18°30”58’ N; 88°29”19’ W). Average maximum temperature in the area is 32.1 °C, and the average minimum is 21.7 °C. Annual rainfall averages 1,180 mm, and average relative humidity (RH) ranges from 76 to 82% (http://smn.conagua.gob.mx).

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Rhipicephalus microplus larva production

A total of 300 engorged adult ticks were collected from at least 25 cattle on the Las Flores Ranch located in the town of Xul-ha, Quintana Roo, Mexico. The ticks were placed in glass tubes with cotton lids and transported to the ITZM Biological Control Laboratory. Here they were washed, dried and weighed. Average tick weight was 200 ± 20 mg. One group of engorged ticks was used in the adult immersion test (AIT) and another group was incubated at 27 ± 1.5 °C and 70 to 80% RH for two weeks to allow oviposition(14). The eggs were transferred to 10 ml glass vials with a cotton lid. Larvae hatched approximately 30 days after collection of engorged females. Larvae from 7 to 14 days of age were used in the larval immersion test.

Vegetal material

Three Leucaena spp. genotypes were included in the study: L. leucocephala (Lam.) de Wit (native); L. leucocephala (Cunningham); and L. leucocephala x L. padilla (KX2). A total of 25 kg of fresh leaves were collected from each genotype. These were dried at 60 ºC for 72 h, and ground with an electric Wiley mill (Thomas Scientific®) to a 3 mm particle size.

Extract production

At the ITZM laboratory the ground Leucaena leaves were submerged in 80% ethanol (absolute ethanol) for 72 h (800 ml methanol and 350 g ground leaves per genotype). Ethanol was used because it is a general polar solvent that extracts compounds of interest (e.g. terpenes and flavonoids) from vegetal material. Each ethanol extract was filtered and evaporated at 45 °C in a rotational evaporator under a vacuum. The resulting crude extracts were transferred to glass flasks and kept at 4 °C until use.

Bioassay with larvae

Efficacy of the three Leucaena genotypes was quantified by larval bioassays using the larvae immersion test as modified by Soberanes et al.(15). Six concentrations (50, 40, 30, 20 and 10%) of each Leucaena genotype extract were tested. Each concentration was transferred to Petri dishes (60 mm x 15 mm diameter), 300 to 500 larvae placed between two sheets of Whatman No. 1 paper,

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and these submerged for 10 min in the extract. Approximately 100 larvae were collected with a brush (No. 4), gently transferred to an envelope of clean filter paper previously marked with identifying information, and this sealed with a metal clip. The control group was exposed to Tween® 20 (2%) and water. Three repetitions were tested per concentration. The envelopes were placed on trays and incubated for 48 h at 27 ± 2 °C and 80 to 90% (RH). After incubation, counts were done of live and dead larvae. Those larvae capable of walking were considered to be alive while those exhibiting an absence of movement, ataxia or movement of appendices were considered dead. Mortality rate was calculated with an established formula(16), as was the efficacy of the different genotypes(17). % Mortality = dead larvae/total larvae x 100 Average % mortality = (mortality 1 + mortality 2 + mortality 3)/3 % Efficacy = (control group-treated group)/control group x 100

Bioassay with adults

Twelve homogeneous groups of ten engorged ticks (average weight = 200 ± 20 mg) each were formed. The same Leucaena spp. genotype extracts were used but only at 20 and 10% concentrations; three repetitions were done per concentration. The treatments were immersed in the corresponding extract and concentration for one minute while the control was immersed in Tween® 20 (2%)(18). The treated ticks were placed in a 24-well culture plate, one per well, and incubated for fifteen days under the temperature and RH conditions described above. Mortality and survival rates were recorded daily using a stereoscope. At day fifteen, the eggs produced in each group were weighed. One hundred of these eggs were placed in glass vials under the same incubation conditions for 21 d, after which hatch rates were estimated for each treatment and these compared to the control.

Thin-layer chromatography (TLC)

Secondary metabolite profiles for the three genotypes were determined using TLC silica gel 60 F264 analytical plates (Merck) and silica gel 60 70-230 mesh for gravity chromatography (Sigma Aldrich). Three chromatographic revealers were used: ceric sulfate (universal revealer); oleum (terpene identification); and stannous chloride (flavonoid and terpene detection)(19).

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Statistical analyses

Established formulas were used to calculate larval mortality(16), and efficacy(17). Reproductive efficiency (RE) and the percentage reduction in estimated reproduction (PRER) were calculated with a reported equation(20). The lethal concentration at 50% (LC50) and its confidence interval at 95% (CI95) were determined with the Probit analysis program. Reproductive efficiency (RE) and PRER were calculated with these formulas: RE = egg mass weight X % larvae hatched/initial tick weight PRER = Control group RE – Treated Group RE/Group Control RE X 100

Results Larval mortality

The 50% Cunningham genotype extract caused 91.7 % mortality of R. microplus larvae and had 92.7 % efficacy. The 50% KX2 genotype extract caused 82.0 % mortality and had 90.7 % efficacy. Activity was notably lower in the Native genotype (54.6 % mortality, 75.6 % efficacy). Calculation of the LC50 found that the KX2 extract required 15.8 % (11.9 ± 19.0) of the concentration to reach this benchmark, while the Cunningham extract required 22.2% (19.2 ± 25.3) and the Native extract 43.7 % (36.0 ± 19.0).

Adult mortality, reproductive efficiency index and reduction in estimated reproduction

Against adult R. microplus, the Native genotype extract exhibited the best performance with 50% mortality at the 20% concentration, as well as a 51.3 % reduction in PRER. The Cunningham genotype produced mortality ≤20% and a PRER of 52.0 %, while the KX2 genotype also caused mortality ≤20% and had a PRER of 40.8 % (Table 1). No differences were observed between the genotypes in the chromatographic profile of the majority metabolites. Identified metabolites included terpenes and flavonoids, mainly of medium polarity (Figure 1).

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Table 1: Mortality rate, reproductive efficiency index (REI) and percent reduction in estimated reproduction (PRER) in adult Rhipicephalus microplus ticks exposed to different concentrations of Leucaena spp. extracts Leucaena spp. Control Native 20% Native 10% Cunningham 20% Cunningham 10% KX2 20% KX2 10%

Mortality (%) 0 50 0 10 20 20 10

REI (%) NA 16.01 45.09 4.04 40.36 42.05 49.84

PRER (%) NA 51.31 13.19 52.04 0.0 44.55 40.86

NA = not applicable.

Figure 1: Thin-layer chromatography of three Leucaena spp. genotypes: A) KX2; B) Native; and C) Cunningham. Hexane:AcOEt (8:2) system, oleum revealer

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Discussion The shrub legume L. leucocephala is distributed throughout the tropics and subtropics(21). It is known to have defense mechanisms against fungi, bacteria and herbivorous insects(22,23), suggesting its use as an alternative in controlling the tick R. microplus. Larval mortality rate was highest at the 50% concentration of the Cunningham (91.7 %) and KX2 (82.0 %) genotype extracts (Table 2). The lowest LC50 was 15.8 % for the 50% KX2 concentration, followed by 22.2 % for the Cunningham genotype. These results are similar to those reported in a study assessing the effect of an acetone L. leucocephala extract against different phases of R. microplus, with a resulting larval mortality rate of 66.79 %(12).

Table 2: Mortality rate and efficacy of Rhipicephalus microplus larvae exposed to different concentrations of ethanol extracts of three Leucaena spp. genotypes Leucaena spp. Control Native Native Native Native Cunningham Cunningham Cunningham Cunningham KX2 KX2 KX2 KX2

Concentration (%) NA 50.0 40.0 30.0 20.0 50.0 40.0 30.0 20.0 50.0 40.0 30.0 20.0

Mortality (%) 0 54.6 49.5 38.7 28.0 91.7 78.4 62.4 29.5 82.0 77.7 64.9 58.4

Efficacy (%) NA 75.65 67.3 62.1 57.0 92.7 82.0 71.0 59.3 90.7 83.0 82.0 72.7

NA = not applicable.

In adult ticks the LC50 for the Native genotype extract was attained with the 20% concentration, which also reduced PRER by 51.3 %. These results differ from a previous report in which no effect was observed against adult ticks(12). Another study assessed the effect of defensive proteins and peroxidase produced by L. leucocephala when under stress versus adult R. microplus ticks, finding that, at a 0.1 mg/ml concentration, the defensive proteins reduced egg production by 8.5 %, larvae hatching by 47.7 % and larval efficacy by 56.3 %(13).

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Other studies have evaluated the effect of extracts from other plants of the Fabaceae family against R. microplus. For example, when tested against adult R. microplus, a 20% concentration of extracts of leaves from Habardia albicans and Cesalpinia gaumeri produced low mortality rates (23 and 30 %, respectively), although the H. albicans extract did exhibit moderate inhibition of oviposition (54.4 %) and larvae hatching (48.7 %); in larvae, however, a 10% concentration caused 90 to 93 % mortality(7). In an evaluation of an ethanol extract of Dalbergia sissoo against adult R. microplus, a 10% concentration was found to cause 85.0 % mortality and 55.9 % inhibition of oviposition, but had no effect on larvae hatching(24). Also at a 10% concentration, ethanol extracts of the leaves of Acacia farnesiana and A. harmandiana were reported to produce low mortality rates in adult R. microplus (4.0 and 5.0 %, respectively)(25). In another study, efficacy against R. microplus larvae was 82 % for a methanol extract of Stylosanthes humilis and 75 % for a methanol extract of S. hamata leaves(26). The biological properties of Leucaena spp. genotypes can be attributed mainly to the presence and abundance of tannins, mimosine, phenols, coumarins and flavonoids, among others. A characterization of the composition of four Leucaena spp. genotypes (Cunningham, K636, native and KX2) found that all four contained different levels of condensed tannins and mimosine(27). In the present study only terpenes and flavonoids were observed; together these constitute the majority metabolites in the evaluated extracts. Terpenes are known to cause mortality in R. microplus(28,29). For instance, a study of essential oil from Lippia sidoides leaves found it to contain 67.6% thymol (a terpene compound), and that the oil caused over 95 % mortality in R. microplus larvae(30). An evaluation of a natural extract of Verbena officinalis identified high flavonoids concentrations and found the extract to cause up to 67 % mortality in vitro against adult R. microplus(31). In conjunction with the present results, these studies support the efficacy terpenes and flavonoids may have in controlling R. microplus. Most of the natural compounds tested for tick control have modes of action that differ from commercial pharmaceutical treatments available for tick control, and may therefore be effective at controlling R. microplus. The present results suggest that ethanol extracts of Leucaena spp. are promising for control of R. microplus larvae but only moderately effective against adults. Studies have been done of the action mechanism of terpenes and flavonoids on arthropods. The monoterpenes contained in plant essential oils (D-limonene, myrcene, terpineol, linalool and pulegone) are known to be neurotoxic to the common fly (Musca domestica) and the German cockroach (Blattella germanica)(32). In addition, thymol may strengthen the action of GABA (gamma-aminobutyric acid) receptors in undefined locations in insects(33). Future research will need to focus on identifying the metabolites contained in ethanol extracts of Leucaena which have activity against R. microplus, be it individually or synergistically.

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Conclusions and implications The ethanol extracts of three Leucaena spp. genotypes were found to have good efficacy against R. microplus larvae and moderate efficacy against adults; they also reduce the percentage reduction in estimated reproduction. Flavonoids and terpenes were identified in the extracts and may be responsible for their acaricide activity. However, identification is still needed of the metabolites that produce this activity, be it individually or synergistically.

Acknowledgements The authors thank the Campus de Ciencias Biológicas y Agropecuarias de la Universidad Autónoma de Yucatán, the Instituto Tecnológico Superior de Felipe Carrillo Puerto and the Instituto Tecnológico de la Zona Maya for access to facilities and equipment. The research reported here forms part of the Masters in Science degree in Sustainable Agroecosystems of GGL.

Literature cited: 1. Rodríguez-Vivas RI, Rosado-Aguilar JA, Ojeda-Chi M, Pérez-Cogollo LC, Trinidad-Martínez I, Bolio-González ME. Control integrado de garrapatas en la ganadería bovina. Ecosist Rec Agropecu 2014;1(3):295-308. 2. Rodríguez-Vivas RI, Gris L, Pérez de León AA, Silva VH, Torres-Acosta J, Fragoso-Sánchez H, et al. Potential economic impact assessment for cattle parasites in México. Rev Mex Cienc Pecu 2017;8(1):61-74. 3. Rodríguez-Vivas RI, Alonso-Díaz MA, Rodríguez-Arévalo F, Fragoso-Sánchez H, Santamaria VM, Rosario-Cruz R. Prevalence and potential risk factors for organophosphate and pyrethroid resistance in Boophilus microplus ticks on cattle ranches from the State of Yucatan, Mexico. Vet Parasitol 2006;336:335-342. 4. Perez-Cogollo LC, Rodriguez-Vivas RI, Ramirez-Cruz GT, Miller RJ. First report of the cattle tick Rhipicephalus microplus resistant to ivermectin in Mexico. Vet Parasitol 2010;168:165169. 5. Miller RJ, Almazan C, Ortiz-Estrada M, Davey RB, George JE, De Leon AP. First report of fipronil resistance in Rhipicephalus (Boophilus) microplus of Mexico. Vet Parasitol 2013;191:97–101.

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6. Arceo-Medina GN, Rosado-Aguilar JA, Rodríguez-Vivas RI, Borges-Argaez R. Synergistic and antagonistic action of fatty acids: sulphides and stilbene against acaricide-resistant Rhipicephalus microplus ticks. Vet Parasitol 2016;228:121–125. 7. Rosado-Aguilar JA, Arjona-Cambranes K, Torres-Acosta JF, Rodríguez-Vivas RI, BolioGonzález ME, Ortega-Pacheco A, et al. Plant products and secondary metabolites with acaricide activity against ticks. Vet Parasitol 2017;138:66-76. 8. Silva WC, Martins JRS, Cesio MV, Azevedo JL, Heinzen H, De Barros NM. Acaricidal activity of Palicourea margcravii a species from the Amazon forest, on cattle tick Rhipicephalus (Boophilus) microplus. Vet Parasitol 2011;179:189–194. 9. Ravindran R, Juliet S, Sunil AR, Ajith Kumar KG, Nair Suresh N, Amithamol KK, et al. Eclosion blocking effect of ethanolic extract of Leucas aspera (Lamiaceae) on Rhipicephalus (Boophilus) annulatus. Vet Parasitol 2011;179:287–290. 10. Koc S, Oz E, Cinbilgel I, Aydin L, Cetin H. Acaricidal activity of Origanum bilgeri P.H. Davis (Lamiaceae) essential oil and its major component, carvacrol against adults Rhipicephalus turanicus (Acari: Ixodidae). Vet Parasitol 2013;193:316–319. 11. Dantas AC, Araujo A, Marques AG, Branco A, Sangioni LA, Guedes JR, et al. Acaricidal activity of Amburana cearensis on the cattle tick Rhipicephalus (Boophilus) microplus. Ciencia Rural 2016;46:536-541. 12. Fernández-Salas A, Alonso-Díaz MA, Acosta-Rodríguez R, Torres-Acosta FJ, SandovalCastro CA, Rodríguez-Vivas RI. In vitro Acaricidal effect of tannin-rich plants against the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Vet Parasitol 2011;175:113-118. 13. Wanderley LF, Rodrigues-Batista KL, de Carvalho JF, da Silva-Lima A, Alves-Landulfo G, dos Santos Soares, et al. The first assessment of the stress inducible defense of Leucaena leucocephala with acaricidal potential effect against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Braz J Vet Parasitol 2017;26(2):171-176. 14. Rosado-Aguilar JA, Aguilar-Caballero AJ, Rodríguez-Vivas RI, Borges-Argaez R, GarcíaVázquez Z, Méndez-González, M. Acaricidal activity of extracts from Petiveria alliacea (Phytolaccaceae) against the cattle tick, Rhipicephalus (Boophilus) microplus (Acari: ixodidae). Vet Parasitol 2010;168:299-303. 15. Soberanes CN, Santamaría VM, Fragoso SH, García VZ. Primer caso de resistencia al amitraz en la garrapata del ganado Boophilus microplus en México. Téc Pecu Méx 2002;40:81-89. 16. Santamaría VM, Soberanes CN. Curso-Taller sobre diagnóstico de resistencia a ixodicidas en garrapatas Boophilus microplus., Jiutepec, Morelos, México 2001;1-121.

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17. Álvarez-Colom O, Neske A, Popich S, Bardón A. Toxic effects of Annonaceous Acetogenins from Annona cherimolia (Magnoliales: Annonaceae) on Spodoptera frugiperda (Lepidoptera: Noctuidae). J Pest Sci 2007;80:63-67. 18. Rosado-Aguilar A, Aguilar-Caballero A, Rodríguez-Vivas RI, Borges R, Méndez M, Dorantes A, et al. Evaluation of three solvents as vehicle of crude methanolic plant extract in the larval immersion test using Boophilus microplus ticks. 21st Int Conf WAAVP, Gent, Belgium 2007;634. 19. Gibbons S. An introduction to planar chromatography. Sarker SD, et al., editors. Natural products isolation. New Jersey, U.S.A. Humana Press; 2006:77–116. 20. Rodríguez-Vivas RI, Cob GL. Técnicas diagnósticas en parasitología veterinaria, Universidad Autónoma de Yucatán, Mérida, Yucatán, México 2005. 21. Geiger CA, Napompeth B, Van Den Beldt R. An update on the status of Leucaena Psyllid in Southeast Asia. In: Shelton HM, et al., editors. Leucaena—opportunities and limitations. In: Proc Int Workshop. Bogor, Indonesia, Canberra 1995;125–128. 22. Eck G, Fiala B, Linsenmair KE, Bin Hashim R, Proksch P. Trade-off between chemical and biotic anti-herbivore defense in the South East Asian plant genus Macaranga. J Chem Ecol 2001;10:1979–1996. 23. Heil M, Baumann B, Andary C, Linsenmair KE, McKey D. Extraction and quantification of “condensed tannins” as valuable measure of plant anti-herbivore defence? Naturwissenschaften 2002;89:519–524. 24. Singh NK, Vemu B, Prerna M, Singh H, Dumka VK, Sharma SK Acaricidal activity of leaf extracts of Dalbergia sissoo Roxb. (Fabaceae) against synthetic pyrethroid resistant Rhipicephalus (Boophilus) microplus. Vet Sci 2016;106:1–6. 25. Chungsamarnyart N, Jiwajinda S, Jansawan W. Larvicidal effect of plant crude-extracts on the tropical cattle tick (B. microplus). Thail Kasetsart J Nat Sci Suppl 1991;25:80–89. 26. Muro-Castrejón FC, Cruz-Vázquez M, Fernández-Ruvalcaba J, Molina-Torres J, Soria Cruz J, Ramos-Parra M. Repellence of Boophilus microplus larvae in Stylosanthes humilis and Stylosanthes hamataplants. Parasitol Latinoam 2003;58:118–121. 27. Ahmed M, Solorio-Sánchez F, Ramírez-Avilés L, Elsaid-Mahdy R, Castillo-Camaal J. Tannins and mimosine in Leucaena genotypes and their relations to Leucaena resistance against Leucaena psyllid and Onion thrips. Agrofor Syst 2016;91:1-8. 28. Ferraz A de BF, Zini JM, Zini CA, Sardá-Ribeiro VL, Bordignon SAL, von Poser G. Acaricidal activity and chemical composition of the essential oil from three piper species. Parasitol Res 2010;107:243–248.

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29. Gomes GA, Monteiro CM, Senra TO, Zeringota V, Calmon F, Matos RS, et al. Chemical composition and acaricidal activity of essential oil from Lipia sidoides on larvae of Dermacentor nitens (Acari: ixodidae) and larvae and engorged females of Rhipicephalus microplus (Acari: ixodidae). Parasitol Res 2012;111:2423–2430. 30. Gomes GA, Monteiro CM, Julião L de S, Maturano R, Souza TO, Zeringóta V, Calmon F. da Silva R, Daemon E, de Carvalho MG. Acaricidal activity of essential oil from Lipia sidoides on unengorged larvae and nymph of Rhipicephalus sanguineus (Acari: ixodidae) and Amblyomma cajennense (Acari: ixodidae). Exp Parasitol 2014;137:41–45. 31. Pulido NJ, Cruz A. Eficacia de los extractos hidroalcohólicos de dos plantas sobre garrapatas adultas Rhipicephalus (Boophilus) microplus. Corpoica Cienc Tecnol Agropec 2013;14:91– 97. 32. Coats R, Karr LL, Drewes CD. Toxicity and neurotoxic effects of monoterpenoids in insects and earthworms. In: Hedin P. editor. Natural occurring pest bioregulators. Am Chem Soc Symp Series 449, 1991:305e316. 33. Priestley CM, Williamson EM, Wafford KA, Satelle DB. Thymol, a constituent of thyme essential oil, is a positive allosteric modulator of human GABAA receptors and a homooligomeric GABA receptor from Drosophila melanogaster. British J Pharmacol 2003;140:1363.

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https://doi.org/10.22319/rmcp.v10i3.4785 Review

Fundaments of the honey bee (Apis mellifera) immune system. Review

Alejandra Larsen ac Francisco José Reynaldi bc* Ernesto Guzmán-Novoa d

Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Argentina.

Centro Científico Tecnológico de la Plata. Consejo Nacional de Investigaciones Científicas

y Técnicas. Argentina. c

Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Argentina.

University of Guelph. School of Environmental Sciences. Ontario, Canada.

* Corresponding author: freynaldi@yahoo.com

Abstract: Honey bees (Apis mellifera) pollinate plants in both natural and managed ecosystems, contributing to food production and sustaining and increasing biodiversity. Unfortunately bee depopulation and colony losses are becoming increasingly common worldwide. Several factors contribute to the decline of bee populations, including pathogens (parasites, fungi, bacteria and viruses), ecosystem alteration or loss, and/or agrochemical use. All of these factors alter the defense mechanisms of the bee immune system. Honey bees have an innate immune system that includes physical barriers and generalized cellular and humoral responses to defend themselves against infectious and parasitic organisms. Pathogens, acaricides, fungicides, herbicides and other pesticides affect the bee immune system and consequently bee health. The defense mechanisms of the bee immune system include 705

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signaling pathways, pathogen recognition receptors and innate immune system effectors. Although A. melliferaâ&#x20AC;&#x2122;s immune system is very similar to that of Drosophila flies and Anopheles mosquitoes, they possess only about a third of the immune system genes identified in these genera. This relatively low number of genes is probably a consequence that A. mellifera has developed social immunity. This defense strategy lowers pressure on the individual immune system of bees. This review article summarizes and discusses the bases of the honey bee immune system. Key words: Immunity, defense mechanisms, immune system regulation, pathogens, Apis mellifera. Received: 26/02/2018 Accepted: 14/06/2018

Introduction Along with other wild pollinators, honey bees (Apis mellifera) contribute to pollinating plants in both natural and managed agriculture systems. In these ecosystems, pollination constitutes an environmental service, which contributes to increasing natural biodiversity, as well as the production of food and fibers for human consumption(1,2). Unfortunately, bee depopulation events and loss of honey bee colonies have occurred worldwide during the last decade, particularly during late winter(3-6). Various factors apparently lead to declines in bee populations, including pathogens (parasites, fungi, bacteria and viruses), ecosystem alteration or loss, and/or the use of agrochemicals. Since all these potential factors can alter defense mechanisms of the beesâ&#x20AC;&#x2122; immune system, it is necessary to first understand how it functions to be able to analyze its response to the different infectious or non-infectious conditions that affect bees. Immune systems in plants and animals involve organs and defense mechanisms that protect them against foreign substances and pathogenic organisms by recognizing them as threats and responding against them. Much of current knowledge on immune systems and their responses has been generated using insects as research subjects; as a result, immunity in insects is very well studied. Many insects are vectors of animal and human diseases, and others cause major damage to agricultural crops. Most insect species live relatively short lives, but they have complex and efficient immune systems. For example, insectsâ&#x20AC;&#x2122; immune systems are more efficient at detecting pathogens and responding to them than are those of vertebrates(7). 706

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The fruit fly Drosophila melanogaster is the most studied insect species and, in addition to many other research areas, studies on this fly have helped to better understand innate immunity in other organisms. Research with fruit flies has generated knowledge on pathogen recognition mechanisms, immune signaling and effector responses against pathogens. Completion of the Drosophila genome sequence in the year 2000 has allowed more potent and specific analyses of immune responses, substantially increasing knowledge of the molecular foundations of immune systems. Not only these studies showed how insect immune systems work, but how the innate immune system of humans function, because many of the basic immune mechanisms are shared by Drosophila and humans. Studies of other insects whose genomes have been sequenced such as A. mellifera, can also contribute to exploration of immune responses at the molecular level. Because their natural pathogens and genetic structure are well known, the honey bee can join several species of flies and moths as important models for researching the genetic mechanisms of immunity and diseases. The honey bee immune system is very similar to that of Drosophila flies and Anopheles mosquitoes, except that honey bees have approximately one third of the immune genes shared by Drosophila and Anopheles, which are grouped into 17 families(8,9). Honey bees however, have more genes for odor receptors, as well as specific genes that regulate pollen and nectar collection, which is consistent with their behavior and social organization(10). The implicit reduction in the number of immune genes in bees may reflect the importance of social defenses (i.e. based on social behavior) and/or their tendency to be attacked by a limited set of pathogens which are highly co-evolved with them(11). Among the similarities of the innate immune systems of honey bees, fruit flies and Anopheles mosquitoes, is that all of them posses the same signaling pathways. Therefore, much of the knowledge that we have about the immune system of A. mellifera has been deduced from the knowledge of dipteran immune systems. Advances in genomics allow study of both the evolution of biological systems and immune systems. The resulting deeper knowledge has proved valuable in understanding, treating and preventing disease in species of social or economic importance. Indeed, the sequencing of the A. mellifera genome has led to prediction of their immune system components, such as the recognition receptors, effectors and pathways involved in host defense(8). The present review of the honey bee immune system covers both general and specific aspects of current knowledge on the innate immune system, its components and regulation, immune responses and social immunity.

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Types of immune systems

Of the two types of immune systems, innate and adaptive, higher vertebrates have both to fight against pathogens, while insects have the innate immune system as their sole line of defense (Table 1). Innate immunity responds to exposure to pathogens or toxic substances with acquired (preexisting) mechanisms, such as physical barriers (e.g. cuticle, mucous membranes, etc.), and cells and chemicals that neutralize toxins and pathogens. The innate immune system in higher vertebrates uses cellular effectors including phagocytes, dendritic cells, natural killer cells and mast cells, among others(7). Humoral effectors consist of supplement system fractions, acute phase proteins, antimicrobial peptides (AMPs), natural antibodies, and the various cytokines that modulate immune response(7). Innate immune system specificity is in part inherited, resulting from coevolution of individual immune systems with myriad pathogens(12). Adaptive, or acquired, immunity refers to specific immune reactions tailored to particular toxins or pathogens. These toxins or pathogens are known as antigens (antibody generators) or immunogens. Adaptive immunity in vertebrates implies the ability to remember specific pathogens and react with production of antibodies specific to each pathogen when an organism is exposed to the same pathogen more than once. One way to differentiate between innate and adaptive immune systems is based on the way an organism encodes the molecules with which it recognizes pathogens. Innate immunity involves encoding these recognition receptors directly into the germline, which is then inherited by offspring. In this sense, the repertoire of receptors identified in studied species is limited and promiscuous. Adaptive immunity requires far more receptors than innate immunity, with a repertoire of adaptive immunity receptors that is broad enough to potentially recognize an infinite number of pathogens(7).

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Table 1: Characteristics of innate and adaptive immunity systems.

Insects

Higher Vertebrates

Innate

Adaptive

CHARACTERISTICS

Specificity

Against structures shared by related microbial groups.

Against microbial and non-microbial antigens.

Receptor diversity

Limited

Very broad

Memory

Null

Yes

Self reactivity

Yes, non-specific collateral damage.

Yes, specific autoimmunity.

COMPONENTS

Humoral effectors

Antimicrobial peptides, thioester linkage proteins, melanization and coagulation proteins.

Complement system. Cytokines.

Antibodies. Cytokines.

Interferon system. Chemokines. Acute phase proteins. Coagulation system.

Cellular effectors

Macrophages, dendritic cells, neutrophils, innate immunity lymphocytes, mastocytes.

Phagocytes. Hemocytes.

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Lymphocytes

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The innate immune system and its components

Physical barriers, coupled with humoral defense mechanisms and different cellular processes, acting in synergy, are powerful tools for neutralizing parasites, pathogens and xenobiotics. Physical barriers The pathogens and xenobiotics that affect insects must first cross the physical barriers of the innate immunity system, such as the exoskeleton, tracheal tubes, and intestinal mucosa. Viruses in particular, often are able to penetrate these barriers with the aid of a vector; for instance, many viruses are transmitted to A. mellifera by the mite Varroa destructor, which pierces these physical barriers, thus facilitating viral infection. Cellular immunity Cellular immunity is provided by hemocytes, cells transported by the hemolymph, which perform processes such as phagocytosis, encapsulation and melanization(13). In insects, hemocytes also synthesize and store humoral effectors such as antimicrobial peptides(14), in association with other sources of immune system soluble effectors such as the salivary glands(15) and the fat body. The latter is the functional analogue of the liver in higher vertebrates since it produces proteins to fight pathogens(16,17). Cellular mechanisms contribute to elimination of foreign agents; in the face of an infectious or external particle, hemocytes can respond by phagocytizing or lysing it, or by engulfing it to neutralize it(13,18). Small foreign agents can be phagocytized by hemocytes for removal. Larger ones (or aggregates of small ones), however, can trigger nodulation or encapsulation, which involves cooperative action among several hemocytes(19). This process requires aggregation and partial disruption of hemocytes on the surface of the agent to be removed(20). Oxygen and nitrogen mediators that affect microorganisms are then released, and process-regulating substances which act as antioxidants are simultaneously generated, minimizing any potential damage from foreign agents. For hemocytes to fulfill their phagocytic and restorative functions, they may have some kind of adhesion molecules that allow them to bind to different surfaces, other cells or each other, which is what happens in nodulation or encapsulation(21,22). Although the number of hemocytes varies in the different stages of bee development, this encapsulation function is unaffected(23). This is notable since in adult bees, including workers, queens and drones, the number of blood cells decreases as they get older(24).

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Insect hemocytes have been identified and classified by their morphological, histochemical and functional characteristics. In bees particularly, hemolymph cytology has been characterized using different methods. Initial studies identified five main hemocyte types(25), 90% of which are represented by plasmatocytes. These in turn have been classified into four subtypes: prohemocytes, clot hemocytes, granular cells and oenocytoids; the latter two related to melanization during and after the encapsulation process(20). Flow cytometry analyses have not found significant morphological differences between hemocytes(26), but have identified two types of plasmatocytes. In another study hemolymph cell groups were classified as proleukocytes, eosinophils, basophils, neutrophils, picnonucleocytes, adipoleukocytes, spherukocytes, granulocytes, macronucleocytes, microleukocytes, and spindle-type cells(27). Still others propose functional classification of hemolymph cells (e.g. adhesion to glass), thus avoiding any possible confusions from morphological classification(21). Melanization is a combination of humoral and cellular processes that occurs during encapsulation or nodulation and healing, and is aimed at dealing with injuries, be they pathogen-mediated or otherwise. This cellular reaction in the insect defense system eliminates large numbers of bacterial cells, parasites and xenobiotics(19). Its main function is to limit agent propagation and retain it for elimination(13). This central and very effective defense strategy is the focus of evasion mechanisms employed by many entomopathogenic microorganisms, confirming its importance as a defense mechanism(19,28). Prophenoloxidase (proPO) is a hemolymph protein that mediates melanization. Activation of proPO in insects occurs through an activation cascade beginning with recognition of pathogen-associated molecular patterns (PAMPs) by pathogen-recognition receptors (PRRs) deployed by hemocytes. These begin an adhesion process on the invading agents, generating an overlapping sheath, and producing and releasing proPO to degranulate or lyse the agents. In conjunction with formation of melanin and its polymerization (along with other proteins) to encapsulate the invading agent, reactive intermediaries of oxygen and nitrogen are produced, such as superoxide anion, hydrogen peroxide(20), and nitric oxide(21,29). These collaborate in agent destruction and induction of melanization. This process has been demonstrated in A. mellifera(29). Bees have but a single proPO gene, whereas Drosophila sp. have three and Anopheles sp. have nine. This proPO-encoded gene is expressed more strongly in adult bees than in larvae or pupae(9).

Humoral and chemical immunity Humoral response is a second category of innate immunity, and the most important defense system of insects, including honey bees. It is mediated by chemicals and antimicrobial peptides (AMPs). These are small, highly conserved proteins, generally between 12 and 50 711

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amino acids in size, which are produced and released into the insect hemolymph in response to bacterial and fungal infections, but can also be synthesized during viral infections(14). These humoral effectors are fundamental to innate immunity in insects. In some pollinating insects, such as Bombus pascuorum, the humoral response is detected within 24 to 48 h postinfection. Humoral effectors can be produced in hemocytes, epithelial cells and salivary glands, but the fat body of the dorsal cavity is the main organ of effectors synthesis(30,31). Over 170 AMPs have been described in insects, although honey bees produce fewer humoral effectors than other insects such as Drosophila and Anopheles(32). Honey bees have four AMP families with broad hemolymph activity: apidaecin, abaecin, hymenoptaecin and defensin. Defensins are small AMPs that act mainly against Gram-negative bacteria such as E. coli, although they do effect Gram-positives and fungi(33). There are 29 different cDNA sequences for defensins, numbered Defensin1 to Defensin 29. Eleven cDNA sequences exist for abaecin, encoding for two different abaecin peptides called AcAb1 and AcAb2. Apidaecin has thirteen cDNA sequences encoding for four peptides: AcAp1 to AcAp4. Finally, there are 34 different cDNA sequences for hymenoptaecin encoding for 13 different peptides(34). In B. pascuorum and B. terrestris, AMPs have been shown to act in synergy to provide greater antimicrobial additive effects; this can involve potentiation in that one AMP can improve anotherâ&#x20AC;&#x2122;s activity. The combination of AMPs increases the spectrum of responses, as well as their specificity, effectiveness and robustness, thus allowing a reduction in the resources allocated the immune system by augmenting the antimicrobial activity of AMPs at low concentrations(35).

Regulation of the immune response

All immune responses involve a sequence of events that can be generally grouped into three stages: 1) recognition, 2) activation of signaling pathways and 3) cellular and humoral effector mechanisms aimed at eliminating pathogens (Figure 1)(36). The immune response is triggered by the recognition process in which PAMPs are identified by PRRs in immune system cells. In response, different signaling pathways are activated, promoting synthesis of the effectors and receptors involved in the humoral and cellular immune response, as well as peptidoglycan recognition proteins (PGRP)(20).

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Figure 1: Immune system regulation

Pathogen recognition

Microorganisms are antigenic mosaics that can be recognized differentially by the innate and adaptive immune systems. The innate immune system recognizes PAMPs, which are preserved and vital protein structures present in defined germ groups; for example, lipoparasaccharides (LPS), lipotheicoic acid, zymosan, glycolipids, glycoproteins or doublestranded RNA(7). The innate immune system also recognizes damage-associated molecular patterns (DAMPs), which are molecules expressed in cells that have suffered infectious or non-infectious damage, such as thermal shock protein. However, in insects, it is more common to refer to microbe-associated molecular patterns (MAMPs), which include socalled virus-associated molecular patterns (VAMPs)(32). These structures act as exogenous

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ligands and are recognized by proteins or PRRs, which are present in soluble form or in immune system cells(12). Multiple PRRs occur in Drosophila; for example, some members of the PGRP family. Of the 13 PRRs in Drosophila, honey bees share four, two of which are synthesized in response to infections (PGRP-S2 for the Toll pathway, and PGRP-LC for the Imd pathway). Other proteins recognize Gram-negative bacteria, such as GNBP1, which recognizes 1,3 glucans, but can also recognize fungi and are involved in recognition of certain Gram-positive bacteria(37,38). These pattern recognition proteins may be involved with serinproteases, which initiate division of Spaetzle and Tollâ&#x20AC;&#x2122;s endogenous ligand in Drosophila; both of these are activated in embryogenesis and immune response(39). Two orthologous genes of the Spaetzle family have been identified in the bee genome(8,32,40,41,42). Recognition of microbial structures triggers two main events: 1) signaling events, which occur when Toll and/or IMD receptors are stimulated, and 2) phagocytosis events. The genes DSCAM and Eater are two examples of genes related to endocytosis in bees. In Drosophila DSCAM is known to be involved in bacteria recognition by hemocytes(42,43). Peptidoglycans, LPS and zymosan also recognize MAMPs. Vitellogenin are carrier proteins of bacterial fragments; they are acquired transgenerationally, producing a kind of sensitization or â&#x20AC;&#x153;primingâ&#x20AC;? of the innate immune system in progeny(44,45).

Signaling pathways

Intracellular signaling pathways translate external signals or stimuli into actions within cells, inducing immune response; for example, by activating a series of genes encoding proteins related to host defense systems (e.g. thioester linkage proteins - TLPs). Signaling pathways depend on large multiprotein complexes that trigger stimuli of cell surface receptors by a specific ligand, and emit an intracellular signal initiating a cascade of enzymatic activity. Receptors made up of transmembrane proteins are associated with enzymes such as protein kinases. These normally phosphorylate the amino acid tyrosine, and are thus called tyrosinases. Onset of this intracellular signaling cascade directs the various biochemical responses that characterize a specific cellular response. Bees have orthologous genes for the central members or components of the four intracellular signaling pathways involved in activating innate immunity effectors (Figure 2), with the Toll and Imd pathways being the most important in insects, including bees.

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Figure 2: Signaling pathways, molecular detail

(Modified from Brutscher et al., 2015)(32).

Toll signaling pathway Toll receptors across the membrane of cells play a critical role in both ontogenic development and the immune system. Only five Toll-related genes have been identified in bees (Toll-1, 6, -2/7, -8, -10); these are also present in the genome of other insects belonging to the orders Diptera, Lepidoptera and Coleoptera, with a few exceptions. The combination of Toll genes present and absent in these insects suggests that these five genes encode the basic set of Toll receptors present in their common ancestor(8,32). Activation pathways involve recruitment of cytoplasmic adapter proteins, which activate kinases that lead to activation of nuclear factors and deregulation of genes that encode immune system effectors, such as AMP growth factors. Detachment of Spaetzle stimulates Toll receptors, which recruit death-domain proteins (DD-death) to assemble a receptor complex. In this process, the adapter protein MyD88 recruits TUBE and activates the protein kinase PELLE (IRAK counterpart) which then recruits the adapter dTRAF0. This complex induces degradation of CACTUS (counterpart of the NF-κB inhibitor protein, IκB) allowing the DORSAL transcription factor (NF-κB’s counterpart) to be transported to the nucleus to 715

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link to regions promoting immune effector genes, inducing their expression. The effectors synthesized when this pathway is activated are mainly AMPs and lysozymes(8,46).

Imd signaling pathway In bees and flies, the immune-deficiency signaling pathway (Imd) activates the RELISH transcription factor (homologue to NF-ÎşB transcription factor). In flies, it controls expression of most AMPs, making this pathway indispensable for immune response against microorganisms. Presence of CACTUS as a transcription factor inhibitor has also been demonstrated. This pathway is highly preserved in bees with possible orthologues for all components. Although this strongly implies that signaling pathways in flies and bees are similar, it does not necessarily mean that they share exactly the same biological functions(8). Microorganism recognition via peptide-glucan recognition protein (PGRP-LC) is the first step in immune response onset via Imd signaling(47). Activation of the Imd pathway also leads to activation of components of the JNK signaling pathway, and there is evidence that the latter controls expression of AMP synthesis through both positive and negative feedback. Possible orthologues of this pathway, such as Basket, JNK and JNK-protein 1 interaction, among others, are known to be present in bees(48).

JAK/STAT signaling pathway The JAK/STAT (Janus-family tyrosinkinases [JAK]/transcription activator proteins [STAT]) signaling pathway in insects is involved in synthesis of effectors similar to the complement system, as well as in proliferation and induction of phagocytosis by blood cells, and antiviral responses(8). In higher vertebrates, this signaling pathway is essential for the synthesis of many cytokines. It is a relatively fast signaling pathway since it directly phosphorylates STATs, which are dimerized transcription factors. These are transported to the nucleus where they stimulate expression of genes that can be induced by the receptor ligand. The only protein that seems to be completely absent in the bee is the JAK /STAT signaling pathway ligand. In bees, there are five Drosophila homologue genes for JAK/STAT pathway components: 1) DOMELESS cytokine receptor (dom), 2) JAK tyrosine kinase (hopscotch), 3) STAT92E transcription factor, 4) negative pathway regulatory proteins such as suppressors of cytokine signaling (SOCS), and 5) protein inhibitor of activated STAT (PIAS). This pathway ends with deregulation of the genes encoding for immune system humoral effectors; for example, the various thioester-carrying proteins (TEPs) in bees. However, no tot genes have been identified, which in Drosophila encode for humoral effectors as a result of severe stress and are produced by activation of this pathway(49,50). In bees, there are also two component orthologues of this pathway: the tyrosine phosphatase Ptp61F and WD40(8). Although the 716

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key ligand for JAK/STAT is unknown, the presence of the cytokine receptor Domelessâ&#x20AC;&#x2122; counterpart, in addition to the presence of other JAK/STAT components, indicate it to be a common mechanism in insects, appearing intact in bees and fruit flies.

RNAi signaling pathway Recognition of VAMPs in bees has been linked to the RNA interference system (RNAi), a physiological mechanism for gene silencing that also functions as a defense mechanism against viral infections by silencing the virus replication cycle. The main RNAi pathway components exist in viral infections in bees; during this process, double-stranded RNAs (dsRNA) are recognized by a dsRNA sensor produced by the dicer-like gene in bees(51). This sensor is related to the PRRs family or RIG-1 cytosolic sensors in mammals (dicer). Once DICER cuts the dsRNA, the resulting small dsRNA fragments, known as small interfering RNAs (siRNA) and microRNA (miRNA), are recognized by the RNA-induced silencing complex (RISC). The latter contains proteins of the AGO2 family (argonaute-2)(51), which it transforms into small single-stranded RNAs (ssRNA). These small ssRNA bind to mRNA transcripts, which contain complementary sequences, thus preventing protein synthesis. Activation of this pathway in bees results in increased expression of the vago gene, an orthologue found in Drosophila, resulting in suppression of viral replication(47,52). Another epigenetic mechanism in bees with antiviral function is DNA methylation, which is part of the antiviral response(52).

Immune response effectors

Recognition of pathogen PAMPs or MAMPs by PRRs, which activates the different signaling pathways, ends with the synthesis or activation of cellular and/or humoral effectors of the immune system. While AMPs are the main post-infection induced effectors, transferrin has been identified in bees and other insects. In higher vertebrates, transferrin is part of the acute phase proteins group, whose immune function is to sequester iron and thus limit bacterial infection(53,54). Like Drosophila and B. mori, honey bees have three members of the transferrin family(55), and their expression pathways would be Imd and Toll(9). Activation of the JAK-STAT signaling pathway results in synthesis of other innate immune system effectors, such as TEPs, which have the C3 fraction of the complement system, a characteristic thioester bond of their counterpart in higher vertebrates. This characteristic bond allows activated proteins to covalently bind to the surface of microorganisms and trigger an immune response(12). In Drosophila, these proteins are synthesized by the fat body, while in Anopheles, they are produced by hemocytes. In the latter, direct evidence has shown the relationship of TEPs to 717

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the protein recognition function in microorganisms and their participation in phagocytosis of Gram-negative bacteria; they are consequently equated with opsonins. Only four C3 counterpart genes encoding for TEPs have been found in the bee genome, compared to 15 in the Anopheles genome and six in Drosophila(8,56,57). Serin proteases (SP) are enzymes involved in various physiological processes such as digestion, development and immune response. Synthesized as zymogens, they participate in activation cascades that result in synthesis of effectors. In mammals the best known representatives of this protein family with immune function are those involved in the coagulation cascade and complement system; in invertebrates, they participate in the acute phase response(8,58). Of the 57 SP-related genomic sequences in the bee genome, 44 correspond to SP and 13 to SP homologues. As is the case with many other genes(8), the 57 SP-related sequences in bees pale before the 204 sequences of Drosophila(59), and the 305 of Anopheles(60). In bees, the Toll signaling pathway recognizes putative snake and eater orthologues related to Spaetzle splitting and pathway activation, which results in the synthesis of effectors such as DROSOMICINE, as occurs in the fruit fly. Bees also have SP genomic sequences similar to other insects, which are related to the prophenoloxidase activation cascade(58). The last regulatory mechanism is that of the SERPINES, which are highly conserved proteins present in the insect hemolymph. These proteins are responsible for eliminating excess protease, maintaining homeostasis, and preventing unregulated activation of immune responses such as melanization or synthesis of the Toll-mediated antimicrobial proteins(61). Seven orthologues have been identified in honey bees, five of which encode SERPINES, the remaining two coding for SERPINE-type proteins(58).

Social immunity

One characteristic of social insects in general, and of bees in particular, is their social life, sharing a nest. Nests usually contain food stores and a high density of individuals living in relative homeostasis. The nests of social insects are therefore attractive sites for the development of various infectious agents(62). However, social insects have developed social immunity(11), which is characterized by cooperative behavior within a colony through different mechanisms, such as the following:

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1) Social fever. Social fever results from bees generating additional heat in the nest. This mechanism is costly for healthy individuals but allows pathogen control in infected hosts. Raising the nest temperature favors the control of the pathogenic fungus Ascosphaera apis(63). 2) Grooming. Grooming is the ability of bees to remove external parasites from their bodies by using their mandibles and legs(36,64). There are two types of grooming behavior, self grooming and social grooming. Social grooming, involves the collaboration of several individuals(65), but self grooming is more common than social grooming. Colonies in which a high proportion of workers express this trait are more resistant to infestations by the mite Varroa destructor than colonies in which fewer members express it. Moreover, the vigor with which a colonyâ&#x20AC;&#x2122;s workers carry out grooming is directly related to the number of mites they remove from their bodies(66,67). Grooming behavior is influenced by genetic factors for which the degree of expression varies between honey bee colonies of different races and stocks(68,69). In several studies, a gene (Neurexin) has been mapped and associated with this behavior(70,71). 3) Hygienic behavior. Hygienic behavior is the ability of worker bees to detect and remove diseased or parasitized brood (larvae and pupae) from comb cells(36). This is a two-step defense mechanism. First, workers uncap cells containing diseased or parasitized larvae or pupae, and then remove them from the nest(36). This social behavior is a defense mechanism that helps to control the fungus A. apis (causal agent of chalkbrood)(72), the bacterium Paenibacillus larvae(73)(etiological agent of American foulbrood), and the mite V. destructor(68). Bees of different genotypes vary in the level of expression of this behavior (73,74,75) . Hygienic behavior is influenced by a group of at least seven genes, meaning it has a more complex genetic coding than previously thought(74), and also appears to be inherited maternally(75). 4) Gathering and use of propolis. Bees collect propolis, resins of trees (mainly from conifers) that have antiseptic and antimicrobial properties. They use them essentially as a prophylactic measure. Propolis is used to coat the interior of brood cells or to mummify any invertebrates or small vertebrates that enter and die inside the colony, preventing or minimizing the development of pathogenic bacteria and fungi(64). In addition, the presence of certain types of propolis inside the colony can promote the expression of genes of the bee immune system(3,76). 5) Decreased contact between congeners. Individuals express this type of altruistic behavior when sick by moving away from the colony to die outside the brood nest(77). 6) Offspring cannibalism. In stressful situations that can cause brood death (e.g. lack of food, extreme temperatures), nurse bees usually cannibalize dead brood to prevent the development 719

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of pathogenic microorganisms such as A. apis. This mechanism also prevents loss of nutrients from the colony. As a defense strategy, social immunity substantially lowers pressure on the immune system of individual bees, thus reducing the number of genes required for defense against infection when compared to the Diptera. This may explain why A. mellifera possesses just one-third of the recognition and immune effector signaling genes of Anopheles and Drosophila(8, 9,11).

Conclusions Honey bees possess an innate immune system, also known as individual immunity. This system includes physical barriers, as well as cellular and humoral responses, which are generalist in nature and allow them to defend themselves against a wide variety of infectious and parasitic organisms. In addition to the various pathogens affecting bees and activating their immune system, xenobiotics such as acaricides, fungicides, herbicides and pesticides, may also exercise effects on bee health and the immune system. Defense mechanisms involve signaling pathways, pathogen recognition receptors and innate immune system effectors. The high-density conditions of honey bee nests, coupled with the presence of food stores, makes them attractive for different pathogens. However, these conditions also promote social immunity, characterized by cooperative behavior within the colony by means of different mechanisms such as social fever, grooming behavior, hygienic behavior, and collection and use of propolis, among others. Social immunity is a defense strategy that greatly diminishes pressure on the immune system of individual bees, resulting in fewer genes related to defense. This may explain why A. mellifera has one-third of the genes linked to recognition and immune effector signaling compared to Anopheles or Drosophila. The immune system of Apis mellifera is influenced by multiple factors, such as pathogens and pesticides, highlighting the importance of continued study of the effects these factors have on immune responses. Future research should focus on studying immune system molecular mechanisms, as well as on the potential application of certain effectors for treatment and/or prevention of pathologies and diseases.

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Acknowledgements and conflicts of interest

This study was funded by a grant from CONICET (PIP Nº 0726). The authors wish to thank Dr. Sguazza Hernán for critical review of earlier versions of the manuscript. The authors declare no conflict of interest.

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https://doi.org/ 10.22319/rmcp.v10i3.4453 Review

Anatomy, physiology, manipulation and veterinary applications of the reticular groove. Review

María-José Martín-Alonso a,b Luis G. Cal-Pereyra c Maximino Fernández-Caso a José-Ramiro González-Montaña a*

Universidad de León. Departamento de Medicina, Cirugía y Anatomía, Facultad de Veterinaria, Tel.: 34.987-29.12.14, Fax: 34.987-29.12.69; Campus de Vegazana s/n. 24071, León, España. b

Universidad de Lleida. Departamento de Ciencia Animal, Lleida, España.

Universidad de La República. Departamento de Patología de la Facultad de Veterinaria. Montevideo, Uruguay.

*Corresponding author: jramirogonzalez@unileon.es

Abstract: Reticular groove closure in ruminants is a primary mechanism, almost exclusive of lactating animals, which makes the passage of food from the orifice of cardia to the abomasum possible, thus avoiding unwanted fermentations in rumen and reticulum. In this review It is described some anatomical and physiological aspects of the reticular groove, given its embryonic and postnatal development, its topographic location, structure, innervation, blood circulation and histology. Also describing the techniques used to study its functioning, both direct and indirect methods. Finally there is a concentrate on handling techniques to manipulate closing reflex of reticular groove and its veterinary applications, in both the stimulation and inhibition, since the possibility to control this reflex is of great interest in the oral administration of various drugs, the treatment of certain diseases, as well as a better utilization of some types of food. Key words: Reticular groove handling, Ruminants, Suckling.

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Received: 29/03/2017 Accepted: 06/07/2018

Introduction

This review has been divided into two parts. Firstly, a brief review is made of some anatomical and physiological aspects of the reticular groove in ruminants, as well as the techniques used to study the functioning of the reticular groove closure reflex. In the second part, those aspects that may stimulate or inhibit the reticular groove reflex, and the manipulation techniques of this reflex, as well as their applications in veterinary medicine. Gastric groove is an anatomical structure, located in the stomach of ruminants. It extends from the orifice of cardia until near the pylorus, through the lesser curvature of reticulum, omasum and abomasum. It is divided into three segments: reticular groove (Sulcus reticuli), omasal groove (Sulcus omasi) and abomasal groove (Sulcus abomasi)(1-3). While some authors only consider two structures: the reticular groove and the omasal groove, ranging from the cardia to the omasoabomasal orifice. Various names have been used for this structure, such as reticular leak, gastric groove, gastric channel, oesophageal channel or oesophageal fluting. The scientific nomenclature represented by the Nomina Anatomica Veterinaria(4) includes the Latin term "Sulcus reticulari". The mechanism of the oesophageal groove is a primary, almost exclusive of lactating animals; which provides ruminant animals the possibility of a physiological gradual adaptation from monogastric to ruminant stomach. When stimulated, muscular tissue contracts, adopting a hollow structure forming a duct along the wall of the reticulum, which connects the esophagus (cardia) to the reticulo-omasal orifice(5). The reticuloomasal orifice remains open allowing the flow of milk(5-7), which is of great interest in new born animals, as it allows the colostrum and milk to pass directly to the abomasum, without falling into the rumen and reticulum, thereby preventing abnormal fermentation. In the early hours of the beast, this powerful reflex allows immunoglobulins from colostrum to pass into the duodenum, where thanks to the high permeability of its mucosa it will be quickly absorbed in order to develop passive immunity that protects the animal from pathogens coming to its digestive tract. Besides, the high energy value of colostrum

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will provide sufficient energy to the animal to combat possible hypothermia and, since it has a high content of magnesium, with laxative action, it will help to expel meconium and facilitate the start of intestinal transit(8,9). The activity of the reticular groove decreases after weaning and as the age of the animal advances, but can be triggered under certain conditions in the adult animal(9).

Anatomical review

Embryology

The ruminant stomach represents the highest evolutionary development of all mammal species(10). It originates from a fusiform dilatation of the primitive gut of the embryo, called primitive stomach. From the lesser curvature derives the reticular, the omasum and the abomasum grooves. Conversely, from the greater curvature derive the rumen, reticulum and greater curvature of abomasum(1). Differentiation of the reticular groove is early in sheep and goat and latter in cattle, showing in this last case at eight weeks of embryonic development. Molinari and Jorquera(11) report that the onset of the leak in the foetuses of ruminants is simultaneous with the differentiation of the rumen and reticulum rudiments. Accordingly, the rotations experienced by these rudiments will affect the reticular groove, passing from a foetal position parallel to the axis, in the right reticular wall, to take a vertical orientation. Thus its forms an angle of 50â ° with the main axis, developing finally a spiral structure of 180â °.

Postnatal development

After birth, proventriculus development depends on the animal feed. At the beginning of the life of the ruminant abomasum size is slightly larger than all the proventriculus. Subsequently when diet begins to be solid, these rapidly increase their size. This development can be divided into three stages(8,9).

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─ From birth to 3rd week of life, the animal is considered a "non-ruminant" because its diet is exclusively dairy. High blood glucose is due to absorption of nutrients (glucose) intestine, and hence the carbohydrate metabolism is typical of a "non-ruminant". ─ Between 3 and 8 wk of life it is considered a transition period. The animal eats small amounts of solid food. Blood glucose decreases and the plasma concentration of volatile fatty acids increases, similarly to the levels of the adult animal. ─ An 8-wk-old animal will be considered as a "true ruminant". This does not occur in those cases when an animal continues exclusively breastfeeding, in which case the proventriculus remain rudimentary till 14 or 15 wk of age (Figure 1).

Figure 1: Reticular groove. 1: oesophagus, 2: left lip, 3: right lip, 4: cardia, 5: reticuloomasal orifice, 6: reticular fundus, 7: ruminoreticular fold, 8: abomasum 9: rumen (Figure taken from Pochón, 2002).

Topographic location

The reticular groove is located in the area formed by the intersection of two imaginary lines. The first, vertical, extends, from the eighth thoracic vertebra to the costochondral junction of the left seventh rib. The other, horizontal, connects the middle third of the left seventh rib and right seventh rib. It is located between the seventh and ninth left rib and the rumen is internally in contact with the rumino-reticular content(10). 732

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Structure

The gastric groove consists of three different parts. The first is the "reticular groove" which is formed by two longitudinal muscular folds or relieves, called right and left lips, and the sulcus of gastric groove. It starts in the cardia, descends in spirals across the lesser curvature of the reticulum (right wall) in caudal direction to the left, continues dextrocaudally to the reticulo-omasal reversing in turn the position of both lips. The right lip describes a rotation around the left one, in clockwise direction; to return back to its initial position to the right, taking the muscle fibers a corkscrew disposition(1,2). From the reticulo-omasal to the omaso-abomasal orifice the gastric groove is called "sulcus of omasum". It runs through the lesser curvature of the omasum. This sulcus is interrupted by a transverse fold (omasum pillar) formed by the convergence of circular muscle fibers that reinforce the omaso-abomasal orifice. The "abomasal veils" mucosal folds of transition, extend from the abomasum to the fore mentioned pillar. In bovine they are lined by tegument from the omasum, while in sheep, both abomasal and omasal face veils are fully glandular(1, 2). The last portion of gastric groove is the "abomasal groove" which runs along the lesser curvature of the abomasum, without ridges and ending at the pyloric part(1).

Innervation of the gastric groove

The control mechanism of the reticular groove is not entirely clear. It is believed to be due to the interaction between a central control and a local control(6,12,13). Central control of motility occurs via the vagus nerve[13]. Local control can be attributed to the myenteric plexus, but the function of this plexus is little known(14). In general, the innervation is provided by the parasympathetic and sympathetic divisions of the autonomous nervous system. The parasympathetic division, efferent innervation of the stomach, consists of the ventral and dorsal vagal trunks, which accompany the oesophagus through the hiatus(10). This efferent branch has excitomotor effects on the reticular groove and inhibitors of the motility of the rumino-reticulum(10). The afferent trigeminal nerve, whose stimuli are albumin, glucose and minerals in milk, copper in ovine and sodium in bovine, reinforced by cortical afferents and the effect produced by suction(15). The trunks of the vagus nerve include viscerosensitive and motor afferent fibers, which will intervene in gastric reflexes through medullary centers, which in turn are influenced by the cerebral cortex and hypothalamus(1,3). Schenk-Saber et al(16)

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have shown that in adult goats and sheep the afferent nerve endings are highly developed in the lining of the reticulum, near the left lip of the reticular groove. These endings, rod, button or arrowhead-shaped, receivers are very important in regulating food passage and intake. Various biologically active peptides, including the vasoactive intestinal peptide (VIP), have been found in neurons of the digestive tract of ruminantâ&#x20AC;&#x2122;s newborns and adults. It is believed that VIP plays a role in mediating non-adrenergic and non-cholinergic relaxation of reticulo-omasal orifice and the abomasum during the act of sucking(7,12). This is accompanied by an increase in the concentration of VIP in the gastric and intestinal venous blood(7). Reid et al(7) found that an arterial infusion of VIP produces a relaxation of the reticulo-omasal and omaso-abomasal orifice, similar to that produced during the act of suckling(10).

Blood supply

The blood supply of the stomach of ruminants, comes from the celiac artery, which is divided into various main branches. Gastric furrow irrigation is provided by the left gastroepiploic artery, reticular accessory artery and reticular branches of the reticular artery (coming from the left ruminal artery). Dorsal and ventral arteries of the reticular groove originate from the reticular artery; supplying the two lips of reticular groove. The veins run parallel to the arteries and drain into the portal vein. The splenic vein, important branch of the portal vein, assures the drainage of the reticular groove. Blood vessels are accompanied by right, left and cranial ruminal lymph node chains, which sometimes may be missing. Auxiliary lymphatic organs are nodes located above the bottom; there are reticulo-omasal and the atrium nodules as well(10).

Histology

According Pochon(10) the structural constitution of reticular groove walls comprises four layers: â&#x201D;&#x20AC; Serous tunic, composed of connective tissue (collagen and elastic fibers) covered by mesothelium.

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─ Muscular layer of mixed origin (smooth and striated). On the lips of the groove the muscular tissue is smooth with its fibers arranged longitudinally(17). On the ground there are two layers of muscle tissue: the outer layer consists of smooth and striated fibers in longitudinal arrangement and the inner layer comprises only smooth muscle and its fibers are arranged perpendicularly to the longitudinal axis of the groove. The myenteric plexus is located between the two layers(9,17). ─ Submucosal tunic, consisting of connective tissue (collagen and elastic fibers), where the submucosal plexus is located(9). ─ Mucosal layer, composed by a stratified squamous epithelium, the muscularis mucosa and lamina propria. It has longitudinal folds on the lips, and its epithelium is darker and folded. Unguiculiform papillae, thick conical buds, with cornified epithelium, slightly curved and even twisted from the base, are found near the reticulum-omasal orifice(10). The existence of mucosal glands, especially in adult animals, and serous and mixed type in pre-ruminants (newborns) has been demonstrated.

Reticular groove physiology

Ruminants begin life similarly to monogastric animals in all matters referred to digestion, absorption and metabolism of the main nutrients. Thus, the drink consumed to pass directly to the abomasum, preventing its penetration in rumino-retículum(18), where the processes of coagulation of casein by rennet action are produced. The transition from the abomasal contents, in a first moment liquid moment, and subsequently solidificated, is slowed so as to allow the action of pepsin and lipase enzymes to reduce protein and lipid to a more suitable form for intestinal digestion(10,19). Alterations in the function of the reticular groove make lots of milk falling into a still immature rumen cavity which will cause significant digestive disturbances short and/or long-term(19). The motility of the reticular groove is initiated by the contraction of smooth and striated fibers of the muscular layer by two movements. The first one, shortening, occurs by joining the right and left lip, allowing direct passage of 30 to 40 % of the liquid volume to the abomasum. The closure is completed with a wave of investment in rotation around the axis that runs along the length of the right lip, allowing the passage of 75 to 90 % of liquid ingested to the abomasum. The reflex of the reticular groove also acts on other organs, being accompanied by transient inhibition of the reticulum and rumen contractions during breastfeeding action(15,17), expansion of the reticulo-omasal orifice, opening omasal groove and abomasal distention(5,10).

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Denac et al(14) studied the effect of vasoactive intestinal peptide (VIP) in muscle fibers incubated in an organic solution derived from the reticular groove, reticulo-omasal orifice and omasal cannel in calves and adult cows. Mechanical muscular activity was quantified by isometric and isotonic transducers. They noted that a relaxation of the longitudinal and circular muscle fibers of the reticular groove occurred, with lowest effect on adult cows, possibly by an involution of specific VIP receptors in the muscles of the reticular groove. Muscle relaxation of the reticulo-omasal and the longitudinal and circular fibers of omasal channel orifice in calves are also produced, the latter being less sensitive to the VIP than those of the reticular groove. Finally they concluded that VIP plays a role in the function of the reticular groove especially in the lactating calf, as an inhibitory, non adrenergic and non cholinergic transmitter. Triggering the closure reflex of the reticular groove can occur by central or peripheral stimuli(9). The reflex is initiated by the action of sucking and drinking, by stimuli produced by the sight of a bottle or food preparations. They do not seem to be affected by neither the type of liquid (water, whole milk, skim milk or whey), nor by the temperature of the milk, nor by the position taken during suction(9). The reflex comes exclusively from the abomasum(9). Contrary to this conclusion, Pochรณn(10) highlights that the temperature of ingested fluid plays an important role in the closing reflex of the reticular groove, being the response more effective when the fluid is offered at body temperature. The afferent fiber comes from the posterior region of the oral cavity after stimulation of oral and pharyngeal receptors activated by mechanical stimuli and certain substances, including some milk components(20). These stimuli are transmitted to the bulbar nucleus via trigeminus nerve. The efferent pathway is formed by cholinergic parasympathetic fibers of the abdominal dorsal vagus nerve that acts stimulating reticular groove lips and inhibiting the motility of proventriculi(8,9). When the pharyngeal receptors are not activated properly food is transferred to rumen and reticulum(8). In the adult animal this reflex, which is vestigial, can be triggered under certain conditions as after severe water deprivation, dehydration or increased plasma osmolality(21). In response antidiuretic hormone is secreted from the neurohypophysis, causing the reticular groove closure. So when the animal drinks water, it goes directly to the abomasum and small intestine to promote its rapid absorption, bypassing the reticulum and the rumen(9). The central nervous influence is demonstrated by the ability of conditioning this reflex by viewing, for example, a bottle in an adult animal(22). Thus the persistence of this reflex in the adult animal depends on its handling.

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Techniques for studying how it works

The attitude of the animal when it extends the neck, shakes its tail and shows an evident satisfaction when suckling vigorously, can give us an idea of progress in the reflex (23). There are multiple experimental methods to study the functioning of the reticular groove in ruminants. A simple classification can be made into direct and indirect methods.

Direct methods

They are those that enable the study of the reticular groove by direct observation from outside the animal. Wester(24) performed a surgical window on the left abdominal wall and the rumen calling it "ruminal fistula". Through this and by direct palpation of the lips of the groove their functioning can be perceived. The aid of a speculum and a light bulb enable a direct observation of its movements(25,26). Laparoscopic techniques. Endoscopy equipment has been used for displaying behaviour of reticular groove in ruminants. This entails making a ruminal fistula, introducing the endoscope fiber optic, and performing the extraction of residue content in the rumen and reticulum (by vacuum pumps or suction) to facilitate visibility of the reticular groove. Cinotti and Gentile(27) and González-Montaña et al(28) have visualized the stimulation of reticular groove though this method.

Indirect methods

They are based on tracking a known substance after being administered to the animal, which will make it possible to estimate the behaviour of the reticular groove.  Animal sacrifice. It is the oldest known method, which consists in the administration of milk and subsequent slaughter of the animals to inspect the contents of the organs(10).  Use of coloured substances. Substances like methylene blue and eosin have been used, to stain the food and in turn the mucosa of the affected organs(29). Ross(30) describes the distribution of coloured solutions in the proventriculi and abomasum after necropsy. Also,

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it is possible to consider how long the dye takes since its intake from food to appearance in stool. It has been found that the dye passed through the reticular groove could be found in stool within 12 hours after being ingested, however this period was lengthened when it fell in the rumen and reticulum.  Ruminal fistula and sampling of its contents. It consists in providing a liquid with a marker substance in order to observe its presence or absence in the rumen depending on the state of the groove. It also enables the removal of fluid entered through the rumen fistula(31). Mikhail et al(26) and González-Montaña et al(28) used methylene blue as marker dissolved in water orally administered to animals.  Abomasal fistula. This surgical technique also allows inspection of the path followed by food. If the reticular groove closure occurs, liquids administered to animals are immediately obtained through the abomasal fistula(26).  X-Ray and contrast agents. It is an old method but today it is still used. X-rays, mainly laterolateral, are taken after ingestion of liquid food with a radio-opaque (barium sulphate) dye to evaluate the performance of the reticular groove(13,32,33). At present solid radiopaque markers are used (polyethylene spheres of 1.5 mm diameter impregnated in barium "BIPS") as a contrast medium in the fluoroscopic proventriculi and abomasum of sheep(34). These spheres are often used in companion animals in the diagnosis of gut motility disorders and intestinal obstructions. Poppi et al(35) established a critical particle size of 1.2 mm in diameter, above which they cannot pass to the omasum and abomasum.  Level of blood glucose and xylose. This method is based on the administration of food with glucose and determination of glucose or xilosemia in a blood plasma sample(36). The amount of glucose administered varies among different authors, Encinas et al(37) used a glucose concentration of 0.625 g/kg BW, unlike others(26) using approximately twice the dose of glucose solution (1 g/ml/kg BW). If the reticular groove closure occurs food is directly absorbed from the abomasum in no more than 30 to 60 min, the glycaemia peak appearing 60 to 90 min after ingestion. On the contrary if the glucose enters the rumen, it will be degraded by the microflora to volatile fatty acids, increasing slowly its concentration in plasma(8,9,33,38,39). Some works estimate that the reticular groove closes when the glucose peak appears in blood between 5 to 15 min postadministration(26,37). Sargison et al(34) suggest that the use of glucose and xylose as markers may be incorrect due to the effects of stress management on the concentration of carbohydrates. Test of xylose absorption was performed to assess the degree of closure of the reticular groove after administration of copper sulphate in sheep. It is based on stimulating the groove and then the application by catheter of D-xylose (0.5 g/kg BW). If the closure appears it will cause an elevation of concentration of xylose in blood(8,9). When xylose is directly applied into the rumen through a rumen fistula, it is fermented and is not detected in blood, but in cases when it was administered orally in the absence of reflex in the groove, the xylose solution reached the reticulum and rumen and near the reticulum-omasal orifice, went to

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the omasum, abomasum and intestine from which it was absorbed(39). Stimulation of the reticular groove causes that when oral administration by catheter 500 ml of a 10% glucose solution takes place, a significant increase in blood sugar occurs, which is found in blood samples taken at 15 min post-application(28). However, this increase is very small when stimulation of the reticular groove has not been successful.  Electromyography. It is based on the implantation of electrodes into the muscular wall of the proventriculi, and the detection, amplification and recording of the action potential generated by the smooth muscle fibers. After stimulation of reticular groove, motility of the rumen and omasum ceases, by contrast the reticulum has small shocks(38).  Determination of strontium and chromium. Strontium chloride (SrCl2) and chromic oxide (Cr2O3) are two markers that, when taken with liquid food, are soluble and its concentration can be determined both in the rumen contents (strontium) and organ mucosae (chrome). Hedde and Ward(31) used strontium (5 mg/kg BW) to evaluate the effectiveness of "ruminal bypass" in calves using various routes of administration.They noted that the reflex of groove was complete in bottle-fed animals and strontium was not detected in rumen samples. However, in those calves receiving strontium in drinking water recovery from the rumen of this substance was complete.  Implantation of thermocouples. It is based on the use of thermocouples catheters housed by laparotomy in the rumen and in the abomasum to measure the temperature with a temperature sensitive device. When the reticular groove closure occurs, a variation in temperature is detected before in the sensor housed at abomasal level than at the one implanted in the rumen(23).  Test based on the detection of carbon 13 of octanoic acid excreted in breath. It is based on identifying a substance with a known reaction, through an enzyme that generates the reaction, wherein as a final part is released 13CO2, which diffuses to the blood, is transported to the lungs, and are exhaled through the expired air. Samples are taken from this air to be later measured in an isotope ratio mass spectrometer(23).

Manipulation of the reticular groove and its use in veterinary medicine

Introduction

The closure reflex of the reticular groove in young ruminant prevents dairy food from passing through the rumen and reticulum, and ensures it goes directly through the

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reticulo-omasal orifice to the abomasum (Figures 2 to 5). Knowledge of physiological factors, as well as the possible pharmacological manipulations of reticular groove reflex, both to stimulate it or to inhibit it, are of great interest in oral administration of certain drugs, in their use for treatment of certain internal diseases, and even in the better use of some foods in adult ruminants as well as in lactating ones(26,40).

Figures 2 to 5: Extremity of the reticular grooves seen by rumen endoscopy performed on the left flank of the sheep. Figures 2: semi-open sphincter. Figure 3: closed sphincter. Figures 4 and 5: closed sphincter and with slight mucosal prolapse.

Figure 2

Figure 3

Figure 4

Figure 5

Handling techniques

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For many years, the effects of diet and management on the reflex of the reticular groove, especially in calves, have been studied. According to most researchers, suction is the most important stimulus as it is considered to cause the closing reflex of the reticular groove(10). "The passage of liquid drunk from a bucket to the abomasum by calves is determined neither by temperature nor composition of the liquid nor by the position of the animal while breastfeeding, not by the act of suckling itself; but it is a result of the action of a kind of behaviour that accompanies the act of suckling"(12).

Conditioning reflex

There are many experiences that suggest that the reflex can be conditioned to a variety of circumstances. The final proof that the reticular groove can be functionally conditioned was obtained when a liquid substance deposited directly into the back of the mouth and pharynx, where there are a number of nerve receptors are found, entered into abomasum, on the condition that the animal should receive visual and auditory stimuli of utensils and equipment used during the regular feed of young ruminants, perhaps olfactory stimuli also influenced(15). However, when these stimuli did not exist the liquid reached rumen and reticulum(41). The strong excitation following the view of the bottle in a conditioned subject, such as an adult sheep, is enough to double or triple the volume of fluid collected from an abomasal fistula after administration in the lower oesophagus. The electromyogram indicates that an effective closure of the reticular groove is produced(22). These results show that the sucking behaviour can be triggered by other stimuli than these of oropharyngeal origin, and its autonomous components do not correspond to more or less complete closure of the reticular groove. In the same line other researchers argue that induction of reticular groove closure, in milk-fed calves, requires a series of determinants, including the drunk fluid must be in contact with the local receptors of the pharynx, which should be consumed voluntarily by the animal, which must not have offensive odour or taste, and the general condition of the animal must not be altered(42,43). When any of these factors is not met, the reticular groove closes incompletely, resulting in passage of the milk into the rumen and reticulum, where it is fermented by local microorganisms(43). Therefore, in some diseases this reflex may be altered, so in lactating animals there may be a failure of the afferent pathway of reflex due to a pharyngitis, the presence of oropharyngeal abscesses(8,9) or infections in the larynx(19). In calves under 14 d old affected by acute catarrhal enteritis non-closure of the reticular groove was observed in

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11.2 % of the animals, dying 11 of the 249 patients. It was possibly due to a ruminal acidosis, due to the increased butyric acid and lactic acid fermentation or vice versa, which can cause neonatal diarrhoea by dysfunction of the reticular groove in milk feeding. These calves had marked dyskeratosis lesions of the ruminal mucosa(43). It seems that a calf or lamb needs to be in a situation of comfort, without stressful situations, for the proper functioning of the stimulus in reticular groove. So it is not surprising that a forced feeding (by catheter or bottle applied to the animal's mouth) uncomfortable for the animal, which can halt stimulus, and cause liquids to pass to reticule and rumen. However, in those animals that feed directly from the motherâ&#x20AC;&#x2122;s nipple, or from a bottle nipple the act of suckling may be considered as the most important stimulus in the closing reflex of the reticular groove(10,22,41), and even a mechanical stimulation by the nipple or pacifier can also contribute(32).

Place of administration of liquids

There are many investigations in which it has been shown that when some substances are administered using an oesophageal tube the reflex of the reticular groove closure can be inhibited(19,20,29,33) . Both Ă&#x2DC;rskov and Benzie (44) and Van Weeren-Keverling Buisman et al(20) cite that as early as in 1951 Comline and Titchen(15) pointed to the nervous receptors located in the mouth and pharynx as responsible for the closure of the reticular groove. Chapman et al(33) found, a more effective treatment when large amounts of fluids are administered to dehydrated calves. In calves to whom various solutions were administered by an oesophageal catheter, it was found that the reticular groove did not lock, even after administering solutions of sodium bicarbonate, copper sulphate and guanidine hydrochloride. However, when commercial glucose solutions, amino acids and electrolytes in significant amounts, at least 2 liters, were applied there was a significant increase in blood glucose. Therefore, the results of this study suggest that the fluids intended to be absorbed in the intestine can be advantageously administered using a oesophageal tube, even if the reticular groove closure does not occur, provided that it implies a considerable amount(33).

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Characteristics of the fluids administered

The stimulation on oropharyngeal and lingual receptors has been attributed to protein and milk salts, which trigger the reflex of reticular groove closure(22). There are many examples of investigations that have worked with fluids of different nature to cause effective closure of the reticular groove(15,41,45-48). The milk supplied at different temperatures and under the same management routine, can effectively stimulate the reticular groove, but the response is more intense when milk is offered at body temperature. Also water orally administered at body temperature is able to cause a partial closure of this structure, though cold water fails to stimulate reflex(10). Some research has shown that water can be recovered from the abomasum, after passing through the reticular groove, when experiences are made in conditioned animals who have been by previously accustomed to suckle from a bucket. So it appears that the conditioned reflex is probably more important than the nature of the administered liquid(10,15,41,44,45,47). Pochรณn(10), cites that Hegland et al in 1957(49), concerned about the effects of diet and management, studied the destination of various fluids, such as whole milk, reconstituted skimmed milk, reconstituted whey and water, and food capsules in fistulised calves fed by regular bucket or bucket with nipple. In those cases, where capsules of varying sizes together with liquid feed was administered, the closing reticular groove leading to these capsules were located in the omasum, however if the calf was not receiving liquid feeding capsules they were placed in the reticulum. It was accepted that a capsule had passed through the reticular groove when after one minute it was neither in rumen nor in reticulum. Therefore, according Hegland et al(49) any of the tested liquids were able to elicit the stimulation of the reticular groove, leading to complete closure in all tested calves during the first 6 weeks of life. They also found that the effectiveness is similar using both methods of feeding (open bucket and bucket with nipple), while feeding bucket-nipple lengthened the reflex in all calves up to 13 wk after birth, while drinking directly from bucket only it was effective in the first 6 weeks of life of the calf. To test the possible effect of whey on the reticular groove 14 dairy calves were fed with a liquid diet(46). Experimental observations were made every two weeks, beginning at 20 wk of age up to 30 wk, and the ingestion of whey made from a bucket, proceeding to palpation of lips of the groove through a ruminal fistula and the whey collection which had passed to the abomasum. The difference between what had been consumed and what had been recovered would be the amount of whey which had reached the rumino-reticular compartment. In most animals 80% of the food was collected, except at 20 wk, which was 53 %. The reflex of groove occurred 15 to 20 sec after ingestion. It was concluded that the use of whey led to the closure of the reticular groove and also the continuous ingestion of increasing amounts of whey, keeping consumption habits (schedules, dosage form and temperature), allowed the maintenance of reflex over time, with consequent

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digestion of milk derivative to intestine. Weight gain was greater than in those animals to whom whey was diverted into the rumen(46).

Temperature of fluids given

It has already been indicated that both water and milk at body temperature can cause to greater or lesser extent, closure of the reticular groove. Cold water does not stimulate this reflex(10). However for Ă&#x2DC;rskov(45) stimuli as temperature and feed composition, dosage form and position of the animal are much less important on the reflex of the reticular groove than the environment surrounding the animal or its mental state at the time of ingestion of liquid food.

Method of administration of fluids

The mere sight of a bottle is enough to trigger the closure of the reticular groove in adult animals, provided they have been accustomed to eating milk or liquids this way(10,22). This reflex is also present if young animals are taught to drink milk food from a bucket with a nipple(10,41). This behaviour would be similar to that produced when an animal suckles its mother's udder. In 1928 it had already been observed, by using rumen fistulae, that milk flowed through the groove to omasum and abomasum using a mechanism of nipple for feeding calves, however when drinking directly from a bucket lots of ingested milk passed to the ruminoreticular cavity(10). A few years later, in 1942, Wise et al(25), working with 17-56 d-old calves, also found more milk in the rumen and reticulum when they were fed using a regular bucket (open) than when they did it with a nipple bucket, located slightly elevated(10). In examining the effect of closure of the reticular groove in the absorption of a combination of sulfamethoxazole-trimethoprim, using calves 6 wk of age, trained to suck through a nipple-bucket with teat, they found that the maximum plasma concentration and persistence sulfamethoxazole time is 7.5 times higher and 6.9 times longer when administered by teat bucket if catheter is used(50), while the trimethoprim is only detected in plasma if calves are fed from buckets with nipple, which is justified in

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both cases by the closure of the reticular groove, with the consequent passage of drugs, given orally, to the abomasum, resulting an increased availability of these drugs(50).

Mineral salts

Various substances have been tested to manipulate the reflex of the reticular groove in ruminants; in some cases applied parenterally and in many others orally administered.

Copper salts

Several authors believe that oral copper salts remain the most effective method. According to Pochรณn(10), after reviewing multiple researches, in ovine the most effective is copper sulphate(21,25,29,30,32,36,38,39,44), but other salts as copper acetate and copper chloride(21,32), zinc sulphate(21,30) are also capable of causing the closure. In adult cattle its effectiveness has also been proven(24), while they have not been particularly effective when used in calves(10,33) nor goats(51). According to some authors(21) copper sulphate solution at 10 % is the most potent agent to stimulate the reticular groove reflex due to stimulation of oropharyngeal receptors in adult sheeps. Copper sulphate at 10 % has also been employed(38) to cause the closure of the reticular groove and to study electromyographically the forestomaches motility in adult sheep. Prior to electromyographic study, they administered a solution at 20 % of glucose orally, before and after application of copper sulphate, and found that all the sheep responded positively to stimulation, altering their glycaemia significantly. To activate the reflex of groove Nicholson and Belkhiri(18) used oral copper sulphate (1 g in 10 ml of water), but later inhibited the reflex with the application of clonidine. In an attempt to test the effect of the administration of copper sulphate and cobalt sulphate in the stimulation of the reticular groove Sargison et al(34) used recently fed sheep (10 in each group), 10 mo old, who were administered barium sulphate as a contrast medium and polyethylene spheres impregnated with barium as solid radiopaque markers and studied under radioscopy. However, others(52) were not able to consistently stimulate the reticular groove with copper sulphate (20 ml at 10 %, via per os). 745

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Salts of sodium

Conversely, sodium salts (chloride, sulphate, bicarbonate, acetate, etc.) appear to have a greater effect in cattle while in sheep they are rarely effective(32,53). De Vuyst(29) caused a reflex of reticular groove closure in adult cattle with a 10 % sodium bicarbonate solution, whereas if this solution was administered via catheter, surpassing the oral mucosa, reflex did not occur. A similar effect was found in experimental research carried out with a 7 % sodium sulphate solution, with a solution of 0.1 % eosin as a marker, which revealed that any food administered afterwards passed through the reticular groove, which means the adult ruminant high quality nutrients could pass to the abomasum through the reticular groove, avoiding destruction by ruminal fermentation(29). Several researchers had previously managed to close the reticular groove in cattle, with high effectiveness(25,30,36,53). Mikhail et al(26) obtained a significant increase in glycaemia in goats by oral glucose administration after stimulating the reticular groove with 1.5 ml of saturated NaCl solution or 10.5 ml of 1.5 %, NaCl solution applied intravenously. The application of sodium bicarbonate or copper sulphate in calves orally, by oesophageal catheter, fails to stimulate the closure of the reticular groove(33), but perhaps may also be influenced by the discomfort caused to the animal when using this method, which may result in an inability to close the groove(10). Bakker(54) administered solutions of hypertonic dextrose, chloride hypertonic saline, hypertonic sodium bicarbonate and magnesium sulphate, and none of them was able to trigger the reticular groove reflex, so he concluded that previous studies that had encouraged closure by using various salts, especially NaCl and HCO3Na, could not be repeated and were caused by the closure of the reticular groove as a result of hypovolemia, rather than the closing of this structure itself.

Glucose solutions

Glucose in concentrations between 5 and 10 % may able to cause the closure of the reticular groove(24), however Riek(53) was unable to achieve this effect. The administration of sugar solutions was not enough to stimulate the closure of the reticular groove when were administered orally in goats(26), perhaps because the animals used in the experience still keep the reflex of the reticular groove closure(51).

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Other salts

In 1997 and 1999 Smith et al(55,56), found that zinc (zinc sulphate and zinc acetate) also stimulates the reflex of reticular groove in sheep, and that this effect depends on concentration of the zinc solution.

Vasopressin

There are many experiences in which vasopressin has been used to trigger this effect(20,26,48,57). In some cases, administration has been exogenous, in others endogenous release in thirsty and dehydrated animals, or after intravenous administration of sodium chloride solutions, has taken place. Water deprivation in goats causes an increase in blood levels of vasopressin (ADH) as a response to thirst(51). Endogenous vasopressin release, after the stimulation of osmoreceptors by hypertonic sodium chloride, is the cause of the closure of the reticular groove and the resulting increase in blood glucose(58). Mikhail et al(26) determined the influence of thirst, administration of sodium chloride in the common carotid artery and vasopressin in the closure of the reticular groove in adult goats. NaCl solutions induce endogenous vasopressin secretion, responsible for the effect on the reticular groove; water deprivation for 48 h also led to the passage of large amounts of water to the abomasum, with hyperglycaemia if water was administered orally with glucose(26). Encinas et al(37) studied the pharmacokinetics of a nonsteroidal anti-inflammatory, sodium meclofenamate, used in sheep for treatment and prophylaxis of allergic diseases and mastitis. This product was orally and intravenously administered to sheep, and determined the influence of reticular groove closure in drug bioavailability. To stimulate the reticular groove, they employed as a pre-treatment solution lysine-vasopressin (0.3 IU/kg iv PV) or 0.9 % NaCl iv 10 min before oral glucose (as a surrogate marker) and sodium meclofenamate. They noted that in sheep to whom lysine-vasopressin was administered, the reticular groove closed in all cases, whereas with 0.9 % NaCl, stimulation only occurred in 2 of 6 sheep. Of course the plasma concentration and removal rate of sodium meclofenamate after oral administration was influenced by the state of the reticular groove, while differences in bioavailability between pretreatments were not found.

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Vasopressin has also been used with positive results in adult cows with acute phosphorus deficiency, to whom phosphate was orally administered. It was determined that the solution was diverted into the abomasum with a faster absorption(59).

Reflex inhibition

Reflex inhibition can also be interesting in some practices with sheep. Inhibition can be achieved administering a local anaesthetic in the oral cavity, with atropine intravenous injections, acting at the level of the efferent pathway(12,60) and with metoclopramide (0.2 mg/kg)(61) which act as an antagonist of dopamine through the cholinergic system. Domperidone has a similar effect although not with the same access intensity in CNS that metoclopramide(62). Some research(18) state that norepinephrine acts on groove motility through a cholinergic mechanism. Nicholson and Belkhiri(18) used clonidine at doses of 2 and 4 g/kg iv in adult sheep as a reticular groove reflex inhibitory by its agonist action to -2 adrenoreceptor, causing inhibition of ruminorreticular motility by acting on the CNS. Orally administered copper sulphate was employed to enable the reflex of the reticular groove. Clonidine at doses of 2 g/kg produced a decrease in reticular motility, complete halt during 10-50 minutes in those animals receiving 4 g/kg. When subsequently used a -2 antagonist idazoxan at 0.1 mg/kg, prior to application of clonidine to prevent inhibition of the groove closure, they observed an increase in the peak concentration of the marker used (xylose), more significant if only idazoxan was used. Hexamethonium exerts a nodal locking effect similar to vagotomy, avoiding the contraction of the reticular groove(10,13).

Use in veterinary medicine

The ability to control this reflex is of great interest in the oral administration of various drugs, to treat certain diseases, as well as in the more efficient use of some food resources. There are many experimental protocols in which it has been demonstrated that treatment with glucose solutions(26,42,63-65), with NSAIDs such as meclofenamate and acetaminophen(37,66,67), with certain antibiotics such as chloramphenicol(57) or sulfamethoxazole-trimethoprim(50), and some antiparasitic are generally ineffective due to product degradation by ruminal microflora or, conversely because of too fast

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circulation through the rumen(68-70). It is therefore of great interest to stimulate furrow closure in the first case and suppressing it in the later. No doubt the therapeutic efficacy of certain substances would considerably improve if these products could go through the stomach directly and reach the abomasum. It has even been postulated that the management of reticular groove could be used to obtain higher economic returns handling cattle feed(23,29,42,44,62). Some researchers have made use of vasopressin at different concentrations for stimulating the reticular groove in ruminants, facilitating the treatment of diseases such as ketosis, diarrhoea and ovine pregnancy toxaemia(26,28,42,64). So, some authors(63,71) obtained better results in the treatment of diseases such as nonspecific diarrhea bovine or primary ketosis in animals with whom vasopressin was used to stimulate the closure of the reticular groove than in those in the control group. El-Hamamsy et al(64) and GonzĂĄlez-MontaĂąa et al(28) used lysine-vasopressin to stimulate the closure of the reticular groove and demonstrated the efficacy for increasing glucose following administration of a glucose solution orally, since glucose went directly to the abomasum avoiding unwanted rumen fermentations. According Ranzini Rodrigues et al(23) performance of calves receiving a source of nondegradable protein is, most of the time, higher than that of animals that do not receive it(72-74) due to the higher flow amino acids that reach the small intestine(23). Therefore, an effective way to avoid protein degradation as it passes through the rumen, thereby preventing the loss of essential dietary amino acids, would be to provide protein sources through the reticular groove. Other authors have demonstrated the effectiveness of using the reflex of the reticular groove in the administration of various liquid supplements in calves (milk, skim milk, soybean bran suspension, fish meal and whey) compared to those receiving concentrated(41,47). Also Standaert et al(75) found increased milk production in cows that were fed with casein through the reticular groove compared to those receiving the same amount of protein, but in a solid form. Conversely, in some cases a reflex inhibition of reticular groove is important from the viewpoint of the administration of various drugs. So management of reticular groove is particularly relevant when used in conjunction with antiparasitic(40,76,77). Mc Ewan and Oakley(40) attributed the failure of certain anthelmintic to the closure of the reticular groove in calves, by reducing the time of exposure to certain parasites such as nematodes. In calves ranging between 125 and 205 kg, the groove is still functional, that is the reason for necropsy evidence of ruminal bypass in half the animals, and that is why antiparasitic efficacy is reduced. The effectiveness of fenbendazole is variable against inhibition of Ostertagia ostertagi larvae in the calf(76,77). Other antiparasitics studied by stimulating the reticular groove are benzimidazole(68), oxfendazole(69) or some coccidiostats as the medium chain fatty acids (MCFAs)(70,78).

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Acknowledgements

We appreciate the assistance of Miss Anna Krutter Abilla for the linguistic revision of this paper.

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https://doi.org/10.22319/rmcp.v10i3.4386 Technical note

Organic matter fertilization improves morphological variables in Nopalea cochenillifera Salm Dyck cv. Miúda grown as forage in Pernambuco, Brazil

Paulina Vazquez Mendoza a* Toni Carvalho de Sousa b Mercia Virginia Ferreira Dos Santos b Oscar Vicente Vazquez Mendoza c Jose Carlos Batista Dubeux Junior d Mario de Andrade Lira b

Universidad Autónoma de Guerrero. Centro Regional de Educación Superior de la Costa

Chica. 41800, Florencio Villarreal, Guerrero. México. b

Universidade Federal Rural de Pernambuco. Departamento de Zootecnia. Pernambuco.

Brasil. c

NOREL México SA DE CV, Querétaro, México University of Florida. North Florida Research and Education Center, Marianna, Florida,

USA.

* Corresponding author: vazmepa@gmail.com 756

Rev Mex Cienc Pecu 2019;10(3):756-766

Abstract: In forage crops such as cactus morphological characteristics respond to management practices such as fertilization. An evaluation was done to determine if organic matter (OM) fertilization (0, 10,000, 20,000 and 30,000 kg OM ha-1 yr-1 bovine manure), mineral fertilization (0, 120, 240 and 360 kg N ha-1 yr-1, using urea) and cut frequency (annual and biennial) influenced cladode length, width and perimeter, and Cladode Area Index (CAI) values in Nopalea cochinillifera Salm Dyck cv. MiĂşda, and how these variables related to productivity. The experimental design was random blocks, using a sub-sub-plot arrangement with four replicates. Fertilization with 30,000 kg OM ha-1 year-1 increased cladode width by 9.8 % and length by 17.8 % compared to the control. Cladode perimeter increased proportionally to OM fertilization level. At the optimum fertilization level (25,970 kg ha-1) the CAI value was 68.29 % higher than the control. Mineral fertilization only affected cladode perimeter at 120 kg ha-1 and only with an annual cut; however, at this fertilization level the CAI value was higher with a biennial cut. Organic matter fertilization increased cladode width and length, and CAI values in N. cochenillifera cv. MiĂşda, while mineral fertilization had only a minimal effect. Biennial cutting frequency results in higher CAI values. Correlations were high between the evaluated variables and dry matter production, highlighting the utility of morphological variables in evaluating productivity. Key words: Forage cactus, Cladode area index (CAI), Fertilization, Cut frequency.

Received: 24/02/2017 Accepted: 22/08/2018

The northeast of Brazil accounts for 18.27 % of the country, and most (62.11 %) of this region is semiarid(1). Annual rainfall distribution (500 mm)(2) is irregular, leading to severe feed shortages in ruminant livestock systems. The cactus species Opuntia sp. and Nopalea sp. can be used as ruminant forage and are adapted to water scarcity, high temperatures and poor soils(3,4,5). They are thus valuable alternative feed sources, especially during the dry season. Opuntia ficus-indica Mill and Nopalea cochenillifera Salm Dyck are the two most widely cultivated cactus species in northeast Brazil(6). Nopalea cochenillifera cv. MiĂşda, commonly known as Nopal Dulce, is a fodder that has the advantage of being resistant to cochineal (Dactylopius opuntiae Cockerell)(7,8). This characteristic, in addition to its 6.2 % crude protein (CP) content, 26 % neutral detergent fiber (NDF) content and 78 % digestibility(9), makes it a promising alternative livestock feed source. As with any cultivated forage species, N. cochenillifera requires adequate soil fertility to produce properly. Fertilization with organic matter and minerals is common practice in

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cactus cultivation to compensate for nutrient extraction by this crop and increase forage production efficiency(10,11). The variables used in forage plant ecophysiology research exhibit different responses depending on plant management. In cacti, cladode morphological characteristics are directly related to fresh and dry matter yields(12), but few studies use morphological characteristics as productivity indicators. The present study objective was to evaluate the effect of organic and mineral fertilization, and cladode harvest frequency on cladode morphological characteristics in Nopalea cochenillifera Salm Dyck cv. Miúda cultivated in the agreste region of Pernambuco, Brazil. The experiment was done between June 2011 and May 2013 at the Experimental Station of the Pernambuco Agronomic Institute, Caruaru Municipality, in the agreste region of the state of Pernambuco. This is a transition zone between humid and semi-arid tropical forest zones. This region’s stony soil supports sparse (˂40 and ˃20% coverage), low-level (˂1.5 m height) vegetation(13). It is in northeast Brazil (8°14’ S; 35°55’ W) at an altitude of 575 m asl and contains Neolithic Regolithic soil(14). During the experimental period precipitation at the site was 1,068.3 mm but varied widely from month to month (Figure 1).

Figure 1: Monthly rainfall (mm) and maximum and minimum temperatures in Caruaru Municipality, Pernambuco, Brazil, from January 2011 to June 2013 Precipitation

250

maxT

minT

150

100

15 10

50 0

5 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun

Rainfall (mm)

2011

2012

758

2013

Temperature (°C)

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Soil samples were collected at the surface and 20 cm depth and chemically analyzed following the soil analysis method of Empresa Brasileira de Pesquisa Agropecuária(15) (Table 1). Table 1: Prefertilization soil chemistry in experimental area in Caruaru Municipality, Pernambuco, Brazil Component pH (water) Phosphorous, mg dm-3† Potassium, mg dm-3 Calcium, mg dm-3 Magnesium, mg dm-3 Manganese, mg dm-3 Zinc, mg dm-3 Iron, mg dm-3 Copper, mg dm-3 †

Mean 4.78 10.45 74.29 428.00 48.62 70.42 12.46 46.20 0.06

SME 0.1 3.81 0.04 0.26 0.05 10.27 1.66 3.06 0.02

Component Sodium, mg dm-3 Aluminum, mg dm-3 Hydrogen, mg dm-3 S.B.¶, cmolc dm-3 CIC§, cmolc dm-3 VÞ, % Carbon, % M¤ , % OM††, %

Mean 11.50 17.98 24.70 2.78 5.46 50.05 1.15 8.15 1.97

SME¶¶ 0.01 0.03 0.14 0.33 0.38 3.21 0.06 1.97 0.10

Mehlich 1; ¶sum of bases; §cation interchange capacity; Þbase saturation; ¤aluminum saturation; †† soil organic matter; ¶¶ standard mean error.

Seven fertilization treatments were evaluated: negative control with no fertilization; fertilization with bovine manure at 10,000, 20,000 and 30,000 kg OM ha-1 yr-1; and mineral fertilization (with added urea) at 120, 240 and 360 kg N ha-1 yr-1. Annual and biennial harvest cutting frequencies were applied. The experimental design was randomized blocks subdivided into plots, with four replicates. The largest plot (14.4-8.0 m) was used to test organic matter levels; subplots (7.2 x 8.0 m) to evaluate cutting frequencies, and other sub-plots (14.4 x 2.0) to assess nitrogen levels. Each experimental unit consisted of six rows of plants. The two side rows and three plants at the ends were considered edges, leaving an effective sampling area of 33.84 m2 containing 282 plants. Planting was done between April and May 2011 by sowing mature N. cochenillifera cladodes in rows with 1.2 m between rows and 0.1 m between cladodes. Overall density was 83,336 plants per hectare. Organic fertilization was done at the time of planting (June 2011) and after the first annual cut (June 2012) using bovine manure containing 1.1 kg-1 N, 3.74 kg-1 P and 16.5 g kg-1 K (determined using AOAC methods)(16). During the first year of cultivation mineral fertilization was done from 5 June to 19 July 2011, and during the second year on 28 June, 23 July and 19 August 2012. Harvest of cladodes was complete, leaving only the mother plant. Cladode length (cm), width (cm) and perimeter (cm) were traced onto a sheet of white paper (A4 size). The outline of two cladodes per sub-subplot were drawn and examined with a leaf area analyzer (Portable Laser Leaf Area Meter CI -202 Bio-Science Inc.®). The CAI was evaluated by adding the area of one plant’s cladodes (m2) (considering

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both sides of the cladode) and dividing it by the soil surface (0.12 m2) occupied by each plant(17). Calculations were done of the correlation coefficient between dry matter (DM) production(18) and the variables of cladode length, width and perimeter, as well as the CAI. The data were analyzed using the MIXED procedure in the SAS software package(19). The Tukey test (P≤0.05) was applied to the factor cut frequency, and polynomial orthogonal contrasts (P≤0.05) were applied to the factors organic and nitrogen fertilization. Cladode length, width and the CAI values exhibited no effects (P>0.05) from mineral fertilization. This could have been due to irregular rainfall during the experimental period (Figure 1), which would affect nutrient absorption, or low soil organic matter content (Table 1). Similar effects have been reported for mineral fertilization on morphological characteristics and biomass production in N. cochenillifera cv. Miúda. In one study these were attributed to the root system, the growth of which responds to rainfall, meaning that irregular rainfall distribution can negatively affect nitrogen absorption efficiency and increase nutrient loss through leaching during excess rainfall or volatilization during its absence(20). Positive effects from nitrogen and mineral phosphate fertilization have been observed in the production of other cactus species such as Opuntia lindheimeri, but only two years after crop establishment(21). Organic fertilization is known to promote crop growth and production(22). In the 30,000 kg ha-1 year-1 treatment, cladode width and length increased proportionally to organic fertilization (R2= 0.26 and 0.47; P≤0.001), and increased 9.8 and 17.8 % (respectively) versus the control (P≤0.05) (Figure 2).

Figure 2: Cladode length and width in Nopalea cochenillifera Salm Dyck cv. Miúda fertilized with bovine manure (Agreste region, Pernambuco, Brazil) Lenght Width

Cladode Length and Width (cm)

Y = 15.74 + 0.103OM

CV=8.5; R² = 0.921; p < 0.000 10 5 0

10000

20000

Organic fertilization (kg ha-1 yr-1)

760

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This is similar to studies showing that cladode length in O. ficus-indica cv. Lisa increased gradually over three consecutive years in response to organic fertilization with bovine manure at 20,000, 40,000 and 60,000 kg ha-1 yr-1, as well as in the control (0 kg ha-1 yr-1 fertilization)(23). In another study using higher fertilization rates (90,000 kg ha-1 yr-1 bovine manure) over a shorter period (600 d), cladode length in O. ficus-indica cv. Gigante increased only 8 % compared to the control(24). In a previous study cladode width in O. ficus-indica did not change (P>0.05) in response to application of bovine manure at 0, 20,000, 40,000 and 60,000 kg ha-1(23). Organic fertilization, mineral fertilization and an annual cut frequency affected cladode perimeter (P≤0.05). This increase became larger (P≤0.05) in a linear manner in response to organic matter fertilization. Cladode perimeter also depended on the interaction between mineral fertilization and cut frequency (Figure 3). This variable increased at 0.339 cm per 1,000 kg OM and 360 kg N per ha-1, and 0.211 cm per 1,000 kg OM and 120 kg N ha-1. The cladode perimeter increase rate was higher with annual cuts (0.211 cm per 1,000 kg OM) than biennials (0.0304 cm per 1,000 kg OM).

Figure 3: Cladode perimeter in Nopalea cochenillifera Salm Dyck cv. Miúda in response to organic fertilization, mineral fertilization and cut frequency (Pernambuco, Brazil) 60

AnnualN120 Y= 46.18+0.211OM, CV=8.09, R2 = 0.66, P=0.04 (….)

Cladode perimeter (cm)

AnnualN360 Y=42.075+0.339OM, CV=8.77, R2=0.65, P=0.004 (_.._.._)

BiennialN120 Y=43.970+0.0304, CV=7.74, R2=0.63, P=0.005 (-.-.-.-)

40 0

10000 20000 Organic fertilization (kg ha-1 yr-1)

30000

In response to organic fertilization level the cladode area index (CAI) exhibited a quadratic trend (P≤0.05) with proportional increases in the 10,000 and 20,000 kg OM ha-1 year-1 treatments (Figure 4). The maximum CAI value was reached at 25,970 kg OM ha-1 year-1;

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organic fertilization levels higher than this did not affect CAI values. This plateau in CAI values may be due to the shading effect, which consists of lack of exposure of the photosynthetically active area and can lead to lower production under high plant densities(25). For instance, in one study spacing between plants and organic fertilization level affected CAI values in Opuntia ficus-indica cv. Gigante, with higher values at spacings of 1 x 0.5 m and organic fertilization levels between 60,000 and 90,000 kg ha-1 yr-1(24). Both this spacing and fertilization level are higher than those used in the present study. Other types of organic fertilizers produce different responses. When a biofertilizer based on bat guano and chopped up cladodes was used at 15,000, 30,000, 45,000 and 60,000 kg ha-1 yr-1 on O. ficus-indica cv. Gigante increases of 30 % in the photosynthetically active area were observed(26). However, this 30 % increase in CAI values is notably less than the 68.9 % (versus the control) observed in the present study at the of 25,970 kg ha-1 yr-1 fertilization level. Figure 4: Cladode area index (CAI) values in Nopalea cochenillifera Salm Dyck cv. Miúda in response to organic fertilization (Pernambuco, Brazil)

Cladode area index

5 4 Y = 1.369 + 0.247OM - 0.0048OM2 CV = 24.72; R² = 0.95; P = 0.0012

3 2 1 0

10 000 20 -1 Organic fertilization (kg ha yr-1)

Cut frequency did affect CAI (P≤0.05). Plants cut at two years had higher (P≤0.05) average CAI values (4.7) than those cut annually (2.2). This difference can be attributed to a smaller residual photosynthetic area in plants subjected to annual cutting than in those cut biennially since a low CAI value represents light interception and slower plant growth(27). The effect of cutting frequency may also be due to other factors. For instance, dry matter production in O. ficus-indica was reported to be higher when secondary cladodes were not cut(28), possibly because plants with secondary-level cladodes have a larger CAI which represents a larger photosynthetic area(29,30).

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Correlation coefficient analyses between productivity and morphological variables identified a high correlation between the evaluated variables, suggesting that measurement of morphological variables can be used as a productivity indicator (Table 2).

Table 2: Correlation coefficients between morphological variables and production (t DM) in Nopalea cochenillifera Salm Dyck cv. Miúda Variables Productionǂ CAI Width Length Perimeter

Productionǂ

CAI 0.94**

Width 0.95** 0.86**

Length 0.82** 0.86** 0.81**

Perimeter 0.91** 0.83** 0.95** 0.88**

** (P<0.01); * (P<0.05). CAI = cladode area index. ǂ Source: Souza TC, 2015(18).

In Nopalea cochenillifera Salm Dyck) cv. Miúda, fertilization with bovine manure increases cladode width and length, and cladode area index (CAI) values, while mineral fertilization with urea has little or no impact on these variables. Cladode area index (CAI) values were higher when using a biennial cut frequency. Correlation was high between morphological and production variables, highlighting the importance of studying morphological variables in this forage crop.

Acknowledgements The research reported here was part of a research visit by PVM under the supervision of Dr. Luis Alberto Miranda Romero and Dr. Mercia Virginia Ferreira dos Santos. This research was financed by the CONACYT, the Universidad Federal Rural de Pernambuco and the Universidad Autónoma Chapingo.

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Ministério da Integração Nacional. Nova Delimitação do Semi-Árido Brasileiro. Secretaria de Políticas de Desenvolvimento Regional. Brasília, D.F. Brasil. 2005.

2. Silva VPR, Pereira ERR, Almeida RSR. Estudo da variabilidade anual e intra-anual da precipitação na região nordeste do Brasil. Rev Bras Metereo 2012;27(2):163-172.

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3. Fuentes-Rodríguez J. Feeding prickly pear cactus to small ruminants in Northern Mexico. I. Goats. J Prof Assoc Cactus Dev 1997;2:23-25. 4. Luttge U. Ecophysiology of crassulacean acid metabolism (CAM). Ann Bot 2004;93:629652. 5. De Waal HO, Zeeman DC, Combrinck JW. Wet faeces produced by sheep fed dried spineless cactus pear cladodes in balanced diets. S Afr J Anim Sci 2006;36:10-13. 6. Galvão Jr. JGB, Silva JBA, Morais JHG, Lima RN. Palma forrageira na alimentação de ruminantes: cultivo e utilização. Acta Vet Bras 2014;8:78-85. 7. Vasconcelos AGV, Lira MA, Cavalcanti VALB, Santos MVF. Seleção de clones de palma forrageira resistentes à cochonilha do carmim (Dactylopius ceylonicus). Rev Bras Zootecn 2009;38:827-831. 8. Neves ALA, Pereira LGR, Santos RD, Voltolini TV, Araújo SA, Moraes SA, Aragão ASL, Costa CTF. Plantio e uso da palma forrageira na alimentação de bovinos leiteiros no Semiárido brasileiro. 1ª ed. Embrapa Gado de Leite, Juiz de Fora M. G. Comunicado Técnico 62. 2010. 9. Batista AM, Mustafa AF, McAllister T, Wang Y, Soita H, McKinnon JJ. Effect of variety on chemical composition, in situ nutrient disappearance and in vitro gas production of spineless cacti. J Sci Food Agric 2003;83:440-445. 10. Dubeux Jr. JCB, Araújo Filho JT, Santos MVF, Lira MA, Santos DC, Pessoa RAS. Adubação mineral no crescimento e composição mineral da palma forrageira clone IPA20. Rev Bras Ciênc Agrár 2010;5:129-135. 11. Santos DC, Farias I, Lira MA, Santos MVF, Arruda GP, Coelho RSB, Dias FM, Melo JN. Manejo e utilização da palma forrageira (Opuntia e Nopalea) em Pernambuco. Empresa Pernambucana de Pesquisa Agropecuária (IPA). Documentos 30. Recife. Brasil. 2006. 12. Neder DG, Costa FR, Edvan RL, Filho LTS. Correlations and path analysis of morphological and yield traits of cactus pear accessions. Crop Breed Appl Biotechnol 2013;13:203-207. 13. Brito CILV, Ffolliot PF, Peixoto PSA. Uma classificação morfo-estructural para descrição e avaliação da biomassa da vegetação da caatinga. Rev Caatinga 2008;21(2):204-213. 14. EMBRAPA (Empresa Brasileira de Pesquisa Agropecuária) Centro Nacional de Pesquisa de Solos. Sistema brasileiro de classificação de solos. 3ed. Rio de Janeiro: Embrapa Solos. 2013.

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15. EMBRAPA (Empresa Brasileira de Pesquisa Agropecuária). Manual de Métodos de Análise de Solo. Centro Nacional de Pesquisa de Solos. 2a ed. Rio de Janeiro, Brasil. 1997. 16. AOAC (Association of Official Agricultural Chemists). Official Methods of Analysis. 15th. ed. Association of Official Agricultural Chemists, Washington, DC. 1990. 17. Nobel PS, Bobich EG. Environmental biology. In: Nobel PS editor. Cacti: Biology and Uses. University of California Press, California, USA. 2002. 18. Souza TC. Sistemas de cultivo para palma forrajeira cv Miúda (Nopalea cochenillifera Salm Dyck) [tesis doctorado]. Brasil, PR: Universidade Federal Rural de Pernambuco; 2015. 19. SAS (Statistical Analysis System). SAS/STAT User´s Guide (Release 9.3). Cary, NC, USA. SAS Inst. Inc. 2012. 20. Cunha DNFV, Gomes ES, Martuscello JA, Amorim PL, Silva RC, Ferreira PS. Morfometria e acúmulo de biomassa em palma forrageira sob doses de nitrogênio. Rev Bras Saúde Produç Anim 2012;13:1156-1165. 21. González CL. Potential of fertilization to improve nutritive value of prickly pear cactus (Opuntia lindheimeri Engelm.) J Arid Environ 1989;16:87–94. 22. Julca-Otiniano A, Meneses-Florián L, Blas-Sevillano R, Bello-Amez S. La materia orgánica, importancia y experiencias de su uso en la agricultura. IDESIA 2006;24:4961. 23. Trejo-Escareño HI, Salazar-Sosa E, López-Martínez JD, Vázquez-Vázquez C. Respuesta del nopal forrajero (Opuntia fícus-indica L.) a la aplicación de estiércol bovino solarizado. AGROFAZ 2014;14:87-95. 24. Donato PER., Pires AJV, Donato SLR, Bonomo P, Silva JA, Aquino AA. Morfometria e rendimento da palma forrageira ‘Gigante’ sob diferentes espaçamentos e doses de adubação orgânica. Rev Bras Ciênc Agrár 2014;9:151-158. 25. Nobel PS. Ecophysiology of Opuntia ficus-indica. In: Mondragon-Jacobo C, PerezGonzalez S editors. Cactus (Optunia spp.) as forage. FAO Plant Protection and Production Paper 169, Rome, Italy. 2002. 26. García CV, Varnero MT, Espinoza M. Efecto de bioabono sobre el área fotosintéticamente activa, producción de cladodios y eficiencia de recuperación de N en un cultivo de tuna (Opuntia ficus-indica L.) en el primer año post-plantación. J Prof Assoc Cactus Dev 2001;4:93-104.

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27. Dubeux JCB, Ben Salem H, Nefzaoui A. Producción y utilización de nopal forrajero en la nutrición animal. En: Inglese P, Mondragon JC, Nefzaoui A, Sáenz C. Ecología del cultivo, manejo y usos del nopal. FAO, Roma, Italia. 2018. 28. Alves RN, Farias I, Menezes C, Simões R, Lira MA, Santos DC. Produção de forragem pela palma após 19 anos sob diferentes intensidades de corte e espaçamentos. Rev Caatinga 2007;20:38-44. 29. Farias I, Lira MA, Santos DC, Filho JJT, Santos MVF, Fernandes APM, Santos VF. Manejo de colheita e espaçamento da palma-forrageira, em consórcio com sorgo granífero, no agreste de Pernambuco. Pesqui Agropecu Bras 2000;35:341-347. 30. Lira MA, Santos MVF, Dubeux Jr. JCB, Mello ACL. Sistema de produção de forragem: alternativas para sustentabilidade da pecuária. Rev Bras Zootec 2006;35:491-511.

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https://doi.org/10.22319/rmcp.v10i3.4911 Technical note

Development and evaluation of equations to predict body weight of Pelibuey ewes using heart girth Alfonso J. Chay-Canul a Ricardo A. García-Herrera a* Rosario Salazar-Cuytún b Nadia F. Ojeda-Robertos a Aldenamar Cruz-Hernández a Mozart A. Fonseca c Jorge R. Canul-Solís d

Universidad Juárez Autónoma de Tabasco, División Académica de Ciencias Agropecuarias.

Carretera Villahermosa-Teapa, km 25, R/A. La Huasteca 2ª Sección, 86280 Villahermosa, Tabasco, México. Tel. DACA: (993) 358-1585, 142-9151, Fax DACA: (993) 142-9150. b

Universidad Autónoma de Yucatán. Facultad de Medicina Veterinaria y Zootecnia, Mérida,

Yucatán, México. c

University of Nevada, Department of Agriculture Nutrition & Veterinary Sciences. Reno, Nevada.

USA. d

Tecnológico Nacional de México, Instituto Tecnológico de Tizimín. Yucatán, México.

* Corresponding author: ricardogarciaherrera@hotmail.com

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Abstract The main objective was to develop equations to predict body weight (BW) using heart girth (HG) in Pelibuey ewes. A second objective was to evaluate this model for precision using an independent dataset. For model develop a data set composed by 366, 2-3-yr-old, non-pregnant and non-lactating ewes; with a mean BW of 45.7 ± 9.16 kg and HG of 87.55 ± 7.93 cm was used. A linear equation was fitted: BW= -47.97 (±2.01) + 1.07 (±0.02)×HG (r2 = 0.86, Root mean square error (RMSE)= 3.46, y n= 366). A second data set composed by 67 animals, with similar characteristics (BW of 38.25 ± 8.62 kg and HG of 80.37 ± 7.03 cm) was used to evaluate the developed equations. For the evaluation, the relationship between observed and predicted values of BW by linear regression, the mean squared error of prediction (MSEP) and root MSEP (RMSEP), and concordance correlation coefficient analysis were used. The proposed equation was highly precise (R2 =0.913) and accurate (Cb=0.996) with a reproducibility index of 0.95. The MEF has indicated a higher efficiency of prediction with higher proportion of the total variance of the observed values been explained by the predicted data (0.91). The partition of the MSEP has indicated a very small mean bias (0.082). The systematic bias has shown that only 1.93 % of the error of prediction was associated with the slope and most of the error was explained by the random component indicating small biases with the predictions. The proposed equation accurately and precisely estimated the BW of non-pregnant and non-lactating Pelibuey ewe using HG and therefore is recommended to be used. Key words: Biometric measurements, Pelibuey sheep, Body weight, Prediction. Received: 21/05/2018 Accepted: 03/08/2018

Body weight (BW) is one of the most accurate measurements to determine livestock growth(1). The further understanding of body growth allows for novel diet optimization approaches with consequences on the improvement of prediction of sales prices(2), as well as management strategies that can improve therapeutic treatments for livestock diseases(2,3). Despite of BW being an important economic trait that can assist feeding and management decision supports; small producers can rarely afford expensive scales necessary to perform such measurement(3,4,5). Although, several techniques to measure or estimate the BW of livestock have been reported, the use if weighing scales, are still the most accurate method, but less preferred by small producers because it’s cumbersomeness, time-consuming efforts, associated cost for implementation, and stress to animals(1,2).

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Recently, it has been reported that the sheep lose a significant amount of BW over a short-term delay prior to weighing as a result of handling operations leading to losses around to 1.8 to 2.9 kg or 3.5 to 5.6 % BW(1). Therefore, it is important to develop alternative practical methods that are of low cost and ease allowing smallholders to monitor the body growth of production animals(4,6). Among alternative method, the use of biometric measurements (BM) such as the heart girth (HG), body length (BL) and withers height (WH) are valuable and rather simple tools used for the estimation of the BW of production animals(5). The HG is highly correlated with the BW of small ruminants (sheep and goats) of different breeds and therefore is used more frequently(6-9). In this regard, Yilmaz et al(8) estimated the BW based on HG in Karya sheep and found medium precision (r2 of 0.63). Others(10) also noted that the HG was related (= 0.79) with the BW of Pelibuey ewe lambs at slaughter. Similarly, Bautista-Diaz et al(11) reported that the HG can be used to predict the BW in Pelibuey ewes (r2= 0.72). Despite of agreements on the correlations of both variables, there are no studies under field conditions designed to evaluate the relationship between BW and HG in Pelibuey sheep. Moreover, the predictive equations reported for small ruminants have rarely been evaluated for Pelibuey sheep. The main objective of current study was to develop equations to predict body weight (BW) using heart girth (HG) in non-pregnant and non-lactating Pelibuey ewes. A second objective was to evaluate this model for precision using an independent dataset. All procedures involving animals were conducted within the guidelines of official techniques of animal care and health in México (NOM-051-ZOO-1995). The experiment was carried out in the commercial farm “El Rodeo”, located at 17° 84” N, 92° 81” W; 10 masl and 14 km from the road Villahermosa-Jalapa, Tabasco, Mexico. A data set composed by 366, of 2 to 3-yr-old, non-pregnant and non-lactating, clinically healthy Pelibuey ewes; with a mean BW of 45.7 ± 9.16 kg was used. A second data set composed by 67 animals, with similar characteristics and mean BW of 38.25 ± 8.62 kg was used to evaluate the developed equations. According to the management of the farm, the ewes were grouped in confinement open-sided facilities provided with a roof and concrete floor. The diet consisted of 66 % forage and 34 % concentrate, with an estimated of metabolizable energy of 12 MJ /kg DM and 10% CP(12). The dietary ingredients were composed of cereal grains (corn or sorghum), soybean meal, tropical grasses hay, vitamins, and minerals. Each ewe had the BW (digital scale; Model EQB, Torrey, Mexico) and heart girth (HG), taken. The HG was measured as the smallest circumference just posterior to the anterior legs, in the vertical plane using a flexible tape fiberglass (Truper®, Truper S.A. de C.V., San Lorenzo, Mexico) as described Bautista-Diaz et al(11). Statistical analyses were performed using SAS(13). Descriptive statistics were obtained with PROC MEANS. The PROC REG was used to develop the predictive equation to estimate BW using HG measurements. Outliers were tested by plotting the studentized residual against the statistical 769

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model-predicted values. Data points were removed if the studentized residual was outside the range of −2.5 to 2.5. The goodness-of-fit of the regression was assessed by the root of the mean square error (RMSE) and the r2. As recommended by Tedeschi(14), additional statistics were used to assess the adequacy of the models, including standard deviation (SD), the mean squared error of prediction (MSEP) and root MSEP (RMSEP), to account for the distance between the prediction and its true value. The mean bias (MB), was used as representation of the average inaccuracy of the model(15). The modelling efficiency factor (MEF), which represents the proportion of the variation explained by the line Y = X, was used as an indicator of goodness of fit(16,17). The coefficient of model determination (CD) was used to assess the variance of the predicted data. The bias correction factor (Cb), a component of the concordance correlation coefficient (CCC)(18), was used as an indicator of the deviation from the identity line, whereas CCC as the reproducibility index simultaneously accounting for accuracy and precision. It was assumed high accuracy and precision when coefficients were >0.80 and low accuracy and precision when coefficients were <0.50. Finally, all calculations were obtained using the Model Evaluation System (http://nutritionmodels.tamu.edu/mes.htm; last accessed mayo 17, 2018;(14). The average, maximum, and minimum values for BW and HG are presented in Table 1. It was observed that the BW of ewes, ranged from 75.00 to 25.55 kg whereas HG ranged from 70 to 114 cm. The correlation coefficient (r) between BW and HG was 0.93, and the regression equation fitted was: BW= -47.97 (±2.00*) + 1.07 (±0.022) × HG (r2= 0.86, RMSE= 3.46, y n= 366).

Table 1: Descriptive analyses of the BW (kg) and HG (cm) recorded in non-lactating and nonpregnant Pelibuey ewes Variables Development BW, kg HG, cm Evaluation BW, kg HG, cm

Mean ± SD

Maximum

Minimum

366 366

45.72± 9.16 87.55± 7.93

75.00 114.00

25.55 70.00

67 67

33.14 ± 8.58 80.37± 7.03

62.00 100.00

21.44 65.00

BW= body weight; HG= heart girth; SD= standard deviation.

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The simultaneous test failed to reject the null hypothesis of an intercept equal to zero and slope equal to one (P>0.05). The proposed equation was highly precise (R2= 0.913) and accurate (Cb= 0.996) with a reproducibility index of 0.95 (Table 2). The MEF have indicated a higher efficiency of prediction with higher proportion of the total variance of the observed values been explained by the predicted data (0.91) (Figure 1).

Table 2: Mean and descriptive statistics of the accuracy and precision of the relationship among observed and predicted values for body weight using the heart girth in Pelibuey ewes Variable1 Mean SD Maximum Minimum r2 CCC Cb MEF CD Regression analysis Intercept (β0) Estimate SE P-value (β0 = 0) Slope (β1) Estimate SE P-value (β1 = 1) MSEP source, % MSEP Mean bias Systematic bias Random error Root MSEP Estimate % of the mean

Obs 33.1 8.58 62 21.4 -------

[Eq. 1] 36.6 7.52 59.0 21.6 0.913 0.95 0.996 0.91 1.19

-------

-1.60 1.53 0.30

-------

1.04 0.04 0.263

-------

0.082 1.93 98.0

-----

8.12 7.61

Obs= observed evaluation data set; CCC is the concordance correlation coefficient; Cb is the bias correction factor; MEF is the modeling efficiency; CD is the coefficient of model determination; MSEP is the mean square error of the prediction.

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Figure 1: Relationships between observed and predicted BW using the HG in non-pregnant and non-lactating Pelibuey ewes

The CD indicated medium to low variability in the predicted data (1.19) whereas the partition of the MSEP have indicated a very small mean bias (0.082) showing a very small inaccuracy with most of the errors concentrated away from the mean. The systematic bias has shown that only 1.93 % of the error of prediction was associated with the slope and most of the error was explained by the random component indicating small biases with the predictions. The current study presents an evaluation of the practicality of use the HG to predict BW of Pelibuey ewes. Although, some articles have been published on this topic in other species and in other sheep breeds; in Pelibuey ewes, there are no reports that evaluate this relationship under field conditions. Moreover, it has been reported that this type of models should be developed for each breed under the conditions of handling and system of production(19). On the other hand, the use of empirical equations developed for the prediction of BW and productive performance, among others, is limited by the lack of evaluation of its predictive capacity with independent data that were used for its development, which makes it difficult to ascertain its accuracy and precision(14).

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Among the more common BM used to predict the BW; the HG it has been used in heifers(6), goats and sheep(9,20). It is in agreement with other authors, indicating that this BM is highly correlated with the BW for different animal species(8). In the same way, Yilmaz et al(8) estimated the BW using the HG in of Karya sheep breed and noticed that the r2 was 0.63. In addition, it was also reported(10) that the HG was related (r= 0.79) with BW of Pelibuey lambs. Under experimental conditions, Bautista-Diaz et al(11) found that the HG was the best predictor of BW in Pelibuey, compared to other BM (r2 = 0.72). This coincides with the results obtained in other study using sheep(21). In other work(21) they found that the HG was highly correlated with BW (r= 0.99 and 0.98, for males and females respectively) in Sudan Nilotic sheep; nonetheless this authors, obtained a non-linear regression (y= axb), the equations had an r2 values of 0.98 and 0.96 for male and female lambs, respectively. The small variance surrounding the slope of the proposed equation indicates that BW can be explained by the variance of HG. The HG itself contributes to the body area where most of organs are housed. It seems that the thoracic circumference of Pelibuey ewe play a bigger role on the determination of BW than body length. The practical implications are that the volume and weight of organs housed in the abdominal cavity may represent better determinants on body mass which determines the bulk of nutrient requirements of maintenance(22). In addition, Kunene et al(3) reported that the relationship between HG and BW had significant rvalues that ranged from 0.33 to 0.86 in of Zulu (Nguni) sheep in different age groups. Also, informed that the HG was a more accurate predictor of BW in young Zulu sheep (<15 mo), therefore concluded that BW in Zulu sheep can be reasonably estimated using the HG. Furthermore, Souza et al(23) concluded that the equations generated using the heart girth and body length can be used to estimate the body weight of male and female meat-type sheep of different breeds and ages. This it agrees with several authors who have reported the importance of HG in BW estimation in sheep of different breeds(7,9,20). On the other hand, in the literature, exist different approaches and techniques to evaluate models, according to Mayer and Butler(17), the main evaluation techniques are based on subjective assessment, comparative graphs, measures of deviation (based on the differences between observed and predicted values) and statistical tests. In the present study, were used the tests and analysis of Model Evaluation Systems described by Tedeschi(14). The parameters for precision and accuracy showed that the equation proposal presented an average accuracy (R2= 0.913 %) and high accuracy (Cb =0.996) and reproducibility (CCC= 0.95) to predict the BW in non-pregnant and non-lactating Pelibuey ewes. The result of the model efficiency (MEF= 0.91) indicates a relatively high value of concordance between the observed values with their predicted that, in a perfect fit, it might be one. The MEF has been reported as the best measure of concordance between the observed and predicted values; however, with respect to CD, on a 773

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perfect fit would be worth one, if its value close to one indicates an improvement in the predictions of the model (CD>1 is an indicator of sub prediction and CD<1 Of on prediction). The CD found in the present study was 1.19, which indicates an underestimation of the BW with a variation of about 2 %(14). The RCCMEP accounted for 9.27 % of the BW observed. Based on the results of the statistical evaluations, the proposed model predicts the in non-pregnant and non-lactating Pelibuey ewes with good precision and accuracy. For Tedeschi(14), the assessment of the adequacy of a model is only possible through a combination of statistical analysis according to the purpose for which the model was conceptualized and developed. It further concluded that the identification and acceptance of inaccuracies of a model is the first step in the evolution model to a more accurate and more confidence. The results of present study may contribute to estimation the BW of Pelibuey ewes and this information may to contribute to updating with the data for BW estimation of some parameters required by nutritional models in order to predict the performance of hair sheep breeds(24,25).

Conflicts of interest The authors state that there are no conflicts of interest.

Acknowledgments The authors are very grateful to Dr. Jose Manuel Piña Gutiérrez who allowed us to use the facilities at Rancho El Rodeo. Financial support: Programa de Fomento a la Investigación, through the project “Eficiencia energética madre/cría en ovinos de pelo” (PFI: UJAT-DACA-2015-IA-02).

Literature cited 1. Wishart H, Morgan-Davies C, Stott A, Wilson R, Waterhouse T. Liveweight loss associated with handling and weighing of grazing sheep. Small Ruminant Res 2017;153:163-170.

2. Wangchuk K, Wangdi J, Mindu M. Comparison and reliability of techniques to estimate live cattle body weight. J Appl Anim Res 2017;46(1):34-352.

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3. Kunene NW, Nesamvuni AE, Nsahlai IV. Determination of prediction equations for estimating body weight of Zulu (Nguni) sheep. Small Ruminant Res 2009;84:41–46.

4. Sebolai B, Nsoso SJ, Podisi B, Mokhutshwane BS. The estimation of live weight based on linear traits in indigenous Tswana goats at various ages in Botswana. Trop Anim Health Prod 2012;44:899-904.

5.Tebug SF, Missohou A, Sabi SS, Juga J, Poole EJ, Tapio M, Marshall K. Using body measurements to estimate live weight of dairy cattle in low-input systems in Senegal. J App Anim Res 2016;46(1):87-93.

6. Oliveira AS, Abreu DC, Fonseca MA, Antoniassi PMB. Short communication: Development and evaluation of predictive models of body weight for crossbred Holstein-Zebu dairy heifers. J Dairy Sci 2013;96:6697–6702.

7. Sowande OS, Sobola OS. Body measurements of west African dwarf sheep as parameters for estimation of live weight. Trop Anim Health Prod 2008;40:433–439.

8. Yilmaz O, Cemal I, Karaca O. Estimation of mature live weight using some body measurements in Karya sheep. Trop Anim Health Prod 2013;45:397-403.

9. Boujenane I, Halhaly S. Estimation of body weight from heart girth in Sardi and Timahdite sheep using different models. Iran J Appl Anim Sci 2015;5(3):639-646 .

10. Hernández-Espinoza DF, Oliva-Hernández J, Pascual-Córdova A, Hinojosa-Cuéllar JA. Descripción de medidas corporales y composición de la canal en corderas Pelibuey: Estudio preliminar. Rev Cien 2012;22:24-31.

11. Bautista-Díaz E, Salazar-Cuytun ER, Chay-Canul AJ, García-Herrera RA, Piñeiro-Vázquez AT, Magaña-Monforte JG, et al. Determination of carcass traits in Pelibuey ewes using biometric measurements. Small Ruminant Res 2017;147:115–119.

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12. AFRC. Energy and protein requirements of ruminants. Agricultural and Food Research Council. CAB International, Wallingford, UK, 1993.

13. SAS 9.3 Software. Institute Inc., Cary, North Carolina, USA. 2010.

14. Tedeschi LO. Assessment of the adequacy of mathematical models. Agric Sys 2006;89:225247.

15. Cochran WG, Cox GM. Experimental Design. John Wiley & Sons, New York, NY. 1957.

16. Loague K, Green RE. Statistical and graphical methods for evaluating solute transport models: Overview and application. J Contam Hydrol 1991;7:51-73.

17. Mayer DG, Butler DG. Statistical validation. Ecol Modell 1993;68:21â&#x20AC;&#x201C;32.

18. Lin LIK. A concordance correlation coefficient to evaluate reproducibility. Biometrics 1989;45:255-268.

19. Fonseca MA, Tedeschi LO, Valadares-Filho SC, De Paula NF, Silva LD, Sathler DFT. Evaluation of equations to estimate body composition in beef cattle using live, linear and standing rib cut measurements. Anim Prod Sci 2017;57:378-390 .

20. Mavule BS, Muchenje V, Bezuidenhout CC, Kunene NW. Morphological structure of Zulu sheep based on principal component analysis of body measurements. Small Ruminant Res 2013;111:23-30.

21. Atta M, El khidir OA. Use of heart girth, wither height and scapuloischial length for prediction of liveweight of Nilotic sheep. Small Ruminant Res 2004;55:233-237.

22. Ferrell CL, Koong JL, Nienaber JA. Effect of previous nutrition on body composition and maintenance energy costs of growing lambs. Brit J Nutr 1986;56:595-605.

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23. Souza S, Leal A, Barioni C, Matos A, Morais J, Araújo M, Neto O, Santos A, Costa, R. Utilização de medidas biométricas para estimar peso vivo em ovino. Arch Latinoam Prod Anim 2009;17(3,4):61-66.

24. Chay-Canul AJ, Espinoza-Hernández JC, Ayala-Burgos AJ, Magaña-Monforte JG, AguilarPérez CF, Chizzotti ML, et al. Relationship of empty body weight with shrunken body weight and carcass weights in adult Pelibuey ewes at different physiological states. Small Ruminant Res 2014;117:10-14.

25. Chay-Canul AJ, Magaña-Monforte JG, Chizzotti ML, Piñeiro-Vázquez AT, Canul-Solís JR, Ayala-Burgos AJ, et al. Energy requirements of hair sheep in the tropical regions of Latin America. Review. Rev Mex Cienc Pecu 2016;7(1):105-125.

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https://doi.org/10.22319/rmcp.v10i3.4810 Technical note

Effectiveness of the smoke of fruits of Guazuma ulmifolia (Sterculiaceae) and vapors of Thymol for control of Varroa destructor infesting Africanized bees William de Jesús May-Itzá a Luis Abdelmir Medina Medina a*

Universidad Autónoma de Yucatán, Facultad de Medicina Veterinaria y Zootecnia,

Departamento de Apicultura, Yucatán, México.

* Corresponding author: mmedina@correo.uady.mx

Abstract: The mite Varroa destructor is a scourge in honey bee colonies worldwide. Conventional chemical-based control treatments can contaminate colony products and cause resistance in the parasite. Plant-source compounds are promising alternatives. The effectiveness of smoke from dried Guazuma ulmifolia fruit and vapors from thymol crystals was evaluated in control of V. destructor in colonies of Africanized bees (Apis mellifera) in Yucatan, Mexico. Three treatments were used during a three-week experimental period. In Group 1, colonies were administered five to eight puffs of smoke from dried G. ulmifolia fruits twice a week. In Group 2, they were administered 4-8 g of thymol crystals once a week. Group 3 was a control and received no treatment. Collections of 200 to 300 adult bees from each colony were done prior to treatment (day 0) and after treatment at 7, 14 and 21 d. These were processed to quantify colony infestation levels and treatment efficacy. Overall V. destructor infestation levels in adult bees decreased in all three groups after 21 d, with differences between treatments. Levels were lowest in Group 2, followed by Group 1 and the control. Efficacy at the end of the treatments was 41 % in Group 1 and 69% in Group 2. Compared to the control, application of thymol crystals provided the most effective alternative control method against 778

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V. destructor. However, regular application of G. ulmifolia fruit smoke also reduced mite infestation levels, and this resource has the advantage of being locally available. Key words: Guazuma ulmifolia, Thymol, Alternative control, Varroa destructor, Africanized bees, Apis mellifera, Yucatan. Received: 14/03/2018 Accepted: 08/08/2018

The Varroa destructor mite remains one of the principal health problems in beekeeping worldwide(1). A serious threat, it negatively impacts the development, survival and productivity of Apis mellifera colonies intended for honey production(2,3) and crop pollination(4). An ectoparasite affecting bee pupae and adults, V. destructor causes a reduction in the body weight of workers at emergence and shortens their lifespan(5). Bee colonies with intense V. destructor infestations can also suffer from increases in viral diseases, mainly deformed wing virus. Transmitted by female V. destructor while feeding on bee pupae and adults, it causes declines in population and honey production in infested colonies; when the mite population grows exponentially the bee colony dies(2,6). In Europe and the United States of America, V. destructor continues to destroy managed colonies and is considered to be one of the factors associated with bee colony collapse and mass mortality, a phenomenon known as colony collapse disorder(2,7). Widespread loss of colonies is negatively impacting honey bee pollination services in various agricultural crops(8). This same phenomenon occurs in Mexico. When V. destructor-infested colonies are not treated, infestation levels quickly increase, reducing honey production(9), and, in conjunction with other diseases, can cause colony collapse and mortality(10). In an effort to control or eliminate V. destructor from honey bee colonies, beekeepers resort to different control methods, including application of approved and prepared pyrethroidbased chemicals(3). However, some also use products such as homemade powders, ointments and wooden strips, which are unauthorized for use in bees and often include acaricides such as amitraz, bromopropylate and coumaphos. These can contaminate honey and other products from bee colonies(11), thus risking their rejection on the international market. In response to this challenge, natural mite control alternatives are being developed. To date various products of plant origin have been tested, such as thymol obtained from Thymus vulgaris (Lamiaceae)(12,13), menthol from Mentha arvensis and Mentha piperita (Lamiaceae)(14,15), as well as formic acid and oxalic acid(12,16). These have the advantages of acceptable efficacy in the presence of larva and pupae, easy application, lower risk of 779

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contaminating honey, wax, pollen and other bee colony products, as well as a reduced likelihood of mites developing resistance to them(3), as occurs with commercial acaricides containing mainly pyrethroids(17). Beekeepers in rural communities of the state of Yucatan, Mexico, have reported the use of various plant-based products to control V. destructor infestations with acceptable results in some regions of the state. Recent data provided by rural beekeepers to the Yucatan State Ministry of Rural Development (Secretaria de Desarrollo Rural del Gobierno del Estado de Yucatán; SEDER-Yucatan) indicate that they have been controlling parasitosis in bee colonies by using the dried fruit of the West Indian elm tree Guazuma ulmifolia (pixoy in Mayan language; Sterculiaceae) as fuel in the bee smoker. Application of the smoke of this fruit has been reported to be sufficient to control V. destructor infestations in bee colonies without the use of other commercial products or control methods. They report that the dry fruit must be collected directly from the tree, that the smoke does not irritate the bees or beekeeper, leaves no scent in honeycombs and does not affect queen bee egg production. However, this information has not been verified under controlled conditions with experimental colonies following research protocols. This is needed to confirm the reported results and, if effective, develop application methods that would allow its use as an alternative mite control technique. The present study objective was to assess the efficacy of dried G. ulmifolia fruit when used as fuel in bee smokers as an alternative for controlling the mite V. destructor in colonies of Africanized bees (Apis mellifera) under conditions simulating those prevalent in rural apiaries in Yucatan, and compare its performance to that of thymol crystals from the T. vulgaris plant, also widely used as an alternative mite control measure. The study was done in the experimental apiary of the Faculty of Veterinary Medicine and Zootechny of the Autonomous University of Yucatan (FMVZ- UADY), where bee colonies are managed following practices similar to those in the state’s honey producing regions. The installations are located in Xmatkuil, Yucatan, 15.5 km south of the city of Merida, Yucatan (20°52’ N; 89°36’ W). Climate in the area is warm sub-humid with summer rains (Aw0). Average annual rainfall is 985 mm, average annual temperature is 26.8°C and average annual relative humidity is 78 %(18). The most important floral resources in this region in terms of nectar and pollen for bee colonies are goldeneye or tajonal (Viguiera dentata), which blooms from January to February, and bastard logwood or ts’iits’ilche’ (Gymnopodium floribundum), which blooms from February to May(19). Under these conditions, bee colonies normally have brood throughout the year, with peaks between February and May(20). The colonies in the experimental apiary are kept in Langstroth-type hives. For the present study the colonies were housed in a single box (brood chamber only) or two boxes (brood chamber and one honey super), distributed similarly among treatments. All the colonies had naturally mated Africanized queens, were heavily populated with adult bees occupying at least eight of the ten honeycombs present in the brood chamber, and contained a similar 780

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number of honeycombs containing brood in different developmental stages (eggs, larvae and pupae), honey and pollen. They were also naturally infested with the V. destructor mite, with no treatment or control methods applied for at least six years prior to data collection. Before implementing the treatments, a preliminary diagnosis was made of each hive to measure V. destructor infestation levels in adult bees. This was done to ensure that the experimental groups had similar infestation levels at the beginning of the evaluations. Evaluation of smoke from G. ulmifolia fruit and thymol crystals as natural alternative products for control of V. destructor was done over a three-week period. The hives were divided into three experimental groups. Group 1 (G1): This group consisted of twelve colonies (ten colonies with a brood chamber and one honey super, and two with only a brood chamber). These colonies were administered smoke from the burning of dried G. ulmifolia fruit. Approximately 220 g of dried G. ulmifolia fruit were placed in a bee smoker, and the smoke applied at the colony entrance and the hives opened to apply smoke between the combs of the brood chamber and honey super (in the case of double colonies). Five to eight puffs were applied to each colony twice a week over the three-week experimental period, the number of puffs varying depending on bee defensive response and hive size. The hives were then closed. This application procedure is similar to that used in routine examinations of colonies. Group 2 (G2): This group consisted of ten colonies (nine colonies with a brood chamber and honey super, and one colony with only a brood chamber). Thymol crystals (96.8% purity) were placed in each hive at seven-day intervals(16). In the brood chamber hives only 4 g crystal were used, while in the double hives 8 g were used. For application, the crystals were placed in disposable plastic lids (250 ml) covered with a wire mesh to prevent the bees from removing the crystals from the colony, which would reduce effectiveness. The lids with the crystals were inserted into the hive entrance using a piece of wire, which allowed for easy insertion and removal. Group 3 (G3): Containing twelve colonies (ten double hives and two with just a brood chamber), this group was a control, receiving no anti-mite treatment during the experimental period. Collections of adult bees (200-300 bees per collection) were taken from each colony to quantify the effectiveness of the G. ulmifolia fruit smoke (G1) and thymol crystals (G2) treatments. Collection was done prior to treatment (day 0) and after application of each treatment at 7, 14 and 21 d. Samples of the adult bees and the mites infesting them were stored in vials containing 80% alcohol, and marked with the collection date, colony number and treatment group. 781

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In the laboratory, the bee samples were placed in plastic containers and 250 ml 80% ethyl alcohol added until the bees were completely covered. These were then mechanically agitated at 180 rpm for 30 min and the alcohol filtered through white gauze to collect any mites. All the mites collected from each adult bee samplewere counted. This methodology successfully removes all mites from the bee body, allowing quantification of infestation level (%) and that for all adult bees (% IAB) in each group, using the formula(21): % IAB (No. mites / No. bees) x 100 At the end of the experimental period (21 d), efficacy of the G. ulmifolia fruit smoke (G1) and thymol crystals (G2) treatments was calculated based on mite infestation levels in adult bees using the formula(22): E = 1- (A x D / B x C) x 100 Where: E= treatment efficacy; A= mite infestation level in control group (G3) before treatment application (d 0); B= mite infestation level in control group (G3) after treatment completion (d 21); C= mite infestation level in treatment group (G1 or G2) before application (d 0); D= mite infestation level in treatment group (G1 or G2) after each treatment (d 7, 14 and 21). Post-treatment V. destructor infestation levels in all three groups were compared with a oneway ANOVA and Tukey multiple comparison test (95% confidence level). Analyses were done with the Statgraphics Centurion ver. XV program(23), and those results expressed as percentages (% infestation) were arcsine transformed (angular transformation)(24). Before the treatments were begun (d 0), V. destructor infestation in adult bees did not differ between the groups (F= 0.00; g.l. 2,31; P=0.99): 13.5 ±5.8 % for G1, 13.3 ±3.2 % for G2 and 13.4 ±3.9 % for G3 (Table 1). This indicates that infestation level distribution was similar in the experimental colonies of the three groups.

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Table 1: Varroa destructor infestation levels in adult bees (%) in the three experimental groups at d 0, and after treatment application on d 7, 14 and 21 Varroa destructor infestation levels in adult bees (%) Group 1 (G. ulmifolia)

Group 2 (T. vulgaris)

Group 3 (Control)

Day 0

13.5 ± 5.8 a

13.3 ± 3.2 a

13.4 ± 3.9 a

Day 7

8.8 ± 2.8 a

10.9 ± 3.8 a

12.7 ± 5.5 a

Day 14

7.7 ± 2.9 a, b

6.4 ± 3.3 a

12.0 ± 6.3 b

Day 21

5.2 ± 2.4 a

2.8 ± 1.4 b

8.8 ± 3.8 c

a,b

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

Seven days after the first application of G. ulmifolia fruit smoke (G1) infestation levels had dropped to 8.8 %, while in the thymol crystals treatment (G2) they had dropped to 10.9 %, and in the control (G3) to 12.7%. No significant differences occurred between the three treatments at this time (F= 2.57; g.l. 2,31; P=0.09). However, after the final application at 21 d infestation levels in G1 had decreased to 5.2 %, those in G2 to 2.8 % and in G3 to 8.8 % (Table 1). The three groups differed from each other (F = 13.73; g.l. 2,31; P=0.0001), with G2 exhibiting the greatest reduction, followed by G1 and G3. Efficacy during the first week was 32 % in G1 and 14 % in G2, but in the second week had increased to 36 % in G1 and 47 % in G2 (Table 2). Total efficacy after the three applications (21 d) of each treatment was 41 % in G1 and 69 % in G2. Compared to Group 3 (Control), Group 1, treated with G. ulmifolia fruit smoke, exhibited a significant reduction in infestation levels at the end of the 21-d experimental period, with 41 % efficacy. However, Group 2, treated with thymol crystals, experienced an even greater reduction in infestation (lower than in Groups 1 and 3), with an overall efficacy of 69 %.

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Table 2: Estimated efficacy (%) of application of G. ulmifolia fruit smoke and thymol (T. vulgaris) crystals in control of V. destructor infestation after application of each treatment Treatment efficacy (%) Treatment

Day 7

Day 14

Day 21 (final)

G. ulmifolia (G1)

T. vulgaris (G2)

Results for Group 1 suggest that frequent application of G. ulmifolia fruit smoke may contribute to lowering V. destructor infestation levels in honey bee colonies when routinely used as fuel to generate smoke during colony management. This treatment’s average efficacy (41 %) exceeded that of other commercial organic products such as Hive-Clean® (made with organic acids (formic, citrus and oxalic), propolis extract, essential oils and sugar syrup). When tested under tropical conditions and with Africanized bees(25), Hive-Clean® has exhibited relatively low efficacy (16.7 %) compared to its rather high efficacy (91.6 %) in a temperate climate (Poland) and with European bees(26). In addition, application of G. ulmifolia fruit smoke had no apparent negative effect on mortality in adult bees or offspring, nor did it repel adult bees. Use of thymol crystals in Group 2 resulted in 69 % efficacy after 21 d, the most effective among the three test groups. This is similar to a previous study of the efficacy of thymol, thymol in gel(13), and formic acid crystals(16), in which the essential oil was found to be an effective alternative method for control of V. destructor in Africanized bee (Apis mellifera) colonies in the environmental conditions of Yucatan. Group 3, the control, also exhibited a reduction in V. destructor infestation levels (13.4 to 8.8 %) over the experimental period, although much less than those in the G1 and G2 treatments. Natural decreases in V. destructor infestation levels in adult bees in the absence of control measures may result from a population dynamic of mites in bee colonies known as the parasite “dilution effect”. In this phenomenon bee population size increases when the availability of food is grater during flowering seasons, thus increasing the number of bees in the colony, diluting the mite population amid a greater number of individuals in the colony and lowering the infestation level in adult bees(27).

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Controlling V. destructor mites in honey bee colonies using plant-origin compounds is preferable to application of conventional pyrethroid-based acaricides or the use of homemade wooden strips and ointments incorporating chemicals such as coumaphos since the latter can leave residues in the honey(28) and may generate resistance in mites(29). Essential oils extracted from different plants have been evaluated as potential insecticides for control of certain parasites(30). The present study is the first report of use of G. ulmifolia fruit smoke as a mite control method in honey bee colonies, although alcohol extracts of G. ulmifolia leaves are known to be toxic to Aedes aegypti mosquito larvae, causing 35% mortality(31). Phytochemical compounds in G. ulmifolia have also been reported to have potential activity in the control of various insects and mites affecting domestic turkeys (Meleagris gallopavo)(32). The present results apparently support first-hand accounts from beekeepers in Yucatan that continual use of dried G. ulmifolia fruit as fuel in bee smokers provides sufficient control of V. destructor in colonies of Africanized bees. Indeed, they claim they use no other method to control this parasite.

When used during routine management of Africanized honey bee colonies in the state of Yucatan, Mexico, the smoke of dried G. ulmifolia fruit proved an effective alternative method for control of the mite V. destructor. Thymol crystals were even more effective at controlling this parasite. However, G. ulmifolia fruits have the advantages of being readily available in the study region, and the smoke from them does not irritate bees or beekeepers, leaves no scent in honeycombs and has no effect on queen bee egg production.

Acknowledgements

The research reported here was supported by a project financed by the Secretaria de Desarrollo Rural (SEDER) del Estado de Yucatán. The authors wish to thank Máximo Francisco Paredes Rodríguez (SEDER) for logistical support and Ligia B. Martín Sosa for field and laboratory technical assistance.

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16. Vicario E, Medina LM. Uso del ácido fórmico y timol en el control del ácaro Varroa jacobsoni en Yucatán, México. Resultados preliminares [resumen]. XIII Seminario Americano de Apicultura. Morelia, Mich. 1999:35-38. 17. Milani, N. Management of the resistance of Varroa mites to acaricides. En: Delaplane KS, Webster T. editores. Mites of the honey bee. Hamilton, USA. Dadant and Sons; 2001:241–250. 18. Orellana LR, Espadas MC, Nava MF. Climas. En: Durán R, Méndez M. editores. Biodiversidad y desarrollo humano en Yucatán. Yucatán, México: CONABIOSEDUMA; 2010:10-11. 19. Alfaro BRG, Ortiz DJJ, Gonzalez AJA. Plantas melíferas: melisopalinología. En: Durán R, Méndez M. editores. Biodiversidad y desarrollo humano en Yucatán. Yucatán, México: CONABIO-SEDUMA; 2010:346-348. 20. Echazarreta CM, Paxton R. Comparative colony development of Africanized and European honey bees (Apis mellifera) in lowland neotropical Yucatan. Mexico. J Apic Res 1997;36:89-103. 21. De Jong D, De Roma A, Goncalves L. Comparative analysis of shaking solutions for the detection of Varroa jacobsoni on adult honey bees. Apidologie 1982;13(3):297-306. 22. Floris I, Cabras A, Garau VL, Minelli EV, Satta A, Troullier J. Persistence and effectiveness of pyrethroids in plastic strips against Varroa jacobsoni (Acari: Varroidae) and mite resistance in a Mediterranean Area. J Econ Entomol 2001;94:806-810. 23. Statgraphics® Centurion XV User Manual. Warrenton Vir, USA: Statpoint Tech. Inc. 2006. 24. Zar JH. Biostatistical analysis. 3rd ed. New Jersey, USA: Prentice-Hall Int.; 1996. 25. Rodríguez-Dehaibes SR, Pardio SV, Luna-Olivares G, Villanueva-Jimenez JA. Two commercial formulations of natural compounds for Varroa destructor (Acari: Varroidae) control on Africanized bees under tropical climatic conditions. J Apic Res 2017;56(1):58-62. 26. Howis M, Nowakowski P. Varroa destructor removal efficiency using BeeVital Hive Clean preparation. J Apic Sci 2009; 53: 15–20. 27. Martin, S. Varroa jacobsoni: monitoring and forecasting mite populations within honey bee colonies in Britain. London, UK: Ministry of Agriculture, Fisheries and Food. 1998. 28. Wallner K, Fries I. Control of the mite Varroa destructor in honey bee colonies. Pestic Outlook 2003;14(2):80-84. 29. Kanga LHB, Adamczyk J, Marshall K, Cox R. Monitoring for resistance to organophosphorus and pyrethroid insecticides in varroa mite populations. J Econ Entomol 2010;103:1797-1802. 787

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30. Zoubiri S, Baaliouamer A. Potentiality of plants as source of insecticide principles. J Saudi Chem Soc 2014;18:925-938. 31. de Mendonça FAC, da Silva KFS, dos Santos KK, Junior KALR, Sant’Ana AEG. Activities of some Brazilian plants against larvae of the mosquito Aedes aegypti. Fitoterapia 2005;76:629-636. 32. López-Garrido SJ, Jerez-Salas MP, García-López JC, Jiménez-Galicia MM, ÁvilaSerrano NY, Sánchez-Bernal EI, Arroyo-Ledezma J, Camacho-Escobar MA. Uso de extractos de árboles para controlar exoparásitos de guajolotes (Meleagris gallopavo) Acta Universitaria 2016;26(6):15-23.

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https://doi.org/10.22319/rmcp.v10i3.4667 Technical note

Yield, agronomic parameters and nutritional quality of Tithonia diversifolia in response to different fertilization levels Julián Mauricio Botero Londoño a Arnulfo Gómez Carabalí b Mónica Andrea Botero Londoño c*

Universidad Industrial de Santander, Programa de Zootecnia. Málaga, Santander, Colombia.

Universidad Nacional de Colombia. Facultad de Ciencias Agropecuarias. Departamento de

Ciencia Animal, Palmira, Valle, Colombia. c

Universidad Industrial de Santander. Facultad de Ingenierías Fisicomecánicas. Bucaramanga,

Santander, Colombia.

*Corresponding author: mabotero@saber.uis.edu.co

Abstract: Tithonia diversifolia is a bushy forage plant with high biomass production, rapid post-harvest recovery, and good feed composition values (especially protein levels). It is a promising alternative raw material for incrementing protein content in livestock feeds, but its response to different fertilization regimes has received limited attention. An evaluation was done of this species’ yield, composition and nutritional quality values at six different fertilization levels. Experimental design was a completely random block design with six treatments to measure nutrient extraction and its relationship to plant agronomic parameters (biomass production, leaf:stem ratio, plant height at cut, stem count per plant), nutritional value and in vitro digestibility. Fertilization produced an overall improvement in T. diversifolia agronomic and composition values: compared to the control, biomass increased five-fold in response to fertilization, protein production four-fold and energy production was significantly higher. The best overall response was observed with fertilization using 28.1 g urea, 15.8 g DAP and 10.1 g KCl after each harvest cut.

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Key words: Feed analysis, Forages, Ruminant nutrition. Received: 21/10/2017 Accepted: 16/08/2018

Cattle ranching requires huge areas with consequently profound environmental impacts such as deforestation, soil degradation from over-grazing and subsequent erosion and nutrient loss(1). Current solutions focus on using closed systems based on production of high biomass forage with nutritional content. These can prove to be economically, ecologically and socially sustainable, but require extensive study of bush forage species(2). Feed costs represent the largest proportion of expenses in livestock production, the principal contributing factor being the high cost of protein-containing raw materials. High protein content forages such as the tree marigold Tithonia diversifolia hold promise as alternatives to costly feed concentrates, and could reduce dependency on them. Inclusion of T. diversifolia in ruminant diets can also contribute to reducing methane production during ruminal fermentation, helping to mitigating the impact of this greenhouse gas(3).

A robust bushy plant belonging to the Compositae (Asteraceae) family, T. diversifolia is widely distributed throughout the tropics from sea level to 2500 m(4,5). It is also highly adaptable to tropical conditions, particularly the coffee-growing regions of Columbia. It is very resistant to permanent harvest conditions, improves nutrient recycling and prevents erosion. It is therefore a promising resource even in hillside conditions. Areas of T. diversifolia reduce the effects of animal trampling on soils, it has high biomass production and is reported to be an ideal forage in cutting and hauling systems on dairy farms(6). Extracts from this species have insecticidal properties, which means it protects plants in proximity, including food and timber crops(7); indeed, it has been used in silvopastoral systems in Antioquia, Colombia(8). Many recent studies of T. diversifolia have focused on its medicinal properties(9-12).

Data on the nutrient requirements of forages is fundamental to developing crops that produce high levels of biomass and are sustainable over time(13). The present study objective was to determine the nutrient extraction potential of Tithonia diversifolia receiving different levels of fertilization, and the effects of fertilization levels on agronomic parameters (biomass production, leaf:stem ratio, plant height at harvest, and number of stems per plant), nutritional value and in vitro digestibility.

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The project was carried out in Andisol soil in the Colombian Coffee Axis, on La Esmeralda farm in the municipality of Circasia, Quindío Department, Columbia (04°38’24” N; 75°38’26” W). Study area elevation is 1,680 m asl, with annual rainfall ranging from 2,000 to 3,000 mm and an average annual temperature of 19 ºC (Barbas Bremen Meteorological Station).

Before formulating the treatments, which were based on fertilization levels, a sample of twelve T. diversifolia plants was collected from a previously established crop of 100 plants which had received no fertilization. This sample was analyzed to quantify biomass production (dry basis) and leaf nutrient content. Soils were also analyzed. Based on these analyses six treatments were formulated: initial nutrient extraction (INE); INE + 25%; INE + 50%; INE + 75%; INE + 100%; and INE + 200%. These were formulated using urea, diammonium phosphate (DAP) and potassium chloride (KCl), and applied after each harvest cut in the following proportions:

T1) no fertilization; T2) 28.3 g fertilizer per plant / cut (16.0 g urea, 8.4 g DAP, 3.9 g KCl) T3) 36.7 g fertilizer per plant / cut (20.0 g urea, 10.8 g DAP, 5.9 g KCl) T4) 45.4 g fertilizer per plant / cut (24.1 g urea, 13.3 g DAP, 8.0 g KCl) T5) 54.0 g fertilizer per plant / cut (28.1 g urea, 15.8 g DAP, 10.1 g KCl) T6) 88.5 g fertilizer per plant / cut (44.3 g urea, 25.7 g DAP, 18.5 g KCl).

At the beginning of the experiment, 80 cm-long cuttings were planted at 20 cm depth, in an area of 1.0 m x 1.0 m blocks with a total of 50 plants per block. The first uniform harvest cut was made at 140 days. After this first cut, fertilizer was applied according to the respective treatments. Four consecutive cuts were made every 50 d at 30 cm above soil surface.

Experimental design was a completely random block design using 50 m² experimental units with 50 plants per unit, and four blocks with six treatments each for a total of 24 experimental units. Thirteen variables were analyzed: nutrient extraction; biomass production; plant height at harvest cut; leaf:stem ratio; stem count per plant; dry matter; crude protein; crude energy; neutral detergent fiber (NDF); acid detergent fiber (ADF); lignin; ash; and in vitro dry matter digestibility (IVDMD). An analysis of variance (ANOVA) was applied, and any differences (P<0.05) analyzed with a Duncan multiple range test to compare means. All statistical analyses were run with the SAS package(14).

Yield and agronomic parameters were analyzed by randomly collecting twelve plants per experimental unit. Each plant was individually measured and weighed, and biomass production measured initially on a fresh basis and then on a dry basis. Measurements were made of the leaf:stem ratio, plant height at cut and stem count per plant.

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Feed composition analysis was done using twelve plants randomly collected from each experimental unit per cut; a total of approximately 2 kg was collected per sample. Samples were weighed and left to dry in the sun. Feed analyses were done following the Weende and Van Soest methodology, and IVDMD quantified with the prececal method and hydrolysis kinetics via enzymatic simulation(15).

Biomass production increased (P<0.05) as fertilization levels increased; for example, at 50 days average biomass production was 79.9 g dry matter (DM) in treatment T1 (no fertilizer) but 304.5 g in T6 (highest fertilization level). Estimated production per hectare/year was 31,075 kg fresh matter (FM) in T1 but 147,408 kg in T6 (Table 1), clearly demonstrating the importance of fertilization in management of T. diversifolia. Correlation values indicated that biomass production (fresh base) was related to levels of N (90.3 %), P2O5 (90.5 %) and K2O (90.9 %).

Table 1: Biomass production per plant at 50 d and estimated annual biomass yield by fertilization treatments (g plant/cut) (kg ha-1 yr) Treatment Biomass Biomass Biomass Biomass DM FM DM FM T1 425.7 f 79.9 f 31,075 f 5,829 f T2 774.5 e 135.7 e 56,538 e 9,908 e d d d T3 1004.5 169.1 73,326 12,347 d T4 1307.1 c 205.9 c 95,416 c 15,027 c T5 1732.6 b 268.6 b 126,481 b 19,609 b T6 2019.3 a 304.5 a 147,408 a 22,229 a CV 6.4 7.2 6.4 7.2 abcdef

FM= fresh matter; DM= dry matter; CV= coefficient of variation. Different letter superscripts in the same column indicate significant difference (P<0.05).

Plant height clearly increased (P<0.05) with greater fertilization, ranging from 90.65 cm in T1 to 154.88 cm in T6, with differences (P<0.05) between all treatments except T5 and T6 (Table 2). Plant height at cut was highly correlated to biomass production (94.6 %).

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Table 2: Agronomic characteristics of T. diversifolia by fertilization treatment Stem Plant Leaf Leaf Leaf width count Leaf:stem Treatment height at length weight (cm) per ratio cut (cm) (cm) (g) plant T1 90.65 e 20.88 e 15.05 f 14.03 f 1.46 a 1.59 e T2 111.88 d 23.38 d 16.84 e 17.08 e 1.10 b 1.99 d T3 129.63 c 25.83 c 18.33 d 19.55 d 0.97 bc 2.37 c T4 141.40 b 28.98 b 20.17 c 23.55 c 0.93 bc 2.97 b T5 151.93 a 37.19 a 22.12 b 27.73 b 0.82 cd 4.12 a T6 154.88 a 37.19 a 23.01 a 29.18 a 0.79 d 4.32 a CV 2.8 2.7 2.9 3.0 10.8 6.6 abcdef

CV= coefficient of variation. Different letter superscripts in the same column indicate significant difference (P<0.05).

Stem count per plant increased (P<0.05) with fertilization from 14.03 stems per plant in T1 to 29.18 in T6 (Table 2). The leaf:stem ratio steadily decreased (P<0.05) as fertilization levels increased (1.46 in T1 to 0.79 in T6). Both these variables respond to the fact that as T. diversifolia develops it generates a greater number of stems (Table 2). This is confirmed by the correlation matrix which produced negative percentages (-85.1 % for plant height at cut, and 85.2 % for stem production). The correlation was also negative (-82 %) for biomass production since this variable increases as the leaf:stem ratio decreases. Leaf diameter and weight also increased (P<0.05) with greater fertilization. Length increased from 20.88 cm in T1 to 37.19 cm in T6, while width grew from 15.05 cm in T1 to 23.01 cm in T6. Leaf weight also increased (P<0.05), from 1.59 g per leaf in T1 to 4.32 g in T6. Treatments T5 and T6 did not differ for the variables leaf length and leaf weight (Table 2).

The parameters leaf length, width and weight are important indicators in most production parameters. Compared to the other analyzed variables, these three had high correlations to biomass production (92 %), agronomic parameters (75 %), and protein and energy content (84 %), and negative correlations to NDF, ADF and lignin (-72 %). Dry matter (DM) content decreased as fertilization increased (P<0.05), dropping from 18.54 % in T1 to 15.07 % in T6, but did not differ from T4 onward (Table 3). In contrast, protein content rose with fertilization levels (P<0.05), from 27.31% in T1 to 30.53 % in T5 and 30.25 % in T6 treatments. This was due to the progressively higher nitrogen content in the fertilization treatments (8.85 g N per plant in T1 to 24.99 g N in T6) since nitrogen is biochemically transformed into protein. Fertilization also affected protein levels (Table 3) in protein content per plant per hectare, which grew substantially (P<0.05) from 22 g per plant in T1 to 92 g in T6. Likewise, estimated protein yield per hectare increased from 1,610 kg protein per year in T1 to 6,726 kg in T6; all the treatments differed (P<0.05). Fertilization level impacted protein 793

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production in T. diversifolia. This is perhaps its greatest contribution to livestock production since protein is the most expensive raw material in feed.

Gross energy also increased (P<0.05) with fertilization from 4,163.0 kcal/kg gross energy in T1 to 4,570.8 kcal/kg in T6, although treatments T4, T5 and T6 differed little if at all (Table 3). This was unexpected because higher fertilization levels produced higher fiber content and lower leaf:stem ratio values. However, the higher nutrient concentrations in response to increased fertilization levels may have been responsible for this.

Table 3: Feed composition analysis of Tithonia diversifolia plants at 50 days by fertilization treatment (%) (kcal/kg) Treatment Dry matter Crude protein Ether extract Ash Gross energy T1 T2 T3 T4 T5 T6 CV abcd

18.54 a 17.59 b 16.87 b 15.79 c 15.45 c 15.07 c 3.8

27.31 c 27.62 c 28.80 b 29.32 b 30.53 a 30.25 a 1.5

3.59 3.1 3.17 3.47 3.26 2.97 5.06

15.57 15.84 14.33 14.05 15.3 15.39 3.53

4163.0 e 4346.5 d 4404.5 c 4500.0 b 4550.5 ab 4570.8 a 6.85

CV= coefficient of variation. Different letter superscripts in the same column indicate significant difference (P<0.05).

Neutral detergent fiber (NDF) was lowest (P<0.05) in T1 (26.80 %), increased in T2 (29.9 %) and then remained essentially unchanged from T3 (31.8 %) to T6 (32.01 %) (Figure 1). This may be due to the lower leaf:stem ratio, as suggested by the negative correlation with this variable (-84 %). Values for ADF steadily increased from 16.91 % in T1 to 26.05 % in T6. Again, this is most probably due to the lower leaf:stem ratio, which had a negative correlation (-83 %).

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Figure 1: Fiber percentages and in vitro DM digestibility in T. diversifolia by fertilization treatment 70 60

(%)

50 40 30 20 10 0

T3 T4 Treatments FDN

FDA

Lignin

DIVMS

Very much like ADF levels, lignin levels increased (P<0.05) steadily with higher fertilization levels. The lower leaf:stem ratio affected this variable as shown by its negative correlation (-75 %). Overall, ADF levels rose from 5.23 % in T1 to 7.39 % in T5, and 7.30 % in T6; treatments T1, T2 and T3 did not differ (P>0.05) and neither did treatments T5 and T6 (Figure 1).

As fertilization levels increased digestibility generally decreased from 62.18 % in T1 to 56.45 % in T6 (P<0.05), although T1 and T2 did not differ and neither did T4, T5 and T6. This variable correlated negatively with NDF (-83 %), ADF (-93 %) and lignin (-88 %) concentrations, but positively (76 %) with the leaf:stem ratio (Figure 1).

Tithonia diversifolia has high biomass production and rapid post-cut recovery, both of which depend on planting density, soils and vegetative condition(4). The present results coincide with biomass production reported for T. diversifolia fertilized with 100 g fertilizer (12-24-12, N-PK) in which production was 0.82 at the 30-day cut, 1.73 at the 60-d and 2.58 at the 85-d(16). In another study addressing the effects of distance between plants (0.5 and 1.0 m), cut frequency (40, 60 and 80 d) and cut height (5, 10 and 15 cm), yields ranged from 0.85 to 5.5 t ha-1 DM, which is at the lower limit of the present results(17). An evaluation of planting area on establishment and production in T. diversifolia found yields of 10.31 t ha-1 DM in a 0.5 m x 1.0 m area, 10.28 t ha-1 DM in a 0.75 m x 1.0 m area, and 13.52 t ha-1 DM in a 1.0 m x 1.0 m area(18); these are consistent with the present results. A study of annual yield and nutritional assessment in five bushy forage species found a 114.2 t ha-1 FM in Thichanthera gigantea, 68.2 in Gliricidia sepium, 16.6 in Erythrina peruviana, 17.4 in Leucaena leucocephala and 9.3 in Moringa oleifera. The yields for E. peruviana, L. leucocephala and M. oleifera exceeded those 795

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of T. diversifolia in the present study, but those for T. gigantea and G. sepium were similar to them(19).

The effects of planting density in T. diversifolia on plant height at 90- and 100-d cuts have been found to produce heights ranging from 158 to 176 cm(18), which are consistent with the present results considering the longer cutting intervals. A study of T. diversifolia production using 60d cuts and a 1.0 m x 2.0 m planting area reported plant heights from 214 to 262 cm (higher than in the present results), leaf weight between 0.42 and 0.66 g (lower than the present results), leaf:stem ratio values from 1.13 to 3.41 (higher than in the present data), and stem counts of 7.6 to 15.4 stems per plant (lower than in the present study)(20); these discrepancies are probably due to the longer cutting intervals and lower plant density used in the present methodology. This influence can also be seen in a study evaluating 44 T. diversifolia introductions aimed at identifying promising plants in which a 1 m planting density was used between sites and 2 m between rows, and cuts were done at 40 cm height every 60 d(21). Average plant height was 246 cm (higher than in the present study), stem count per plant was 12.08 (lower than the present study), average leaf weight was 0.52 g (lower than in the present results), and the leaf:stem ratio was 1.70 (higher than in the present study); the differences between the studies are probably due to lower planting density and higher luminosity.

Cut height and frequency have a significant effect on crude protein content in T. diversifolia; for instance, cutting at 20 cm height and at shorter intervals is reported to produce the highest protein concentrations(16). The present protein content results are consistent with the 28.5 to 29.8 % reported at 30 daysâ&#x20AC;&#x2122; regrowth(5,22), but higher than those reported in other studies using various cut intervals, plant heights at cutting and fertilization regimes(23,24,25). Application of biofertilizers and irrigation significantly increases nutrient accumulation(26).

The gross energy content in the present results is higher than reported for the forage bushes M. oleifera (3,764 kcal/kg), Gmelina arborea (3,755 kcal/kg) and T. diversifolia (3,912 kcal/kg)(27). Neutral detergent fiber (NDF) contents reported for T. diversifolia are higher than those found in the present study, possibly due to the longer cut intervals employed. The values nearest the present results are 33.3 to 34.5 % at a 56-d interval(28), but many others are higher, such as 43.9 to 54.5 %(21), 35.2 %(23), and 55.5 %(29). The same is true of ADF values, with previous reports being higher than the present results: 45.8 to 56.7 %(21,29), with 26.3 and 27.7 % at a 56-d interval(28). In a study of lignin concentrations in fodder from Morus alba (4.8 %), Erythrina poeppigiana (4.8 %), T. diversifolia (4.4 %) and Hibiscus rosa-sinensis (5.2 %), the lowest values were in T. diversifolia, which were even lower than in the present study(23).

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In vitro dry matter digestibility (IVDMD) values for T. diversifolia in the present study were higher than reported for M. oleifera (52.44 %), T. gigantea (37.18 %) and L. leucocephala (51.99 %), but lower than those for Morus alba (79.76 %)(30). In an evaluation of ten potential forage species for ruminants (Albizia niopoides, G. sepium, L. leucocephala, Samanea saman, Acacia farnesiana, Mimosa pigra, M. oleifera, Brosimun alicastrum, Cordia dentata and Guazuma ulmifolia) both C. dentata and G. sepium had IVDMD values largely similar to those of the present results for T. diversifolia, although they ranged from 39.1 to 74.8 %(31).

Tithonia diversifolia demonstrated a high nutrient absorption capacity as shown in its greater biomass production as fertilization levels increased. Indeed, this variable increased fivefold between treatment T1 (no fertilizer) and T6 (the highest fertilizer level – 88.5 g per plant). Fertilization also had a significant impact on this crop’s agronomic characteristics, such as accentuated plant growth, higher stem counts per plant, and greater leaf length, width and weight. Leaf characteristics were found to be highly correlated to other productive characteristics, making them an easy-to-measure benchmarks for determining productive characteristics and nutritional properties in T. diversifolia.

Application of fertilizers in Tithonia diversifolia significantly increased leaf content of protein, energy and ash, combined with increased biomass production. The present results suggest that optimizing crop performance requires interpretation of soil analyses and plant nutrient extraction capacity, which allow calculation of optimal fertilization levels. Optimal levels result in the highest biomass production, nutrient contents and digestibility. Based on the present results the optimal fertilization level was that in treatment T5 (28.1 g urea, 15.8 g DAP, 10.1 g KCl) which had the best biomass and nutrient yields. This treatment is preferable to T6 since they did not differ in most variables. The data provided here on optimum fertilization levels for Tithonia diversifolia is important to promoting cultivation of this high protein content forage, which can help to reduce the need for protein concentrates in livestock feed systems.

Acknowledgements The research reported was supported by the Universidad Nacional de Colombia. The authors thank the Chemistry Laboratory of the Universidad Nacional de Colombia, Palmira, for sample analysis, and Ganadería Agroforestal La Esmeralda for allowing access to their farm in Circasia (Quindío, Colombia).

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Rev. Mex. Cienc. Pecu. Vol. 10 Núm. 3, pp. 522-800, JULIO-SEPTIEMBRE-2019