RMCP Vol. 14 Num. 2 (2023) : April-June (English Version)

Edición Bilingüe
Bilingual Edition
ISSN: 2448-6698
Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 14 Núm. 2, pp. 260-487, ABRIL-JUNIO-2023
Rev. Mex. Cienc. Pecu. Vol. 14 Núm. 2, pp. 260-487, ABRIL-JUNIO-2023

REVISTA MEXICANA DE CIENCIAS PECUARIAS Volumen 14 Numero 2, Abril-Junio
2023. 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 de Investigación Regional Sureste, Calle 6 No. 398 X 13, Avenida
Correa Racho, Col. Díaz Ordaz, Mérida Yucatán, C.P. 97130.
Editor responsable: Arturo García Fraustro Reservas de Derechos al Uso Exclusivo número
04-2022-033116571100-102. ISSN: 2448-6698, otorgados por el Instituto Nacional del
Derecho de Autor (INDAUTOR).
Responsable de la última actualización de este número: Arturo García Fraustro, Campo
Experimental Mocochá, Km. 25 Antigua Carretera Mérida–Motul, Mocochá, Yuc. C.P. 97454.
http://cienciaspecuarias. inifap.gob.mx, la presente publicación tuvo su última actualización
en marzo de 2022.
2° Concurso de Dibujo Infantil INIFAP 2022
Futuros Investigadores
Autor: Jesús Itzae Ávalos Franco
Edad: 6 años, Baja California Sur DIRECTORIO
Título: Trabajo de campo
FUNDADOR
John A. Pino
EDITOR EN JEFE EDITORES ADJUNTOS
Arturo García Fraustro Oscar L. Rodríguez Rivera
Alfonso Arias Medina
EDITORES POR DISCIPLINA
Dra. Yolanda Beatriz Moguel Ordóñez, INIFAP, México Dr. Juan Ku Vera, Universidad Autónoma de Yucatán, México
Dr. Ramón Molina Barrios, Instituto Tecnológico de Sonora, Dr. Ricardo Basurto Gutiérrez, INIFAP, México
Dr. Alfonso Juventino Chay Canul, Universidad Autónoma de Dr. Luis Corona Gochi, Facultad de Medicina Veterinaria y
Tabasco, México Zootecnia, UNAM, México
Dra. Maria Cristina Schneider, Universidad de Georgetown, Dr. Juan Manuel Pinos Rodríguez, Facultad de Medicina
Estados Unidos Veterinaria y Zootecnia, Universidad Veracruzana, México
Dr. Feliciano Milian Suazo, Universidad Autónoma de Dr. Carlos López Coello, Facultad de Medicina Veterinaria y
Querétaro, México Zootecnia, UNAM, México
Dr. Javier F. Enríquez Quiroz, INIFAP, México Dr. Arturo Francisco Castellanos Ruelas, Facultad de
Dra. Martha Hortencia Martín Rivera, Universidad de Sonora Química. UADY
URN, México Dra. Guillermina Ávila Ramírez, UNAM, México
Dr. Fernando Arturo Ibarra Flores, Universidad de Sonora Dr. Emmanuel Camuus, CIRAD, Francia.
URN, México Dr. Héctor Jiménez Severiano, INIFAP., México
Dr. James A. Pfister, USDA, Estados Unidos Dr. Juan Hebert Hernández Medrano, UNAM, México
Dr. Eduardo Daniel Bolaños Aguilar, INIFAP, México Dr. Adrian Guzmán Sánchez, Universidad Autónoma
Dr. Sergio Iván Román-Ponce, INIFAP, México Metropolitana-Xochimilco, México
Dr. Jesús Fernández Martín, INIA, España Dr. Eugenio Villagómez Amezcua Manjarrez, INIFAP, CENID
Dr. Maurcio A. Elzo, Universidad de Florida Salud Animal e Inocuidad, México
Dr. Sergio D. Rodríguez Camarillo, INIFAP, México Dr. José Juan Hernández Ledezma, Consultor privado
Dra. Nydia Edith Reyes Rodríguez, Universidad Autónoma del Dr. Fernando Cervantes Escoto, Universidad Autónoma
Estado de Hidalgo, México Chapingo, México
Dra. Maria Salud Rubio Lozano, Facultad de Medicina Dr. Adolfo Guadalupe Álvarez Macías, Universidad Autónoma
Veterinaria y Zootecnia, UNAM, México Metropolitana Xochimilco, México
Dra. Elizabeth Loza-Rubio, INIFAP, México Dr. Alfredo Cesín Vargas, UNAM, México
Dr. Juan Carlos Saiz Calahorra, Instituto Nacional de Dra. Marisela Leal Hernández, INIFAP, México
Investigaciones Agrícolas, España Dr. Efrén Ramírez Bribiesca, Colegio de Postgraduados,
Dr. José Armando Partida de la Peña, INIFAP, México México
Dr. José Luis Romano Muñoz, INIFAP, México Dra. Itzel Amaro Estrada, INIFAP, México
Dr. Jorge Alberto López García, INIFAP, México
Dr. Alejandro Plascencia Jorquera, Universidad Autónoma de
Baja California, México
TIPOGRAFÍA Y FORMATO: Oscar L. Rodríguez Rivera
Indizada en el “Journal Citation Report” Science Edition del ISI . Inscrita en el Sistema de Clasificación de Revistas Científicas y
Tecnológicas de CONACyT; en EBSCO Host y la Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
(RedALyC) (www.redalyc.org); en la Red Iberoamericana de Revistas Científicas de Veterinaria de Libre Acceso
(www.veterinaria.org/revistas/ revivec); en los Índices SCOPUS y EMBASE de Elsevier (www.elsevier. com).
I
REVISTA MEXICANA DE CIENCIAS PECUARIAS
La Revista Mexicana de Ciencias Pecuarias es un órgano trimestral en formato bilingüe Español e Inglés. El costo
de difusión científica y técnica de acceso abierto, revisada total por publicar es de $ 7,280.00 más IVA por manuscrito
por pares y arbitrada. Su objetivo es dar a conocer los ya editado.
resultados de las investigaciones realizadas por cualquier Se publica en formato digital en acceso abierto, por lo que
institución científica, relacionadas particularmente con las se autoriza la reproducción total o parcial del contenido de
distintas disciplinas de la Medicina Veterinaria y la los artículos si se cita la fuente.
Zootecnia. Además de trabajos de las disciplinas indicadas
El envío de los trabajos de debe realizar directamente en el sitio
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II
I
REVISTA MEXICANA DE CIENCIAS PECUARIAS
REV. MEX. CIENC. PECU. VOL. 14 No. 2 ABRIL-JUNIO-2023
CONTENIDO
Contents
ARTÍCULOS
Articles
Pág.
Producción de anticuerpos séricos en respuesta a la vacunación contra los virus de

la rinotraqueitis infecciosa bovina y la diarrea viral bovina con una vacuna comercial
Production of serum antibodies in response to vaccination against infectious bovine rhinotracheitis
and bovine viral diarrhea viruses with a commercial vaccine
Jorge Víctor Rosete Fernández, Guadalupe Asunción Socci Escatell, Abraham Fragoso Islas, Sara
Olazarán Jenkins, Ángel Ríos Utrera........................................................................................260
Characterization of fetal bovine serum obtained from the meat industry for cell culture
Caracterización del suero bovino fetal proveniente de la industria cárnica mexicana en el cultivo
celular
Francisco Javier Preciado-Gutiérrez, David Masuoka-Ito, José Luis Barrera-Bernal, Bryan Ivan Martín
del Campo-Téllez, Vicente Esparza-Villalpando, Ricardo Ernesto Ramírez-Orozco ...………….........277
Ixodicide action of natural products from native Mexican plants

Acción ixodicida de productos naturales de plantas nativas mexicanas
Javier Sosa-Rueda, Fabiola Villarauz, Vanihamin Domínguez-Meléndez, Ida Soto-Rodríguez,
Fernando C. López-Fentanes, David I. Martínez-Herrera, Álvaro Peniche-Cardeña, Francisco
Cen-Pacheco ..............…………………………………………...............................................................292
Efecto de Xoconostle (Opuntia matudae Scheinvar) sobre la concentración de metano

y las variables ruminales durante una fermentación in vitro de rastrojo de maíz
Effect of Xoconostle (Opuntia matudae Scheinvar) on methane concentration and ruminal variables
during in vitro fermentation of corn stover
José Jesús Espino-García, Isaac Almaraz-Buendía, J. Jesús Germán Peralta-Ortiz, Abigail Reyes-
Munguía, Iridiam Hernández-Soto, Lucio González-Montiel, Rafael Germán Campos-Montiel …….309
Factores que afectan la tasa de preñez mediante transferencias de embriones por

fertilización in vitro en novillas multirraciales en condiciones de trópico colombiano
Factors affecting the rate of pregnancy by embryo transfers (ET) by in vitro fertilization in
multibreed heifers under Colombian tropical conditions
Heli Fernando Valencia Ocampo, Nancy Rodríguez Colorado, Tatiana Mantilla ...........................326
III
Estructura y variabilidad genética del bisonte americano (Bison bison) en México
Genetic structure and variability in American bison (Bison bison) in Mexico
Joel Domínguez-Viveros, Guadalupe Nelson Aguilar-Palma, Rafael Villa-Angulo, Nancy Hernández-
Rodríguez, José Manuel Pérez-Cantú, Flora Moir, Pedro Calderón-Domínguez .…………………………339
Composición química del rastrojo de tres cultivares de maíz esterilizados y colonizados

por micelio de Ganoderma lucidum
Stover chemical composition in three corn cultivars after sterilization or colonization with
Ganoderma lucidum mycelia
Liz Sarahy Pérez-Martell, Juan de Dios Guerrero-Rodríguez, Daniel Claudio Martínez-Carrera, Javier
Francisco Enríquez-Quiroz, Efraín Pérez-Ramírez, Benito Ramírez-Valverde ...............................349
Nutrient concentrations, in vitro digestibility and rumen fermentation of agro-industrial

residues of Cannabis sativa L. as a potential forage source for ruminants
Concentraciones de nutrientes, digestibilidad in vitro y fermentación ruminal de residuos
agroindustriales de Cannabis sativa L. como fuente potencial de forraje para rumiantes
Elia Esther Araiza-Rosales, Esperanza Herrera-Torres, Francisco Óscar Carrete-Carreón, Rafael
Jiménez-Ocampo, Daniel Gómez-Sánchez, Gerardo Antonio Pámanes-Carrasco………………………..366
REVISIONES DE LITERATURA
Reviews
Importancia de Haematobia irritans en la ganadería bovina de México: Situación actual

y perspectivas. Revisión
Importance of Haematobia irritans in cattle in Mexico: Current situation and perspectives. Review
Roger Iván Rodríguez Vivas, Carlos Cruz Vázquez, Consuelo Almazán, Juan José Zárate Ramos..384
NOTAS DE INVESTIGACIÓN
Technical notes
Curvas de crecimiento en bovinos Limousin de raza pura y cruzados

Growth curves in purebred and crossbred Limousin cattle
Joel Domínguez-Viveros, Antonio Reyes-Cerón, Carlos Enrique Aguirre-Calderón, Ricardo Martínez-
Rocha, Carlos Luna-Palomera, Nelson Aguilar-Palma ……….......................................................412
Relationship between body measurement traits, udder measurement traits and milk
yield of Saanen goats in Capricorn district of South Africa
Relación entre rasgos de mediciones corporales, rasgos de mediciones de la ubre y producción de
leche de cabras Saanen en el distrito de Capricorn de Sudáfrica
Thlarihani Cynthia Makamu, Molabe Kagisho Madikadike, Kwena Mokoena,
Thobela Louis Tyasi …………………………………………………………………………………………………………..423
IV
Análisis genético del bovino Criollo Mixteco de Oaxaca
Genetic analysis of Oaxacan Mixteco Creole cattle
Miguel Ángel Domínguez Martínez, Víctor Hernández Núñez, Araceli Mariscal Méndez, Amparo
Martínez Martínez, Gisela Fuentes-Mascorro ……......................................................................434
Influence of the cut intervals on hay quality of Panicum maximum cv. BRS Tamani in
brazilian Cerrado
Influencia de los intervalos de corte en la calidad del heno de Panicum maximum cv. BRS Tamani
en el Cerrado brasileño
Eva Nara Oliveira Gomes, Alexandre Menezes Dias, Luciana Junges, Luís Carlos Vinhas Ítavo,
Gelson dos Santos Difante, Juliana Oliveira Batistoti .....................................................………...450
Evaluación de la seroconversión de cerdas con el uso de un inóculo a diferentes dosis y

vehículos contra la diarrea epidémica porcina
Evaluation of sow seroconversion with the use of inoculum at different doses and vehicles against
porcine epidemic diarrhea
Nancy Paulina García Cano Rubí, Francisco Ernesto Martínez-Castañeda, Elein Hernández Trujillo,
Rosa Elena Sarmiento Silva, Rolando Beltrán Figueroa, Montserrat Elemi García-Hernández, María
Elena Trujillo-Ortega .............................................................................................................466
Efecto de la fuente de selenio en el comportamiento productivo, contenido de selenio

en suero y músculo, y nivel sérico de albúmina, α-, β- y ∂-globulinas en ovinos Pelibuey
Effect of selenium source on productive behavior, serum and muscle selenium content, and serum
level of albumin, α-, β- and ∂-globulins in Pelibuey sheep
Lino Rigoberto Cárdenas-Ramírez, Carlos Sánchez del Real, Agustín Ruíz-Flores, Gabriela Pérez-
Hernández, Reyes López-Ordaz, Claudio Vite-Cristóbal, Rufino López-Ordaz ..............................476
V
Actualización: marzo, 2020
NOTAS AL AUTOR
La Revista Mexicana de Ciencias Pecuarias se edita 6. Los manuscritos de las tres categorías de trabajos que
completa en dos idiomas (español e inglés) y publica tres se publican en la Rev. Mex. Cienc. Pecu. deberán
categorías de trabajos: Artículos científicos, Notas de contener los componentes que a continuación se
investigación y Revisiones bibliográficas. indican, empezando cada uno de ellos en página
aparte.
Los autores interesados en publicar en esta revista
deberán ajustarse a los lineamientos que más adelante se Página del título
indican, los cuales en términos generales, están de Resumen en español
acuerdo con los elaborados por el Comité Internacional de Resumen en inglés
Editores de Revistas Médicas (CIERM) Bol Oficina Sanit Texto
Panam 1989;107:422-437. Agradecimientos y conflicto de interés
Literatura citada
1. Sólo se aceptarán trabajos inéditos. No se admitirán
si están basados en pruebas de rutina, ni datos
7. Página del Título. Solamente debe contener el título
experimentales sin estudio estadístico cuando éste
del trabajo, que debe ser conciso pero informativo; así
sea indispensable. Tampoco se aceptarán trabajos
como el título traducido al idioma inglés. En el
que previamente hayan sido publicados condensados
manuscrito no es necesaria información como
o in extenso en Memorias o Simposio de Reuniones o
nombres de autores, departamentos, instituciones,
Congresos (a excepción de Resúmenes).
direcciones de correspondencia, etc., ya que estos
2. Todos los trabajos estarán sujetos a revisión de un datos tendrán que ser registrados durante el proceso
Comité Científico Editorial, conformado por Pares de de captura de la solicitud en la plataforma del OJS
la Disciplina en cuestión, quienes desconocerán el (http://ciencias pecuarias.inifap.gob.mx).
nombre e Institución de los autores proponentes. El
8. Resumen en español. En la segunda página se debe
Editor notificará al autor la fecha de recepción de su
incluir un resumen que no pase de 250 palabras. En
trabajo.
él se indicarán los propósitos del estudio o
3. El manuscrito deberá someterse a través del portal de investigación; los procedimientos básicos y la
la Revista en la dirección electrónica: metodología empleada; los resultados más
http://cienciaspecuarias.inifap.gob.mx, consultando importantes encontrados, y de ser posible, su
el “Instructivo para envío de artículos en la página de significación estadística y las conclusiones principales.
la Revista Mexicana de Ciencias Pecuarias”. Para su A continuación del resumen, en punto y aparte,
elaboración se utilizará el procesador de Microsoft agregue debidamente rotuladas, de 3 a 8 palabras o
Word, con letra Times New Roman a 12 puntos, a frases cortas clave que ayuden a los indizadores a
doble espacio. Asimismo se deberán llenar los clasificar el trabajo, las cuales se publicarán junto con
formatos de postulación, carta de originalidad y no el resumen.
duplicidad y disponibles en el propio sitio oficial de la
9. Resumen en inglés. Anotar el título del trabajo en
revista.
inglés y a continuación redactar el “abstract” con las
4. Por ser una revista con arbitraje, y para facilitar el mismas instrucciones que se señalaron para el
trabajo de los revisores, todos los renglones de cada resumen en español. Al final en punto y aparte, se
página deben estar numerados; asimismo cada deberán escribir las correspondientes palabras clave
página debe estar numerada, inclusive cuadros, (“key words”).
ilustraciones y gráficas.
10. Texto. Las tres categorías de trabajos que se publican
5. Los artículos tendrán una extensión máxima de 20 en la Rev. Mex. Cienc. Pecu. consisten en lo
cuartillas a doble espacio, sin incluir páginas de Título, siguiente:
y cuadros o figuras (los cuales no deberán exceder de
a) Artículos científicos. Deben ser informes de trabajos
ocho y ser incluidos en el texto). Las Notas de
originales derivados de resultados parciales o finales
investigación tendrán una extensión máxima de 15
de investigaciones. El texto del Artículo científico se
cuartillas y 6 cuadros o figuras. Las Revisiones
divide en secciones que llevan estos
bibliográficas una extensión máxima de 30 cuartillas y
encabezamientos:
5 cuadros.
VI
Introducción referencias, aunque pueden insertarse en el texto
Materiales y Métodos (entre paréntesis).
Resultados
Reglas básicas para la Literatura citada
Discusión
Conclusiones e implicaciones Nombre de los autores, con mayúsculas sólo las
Literatura citada iniciales, empezando por el apellido paterno, luego
iniciales del materno y nombre(s). En caso de
En los artículos largos puede ser necesario agregar apellidos compuestos se debe poner un guión entre
subtítulos dentro de estas divisiones a fin de hacer ambos, ejemplo: Elías-Calles E. Entre las iniciales de
más claro el contenido, sobre todo en las secciones de un autor no se debe poner ningún signo de
Resultados y de Discusión, las cuales también pueden puntuación, ni separación; después de cada autor sólo
presentarse como una sola sección. se debe poner una coma, incluso después del
b) Notas de investigación. Consisten en penúltimo; después del último autor se debe poner un
modificaciones a técnicas, informes de casos clínicos punto.
de interés especial, preliminares de trabajos o El título del trabajo se debe escribir completo (en su
investigaciones limitadas, descripción de nuevas idioma original) luego el título abreviado de la revista
variedades de pastos; así como resultados de donde se publicó, sin ningún signo de puntuación;
investigación que a juicio de los editores deban así ser inmediatamente después el año de la publicación,
publicados. El texto contendrá la misma información luego el número del volumen, seguido del número
del método experimental señalado en el inciso a), (entre paréntesis) de la revista y finalmente el número
pero su redacción será corrida del principio al final del de páginas (esto en caso de artículo ordinario de
trabajo; esto no quiere decir que sólo se supriman los revista).
subtítulos, sino que se redacte en forma continua y
coherente. Puede incluir en la lista de referencias, los artículos
aceptados aunque todavía no se publiquen; indique la
c) Revisiones bibliográficas. Consisten en el
revista y agregue “en prensa” (entre corchetes).
tratamiento y exposición de un tema o tópico de
relevante actualidad e importancia; su finalidad es la En el caso de libros de un solo autor (o más de uno,
de resumir, analizar y discutir, así como poner a pero todos responsables del contenido total del libro),
disposición del lector información ya publicada sobre después del o los nombres, se debe indicar el título
un tema específico. El texto se divide en: del libro, el número de la edición, el país, la casa
Introducción, y las secciones que correspondan al editorial y el año.
desarrollo del tema en cuestión.
Cuando se trate del capítulo de un libro de varios
11. Agradecimientos y conflicto de interés. Siempre autores, se debe poner el nombre del autor del
que corresponda, se deben especificar las capítulo, luego el título del capítulo, después el
colaboraciones que necesitan ser reconocidas, tales
nombre de los editores y el título del libro, seguido del
como a) la ayuda técnica recibida; b) el
país, la casa editorial, año y las páginas que abarca el
agradecimiento por el apoyo financiero y material,
capítulo.
especificando la índole del mismo; c) las relaciones
financieras que pudieran suscitar un conflicto de En el caso de tesis, se debe indicar el nombre del
intereses. Las personas que colaboraron pueden ser autor, el título del trabajo, luego entre corchetes el
citadas por su nombre, añadiendo su función o tipo de grado (licenciatura, maestría, doctorado), luego el
colaboración; por ejemplo: “asesor científico”, nombre de la ciudad, estado y en su caso país,
“revisión crítica de la propuesta para el estudio”, seguidamente el nombre de la Universidad (no el de
“recolección de datos”, etc. Siempre que corresponda, la escuela), y finalmente el año.
los autores deberán mencionar si existe algún
conflicto de interés. Emplee el estilo de los ejemplos que aparecen a
continuación, los cuales están parcialmente basados
12. Literatura citada. Numere las referencias en el formato que la Biblioteca Nacional de Medicina
consecutivamente en el orden en que se mencionan de los Estados Unidos usa en el Index Medicus.
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 Revistas
paréntesis, sin señalar el año de la referencia. Evite
hasta donde sea posible, el tener que mencionar en el Artículo ordinario, con volumen y número. (Incluya el
texto el nombre de los autores de las referencias. nombre de todos los autores cuando sean seis o
Procure abstenerse de utilizar los resúmenes como menos; si son siete o más, anote sólo el nombre de
referencias; las “observaciones inéditas” y las los seis primeros y agregue “et al.”).
“comunicaciones personales” no deben usarse como
VII
I) Basurto GR, Garza FJD. Efecto de la inclusión de grasa XI) Olea PR, Cuarón IJA, Ruiz LFJ, Villagómez AE.
o proteína de escape ruminal en el comportamiento Concentración de insulina plasmática en cerdas
de toretes Brahman en engorda. Téc Pecu Méx alimentadas con melaza en la dieta durante la
1998;36(1):35-48. inducción de estro lactacional [resumen]. Reunión
nacional de investigación pecuaria. Querétaro, Qro.
Sólo número sin indicar volumen.
1998:13.
II) Stephano HA, Gay GM, Ramírez TC. Encephalomielitis,
XII) Cunningham EP. Genetic diversity in domestic
reproductive failure and corneal opacity (blue eye) in
animals: strategies for conservation and
pigs associated with a paramyxovirus infection. Vet
development. In: Miller RH et al. editors. Proc XX
Rec 1988;(122):6-10.
Beltsville Symposium: Biotechnology’s role in
III) Chupin D, Schuh H. Survey of present status ofthe use genetic improvement of farm animals. USDA.
of artificial insemination in developing countries. 1996:13.
World Anim Rev 1993;(74-75):26-35.
Tesis.
No se indica el autor.
XIII) Alvarez MJA. Inmunidad humoral en la anaplasmosis
IV) Cancer in South Africa [editorial]. S Afr Med J y babesiosis bovinas en becerros mantenidos en una
1994;84:15. zona endémica [tesis maestría]. México, DF:
Universidad Nacional Autónoma de México; 1989.
Suplemento de revista.
XIV) Cairns RB. Infrared spectroscopic studies of solid
V) Hall JB, Staigmiller RB, Short RE, Bellows RA, Bartlett oxigen [doctoral thesis]. Berkeley, California, USA:
SE. Body composition at puberty in beef heifers as University of California; 1965.
influenced by nutrition and breed [abstract]. J Anim
Sci 1998;71(Suppl 1):205. Organización como autor.
Organización, como autor. XV) NRC. National Research Council. The nutrient
requirements of beef cattle. 6th ed. Washington,
VI) The Cardiac Society of Australia and New Zealand. DC, USA: National Academy Press; 1984.
Clinical exercise stress testing. Safety and performance
guidelines. Med J Aust 1996;(164):282-284. XVI) SAGAR. Secretaría de Agricultura, Ganadería y
Desarrollo Rural. Curso de actualización técnica para
En proceso de publicación. la aprobación de médicos veterinarios zootecnistas
responsables de establecimientos destinados al
VII) Scifres CJ, Kothmann MM. Differential grazing use of
sacrificio de animales. México. 1996.
herbicide treated area by cattle. J Range Manage [in
press] 2000. XVII) AOAC. Oficial methods of analysis. 15th ed.
Arlington, VA, USA: Association of Official Analytical
Chemists. 1990.
Libros y otras monografías
XVIII) SAS. SAS/STAT User’s Guide (Release 6.03). Cary
Autor total. NC, USA: SAS Inst. Inc. 1988.
VIII) Steel RGD, Torrie JH. Principles and procedures of XIX) SAS. SAS User´s Guide: Statistics (version 5 ed.).
statistics: A biometrical approach. 2nd ed. New Cary NC, USA: SAS Inst. Inc. 1985.
York, USA: McGraw-Hill Book Co.; 1980.
Publicaciones electrónicas
Autor de capítulo.
XX) Jun Y, Ellis M. Effect of group size and feeder type
IX) Roberts SJ. Equine abortion. In: Faulkner LLC editor. on growth performance and feeding patterns in
Abortion diseases of cattle. 1rst ed. Springfield, growing pigs. J Anim Sci 2001;79:803-813.
Illinois, USA: Thomas Books; 1968:158-179.
http://jas.fass.org/cgi/reprint/79/4/803.pdf.
Accessed Jul 30, 2003.
Memorias de reuniones.
XXI) Villalobos GC, González VE, Ortega SJA. Técnicas
X) Loeza LR, Angeles MAA, Cisneros GF. Alimentación
para estimar la degradación de proteína y materia
de cerdos. En: Zúñiga GJL, Cruz BJA editores.
orgánica en el rumen y su importancia en rumiantes
Tercera reunión anual del centro de investigaciones
forestales y agropecuarias del estado de Veracruz. en pastoreo. Téc Pecu Méx 2000;38(2): 119-134.
Veracruz. 1990:51-56. http://www.tecnicapecuaria.org/trabajos/20021217
5725.pdf. Consultado 30 Ago, 2003.
VIII
XXII) Sanh MV, Wiktorsson H, Ly LV. Effect of feeding ha hectárea (s)
level on milk production, body weight change, feed h hora (s)
conversion and postpartum oestrus of crossbred i.m. intramuscular (mente)
lactating cows in tropical conditions. Livest Prod Sci i.v. intravenosa (mente)
2002;27(2-3):331-338. http://www.sciencedirect. J joule (s)
com/science/journal/03016226. Accessed Sep 12, kg kilogramo (s)
2003.
km kilómetro (s)
13. Cuadros, Gráficas e Ilustraciones. Es preferible L litro (s)
que sean pocos, concisos, contando con los datos log logaritmo decimal
necesarios para que sean autosuficientes, que se Mcal megacaloría (s)
entiendan por sí mismos sin necesidad de leer el texto. MJ megajoule (s)
Para las notas al pie se deberán utilizar los símbolos
m metro (s)
convencionales.
msnm metros sobre el nivel del mar
14 Versión final. Es el documento en el cual los autores µg microgramo (s)
ya integraron las correcciones y modificaciones µl microlitro (s)
indicadas por el Comité Revisor. Los trabajos deberán
µm micrómetro (s)(micra(s))
ser elaborados con Microsoft Word. Las fotografías e
imágenes deberán estar en formato jpg (o mg miligramo (s)
compatible) con al menos 300 dpi de resolución. ml mililitro (s)
Tanto las fotografías, imágenes, gráficas, cuadros o mm milímetro (s)
tablas deberán incluirse en el mismo archivo del texto. min minuto (s)
Los cuadros no deberán contener ninguna línea ng nanogramo (s)Pprobabilidad (estadística)
vertical, y las horizontales solamente las que delimitan p página
los encabezados de columna, y la línea al final del PC proteína cruda
cuadro.
PCR reacción en cadena de la polimerasa
15. Una vez recibida la versión final, ésta se mandará para pp páginas
su traducción al idioma inglés o español, según ppm partes por millón
corresponda. Si los autores lo consideran conveniente % por ciento (con número)
podrán enviar su manuscrito final en ambos idiomas.
rpm revoluciones por minuto
16. Tesis. Se publicarán como Artículo o Nota de seg segundo (s)
Investigación, siempre y cuando se ajusten a las t tonelada (s)
normas de esta revista. TND total de nutrientes digestibles
17. Los trabajos no aceptados para su publicación se UA unidad animal
regresarán al autor, con un anexo en el que se UI unidades internacionales
explicarán los motivos por los que se rechaza o las vs versus
modificaciones que deberán hacerse para ser xg gravedades
reevaluados.
Cualquier otra abreviatura se pondrá entre paréntesis
18. Abreviaturas de uso frecuente: inmediatamente después de la(s) palabra(s)
cal caloría (s) completa(s).
cm centímetro (s) 19. Los nombres científicos y otras locuciones latinas se
°C grado centígrado (s) deben escribir en cursivas.
DL50 dosis letal 50%
g gramo (s)
IX
Updated: March, 2020
INSTRUCTIONS FOR AUTHORS
Revista Mexicana de Ciencias Pecuarias is a scientific

journal published in a bilingual format (Spanish and
English) which carries three types of papers: Research
Articles, Technical Notes, and Reviews. Authors interested Title page
in publishing in this journal, should follow the below- Abstract
mentioned directives which are based on those set down Text
by the International Committee of Medical Journal Editors Acknowledgments and conflict of interest
(ICMJE) Bol Oficina Sanit Panam 1989;107:422-437. Literature cited
1. Only original unpublished works will be accepted.
Manuscripts based on routine tests, will not be 7. Title page. It should only contain the title of the
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statistical analysis. Papers previously published as the title translated into English language. In the
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than 250 words should be included. This abstract
3. Papers will be submitted in the Web site should start with a clear statement of the objectives
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“Guide for submit articles in the Web site of the The more significant results and their statistical value
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Figures (8 maximum and be included in the text). Conclusions and implications
Technical notes will have a maximum extension of 15 Literature cited
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exceed 30 pages and 5 Tables and Figures. sections to make the content clearer. Results and
6. Manuscripts of all three type of articles published in Discussion can be shown as a single section if
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cases of special interest, complete description of a
limited investigation, or research results which
X
should be published as a note in the opinion names(s), the number of the edition, the country, the
of the editors. The text will contain the same printing house and the year.
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summarize, analyze and discuss an outstanding topic. of the chapter, the editors and the title of the book,
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needed that relate to the description of the topic in
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11. Literature cited. All references should be quoted in

their original language. They should be numbered
Journals
consecutively in the order in which they are first
mentioned in the text. Text, tables and figure Standard journal article (List the first six authors
references should be identified by means of Arabic followed by et al.)
numbers. Avoid, whenever possible, mentioning in the
text the name of the authors. Abstain from using I) Basurto GR, Garza FJD. Efecto de la inclusión de grasa
abstracts as references. Also, “unpublished o proteína de escape ruminal en el comportamiento
observations” and “personal communications” should de toretes Brahman en engorda. Téc Pecu Méx
not be used as references, although they can be 1998;36(1):35-48.
inserted in the text (inside brackets).
Issue with no volume
Key rules for references
II) Stephano HA, Gay GM, Ramírez TC. Encephalomielitis,
a. The names of the authors should be quoted reproductive failure and corneal opacity (blue eye) in
beginning with the last name spelt with initial capitals, pigs associated with a paramyxovirus infection. Vet
followed by the initials of the first and middle name(s). Rec 1988;(122):6-10.
In the presence of compound last names, add a dash
between both, i.e. Elias-Calles E. Do not use any III) Chupin D, Schuh H. Survey of present status of the
punctuation sign, nor separation between the initials use of artificial insemination in developing countries.
of an author; separate each author with a comma, World Anim Rev 1993;(74-75):26-35.
even after the last but one.
No author given
b. The title of the paper should be written in full,
followed by the abbreviated title of the journal without IV) Cancer in South Africa [editorial]. S Afr Med J
any punctuation sign; then the year of the publication, 1994;84:15.
after that the number of the volume, followed by the
number (in brackets) of the journal and finally the Journal supplement
number of pages (this in the event of ordinary article).
V) Hall JB, Staigmiller RB, Short RE, Bellows RA, Bartlett
c. Accepted articles, even if still not published, can SE. Body composition at puberty in beef heifers as
be included in the list of references, as long as the influenced by nutrition and breed [abstract]. J Anim
journal is specified and followed by “in press” (in Sci 1998;71(Suppl 1):205.
brackets).
d. In the case of a single author’s book (or more
than one, but all responsible for the book’s contents),
the title of the book should be indicated after the
XI
Organization, as author Organization as author
VI) The Cardiac Society of Australia and New Zealand. XV) NRC. National Research Council. The nutrient
Clinical exercise stress testing. Safety and requirements of beef cattle. 6th ed. Washington,
performance guidelines. Med J Aust 1996;(164):282- DC, USA: National Academy Press; 1984.
284. XVI) SAGAR. Secretaría de Agricultura, Ganadería y
In press Desarrollo Rural. Curso de actualización técnica para
la aprobación de médicos veterinarios zootecnistas
VII) Scifres CJ, Kothmann MM. Differential grazing use of responsables de establecimientos destinados al
herbicide-treated area by cattle. J Range Manage [in sacrificio de animales. México. 1996.
press] 2000.
XVII) AOAC. Official methods of analysis. 15th ed.
Books and other monographs Arlington, VA, USA: Association of Official Analytical
Chemists. 1990.
Author(s)
XVIII) SAS. SAS/STAT User’s Guide (Release 6.03). Cary
VIII) Steel RGD, Torrie JH. Principles and procedures of NC, USA: SAS Inst. Inc. 1988.
statistics: A biometrical approach. 2nd ed. New
York, USA: McGraw-Hill Book Co.; 1980. XIX) SAS. SAS User´s Guide: Statistics (version 5 ed.).
Cary NC, USA: SAS Inst. Inc. 1985.
Chapter in a book
Electronic publications
IX) Roberts SJ. Equine abortion. In: Faulkner LLC editor.
Abortion diseases of cattle. 1rst ed. Springfield, XX) Jun Y, Ellis M. Effect of group size and feeder type
Illinois, USA: Thomas Books; 1968:158-179. 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.
Conference paper
Accesed Jul 30, 2003.
X) Loeza LR, Angeles MAA, Cisneros GF. Alimentación
XXI) Villalobos GC, González VE, Ortega SJA. Técnicas
de cerdos. En: Zúñiga GJL, Cruz BJA editores.
Tercera reunión anual del centro de investigaciones para estimar la degradación de proteína y materia
forestales y agropecuarias del estado de Veracruz. orgánica en el rumen y su importancia en rumiantes
Veracruz. 1990:51-56. en pastoreo. Téc Pecu Méx 2000;38(2): 119-134.
http://www.tecnicapecuaria.org/trabajos/20021217
XI) Olea PR, Cuarón IJA, Ruiz LFJ, Villagómez AE. 5725.pdf. Consultado 30 Jul, 2003.
Concentración de insulina plasmática en cerdas
alimentadas con melaza en la dieta durante la XXII) Sanh MV, Wiktorsson H, Ly LV. Effect of feeding
inducción de estro lactacional [resumen]. Reunión level on milk production, body weight change, feed
nacional de investigación pecuaria. Querétaro, Qro. conversion and postpartum oestrus of crossbred
1998:13. lactating cows in tropical conditions. Livest Prod Sci
2002;27(2-3):331-338.
XII) Cunningham EP. Genetic diversity in domestic
animals: strategies for conservation and http://www.sciencedirect.com/science/journal/030
development. In: Miller RH et al. editors. Proc XX 16226. Accesed Sep 12, 2003.
Beltsville Symposium: Biotechnology’s role in 12. Tables, Graphics and Illustrations. It is preferable
genetic improvement of farm animals. USDA.
that they should be few, brief and having the
1996:13.
necessary data so they could be understood without
reading the text. Explanatory material should be
Thesis
placed in footnotes, using conventional symbols.
XIII) Alvarez MJA. Inmunidad humoral en la anaplasmosis
y babesiosis bovinas en becerros mantenidos en una 13. Final version. This is the document in which the
zona endémica [tesis maestría]. México, DF: authors have already integrated the corrections and
Universidad Nacional Autónoma de México; 1989. modifications indicated by the Review Committee. The
works will have to be elaborated with Microsoft Word.
XIV) Cairns RB. Infrared spectroscopic studies of solid Photographs and images must be in jpg (or
oxigen [doctoral thesis]. Berkeley, California, USA:
compatible) format with at least 300 dpi resolution.
University of California; 1965.
Photographs, images, graphs, charts or tables must
be included in the same text file. The boxes should
not contain any vertical lines, and the horizontal ones
only those that delimit the column headings, and the
line at the end of the box.
XII
14. Once accepted, the final version will be translated into MJ mega joule (s)
Spanish or English, although authors should feel free m meter (s)
to send the final version in both languages. No µl micro liter (s)
charges will be made for style or translation services. µm micro meter (s)
15. Thesis will be published as a Research Article or as a mg milligram (s)
Technical Note, according to these guidelines. ml milliliter (s)
mm millimeter (s)
16. Manuscripts not accepted for publication will be min minute (s)
returned to the author together with a note explaining ng nanogram (s)
the cause for rejection, or suggesting changes which
P probability (statistic)
should be made for re-assessment.
p page
CP crude protein
PCR polymerase chain reaction
17. List of abbreviations:
pp pages
cal calorie (s) ppm parts per million
cm centimeter (s) % percent (with number)
°C degree Celsius rpm revolutions per minute
DL50 lethal dose 50% sec second (s)
g gram (s) t metric ton (s)
ha hectare (s) TDN total digestible nutrients
h hour (s) AU animal unit
i.m. intramuscular (..ly) IU international units
i.v. intravenous (..ly) vs versus
J joule (s) xg gravidity
kg kilogram (s)
The full term for which an abbreviation stands should
km kilometer (s) precede its first use in the text.
L liter (s)
log decimal logarithm 18. Scientific names and other Latin terms should be
written in italics.
Mcal mega calorie (s)
XIII
La Revista Mexicana de Ciencias Pecuarias
Expresa sus más sinceras condolencias por el sentido fallecimiento del
Dr. Raymundo Martínez Peña
Nació el 20 de septiembre de 1939 en Nogales, Veracruz; se desempeñó

como Editor en Jefe de la Revista Técnica Pecuaria en México durante el
periodo de 1971 a 1985.
Falleció el 20 de marzo de 2023.
Descanse en paz.
XIV
https://doi.org/10.22319/rmcp.v14i2.5657
Article
Production of serum antibodies in response to vaccination against

infectious bovine rhinotracheitis and bovine viral diarrhea viruses with a
commercial vaccine
Jorge Víctor Rosete Fernández a
Guadalupe Asunción Socci Escatell b
Abraham Fragoso Islas a
Sara Olazarán Jenkins a
Ángel Ríos Utrera c*
a
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Sitio
Experimental Las Margaritas. Km. 9.5 carretera Hueytamalco-Tenampulco, Hueytamalco,
Puebla, México.
b
INIFAP. CENID Salud Animal e Inocuidad. Ciudad de México, México
c
Universidad Veracruzana. Facultad de Medicina Veterinaria y Zootecnia. Veracruz,
Veracruz, México.
*Corresponding author: ariosu@hotmail.com
Abstract:
The objective was to determine the prevalence of serum antibodies (PSA) in response to
vaccination against infectious bovine rhinotracheitis (IBR) and bovine viral diarrhea (BVD)
viruses in dairy cows under subtropical conditions. A commercial polyvalent vaccine with
inactivated BVD virus and modified active viruses of IBR, parainfluenza 3 and bovine
respiratory syndrome was used. Two groups were formed: vaccinated (VEG) and
unvaccinated (UEG) experimental group, which were homogeneous in PSA against IBR
and BVD before vaccination. VEG was immunized on d 0 and d 30 after the first
260
Rev Mex Cienc Pecu 2023;14(2):260-276
vaccination (booster vaccine). To detect antibodies, serum samples were collected 30 d

after the first and second vaccinations. Serum antibodies against IBR and BVD were
determined by the ELISA test. The average PSA against IBR and BVD before vaccination
was 16 (18 % in VEG vs 14 % in UEG; P>0.05) and 8 % (10 % in VEG vs 6 % in UEG;
P>0.05), respectively. The first and second vaccinations against IBR induced the formation
of antibodies 30 d after their application; with the first vaccination, PSA in vaccinated cows
was 36 percentage units higher (P<0.05) than in unvaccinated cows (58 vs 22 %) and with
the booster vaccine, PSA in vaccinated cows was 66 percentage units higher (P<0.05) than
in unvaccinated cows (94 vs 28 %). The commercial vaccine did not induce the production
of antibodies against BVD with either immunization.
Keywords: Infectious bovine rhinotracheitis, Bovine viral diarrhea, Polyvalent vaccine,

Modified active virus, Inactivated virus, Serum antibodies, Dairy cows.
Received: 06/04/2020
Accepted: 29/06/2020
Introduction
Infectious bovine rhinotracheitis (IBR), also known as infectious pustular vulvovaginitis, is

a disease that affects domestic cattle and wild ruminants and is caused by Bovine
Herpesvirus Type 1 (BHV-1), a member of the genus Varicellovirus of the family
Herpesviridae. This virus causes respiratory (virus subtype 1.1 ), genital (virus subtype 1.2)
and neurological (virus subtype 1.3) infections; however, although several subtypes have
been distinguished, there is only one antigenic type important in the reproduction of cattle,
BHV-1(1). The importance of detecting the BHV-1 type in cattle herds is that it causes a
decrease in cow productivity, due to reproductive failures, and respiratory conditions in
young calves(2). IBR can go unnoticed when the animals do not show clinical signs, but the
virus remains latent, lodged in target organs, so that if the infection is acquired via the
genitals, it replicates in the vaginal or preputial mucosa and establishes itself in the sacral
nodes. Stress due to calving, transport or handling induces the reactivation of the infection
and animals, even without signs of the disease, eliminate the virus into the environment,
acting as apparently healthy carriers, which constitute the main risk factor for the disease(1).
In Mexico, IBR has been identified in dairy herds of the highlands(3) and its relationship
with reproductive problems has been documented(4-7). In herds of the humid tropics, the
261
presence of IBR has been identified and its prevalence and incidence have been studied(8,9),
but it has not been reported whether the production of antibodies in response to vaccination
is appropriate, so it is convenient to perform this type of study in cattle of the tropics and
subtropics, to control the transmission of the disease.
Additionally, bovine viral diarrhea (BVD) has also been identified as a disease that affects
reproduction in cows, which show low fertility, estrus repetition, embryo resorption and
abortions(6,10). After birth, pneumonia, conjunctivitis and ulcers in the nose and mouth are
frequent in calves(6,11,12), so vaccination has been used to control BVD in cattle herds(13), but
one must be certain about the effectiveness of the vaccine by measuring antibodies
generated in the herd, because the recommended type of vaccine has been that of
inactivated virus, for not causing abortion in pregnant cows or heifers and not generating
vaccine infection in animals(14). In herds of the humid tropics, the presence of BVD has
been identified, studying the prevalence and incidence of this disease(9,15); however, the
production of antibodies in response to vaccination, to ensure immune protection and
establish a control mechanism, has not been determined.
The objective was to determine the prevalence of serum antibodies in response to

subcutaneous vaccination against infectious bovine rhinotracheitis (IBR) and bovine viral
diarrhea (BVD) viruses in dairy cows under subtropical conditions in Mexico.
Material and methods
Study location
The study was carried out in a dairy herd of the Las Margaritas Research Station, belonging
to the National Institute for Forestry, Agricultural and Livestock Research (INIFAP, for its
acronym in Spanish), located in the eastern region of the state of Puebla at 20° 00' 07.86'' N
and 97° 18' 19.08'' W, at 545 m asl, with an average annual temperature of 21°C and annual
rainfall of 2,500 mm.
Experimental groups
One hundred female bovines of the Brown Swiss and Holstein breeds under rotational
grazing of African Star grass (Cynodon plectostachyus), which had never been vaccinated
262
against IBR and BVD, were used. The females were divided into two experimental groups
with 50 individuals each. One group was applied a commercial vaccine; the other group
consisted of unvaccinated control females. The conformation of the two experimental
groups was similar, so that the vaccinated group had 33 lactating cows (7 pregnant and 26
open), 10 pregnant dry cows and 7 heifers, while the unvaccinated group consisted of 34
lactating cows (7 pregnant and 27 open), 9 pregnant dry cows and 7 heifers. In addition, to
homogenize the two groups in terms of prevalence of serum antibodies, they underwent a
first serological diagnosis of antibodies against IBR and BVD viruses before vaccination.
Care was taken that the body condition (1=emaciated; 5=obese) did not fall below 2.5 units.
Vaccination protocol
The vaccinated experimental group was immunized for the first time on day 0;
subsequently, it was immunized on d 30 with a booster vaccine, subcutaneously applying 2
ml of a commercial polyvalent vaccine at each immunization. However, the vaccination
protocol recommended by the laboratory consists of two subcutaneous applications of 2 ml
each with an interval of 21 d. The vaccine used in the two immunizations was from the
same batch. This consisted of two independent fractions; a lyophilized preparation of
chemically modified active strains of the viruses of IBR, parainfluenza 3 and bovine
respiratory syncytial syndrome, and a liquid preparation (diluent) of inactivated BVD virus
types 1 and 2 (cytopathic and noncytopathic strains). The viral antigens were propagated in
a cell line established by the laboratory. In addition, the vaccine contained a combination of
adjuvants, not described by the manufacturer, including Amphigen as an immune response
enhancer. Since its acquisition, the vaccine was kept at 4 °C until the time of its application.
During vaccination, it was kept in the shade in a cooler with abundant refrigerants (5 to 7
°C), according to the manufacturer’s specifications. During the development of the
experiment, no animal showed clinical signs attributable to diseases related to the vaccine
or any other disease.
Post-vaccination blood sampling and serum collection
Thirty (30) days after each immunization, blood samples were taken from the two
experimental groups to determine antibody production in response to the first vaccination
and booster vaccine. Blood samples were collected in 6 ml vacuum tubes that contained
clot-separating gel. The samples were centrifuged at 4,000 rpm for 10 min to obtain blood
263
serum. The collected sera were deposited in 6 ml polypropylene vials and then frozen at -20
°C until laboratory analysis.
Laboratory analysis
The serological diagnosis for the detection of antibodies against IBR and BVD viruses was
carried out with the CIVTEST BOVIS IBR and CIVTEST BOVIS BVD/BD P80 kits
(Laboratorios Hipra, S.A., Mexico), based on the ELISA test, whose sensitivity and
specificity is 96.3 and 99.5 %, respectively. In both serological diagnoses, the reading was
performed at an optical density of 450 nanometers, in a BioTek ELx800 spectrophotometer
(BioTek Instruments, Inc., USA).
Response variables and statistical analyses
For each disease, three response variables were analyzed: 1) Prevalence of serum
antibodies before vaccination (day 0); 2) Prevalence of serum antibodies 30 d after the first
vaccination (d 30), which was considered as the production of antibodies in response to the
first vaccination; and 3) Prevalence of serum antibodies 30 d after the second vaccination (d
60), which was considered as the production of antibodies in response to the booster
vaccine. Response variables were treated as binary variables, so antibody prevalence was
recorded as 1 when a female had serum antibodies on day 0 before vaccination, 30 d after
the first vaccination, or 30 d after the booster vaccine; otherwise, the prevalence of serum
antibodies was recorded as 0. The information was analyzed with the GENMOD procedure
(PROC GENMOD) of the SAS program, fitting a logistic regression model that included
the fixed effect of vaccination or treatment (vaccinated experimental group, unvaccinated
experimental group), in a binomial distribution and applying a logit link function. The
convergence criterion was 10-8 in the six statistical analyses. In preliminary analyses, it was
determined that the status of the female (in lactation, dry, heifer) did not affect any of the
response variables analyzed (P>0.05), so it was not included in the definitive model.
Results
The statistical significance of the treatment effect, by response variable and disease, is
shown in Table 1. Prior to vaccination, the prevalence of serum antibodies against the IBR
264
virus, as well as against the BVD virus, was similar (P>0.05) in the vaccinated and
unvaccinated experimental groups, so the two groups were homogeneous in antibodies
against IBR and BVD viruses prior to vaccination. Treatment affected (P<0.001) the
production of antibodies against the IBR virus at the first and second (booster vaccine)
vaccinations; however, contrary to what was expected, it did not affect (P>0.05) the
formation of antibodies against the BVD virus at either immunization.
Table 1: Statistical significance of treatment effect, by response variable and disease

Response variable (prevalence of antibodies)
At the booster
Disease Before vaccination At the first vaccination
vaccine
IBR 0.5850 0.0002 <0.0001
BVD 0.4588 0.1769 0.1042

IBR= infectious bovine rhinotracheitis; BVD= bovine viral diarrhea.
Table 2 presents the prevalences of serum antibodies against the IBR virus and their
standard errors and 95 % confidence intervals, before vaccination, by experimental group.
The prevalences were 18 and 14 % for the vaccinated and unvaccinated experimental
groups, respectively; the average prevalence of the two groups was 16 %.
Table 2: Prevalences (%) of serum antibodies against infectious bovine rhinotracheitis

virus and their standard errors and 95% confidence intervals, before vaccination, by
experimental group
Experimental Number of Positive Prevalence of Confidence
group animals animals antibodies interval
Vaccinated 50 9 18.0 ± 5.4 a 9.1 - 31.9
Unvaccinated 50 7 14.0 ± 4.9 a 6.3 - 27.4
Total 100 16 16.0 ± 5.5 9.7 - 25.0
a
Prevalences with the same literal are not different (P>0.05).
The prevalences of serum antibodies against the BVD virus and their standard errors and
95% confidence intervals, before vaccination, by experimental group, are shown in Table 3.
The prevalences for the vaccinated and unvaccinated experimental groups were 10 and 6 %,
respectively; the average prevalence of the two groups was 8 %.
265
Table 3: Prevalences (%) of serum antibodies against bovine viral diarrhea virus and their
standard errors and 95% confidence intervals, before vaccination, by experimental group
Experimental Number of Positive Prevalence of Confidence
group animals animals antibodies interval
Vaccinated 50 5 10.0 ± 4.2 a 4.2 - 21.9
a
Unvaccinated 50 3 6.0 ± 3.4 1.9 - 17.0
Total 100 8 8.0 ± 3.8 3.0 - 19.4
a
Table 4 shows the prevalences of serum antibodies against the IBR virus at the first and
second vaccinations. The first and second vaccinations against the IBR virus induced the
production of antibodies 30 d after their application; with the first vaccination, the
prevalence of serum antibodies in vaccinated cows was 36 percentage units higher (P<0.05)
than in unvaccinated cows (58 vs 22 %); with the booster vaccine, the prevalence of serum
antibodies in vaccinated cows was 66 percentage units higher (P<0.05) than in
unvaccinated cows (94 vs 28 %).
Table 4: Prevalences (%) of serum antibodies against infectious bovine rhinotracheitis

virus and their standard errors and 95% confidence intervals, by experimental group
First immunization Booster immunization
Experimental No. of
Positive Prevalence CI Positive Prevalence CI
group animals
44.1 - 83.0 -
a
Vaccinated 50 29 58.0 ± 7.0 70.8 47 94.0 ± 3.4 ª 98.1
12.6 - 17.3 -
Unvaccinated 50 11 22.0 ± 5.9 b 35.5 14 28.0 ± 6.4 b 41.9
CI= confidence interval.
ab
Prevalences with different literals are different (P<0.05).
The prevalences of serum antibodies against the BVD virus at the first and second
vaccinations are shown in Table 5. Antibody production in vaccinated females was not
satisfactory with either immunization; with the initial vaccine, the prevalences of serum
antibodies for the vaccinated and unvaccinated experimental groups were 14 and 6 %,
respectively; with the booster vaccine, they were 16 and 6 %, respectively.
266
Table 5: Prevalences (%) of serum antibodies against bovine viral diarrhea virus and their
standard errors and 95% confidence intervals, by experimental group
First immunization Booster immunization
Experimental No. of
Positive Prevalence CI Positive Prevalence CI
group animals
6.8 - 8.2 -
a
Vaccinated 50 7 14.0 ± 4.9 26.6 8 16.0 ± 5.2 ª 28.9
1.9 - 1.9 -
a a
Unvaccinated 50 3 6.0 ± 3.4 17.0 3 6.0 ± 3.4 17.0
a
Discussion
Infectious bovine rhinotracheitis
In the present study, the average prevalence of serum antibodies against the IBR virus was
16.0 %, which is considerable for a herd with no history of vaccination; therefore, the
animals should be included in a vaccination program to protect them from the disease and
avoid reproductive problems. The prevalence of serum antibodies against the IBR virus
observed in this study was lower than those observed in grazing cattle in the state of
Veracruz, with values of 58.6(16) and 76.3 %(17); however, it is higher than that observed
(5.3 %) in Zebu, Brown Swiss and Holstein cattle in Tizimín, Yucatán(18). These
prevalences identified in cattle kept in a tropical climate, as well as those observed in the
Mexican highlands, particularly that of the state of Hidalgo, which was 35.2 %(19), highlight
the circulation of the virus in the herd with the risk of the animals getting sick and the need
for its control in Mexico. In a literature review that summarized information from Mexican
studies published from 1975 to 2016, a prevalence of antibodies against the IBR virus of
56.4 % was estimated(20).
Outside Mexico, in dairy herds of Toca-Boyacá and Caquetá, Colombia, prevalences of

35.7(21) and 90.0 %(22), respectively, were found; in Valle de Cauca, in beef cattle, a
prevalence of 69.8 %(23) was found. In Peru and Chile, prevalences of 29.0(24), 67.6(25),
36.0(26) and 76.0 %(27) have been found. Due to its detection in these and many other
countries, the IBR virus (BHV-1) is considered one of the most widely distributed
pathogens in the world(28). Nevertheless, despite the fact that there are multiple biological
products for the immunization of animals(29), it is thought to be one of the largest generators
of economic losses in livestock production, both beef and milk. Asymptomatic cattle are
267
the most important reservoir because they can excrete the virus intermittently and transmit
it to healthy cattle(30).
With the first vaccination, the prevalence of antibodies in vaccinated cows (58 %) was
higher (P<0.05) than the prevalence of antibodies observed in unvaccinated cows (22 %).
This allows to interpret that there was production of antibodies in response to the first
vaccination against the IBR virus, but relatively mild. However, with the booster vaccine,
the prevalence of antibodies in vaccinated cows increased considerably up to 94 %, a
prevalence that was much higher (P<0.05) than that found in unvaccinated cows (28 %), so
the substantial increase in antibodies reinforces the interpretation of a favorable antibody
production with the second immunization; therefore, it can be inferred that the modified
active virus of the commercial vaccine did produce immune protection against the IBR
virus. With this vaccination protocol (initial vaccine + booster vaccine), it is considered
feasible to protect cattle against the IBR virus, particularly females, which are the ones that,
due to the disease, suffer from genital tract infections, such as infectious pustular
vulvovaginitis, metritis(8), mastitis, abortions, repetition of services, fetal infection and
anestrus(31,32).
The magnitude of antibody production observed in the present study was not achieved in a
study where an intranasal vaccine of attenuated viruses of IBR and PI3 (TSV-2) was used
in a single dose, which was poorly immunogenic, since only 33.3 % of the animals
produced antibodies 28 d after vaccination(33); it is likely that, with the intranasal route, a
second or more immunizations will be required to achieve a better immune response. In
another study where a vaccine with modified active virus was used, the percentage of
abortions in vaccinated females was 5 % and in unvaccinated 73 %(34), so it can be inferred
that the vaccine substantially prevented abortion. Something similar happened in the
present study, since no abortion was observed; nevertheless, in the cited study(34), it was not
determined if the low percentage of abortions (5 %) was due to the effect of vaccination, so
it could be due to other factors. Consequently, it seems better to use a vaccine with
modified active virus to protect against the IBR virus, especially in females of reproductive
age, since when the virus replicates within the host cells, protective immunity increases(35).
In Argentina, a study was carried out where two types of intradermal vaccines made with
inactivated BHV-1 that contained the sequence of the secreted version of glycoprotein D
were used, one made with adjuvant and the other not, which were applied with a booster at
20 and 33 d, assuming that both increased the humoral immune response; however, only
the one that had adjuvant improved the cellular immune response(36).
268
Bovine viral diarrhea
The average prevalence of serum antibodies against the BVD virus obtained in the present
study (8 %) was lower than those reported (69.0 and 60.3 %) by other authors(37,38) for
grazing cattle in the state of Veracruz. In studies conducted in Mexico with dairy cows in
the states of Hidalgo and Aguascalientes, prevalences of 32.8(7) and 48.6 %(19) were found.
In a literature review that summarized information from Mexican studies published from
1975 to 2016, a prevalence of antibodies against the BVD virus of 59.3 % was estimated(39).
Contrary to what was expected, in the present study it was evident that vaccinated females
did not satisfactorily produce antibodies against the inactivated BVD virus, since a
prevalence of serum antibodies of not less than 94 % was expected as a humoral response,
as in immunization against the IBR virus, so with the commercial vaccine evaluated, it is
not possible to ensure immunological protection in cattle, particularly in cows, which are
the ones that, due to the disease, suffer from infections that affect reproduction(6,10), and
replacement heifers, which can become infected from birth, suffering from pneumonia,
conjunctivitis and ulcers in the nose and mouth(6,11,12), they can even die due to the
infection, which very often goes undetected and undiagnosed. Therefore, although in recent
years vaccines with inactivated virus have been improved by adding potent adjuvants, the
low production of antibodies in this study in response to vaccination with inactivated virus
makes it necessary to try other control options, as it has been suggested that a good strategy
to overcome weak production of antibodies in response to vaccination with inactivated
virus is the alternation of repeated immunizations with inactivated virus vaccines and
modified active virus vaccines, or vice versa(40), as demonstrated in an experiment where a
vaccination protocol for heifers was used, which consisted of initially immunizing with
inactivated virus, four weeks later with modified active virus, and subsequently
revaccinated annually with inactivated virus, improving the immune response
considerably(41).
On the other hand, it has been reported that vaccination against BVD as the only control
measure is not sufficient to prevent the circulation of field virus in cattle(42,43,44), since the
elimination of persistently infected (PI) animals should be included as an important action
and effective vaccination strategies should be used to reduce this type of animals, and thus
control BVD more efficiently(45) after a program to detect PI animals from birth(46), as these
animals are immunotolerant to homologous non-cytopathogenic viruses(47). Therefore, the
failure to observe adequate antibody production in vaccinated animals could be due to the
fact that the experimental herd used had a significant proportion of PI animals, which does
not develop antibodies, since the immune system does not consider the virus as a foreign
agent to the organism(47). Another reason could be that the inactivated virus vaccine
269
induced short-lived antibody production, as it does not promote immunological memory

compared to active virus vaccines(48); consequently, it is very likely that antibodies induced
by the inactivated BVD virus vaccine were at undetectable levels at the time the ELISA test
was performed, considering that, in a study in which two groups of animals were
immunized with two inactivated virus vaccines, a prevalence of serum antibodies of 0 and
12.5 % was obtained after using an ELISA kit to detect antibodies against the p80 protein
of the virus (as in the present study); conversely, when an ELISA kit for antibodies against
the whole virus was used, antibodies were detected in 80 and 100 % of the animals(49).
However, the authors of the study(49) commented that there is a discrepancy in the results
obtained, since there are previous studies in which the detection of antibodies was achieved
with this ELISA kit, arguing that the p80 protein is mostly expressed during viral
replication, but replication does not occur if inactivated virus vaccine is applied. Therefore,
if the ELISA test adequately detects specific antibodies against the p80 protein of the BVD
virus, and knowing that the highest proportion of antibodies present in the serum are of the
IgG class, which are the ones that increase significantly after a natural infection or
vaccination, regardless of whether it is a vaccine of active or inactivated virus, the ELISA
test used in the present study is considered to be effective in detecting antibodies against
the BVD virus induced by the inactivated virus vaccine.
Additionally, it has been mentioned that the safety and efficacy of inactivated vaccines may
be attributable to several factors, among which are the type of strain, the inactivation
technique, the viral titer and the adjuvant used(50), so perhaps some of these factors may
also have influenced the observed antibody production. Finally, in this work it was
expected that immunization would induce the formation of antibodies in animals to
establish a “herd immunity”, which would prevent the virus from circulating in animals
and, thus, prevent the manifestation of clinical signs of the diseases, since the proportion of
infected animals was known before applying the treatments.
Conclusions and implications
The commercial polyvalent vaccine induced the production of satisfactory levels of

antibodies against the IBR virus; nevertheless, it was necessary to apply a second booster
immunization to increase the percentage of animals with serum antibodies (more than
90 %). On the contrary, this vaccine did not induce an adequate production of serum
antibodies against the BVD virus, so it is of utmost importance to find out if there are PI
animals within the herd studied. It is possible that by applying the vaccine with inactivated
virus, with modified active virus boosters, or vice versa, the antibody production in
response to the vaccination against the BVD virus is improved.
270
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276
Article
Characterization of fetal bovine serum obtained from the meat industry

for cell culture
Francisco Javier Preciado-Gutiérrez a
David Masuoka-Ito b
José Luis Barrera-Bernal b
Bryan Ivan Martín del Campo-Téllez b
Vicente Esparza-Villalpando b
Ricardo Ernesto Ramírez-Orozco c*
a
Universidad Autónoma de Aguascalientes. Centro de Ciencias de la Salud. Maestría en
Investigación Biomédica. Aguascalientes, México.
b
Universidad Autónoma de Aguascalientes. Centro de Ciencias de la Salud. Unidad Médico-
Didáctica, Departmento de Estomatología. Aguascalientes, México.
c
Universidad Autónoma de Aguascalientes. Centro de Ciencias de la Salud. Drepartamento de
Nurición. Aguascalientes, México.
* Corresponding author: dcmrero@gmail.com
Abstract:
The present work aims to characterize a fetal bovine serum (FBS) obtained from the meat
industry for use in cell culture. FBS is the most widely used supplement for cell culture
since its complex composition provides the necessary nutrients for the growth of most cells.
It is a by-product of the meat industry, and its availability and production depend mainly on
two uncontrollable external factors, climatic conditions and changes in beef consumption.
According to the strict quality features of the International Serum Industry Association
(ISIA), tests for total proteins, osmolarity, presence or absence of pathogenic biological
agents, pH, DNA concentration, biological contaminants, negative results, and cell viability
277
were performed. The characterization of the serum in the DNA and total protein
concentration tests showed significant differences. Additionally, osmolarity and pH did not
present significant differences between groups. Regarding the viability test, no
complication for cell growth was observed despite the differences found in the
characterization. The results showed that the serum obtained from the meat industry could
maintain cell cultures and allow cell proliferation compared to commercial serum.
Furthermore, if FBS is not available, some kinds of plasma can be used as a surrogate to
maintain cell cultures.
Key words: Fetal bovine serum, Supplement, Production, Sterile, International Serum
Industry Association.
Introduction
Cell culture started at the beginning of the 20th century as a method to study the behavior
of animal cells outside of the systemic variations that can occur in vivo. Therefore, cell
culture can be defined as acquiring animal cells and their propagation in vitro(1). To
preserve most of their physiological, biochemical, and genetic properties in an artificial
environment, freezing, thawing, seeding or trypsinization techniques are necessary to allow
the maintenance, survival, and multiplication of cells of specific organs(2,3).
Cell cultures are used in basic and applied research and can be classified into three types:
monolayer, suspension, and three-dimensional(4,5). Usually, cell cultures are worked in a
completely sterile environment, avoiding contaminations(6). In addition, the same sterile
conditions must be maintained with all the reagents that are involved with the culture
medium (e.g. FBS, antimycotics, among others)(7,8).
FBS is the main culture media supplement since it provides more than 1,000 nutritional
components for cells. These include amino acids, proteins, vitamins (particularly fat-
soluble vitamins such as A, D, E, and K), carbohydrates, lipids, hormones, growth factors,
minerals, and trace elements(9). In addition, serum buffers inactivate the culture medium
proteolytic enzymes, increase the average viscosity, and maintain the conditions for the
growth surface of the culture container(10,11).
278
The increased use of FBS for research, diagnostics, and pharmaceutical manufacturing have
made it a global business that represents a significant economic impact (exp. 17,724.63
Mexican pesos for a unit of 500 mL SIGMA® brand). The global availability and demand
may create opportunities for the production of the reagent(12). Mexico has 35 million heads
of livestock, of which approximately 13 million are raised on a free grazing basis, giving
the opportunity that one out of every eight cows that are sent to the slaughterhouse arrives
pregnant(13-15). In this context, Mexican serum production can be suitable for producing this
essential component for cell cultures(16,17). Because sometimes there are problems with the
acquisition of the serum due to border closure or sanitary regulation problem that prevents
the importation of potentially contaminated reagents. The purpose of this project was, as a
first stage, to characterize FBS obtained from the meat industry in Mexico and compare it
with a commercial serum according to the tests requested by the International Serum
Industry Association (ISIA) and evaluate its suitability for its use in cell culture.
This in vitro study consists of obtaining FBS for cell culture using the parameters of a
commercial serum as a control. The FBS characterization included microfiltration, pH
levels, osmolarity, total protein concentration, presence/absence of Mycoplasma sp., cell
proliferation, and DNA concentration. A commercial FBS was used as a control (F2442,
Fetal Bovine Serum, Mammalian and insect Cell Culture Tested, 17L436 from SIGMA®).
Following the guidelines of the ethics regulation for the use of animals in teaching and
research at the Autonomous University of Aguascalientes (CEADI-UAA) and the Official
Mexican Standard NOM-024-ZOO-1995, "Specifications and zoosanitary characteristics
for the transport of animals, their products and by-products, chemical, pharmaceutical,
biological and food products for use in or consumption by animals”.
Obtaining bovine blood and separation of serum from fetal bovine blood
One lot of serum was collected from FREASA (Frigorífico y Empacadora de

Aguascalientes), by trained personnel. Later, in freezing conditions -20 °C, it was
transferred to the laboratory processing. Blood was obtained by cardiac puncture
technique(18), and it was collected in sterile 30 ml Falcon® tubes and a 500 ml blood
containment bag with anticoagulant (500 ml ACD BLORECEP bag with 2.20 g trisodium
citrate, 0.80 g citric acid, 2.45 g dextrose, and pyrogen-free H2O).
The blood was centrifuged at 3,000 rpm for 5 min(19,20). Subsequently, the serum was
extracted and placed in 30 ml Falcon® tubes. Finally, serum aliquots were made in tubes,
and they were stored at -20 °C until use. When performing the blood component separation
process, two samples were obtained: serum (centrifugation of blood sample without
279
anticoagulant) and plasma (centrifugation of blood sample with anticoagulant). In short, the
difference between both is the presence of the proteins responsible for coagulation
processes(21,22). Based on the samples collected, the experimental groups were as follows:
Control group was commercial FBS (C-FBS), experimental groups were serum obtained
from the meat industry (E-FBS), and plasma obtained from the meat industry (E-Plasma).
Filtration of the FBS obtained for the elimination of cellular components
The elimination of the cellular components was carried out with the microfiltration method.
For this, 0.2 µm syringe filters were used. The same filtration process was applied to all the
groups [C-FBS (F1), E-FBS (F1), and E-Plasma (F1)], and all the samples were placed in
new sterile 2 ml tubes.
Evaluation of sterility with microbiological tests
Soy broth (BD Bioxon®, Becton Dickinson de Mexico) was used for sterility assay. The
instructions according to the manufacturer were followed. The sterility test consisted in
preparing six tubes with soy broth, to which 200 µl of unfiltered C-FBS, E-FBS, and E-
Plasma, an additional positive control (human saliva) was used. Once the tubes were
prepared, they were placed in the bacteriological oven at 37 °C for 24 h.
Assessment of the presence/absence of Mycoplasma sp.
The Mycoplasma sp. presence/absence test was performed on selective growth with
Mycoplasma sp. agar (MO660-500G from SIGMA). 100 ml of the medium was prepared
for 15 small Petri dishes. Similarly, seven boxes with sterile swabs were seeded following
the labeling, with the difference that the positive control was a scraping of the facial
epidermis. Once the boxes were prepared, they were placed in the bacteriological oven at
37 °C for 24 h.
Evaluation of pH
The pH test was carried out with reagent strips (Hydrion® 9400, Plastic pH Indicators
Strips, pH range 5.0-9.0). It is based on a colorimetric scale. The same groups previously
described were evaluated; additionally, the FBS groups were evaluated with culture media
dilution.
280
Preparation of dilution samples for total protein concentration and DNA

concentration measurements
For the presence of DNA components in the FBS, the samples dilute at a final
concentration of 1,030 µg/mL, which was a dilution factor (DF) of 32, which means that
the FBS concentration was diluted 32 times. To obtain the DF, the following calculation
was performed: DF= CI / CF; where: DF= dilution factor, CI= initial concentration, CF=
final concentration. With the DF, it was proceeded to process the corresponding test
samples.
Total protein concentration evaluation
Total protein measurement was based on Pierce's colorimetric method, and the PierceTM
BCA Protein Assay Kit (ref. 23227) from Thermoscientific® was used. Following the
protocol proposed by the manufacturer, the working reagent, the samples, and the
calibration curve were prepared. Briefly, 25 µL of each standard or unknown was pipetted
per replica (three replicates) into each well of the plate. Later, 200 µL of the working
reagent was added and incubated at 37 °C for 2 h. Finally, readings were carried out at 620
nm, in the 96-well plate reader Multiskan FC (SN 357-914771) from Thermoscientific®.
DNA concentration evaluation
To quantify the DNA concentration with Nanodrop, the Phenol-Chloroform extraction

method was carried out, which is divided into three phases:
1. Cell lysis: where the sample was centrifuged for 5 min at 270 xg, and then the
supernatant was discarded. The pellet is resuspended in 100 µL of PBS. The sample was
incubated at -80 °C for 30 min and transferred to the sonicator at 20 kilohertz and applied
in two batches of 10 sec to break the cells. Once the lysis was done, the samples were
centrifuged at 10,000 xg for 20 min. Finally, the supernatant was moved to a new sterile
Eppendorf® tube to continue with the second phase.
2. Phase separation: 250 µL of Phenol-chloroform was added to the obtained supernatant
and was passed through a continuous vortex process. They were centrifuged at 10,000 xg
for 5 min to finally observe a separation of two phases, from which, the upper part was
taken, and proceeded to next phase.
3. DNA purification: 200 µL of chloroform were added to the samples and were
centrifuged at 10,000 xg for 10 min to obtain a supernatant and transferred to a new tube.
Then, 1/10 volume of 3 molar sodium acetate and two volumes of 100% ethanol were
added to the supernatants. Subsequently, they were left to precipitate overnight at -80 °C.
After finishing, the samples were centrifugate at 10,000 xg for 30 min at 4 °C, the
supernatant was extracted, and 100 µL of ethanol at 70% was added to the remaining
281
content in the tube, and was centrifuged for 10 min at 10,000 xg (the 2 previous steps of
70 % ethanol and centrifugation were repeated 2 to 3 times to clean the genetic material).
Finally, the largest possible supernatant was removed and the remaining ethanol was
allowed to evaporate. Genetic material was resuspended in 100 µL of Milli-Q grade water
and nucleic acid readings were performed on the Thermoscientific Nanodrop 2000
spectrophotometer.
Osmolarity assessment
Osmolarity was measured on a Model 5004 Automatic Osmometer. Briefly, the protocol
consisted of connecting and calibrating the equipment, selecting the reading range to be
measured, placing 100 µL of the sample in an Eppendorf® tube, and reading.
Evaluation of cell proliferation
For the cell proliferation test, a low glucose DMEM culture medium with L-Glutamine and
Sodium Pyruvate (Biowest®) was used. In addition, the antibiotic/antifungal solution of
Sigma® (A5955) was added, and finally, it was supplemented with the commercial FBS
(the control serum) Sigma® (F2442). Three types of media were prepared for this
evaluation, commercial serum (C-FBS), serum obtained from the meat industry (E-FBS),
and the last with plasma (E-Plasma).
The cell viability test was carried out with the Abcam® brand MTT Assay Kit (Cell
Proliferation) method. The experiment was performed on 96-well plates and read on the
Thermoscientific® Multiskan FC 96-well plate reader (SN 357-914771) at 620 nm. The
hFOB 1.19 ATCC line of osteoblasts was used to measure cell viability, osteoblasts are a
type of cell without specific requirements for growth and proliferation, although standard
conditions for cell culture were performed in this case: 37 °C in an atmosphere of 5% CO2,
95 % air(9). Measurements at 3, 7, 14, and 21 d were performed to observe how the cells
were growing in the plate from an initial seeding of 1,000 cells.
Statistical analysis
For the statistical analysis, means, medians, and standard deviation were calculated, the
normality distribution of data was evaluated with Q-Q plot, and homogeneity of variance
with Levene’s test, if the statistical assumption were made a two-tailed or one-way
ANOVA test was performed with the statistical program R version 4.0.3. To evaluate the
proliferation cell kinetic, a Likelihood ratio (LR) test was used to compare the kinetic
curves, a confidence level of 95 % was considered.
282
Results
A general characterization was performed on E-FBS to verify the serum status. Table 1
shows the results obtained. The sample was taken from a fetus; then some parameters may
be outside the reference limits established for a fully grown organism.
Table 1: Characteristics of the E-FBS obtained from the meat industry

Analyte Value Reference value
Color Ambar Ambar
Glucose, mg. dL 37 80-120 mg. dL
Creatinine, mg. dL 2.73 1.2-1.9 mg. dL
Uric acid, mg. dL 2.0 1.21-3.47 mg. dL
Phosphorus, mg. dL 10.5 2.5-5.0 mg. dL
Calcium, mg. dL 16 12.0-14.0 mg. dL
Bilirubin, mg. dL 0.8 0.2-0.5 mg. dL
TGP, U/L 8 11-40 U/L
ALP, U/L 280 86-285 U/L
Total proteins, g. dL 3.81 6.0-8.0 g. dL
Albumin, g. dL 2.63 2.5-3.5 g. dL
Serum iron, Ug. dL 169 37-170 Ug. dL
Serum amylase, U/L 49 30-110 U/L
Globulin, g. dL 1.18 2.5-4.5 g. dL
Atherogenic index 3.6 0-5
Summary of the FBS properties obtained from the meat industry. The test was carried out by an external
company and served as support for the results obtained. TGP= alanin-aminotrasnferase; ALP= alkaline
phosphatase.
Evaluation of sterility with microbiological tests
For microbiological tests, two measurements were made. One at the time the serum was
obtained and 1 mo after stored at -20 °C. Figure 1A shows the absence of microbiological
growth (translucent) in the experimental groups after 24 h at 37 °C incubation period. The
same negative result was obtained after one month of storage, in the saliva group turbid
appearance characteristic of microbiological growth can be observed. The Mycoplasma sp.
evaluation is shown in Figure 1B (E-FBS), Figure 1C (E-Plasma), and Figure 1D (C-FBS),
since from the previous evaluation the presence of Mycoplasma sp. Is not found in the agar,
except for the skin group (Figure 1E).
283
Figure 1: Microbiological tests
The absence of microbial growth for experimental groups (A), the absence of Mycoplasma sp. growth for
experimental groups (B, C and D), Mycoplasma sp. growth from skin sample (E).
DNA concentration
According to the data obtained (Table 2), in the DNA concentration, there were significant
differences between groups and the filtered or non-filtered factor (P<0.05). In the filtering
section, there are statistical differences between groups. Nevertheless, in the non-filtered
area, there are no differences between the E-Plasma and C-FBS groups, but statistically
significant differences were observed between the E-Plasma/E-FBS and E-FBS/C-FBS
groups.
Total protein concentration
Table 2 shows how the total protein concentration presents significant differences between
the groups (P<0.05). In the filtered section, differences between C-FBS/E-FBS and E-
Plasma/C-FBS can be seen. However, in the non-filtered area, no differences can be
observed between the E-Plasma/E-FBS groups, but they are observed between E-Plasma/C-
FBS and E-FBS/C-FBS groups.
pH evaluation
The pH obtained from the groups before filtering was 7.0 (C-FBS and E-FBS) except E-
Plasma, which presented a pH of 6.0. The pH of the 3 groups after filtering was 7.0.
284
Osmolarity
The obtained osmolarity values were similar between the three experimental groups
regardless of the filtration factor (Table 2). Therefore, no significant differences were seen
at P>0.05.
Table 2: Summary of statistical data

Variable Exper. Mean ± SD ANOVA 1 vs 1 Cohen Tukey
evaluation Groups P-value comparison d P-value
C-FBS 9.46 ± 0.358
C-FBS vs 1.3400 0.21777
E-FBS 2.88 ± 0.303 E-FBS
E-Plasma 9.60 ± 0.860
DNA E-FBS vs 3.0400 0.00434
Concentratio C-FBS(F1) 6.16 ± 0.167 <0.00001 E-Plasma
n
(ng/µL) E-FBS(F1) 7.50 ± 2.04
E-Plasma vs -1.7000 0.10154
E-Plasma 4.46 ± 0.288 C-FBS
(F1)
C-FBS 0.744 ± 0.0322 C-FBS vs - 0.00172
E-FBS 0.06688
E-FBS 0.677 ± 0.0238 <0.001 E-FBS vs - 0.96217
E-Plasma 0.00390
Total protein E-Plasma 0.674 ± 0.00494 E-Plasma vs - 0.00108
concentration C-FBS 0.07078
(g/dL) C-FBS(F1) 0.666 ± 0.0324 C-FBS(F1) vs - 0.00414
E-FBS(F1) 0.06418
E-FBS(F1) 0.602 ± 0.0278 E-FBS(F1) vs 0.06956 0.00244
E-Plasma (F1)
E-Plasma 0.671 ± 0.00729 <0.001 E-Plasma (F1) 0.00538 0.93867
(F1) vs C-FBS(F1)
C-FBS 253 ± 14.7
C-FBS vs N/A N/A
E-FBS 242 ± 6.88 E-FBS
E-Plasma 252 ± 9.92 N/A
E-FBS vs N/A
Osmolarity C-FBS(F1) 245 ± 2.07 0.48 E-Plasma
(mOsm/Kg
H2O) E-FBS(F1) 246 ± 3.91
E-Plasma vs N/A N/A
E-Plasma 257 ± 26.5 C-FBS
(F1)
C-FBS 0.131 ± 0.00829
C-FBS vs N/A N/A
E-FBS
E-FBS 0.124 ± 0.00593
0.08 E-FBS vs N/A N/A
Cell viability E-Plasma
285
(%) E-Plasma 0.121 ± 0.00439

E-Plasma vs N/A N/A
C-FBS
All the comparisons were made with independent methods; One-way or two-way ANOVA analysis was
applied independently for each variable according to the relationship of the factors: filtered or unfiltered and
the experimental groups. The 1vs1 comparisons were made with the posthoc Tukey method. C-FBS: Control-
FBS; E-FBS: Experimental-FBS; E-Plasma: Experimental-Plasma sd; Standard deviation. Significance: P-
value<0.05.
Cell viability
Cell growth among the experimental groups was evaluated from the percentage of cell
viability through time (Figure 2). The comparison was made using a Likelihood Ratio Test
with a value of LR=0.031124 and P=0.999. No significant differences were observed
between groups.
Figure 2: Cell viability curve
The graph shows how the cell growth trend at 21 d is positive. In addition, it can be seen how the
percentage of viability of the cells at 3, 7, and 14 d is similar between the groups.
Discussion
The overall aim of the article was to the FBS characterization obtained from the meat
industry for use in cell culture and to compare it with a commercialized FBS. As is already
known, Mexican livestock is suitable for obtaining and producing this reagent because
Mexico has been free of the main quarantine diseases since its appointment in 2016 to
date(17,23).
286
To obtain the FBS, the blood to produce this reagent must be from fetuses, and it is
obtained by cardiac puncture to avoid risks of contamination and must be taken by highly
trained personnel(18). The main reason for obtaining the sample from that gestational stage
is because the fetus is protected by the “placental barrier”, which is a natural protection that
defends the developing organism from any infection(24). Another aspect that helps the
defense of the fetus is that at the time of fertilization and the ovum reaches the stage of
implantation in the womb, most of the signaling processes to be carried out are
inflammation, which means that immune cells are always present at all times, increasing
defenses against contaminants, confirming that obtaining serum from fetuses for cell
culture is indeed the best option(25).
One of the limitations of the present study was the tests chosen for this initial stage of
serum characterization. There were based on the Certificate of Analysis Guidance (CoA)
described for ISIA(26), however, the virus testing (Cytopathic, hemadsorbing, and bovine
viral diarrhea virus) IgG and GGT were not performed at this stage because the main
objective was the performance testing.
The osmolarity test measures the concentration of nutrient solutes in the reagent.
Maintaining adequate levels (within standards) is of vital importance since presenting
adequate levels allows the cells of the cell culture to grow quickly and avoid morphological
malformations due to a lack of nutrients. Ryan JM.(27) used different concentrations of FBS
to verify the useful life of cultured chicken cells (5%, 10%, 20%, and 30%), and Kwon, et
al(28), verified the effect of the concentration of FBS on the efficiency of cell
reprogramming for the generation of pluripotent stem cells (5%, 10%, 20%, and 30%), with
the result that a good concentration of FBS allows for good proliferation and maintenance
performance of the cells of interest. For this reason, it is pertinent to have a serum with its
established concentration (260 - 340 mOsm / Kg H2O) since a low or no FBS
supplementation would jeopardize the cells.
Focusing on the final cell viability test, the three groups at 21 d of growth maintain the
same viability percentage. In this context, it can be assumed that plasma could be used to
supplement culture media. Although, it may not be correct in all cases because of the
presence of fibrinogen. Fibrinogen is a zymogen, which is an inactive enzyme precursor
that participates mainly in the coagulation cascade. Its primary function is the formation of
fibrin for the creation of the clot that "covers" the injured blood vessel. It is also known as a
proenzyme that does not require a protein activator, but simply with a biochemical change
in the environment, can be activated(29,30). Fibrinogen not only has this function, it is also
involved in processes such as platelet distribution, adhesion and signaling, the proliferation
of fibroblasts and endothelial cells, healing, and inflammatory response; In addition, it is
capable of binding to proteins such as fibronectin (facilitating its incorporation into the
extracellular matrix), growth factors for fibroblasts (FDF-2, β-FGF) and vascular
287
endothelium (VEDF) that stimulate angiogenesis, and interleukin-1β that intervenes in

inflammation, therefore, it is a proenzyme with a wide field of action(24,25). That is why
serum is still the best option to supplement culture media compared to plasma.
Finally, significant differences were found in some of the characterization tests carried out
but not in the viability test. However, none of these data has a significant effect when
supplementing the culture medium with serum obtained from the meat industry (E-FBS).
Therefore, although the project is only focused on the first stage of characterization, it can
be suggested the serum obtained can be used to supplement cell cultures. It is intended to
carry out a second stage of the project in which the missing characterization tests are
carried out, among which are the measurement of endotoxins, hemoglobin, hormones, and
vitamins, additionally, the evaluation in different cell lines it is necessary to verify the
performance of the E-FBS.
Conclusion and implications
According to the results, it can be concluded that the serum obtained from the meat industry
did not show significant differences in the aspects of cell maintenance and proliferation
compared to the commercial serum. Although plasma is not the commonly used
supplement for cell cultures, if FBS is not available, plasma could be used in emergency
cases as a substitute to maintain certain cell cultures. Although the FBS obtained from the
meat industry has not undergone all the requested standard tests, since it is in the first stage
of characterization, it can be suggested that the serum produced with Mexican livestock can
be used to supplement cell cultures.
Acknowledgments
This work was elaborated thanks to the facilities of the Research and Development
Laboratory in Molecular Diagnosis and Biomaterials of the Universidad Autónoma de
Aguascalientes; we also want to thank the Consejo Nacional de Ciencia y Tecnología
“National Council of Science and Technology” (CONACYT).
Conflict of interest
The authors declare no conflict of interest.
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291
Article
Javier Sosa-Rueda a
Fabiola Villarauz b
Vanihamin Domínguez-Meléndez c
Ida Soto-Rodríguez b
Fernando C. López-Fentanes b
David I. Martínez-Herrera a
Álvaro Peniche-Cardeña a
Francisco Cen-Pacheco b*
a
Universidad Veracruzana. Facultad de Medicina Veterinaria y Zootecnia. Miguel Ángel de
Quevedo s/n, 91710, Veracruz, Veracruz, México.
b
Universidad Veracruzana. Facultad de Bioanálisis. Iturbide s/n, 91700, Veracruz, Veracruz,
México.
c
Universidad Veracruzana. Centro de Estudios y Servicios en Salud, Veracruz, México.
*
Corresponding author: fcen@uv.mx
Abstract:
This work determined the acaricidal effect of 18 Mexican plants against Rhipicephalus
microplus. The results of the larvicidal assay revealed that 5 methanolic extracts produced
high activity (86-100 % mortality), 3 extracts exhibited relatively high activity (71-85 %
mortality), 2 extracts displayed moderate activity (56-70 % mortality), 2 extracts presented
low activity (31-55 % mortality) and 6 extracts showed non-significant acaricidal activity (0-
30 % mortality). Extracts inducing >56 % mortality were subsequently assayed against
292
engorged ticks of R. microplus by adult immersion test at a concentration of 5.0% w/v. In

general terms, the results on larvae and adult ticks indicated that the methanolic extracts of
Annona globiflora, Annona scleroderma, Litchi chinensis and Azadirachta indica showed
the greatest activities. The crude extract of A. indica was subjected to chromatographic
purification, which has led to the isolation of 3-O-butyl-(-)-epigallocatechin (1), 3-O-butyl-
(-)-epicatechin (2), (-)-epigallocatechin (3), (+)-gallocatechin (4), (-)-epicatechin (5), β-
sitosterol (6), stigmasterol (7), stigmasterol glucoside (8), triolein (9), azadirachtin A (10),
and the octadecanoic acid-tetrahydrofuran-3,4-vinyl ester (11). The isolated compounds'
chemical structures were identified by the interpretation of NMR and HRESI-MS
spectroscopic data. The isolated compounds were assayed against engorged ticks of R.
microplus at a concentration of 6 mM. Based on the results obtained, it was concluded that
3-O-butyl-(-)-epigallocatechin (1), 3-O-butyl-(-)-epicatechin (2), azadirachtin A (10), and
octadecanoic acid-tetrahydrofuran-3,4-vinyl ester (11) show the highest effectiveness.
Keywords: Mexican plants, Acaricidal screening, Azadirachta indica, Ixodicide metabolites.
Introduction
Rhipicephalus (Boophilus) microplus (R. microplus), is distributed in tropical and subtropical

latitudes worldwide and is responsible for severe economic losses in livestock farming in
countries in America, Africa, Asia, and Australia(1). In México, R. microplus is widely
distributed, infesting several host species(2). This ectoparasite produces smaller weight gain
and reduction of milk production, anemia, hide damage, and even mortalities in cattle. It is
also an important vector of pathogens such as Babesia bovis, B. bigemina and Anaplasma
marginale(3). Currently, tick control mostly consists of the use of acaricides, tick-resistant
animals, anti-tick vaccines, and biological control(4). Among them, acaricides are the most
common control method since it offers quick and cost-effective suppression of tick
populations. However, the indiscriminate use of chemical substances (synthetic pyrethroids,
organophosphates, macrocyclic lactones and amidines) to control tick plagues has promoted
multi-resistance in this ectoparasite(5-7). Further, the accumulation of pesticides in animal
tissues causes human exposure through the consumption of derived animal products(8).
Therefore, it is necessary the development new substances with novel mechanisms of action
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and/or less toxic than those currently used. In this sense, natural products emerge as an eco-
biological alternative for tick control due to their low costs and toxicity(9-12).
The chemistry of natural products has been one of the sources of inspiration for the
development of new drugs over many decades, either directly as drugs or as lead structures
that were further optimized by medicinal chemists(13,14). Within the wide range of natural
sources, traditional herbal medicine has been one of the most prolific producers of bioactive
metabolites. Indeed, phytochemical studies of medicinal plants have led to the development
of over 50 % of the active pharmaceutical ingredients that are currently marketed(15-17).
Azadirachta indica A. Juss, commonly known as the “neem” tree in Latin America, is a well-
known curative plant with a wide range of pharmacological activities and beneficial health
properties(18-20). A variety of metabolites with high structural diversity have been isolated
from the “neem” tree, some of which have shown important bioactivities, like antioxidant,
cytotoxic, bactericide or larvicide effects(21-24). This study evaluates the acaricidal activity of
18 extracts from Mexican plants, against larvae and engorged ticks of R. microplus.
Additionally, a phytochemical study into the bark of A. indica, collected in spring 2018 in
Veracruz state (México), led to the isolation of 11 natural metabolites from the “neem” tree.
Their structures were determined based on detailed spectroscopic studies. The isolated
compounds were evaluated against engorged female ticks.
Plant material
Eighteen (18) plant species were collected in spring 2018 in the region of Sotavento of
Veracruz State, Mexico. Taxonomists of the Institute for Biological Research (CIB) of
Veracruz University identified the plants (Table 1). After collection, the plant material was
dried at room temperature for two weeks and then triturated.
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Table 1: Plant species studied
Plants Part used Voucher Geographic coordinates

Annona globiflora Seeds 10750UV 19º 42’ 42.6’’ N, 96º 28’ 7.2’’ W
Annona scleroderma Seeds 23839UV 19º 9’ 39.4’’ N, 96º 13’ 5.7’’ W
Litchi chinensis Seeds 23764UV 19º 10’ 26.8’’ N, 96º, 13’ 27.3’’ W
Inga jinicuil Seeds 11859UV 19º 29’ 15.8’’ N, 96º 5’ 26.5’’ W
Ensete ventricosum Seeds 11244UV 18º 17’ 15.1’’ N, 95º 18’ 51.3’’ W
Azadirachta indica Bark 23765UV 19º 10’ 26.4’’ N, 96º 13’ 22.8’’ W
Salvia hispanica Seeds 11164UV 18º 38’ 3’’ N, 97º 0’ 45’’ W
Sterculia apetala Seeds 11165UV 18º 17’ 15.1’’ N, 95º 18’ 51.3’’ W
Citrus sinensis Rind 10500UV 18º 39’ 39’’ N, 96º 56’ 18’’ W
Citrus paradisi Rind 23762UV 19º, 10’, 26.6’’ N, 96º 13’ 27.3’’ W
Citrus latifolia Rind 23766UV 19º, 10’ 30.5’’ N, 96º 13’ 28.8’’ W
Citrus medica Rind 23768UV 19º 33’ 50.4’’ N, 96º 56’ 27.6’’ W
Mimosa pudica Whole plant 12879UV 18º 3’ 50.5’’ N, 94º 22’ 13.4’’ W
Heliotropium indicum Whole plant 21157UV 18º 34’ N, 95º 4’ W
Momordica charantia Whole plant 12161UV 19º 52’ 47’’ N, 96º 67’ 86’’ W
Tagetes erecta Whole plant 20090UV 19º 43’ 50.3’’ N, 96º 43’ 40.7’’ W
Tridax procumbens L. Whole plant 21537UV 19º 18’ 29’’ N, 96º 22’ 14’’ W
Randia aculeata Roots 20326UV 19.3º 41’ 10.1’’ N, 96.3º 8’ 36.9’’ W
Plant extraction
Plant material was extracted four times by cold maceration for 3 h at room temperature using
1 L of methanol for 300 g of plant material, each time. Afterward, the solvent was removed
in vacuum in a rotary evaporator (Buchi Rotavapor R-3, Switzerland).
Isolation procedure of the compounds
The methanolic extract of A. indica (117 g, 2.8 % dry weight) was fractionated by liquid-
liquid extraction following the Kupchan method. Briefly, the extract was suspended in a
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methanol/water mixture (MeOH/H2O; 1 L, 1:1) and was successively separated with hexane
(Hex; 3 × 1 L), dichloromethane (DCM; 3 × 1 L), and ethyl acetate (EtOAc; 3 × 1 L) (Sigma-
Aldrich, St. Louis Mo., USA) to obtain four fractions of increasing polarity(25-27). The
dichloromethane fraction (21.0 g) was subjected to silica gel 60 column chromatography (5
cm of internal diameter and 35 cm of length) (Merck, Darmstadt, Germany) with Hex:EtOAc
(6:4), and, subsequently, in a medium-pressure Lobar LiChroprep-Si60 column (Merck,
Darmstadt, Germany) with Hex:Acetone (7:3) as the eluent. The fractions collected between
6-11 min and 108-175 min were pooled together (3B and 3F, 44 and 58 mg, respectively).
Final purification was performed on an HPLC with a µ-Porasil column (Waters, Wexford,
Ireland), using Hex/DCM/Acetone (5:2:3) as eluent to afford β-sitosterol (6) (32.9 mg)
stigmasterol (7) (39.7 mg), stigmasterol glucoside (8) (9.9 mg) and azadirachtin A (10) (28.4
mg) in fraction 3F. On the other hand, for fraction 3B, Hex/EtOAc (9:1) was used to yield
trilinolein (9) (9.3 mg) and octadecanoic acid-tetrahydrofuran-3,4-vinyl ester (11) (21.7 mg).
The ethyl acetate fraction (56.4 g) was chromatographed using Sephadex LH-20 column (5
× 35 cm; eluent: MeOH) (Merck, Darmstadt, Germany). The second fraction (4B 120 mg)
was purified on an HPLC with a µ-BondapakTM C-18 (1.9 × 15 cm) (Waters, Wexford,
Ireland) column using MeOH/H2O (2:3) to yield 3-O-butyl-(-)-epigallocatechin (1) (23.3 mg)
and 3-O-butyl-(-)-epicatechin (2) (24.7 mg). The 4D fraction (2.0 g) was processed by
medium-pressure chromatography, using Lobar LiChroprep-RP18 (eluent: MeOH/H2O
(7:3)). Finally, HPLC was performed on a µ-BondapakTM C-18 column using MeOH/H2O
(2:3) to provide three pure compounds (-)-epigallocatechin (3) (34.8 mg), (+)-gallocatechin
(4) (27.9 mg) and (-)-epicatechin (5) (25.7 mg) (Figures 1 and 2).
Figure 1: Isolation procedure followed for compounds 1-11
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Figure 2: Metabolites 1-11 from Azadirachta indica. In pink: the introduction of an O-

butyl ether group, at C-2, in flavonoids 1 and 2 showed the best acaricidal activities
General experimental chemical procedures
NMR spectroscopy was performed on Bruker AVANCE 600 MHz instruments using CDCl3
and CD3OD at 298 K. The NMR data were acquired using standard pulse sequences. NMR
data were processed using MestReNova software (v 11.01, Santiago de Compostela, Spain).
HPLC separations were carried out with the HPLC Breeze 2 system (Waters, Wexford,
Ireland) equipped with a UV detector. All of the solvents used were HPLC-grade. HPLC was
297
monitored by thin layer chromatographic (TLC), performed on AL Si gel Merck 60 F254

(Kenilworth, NJ, USA). TLC plates were visualized by UV light (365 nm) and
phosphomolybdic acid solution of 10 % wt in ethanol.
Tick collection
One thousand engorged females of R. microplus were collected from six naturally infested
cattle on a farm located in the municipality of Puente Nacional, Veracruz, México (19°19’N;
96°28’W). These cattle had not been treated with acaricides for 45 d before the collection of
ticks. Seven hundred (700) engorged females were used in the adult immersion test, and three
hundred were placed in Petri dishes and incubated at 28 °C and 80 % relative humidity for
two weeks to provide optimal conditions for oviposition. After, the eggs were mixed and
transferred to twenty 10-mL glass vials closed with a swab of cotton for approximately 30 d,
at 28 °C and 80 % relative humidity(28).
Preparation of control solutions
For the positive control, the commercial compound Taktic® (12.5%; Intervet, Mexico) was
used to prepare a discriminatory dose of amitraz at 0.0002%. For the negative control, an
aqueous solution with 1.0% ethanol and 0.02% Triton X-100 was prepared(29). In both cases,
the final volume used was 750 μL.
Concentration of the tested samples
Methanolic extracts of the 18 plants were assayed at the concentration of 5.0% w/v (37.5 mg
in 750 μL for larvae assay and 250 mg in 5 mL for adult assay). For the bio-guided
purification of A. indica were used different concentrations (≤ 5.0% w/v), depending on the
degree of purity of the fraction to be tested. On the other hand, compounds 1-11 were tested
at a concentration of 6 mM. The final volume used in the larval immersion test was 750 μL,
whereas in the adult immersion test it was 5 mL(24).
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Larval immersion test
Approximately 100 larvae of R. microplus were immersed for 10 min in 750 μL of each
dilution to be tested using paintbrushes. Then, they were placed on filter paper envelopes and
kept at 28 °C and 80 % relative humidity for 24 h. The control group was treated with 1.0%
ethanol and 0.02% Triton X-100 aqueous solution. After 24 h, dead and alive larvae were
registered, and mortality percentages were calculated(30). One experiment with three
replicates was used for each test.
Adult immersion test
Ten engorged female ticks with homogeneous weights (approximately 200 ± 20 mg each)
were immersed for 10 min in 5 mL final volume of each solution to be tested and then dried
on Whatman nº 1 filter paper. The ticks were placed in Petri dishes and kept at 28 °C and
80 % relative humidity for 24 h. After a week, the numbers of live or dead engorged females
were recorded and mortality percentages calculated. One experiment with three replicates
was used for each test as well as for the negative control (1.0% ethanol and 0.02% Triton X-
100 solution)(30,31).
Determination of acaricidal activity
The larval mortality was corrected using Abbott’s formula as recommended by the FAO(32).
Thus, the corrected mortality (CM) was calculated as follows: CM = [(test mortality % −
control mortality %)/100 − control mortality %] × 100, if the mortality in control was above
7 %, the bioassay test was annulled and repeated.
In this study, acaricidal activity of the extracts was classified as follows: High: (86-100 %
mortality); relatively high: (71-85 % mortality); moderate: (56-70 % mortality); low: (31-
55 % mortality); and non-significant: (0-30 % mortality)(33-35).
299
Results
To explore the acaricidal potential of 18 Mexican plants, initially, their methanolic extracts
were assessed for larvicidal activity at a cut-off concentration of 5.0 % w/v. The results
revealed that 5 extracts produced high activity (86-100 % mortality), 3 extracts exhibited
relatively high activity (71-85 % mortality), 2 extracts displayed moderate activity (56-70 %
mortality), 2 extracts presented low activity (31-55 % mortality) and 6 extracts showed non-
significant acaricidal activity (0-30 % mortality). Extracts inducing >56 % mortality were
subsequently assayed against engorged ticks of R. microplus by adult immersion test at a
concentration of 5.0 % w/v. These results showed that Annona globiflora, Annona
scleroderma, Litchi chinensis and Azadirachta indica have the best activities in both larvae
and adult ticks (Table 2). A bio-guided purification was carried out to identify the active
principles responsible for the acaricidal activity in the methanolic extract of A. indica.
Initially, the results of the larvicidal assay of the Kupchan fractions, Hex, DCM, EtOAc, and
MeOH/H2O, showed that the activity was predominantly found in the DCM and EtOAc
fractions. Thus, a chromatographic study was performed on them to identify the active
compounds. Six metabolites were found from the dichloromethane fraction: β-sitosterol (6),
stigmasterol (7), stigmasterol glucoside (8), trilinolein (9), azadirachtin A (10), and the
octadecanoic acid-tetrahydrofuran-3,4-vinyl ester (11). In addition, 3-O-butyl-(-)-
epigallocatechin (1), 3-O-butyl-(-)-epicatechin (2), (-)-epigallocatechin (3), (+)-
gallocatechin (4), (-)-epicatechin (5) were isolated from the ethyl acetate fraction (Figure 2).
Table 2: Acaricidal effect of the extracts from Mexican plants at a cut-off concentration of
5.0% w/v
Larva mortality Adult mortality
Plant Key
(%) (%)
Annona globiflora AGS 100 100
Annona scleroderma ASS 100 100
Litchi chinensis LCS 91.6 ± 2.2 66.7 ± 5.8
Inga jinicuil IJS 89.3 ± 4.8 26.7 ± 15.3
Ensete ventricosum EVS 41.5 ± 13.7 NT
Azadirachta indica AIC 84.9 ± 4.3 53.3 ± 11.5
Salvia hispanica SHS 32.7 ± 12.4 NT
Sterculia apetala SAS 1.4 ± 2.5 NT
Citrus sinensis CSR 74.3 ± 7.9 10 ± 10
Citrus paradisi CPR 84.4 ± 7.1 3.3 ± 5.8
300
Citrus latifolia CLR 89.3 ± 4.2 16.7 ± 11.5

Citrus medica CMR 56.7 ± 9.2 6.7 ± 5.8
Mimosa pudica MPW 58.5 ± 9.1 0
Heliotropium indicum HIW 0 NT
Momordica charantia MCW 3.5 ± 6.1 NT
Tagetes erecta TEW 0.3 ± 0.4 NT
Tridax procumbens L. TPW 0 NT
Randia aculeata RAR 1.1 ± 1.0 NT
Amitraz1 - 63.2 ± 5.5 56.7 ± 5.8
± Standard deviation. 1Tested at the concentration of 0.0002%. NT: not tested.
Regarding the acaricidal assay of the isolated compounds on engorged female ticks, the
results indicated that the flavonoids 3-O-butyl-(-)-epigallocatechin (1) and 3-O-butyl-(-)-
epicatechin (2) caused mortality (36.7 and 43.3 %, respectively), while the other evaluated
flavonoids, 3-5, showed no activity at the concentration of 6 mM. In the same way, the
compounds azadirachtin A (10) (66.7 %) and the octadecanoic acid-tetrahydrofuran-3,4-
vinyl ester (11) (46.7%) show good efficacy (Table 3). Finally, it was observed that
compounds 3-7 did not induce adulticidal activity. Compounds 8 and 9 could not be tested,
due to a lack of sample material.
Table 3: Acaricidal activity of compounds 1-11

Adult mortality Adult mortality
Compound Compound
(%) (%)
1 36.7 ± 5.8 7 0
2 43.3 ± 5.8 8 NT
3 0 9 NT
4 0 10 66.7 ± 5.8
5 0 11 46.7 ± 5.8
6 0 Amitraz1 56.7 ± 5.8
± Standard deviation. Compounds were tested at a concentration of 6 mM.
1
Tested at the concentration of 0.0002%. NT= not tested.
301
Discussion
Identifying the trusted range of the assay is essential to the conclusions of this investigation,
especially when the resistance of ticks does not depend only on intrinsic factors such as their
genetics and physiology, but also on the biotic and abiotic factors at the time of collection.
That is why the standard error (SE) analysis (supplementary material) was performed, in
which a variation ≤2.8 % was observed in the five extracts that generate high mortality (86-
100 %). However, the SE becomes inversely proportional to mortality, that is, the less activity
the standard error increases. The SE analysis suggests that the variation in future evaluations
of the extracts of the 18 plants examined will be less than 5% at a concentration with high
mortality (Figure 3).
Figure 3. Standard error of the larvicidal activity of the most active methanolic extracts
In general terms, the combined results of the adulticidal and larvicidal activity indicated that
the methanolic extracts of Annona globiflora, Annona scleroderma, Litchi chinensis and
Azadirachta indica have the best effectiveness. Annona genus has become a proliﬁc producer
of interesting compounds with great biological activity(36,37). The acaricidal activities of the
extracts of A. globiflora and A. scleroderma are possibly related to the presence of
acetogenins, the main chemical constituents of the Annonaceae family, which have been
found to have potent pesticidal activity against a variety of arthropods(38). These results agree
with the larvicidal activity reported for ethanolic extracts of the seeds of A. squamosa against
R. microplus(39). In the case of the seeds of L. chinensis, previous studies have shown that
this plant displays significant antimicrobial, antioxidant, and anticancer activities(40), though
this is the first report of its acaricidal activity. A large number of compounds with significant
structural and pharmacological diversity have been identified from A. indica. However, the
acaricidal activity of the “neem” tree has been attributed to the presence of azadirachtin A
302
(10), although there are some reports that contradict the above(41,42). Eleven major compounds
were isolated from the stem bark of Azadirachta indica, among which is the azadirachtin A
(10). The acaricidal assay by adult immersion test of these compounds revealed that, as well
as azadirachtin A (10), some other compounds showed an acaricidal effect, such as 3-O-
butyl-(-)-epigallocatechin (1), 3-O-butyl-(-)-epicatechin (2) and octadecanoic acid-
tetrahydrofuran-3,4-vinyl ester (11).
About flavonoids 1-5, the results indicated that only the flavonoids 3-O-butyl-(-)-
epigallocatechin (1) and 3-O-butyl-(-)-epicatechin (2) caused mortality (36.7 and 43.3%,
respectively) at a concentration of 6 mM. Based on these results, it seems clear that the butyl
ether fragment is essential for the activity of 1 and 2. Such butyl ethers fragments lead to an
increase in the liposolubility properties of these metabolites concerning structurally-related
compounds 3-5. In effect, lipophilicity is one of the most important physical properties in
drug discovery, since it intervenes in the pharmacodynamics, pharmacokinetics, and toxicity
of many compounds(43). For example, Echeverría et al(44) and Cen-Pacheco et al(24) reported
that the bactericide and acaricidal activity of the flavonoids is associated with a narrow range
of lipophilicity values (LogP between 1.5 and 3.0).
The evaluation of 18 Mexican plants against larvae and adult ticks of R. microplus indicated
that the methanolic extracts of A. globiflora, A. scleroderma, L. chinensis, and A. indica or
their mixtures have great potential to be used as an alternative in the control of R. microplus.
In general, the pesticide activities of A. indica are associated with azadirachtin A (10), which
is the best-known insecticidal compound of this plant. However, here was report three new
adulticidal compounds of the “neem” tree identified as 3-O-butyl-(-)-epigallocatechin (1), 3-
O-butyl-(-)-epicatechin (2), and octadecanoic acid-tetrahydrofuran-3,4-vinyl ester (11), the
above indicates that the bark of A. indica is a great source of acaricidal compounds with great
structural diversity. The use of these compounds may represent a new strategy for the control
of R. microplus zoonoses. From the chemical point of view, it is also important to highlight
that 1 and 2 have a butyl ether group which, in addition to being uncommon in nature,
increases liposolubility and their acaricidal activity.
303
Acknowledgments
This work was supported by the Gobierno del estado de Veracruz de Ignacio de la Llave and
by the Consejo Veracruzano de Investigación Científica y Desarrollo Tecnológico ‒
[COVEICyDET, grant number 14 1953/2021]. J.S.R. thanks the CONACyT foundation for
a grant (1075240). To Dr. Fernando Nicolalde-Morejón for the identification of the plants.
Conflict of interest
The authors declare no conflict of interest.
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308
Article
Effect of Xoconostle (Opuntia matudae Scheinvar) on methane

concentration and ruminal variables during in vitro fermentation of corn
stover
José Jesús Espino-García a
Isaac Almaraz-Buendía a
J. Jesús Germán Peralta-Ortiz a
Abigail Reyes-Munguía b
Iridiam Hernández-Soto a
Lucio González-Montiel c
Rafael Germán Campos-Montiel a*
a
Universidad Autónoma del Estado de Hidalgo (UAEH). Instituto de Ciencias
Agropecuarias. Avenida Universidad Km. 1 s/n Exhacienda Aquetzalpa, 43600. Tulancingo
de Bravo, Hidalgo, México.
b
Universidad Autónoma de San Luis Potosí (UASLP). Facultad de Estudios Profesionales
Zona Huasteca. San Luis Potosí, México.
c
Universidad de la Cañada (UNCA). Oaxaca, México.
*Corresponding author: rcampos@uaeh.edu.mx
Abstract:
The effect of the addition of xoconostle on in vitro ruminal fermentation of corn stover was
determined in order to reduce methane emission. Previous studies have shown that
xoconostle contains bioactive compounds with potential antimicrobial activity that enhance
ruminal fermentation. Zero point zero percent, 2.0 %, 4.0 % and 6.0 % of xoconostle were
added. The following were determined: chemical composition of the substrates, phenolic
309
compounds, antioxidant capacity, in vitro disappearance of dry matter (IVDDM), the

production of volatile fatty acids (VFAs) and the kinetic variables of gas production. The
volume of methane was measured using the technique of capturing carbon dioxide in sodium
hydroxide solution. The content of protein, ether extract, total phenols and antioxidant
activity significantly increased (P<0.05) with the addition of xoconostle. The IVDDM also
increased with the addition of xoconostle. Regarding the production of propionic acid, it
increased significantly (P<0.05) with 6.0 % of xoconostle. The kinetic parameters obtained
by the best fit of the experimental data showed a higher digestion rate and lower methane
production with the addition of 4.0 and 6.0 % of xoconostle. The use of xoconostle as an
additive in ruminant diets decreases methane production in vitro so it can be an alternative to
mitigate the increase in the greenhouse effect and benefit the cultivation of a commercially
not very appreciated fruit.
Keywords: Xoconostle, Corn stover, Ruminal fermentation, Climate change, Enteric

methane.
Introduction
According to the UN, in studies carried out between 2009-2019 by the Economic
Commission for Latin America and the Caribbean (ECLAC), Latin American countries are
very vulnerable to the effects of climate change, so it is urgent to adopt short-term measures
to reduce their impact on ecosystems(1). The natural balance of greenhouse gases (GHGs),
which are found in greater proportion, carbon dioxide (CO2) and methane (CH4), has
experienced an imbalance in recent decades due to various anthropogenic activities resulting
in an accumulation in the atmosphere, CO2 increased from 315 ppm in 1960 to 410 ppm in
2019(2); CH4 from 1,770 ppb in 2000 to 1,860 ppb in 2019(3). The proportion of CH4
accumulated per year is lower than that of CO2, but its global warming potential is 25 times
higher(4). Of the anthropogenic sources of CH4, the agricultural sector is one of the largest
contributors, the emission of enteric CH4 as a result of the digestive process of ruminants is
approximately 115 million tons per year and corresponds to 20 % of global emissions(5). Of
the strategies used to mitigate enteric CH4 emissions, the incorporation of additives in animal
feed appears to be the most promising for its practicality and economy.
Several phenolic compounds contained in some vegetables have potential antimicrobial

activity, which can improve ruminal fermentation and decrease the emission of CH4(6).
310
Among the endemic plants of Mexico that have this characteristic, some species of the genus
Opuntia that produce acid fruits, known as xoconostles, are an important source of phenolic
compounds, with potential antimicrobial activity(7,8,9). Despite the extensive number of
studies regarding the reduction of GHGs caused by methanogenesis in ruminants, studies
using cacti as forage are few, and it can be considered that there are no studies to date that
relate the decrease of ruminal CH4 with secondary metabolites of xoconostle. It is important
that livestock producers have access to technologies that allow them to reduce CH4 emissions
in a way that ensures animal safety and welfare, and that, on the other hand, is economically
viable. Therefore, the objective of this research was to evaluate the effect of xoconostle on
the in vitro fermentation of corn stover (Zea mays).
Area of study
The study was conducted in the Multidisciplinary, Animal Nutrition and Special Analysis
laboratories of the Institute of Agricultural Sciences of the Universidad Estatal Autónoma de
Hidalgo (UAEH, for its acronym in Spanish), in Tulancingo de Bravo, Hidalgo, Mexico.
Collection and preparation of samples
Fruits of xoconostle (Opuntia matudae Scheinvar cv. Rosa) in a state of commercial maturity,
harvested in the state of Hidalgo, Mexico (Figure 1), were cut into slices and dehydrated for
72 h in an air flow oven (Felisa 242 A, Mexico) at 60 °C. It was subsequently ground (Weg
Crusher, Mexico) and passed through a 2 mm diameter mesh. The corn stover was obtained
from the University Ranch of the Autonomous University of the State of Hidalgo and was
dehydrated and pulverized in the same way as the xoconostle.
Figure 1: Fruits of xoconostle (Opuntia matudae Scheinvar cv. Rosa)
311
The ruminal fluid was obtained from two sheep (Hampshire, 54 kg live weight ± 2.4) via
cannula in the rumen. All surgical procedures were carried out based on the protocol of the
Institutional Ethics Committee for the Care and Use of Laboratory Animals of the UAEH
and in accordance with the guidelines of the Law of Protection and Decent Treatment for
Animals of the government of the state of Hidalgo, Mexico(10). The sheep received
commercial antiparasitic (Ivermectin, Bayer. 200 mcg/kg live weight), vitamins ADE
(Vigantol ADE fuerte 2 mL) and were fed ad libitum with corn stover and mineral premix
(Multi-Brick Triple, Malta-Cleyton) for 15 d prior to taking the ruminal fluid.
Physicochemical characterization
The physicochemical characterization of the substrates (corn stover and xoconostle) and of
the established treatments: 100 % corn-0 % xoconostle (0%Xoco); 98 % corn-2 % xoconostle
(2%Xoco); 96 % corn-4 % xoconostle (4%Xoco) and 94 % corn-6 % xoconostle (6%Xoco)
was carried out by proximate analysis, determining: moisture content; mineral content (Mi);
ether extract (EE); crude protein (CP); crude fiber (CF)(11). The proportion of neutral
detergent fiber (NDF) and acid detergent fiber (ADF) was determined as described by Van
Soest(12), digestions were carried out using filter bags (Ankon F57 USA). In both cases, a bag
without substrate was used as a blank to perform the calculations.
Determination of total phenols
The total phenol content was determined using the Folin-Ciocalteu(13) method, the calibration
curve was constructed from a standard solution of gallic acid (1 g/L H2O), and 0-20 ppm
dilutions were obtained. To 250 μL of each dilution, 5 mL of Folin-Ciocalteu 1/10 was added
in a light-tight container and left to stand for 8 min. Subsequently, 4 mL of Na2CO3 7.5 %
was added and it was kept 2 h in the dark. The absorbance was read at 765 nm (A765)
(Spectronic-Genesys 5, USA) using distilled water as a blank. Approximately 150 mg of
sample, corn stover or xoconostle, was placed in 2 mL vials adding 1.5 mL of methanol and
100 μL of NaF 2 mM in order to inhibit polyphenol oxidase (PPO). They were placed in a
light-tight container and stirred at room temperature for 30 min. They were centrifuged at
10,510 xg (Hermle Z36 HK, Germany) for 20 min at 4 °C. Aliquots of 250 μL of supernatant
were treated in the same way as dilutions to construct the calibration curve, absorbance was
read at 765 nm (A765) and the corresponding calculations were made.
Determination of antioxidant capacity
ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) method
312
The ABTS•+ radical was obtained after the reaction of 10 ml of ABTS solution (7 mM) with
10 ml of potassium persulfate solution (2.45 mM). The reaction was carried out at room
temperature and in the dark with constant stirring for 16 h(14). Once the ABTS•+ radical was
formed, it was diluted with ethanol (20 %) until the absorbance 0.7 (±0.1) was adjusted to
754 nm (A754) using ethanol (20 %) as a blank. As a reference antioxidant, dilutions of
ascorbic acid 0-100 ppm and gallic acid 0-10 ppm were used to obtain the calibration curves.
Approximately 1 g of sample was suspended in 9 mL ethanol (50 %) in light-tight centrifuge
tubes, and they were stirred at room temperature for 30 min. It was centrifuged at 10,510 xg
at 4 °C for 20 min; 200 μL of supernatant, dilution of ascorbic or gallic acid was added with
2 mL of standardized ABTS•+, allowed to stand 6 min and A754 was determined using ethanol
(20 %) as a blank. The calibration curves were used to convert the absorbances obtained in
the samples to ascorbic acid equivalents (AAE) or gallic acid equivalents (GAE) respectively.
DPPH (2,2-diphenyl-1-picrylhydrazyl) method
The method developed by Brand-Williams(15) with some modifications to that described by

Kim(16) was used, where the absorbance of the DPPH• radical 200 μM in methanol (80 %)
stirred for 2 h in a light-tight container and adjusted its absorbance to 515 nm (A515) to
0.7±0.1 with methanol (80 %) was measured after the reaction with a reference antioxidant.
As a reference antioxidant, dilutions of ascorbic acid 0-50 ppm and gallic acid 0-5 ppm were
used to obtain the calibration curves. Approximately 1 g of sample was suspended in 9 mL
ethanol (50 %) in light-tight centrifuge tubes and stirred at room temperature for 30 min. It
was centrifuged at 10,510 xg at 4 °C for 20 min; 0.5 mL of supernatant or ascorbic acid
dilution was added with 2.5 mL of standardized DPPH•, allowed to stand for 1 h and A515
was determined using methanol (80) as a blank.
pH and in vitro degradation of dry matter (IVDDM)
The pH was measured (HANNA, HI2211, Romania) at the end of the incubation time and
the contents of each bottle were transferred to 50 mL polysulfone tubes, which were
centrifuged (HERMLE, Z326K, Germany) at 15,130 xg for 15 min. The supernatant was
removed by decantation and the solid material was dried at 65 °C for 48 h. The IVDDM was
calculated as the difference between the weight of the initial dry matter and the weight of the
residual dry matter and was expressed as g/100 g DM.
Determination of volatile fatty acids (VFAs)
At the end of fermentation, 1.6 mL of the liquid fraction of the fermentation bottles was
placed in 2.5 mL vials containing 0.4 mL of HPO3 25 % w/v, they were stored at 5 °C.
Subsequently, they were centrifuged at 15,130 xg 15 min. The concentration, millimoles/liter
313
(mM L-1) of VFAs was determined in a gas chromatograph Claurus 500, Perkin Elmer, USA,
provided with autosampler, 15 m capillary column (ELITE-FFAP, Perkin Elmer, USA) and
flame ionization detector (FID). The carrier gas was N2 at 60 psi, H2 and extra dry air were
used to generate the flame. The temperatures of the oven, injector and column were 120, 250
and 250 °C, respectively. The retention time was 1.22, 1.55 and 2.02 min for acetic, propionic
and butyric acids, respectively. Previously, a calibration curve was constructed with standard
solutions of acetic, propionic and butyric acids(17).
Fermentation and gas production
In glass bottles with a capacity of 125 mL, 0.5 g of substrate corresponding to each corn
stover-xoconostle treatment was deposited. The ruminal fluid was filtered through eight
layers of gauze and stored at 39 °C under anaerobic conditions until use. To each bottle and
under continuous flow of CO2, 40 mL of culture medium and 4 mL of ruminal fluid were
added. Per liter of solution the culture medium contains: 1 g NH4HCO3; 8.74 g NaHCO3;
1.43 g Na2HPO4; 1.55 g KH2PO4; 0.15 g MgSO4.7H2O; 0.017 g CaCl2.2H2O; 0.013 g
MnCl.4H2O; 0.0013 g CoCl.6H2O; 0.01 g FeCl3; 1.29 mL of 0.1 % resazurin solution as an
indicator and 37 mL of reducing solution containing 0.21 g Na2SO4 and 1.5 mL of 0.1N
NaOH solution. The bottles were hermetically closed (Manual crimper, Wheaton, USA) by
means of a silicone stopper and a vial capsule with a removable center. Similar containers
with only ruminal inoculum were included as blanks. The bottles were incubated in a water
bath at 39 °C. The volume of gas produced (ml) inside each bottle at 1, 2, 3, 4, 5, 6, 7, 8, 10,
12, 14, 16, 18, 22, 26, 30, 42, 54, 66, 78, 92 h of incubation was recorded by displacement
of water volume by puncturing through the silicone stopper using a hypodermic needle
coupled to a graduated glass column containing water. After each measurement, the gas was
released by equalizing the internal and external pressure of the bottles(18).
The rate at which gas production takes place depends on the characteristics of the rumen
microbiota present(19); as well as on the type of substrate, pH, Redox potential(20), resulting
in different kinetic profiles. The mathematical description of these profiles allows comparing
characteristics of the substrates or the fermentation environment. The fit of the experimental
data to the Logistic model using the Sigma Plot 12© software allowed obtaining Equation 1;
where y (mL g-1 DM) denotes the amount of accumulated gas produced per gram of dry
matter (DM) at time t (h) during incubation. A (mL g-1 DM) represents the maximum gas
production at infinite time. to (h) is the incubation time in which half of A has been produced
and b is a dimensionless constant that determines the characteristic profile and therefore the
inflection point of the curve(21).
𝐴
𝑦= 𝑡0 𝑏
Eq. (1)
1+( )
𝑡
314
The indicator inflection point of the lag phase (L) or Equation 2 resulting from dy/dt is:
1⁄
𝑏−1 𝑏
𝐿 = 𝑡0 [ ] Eq. (2)
𝑏+1
Considering the disappearance of substrate (P) as a kinetics of the first order, the digestion
rate of the substrate (S) (Equation 3) for values of b>1 increases until reaching a maximum
(Smax) when the size of the microbial population no longer limits the fermentation of the feed.
The time in which Smax is reached is given by the resolution of dS/dt=0 (Equation 4)(21).
1 𝑑𝑃 𝑏𝑡 𝑏−1
𝑆= = Eq. (3) 𝑡𝑆𝑚𝑎𝑥 = 𝑡𝑜(𝑏 − 1)1/𝑏 Eq. (4)
𝑃 𝑑𝑡 𝑡𝑜𝑏 +𝑡 𝑏
Methane determination
The volume of CH4 was measured using the technique described by Torres-Salado(22) with
the following modifications: the biodigester bottle was coupled by means of a Taygon® hose
(2.38 mm internal Ø and 30 cm long) with hypodermic needles (20 G x 32 mm) at the ends
to an inverted vial and fully filled with NaOH 2N. The gas originated by the fermentation of
the substrate flows through the NaOH 2N, where CO2 reacts and forms sodium carbonate.
The residual gas is insoluble in the solution and corresponds to CH4, where the amount is
quantified according to the ml of NAOH 2N displaced through another hypodermic needle
placed in the silicone stopper as an outlet valve and measured with a graduated cylinder.
Statistical analysis
A completely randomized design was used, the statistical model used was:
Yij = µ + t i + εij
Where:
Yij Response variable of the ij-th experimental unit;
μ Effect of the overall mean;
ti Effect of the i-th treatment;
εij Effect of the experimental error associated with the i-th experimental unit.
Each treatment had five independent repetitions, with the experimental unit being a bottle
with 500 mg of substrate. Data analysis was performed using ANOVA and comparison of
means with the Tukey test adjusted to a significance level α=0.05.
315
Results and discussion
Physicochemical characterization and antioxidant capacity of the

substrates used
The concentration of moisture, CP, EE and ash in Xoconostle (Table 1) is similar to that
reported by Sánchez-González(23), where the variation in these nutrients depends on maturity
and culture conditions. Information about the proportion of NDF, ADF is scarce to null
because this fruit is mainly used in human food, where the relevance is due to its beneficial
and antioxidant properties, as reported by Morales and Espinoza-Muñoz(24,25). However,
being a cactus fruit, the concentration of NDF is similar to the prickly pear (52 % vs
40.74 %) based on what is reported in the NRC(26). The content of phenolic compounds and
antioxidant activity in the corn stover used in this study (Table 1) is close to that reported by
Vázquez-Olivo(27) for corn stover with values of 219 GAE/100 g sample. Regarding
xoconostle, the content of total phenols is consistent with what has been reported by other
studies(28,29).
Table 1: Physicochemical characterization and antioxidant capacity of the substrates used

Corn stover (%DB) Xoconostle (%DB)
Dry matter 92.97 ± 0.003 87.27 ± 0.002
Minerals 7.45 ± 0.001 13.13 ± 0.002
Crude protein 3.46 ± 0.004 4.82 ± 0.004
Ether extract 0.78 ± 0.020 4.07 ± 0.008
Nitrogen-free extract 88.31 ± 0.002 77.98 ± 0.001
Neutral detergent fiber 68.05 ± 0.008 40.74 ± 0.024
Acid detergent fiber 36.99 ± 0.016 30.34 ± 0.067
Total phenols 246.93 ± 0.206 mg GAE 100 g-1 740.59 ± 0.461 mg GAE 100 g-1
4.94 ± 0.013 mg GAE 100 g-1 21.42 ± 0.028 mg GAE 100 g-1
DPPH
Antioxidant 22.77 ± 0.037 mg AAE 100 g-1 66.80 ± 0.076 mg AAE 100 g-1
activity 10.87 ± 0.050 mg GAE 100 g-1 19.73 ± 0.260 mg GAE 100 g-1
ABTS
99.74 ± 0.048 mg AAE 100 g-1 191.43 ± 0.273 mg AAE 100 g-1
Average values ±SD of three repetitions. GAE (gallic acid equivalents) AAE (ascorbic acid equivalents).
The physicochemical characterization of the treatments is shown in Table 2, where it is

observed that the content of DM decreases when the content of xoconostle increases, this is
because the moisture contained in the fruit is greater than that of corn stover, on the contrary,
the concentration of Mi and CP increases proportionally in the treatments since, when
replacing stover with xoconostle, the latter contains a higher percentage of these components,
as shown in Table 1. Regarding the EE, when the amount of stover substituted for xoconostle
increases, the proportion of this fraction in the treatments increases substantially, since the
magnitude of the extract determined in the xoconostle is 4.21 times greater than in that of
316
corn stover. Some varieties of xoconostle such as Opuntia matudae Scheinvar cv. Rosa
contain saturated fatty acids such as palmitic and myristic; and polyunsaturated as oleic and
linoleic(24). Despite the increase in EE (Table 2), the concentration of lipids in the treatments
of this study is considered not to influence methanogenesis since its effect has been observed
from 50 g kg-1 of dry matter in the diet(30). The NFE includes assimilable carbohydrates and
crude fiber, this parameter decreases when the amount of corn stover replaced with
xoconostle increases since the latter, despite containing more simple carbohydrates than corn
stover, has a lower proportion of structural carbohydrates, 18 % less ADF. This decrease in
ADF results in better digestibility of the feed, since the supply of non-structural
carbohydrates, up to a certain limit, reduces the lag phase and improves the energy content
of the diet being decisive in the production of ruminal bacterial protein. The increase in the
percentage of xoconostle as a substitute also increased TF content and antioxidant activity.
The presence of phenolic compounds could have an effect on some rumen microorganisms;
it has been shown that these secondary metabolites of plants have an inhibitory effect, Diaz-
Solares and others evaluated the content of phenols and flavonoids as well as the
antimicrobial capacity of extracts of Morus alba leaves, finding abundant presence with
activity against S. aureus, E. coli, P. aeruginosa, K. pneumoniae and β hemolytic S.,
suggesting its use in animal feed(31). Hayek and Ibrahim(7) report inhibitory effect of aqueous
extracts of xoconostle on E. coli. It has been shown that phenolic compounds contained in
some vegetables improve ruminal fermentation and decrease methane production(32,33,34).
Table 2: Physicochemical characterization of treatments

0% Xoco 2% Xoco 4% Xoco 6% Xoco
(%DB)
Dry matter 92.97 ± 0.003a 92.85 ± 0.000b 92.74 ± 0.005c 92.62 ± 0.003d
Minerals 7.45 ± 0.001ª 7.57 ± 0.000b 7.68 ± 0.005c 7.80 ± 0.003d
Crude protein 3.46 ± 0.004ª 3.49 ± 0.000b 3.52 ± 0.001c 3.54 ± 0.000d
Ether extract 0.78 ± 0.020ª 0.85 ± 0.000b 0.91 ± 0.003c 0.98 ± 0.002d
Nitrogen-free extract 88.31 ± 0.002ª 88.09 ± 0.001b 87.89 ± 0.010c 87.67 ± 0.006d
Neutral detergent fiber 68.05 ± 0.008ª 67.48 ± 0.003b 66.94 ± 0.028c 66.37 ± 0.016d
Acid detergent fiber 36.99 ± 0.016ª 36.85 ± 0.000b 36.72 ± 0.006c 36.58 ± 0.004d
Total phenols,
mg GAE 100 g-1 246.93±0.206ª 257.30±0.051b 266.97±0.505c 277.34±0.287d
GAE 4.94 ± 0.013ª 5.29 ± 0.002b 5.61 ± 0.017c 5.96 ± 0.010d
DPPH
Antioxidant AAE 22.77 ± 0.037ª 23.73 ± 0.005b 24.56 ± 0.045c 25.48 ± 0.026d
activity GAE 10.87 ± 0.05ª 11.06 ± 0.001b 11.23 ± 0.009c 11.42 ± 0.005d
ABTS
AAE 99.74 ± 0.048ª 101.67±0.010b 103.460.094c 105.39 0.053d
Average values ± SD of five repetitions. GAE (gallic acid equivalents); AAE (ascorbic acid equivalents).
abcd
Values on the same row with different superscript are different (P<0.05).
317
pH and IVDDM
The pH values of the ruminal fluid of the treatments at the end of fermentation showed no
significant difference (P>0.5) (Table 3). The use of a buffer solution based on bicarbonate
and phosphates in the culture medium probably maintained the pH with values above 6.0
during the fermentation time favoring the digestibility of the DM. pH values below 6.0 inhibit
the development of cellulolytic bacteria (i.e., diets with high concentrate content) and result
in longer times in the lag phase (L) and a decrease in the IVDDM(35,36). One of the quality
parameters of forages is the IVDDM, as it indicates the efficiency with which ruminants can
metabolize them. In this study, the treatment of 6% Xoco had a higher IVDDM than the
control (P<0.05). The treatment of 4% Xoco did not present a statistic difference with respect
to the control, but it did show a tendency to increase, there was no significant difference
(P>0.05) between both treatments, this increase in the IVDDM due possibly to the addition
of non-structural carbohydrates of the xoconostle caused a better fermentation, resulting in a
lower lag phase and a higher digestion rate (Table 3 ). In vitro digestibility of NDF
(IVDNDF) showed no difference between treatments (P>0.05), which could indicate that the
bioactive compounds of xoconostle have no activity on cellulolytic bacteria and that the
degradation of structural carbohydrates is not affected. Regarding total nitrogen, there was
no significant difference between the treatments (P>0.05) but, the concentration at the end
of the experiment probably increased as a result of the bacterial action on the proteins
solubilizing nitrogen.
Table 3: Response variables after 92 h of fermentation

pH 6.43 ± 0.059 6.43 ± 0.019 6.44 ± 0.019 6.43 ± 0.019
b
IVDDM, % 73.97 ± 4.73ª 72.76 ± 3.07a 78.99 ± 4.70bc 80.44 ± 4.71c
IVDNDF, % 89.31 ± 1.94 88.67 ± 1.27 88.61 ± 2.55 89.69 ± 2.48
Nitrogen Initial 0.0338 ± 0.003 0.0338±0.002 0.0338±0.001 0.0339±0.002
Total, mg d l-1 Final 0.036 ± 0.002 0.04 ± 0.003 0.04 ± 0.003 0.04 ± 0.002
Average values ±SD of five repetitions.
abc
Values in the same row with different superscript are significantly different (P<0.05).
Production of volatile fatty acids (VFAs)
The concentration of VFAs (mM L-1) in the ruminal fluid as a result of fermentation is shown
in Table 4, where it is observed that the treatment of 6% Xoco has a difference in the amount
of propionic acid generated (P<0.05), which suggests that xoconostle has an effect on the
rumen microbiota directing ruminal fermentation towards a decrease in available H+,
necessary for the production of CH4(33), another possibility is that the metabolism of phenolic
compounds contained in xoconostle increases the synthesis of propionic acid, which also
318
decreases the production of methane but without having a direct effect on rumen
microorganisms (Ku Vera, et al)(37).
Table 4: Concentration and proportion of VFAs originated by in vitro fermentation of corn

stover and xoconostle
-1
Acetic, mM L 35.6 ± 2.43 35.8 ± 0.862 33.1 ± 0.100 33.8 ± 02.34
-1 a a a
Propionic, mM L 14.8 ± 0.898 14.5 ± 0.412 13.8 ± 0.350 16.85 ± 0.845b
Butyric, mM L-1 5.2 ± 0.340 5.1 ± 0.186 4.9 ±0.165 5.4 ± 0.793
-1
Total, mM L 55.6 55.4 51.8 56.05
Acetic, % 64.0 64.6 63.9 60.3
Propionic, % 26.6 26.2 26.6 30.1
Butyric, % 9.4 9.2 9.5 9.6
Acetic/Propionic 2.40 2.46 2.40 2.00
Average values ±SD of three repetitions.
ab
Values in the same row with different superscript are significantly different (P<0.05).
Gas production
The production profiles of total accumulated gas and methane gas (Figure 2a and 2b) show
that the treatments have a sigmoid behavior during the 92 h of incubation following the
logistic behavior(38), the mathematical model used(21) allowed a good fit of the data (R2
>0.99). Regarding the accumulated volume of total gas, the treatment of 2% Xoco was
different (P<0.05), observing a greater production of gas compared to treatments of 4 and
6% Xoco (Table 5), probably increasing the amount of phenolic compounds contained in the
xoconostle has a consequence on the activity of the rumen microbiota, similar results were
determined in previous studies(39). The incorporation of phenolic compounds such as tannins
in ruminal fermentation has an effect on the microbiota since they can bind to the cell wall
of microorganisms, causing morphological changes or secretion of extracellular enzymes or
they can bind to enzymes causing changes in their metabolism(40). The production profiles of
CH4 (Figure 2b) show that the volume produced tends to decrease in response to the
incorporation of xoconostle, with the treatment of 6% Xoco being where the effect is most
evident. There are several studies that address the reduction of enteric methane from the
incorporation of phenolic compounds, such as that by Tiemann(41), which evaluated the effect
of two legumes rich in condensed tannins on methane emissions in lambs, achieving a
reduction of up to 24 % but reducing the digestibility of dry matter. In this study, an 8.5 %
reduction was achieved in the treatment of 6% Xoco without having this negative effect.
319
Figure 2: Volume of total gas (a) and methane (b) during 92 h of in vitro fermentation
The kinetic parameters resulting from the fit to the Logistic model are shown in Table 3 and
through Equation 4, they determine tsmax for the study treatments, being 5.3, 2.8, 0.9 and 1.1
h for 0% Xoco, 2% Xoco, 4% Xoco and 6% Xoco respectively (Figure 3). A reduction in the
time to reach the maximum digestion rate of the substrate represents better digestibility and
therefore lower methane production.
Table 5: Kinetic parameters obtained from the fit to the Logistic model
-1 a b a
Atotal gas, ml g DM 334.88 395.48 356.84 351.26a
ACH4, ml g DM-1 193.60a 188.70a 177.2a 184.3a
b (-) 1.1978a 1.0031b 0.9474b 0.9325b
to, h 21.142a 33.902b 20.598a 19.97a
Smax, h-1 0.026a 0.037b 0.049c 0.052d
tSmax, h 5.32a 2.84b 0.90c 1.10bc
L, h 2.76a 1.41b 0.44b 0.54b
R2 0.9938 0.9942 0.9945 0.9947
Atotal gas (total gas CO2 + CH4); ACH4 (methane produced); S (digestion rate); tSmax (time to reach Smax); L (lag
phase).
abcd
Values on the same row with different superscript are significantly different (P<0.05).
320
Figure 3: Change in digestion rate (S) during the 92 h of fermentation
The addition of xoconostle in an in vitro fermentation of corn stover increased the digestion
rate and reduced the lag phase, which translates into an improvement in the digestibility of
the substrate. The bioactive compounds of xoconostle increase propionic acid production by
13.1 % when 6 % of xoconostle is added and reduce methane production by 8.5 % with the
addition of 4 % of xoconostle.
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Article
Factors affecting the rate of pregnancy by embryo transfers (ET) by in

vitro fertilization in multibreed heifers under Colombian tropical
conditions
Heli Fernando Valencia Ocampo a*
Nancy Rodríguez Colorado a
Tatiana Mantilla a
a
Universidad Francisco de Paula Santander Ocaña. Sede El Algodonal Km 1 Vía Acolsure;
Facultad de Ciencias Agrarias y del Ambiente. Ocaña, Colombia.
*Corresponding author: hfvalenciao@ufpso.edu.co
Abstract:
Embryo transfer (ET) is currently considered a biotechnological tool with great importance
to multiply and obtain individuals with productive potential and high genetic merit. The
objective of this study was to determine the influence of factors such as THI, CL size, embryo
development status and weight on the rate of pregnancy by embryo transfer by in vitro
fertilization in multibreed heifers. Eight hundred forty heifers were selected as recipients,
with an average age of 3 years, a weight of 346.5 ± 33.4 kg of live weight, to which a
synchronization protocol for ET was applied, after recording the stage of the embryo,
ultrasound monitoring and the environmental variables were monitored. The information was
analyzed using a logistic regression model to determine the correlation between the
independent variables and the dichotomous response variable pregnancy rate. The influence
of corpus luteum (CL) size was determined given the significant differences (P<0.05) in the
sizes of CL1 and CL2. Differences (P<0.05) were also found with the stages of BL and BX.
In contrast, no statistical differences (P>0.05) were found in the other variables. The present
study showed the impact of CL size and embryo development on the success of the ET
technique.
326
Key words: Embryo transfer, Humidity-temperature index (THI), In vitro fertilization,

Corpus luteum (CL).
Introduction
Embryo transfer (ET) is currently considered a biotechnological tool with great importance
to multiply and obtain individuals with productive potential and high genetic merit, capable
of improving performance in bovine production systems(1). Several studies show the impact
of the factors that influence the effectiveness of the technique, the intrinsic factors which are
specific to the animal and are directly related to its physiology, as well as factors related to
the embryo [size of the corpus luteum (CL), stage of embryo development, bulls used for
fertilization, among others]; and extrinsic factors that are related to the environment that
surrounds the animals and that somehow affects the physiology of the animal (environment,
nutrition, management, among others)(2).
Climatic conditions at levels outside the resting state of the animal frame the productivity of
the individual, destabilizing the comfort conditions and subjecting it to alterations of
physiological functioning that leads it to an environment of heat stress (HS)(3). Over the years,
many studies have established that the temperature-humidity index (THI) can specify, as a
function of the combination of the variables temperature and relative humidity, the degree of
HS suffered by animals(4). The value of the THI varies between authors, but similarity is
found when specifying that the values > 72 present a HS in animals. In this sense, different
thresholds are considered to characterize the state of animal comfort, according to the value
of the THI, the HS is characterized as: comfort (<68), mild discomfort (68 - 72), discomfort
(72 - 75), alert (75 - 79), danger (79 - 84) and emergency (> 84)(5). However, the improvement
in the technique and the results of pregnancy rates remain the subject of research considering
the number of factors that influence the success of biotechnology(6).
The objective of this study was to determine the influence of factors such as THI, CL size,
embryo development status and weight on the rate of pregnancy by embryo transfer by in
vitro fertilization in multibreed heifers.
327
Materials and methods
The study was conducted in Colombia, in the municipality of Puerto Boyacá, geographically
located at 5°58 ́34 ́ ́ N and 74°35 ́15 ́ ́ W, belonging to the department of Boyacá. An
observational experiment was carried out in order to measure the variables THI, CL size,
embryo development status and weight, it was also estimated how they can affect the rate of
pregnancy obtained by ET by in vitro fertilization. Recipient multibreed heifers (n= 840)
with an average age of 3 yr, with an average weight of 346.5 kg, were selected; sanitary
management was carried out prior to reproductive work, vitaminization, deworming,
vaccination against reproductive diseases, antibiotic therapy and bath for external parasites.
In the same sense, a control weighing was made every month, thus observing the weight gain
of the animals. The feeding was implemented by extensive grazing, with the following
species predominating: guinea grass (Megathyrsus maximus), sweet brachiaria (Brachiaria
humidicola), para grass (Brachiaria mutica), water at will and mineralized salt at 8 %
phosphorus, in this way a homogeneous management was provided for all animals. Variables
such as breed were not evaluated given the conditions of high variability in the breed
component of the recipient females.
The reproductive works began in March in continuous activities until December 2020; before
starting the works, the recipients were examined rectally and by ultrasound, selecting animals
that did not have anatomical problems of uterus, cervix and ovaries that limited the response
to the transfer. The synchronization protocol for the recipients that was implemented was a
combination of progesterone with estradiol benzoate as follows: on day 0 a bovine
progesterone-releasing intravaginal device (0.6 g of progesterone P4) plus estradiol benzoate
(2 mg). The device was removed on d 80 and 300 IU of eCG (equine chorionic gonadotropin),
150 μg of D-cloprostenol (synthetic analog of prostaglandin F2α) and 1 mg of estradiol
cypionate were applied(7).
The embryo transfer was performed on d 17 of the start of the synchronization protocol, for
this, palpation was first performed by transrectal ultrasonography in order to detect the
presence of a corpus luteum in the ovary. Subsequently, for the execution of the transfer
technique, 3.5 to 4 mL of lidocaine (epidural anesthesia) was applied to the recipient, the
vulvar area was cleaned and then the embryo was placed in the most distal point of the uterine
horn ipsilateral to the ovary that presents the corpus luteum(8). The embryos used were by in
vitro fertilization obtained and packed by professionals from the laboratory BIOEMBRIO
FIV S.A.S. of about 7 d, who determined the stage of the embryo when packed. The embryos
used in this study were of the Girolando breed (F1 and 3/8 Gyr X 5/8 Holstein). The technique
was applied by a single veterinarian with experience, avoiding variability in the procedure.
The detection of pregnancy was performed on d 28 after the transfer process, for which a
Chison eco2-vet ultrasound machine with an L7V-A 6.5MHz linear transducer was used,
328
where it was determined if there was presence of the gestational vesicle in the uterus by
ultrasound for the positive diagnosis.
Meteorological data
The environmental data were obtained from the Institute of Hydrology, Meteorology and
Environmental Studies (IDEAM), through a meteorological station near the study area. The
value of the index was calculated with the temperature and relative humidity data provided
by that station. The temperature-relative humidity index (THI) was calculated using equation
1 reported by Habeeb(5):
THI = (1.8*At+32) − [(0.55−0.0055* RH) × (1.8* At −26)]

Where: RH= Relative air humidity (%) and At= Air temperature (ºC).
Data analysis
The data were analyzed using the statistical program Epi Info version 7(9), a logistic
regression analysis was used to reveal the model of the relationship between independent
variables (CL size, embryo stage, THI and precipitation) and the dichotomous response
variable (pregnancy rate) which was obtained using embryo transfer; this statistical model
uses Odds Ratios (OR). Standardized measures that allow comparing the level of influence
or strength of the independent variables on the dependent variable, for the level of
significance of the tests, P<0.05 was accepted. The GLM is described as Y= 𝛽0 + α + e.
Where Y is the response variable 1= pregnant and 0= not pregnant, 𝛽0 is the intercept, 𝛼 is
the effect of the categorical variables under study and 𝑒 is the statistical error. Likewise, the
continuous variable of weight was analyzed by descriptive statistics and an analysis of
variance ANOVA, observing the degree of significance that this variable presented when
associated with the response variable.
Results
A total of 2,259 synchronizations were performed and an overall response to the fixed time
synchronization protocols of 75.6 % was obtained, which allowed carrying out a total of
1,710 embryo transfers by in vitro fertilization, where an overall pregnancy rate of 29 % was
achieved.
329
Effect of CL size on pregnancy rate
The classification of the CL according to size allowed obtaining three categories CL1: <15
mm , CL2: 15-25 mm; CL3: >25 mm in diameter(10). Table 1 shows that when associating
the pregnancy rate obtained with the size of the corpus luteum, significant differences
(P<0.05) are found for the sizes of CL1 and CL2. After the transfer, it was obtained that the
probability of pregnancy (odds ratio, OR) is 0.46 for CL1; 0.62 for CL2 and 0.67 for CL3.
This showed that there is 0.21 and 0.16 times more of obtaining a pregnancy with a CL3 and
CL2 respectively, compared to CL1; in this way it is more likely to increase pregnancy rates
when transferring embryos with larger sizes of CL 3 and 2 compared to CL1.
Table 1: Association of pregnancy rate with CL size

Pregnancy 95 % Confidence
Variable Odds ratio P-value
(%) interval
Size CL1 25 0.4629 0.2636 0.8128 0.0073
Size CL2 31 0.6264 0.4051 0.9688 0.0355
Size CL3 32 0.6709 0.4342 1.0367 0.0722
Constant * * * 0.1255
Underlined values present significant differences (P<0.05).
Effect of embryo stage on pregnancy rate
Embryo stage refers to the time and development of the embryo considering the classification
guidelines established by the International Embryo Transfer Society (IETS)(11); identified
with the letters BI: initial blastocyst about 5 d of development, BL: blastocyst with 6 d of
development, BX: expanded blastocyst with 7 d of development and BN: blastocyst in
hatching, it is breaking the zona pellucida.
The analyses carried out to determine the effect of embryo stage on pregnancy show
significant differences (Table 2) with the stages of BL and BX with P values of 0.0136 and
0.0000, respectively. It is also observed that the OR probabilities of obtaining a pregnancy
by transferring embryos with development stages of BX and BL are 0.9 and 0.5 times more
respectively, compared to BI. This may infer that, the greater development in embryos with
the zona pellucida intact reflects their greater activity and viability, generating better
pregnancy rates than when transferring late embryos, compromising fertility.
330
Table 2: Association of pregnancy rate with the effect of embryo stage

95 % Confidence
Variable Odds ratio P-value
interval
Embryo stage BI 0.8290 0.5569 1.2342 0.3557
Embryo stage BL 1.4129 1.0737 1.8594 0.0136
Embryo stage BN 0.3060 0.0385 2.4337 0.2630
Embryo stage BX 1.7750 1.3727 2.2952 0.0000
Constant * * * 0.0000
BI= initial blastocyst about 5 days of development; BL= blastocyst with 6 d of development; BX= expanded
blastocyst with 7 d of development; BN= blastocyst in hatching.
Underlined values present significant differences P<0.05.
Effect of THI on pregnancy rate
Regarding the association of THI with pregnancy rates, no significant differences were found
in the percentage of pregnancy with respect to the different values of THI at the time of
transfer (P>0.05) (Table 3), although it was determined that, in the area where the study was
carried out, the animals were in a range of 78 to 83 according to the index framed in danger
zone according to the Armstrong classification, given the influence of a highly stressful
environment where genetic and adaptive conditions can influence the response obtained.
Table 3: Association of pregnancy rate with the Temperature-Humidity Index (THI)

Odds 95 % Confidence
Variable P-value
ratio interval
THI 78 transfer day 1.0818 0.000 >1.0E12 1.0000
Constant * * * 1.0000
331
Effect of precipitation on pregnancy rate
According to the rainfall regime occurred during the period of study and reflected in the data
of the meteorological station, it is classified as a period of transition of dry weather with the
first rainy season of the year in March, obtaining a value of 184 mm per month; it can also
be inferred that the months of greatest precipitation were April and September, with values
of 380 and 578 mm, respectively. Likewise, the months with dry season were October,
November and December, with values of 1, 71 and 0 mm respectively, of the year 2020.
Table 4 shows the analysis of association of the pregnancy rate with precipitation, choosing
to generate 4 categories that frame the months according to rainfall regime 1: rainfall 0 to
100 mm, 2: 101 to 200 mm, 3: 201 to 300 mm, and 4: > 301 mm. Taking into account the
analyses carried out, no significant differences (P>0.05) were found between the
precipitation variable with the response variable, but given the ORs, as the value is >1, the
months categorized with the highest precipitation 3 and 4 are more likely to improve
pregnancy rates compared to categories 1 and 2, being the least rainy months.
Table 4: Association of pregnancy rate with precipitation
Variable Odds ratio 95 % Confidence interval P-value
Precipitation 1 0.8368 0.0000 >1.0E12 1.0000
Precipitation 2 0.9175 0.0000 >1.0E12 1.0000
Precipitation 3 1.1640 0.0000 >1.0E12 1.0000
Precipitation 4 1.0937 0.0000 >1.0E12 1.0000
Constant * * * 1.0000
Effect of recipient female weight on pregnancy rate
The average weight of the recipient females at the time of transfer showed an average of 344
± 107 kg for animals that were not pregnant and 337 ± 42 kg for animals that became
pregnant; the analysis of variance showed no statistical differences (P>0.05) that associate
weight with pregnancy obtained (Table 5).
332
Table 5: Analysis of variance of weight associated with pregnancy rate
Variation Sum of squares df Mean squared F statistic
Between 14888.97033 1 14888.97033 1.79175
Within 12215293.50793 1470 8309.72347
Total 12230182.47826 1471

P-value= 0.18071.
Discussion
The results of the present study determined that the size of the CL has an effect on gestation
rates after embryo transfer, it was observed that the pregnancy rate was higher as the CL size
was larger. Gestation rates were higher in recipients with CL2 and 3 (31 % and 32 %
respectively) compared to recipients with CL1 (25 %). Similar results were presented by
Alkan et al(10) as they showed in their study that the diameter of CL had significant effects
on the pregnancy rate during embryo transfer in meat heifers. Similarly, Baruselli et al(7)
determined that the effect of CL size on progesterone concentration and conception rate in
embryo recipients is established, since larger CLs secrete more P4 and this can have a
positive effect on pregnancy recognition and, consequently, on effectiveness rates in ET
programs. In contrast, other researchers(12) found no statistically significant effect for the
physical traits of CL size and quality of the recipients on conception rate [CL volume (P=
0.20), CL side (P= 0.14)]. Similarly, in their study, Vieira et al(13) observed that there were
no significant differences in the effect produced by the size of the CL on the percentage of
pregnancy obtained.
In this study, significant differences were found for the variable of embryo development
stage, obtaining better results when transferring embryos in blastocyst and expanded
blastocyst. This can be explained since when transferring an embryo whose development has
been faster, it could express more quickly the factors of recognition of pregnancy, compared
to one of the same time and of smaller size. This may be supported by a study(13) in which it
was observed that the degree of development of the embryo had an important impact on
pregnancy outcomes in the recipients. In contrast, several authors(14) did not identify
significant differences in pregnancy rates depending on the embryo development status BI,
BL and BX. Bényei et al(15) reported that the effect that statistical analysis did not reveal
significant differences in pregnancy obtained taking into account the stage in embryo
development.
333
The statistical analysis of the present study found no significant differences in the effect of
THI on pregnancy rate. In contrast, Silva et al(16) observed that from a THI greater than 72,
Holstein cows begin to decrease milk production influenced by climatic variables. Other
studies(17) conclude that the effects of the low pregnancy rate are influenced by the prolonged
heat loads to which animals are exposed in their productive environment than to the THI on
the day of service; in their study conducted in Queensland, Australia with lactating Holstein
animals, THI 72 was established as the threshold for triggering negative reproductive effects,
obtaining reduced conception rates due to the prolonged exposure to heat stress 5 and 1 week
before and after service. Likewise, in their study with lactating cows, Schüller et al(18) found
that the conception rate decreases as the animals are in a heat stress at the time of service as
their prolonged exposure to these conditions. They also determined that 1 hour of exposure
with a THI of 73 was enough for the conception rate to drop by 5 %.
In this experiment it was concluded that constant exposure to heat stress with THI 73 acts
with negative effects on reproduction 42 and 31 d before and after the day of service
respectively, decreasing pregnancy rates caused by heat stress. Cordeiro et al(19) in their study
demonstrated that heat stress negatively affects the conception rates of crossbred (Bos taurus
× Bos indicus) cows transferred to northern Brazil, where they had a decrease in conception
rates when THI reached 75.7; in contrast, the month that obtained the highest conception rate
presented the most favorable climate during the experiment (THI 73.1). It has been
mentioned(20) that high THI levels have a negative effect on the resumption of ovarian activity
and reproductive behavior in Bos indicus cows kept in grazing, especially if a high THI
occurs during the last trimester of gestation.
Precipitation variability in animal production systems in tropical areas can have negative
effects on forage growth and quality; representing an important economic condition by
altering the productive and reproductive performance of the animal due to the low availability
of nutrients(21). In this study, no significant effect was found on the rate of pregnancy by
embryo transfer associated with the rainfall regime occurred at the time of the experiment.
In this sense, Mulliniks et al(22) obtained similar results as they concluded that the
precipitation regime of the dry season and rainy season did not significantly influence
(P>0.46) the reproductive activity and performance of the animals under study; also
indicating that, given the variability in annual precipitation, animals that have a body
condition score of 4 to 4.5 (rating from 1 to 9) may have reproductive performance similar
to females with better body condition scores. Added to this, in the study conducted by
Fernandes et al(23), no significant differences (P>0.05) were found, obtaining a similar
gestation rate between the rainy and dry seasons with values of 42.3 vs 45.8 %, respectively
(P>0.05). In contrast, Scasta et al(24) determined a positive impact that precipitation has on
forage yield, which would indicate that cold and wet conditions offer a greater level of
nutrition for grazing; thus, the different levels of precipitation experienced can be decisive
during the key moments of gestation.
334
The statistical analysis of the study showed that there was no significant difference (P>0.05)
in the variable of the weight of the recipient female with respect to the pregnancy rate. Similar
data were obtained(23) when determining that there was no difference (P>0.05) in body weight
(346.5 ± 33.4 kg) of the recipients with respect to the gestation rate. They also determined
that individual variation in the potential to achieve daily weight gain (DWG) above 250 g/d
was the main factor affecting pregnancy rates as DWG increases to 350 g/d, so they obtained
this range in their experiment as an optimal threshold to improve results in the averages of
pregnancies achieved. In addition, they concluded that pregnancy rates in embryo recipients
reared under grazing in tropical climates could be improved by selecting females according
to their potential for body weight gain. Contrary to this, in their study, Shorten et al(25) found
significant difference (P<001) as they observed better pregnancy rates with higher body
weight before mating (364 ± 77 kg) in females of the Angus breed.
The study shows a significant effect of the size of the CL on the pregnancy rate, observing
greater probabilities of a gestation when the embryo is transferred in the ipsilateral horn to a
CL3 and 2, presenting a greater probability of not becoming pregnant when the embryo is
transferred in a recipient that has a CL1 < 15 mm in diameter. In addition to this, there is a
greater probability of obtaining a pregnancy by transferring embryos in a development stage
of expanded blastocyst BX and blastocyst BL compared to developments of initial blastocyst
BI and blastocyst in hatching BN. Variables such as THI, animal weight and precipitation
did not present statistical differences that demonstrate the marked influence of the association
of these variables with the pregnancy rate obtained. It is recommended to carry out further
research to obtain more information about the effects of factors such as the breed component
of recipients and embryos that influence the effectiveness of the embryo transfer technique.
Acknowledgements
To the technical and scientific team of the project Identification and analysis of genetic,
nutritional and health factors that affect the rates of gestation by in vitro embryos in cattle in
the department of Norte de Santander with Agreement number 00120, Government of Norte
de Santander and to the Francisco de Paula Santander Ocaña University. In the same way, to
all the producers who opened the doors to research in their production systems and allowed
the timely collection of samples and information for the development of the study.
335
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338
Article
Genetic structure and variability in American bison (Bison bison) in

Mexico
Joel Domínguez-Viveros a
Guadalupe Nelson Aguilar-Palma a*
Rafael Villa-Angulo b
Nancy Hernández-Rodríguez c
José Manuel Pérez-Cantú c
Flora Moir d
Pedro Calderón-Domínguez d
a
Universidad Autónoma de Chihuahua. Facultad de Zootecnia y Ecología. Periférico
Francisco R. Almada Km. 1, CP 31453. Chihuahua, Chihuahua, México.
b
Universidad Autónoma de Baja California. Instituto de Ingeniería. Baja California, México.
c
Fondo Cuenca Los Ojos, Rancho El Uno. Chihuahua, México.
d
Fondo Mexicano para Conservación de la Naturaleza. Chihuahua, México.
*Corresponding author: naguilar@uach.mx
Abstract:
Controlling for genetic variables to managing conservation populations. Single nucleotide

polymorphism (SNP) genetic markers were used to analyze genetic structure and variability
in an American bison population in the state of Chihuahua, Mexico. A total of 174 individuals
were sampled and analysis done of 42,366 SNP distributed in 29 chromosomes. Estimates
were done of expected (He) and observed (Ho) heterozygosity, polymorphic information
content (PIC), the fixation index (FST), the Shannon index (SI), linkage disequilibrium (LD),
339
kinship relationships (Rij; %), and effective population size (Ne). A genetic structure analysis
was run to infer how many lines or genomes (k) define the studied population. A panel with
2,135 polymorphic SNPs was identified and selected, with an average of 74 SNP per
chromosome. In the exclusion process, 84.5 % were monomorphic, 8.5 % had a usable
percentage less than 90 %, 6.3 % had a minor allele frequency less than 0.01 and 0.70 %
exhibited Hardy-Weinberg disequilibrium (P<0.05). Estimated values were 0.30 for the SI,
0.187 for Ho, 0.182 for He, -0.029 for the FST, and 0.152 for PIC. Of the 15,051 Rij estimates
generated, the average value was 7.6 %, and 45.1 % were equal to zero. The Ne was 12.5,
indicating a possible increase of 4 % in consanguinity per generation. Three genetic lines
were identified (proportions = 0.730, 0.157 and 0.113), and, given the study population’s
origin, are probably associated with natural selection or genetic drift. Genetic variability, as
well as Rij levels, must be considered in conservation schemes.
Key words: Heterozygosis, Genetic resources, Effective population size, Consanguinity,

Conservation, SNP.
Introduction
The Bison genus (bison) is native to Asia and central Europe, but migrated to the American
continent via the steppe bison (Bison priscus) and the giant bison (Bison latifrons). Current
populations of American bison (AB; Bison bison) are the product of adaptation, evolution
and natural selection; there are two allopatric subspecies, the plains bison (Bison bison bison)
and the mountain bison (Bison bison athabascae). Historical and archaeological data suggest
that the AB developed on the North American prairies, with estimated populations as high as
60 million individuals. During the 19th Century, bison hunting for food and hides decimated
the population, bringing it near extinction(1,2,3,4).
In Mexico, there are historical accounts of AB in the states of Chihuahua, Coahuila, Durango
and Sonora; the Janos-Hidalgo herd was a transboundary herd that moved between
Chihuahua and New Mexico(5,6). There is currently a conservation herd at El Uno Ranch, in
the Janos Biosphere Reserve (Chihuahua, Mexico), which was created with 23 individuals
from Wind Cave National Park in the United States(7). As a genetic resource, the AB exhibits
the time and space components, as well as use and option values of biodiversity. The time
340
and space components are determined by evolution and changes in species richness, relative
abundance and dominance. The use value consists of the benefits provided by the resource,
and the option value is defined by a genetic resource’s role in or contribution to ecosystem
stability(8,9).
Population biodiversity is the product of adaptation to and integration into ecosystems driven
by evolutionary forces and population genetics (e.g., natural selection, genetic drift and
migration). Genetic diversity is a component of biodiversity and comprises differences in
heritable genetic material. Genetic variability is a measure of genotype differentiation as a
function of population size and the criteria used to define inheritance of genetic material.
Determined by its evolutionary history, a population’s genetic structure expresses the genetic
diversity it harbors and this is distributed within the population. Loss of genetic diversity is
the main challenge in at-risk populations, and is therefore a vital concept in the design of
conservation schemes(10,11). The present study objective was to analyze the genetic structure
and variability of the AB herd at El Uno Ranch using simple nucleotide polymorphism (SNP)
genetic markers.
The AB herd at El Uno Ranch exists in a wild environment with almost no human contact,
and is isolated and protected from populations of other bovids or other species that could
alter its normal development. All ranch personnel are specialized and facilities are
exclusively for managing the herd. A herd census and identification is done annually. For the
present study, 174 animals (80 % of the total herd) were sampled: 102 females and 72 males
born in 2012. Blood deposited on specialized cards in the GeneSeek Laboratory of Neogen®
Corporation was used for DNA extraction. Analyses were done of 42,366 SNP genotypes
distributed in 29 chromosomes and defined in the GGP Bovine 50K chip. During editing,
loci were discarded if they had a usable percentage (UP) <90 %, were monomorphic, had a
minor allele frequency (MAF) <0.01 and/or were in Hardy-Weinberg disequilibrium (HW;
P<0.05).
After editing, the SNPs panel was used to estimate six genetic variability indicators: expected
heterozygosity (He); observed heterozygosity (Ho); polymorphic information content (PIC);
the fixation index (FST); the Shannon index (SI) and linkage disequilibrium (LD)(12,13). The
LD was evaluated based on the correlation (r2) between haplotype frequencies through
loci(14). Correlation (r2) values range from zero to one, with values near zero indicating an
absence of LD and independent segregation and those near 1 indicating non-random
association between loci. The kinship relationship (Rij; %) was estimated using all the
341
sampled individuals and the effective population size (Ne) based on adjusted average r2 via
the Waples method(14). Estimates of r2 were done using the FSTAT program(15); the GenAlex
program(16) was used to estimate He, Ho, PIC and the FST; the LDNE program(17) was used
to analyze Ne; and ML-Relate(18) was applied to estimate Rij.
Genetic structure was elucidated with the Structure genetic analysis program(19). This uses
Bayesian grouping to infer the number of lines or genomes (k) within a population by using
genetic markers for genotype analysis. The procedure assumes that individuals are of pure
ancestry (k= 1) vs. ancestry of two or more lines (k ≠ 1), and proportionally assigns a genome
to each line. Use of Bayesian clustering to infer k is derived from the a posteriori probability
distribution generated by the Markov Chain-based Monte Carlo method. Five possible lines
were evaluated in the present study, and individuals were assigned to them probabilistically.
The number of lines (k) that provides the best fit is derived from the logarithmic likelihood
of each sampling step, and the maximum or optimal value was obtained with the approach
of Evanno et al.(20) and the Structure Harvester program(21).
Editing produced a panel with 2,135 identified and selected SNPs (5.04 % yield versus total
number of evaluated SNPs), with an average of 74 SNPs per chromosome. A total of 40,231
SNPs were discarded: 84.5 % were monomorphic; 8.5 % by UP<90 %, 6.3 % by MAF<0.01,
and 0.70 % by HW in disequilibrium. The panel of selected SNPs had a SI of 0.30, a Ho of
0.187, a He of 0.182, a FST of -0.029, and a PIC of 0.152 (Table 1). The He, Ho and PIC
values determine genetic marker viability in genetic variability studies. All SI estimates were
nearer zero than one, which is associated with homogeneity in the population and reduces
uncertainty when predicting the probability of assignment of an individual to a population.
In all the chromosomes FST had values ranging from -0.002 to -0.062. This indicator measures
levels of heterozygosis and homozygosis, and produces values between -1 and 1. Positive
values indicate a heterozygote deficit and negative ones an excess. Values near zero are a
sign of stability in the homozygous/heterozygous relationship. The average Rij value was
7.61 % based on 15,051 estimates from 174 individuals. Within the 0 to 100 % range of this
indicator, the present results could be classified into five strata: 45.1 % of the estimates were
equal to zero; 14.5 % were from 0.01 to 4.9; 27.3 % from 5.0 to 19.9; 11.8 % from 20.0 to
49.9; and 1.3 % were equal to or greater than 50.0. An individual’s consanguinity (F) is half
the Rij of its parents. Estimates of Rij can therefore be used to select subpopulations for
reproduction and conservation schemes, with a view to maintaining F levels.
342
Table 1: Number of SNPs, and genetic variability estimators per chromosome

Cr ni nf PIC Ho He FST SI r2
1 2587 88 0.155 0.194 0.186 -0.034 0.304 0.022
2 2199 52 0.153 0.193 0.186 -0.029 0.302 0.019
3 2072 56 0.211 0.263 0.260 -0.011 0.403 0.023
4 1933 170 0.117 0.140 0.136 -0.035 0.241 0.222
5 2173 160 0.105 0.126 0.122 -0.025 0.215 0.205
6 2056 70 0.193 0.240 0.234 -0.025 0.372 0.020
7 1858 232 0.195 0.240 0.226 -0.062 0.376 0.324
8 1832 47 0.186 0.228 0.225 -0.024 0.359 0.021
9 1818 57 0.230 0.298 0.289 -0.032 0.437 0.026
10 1736 205 0.089 0.107 0.103 -0.028 0.189 0.288
11 1766 52 0.143 0.174 0.170 -0.019 0.282 0.019
12 1418 49 0.218 0.281 0.270 -0.034 0.415 0.031
13 1544 65 0.151 0.192 0.187 -0.020 0.295 0.127
14 1483 61 0.156 0.190 0.189 -0.013 0.307 0.105
15 1395 59 0.176 0.221 0.212 -0.034 0.342 0.022
16 1302 40 0.194 0.247 0.235 -0.044 0.372 0.024
17 1233 41 0.195 0.246 0.239 -0.021 0.375 0.026
18 1219 33 0.210 0.267 0.255 -0.039 0.399 0.028
19 1218 65 0.124 0.147 0.146 -0.016 0.251 0.210
20 1335 50 0.192 0.238 0.232 -0.031 0.370 0.026
21 1183 33 0.185 0.235 0.228 -0.032 0.358 0.024
22 1017 23 0.197 0.242 0.239 -0.017 0.379 0.029
23 943 35 0.223 0.274 0.275 -0.010 0.425 0.074
24 1081 56 0.162 0.197 0.198 -0.002 0.317 0.113
25 749 115 0.083 0.099 0.094 -0.031 0.179 0.504
26 879 145 0.096 0.108 0.107 -0.015 0.204 0.256
27 724 26 0.248 0.313 0.308 -0.024 0.466 0.036
28 785 21 0.209 0.265 0.263 -0.015 0.401 0.022
29 828 29 0.200 0.254 0.247 -0.025 0.383 0.026
Cr= chromosome; ni= number of evaluated loci; nf= number of polymorphic loci; PIC= polymorphic
information content; Ho= observed heterozygosis; He= expected heterozygosis; FST= fixation index; SI=
Shannon index; r2= average correlation between haplotype frequency through loci.
In the present results Ne was 12.5, and overall average r2 was 0.099, with a range of 0.019 to
0.504 (Table 1). Genetic structure analysis using five lines identified three lines in the study
population, with proportions of 0.730, 0.157 and 0.113 (Figure 1). Based on Ne, the possible
change or increase in inbreeding levels per generation is 4.0 % (ΔF = 1/(2*Ne)). In small
populations managed for conservation, increases in F levels indicate loss of genetic
variability. This can drive consanguineous depression which can affect population viability,
343
survival, reproduction, disease resistance, and environmental stress, among other

factors(22,23). A Ne ≥50 is recommended for populations under conservation management(10),
with the aim of keeping any increase in inbreeding at or below 1 % per generation. For
example, a study of a European bison population reported estimated Ne values of 7.0 to 28.4
through five generations(24). However, population increases did not result in higher Ne values,
highlighting the fact that Ne may be influenced by founding population size and that low Ne
levels may be associated with genetic drift and greater loss of diversity. A population’s
evolutionary potential depends on its genetic variability and Ne values; if Ne is low then
genetic drift is strong and may negatively impact its evolutionary potential(25).
Figure 1: Structure and composition of El Uno Ranch American bison population based on
five lines (k= 1, 2, 3, 4, 5).
Each vertical line represents an individual and segment color represents the proportion of each group.
344
In similar studies, the Bovine SNP50K chip was used in three bison populations (one
European and two American), producing SNP percentages of 1.8, 2.6 and 2.9, and He
estimates of 0.135, 0.197, and 0.199(26,27). A SNP percentage in the same range (2.8 %) was
reported for B. bonasus(28), although higher values (9.35 %) have also been reported for
European bison, with an accompanying Ho of 0.306 and He of 0.250(29). Another study of
European bison identified 1,536 SNPs, distributed at 8 to 136 SNPs per chromosome with an
average of 51.2(30).
The current AB population in the United States of America is derived from a genetic
bottleneck process with significant variability and genetic structure(31,32). Three genetic lines
were defined for the present study population. Given the origins of the El Uno Ranch herd,
the line corresponding to the highest proportion probably corresponds to plains bison. A
complementary line may be a contribution of the mountain bison and a third was likely
generated by separation and development of the studied population. Any differentiation in
the study population may have been caused by the genotype-environment interaction,
although its adaptation and contribution to the ecosystem may also have had an effect.
Genetic isolation between subpopulations affects some demographic and evolutionary
processes; the consequent reduced gene flow can lead to accumulation of genetic differences
between subpopulations(33). Overall, differences within populations can derive from the
genetic diversity of the founding ancestors and their relative contributions, as well as Ne and
its evolution over time(32). Genetic substructure does not always coincide with obvious
morphological or geographic differences between subpopulations. Data from genetic markers
and complementary analyses are required to draw contrasts between populations, identify
possible sources of genetic material, and/or, where appropriate, define any possible
differentiation. For example, a study of eleven bison populations identified genetic
differentiation grouped into eight clusters(32), while another study identified two genetically
distinct subpopulations within the Yellowstone National Park herd(33). Finally, an analysis of
genetic structure in twelve bison herds identified three lines or genetic substructures (average
constitution = 0.412, 0.303 and 0.285)(34).
Three genetic lines were identified within the El Uno Ranch American bison herd. Two may
be associated with the source populations while a third is probably linked to the separation
process and the effects of natural selection or genetic drift. The present results highlight the
need to consider genetic variability and parentage levels when designing reproduction and
conservation plans.
345
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Article
Stover chemical composition in three corn cultivars after sterilization or

colonization with Ganoderma lucidum mycelia
Liz Sarahy Pérez-Martell a
Juan de Dios Guerrero-Rodríguez a*
Daniel Claudio Martínez-Carrera a
Javier Francisco Enríquez-Quiroz b
Efraín Pérez-Ramírez a
Benito Ramírez-Valverde a
a
Colegio de Postgraduados-Campus Puebla. Boulevard Forjadores de Puebla No. 205,
Santiago Momoxpan, 72760, San Pedro Cholula, Puebla, México.
b
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Campo
Experimental La Posta, Km. 22.5 Carretera Federal Veracruz-Córdoba. 94277, Medellín,
Veracruz, México.
*Corresponding author: rjuan@colpos.mx
Abstract:
Nutritional quality in grain by-products such as corn stover can be improved with processes
such as steam sterilization and fungus inoculation. The stover of two native corn cultivars
and one commercial hybrid cultivar were steam sterilized or inoculated with mycelium of the
white-rot fungus Ganoderma lucidum. The experimental design was completely random with
a 3x4 factorial arrangement, one additional treatment and four replicates. The four treatments
were untreated stover, sterilization and immediate drying, sterilization and drying after 15 d,
and colonization with G. lucidum for 15 d; pure mycelia were also analyzed to establish
values for the fungus. Five variables were measured: in vitro dry matter digestibility
(IVDMD), neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin and crude
349
protein (CP). The three cultivars differed (P<0.0001) in terms of digestibility, with cultivar
A having the highest values. Digestibility was lowest (P<0.05) in the G. lucidem-colonized
stovers (P<0.05), intermediate in the untreated stovers and highest in the sterilized stovers.
Contents of NDF, ADF, lignin, and CP differed (P<0.0001) between the cultivars and
treatments (P<0.0001). Cultivar A had less NDF than the other cultivars. The untreated
stovers had less NDF than the sterilized and G. lucidem-colonized stovers. For both ADF and
lignin, the untreated stovers had the lowest values, the sterilized stovers had intermediate
values and the colonized stovers had the highest. Crude protein (CP) differed between the
cultivars (P<0.0001), and the colonized stovers had the highest values (P<0.05). Inoculation
of corn stover with Ganoderma lucidum mycelia did not improve digestibility after fifteen
days colonization, but slightly increased crude protein content.
Keywords: Digestibility, White-rot fungus, Landrace corns, Hybrid corns.
Introduction
Corn stover is a common ingredient in ruminant diets in arid and tropical regions, mainly
when dry or cold conditions restrict plant growth and reduce green forage availability.
However, ruminants can make only limited use of corn stover because it has low nutrient
concentrations and low digestibility(1). Among the many methods of improving stover
digestibility is the use of edible or functional fungi, which can transform plant tissue cell
structure(2).
White-rot fungi have been applied as an alternative for improving stover nutritional quality(2).
For example, species in the Pleurotus genus can degrade lignin through their multienzymatic
system which acts on complex molecules that are difficult for ruminants to degrade(3,4,5).
Species such as Ceriporiopsis subvermispora, Lentinula edodes, Pleurotus eryngii and
Pleurotus ostreatus, increase substrate crude protein content but reduce nutrient
concentrations(6). Strains of Pleurotus florida, P. ostreatus, P. pulmonarius and P. sajor-caju,
have been used to treat corn stover(7); indeed, P. sajor-caju is known to increase crude protein
content and metabolizable energy while reducing lignin content.
350
The white-rot fungus Ganoderma lucidum also degrades lignin, normally within three to five
months post inoculation(8). However, this fungus produces polysaccharides such as mannose,
xylose, arabinose, galactose, glucose and rhamnose(9-12), as well as chitin(13), all of which can
increase fiber content. In addition, when used in corn stover it can cause the autoclave
sterilization process to produce net negative results since nitrogenous compounds in the
stover are solubilized(6). The fungus strain used to improve stover quality is therefore vital
because each requires a carbohydrate source and a certain time to colonize the substrate, and
its long-term consumption must be safe for animals(14). To date, no data is available on the
effects of G. lucidum on corn stover during short colonization times. The present study
objective was to analyze the chemical composition of corn stover from two landrace cultivars
and one hybrid, when unprocessed, sterilized and dried at two different times, or inoculated
with G. lucidum.
Corn cultivars
Two landrace corn cultivars (named A and C) and the hybrid cultivar Aspros 1503® (assigned
the letter B), all with white grains, were planted under natural rainfall conditions, in the
municipality of Cuautinchan, Puebla state, Mexico. The three cultivars were planted in a 200
m x 50 m area, following a randomized block design, with four replicates. The landraces
were selected from the previous harvest of two producers located in the same municipality,
both known for conserving and improving corn cultivars following traditional practices. The
hybrid seeds, also from the previous year’s harvest, were purchased from an Aspros seed
distributor in the same municipality.
Stover preparation
The corn grain was harvested 170 d after planting. At this time, five plants were taken from
each replicate, and ground in a two-centimeter mill with sieve. Average stover dry matter
(DM) yield was 300 g per replicate. The material for each replicate was divided into four
parts corresponding to the four treatments: untreated stover (untreated); sterilized stover
dried immediately (sterilized/dried); sterilized stover stored in moisture for 15 d
(sterilized/dried at 15 d); and sterilized stover inoculated with G. lucidum (G. lucidum-
colonized).
351
Sterilization was done with an autoclave (All American®) at 121.5 °C for 25 min. To evaluate
nutrient leaching, immediately after sterilization one portion of sterilized stover was dried at
60 °C to constant weight in a forced-air oven (Thermo Scientific® Model 3478); this is the
sterilized/dried treatment. To evaluate the effect of storage in moisture, which can generate
hydrolysis reactions that modify stover nutritional quality, a second portion of sterilized
stover was placed in Petri dishes (20 g DM per dish) for 15 d and then dried; this is the
sterilized/dried at 15 d treatment. A third portion of sterilized stover (20 g DM) was placed
in a Petri dish and inoculated with G. lucidum (G. lucidum-colonized).
Fungus strain preparation and stover inoculation
The fungus was G. lucidum CP-145, obtained from the Center for Biotechnology of Edible,
Functional, and Medicinal Fungi (Centro de Biotecnología de Hongos Comestibles,
Funcionales y Medicinales) of the Colegio de Postgraduados, Puebla Campus. The strain was
cultured in Petri dishes in potato dextrose agar (PDA) culture medium for 8 d, sufficient time
for growth of mycelium. Five discs (5 mm diam.) with mycelia were used to inoculate the
stover. The inoculated stover was monitored every three days until 15 d post inoculation, the
time required for maximum mycelial colonization and the average reported time required to
attain the highest fungal enzymatic activity(15).
Stover processing
All samples were dried in a forced-air oven at 60 °C to constant weight and ground in a
cyclone mill (Foss Tecator®) with 1 mm mesh. The ground samples were stored in resealable
plastic bags until nutritional quality analysis.
A separate culture was done of G. lucidum alone to quantify the direct contribution it might
make to stover nutritional quality. Before inoculation with G. lucidum, potato dextrose broth
(PDB; Difco™) (24 g L-1) was sterilized in an autoclave (All American®) at 121.5 °C and 15
lb ppm-2 for 25 min. The medium was inoculated by depositing five circles (5 mm diam)
from an eight-day-old G. lucidum colony grown on PDA medium. It was incubated in an
orbital shaker (Thermo Scientific®) at 120 rpm and 27 ºC for 20 d. After harvesting, mycelia
were recovered by filtering through Whatman #1 filter paper placed in a Buchner funnel in a
Kitazato flask connected to a vacuum pump. The recovered biomass was placed in aluminum
trays and dried at 60 °C in a drying oven (Felisa®) for 48 h. It was ground in a ceramic mortar
and stored in a resealable plastic bag for subsequent nutritional quality analysis.
352
Evaluated variables
Five variables were measured in the stover treatments and the G. lucidum culture: neutral
detergent fiber (NDF)(16); acid detergent fiber (ADF)(16); lignin(16); in vitro dry matter
enzymatic digestibility (IVDMD)(17,18); and crude protein (CP)(19).
Experimental design and statistical analysis
A completely randomized experimental design was applied including the three cultivars (A,
B and C) and the four treatments (untreated, sterilized/dried, sterilized/dried at 15 d and G.
lucidum-colonized), as well as the pure fungus culture(20). After verifying data normality, an
ANOVA was run. Differences between means were identified with a Tukey test, at an α=0.05
significance level. Data analyses were run with the SAS ver. 9.0 statistical program(21).
Results
In vitro dry matter digestibility (IVDMD) differed (P<0.0001) between the corn cultivars due
mainly to treatment; there was also an interaction (P<0.0001) between two cultivars in terms
of treatment (Figure 1).
Figure 1: In vitro dry matter digestibility (IVDMD) of the three evaluated corn stovers in
four treatments, including colonization with the white rot fungus Ganoderma lucidum
Bars represent honestly significant difference in the comparison of means.

abc
Different lowercase letters on bar points indicate significant difference (P≤0.05).
353
The untreated stover had IVDMD values intermediate (63.7 %) to those of the sterilized
(70.4 %) and colonized stovers (58.7 %). Cultivar A was 7.5 % more (P<0.05) digestible
than cultivar B and 7.1 % more than cultivar C. Both the sterilized/dried, and sterilized/dried
at 15 d treatments had digestibility 6.7 % higher (P<0.05) than the untreated stovers. Among
the untreated stovers, cultivar A had the highest (P<0.05) digestibility (73 %) followed by
cultivars B (69.3 %) and C (68.9 %).
Compared to the untreated and sterilized stovers, the G. ludicum-colonized stovers had lower
(P<0.05) digestibility values: 62.0 % for cultivar A; 57.1 % for B; and 57.0 % for C. Among
the colonized treatments, cultivar A had higher (P<0.05) digestibility than cultivars B and C.
Pure cultured G. ludicum exhibited the overall lowest digestibility (14.7 %) (Figure 1).
Neutral detergent fiber (NDF) differed between cultivars (P<0.0001), mainly in response to
treatment (Figure 2); an interaction (P<0.0001) was apparent in cultivar C. Among the
untreated stover treatments, cultivar A had a NDF content 9 % lower (P<0.05) than that of
cultivar B and 5 % lower than that of cultivar C; cultivar B had the overall highest (P<0.05)
NDF content. Neutral detergent fiber (NDF) content generally increased (P<0.05) in
response to sterilization and G. ludicum colonization, an effect most notable in the landraces
(cultivars A and C).
Figure 2: Neutral detergent fiber (NDF) concentration of the three evaluated corn stovers
in four treatments, including colonization with the white rot fungus Ganoderma lucidum

abc
354
The G. lucidum-colonized stovers had higher (P<0.05) NDF concentrations than the other
treatments. Among the sterilized/dried treatments , cultivar A had a lower concentration
(68.6 %) than cultivars B and C, which did not differ (P>0.05). The G. lucidum-colonized
stovers exhibited higher NDF (P<0.05) than the untreated stovers; 8.6 % higher in cultivar
A, 3.2 % higher in B and 8.1 % higher in C. Of note is that the colonized cultivar B stover
had a NDF that did not differ from those of the sterilized/dried treatments. Also, among the
cultivar C stovers NDF did not differ between the colonized and sterilized/dried treatments,
but both differed (P<0.05) from the sterilized/dried at 15 d treatment. In contrast, in cultivar
A NDF in the colonized treatment did not differ from the sterilized/dried at 15 d treatment
but did differ from the sterilized/dried treatment (Figure 2). The pure G. lucidum had NDF
content (88.2 %) much higher (P<0.05) than the in the stover treatments (Figure 2).
Acid detergent fiber (ADF) differed among the cultivars (P<0.0001), with a clear effect from
treatment (P<0.0001)(Figure 3), in addition to an interaction effect (P<0.0001). The
untreated stovers did not differ in terms of ADF and all had the lowest (29.0 to 29.9 %) values
among the treatments. The sterilized/dried stovers had higher (P<0.05) ADF concentrations
than the untreated stovers; 5.1% higher than cultivar A, 2.8 % higher than B and 5.7 % higher
than C. In both the sterilized/dried and sterilized/dried at 15 d treatments, ADF differed
minimally (3 %) between cultivars B and C. The sterilized/dried at 15 d treatment had a
higher (P<0.05) ADF content than the untreated stovers; 2.9 % higher in cultivar A, 2.2 %
higher in B and 4.4 % higher in C. When comparing the sterilized/dried treatment with the
sterilized/dried at 15 d treatment, difference (P<0.05) was observed only in cultivar A
(2.2 %). In the sterilized/dried treatment, ADF in cultivar C was 2.4 % higher (P<0.05) than
in cultivar A (Figure 3).
Figure 3: Acid detergent fiber (ADF) concentration of the three evaluated corn stovers in
four treatments, including colonization with the white rot fungus Ganoderma lucidum

abc
355
When colonized with G. lucidum, all three stovers exhibited higher (P<0.05) ADF
concentration than the untreated stovers; 7.0 % higher in cultivar A, 8.5 % in B and 9.6 % in
C. When compared to the sterilized/dried treatment, the colonized cultivar A stover had
1.9 % more ADF, cultivar B had 5.7 % more and cultivar C had 3.9 % more (P<0.05). The
colonized stover also had higher (P<0.05) ADF than the sterilized/dried at 15 d treatment:
4.1 % higher in cultivar A, 6.3 % higher in B and 5.2 % higher in C. Among the colonized
stovers, only cultivar C had higher (P<0.05) ADF than cultivar A (3.5 %). The pure G.
lucidum contained 68 % ADF, far more than all the stover treatments (P<0.05) (Figure 3).
Lignin content differed (P<0.0001) between the three cultivars, with a notable effect
(P<0.0001) from treatment and a slight but significant (P<0.0001) interaction (Figure 4). The
untreated stovers had the lowest (P<0.05) lignin values (2.1-2.5 %), with only a 0.4 %
difference (P<0.05) between cultivars C and B (Figure 4). The sterilized/dried stovers had
higher (P<0.05) lignin contents than the untreated stovers; 1.0 % more in cultivar A, 1.4 %
more in B and 0.7 % more in C. Lignin content did not differ between the sterilized/dried
stovers. This variable was also higher (P<0.05) in the sterilized/dried at 15 d treatment than
in the untreated stovers (0.9 % higher in cultivar A, and 1.5 higher in B), although it did not
differ (P>0.05) in cultivar C (0.4 %). Indeed, in the sterilized/dried at 15 d treatment lignin
content in cultivar C was 0.5 % lower than in cultivar A and 0.8 % lower than in cultivar B
(Figure 4).
Figure 4: Lignin content of the three evaluated corn stovers in four treatments, including
colonization with the white rot fungus Ganoderma lucidum

abc
356
Lignin content was highest (P<0.05) overall in the G. lucidum-colonized treatment in all
three cultivars. Compared to the untreated stovers, colonization with G. lucidum increased
lignin content by 1.9 % in cultivar A, 2.5 % in B and 1.6 % in C. The colonized stover also
had higher lignin content than both the sterilized treatments: about 1% higher for cultivars A
and B (both treatments); 0.9 % higher than the cultivar C sterilized/dried treatment, and
1.3 % higher than the cultivar C sterilized/dried at 15 d treatment (Figure 4). Within the G.
lucidum-colonized treatment, lignin content was 0.5 % lower in cultivar C than in cultivar B.
The highest overall lignin content (6.4 %) was in the pure G. lucidum (Figure 4).
Crude protein (CP) content differed (P<0.0001) between cultivars, which was affected
(P<0.0001) by treatment and exhibited an interaction effect (P<0.0001) (Figure 5). This
variable was lowest (P<0.05) in untreated cultivars A (1.1 %) and B (2.0 %); however,
untreated cultivar C had 3.0 % CP, higher (P<0.05) than the other two cultivars. Most of the
sterilized stovers had CP content higher than the corresponding untreated stover; again, the
exception was cultivar C for which this variable was lower (P<0.05) in the sterilized/dried
treatment than in the untreated stover. The G. lucidum-colonized stovers all had higher
(P<0.05) CP content than the untreated and the sterilized stovers: 7.2 % in cultivar A, 4.2 %
in B and 3.8 % in C. The pure G. lucidum had the highest (P<0.05) CP content (9.4 %) overall
(Figure 5).
Figure 5: Crude protein (CP) content of the three evaluated corn stovers in four treatments,
including colonization with the white rot fungus Ganoderma lucidum

abc
357
Discussion
The sterilized stovers lost hydrosoluble substances. This coincides with previous reports of
losses of carbohydrates, soluble proteins, organic and inorganic acids, and minerals caused
by sterilization with high-pressure steam(5,6). This would explain the lower fiber content in
the unsterilized stovers, since the absence of leaching, and consequent nutrient loss, would
have allowed them to maintain a higher soluble substances content by weight (mainly cellular
contents). Sterilizing by autoclave would therefore have caused a dilution effect in stover
NDF content.
The three evaluated cultivars varied in terms of cell wall and cellular contents. Cultivar A
had the lowest amount of cell wall (perhaps due to thinner walls or a different chemical
composition), while the hybrid (cultivar B) was more fibrous, a possible genetic
differentiation related to plant architecture (i.e. erect leaves). Hybrid corns have been
developed with erect leaves to increase sowing density, capture more solar radiation and
therefore increase yield(22,23). This trait also implies increased leaf venation, and changes in
venation pattern and sclerenchyma(24), which result in higher fiber content. Inter-cultivar
differences in cell wall fiber content, even among hybrids, is widely reported and confirms
diversity between cultivars(15,25).
The stovers colonized with G. lucidum contained more fiber than the untreated stovers, as
shown in NDF values. This fungus was also found to be quite fibrous. Several reports indicate
that in G. lucidum fibrous compounds are synthesized in the mycelium and contain high
concentrations of polysaccharides such as mannose, xylose, arabinose, galactose, glucose
and rhamnose(9,10,11,12), as well as chitin(13). This suggests that in the colonized stover NDF
values were higher due to the combination of the fungus’s insoluble compound content and
the stover fiber content. Some cell wall degradation by the fungus was expected, but the
mycelial development time and evaluated colonization time used in the present study may
have been insufficient to permit significant lignin solubilization. For example, when
inoculated onto a mixture of maple, chestnut and blackberry with other ingredients, G.
lucidum did not degrade lignin until three to five months post inoculation(8). Only 15 d
colonization was allowed in the present study, which is apparently not enough time to detect
degradation of cell wall components. It is possible that the fungus began by metabolizing
stover cell content, which could explain the lack of any significant decrease in NDF content.
This is supported by previous studies of fungus-inoculated corn stovers. Various maize
hybrid stovers inoculated with fungi such as Sporotrichum pulverulentum, Bjerkandera
adusta and Trametes trogii, were found to have higher concentrations of the β-glucosidase
and exoglucanase enzymes during the first fifteen days’ incubation(15). This indicates a
preference for cell parts containing reducing sugars, including those easily available in cell
358
contents. Several fungi, including brown-rot fungi (Serpula lacrymans, Coniophora puteana
and Gloeophyllum trabeum, among others) consume large amounts of fermentable sugars(26),
which are very accessible in the cell content. White-rot fungi are also known to consume a
higher proportion of non-structural sugars during initial colonization(27).
Comparing the present results obtained with G. lucidum with previous research is challenging
because many studies have utilized substrates composed of 80 % corn stover mixed with
other grain-derived ingredients(28). In addition, fungus growth times are not mentioned in
some studies. However, decreases in NDF of up to 5 % have been reported in an inoculated
substrate (59.76 %) versus an uninoculated control (64.94 %)(28); this is a larger decrease than
observed in the present results.
The principal difference between the evaluated cultivars was NDF content, since both ADF
and lignin content differed little between them (Figures 3 and 4). Therefore, the main
differences between cultivars were due to the contents of hemicelluloses and other soluble
substances in the neutral detergent fiber. Sterilization increased fiber content in the treated
corn stovers, and inoculation raised it higher in response to higher ADF and lignin contents.
Acid detergent fiber (ADF) levels in untreated cultivar A stover were lower than in the
sterilized/dried treatment, implying that hydrolysis may have occurred in the cell wall, which
contributed to the interaction effect. The cultivar C stover had the highest lignin content,
which did not change significantly after sterilization and drying after 15 d, and probably
contributed to the interaction effect. These data suggest that the evaluated cultivars differ in
terms of cell wall composition.
Both ADF and lignin contents were highest in the stovers colonized with G. lucidum. Some
edible fungi can degrade lignin in forages via enzymes and other compounds(2), although this
was not observed in the G. lucidum-colonized corn stovers evaluated here. One possible
explanation is that this fungus contains chitin(13) and traces of lignin(9-12), which would have
caused the colonized stovers to exhibit higher values for these two variables. This contrasts
with a previous study in which inoculation of corn stover (14.9 % initial lignin content) with
G. lucidum reduced lignin values by 6 %, although fungus growth times and stover
preparation methods are not specified(29).
In vitro dry matter digestibility (IVDMD) was higher in the sterilized/dried and
sterilized/dried at 15 d treatments than in the untreated and G. lucidum-colonized stovers.
The stovers in both of the sterilized treatments exhibited higher values for the three evaluated
fibers, but the pressure, temperature, and possibly the water vapor, involved in the
sterilization technique increased their digestibility. Pressurized steam treatment of corn
stover has been reported to improve digestibility by reducing polymerization, breaking down
359
bonds between hemicellulose, cellulose and lignin, hydrolyzing hemicellulose, increasing

porosity, and changing the cellulose crystalline structure(30).
In cultivar C, sterilization followed by humid storage for 15 d prior to drying resulted in more
hydrolysis than with sterilization followed immediately by drying, which contributed to the
interaction effect. This may have occurred due to differences in composition of the
synthesized hemicellulose compounds. Sterilization of substrates at temperatures greater than
100 °C and in moisture for more than 45 min degrades macromolecule structure and function
due to denaturation and hydrolysis(31,32). Temperatures higher than 85 °C partially break
hydrogen bonds in the lignin-cellulose complex, solubilizing simple sugars(33), which may
explain the increased digestibility in the sterilized treatments.
In the untreated stovers, digestibility was lower in cultivars B and C than in A, highlighting
inter-cultivar differences in digestibility(15). The overall lowest digestibility values were
observed in the G. lucidum-colonized stovers because fungus mycelium components such as
chitin, lignin and structural polysaccharides apparently negatively influenced digestibility(9-
12)
.
Colonization with G. lucidum increased CP content in all three evaluated corn stovers.
Several proteins have been isolated from G. lucidum mycelia, including LZ-8, which contains
110 amino acids(34). A polysaccharide-protein complex has also been found in G. lucidum
which contains various essential amino acids(35). These amino acids may have increased CP
content in the colonized stovers. Inoculation with other fungi, such as strains of Ceriporiopsis
subvermispora, Lentinula edodes, Pleurotus eryngii or Pleurotus ostreatus, has also been
reported to increase CP content by approximately 30%(6); in the present study this was
notable in cultivar Criollo A.
Compared to the untreated stovers, both the sterilized/dried and sterilized/dried at 15 d

treatments had higher CP values. This probably occurred due to hydrolysis of cell wall
compounds, since substrates sterilized at temperatures above 100 °C with moisture are known
to lose macromolecule structure and function(31,32). The cultivar C stover in the
sterilized/dried at 15 d treatment was the one exception in that CP content decreased, possibly
because its components were more soluble, which contributed to the interaction effect.
Further study is needed to better understand the causes of this loss.
360
The three untreated stovers differed in terms of NDF and CP contents, but not in ADF and
lignin. Digestibility among the untreated stovers was highest in cultivar A. Steam sterilization
increased NDF and ADF content. It also raised lignin content, but not in cultivar C, which
exhibited the most pronounced negative interaction. Stover crude protein content differed in
response to sterilization, increasing it in one, leaving it unchanged in others, and even
lowering it another. Digestibility generally increased after sterilization, indicating the strong
effect of high temperature and pressure. Colonization with G. lucidum for 15 d did not
improve digestibility, but rather lowered it by increasing fiber concentrations. In contrast, CP
content increased after G. lucidum colonization (7.2 % for cultivar A, 4.2 % for B and 3.8 %
for C), raising it above CP contents in both the untreated and sterilized treatments.
Colonization with the white-rot fungus Ganoderma lucidum increased crude protein content
in the three evaluated corn stovers but did not affect in vitro digestibility. Longer fungus
growth times would be required to detect changes in the corn stover cell wall, mainly via
solubilization of lignin and other elements, and any consequent improvement in digestibility.
Acknowledgements
The Consejo Nacional de Ciencia y Tecnología (CONACYT) granted a doctoral sholarship

to LSPM.
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Article
Nutrient concentrations, in vitro digestibility and rumen fermentation of

agro-industrial residues of Cannabis sativa L. as a potential forage source
for ruminants
Elia Esther Araiza-Rosales a
Esperanza Herrera-Torres b
Francisco Óscar Carrete-Carreón c
Rafael Jiménez-Ocampod
Daniel Gómez-Sánchez e
Gerardo Antonio Pámanes-Carrasco f*
a
Universidad Juárez del Estado de Durango. CONACYT. Facultad de Medicina Veterinaria
y Zootecnia. Carretera Durango-Mezquital km 11.5, Durango, Dgo. México.
b
Tecnológico Nacional de México, Instituto Tecnológico del Valle del Guadiana. México.
c
Universidad Juárez del Estado de Durango. Facultad de Medicina Veterinaria y Zootecnia.
d
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. México.
e
Instituto de Investigación del Aprovechamiento de la Cannabis sativa. México.
f
Universidad Juárez del Estado de Durango. CONACYT. Instituto de Silvicultura e Industria
de la Madera. México.
*Corresponding author: gerardo.pamanes@gmail.com
Abstract:
This study aimed to determine the concentration of CP, EE, NSC, fibers, TPC, CT, CBD,
THC, in vitro digestibility of dry matter and rumen fermentation parameters of agroindustrial
366
residues of Cannabis sativa L. from two extractive processes of cannabinoids, as a potential

source of forage in ruminants feeding. The flower of Cannabis sativa was exposed to cold-
press extraction (CPC) and alcoholic extraction (AEC) process; vegetative residues obtained
after extractions were compared to raw flower as a control (RFC) using a completely
randomized design and Tukey’s test for means comparison. Extractive processes decreased
EE, TPC and cannabinoids (CBD and THC). Otherwise, fibers, NSC and digestibility,
increased after the extractive processes in CPC and AEC. Similarly, in vitro degradability
increased after both extractive processes above 120 % as well as latency period. Additionally,
protozoa increased with CPC but no changes were observed in AEC. Likewise, no changes
were observed in cellulolytic bacteria in CPC and AEC. However, total bacteria were reduced
after both extractions. Moreover, N-ammonia in ruminal fermentations decreased with CPC
and AEC whereas total volatile fatty acids increased. In addition, gas production increased
above 75 % in CPC and AEC; however, no changes were observed in latency period.
Furthermore, methane and CO2 production increased above 80 and 60 %, respectively for
CPC and AEC; these augmentations are positively associated with improvements in the
ruminal fermentations. In conclusions, the agroindustrial residue of Cannabis sativa L.
obtained after the analyzed extractive processes may arise as a potential forage source in
ruminants feeding.
Keywords: Hemp, Methane, Degradability, Ruminal fermentation, Gas production kinetics.
Introduction
The general acceptance and regulatory outlook concerning to Cannabis spp. crops have
changed in the past few years. This plant is no longer treated as a solely source of
psychotropic agents; biological compounds have been focused on therapeutically approaches
successfully(1). In fact, the cannabidiol (CBD) and tetrahydrocannabidiol (THC) are the main
bioactive compounds contained in the plant; these compounds are synthetized mostly in the
flower and leafs(2). Due to the latter, pharmaceutical industry has been attempting in updating
and becoming purer the extractive compounds of Cannabis spp. for their use in medicines
and drugs (1,3). In fact, some crops of Cannabis sp. have been used as a supplier of hemp for
textile industry(4), for production of biofuel(5) and as a component in the automotive
industry(6). In addition, some livestock farmers use seeds as additives in animal feeding(7).
367
However, the extractive methods of bioactive compounds of the plant generate agricultural
residue which may contain minimal concentrations of cannabinoids but considerable contents
of fiber, cellulose and hemicellulose; these contents may arise as an important forage source
in animal feeding, mainly ruminants. Worldwide information about production and
plantations of Cannabis sp. is limited due to legal traits. Nevertheless, reports from the USDJ
affirmed that the hemp production reached 10,000 t in Mexico in 2006; even though
plantations above 31,000 ha were eradicated(8). Accordingly, an increasing production of
Cannabis sp. for extraction of cannabinoids with medical purposes may represent a
substantial expansion of agricultural residue that may potentially be used as a forage source
in ruminants feeding (9). However, published information about the nutritional value of by-
products of hemp in animal nutrition is limited. Furthermore, this study aimed to determine
the concentration of crude protein (CP), ether extract (EE), non-structural carbohydrates
(NSC), fibers, total phenolic compounds (TPC), condensed tannins (CT), cannabidiol (CBD),
delta-9-tetrahydrocannabinol (THC), in vitro digestibility of dry matter and rumen
fermentation parameters of agro-industrial residues of Cannabis sativa L. from two
extractive processes of cannabinoids as a potential source of forage in the ruminants feeding.
Material y methods
Study area
This study was carried out in the Faculty of Veterinary Medicine and Husbandry of the
Durango State Juarez University, located in Durango, Mexico.
Ingredients and feedstuffs
All vegetative material was donated by the IIAC (Institute for Research and Exploitation of
Cannabis) located in Durango, Mexico. The flower of Cannabis sativa L. was processed by
two methods of extraction, which were performed by the IIAC. Briefly, samples of the flower
of Cannabis sativa L. were cold-pressed for oil obtaining; the process did not exceed 35 ºC
and no solvents were used. The obtained residues after cold-pressing were named CPC due
to the cold-presses Cannabis sativa L. flower cake obtained. On the other hand, other samples
of the flower of Cannabis sativa L. were exposed to an alcoholic extraction, which was
carried out at room temperature for 5 h. Later, the by-product or residue was obtained by
filtering the solvent; this treatment was named AEC (alcoholic extraction of Cannabis sativa
368
L. flower cake). Thus, using raw flower of Cannabis sativa L. (RFC) as a control, it was
compared to agro-industrial residues obtained after extractive processes. Afterwards,
samples of agro-industrial wastes of Cannabis sativa L. were dried in a forced air oven
(Felisa, Model FE-294AD) at 55 ºC for 48 h and were ground in a miller (Thomas Wiley
Miller Lab, Model 4) at a 1 mm particle size. Consequently, samples were stored for further
analyses.
Chemical composition
Samples were analyzed for dry matter (DM), ether extract (EE), crude protein (CP) and ashes
according to standardized procedures(10). Cellulose, hemicellulose, neutral detergent fiber
(NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were evaluated according
to proposed by Van Soest et al(11). Non-structural carbohydrates (NSC) were estimated with
the following equation:
NSC= 100 – (CP+EE+NDF+A); where NSC= non-structural carbohydrates (% DM); CP=

crude protein (% DM); EE= ether extract (% DM); NDF= neutral detergent fiber (% DM);
A= ashes (% DM).
In vitro dry matter digestibility (IVDMD) was estimated using the DAISYII® equipment
(ANKOM Technology Corp., Macedon, NY) and according to manufacturer procedures(12).
Metabolizable energy was calculated according to the following equation(13):
ME = [2.20 + 0.136(GP24) + 0.0057(CP) +0.0029(EE)2]/4.184; where ME= metabolizable

energy (MCal/kg); GP24= in vitro gas production at 24 h (ml/g); CP= crude protein (g/kg);
EE= ether extract (g/kg).
Secondary metabolites
Dry samples for each treatment were exposed to alcoholic extraction (0.5 g dissolved in 45
ml of 70% ethanol-water solution) during overnight. Afterwards, samples were filtered and
vacuum-evaporated (at 40 ºC) until total removal of ethanol solution and leave them dry
overnight. Yields of concentrated extractions were calculated based on dry matter. Dry
samples were stored for further secondary metabolites analyses.
369
Analysis of condensed tannins (CT)
Briefly, samples for each treatment were diluted (0.5 g dissolved in 45 ml of 70% ethanol-
water solution) and let them extract during overnight. Later, 50 µL aliquots were mixed with
a 4% solution of vainillina-methanol and concentrated HCl according to Heimler et al(14).
Absorbance was measured at 500 nm using catequine as standard. Yield of CT was estimated
with the final concentration in solution and yield in dry matter.
Analysis of total phenolic compounds (TPC)
Briefly, samples for each treatment were diluted (0.5 g dissolved in 45 ml of 70% ethanol-
water solution) and let them extract during overnight. Total phenolic compounds were
estimated through the Folin-Cioucalteau method adapted by Dewanto et al(15) using gallic
acid as standard and measuring absorbance at 760 nm for every diluted sample. Yield of TPC
was estimated with the final concentration in solution and yield in dry matter.
Analysis of cannabinoids
For the detection of cannabinoids (specifically CBD and THC) was used the method of thin
layer chromatography (TLC), according to procedures proposed by Novak et al(16). This trial
was conducted at the facilities of the IIAC.
In vitro dry matter degradability (IVDMD)
For this analysis, 1 g (DM) of sample from each experimental treatment was placed in nylon
bags (F57, ANKOM Technology, Corp., Macedon, NY) into glass modules equipped with
electronic transducers for pressure measuring according to manufacturer’s procedures
(ANKOM, USA) and incubated in triplicate with buffer solutions (CaCl2 13.2% w/v; MnCl2
10% w/v; CoCl2 1% w/v; FeCl3 8% w/v; NaHCO3 39% w/v) and ruminal inoculum in a 2:1
ratio, according to Theodorou et al(17). Ruminal inoculum was obtained from two fistulated
Angus steers fed with alfalfa hay based diet before the morning feeding; ruminal liquor was
extracted from rumen and immediately placed into a thermal container preheated at 39 ºC
and then it was transported to the lab for further analysis. The Nylon bags were incubated
370
individually for each fermentation time (0, 3, 6, 12, 24, 36, 48, 72 and 96 h). Bags were
removed from modules and washed until attaining crystalline water. They were placed in the
oven at 55 ºC for 48 h. Changes in dry matter were registered and digestibility data was fitted
into the Gompertz function for kinetics parameters estimation according to Murillo et al(18).
−(𝑘 𝑡)
𝐷𝑒𝑔 = 𝐴𝑑 𝑒 −𝐿𝑑 𝑒 𝑑
Where: Deg= dry matter degradability (% DM); Ad= maximum degradability (% DM); kd=
degradability constant rate (h-1); Ld= latency time before degradability begins (h).
In vitro gas production and ruminal fermentation parameters
For in vitro gas production, 1 g of sample from each treatment previously dried was placed
into glass modules equipped with pressure transducer (ANKOM, USA) with 120 mL a mix
of buffer solutions (CaCl2 13.2% w/v; MnCl2 10% w/v; CoCl2 1% w/v; FeCl3 8% w/v;
NaHCO3 39 % w/v) and ruminal inoculum in a 2:1 and were incubated at 39 ºC for 24 h by
triplicate according Theodorou et al(17); ruminal inoculum was obtained from two fistulated
Angus steers fed with alfalfa hay based diet before the morning feeding; ruminal inoculum
was extracted from rumen and immediately placed into a thermal container preheated at
39 ºC and then it was transported to the lab for further analysis. Thus, changes in gas volume
were registered at 0, 3, 6, 12, 24, 36, 48, 72 and 96 h and data was fitted into the Gompertz
function for kinetics parameters estimation according to Murillo et al(18).
−(𝑘𝑔 𝑡)
𝐺𝑃 = 𝐴𝑔 𝑒 −𝐿𝑔 𝑒
Where: GP= gas production (ml); Ag= maximum gas production (ml); kg= gas production
constant rate (h-1); Lg= latency time before gas production begins (h). Meanwhile, two 10 mL
aliquots of in vitro ruminal fermentation were destined for determination of in vitro ruminal
parameters after 24 h of fermentation time, were processed with metaphosphoric acid (25%
w/v) and sulfuric acid (50% v/v) for volatile fatty acids (VFA) and nitrogen-ammonia (N-
NH3), respectively and according to Galyean(19).
Likewise, the same number of experimental treatments were incubated in glass modules
(ANKOM, USA) until 24 h of fermentation time(17). Once the time was elapsed, modules
pressure release valve was opened and released gas was measured for methane and CO2
compositions according to procedures proposed by Herrera Torres et al(20) using the portable
gas analyzer GEM5000 (Landtec, USA).
371
Determination of rumen bacteria and protozoa
This assay was determined by weighing 1 g of sample from each treatment previously dried
and placed into glass modules equipped with pressure transducer (ANKOM, USA) with 120
mL a mix of buffer solutions and ruminal inoculum in a ratio 2:1, and were incubated at
39 ºC for 24 h by triplicate(17); ruminal inoculum was obtained from two Angus steers fed
with alfalfa hay based diet. Afterwards, concentrations of total and cellulolytic bacteria were
determined according to Dehority(21), protozoa were analyzed according to Ogimoto and
Imai(22), whereas fungi were determined according to Joblin(23) for each treatment. Briefly,
culture mediums were prepared in sterile Petri dishes with nutritive agar (BD, Bioxon, USA)
for total bacteria determinations and nutritive agar (BD, Bioxon, USA) plus
carboxymethylcellulose (SIGMA, USA), as a cellulose source for cellulolytic bacteria
determinations. Inoculum was obtained from in vitro ruminal fermentations of treatments
and dilutions were carried out until 10-6 was reached. After, dilutions with inoculum were
placed in previously labeled Petri dishes and incubated under CO2 atmosphere at 39 ºC. Total
bacteria dishes were incubated for 48 h whereas cellulolytic bacteria dishes were incubated
for 72 h. Once the incubation time was elapsed, Petri dishes were opened and microorganisms
were measured using the most likely number technique (24). For fungal determination, culture
medium was prepared with PDA agar (3 %) and placed into plates under sterile conditions.
Later, 10 mL of in vitro ruminal inoculum was mixed with peptone solution (1:9 ratio) and
dilutions were prepared with 1 ml of inoculated solution until 10-5 was reached and placed in
plates previously labeled. Immediately, a sample of each dilution is flushed with CO2 and
incubated under CO2 atmosphere at 39 ºC for 120 h for identification. For protozoa
determination, in vitro ruminal inoculum was filtered through four layers of cheese clothes
and obtained filtered was placed in a separation funnel during 15 min approximately, until
protozoa precipitated (a whitish ring appeared at the bottom of the funnel). Later, 1 mL of
the obtained sample with protozoa was mixed with 4 mL of ruminal inoculum (previously
incubated at 39 ºC and flushed under CO2 conditions) in 18 x 150 mm culture tubes and
placed one more time in the incubator; this last step was repeated five times for each dilution
until 10-5 was reached. Consequently, protozoa number was determined through direct
quantification in Neubauer chamber using a contrasting microscope (Collegiate 400) and an
amplification of 400x.
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Statistical design
Obtained data for all agroindustrial residues were analyzed through a completely randomized
design using the GLM procedure in SAS(25). Means comparison was evaluated with the
Tukey multiple range test declaring significances at P<0.05.
The chemical composition and dry matter digestibility for treatments are shown in Table 1.
The used method for the extraction of cannabinoids affected mainly contents of ash, EE, and
fibers (NDF and ADF). In fact, cannabinoids are separated from the flower in an organic
phase, which is removed within the EE fraction(3). As a consequence, there is a reduction of
95 % and 40 % in EE for AEC and CPC treatments after extraction, respectively (P<0.05).
Otherwise, ash content increased 17 and 8 % in AEC and CPC, respectively when compared
to the control (RFC) (P<0.05). These augmentations are positively associated with a
redistribution of the chemical components after the extractive process; remained values of
NDF, ADF and NSC represent higher fractions in the chemical composition while reducing
the EE fraction once it was extracted. On the other hand, fiber content in CPC remains similar
to the control. Cold press extraction removed more fiber fractions when compared to
alcoholic extraction technique. Cold press is one of the most used methods for oil extraction;
this method requires less energy than others and it is considered as an ecofriendly process(26).
However, the mechanical force applied in the process may remove higher fiber fractions than
any other process. Furthermore, others(27) presented similar contents of fiber in their study.
Likewise, CP content was similar among treatments (P>0.05). However, it is assumed that a
little fraction of soluble protein is removed in the extraction process; the remaining protein
may be lower in amount but similar in proportion after redistribution of nutrients after the
extraction. Similarly, it was observed this behavior in rapeseeds in cold-press extraction
processes(28). Additionally, another research(27) reported similar concentrations of protein in
hempseed cake after cold press extraction. Otherwise, NSC increased in AEC and CPC
(P<0.05); these augmentations are linked with a dilution effect due to a reduction in fractions
of fiber as explained earlier. In fact, Jarrell(29) introduced this term related to plant nutrition
studies in the early 80s. Moreover, extractive processes reduced TPC and CT (P<0.05).
Secondary metabolites as phenolic compounds and condensed tannins are extracted in
alcoholic solutions(14). Secondary metabolites play a very important role in methane
mitigation in ruminants. Thus, different mechanism of action was observed directly or
indirectly in ruminants’ fermentation or ruminants’ microorganisms depending on the
concentrations of certain metabolites (saponins, condensed tannins, phenolic compounds,
373
etc.)(30). Therefore, the importance in evaluating the concentrations of TPC and CT. Fiber
fractions (NDF and ADF) increased after alcoholic extraction process (P<0.05). These
changes are positively connected to a higher extraction of crude fat in EE. Thus, the more EE
is extracted in AEC the higher the fiber compositions are. Additionally, the IVDMD
increased about 142 and 97 % in AEC and CPC, respectively when compared to the control
(RFC) (P<0.05). Moreover, cannabinoids were also affected by the extractive processes
(P<0.05). Both, CBD and THC decreased their concentrations substantially. In this way,
concentrations of CBD were reduced 73 and 28 % in AEC and CPC, respectively. Similarly,
concentrations of THC decreased 99 and 88 % in AEC and CPC, respectively. Alcoholic
extraction is more efficient in the extraction of cannabinoids when compared to a cold-press
extraction. Apparently, a polar extraction may be more effective and less expensive when
compared to an extractive process using mechanical force, which may remove other nutrients
with no interest(26). The published information regarding to harmless consumption of CBD
and THC in animals is very limited. However, Kleinhenz(31) offered industrial hemp to calves
as a feed resource. These authors administered 1142 and 120 mg of CBD and THC to calves
weighing approximately 215 kg of live weight, respectively; no changes were observed in
behavior, feeding intake or chemistry of blood serum (glucose, BUN, creatinine and total
protein) in animals. Moreover, THC was not detected in blood plasma; CBD was totally
metabolized and was not detected after 48 h. Likewise, Cornette(32) administered 5 mg of
CBD/ kg live weight and observed no changes in behavior as well. Hence, according to the
latter, AEC presented in this study, can be offered harmlessly until 2 kg per animal per day
(animals weighting approximately 215 kg live weight) and no effects on any of the chemical
parameters or behavior would be expected. Nevertheless, no information on meat safety and
hemp consumption in ruminants is available. In agree to the results given in the present study,
lower concentrations of cannabinoids (CBD and THC) and higher concentrations of NSC led
to a higher IVDMD and ME. In fact, other study(16) reported antimicrobial activity of
cannabinoids, which may affect directly the digestibility. Consequently, IVDMD increased
approximately two-fold when compared to control (RFC). In addition, these changes could
not be attributed to variations in TPC and CT since their concentrations are similar among
AEC and CPC treatments; changes in IVDMD in AEC and CPC could not be correlated to
TPC and CT concentrations in both treatments.
374
Table 1: Chemical composition and in vitro digestibility of agro-industrial residues of

Cannabis sativa L
Variable RFC AEC CPC P SEM
%, DM
b
Ash 11.5±0.25 13.5±0.25a 12.5±0.04ab 0.001 1.33
CP 21.2±0.25a 20.9±0.29a 20.2±0.0.28a 0.10 0.12
EE 12.3±0.06a 0.5±0.04b 7.5±0.0b <0.001 0.08
NDF 27.9±0.39b 32.2±0.33a 28.0±0.86b 0.003 0.58
ADF 16.5±0.02b 18.9±0.69a 15.5±0.19b 0.015 1.47
ADL 2.2±0.15b 2.5±0.08a 2.3±0.006ab 0.002 0.07
HEM 11.5±0.31b 13.3±2.23a 12.4±0.28a <0.001 1.06
CEL 14.4±0.14a 16.4±0.36a 13.1±0.11a 0.009 1.24
NSC 26.5± 0.16b 32.7± 0.82a 31.1± 1.0a <0.001 0.77
TPC, mg/g DM 15.7±0.10a 14.8±0.08b 15.4±0.05b <0.001 0.06
CT, mg/g DM 6.7±0.09a 4.6±0.16c 5.4±0.05b <0.001 0.09
IVDMD, % 23.9±1.11c 57.9±1.58a 47.2±2.74b <0.001 1.58
ME, Mcal 1.4 ± 0.04c 1.9±0.07a 1.6 ±0.06b <0.001 0.05
CBD, g/kg 1.8± 0.01a 0.5± 0.003c 1.3± 0.006b <0.001 0.03
THC, g/kg 10.9± 0.07a 0.08± 0.001c 1.4± 0.007b <0.001 0.001
RFC= raw flower Cannabis sativa L.; AEC= alcoholic extracted Cannabis sativa L. flower residue; CPC=
cold-pressed Cannabis sativa L. flower residue; SEM= standard error of the difference among means; CP=
crude protein; EE= ether extract; NDF= neutral detergent fiber; ADF= acid detergent fiber; ADL= acid
detergent lignin; HEM= hemicellulose; CEL= cellulose; NSC= non structural carbohydrates; TPC= total
phenolic compounds; CT= condensed tannins; IVDMD= in vitro dry matter digestibility; ME= metabolizable
energy; CBD= cannabinol; THC= tetrahydrocannabinol.
abc
Means with different letters in the same row indicate differences (P<0.05).
In vitro ruminal degradability for treatments is presented in Table 2. Extractive processes

affected degradability; AEC and CPC increased maximum degradability (Ad parameter)
above of 120 % in both treatments when compared to control (P<0.05). Likewise, extractive
processes increased degradability specific rate (kd parameter) about 120 % for both
treatments whereas latency period (Ld parameter) increased 140 % for both treatments
(P<0.05). Augmentations in the kinetics parameters may be positively associated with a
reduction in the cannabinoids content and changes in the NSC; this effects were observed
earlier in the IVDMD. Higher values in Ad, kd and Ld are presented in the treatment with
lower values of cannabinoids (AEC). According to this, Semwogerere et al(33) reported that
digestibility of hempseed cake is lower than the digestibility of canola meal and soybean
meal. In fact, it was affirmed that hempseed cake increased the rumen retention time which
is comparable with the fermentation time in in vitro assays(27); this effect was observed in
this study. Actually, AEC and CPC presented higher values in degradability specific rate (kd
parameter) which led to reach the asymptotic value (Ad) in a shorter time; RFC would need
375
more time to reach Ad which is consistent with previous studies(27). Otherwise, more time is
necessary for microorganisms to begin degradation of substrate as observed in values of
latency time (Ld parameter) for AEC and CPC. The latter may be associated with changes in
microorganisms’ populations (protozoa and bacteria) which will be discussed later.
Table 2: In vitro ruminal degradability parameters of agroindustrial residues of Cannabis

sativa L. after two extractive methods
Parameter RFC AEC CPC P SEM
c a b
Ad, % 24.1±0.29 57.6±0.01 54.6±0.81 <0.001 0.502
b a a
Ld, h 1.9±0.01 4.7±0.04 4.6±0.16 0.004 0.096
b a a
kd, %/h 0.14±0.005 0.31±0.005 0.32±0.000 <0.001 0.002
RFC= Raw flower Cannabis sativa L.; AEC= alcoholic extracted Cannabis sativa L. flower residue; CPC=
cold-pressed Cannabis sativa L. flower residue; SEM= standard error of the difference among means; Ad:
maximum degradability; kd= specific rate of degradability; Ld= latency period before the degradation begins;
(lag phase).
abc
The population of microorganisms after ruminal fermentation of different residues is shown

in Table 3. Protozoa increased after the extractive process in CPC when compared to control
(P<0.05); no changes were observed in AEC (P>0.05). Contrariwise, no traces of fungi were
observed in the control (RFC); nevertheless, fungi increased after the extractive process
(P<0.05). Otherwise, total bacteria were reduced after the extractive process in both
treatments (P<0.05). However, no changes were registered in cellulolytic bacteria (P>0.05).
Novak et al(16) affirmed that extracts of hemp exhibited antimicrobial activity in most
bacterial habitats from human and animal but these changes were not observed in fungi;
conversely, extracts from hemp showed antifungal activity in in vitro assays reported
previously(34). Additionally, reductions in total bacteria are associated with an increase in the
protozoa populations. This is in accordance with others(35), who claim that the bacteria
predation is caused mainly by protozoa activity.
376
Table 3: Microorganisms populations in in vitro ruminal fermentation of agroindustrial

residues of Cannabis sativa L. after two extractive methods
Microorganisms RFC AEC CPC P SEM
Protozoa, mL-1 x104 7.3±0.07b 15.8±0.30ab 26.6±0.37a 0.007 0.22
Cellulolytic bacteria, mL-1
142.1±2.88a 127.6±14.77a 115.6±7.21a 0.232 7.84
x105
Total bacteria, mL-1 x105 187.2±20.20a 100.6±6.74b 115.2±6.92b 0.006 10.57
Fungi, CFU ND 7.2±0.57a 6.3±1.15a 0.001 0.60
RFC= raw flower Cannabis sativa L.; AEC= alcoholic extracted Cannabis sativa L. flower residue; CPC=
cold-pressed Cannabis sativa L. flower residue; CFU: colony forming units; ND= non-detected; SEM=
standard error of the difference among means..
abc
Means with different letters in the same row indicate statistical differences (P<0.05).
Ruminal fermentation parameters and gas production kinetics are shown in Table 4. No
changes were observed in pH (P>0.05). Nevertheless, extractive processes in AEC and CPC
affected concentrations of N-ammonia and TVFA (P<0.05). A reduction in the N-ammonia
is positively associated with a defaunation of ruminal bacteria due to the presence of
protozoa. In fact, Wang et al (36) reported a reduction in the protozoa populations when using
hempseed oil in ruminal fermentations. Thus, it is expected that RFC promote a reduction of
protozoa and an increase in bacteria populations; the latter encourages an increase in the N-
ammonia. Furthermore, a reduction in the deamination of protein would be expected as a
consequence of an increase of bacteria which would lead to a reduction in the N-ammonia(37).
These asseverations were observed in this study. Protozoa increased with both treatments and
a reduction of total bacteria is observed (P<0.05). Additionally, protozoa play an important
role in the synthesis of some volatile fatty acids. As a matter of fact, some others(38) affirmed
that the ability of protozoa to digest fatty acids could divert more carbon towards volatile
fatty acids synthesis; these changes were observed in the present study in AEC and CPC with
the higher protozoa populations. Regarding to gas production kinetics, Ag increased above
87 and 77 % in AEC and CPC, respectively (P<0.05). These changes suggest a better
nutrients utilization of microorganisms which led to a higher fermentation process and a
higher gas production; hence, it is expected a higher gas production rate. However, no
changes were observed in latency period (Lg) and gas production specific rate (kg) (P>0.05)
but similar trends to those in degradability are observed.
On the other hand, methane production increased 81 and 97 % in AEC and CPC, respectively
(P>0.05). Likewise, CO2 increased about 60 % in both treatments when compared to control
(P<0.05). Apparently, the reduction of cannabinoids and the increase in the NSC led to an
augmentation of the fermentation process, which increased methane production. Whereas,
the CH4: CO2 ratio increased 18 % in CPC when was compared to control (P<0.05). This
ratio indicates the volume of methane produced divided into the volume of CO2 present in
377
the process; higher values of this variable suggest that more methane is being synthetized
through the CO2 reduction pathway(18). Thus, these changes in CH4: CO2 ratio may suggest
that more methane is being synthetized through the CO2 pathway and that the increases in
methane production are not only associated to an improvement in the ruminal fermentation
and an extension of the total gas production. In fact, the presence of cannabinoids may result
as inhibitors in methanogenesis through the CO2 reduction pathway since lower values of
CH4: CO2 ratio are presented with higher values of cannabinoids. The higher the
cannabinoids are the lower the protozoa; methane synthesis and protozoa number is
positively associated(39). Thus, if there is a reduction in protozoa a reduction in methane
would expected as well. An increase in the protozoa may affect the bacteria including the
methanogens. Regarding to the latter, it was reported(33) that hempseed oil is more efficient
in the inhibition of methanogens since it has more terpenes, polyphenols and lignin.
Similarly, Embaby et al(40) found a 10 % reduction in methane production when using
hempseed oil and compared to corn oil. However, Patra et al(41) affirmed that reductions in
methane production are associated to the presence of polyunsaturated fatty acids (PUFA)
than the presence of cannabinoids; these fatty acids were not evaluated in this study.
According to the findings in this study, raw flower in RFC inhibits not only degradability but
methane synthesis and protozoa populations as well. Nevertheless, these affections are highly
associated with the nutrients utilization from microorganisms which may lead to a better
ruminal fermentation process as exposed earlier in this study.
Table 4: Ruminal fermentation parameters and gas production kinetics of agroindustrial

residues of Cannabis sativa L. after two extractive methods
Variable RFC AEC CPC P SEM
pH 6.8±0.11a 6.9±0.10a 6.8±0.12a 0.251 0.13
N-NH3, mg/dL 15.2±0.08 a
10.9±0.11 c
13.8±0.11 b <0.001 0.29
TVFA, mM 0.11±0.007b 0.16±0.003a 0.16±0.008a <0.001 0.05
b
Ag, ml/g DM 41.82±8.89 78.46±5.13 a
74.27±2.08 a
<0.009 4.93
a a a
Lg, h 2.6±0.64 3.6±0.35 3.24±0.37 0.384 0.15
a a a
kg, %/h 0.09±0.05 0.22±0.01 0.24±0.02 0.063 0.02
CH4, ml/g DM 3.7±0.15 b
6.7±0.29 a
7.3±0.20 a <0.001 0.22
CO2, ml/g DM 32.5±2.470b 52.8±0.89a 52.3±1.52a <0.001 1.75
CH4/CO2 ratio 0.11±0.004 b
0.12±0.003 ab
0.13±0.005 a 0.023 0.004
RFC= Raw flower Cannabis sativa L.; AEC= alcoholic extracted Cannabis sativa L. flower residue; CPC=
cold-pressed Cannabis sativa L. flower residue; SEM= standard error of the difference among means; N-NH3:
N-ammonia; TVFA= total volatile fatty acids; Ag: maximum gas production; kg= specific rate of gas
production; Lg= latency period before the gas production begins (lag phase).
abc
378
Both residues obtained after the two extractive processes offer acceptable nutritional
properties in animal feeding. Residues obtained after the alcoholic extraction offered a better
nutrient utilization of microorganisms present in the ruminal fermentation which led to
increase in IVDMD and fermentation parameters. Therefore, the agroindustrial residue of
Cannabis sativa L. obtained after the extractive processes may arise as a potential forage
source in ruminants feeding. However, more in vitro and in vivo assays using these agro-
industrial residues as a part of a ration are highly recommended, considering potential
secondary effects of cannabinoids on animal health and food safety.
Acknowledgments
The authors would like to thank to the Durango State Council of Science and Technology for its
support through the grant #2020-123. Authors would like to thank also to the Institute for
Research and Exploitation of Cannabis for the availability in vegetative material and the facilities
used in some analyses.
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383
Review
Importance of Haematobia irritans in cattle in Mexico: Current situation

and perspectives. Review
Roger Iván Rodríguez Vivas a*
Carlos Cruz Vázquez b
Consuelo Almazán c
Juan José Zárate Ramos d
a
Universidad Autónoma de Yucatán. Facultad de Medicina Veterinaria y Zootecnia.
Carretera Mérida-Xmatkuil Km 15.5, Mérida, Yucatán, México.
b
Tecnológico Nacional de México. Instituto Tecnológico El Llano, Aguascalientes, México.
c
Laboratorio de Inmunología y Vacunas; Facultad de Ciencias Naturales, Universidad
Autónoma de Querétaro, Querétaro, México.
d
Universidad Autónoma de Nuevo León. Facultad de Medicina Veterinaria y Zootecnia,
Campus de Ciencias Agropecuarias. General Escobedo, Nuevo León, México.
*Corresponding author: rvivas@correo.uady.mx
Abstract:
The horn fly Haematobia irritans is a cosmopolitan hematophagous ectoparasite of great

importance in livestock. In Mexico, H. irritans is distributed across the country, and is found
during the whole year. The fluctuation of H. irritans population is related with climate
conditions. Despite its wide distribution, the effects on animal health, and its negative impact
on meat and milk production, little data exists on its infestation and epidemiology is limited.
This paper is a review on the current situation of H. irritans in cattle in Mexico, its economic
impact, control methods, perspectives, and research opportunities.
Keywords: Haematobia irritans, Horn fly, Epizootiology, Control.
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Introduction
Horn fly Haematobia irritans (Linneaus, 1758) is a dipteran belonging to the Muscidae
family. This fly is a widely distributed hematophagous ectoparasite of cattle, that negatively
impacts beef and dairy production(1). The direct effects of H. irritans parasitism include blood
loss and skin damage, as well as constant restlessness of infested animals, which cause
reduction in production of meat and milk(2). The impact of H. irritans on animal production
is related to infestation levels, which depends of animal characteristics and regional
environmental conditions(3). In Mexico, H. irritans is geographically distributed in the
country during the whole year(2).
Control of H. irritans infestations is mainly attempted by use of insecticides from chemical

families such as pyrethroids, organophosphates, phenylpyrazolones, growth regulators, and
insect growth inhibitors, among others. However, over frequent and incorrect use of these
insecticides has led to the selection of insecticide-resistant H. irritans populations. Currently,
resistance to pyrethroids and organophosphates in H. irritans is known(2).
Environmentally sustainable control strategies include cultural management of manure and

biological control measures, such as the use of natural enemies, entomopathogenic agents
and botanically-sourced repellents and pesticides. Immunological control by vaccination can
prevent or reduce the insect hematophagy, but experimental results of this method are still
preliminary and vaccines against fly infestation do not exist(4). Integrated pest control (IPC),
the combined and rational application of existing methods, is the most effective method of
horn fly reduction(2)
This paper presents a review on the current situation of H. irritans in Mexico, the economic
impact in cattle, the available control methods, and perspectives and research opportunities.
Direct and indirect effects of H. irritans on cattle
Direct damage. Female and male H. irritans feed from 20 to 38 times a day, consuming
small portions of blood in each feeding, with an average of 10 µl per day per fly(5). By
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piercing the host’s skin, this hematophagous action produces damage and a reduction in skin
quality(3). Skin damage includes blackheads and orifices, in which most damage is apparently
due to dermal inflammatory responses (Figure 1). Eosinophilic infiltration, eosinophilic
folliculitis, and furunculosis with alopecia can occur at the feeding site(6).
Figure 1: Severe skin damage, including alopecia and hypercheratosis, in a cow without
ectoparasite treatment during the season of highest intensity of Haematobia irritans in the
northern Gulf of Mexico region
Source: C. Almazán
Disease Transmission. Haematobia irritans is the intermediate host of Stephanofilaria

stilesi, a nematode that causes skin lesions in cattle and is reported in cattle in Canada and
the western and southwestern United States of America (USA). This fly can also
mechanically transmit several species of Staphylococcus bacteria, which can cause mastitis
in dairy cows(7). In addition, it is involved in the mechanical transmission of other pathogens
such as Trypanosoma vivax and T. evansi, Francisella tularensis, Corynebacterium
pseudotuberculosis, Parabronema skrjabini and Anaplasma marginale(8-10).
Economic impact
The weight loss due to H. irritans infestations in beef cattle has been estimated in 3.25
kg/cow in Brazil in average per year(11), and 0.028 kg/cow/d (305 d of lactation, 8.54 kg per
cow) in Argentina(12). In a study done in the USA, heifers treated against horn fly exhibited
14 % more weight gain than untreated control(13). Control measures also benefited cows,
which won 14.4 kg more after treatment(14).
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Milk production also drops due to H. irritans infestation, with reductions of approximately
27 kg of milk/cow/yr have been reported on dairy farms in the US(15). Indeed, in 2016 an
estimation of US 1.75 billion dollars due to the direct effects of H. irritans on dairy cows
was reported. This parasite also generates the additional expense of US 60 million dollars
annually for chemical control(16). Based on Mexico’s potential at risk cattle population,
estimated annual losses attributed to H. irritans amount to US$ 231.66 million(1). However,
evaluations have not been done of losses generated through reduced pregnancy rates,
transmitted pathogens and the need for additional control measures.
Life cycle of H. irritans
In this review, the horn fly is referred to as H. irritans. However, it has been suggested that
there are actually two morphologically similar subspecies of horn fly, H. irritans irritans and
H. irritans exigua (buffalo fly). The former is distributed in Europe and America, and the
latter in Asia and Australia(17).
Tropical and subtropical climates with average temperatures of 20 to 30 °C and relative

humidity from 65 to 90 % are extremely propitious for development of H. irritans(17). In
Mexico, H. irritans is also distributed in temperate climates(18).
The adult H. irritans flies are 3 to 4 mm long, gray in color with dark stripes on the thorax,
and with a pair of dark reddish compound eyes. It exhibits sexual dimorphism; the eyes are
more separated and smaller in females than in males, and males have a slightly folded
abdomen. On the host animal, this fly normally perches facing the ground(17).
The H. irritans host range is ample and its main host is cattle, although it also parasitizes
sheep, horses, canines, water buffalo, bison and humans(19). Animal color influences fly
preference, with black animals attracting greater numbers of flies(2). In cattle, bulls prove
more attractive to H. irritans than steers or cows. The fly spends most of its life on the host,
mostly feeding but also reproducing. Females may leave their host to lay eggs in extremely
fresh manure; in fact, for H. irritans fecal attraction begins to disappear about 10 min post-
defecation. To lay eggs, females spend postrated on fresh fecesone to ten minutes on feces(2).
Fly distribution on a host changes during the day. In the early daylight hours they tend to
concentrate on the shoulders and back, then move to the abdomen midline and the sides in
the afternoon, returning to the shoulder and back area at night(2). Average adult lifespan is
six to eight weeks. Longevity is inversely related to low temperatures, which negatively
influence ovary development, mating, larval development, and adult emergence(17). After
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emergence, adults require three days for their reproductive organs to fully mature. Adults
mate three to five days after emergence and oviposition occurs three to eight days after(20).
In the Americas, air temperature is the main climatic factor affecting the H. irritans life cycle
and the humidity/temperature relationship is essential for fly reproduction(18). Temperature
influences infestation seasonality and is related to presentation of total or partial facultative
diapause. In both, tropical and temperate climate regions, fly population decline during
winter without entering into total diapause, while in temperate-cold climate regions diapause
invariably occurs in winter(18). Unsurprisingly, fly populations have been reported during the
whole year in Mexico’s humid tropics(21).
Regardless of the time of the day, H. irritans lays eggs in fresh feces, usually within the first
two minutes after feces are excreted. One female can lay up to 400 eggs, which are deposited
in groups of 20-25(22). Eggs are oval-cylindrical in shape, slightly curved with a longitudinal
medial groove, and yellow or white in color when laid, and become dark after. They range in
size from 1.0 to 0.5 mm long by 0.34 to 0.39 mm wide(17). To hatch, a temperature of 24 -
26 ºC and relative humidity near 100% is needed. Hatching normally occurs after a period of
20 to 48 h of incubation(20).
H. irritans larvae are yellowish-white in color, measuring 7 mm long. They present with a
pair of posterior spiracles showing a “D” shape(20). Larvae have three developmental stages
(L1, L2 and L3). Development from L1 to L3 requires four to eight days, and pupation six to
eight days. Both L2 and L3 larvae have anterior spiracles while L1 larvae lack them. The
posterior spiracles allow differentiation between L2 and L3 stages: L2 larvae have two
openings in the spiracles while L3 larvae have three. The larvae feed on bacteria in feces(23).
Development of pupa requires six to eight days(23). The pupal stage is surrounded by the
exoskeleton from L3, which darkens and hardens, forming a capsule called puparium(23).
Pupa development requires humidity and temperature conditions similar to those for larval
development. After seven to eight days adults emerge and search immediately for a host to
feed(20). Diapause occurs at temperatures below 23 °C and pupae can survive prolonged
periods of exposure to temperatures as low as -5 °C(22). Under normal conditions, the life
cycle is completed in 10 to 20 days(3).
Geographic distribution and population dynamics. In Mexico, H. irritans was reported

for the first time in the state of Veracruz in 1984. It is currently known to be distributed
throughout the country, mainly in association with livestock in extensive systems which
facilitate its life cycle(2,24).
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The population dynamics of H. irritans is related to regional climatic conditions, and flies
are seen during the whole year in tropical climates. The intensity of H. irritans during the
differs regionally, but always tends to show seasonality. Two population peaks can generally
be observed between late spring and early autumn. In addition to regional climate, abundance
can also respond to other environmental and management factors that may cause population
fluctuations during a year and even between different years(25). Populations do not develop
significantly at altitudes higher than 1,800 m asl. In Mexico, the population dynamics of H.
irritans generally exhibit a bimodal behavior, with a wide intra-annual fluctuations.
Infestation seasonality is associated with temperature and relative humidity and the
infestation index varies more in tropical than in temperate regions and decreases at high
altitude(26,27,28).
The highest infestation rates of H. irritans are found from late spring to early autumn, and
up to three population peaks occur in certain areas. During the summer months, infestation
index values can exceed 4,000 flies per animal, while in less propitious periods it can drop
to 200 to 450 flies per animal. Insecticide application and H. irritans resistance, as well as
grazing and excreta management, may affect the index estimation in a herd(28). In temperate
climates, H. irritans population dynamics is bimodal and is considered seasonal, with
increases from late spring to early autumn, and peak infestation rates in summer. Facultative
diapause may occur during winter in temperate climates, therefore animal infestations are not
observed(26,27).
Several generations of H. irritans may be produced in a year. In cold climates, 7 to 9

generations a year have been estimated, while in warm climates, the number of generations
can range from 8 to 14(22). in a semi-arid region of Brazil, thirty generations per year have
been reported(20). In Mexico, information on the number of generations produced by H.
irritans per year does not exit. This information is essential to understand the parasite
behavior and to elaborate control strategies.
Host resistance. Bos indicus breeds are less susceptible to ectoparasite infestation than B.
taurus breeds(29). Significant differences on H. irritans density between different B. taurus
breeds have been observed. For example, the Chianina breed is more resistant to fly
infestation than Angus, Hereford and Charolais breeds(30). In Brazil, it was found that Guzerat
x Holstein cross cattle had higher infestation levels than pure breed Guzerat cattle(31). A study
of infestation resistance done in southern Mexico reported fly counts on B. indicus animals
to be equal or lower than on B. taurus animals(2). Within the same herd, H. irritans infestation
is not homogeneous, with more than 50% of a fly population parasitizing only 15-30% of the
animals, which suggests that some animals are more susceptible to fly infestation(32).
Susceptibility to H. irritans infestations is influenced by animal color (dark-colored animals
are more susceptible), size (large animals have higher levels), hair density, and sebum
production (infestation is higher in animals with lower hair density and sebum production),
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and hormones (higher testosterone levels favor higher infestation). Also, natural resistance,
such as individual immune response and coagulation system, can influence infestation
levels(33).
Estimating infestation as number of flies and the economic threshold (ET). Establishing
the ET in H. irritans infestations requires estimation of the quantity of flies on the animals
that would cause economic losses. Economic losses is understood as an amount of damage
that would justify the cost of artificial pest control, while ET is the parasite population
intensity that requires control measures to prevent losses that would exceed the cost of the
control intervention(2). Quantifying fly counts on animals is done using two methods: direct
visual (DV) or indirect digital (IDV; i.e. photographs or video). In both methods, fly counts
or images are obtained by trained persons at a distance of 1 to 4 m from an animal. Longer
distances (5 - 10 m) can be used depending on animal docility(34-36), using binoculars(37). In
order to obtain the most accurate ET, counting should be done when flies are most visible on
the animal and there is enough natural light. Accuracy may be lower during warmer time of
the day since a high proportion of horn flies move to the lower abdomen. When ET is done
on different days, it should be done at the same time, from 06:00 to 12:00 h(16,38,39). In other
reports, counts have been done from 15:30 to 19:00 h(36).
Whether with DV or IDV, counts must be done by trained personnel. Counts are normally
done on one side of the animal and then multiplied by two to produce the total number of
flies per animal, but counting can also done on both sides of an animal by two persons
simultaneously(34-36). Fly counts can be underestimated when fly density is extremely high.
If fly density on the scapular, interscapular and costal regions is ≤25, they are counted
individually but when it is ≥25 it is recommended to count in groups of five(40).
Horn fly density is usually highest in the scapular, interscapular and costal regions(40) (Figure
2). Also, the back, flanks, legs and both sides of the head can also be considered(35).
Quantification of fly infestation can be done in confined, semi-confined or free-ranging
animals(16,34,36).
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Figure 2: Haematobia irritans infestation on the upper neck and scapular areas of a dark-
skinned bull.
Source: Ma. Lorena Torres-Rodríguez
The use of photographs and videos (e.g. VID)(35,36) and videos alone(41) provide the
opportunity to very accurately count flies in the recorded images since counting using images
is less prone to estimation errors and does not require intensive labor(36). However, the DV
method is faster and more efficient, and sufficiently accurate to identify changes in H. irritans
population density(39).
Several studies worldwide have estimated that the ET of H. irritans in beef cattle is ≥200
flies per animal(16,42). Exceeding this ET can lead to losses; for instance, it has been estimated
that with infestations higher than 200 flies/animal losses of 520 ml milk per day and 28 g live
weight per animal per day are produced(43). Calves and dairy cows cannot tolerate large
numbers of flies without experiencing harm. The ET in dairy cows is considered to be no
more than 50 flies/animal. The ET can vary between breeds and sexes. For instance, in
Holstein breed, the ET is 80-100 flies per animal(44), while beef cattle can tolerate more than
200 flies per animal, although bulls can tolerate even more(45). In Mexico, the ET is generally
estimated by DV. The highest reported fly counts in the country are 120 flies/animal in
central Mexico and 300 flies/animal in the southeast, both of which occurred during periods
of maximum rainfall(2).
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Chemical control and application methods
Chemical control
The most widely used method to control infestations by H. irritans in cattle is the use of
insecticides. These insecticides are divided in nine main families:
Organophosphates (OPs). Phosphoric acid derivatives interfere with nerve function at the
synaptic level by inactivating acetylcholinesterase (AChE), which reacts with serine residues
located at the site of AChE catalysis. Because acetylcholine is not hydrolyzed, OPs
accumulate excessively, generating an increase in stimulation with an eventual insect
paralysis(46). This mechanism makes OPs highly toxic to animals and humans. OPs are
effective against animal ectoparasites such as flies, fleas, lice, mites and ticks, and were the
first insecticides used to control H. irritans. The most commonly applied OPs compounds
are diazinon and ethion, both generally used to control pyrethroid- resistant H. irritans
populations(33). Ear tags containing 21.4 % diazinon produced an 87 % reduction of H.
irritans in grazing cattle in Tuxpan, Veracruz, for up to 90 d(47).
Pyrethroids (Ps). Ps are derived from pyrethrins and are natural insecticides found in
Chrysanthemum cinerariaefolium flowers. They are classified into TI and TII pyrethrins. TI
pyrethrins lack the α-cyano group located at the phenyl-benzyl alcohol position of TII
pyrethrins. Natural pyrethrins are sensitive to sunlight, while synthetic Ps are not(48). Target
sites for Ps are the sodium and chloride channels at the point where they inhibit transmission
of nerve impulses in insects, causing changes in membrane permeability(33). The TI Ps change
the arrangement of sodium channels in neuronal membranes in response to stimuli, while
TIIs affect chloride channels, including those dependent on gamma amino butyric acid
(GABA), resulting in membrane depolarization and suppression of the action potential(48).
Insects have a large number of sodium channels sensitive to their structures and body
temperature, making Ps highly toxic, in comparison to mammals, where toxicity is
minimal(48).
Phenylpyrazolones. These are phenyl pyrazole-type chemical components, and the principal
one used in fly control is fipronil. These pesticides act on GABA receptors, blocking chloride
channels. They also block two types of chloride channel glutamate activators found only in
invertebrates, causing arthropod paralysis and eventually death. A 1 % fipronil-based
backsplash formulation shown >80 % efficacy against H. irritans up to 21 d after
treatment(49).
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Macrocyclic lactones (MLs). The MLs are divided into three families: a) the avermectins,
which are fermentation products of Streptomyces avermitilis, for example, ivermectin,
doramectin and eprinomectin; b) the milbemycins, derived from fermentation of S.
cyanogriseus, for example, moxidectin; and c) spinokines, derived from Saccharopolyspora,
for example spinosad(50). The MLs irreversibly interact with GABA and chloride channel
glutamate receptors, increasing membrane conductivity and causing paralysis in insects and
mites(51). Because they are effective against endo- and ectoparasites, they are known as
endectocides. The chemical composition of avermectins and milbemycins is not altered
during passage through the digestive tract and they are excreted intact, meaning that they
continue affecting larval development in the manure of treated animals. However, they are
also eliminated in milk, which is their main disadvantage(33). In grazing cattle in Tuxpan,
Veracruz, injectable ivermectin has shown >90 % efficacy on fly reduction for up to 90 d
after treatment(47).
Growth regulators (GR). In insects, GRs accelerate or inhibit essential physiological

processes required for normal development of adult insects and/or progeny. They are not
necessarily toxic, but cause abnormalities that compromise insect survival(2). For example,
insect-specific juvenile hormones (JH), which are ecdysone analogues, normally decline in
each evolutionary phase, allowing development of adults. Constant JH levels block
maturation in insects(52). The GRs metropene and cyromazine are non-toxic to mammals and
are applied via bolus or food supplement in cattle.
Growth inhibitors (GI). GIs block polymerization of N-acetylglucosamine, thus preventing

synthesis of chitin, an essential insect exoskeleton component, and as consequence
emergence of H. irritans does not occur(53). This group includes benzoyl-phenyl ureas such
as diflubenzuron, lufenuron, and triflumuron, of which diflubenzuron is the most widely used
against H. irritans. These products act against eggs and larvae, not against adult phases. They
are usually administered orally, as bolus or as a supplement in mineral salts. Also, spray and
powder formulations exist. In the US and Brazil, diflubenzuron produced from 90 to 99 %
reduction of H. irritans 20 to 33 days after treatment(53). In Mexico, oral diflubenzuron is
used (1 g/animal/day) with good results for H. irritans control.
Pyrrole derivatives. Halogenated pyrroles are aromatic organic compounds produced by

Streptomyces. They are also known as proinsecticides, because, once inside the insect, they
are activated by oxygenases such as cytochrome p450 to form more toxic metabolites.
Pyrrole targets the mitochondria, affecting oxidative phosphorylation, breaking the proton
gradient and preventing production of ATP(54). A member of this group is chlorfenapyr, the
first insecticide used at 30 % in ear tags to control H. irritans, and widely used as an
alternative treatment for pyrethroid-resistant H. irritans(33).
393
Repellents. Some plant extracts and essential oils, mainly nitrogenous compounds, alkaloids,
phenolics, protein inhibitors and essential oils(55), exhibit insect repellent activity. They
represent a replacement for use of conventional insecticides in organic production units, or
an alternative to conventional pest control methods that can help mitigate insecticide
resistance. One limitation of extracts or essential oils is their short repellence effect. For
example, the essential oils of lemongrass (Cymbopogon citratus), geranium (Geranium
odoratissimum), and peppermint (Menta piperita) at 5 % concentration in sunflower oil,
exhibited repellence during 8 to 24 h(56) versus H. irritans.
Attractants. These are volatile substances detectable by insects over large distances and
emitting alarm or reproductive signals. In the case of H. irritans, they are pheromones or
chemical messengers found in the cuticular wax of females. This cuticular wax is composed
of 21- to 29-carbon chains which function as copulation stimulants for males(2). Synthetic
pheromones have been applied in traps treated with insecticides to attract insects, but in this
way, they have functioned as physical rather than chemical control method(57).
Application methods
Several methods of application of insecticides to control H. irritans exist. The method of

choice depends on factors such as farm type, production system (intensive, extensive, mixed),
beef and dairy cattle, or both), excreta management, infrastructure, facilities, and the
technical personnel in charge of insecticide application(2). The most common methods of
application of insecticides are described below(3,58,59).
Insecticide-impregnated ear tags: These are plastic ear tags containing one or more
insecticides in the tag matrix. As the tag moves small amounts of insecticide are released and
distributed through the animal’s hair. Ear tags are currently available containing Ps, OPs,
MLs, and Ps/OPs mixtures. All adult animals in a herd should be tagged, and tags should be
removed if no efficacy is observed.
Powders: Powdered insecticide is placed in sacks or bags from which small amounts are
released through filters when an animal is in contact with the bag. Using this method requires
that bags are suspended near water intakes and arranged in a way that ensures that the dust
falls onto the animals. Powdered insecticide is also used to treat manure.
Dorsal pour-on: Dorsal pour-on insecticides are applied along an animal’s back line, at a
weight-dependent dose. This is one of the most widely used methods for cattle in Mexico.
394
Sprays: Spray treatments effectively control flies, but the insecticide must be applied on the
entire animal. This method increases animal handling and stress in animals, which is a
disadvantage. However, it is effective when small numbers of animals are treated. Spraying
is a common method used in Mexico.
Oral larvicides: These are directly applied in food, mineral blocks, or as food supplements.
Oral insecticides include MLs, GRs and GIs. They pass through the gastrointestinal tract and
are excreted in the feces where they prevent larval development. One challenge with this
technique is ensuring that sufficient active agent is applied, because underdosing may allow
fly infestation levels above the ET. A solution to this challenge is to use slow-release boluses,
which remain in the reticulum, and continuously release the product.
Injection or systemic: Although the vast majority of insecticides are applied topically,
intramuscular injection is effective for applying of MLs such as ivermectin. This is a very
common method used to control ticks, flies and gastrointestinal nematodes in beef cattle.
VetGun®: This novel insecticide administration method involves firing an insecticide-loaded
gelatin capsule (VetCap®) from a special gun. The capsule is very fragile and breaks upon
impact with the skin, releasing the insecticide which begins disseminating through the
animal’s hair and skin. Capsules can be shot onto an animal from 5 to 10 m away, although
it does not ensure the insecticide adequately covers both sides of the animal. This technology
is not yet commercially available in Mexico, but may become more available in the near
future.
Bioinsecticides. These are extracts or essential oils from plants that have efficacy on the
control of H. irritans. For example, the development of H. irritans in feces was inhibited by
an extract from neem (Azadirachta indica) containing azadirachtin administered orally to
cattle at doses of ≥0.03 mg per kg body weight per day in a food supplement of neem seeds
at ≥ 10 mg of seeds per kg(60). Other botanical compounds with good efficacy against H.
irritans are p-anisaldeide, extracted from plants such as Pimpinella anisum and Cuminum
cyminum(61), and essential oils of Carapa guianensis(62), Eucalyptus polybractea(63), and
Pelargonium spp.(56).
In a study conducted in dairy cows naturally infested with H. irritans in Mexico, a reduction
of infestation from 9.5 to 68.0 % was found after spraying 20% Larrea tridentata leaf
extract(64). Further research is needed in Mexico to identify bioactive molecules in extracts
from native plants from different regions in the country, and to develop vehicle formulation
and application methods in cattle.
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Insecticide resistance in H. irritans
Insecticide resistance is a genetic-evolutionary response of insect populations exposed to

continuous stress due to frequent insecticide exposure. In the field, resistance is suspected
when a previously effective product no longer demonstrates the same effect; this applies as
long as the application and dose have been optimal(42). Because the H. irritans life cycle lasts
few days, control treatments are carried out at short intervals, leading to a progressive
increase in the frequency of resistant individuals and eventual loss of insecticide biological
effectiveness(20).
Several resistance mechanisms in H. irritans are known. They include changes in insect
behavior to avoid insecticide exposure, detoxification by overexpression of the cytochrome
p450 enzyme, and insensitivity at the site of action due to mutations in the sodium channel(42).
Resistance to Ps in H. irritans is associated with resistance to knockdown due to mutations
in the sodium channel (known as kdr or super kdr) which prevent or reduce interaction with
the sodium channel(65). Resistance to OPs arises from point mutations that produce changes
in acetylcholine’s structure, conformation and site of action. These changes have been found
in the active site of AChE in OPs-resistant mosquitoes, and are known to result in decreased
AChE sensitivity(33).
Insecticide resistance is most commonly diagnosed using a bioassay in which recently

captured flies are exposed to filter papers impregnated with insecticide at lethal
concentrations (LC) of 0, 50 and 99 %, using acetone as a diluent. Three replicates are done
and after one hour of exposure, the percentage of mortality is recorded for each concentration
in comparison with the control (100 % acetone)(66). Resistance to Ps can be identified
molecularly. A fragment of the gene that codes for the sodium channel of a single individual
is amplified and the resulting sequences analyzed, identifying whether the mutations are kdr
or super-kdr type. For OPs, PCR is used to identify a point mutation where a glycine is
substituted for an alanine at position 262 of the AChE amino acid sequence. With this
method, fly resistance can be detected in the field(67).
H. irritans resistant to insecticides has been documented in the US since the 1960s.
Resistance to Ps in H. irritans populations controlled by Ps ear tags was first reported in the
1980s in Florida, US. The first study of resistance in H. irritans in Mexico was done on the
Gulf of Mexico, finding high resistance to fenvalerate and less resistance to OPs(68). A study
to test the susceptibility of H. irritans to cypermethrin and diazinon in the state of
Tamaulipas, Mexico, detected the presence of both kdr and super-kdr genes. The super-kdr
gene was only identified at one ranch, but kdr frequency ranged from 43 to 78 % in the
remaining studied places(66). Another study done in Tamaulipas used filter paper tests to
396
confirm that Ps resistance in H. irritans was distributed across the state, but simultaneous
resistance to Ps and diazinon was only found in populations in the south(69). Similar studies
showed high resistance to Ps and low resistance to diazinon in northern Veracruz central
Nuevo León(70). In Guerrero, resistance to both OPs and Ps was found in 100 % of 30 sampled
farms(71). Currently, the geographical distribution of H. irritans resistant to the main
insecticide families is unknown in Mexico, highlighting the need for a national-level
resistance survey.
Alternative control methods
Physical control. Physical control of H. irritans involves trapping adult flies as they search
for new cattle hosts. Some traps are cylinders or inverted cups in shaped, and are covered
with a sticky material, others are like black balls and emit violet light, and others are
impregnated with attractants such as pherohormones(72). Another kind of physical control is
the walk-through trap, which consists in a dark tunnel. As the animals walk through the dark
tunnel, flies separate from it seeking lighted areas on the roof, where they are trapped and die
within 2 to 12 h. Some walk-though traps are equipped with an electric suction system to
vacuum the flies. However, this requires electric installation and this increases the operation
costs(34). The use of traps is very limited in Mexico, therefore this is an area of opportunity
for development and evaluation. Physical control of H. irritans reduces the use of insecticide,
and selection of insecticide-resistant horn-fly populations.
Biologic control. The use of natural enemies of H. irritans is a widely explored way to
control its populations(73-76). Natural enemies of H. irritans include Pteromalid parasitoids,
such as the genera Muscidifurax spp. and Spalangia spp., that parasitize fly pupae;
entomopathogenic bacteria such as Bacillus thuringiensis; entomopathogenic nematodes
such as Steinernema spp. and Heterorhabditis spp.; and entomopathogenic fungi such as
Beauveria spp., Metarhizium spp. and Isaria spp. Dung beetles of the Scarabaeidae family
also play an important role in biological control of H. irritans by degrading cattle feces and
incorporating them into the soil, thus preventing development of the non-parasitic phase of
H. irritans(50). Biological control strategies pose minimal risk to non-target invertebrates and
vertebrates (including birds, and mammals), while reducing insecticides and development of
horn-fly resistance(77,78).
Parasitoids attack any fly species and are available in the Mexican and international markets
for use in livestock production systems. They are sold in cloth bags or plastic containers
containing housefly pupae parasitized by one or two genera of wasps (Muscidifurax and/or
Spalangia). These are placed in paddocks and pens 48 hours before emergence of the adult
397
parasitoids, which easily establish themselves in environments with moderate chemical

product use. Various parasitoid species have been reported in Mexico, the most frequent
being Spalangia endius, S. nigroaenea, and Muscidifurax raptor(73).
When applied directly to manure, entomopathogenic bacteria such as B. thuringiensis is

useful in controlling larval-stage H. irritans. However, limited data is available on its use in
the field. Entomopathogenic nematodes (Steinernema spp. and Heterorhabditis spp.) have
been presented as an alternative method of biological control, but further research is required
to evaluate their use in the field(74,76).
In Mexico, various isolates of B. bassiana (Bb), M. anisopliae (Ma), and Isaria fumosorosea
have been evaluated in vitro versus H. irritans(75). A study performed under controlled
conditions in the dry tropics, evaluated different formulations on cattle, found out that five
M. anisopliae strains controlled 94 to 100 % of infestation after 12 to 13 days’ post-treatment,
while three I. fumosorosea strains decreased generation of immature phases from 90 to 98 %
up to 13 days’ post-treatment(79).
An aqueous formulation of the Mexican strain Ma134 of M. anisopliae evaluated in dairy

cattle naturally infested with H. irritans in a semi-arid climate controlled 68 % of the
infestation after four weeks’ treatment(80). Strain Ma135 was evaluated against natural
infestations of Stomoxys calcitrans and H. irritans in dairy cattle in a combined
grazing/corral system, lowering the S. calcitrans infestation by 69% and the H. irritans
infestation by 58 % at six weeks’ treatment(81). The main disadvantage of entomopathogenic
fungi treatments is that ultraviolet rays deactivate conidia. Therefore, application of
entomopathogenic fungi must be done before sunrise to maintain its efficacy.
Dung beetles form the Scarabaeidae family degrade organic matter in feces, competing with
H. irritans for space and organic matter. During their mating process, these beetles bury feces
in the soil, preventing horn flies from development. Under laboratory conditions, the
Aphodius lividus beetle is capable of reducing H. irritans emergence by 98 to 100 %(82). A
study performed in North America found that a density 40> of Digitonthophagus gazelle
adult beetles in cattle feces reduced the emergence of H. irritans from 38 to 56 %(83).
However, it is known that dung beetle populations are negatively affected by MLs, such as
ivermectin and doramectin(59,84). For example, use of 10 % moxidectin in cattle reduces
reproductive capacity in the dung beetle Onthophagus landolti(85). The challenge is to use
selective treatments that generate lower ML excretion levels, and consequently lower the
impact on dung beetle populations.
398
Immunological control. The need for horn-fly control methods friendy with the
environment and public health has encouraged research on the immune response of cattle to
H. irritans antigens for anti-horn fly vaccines, analogous to the approach used with ticks. It
has been demonstrated that 200 flies per animal produce a weak antibody response to
antigens from fly saliva, increasing when the flies are removed from the animals. This
suggests a modulation effect of the antigens in the H. irritans salivary glands(86). Another
study identified a correlation between reductions in egg counts and levels of antibodies
against H. irritans fed with blood from bovines immunized with antigens from H. irritans
intestine; however, the fly mortality was not significant(87).
Vaccination of cattle with recombinant proteins such as thrombostasin, a coagulation-

inhibiting protein identified in the salivary glands of H. irritans(88), and hematobin, an
immunomodulatory protein from saliva, produced a decrease in blood consumption by flies,
and decreased development of eggs, and adult flies. Experimental vaccination with
recombinant hematobin increased the anti-hematobin IgG response in cattle and reduced fly
numbers in 30 % compared to controls(4). So far, very few recombinant proteins have been
evaluated and a recombinant vaccine against H. irritans does not exist yet.
Functional genomics and proteomics studies offer an opportunity to discover new candidate
vaccine antigens that can then be expressed and produced in recombinant proteins to be used
alone or in combination as part of vaccination and challenge trials against H. irritans
infestations in cattle. In Mexico, candidates for H. irritans vaccine development were
identified via gene silencing using RNA interference (RNAi) in a cDNA library constructed
from abdominal tissues of partially fed H. irritans. The RNAi of the protease inhibitor
functional group produced high mortality and vitellogenin, ferritin, and ATPase, as well as
components of the proteasome, immune response and 5'-NUC produced reduction of
oviposition of. However, these candidates have not been evaluated in immunization against
H. irritans and infestation trials(89).
Little research on identification of candidates for development of vaccines against H. irritans

has been performed and so far, the results are preliminary. Therefore, the immunological
control of horn fly is not an alternative in the short-term. The recent sequencing, assembly
and annotation of the H. irritans genome(90) will be useful on identification of new candidate
antigens for vaccine development.
399
Cultural, tactical, strategic, and selective control
Cultural control is the implementation of good management practices such as the removal
and proper disposal of fresh excreta from pens and stables, which interrupts the horn fly life
cycle and prevents development of new populations(42).
Tactical control is an immediate action triggered by harmful infestation levels. Effective

tactical control requires monitoring of the fly population every 8 to 10 d with immediate
treatment when infestation levels exceed the ET(2).
Selective control is to apply treatment of only those animals with the highest fly infestation
levels in a herd. Several trials applying different insecticides to 25 % and 50 % of the herd
reduced infestation levels of H. irritans in the herd with a low cost; however, more frequent
treatments were required due to fly infestation persistence(91).
Strategic control is based on knowledge of the epidemiology and biology of H. irritans in a

given region. In this case, limitation of treatments during highest infestation and economic
damage are applied to prevent peaks in fly populations. This approach can be implemented
once a pre-established maximum fly infestation level is exceeded based on weekly
evaluations(2).
Integrated control
Integrated pest control (IPC) considers the association between the environment and
population dynamics of parasite species, using a combination of compatible techniques and
sustainable methods to maintain parasite populations below the ET. Application of IPC is
generally associated with a drastic decrease on frequency of treatments and as consequence
the genetic selection pressure and resistant parasites decrease(1). Although different strategies
to control H. irritans have been explored worldwide, no research has been done on
integrating strategies, in contrast to other parasites such as ticks(92). In Mexico as in other
countries, the main challenge on H. irritans control is to design and establish effective IPC
that include chemical and non-chemical strategies.
400
Conclusions
Based on the information presented and discussed on the situation and prospects for the study
of H. irritans in cattle in Mexico, it is concluded that:
Horn fly H. irritans is an obligate ectoparasite of cattle, that is distributed across Mexico,
during the year, with peaks in summer or in rainy season. This parasite is responsible for
significant economic losses in cattle systems, highlighting the need to study its population
dynamics in different regions of the country, to establish effective control strategies and
prevent population peaks.
Chemical methods are the most common approach to control H. irritans infestations.
Insecticides used to control these flies include OPs, Ps, ML, GR, GI, and pyrroles, as well as
repellent and attractant products. Insecticides are applied using various methods and
application ways. The frequent use of insecticides selects genetic resistant populations of H.
irritans. In Mexico, populations of H. irritans resistant to OPs and Ps have been reported in
the states of Tamaulipas, Veracruz, Nuevo León, Guerrero and in southeastern Mexico.
Biological control is a promising alternative from which entomopathogens fungi is the most
useful method. The species B. bassiana, M. anisopliae and I. fumosorosea have been shown
efficacy against H. irritans in Mexico. Another approach of biocontrol is the use of dung
beetles that degrade organic matter in feces and compete for resources blocking development
of immature H. irritans stages. The frequent application of MLs for control of endo and
ectoparasites negatively affects dung beetle development, therefore, rational use of ML in
cattle systems to preserve natural regulators of H. irritans populations is needed.
Research is required on several areas to find other ways to control H. irritans, emphasizing
on the identification and development of new bio-insecticides, and the use of integrated
control strategies.
Studies are required to identify and develop new bioinsecticides for the control of H. irritans
in cattle.
The use of different integrated control strategies for H. irritans has been little explored
worldwide and in Mexico.
401
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Technical note
Joel Domínguez-Viveros a
Antonio Reyes-Cerón b
Carlos Enrique Aguirre-Calderón c*
Ricardo Martínez-Rocha d
Carlos Luna-Palomera e
Nelson Aguilar-Palma a
a
Universidad Autónoma de Chihuahua. Facultad de Zootecnia y Ecología. Chihuahua,
México.
b
Asociación Mexicana de Criadores de Ganado Limousin. Zacatecas, México.
c
Instituto Tecnológico de México. Instituto Tecnológico de El Salto. El Salto, Durango,
México.
d
Universidad Nacional Autónoma de México. Facultad de Estudios Superiores Cuautitlán.
Ciudad de México, México.
e
Universidad Juárez Autónoma de Tabasco. División Académica de Ciencias
Agropecuarias. Tabasco, México.
*Corresponding author: carlos.ac@salto.tecnm.mx
Abstract:
The objective was to fit a non-linear model (NLM) to evaluate the growth curve in purebred
(PB) Limousin cattle and in five degrees of crossbreeding (DCBs: 1/2, 3/4, 7/8, 15/16, 31/32
Limousin). Live weight, the birth weight interval at 500 d of age, was analyzed. Four NLMs
were evaluated: Brody, Bertalanffy, Gompertz, and logistic. Growth parameters were
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estimated: adult weight (ADW); growth rate (GR); age (AIP; months) and weight (WIP; kg)
at inflection point; age (months; A50M) to reach 50 % maturity and degree of maturity at 15
mo (DM15). The growth curve in DCB was characterized using the NLM selected for BP.
The best-fitting model was Bertalanffy. The ADW for purebred (PB) males was 566.1, for
crossbred (CB) males it was in the range of 446.9 to 527.4; for CB females it was in the range
of 374.5 to 419.9, and for PB females, it was 443.0. The NLMs exhibited correlations below
-0.75 between ADW and GR. In PB heifers, AIP was estimated at 3.7, and WIP, at 131.2; in
CB heifers, AIP and WIP were in the ranges of 2.9 to 3.7 and 110.9 to 124.4, respectively.
A50M for PB females was 10.6, and for CB females, within the range of 8.9 to 10.5. DM15
for CB females, the average was 90.5 %, and 87.9 % for PB females. PB males reach A50M
at the age of 13 mo.
Key words: Bos Taurus, Crossover, Growth parameters, Heterosis, Nonlinear models.
The Limousin breed, originated in France as a pure breed or in crossbreeding schemes(1,2) has
productive, reproductive, and adaptive qualities that have allowed its distribution in a large
number of countries and production systems(3,4,5); it has also been used in the development
of synthetic breeds(6). It arrived in Mexico through imports from Canada and the United
States in the 1970s; the Mexican Association of Limousin Cattle Breeders (AMCGL, in
Spanish) was established in 1989(7,8). It is currently distributed in 17 states, especially as a
pure breed, although it is also used in open crossbreeding schemes and as a basis for the
makeup of synthetic breeds, such as Limousan (5/8 Limousin and 3/8 Angus)(9) and
Brahmousin (5/8 Limousin and 3/8 Brahman)(10).
The AMCGL coordinates the genealogical record of breed purity and purity degrees, as well
as the production records that define the breed selection criteria and objectives(7). Productive
data associated with growth include live weight at birth and at 120, 210, and 365 d of age,
with measurements at the plus or minus 45-d interval of the specified age. Live weight
measurements generate a distribution of observations throughout the life of the animal, which
together can be used to characterize and evaluate the growth curve. Non-linear models
(NLM) characterize and analyze the animal growth curve based on the biological
interpretation and applications of the regression coefficients, as well as growth parameters
derived from the regression coefficients(11,12,13). Regression coefficients and growth
parameters play an important role in decision making for management, nutrition, breeding,
and genetic improvement programs(14,15,16,17). Based on the above, the objective of the present
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study was the selection and adjustment of a NLM to describe and evaluate the growth curve
in Limousin cattle from Mexico.
The database consisted of live weight measurements in the weight interval from birth to 500
d of age in Limousin cattle (PB; purebred). In order to define the growth curve, four NLMs
were evaluated: Brody (BRO), von Bertalanffy (BER), Gompertz (GOM), and logistic
(LOG), all of which are made up of three regression coefficients (β1, β2, and β3)(12,13,18). In
the NLM equations (Table 1), yi represents the live weight (kg) measured at time t; β1, is the
asymptotic value when t tends to infinity, interpreted as the adult weight parameter (AWP);
β2, is an adjustment parameter when y ≠ 0 and t ≠ 0; and β3, is the growth rate (GR),
expressing weight gain as a proportion of total weight. The BER, GOM, and LOG models
are characterized by describing growth based on a sigmoid curve, for which age (AIP;
months) and weight (WIP; kg) at the inflection point were calculated. The BRO model
exhibits a growth curve with a constant GR and no inflection point. The regression
coefficients were used to estimate the age at 50 % maturity (A50M), the degree maturity
attained at 15 months (A15M) of age(19,20), as well as the correlation (rac) between GR and
ADW.
Table 1: Nonlinear models evaluated in purebred and crossbred Limousin cattle

Model Equation
Logistic yi = β1 / (1 + β2*(exp(-β3*t))) + ei
Bertalanffy yi = β1*((1 - β2*(exp(-β3*t)))**3) + ei
Gompertz yi = β1*(exp(-β2*(exp(-β3*t)))) + ei
Brody yi = β1*(1 - β2*(exp(-β3*t))) + ei
yi= live weight in kg, measured at time t; β1= asymptotic value; β2= integration constant; β3= slope of the
growth rate curve.
Analyses were performed for each sex, using the Gauss-Newton method of the NLIN
procedure of the SAS statistical analysis software(21). The selection of the best-fitting model
was based on(18,19): Akaike information criterion [AIC= n*nl(sse/n) + 2k]; Bayesian
information criterion [BIC= n*nl(sse/n) + k*nl(n)]; coefficient of determination [R2= (1 -
𝑠𝑠𝑒
(sse/tss))]; and, overall standard error or model (OSE= √ . Where: n = total number of
𝑛−𝑝−1
data; sse= sum of squares of the error; tss = total sum of squares; k= number of parameters
in the model; nl = natural logarithm. For AIC and BIC, the model with the lowest value was
considered the best fit.
The AMCGL managed a herd register with various degrees of purity (DCB) for the purpose
of increasing the Limousin cattle population through absorbing crossbreeding, based on
crossbred cows and PB sires. With the model selected as the best fit in the PB population,
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the growth curve was characterized in populations defined by five DCB or generations: first
(D1) with ½ Limousin; second (D2) with ¾ Limousin; third (D3) with 7/8 Limousin; fourth
(D4) with 15/16 Limousin; and, fifth (D5) with 31/32 Limousin. Table 2 describes the
database analyzed in terms of BP and DCB.
Table 2: Live weight database, analyzed across genetic and sex groups, with measurements
from birth to 500 d of age
Sex / Group D1 D2 D3 D4 D5 Purity
Males 1963 1489 1607 3428 6224 31784
Females 2220 2296 2449 4784 7382 35695
Genetic groups: PG, 1/2 Limousin; SG, 3/4 Limousin; TG, 7/8 Limousin; CG, 15/16 Limousin; QG, 31/32
Limousin. Breed purity (> 63/64 Limousin).
In model selection, within sex with AIC and across sex with BIC, the best fitting model was
BER, followed by BRO and GOM; in all models the R2 was greater than 95 % (Table 3).
Table 4 shows the results for the regression coefficients and product growth parameters of
the evaluated NLMs. ADW estimation was higher for PB vs CB, in contrast, the GR was
higher in CB. The genetic improvement scheme for Limousin cattle in Mexico includes
weaning weights adjusted to 205 d(7), with potential significance in the growth curves, given
that the inflection point is located in the pre-weaning period. The BRO model was second in
the model ranking; however, it exhibited outlier results for ADW, A50M and DM15. All
models had a rac below -0.75 (Table 4), which indicates that high ADWs do not derive from
high GRs. Figure 1 for males and Figure 2 for females depicts the growth based on the BER
model for all genotypes evaluated.
Table 3: Statistics used for selection of the best-fit nonlinear model

Statistics Brody Gompertz Logistic Bertalanffy
Males
R2 96.7 96.7 96.6 96.7
OSE 40.3 40.3 40.9 40.3
AIC 236935.8 237022.3 237856.6 236896.4
BIC 236960.9 237047.5 237881.7 236921.2
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Females
R2 96.8 96.8 96.7 96.8
OSE 35.6 35.7 36.1 35.6
AIC 255208.7 255253.0 256169.4 255127.7
BIC 255234.2 255278.4 256194.9 255153.1
AIC= Akaike information criterion; BIC= Bayesian information criterion; R2= coefficient of determination;
OSE= overall or model standard error.
Table 4: Regression coefficients and growth parameters derived from nonlinear models
evaluated in purebred and crossbred Limousin cattle
item β1 β2 β3 rac AIP WIP A50M DM15
Purebred males with all the evaluated nonlinear models

Brody 1645.9 0.9778 0.000618 -0.99 -- -- 36.2 26.0
Gompertz 491.0 2.5475 0.00583 -0.92 5.3 180.6 7.4 83.1
Logistic 408.2 8.8538 0.0117 -0.76 6.2 204.1 6.2 98.5
Bertalanffy 566.1 0.5949 0.00400 -0.96 4.8 167.7 13.0 81.2
Purebred females with all evaluated nonlinear models
Brody 715.1 0.9508 0.00151 -0.99 -- -- 14.2 51.8
Gompertz 402.9 2.396 0.00656 -0.90 4.4 148.2 6.3 88.2
Logistic 352.3 8.0113 0.0124 -0.71 5.6 176.1 5.6 99.1
Bertalanffy 443.0 0.5666 0.00477 -0.95 3.7 131.2 10.6 87.9
Males in degree of purity with Bertalanffy model
D1 527.4 0.5896 0.00394 -0.97 4.8 156.3 13.1 80.7
D2 522.4 0.5858 0.00418 -0.96 4.5 154.8 12.3 83.0
D3 514.5 0.5876 0.00410 -0.96 4.6 152.4 12.6 82.3
D4 446.9 0.5705 0.00481 -0.95 3.7 132.4 10.5 88.1
D5 467.1 0.5724 0.00469 -0.96 3.8 138.4 10.8 87.3
Females in degrees of purity with the Bertalanffy model
D1 391.4 0.5563 0.00511 -0.94 3.3 115.9 9.7 90.0
416
D2 374.5 0.5446 0.00563 -0.94 2.9 110.9 8.7 92.6

D3 419.9 0.5619 0.00476 -0.95 3.7 124.4 10.5 87.9
D4 399.2 0.5572 0.00509 -0.95 3.4 118.3 9.8 89.9
D5 379.9 0.5471 0.00551 -0.94 3.0 112.5 8.9 92.1
Degrees of purity: D1, 1/2 Limousin; D2, 3/4 Limousin; D3, 7/8 Limousin; D4, 15/16 Limousin;
D5, 31/32 Limousin. Regression coefficients: β1, β2, and β3. Where: β1 is the asymptotic value,
interpreted as the adult weight parameter; β2 is an adjustment parameter, and β3 is the growth rate,
expressing weight gain as a proportion of total weight. Age (AIP; months) and weight (WIP; kg) at
the inflection point. A50M, age at 50 % of maturity. DM15, degree of maturity (%) at 15 months of
age. rac, correlation between β1 and β3.
Figure 1: Growth curves for Limousin males. Purity, purebred animals; D1, 1/2 Limousin;
D2, 3/4 Limousin; D3, 7/8 Limousin; D4, 15/16 Limousin; D5, 31/32 Limousin
Figure 2: Growth curves for Limousin females. Purity, purebred animals; D1, 1/2
Limousin; D2, 3/4 Limousin; D3, 7/8 Limousin; D4, 15/16 Limousin; D5, 31/32 Limousin
417
In purebred Limousin cattle with three different production systems, Igarzabal et al(3)
reported GOM as the best fitting model. In crossbreeding schemes of Limousin with Angus,
Hereford, and MARC III, Zimmermann et al(17) used the BRO model to characterize the
growth curve and evaluate live weight at maturity; in Limousin x Friesian cattle, they
represented growth based on the GOM model(22). In the Madrasin breed, product of the
crossbreeding of Limousin with Madura, the growth described a sigmoid type curve,
characterized with the LOG model(23). Growth curves evaluated with the BER model were
reported in Holstein(24), Pyrenean, and Blonde cattle(3).
In Mexico, several studies have discussed contrasts in the type of growth curve across breeds.
For growth curves without an inflection point, in five zebu breeds in tropical cattle ranching,
Domínguez-Viveros et al(25) reported that the best fitting NLMs were Brody, Meloum III and
Mitscherlich; the BRO model in particular was selected as the one with the best fit in
Romosinuano cows(20), as well as in Tropicarne(19) and Salers cattle(26). For sigmoid growth
curves, Contreras et al(27) in Jersey, Holstein, and Jersey-Holstein crossbred cows, the
selected MNLs were GOM, LOG, and BER, respectively; the BER model has been reported
for Hereford cattle(26).
The incorporation of replacement heifers at the reproductive phase is of transcendence for

the genetic progress and profitability of the herd. This procedure is carried out in three
stages(28): at the onset of pituitary maturation, triggered at a certain age and weight; followed
by the development of the ovaries and body growth; maturation of the uterus as a
consequence of pituitary development and its hormonal influence on body growth and
ovarian activity, allowing the heifer to mate and develop gestation. Several studies have
analyzed the influence of growth parameters on reproductive variables(20,24,29); Thus, the
inflection point has been associated(13,30,31) with the onset of the reproductive phase. Age at
first calving is an indicator of the time it takes for an animal to reach sexual maturity and
reproduce for the first time, and mating at approximately 15 mo, with age at first calving of
approximately 24 mo, has positive effects on cow longevity and productivity(32,33). Based on
the BER model, differences are observed in CB vs PB females for the components of the
growth curve (Table 4), which can be attributed to genetic differences across breeds and
heterosis effects resulting from the crossing scheme. In PB heifers, AIP was estimated at 3.7
mo with a WIP of 131.2 kg; in CB, AIP and WIP were in the ranges of 2.9 to 3.7 mo and
110.9 to 124.4 kg, with average values of 3.3 and 116.4, respectively. A50M in females, was
estimated to be at 10.6 mo in PB, and within the range of 8.9 to 10.5 mo for CB, with an
average value of 9.5 months. For DM15 in females, the average value was 90.5 % for CB,
while the estimate for PB was 87.9 %. In contrast, among females from other populations
and based on the BER model: Contreras et al(27) for Holstein, Jersey and crossbreds estimated
AIP (months) and WIP (kg) in the ranges of 7.4 to 9.8 and 115.0 to 151.7, respectively; in
five zebu breeds, Domínguez-Viveros et al(25) reported AIP and WIP estimates in the ranges
418
of 3.9 to 11.7 and 107.2 to 230.9, respectively; in Romosinuano(20), Tropicarne(19), and

Siboney(29). AIP - WIP results were 15.5 - 132.5, 7.7 - 180.5 and 5.9 - 152.4, respectively.
As for the males, the selection of stallions is carried out among PB, and they are incorporated
for reproduction from one year of age; however, lowering the age of entry into reproduction
reduces the generation gap and has an impact on genetic progress(34). The growth curve can
influence the development of the reproductive phase; in Bos taurus breeds, the physiological
events associated with reproduction begin at six to eight months; maturity and reproductive
capacity are determined by the quality of the semen, with variations due to the effects of live
weight, growth rate, scrotal circumference, among other factors(35). The results indicate that
PB males reach 50 % maturity at 13 mo of age, with values above 80 % at 15 mo of age
(Table 4). In contrast(26), Hereford and Salers cattle reported maturity levels of 68.2 % and
76.6 % at one year, respectively. On the other hand, growth curve indicators are associated
with profitability in production; the GR has an effect on age and slaughter weight; degree of
maturity is important for efficiency and carcass composition(16,22). Various authors(15,36,37)
have assessed the differences and derivations of the growth curve in relation to production
for purebred males and various crosses.
The best fitting model was von Bertalanffy, which described a sigmoid growth curve, with
differences in growth parameters across the evaluated genotypes. Tipping point estimates are
within the context of pre-weaning growth.
Acknowledgments
The authors are grateful to the Mexican Association of Limousin Cattle Breeders (Asociación
Mexicana de Criadores de Ganado Limousin) for having provided the database analyzed
herein.
Literature cited:
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2010;88(2):460-466.
3. Igarzabal A, Oregui LM, Mandaluniz N, Amenabar ME, Ruiz R, Neiker AB. Estudio de
las curvas de crecimiento del ganado vacuno en los principales sistemas de producción
del País Vasco. ITEA 2005;26(I):222-224.
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Technical note
Relationship between body measurement traits, udder measurement

traits and milk yield of Saanen goats in Capricorn district of South Africa
Thlarihani Cynthia Makamua
Molabe Kagisho Madikadikea
Kwena Mokoena
Thobela Louis Tyasi a*
a
University of Limpopo. Department of Agricultural Economics and Animal Production.,
Limpopo Province, South Africa.
*Corresponding author: louis.tyasi@ul.ac.za
Abstract:
The association between body measurements and udder measurements can be used towards
the improvement of milk yield. The objective of the study was to investigate the correlation
between udder measurement traits and milk yield. The study was conducted at Sikline village
in Mankweng, Capricorn district of Limpopo province, South Africa where a total number
of 30 lactating Saanen goats were used. Pearson’s correlation technique was used for data
analysis. The results showed a significant (P<0.05) correlation between distance between
teats and milk yield (r= 0.45) and a highly negative significant (P˂0.01) correlation between
teat diameter and milk yield (r= -0.57). Body weight and milk yield (r= 0.54) had a highly
positive significant correlation (P˂0.01). The finding of the current study implies that body
weight and distance between teats can be used to improve the milk yield in Saanen goats.
The finding of the study might be used to predict the milk yield of Saanen goats. However,
further studies need to be conducted on relation of body and udder measurements and milk
yield using higher sample size.
Keywords: Correlation, Body weight, Teat diameter, Udder circumference, Milk yield.
423
Saanen goats can adapt to different climatic conditions, and they are characterised as medium
to large sized goats with high milk yield(1). Body and udder measurements of an animal are
of importance to the farmers as it can be used for feeding, administering of medication,
selection for breeding and management on the farm(2). Kouri et al(3) reported that body weight
and udder measurements play a significant role on milk yield. Saanen goats are docile with
high milk yield of about 2.2 kg/d(4). There is lack of knowledge on communal farmers to
which traits can be used to improve milk yield(5).
The relationship between the udder and body measurements of different animals have been
investigated; Arcos-Alvarez et al(6) reported that there is a correlation between udder
measurements, the volume of the udder, and the daily milk yield of Pelibuey ewes. Adewumi
et al(7) highlighted that in a traditional rearing system of small stock, results revealed that
partial milk yield could be determined based on udder size and teat length in extensively
reared does and ewes and heart girth of kids could be used to indicate the doe’s milk
production. Therefore, milk yield can be predicted from body measurement traits and udder
measurement traits. However, as far as known, information on using body measurement traits
and udder measurement traits to improve the milk yield of Saanen goats in South Africa is
limited and not conclusive. Hence, the objective of this study was to determine the correlation
between body measurement traits, udder measurement traits, and milk yield of Saanen goats.
The outcomes of this study will help communal farmers to identify body measurement traits
and udder measurement traits that can be used to improve milk yield of Saanen goats.
The study was conducted at Sikline village in Mankweng situated at 23°53′24″S latitude and
29°45′25″E longitude, Capricorn district of Limpopo Province, South Africa. The ambient
temperature around the study area ranges between 16 and 27 ℃ in summer while in winter it
ranges between 8 and 22 ℃. The area receives a mean annual rainfall of 450 mm(8). A total
of 30 lactating Saanen goats reared under an extensive farming system between the ages of
3 to 5 yr were used.
The animals were kept under an extensive farming system. Whereby, they were released into
the veld to browse in the morning and collected back to their kraal in the evening. Clean
water was provided ad-libitum.
A cross-sectional design was used for measuring body measurement traits and udder
measurement traits in this study. Milking records for 2 wk that took place 7 d after kidding
were collected by the farmer.
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Body measurements were recorded using a tape measure calibrated in centimeters while body
weight was taken using a weighing scale once in the morning before grazing. Body
measurement traits were taken as described by Pesmen and Yardimci(9). Briefly, Wither’s
height (WH) was measured as the distance from the surface of a platform to the withers,
Body length (BL) was measured as the distance from the occipital joint to the first caudal
vertebra, Heart girth (HG) was measured behind the scapula, Sternum height (SH) was
measured as the distance between the floor and the ventral surface of the sternum, and Rump
height (RH) was measured as a distance between the floor and the dorsal edge of the pelvic
girdle.
The following external udder measurement traits: Udder length before milking and after
milking (ULB & ULA); Udder width before milking and after milking (UWB & UWA);
Udder circumference before milking and after milking (UCB & UCA); Distance between
teats before milking and after milking (DBTB & DBTA); Teat diameter before milking and
after milking (TDB & TDA); Teat length before milking and after milking (TLB & TLA)
were measured using a tape measure (cm)(10). The milking of goats was performed twice a
day for 2 wk, in the morning and afternoon. The udder measurements were collected before
and after milking.
Statistical Package for Social Sciences (IBM SPSS, 2020) version 27.0 software was used
for analysing the data. Descriptive statistics including mean, standard deviation, standard
error, and coefficient of variance were calculated. Pearson’s correlation was used to
determine the relationship between measured traits. A probability of 5% was used for
significance and 1% for high significance between traits.
Descriptive statistics of body measurement traits, udder measurement traits, and milk yield
of Saanen goats are shown in Table 1. The mean value of MKY was found to be 47.69 L, the
highest mean value was displayed by HG and BL, while TDA had the lowest mean value.
The highest coefficient of variation was recorded by TLB whereas WH recorded the lowest
value. The results highlight that the Saanen goats had an average of 74.27 kg BW; the results
are higher than the results that were obtained by Adewumi et al(7) where average body weight
was 19.20 and 23.09 kg for goats and sheep respectively.
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Table 1: Descriptive statistics of measured traits (cm)

Traits Mean SD SE CV
BL 76.13 7.92 1.45 10.40
RH 75.37 5.43 0.99 7.20
WH 74.80 4.37 0.80 5.84
SH 49.03 3.85 0.70 7.84
HG 86.47 7.97 1.46 9.22
BW, kg 74.27 15.30 2.79 20.60
ULB 24.40 1.89 0.34 7.73
ULA 20.07 3.53 0.64 19.60
UWB 26.60 1.96 0.36 7.36
UWA 20.77 2.96 0.54 14.23
UCB 57.43 4.01 0.73 6.97
UCA 37.17 5.40 0.99 14.52
DBTB 11.27 2.07 0.38 18.34
DBTA 9.43 1.41 0.26 14.91
TDB 3.77 1.45 0.27 38.62
TDA 3.17 1.20 0.22 37.74
TLB 5.27 2.55 0.46 48.33
TLA 4.20 1.58 0.29 37.72
MKY, L 47.69 11.21 2.80 23.50
Body length (BL), Rump height (RH), Wither’s height (WH), Sternum height (SH), Heart girth (HG), Udder
length before milking (ULB), Udder length after milking (ULA), Udder width before milking (UWB), Udder
width after milking (UWA), Udder circumference before milking (UCB), Udder circumference after milking
(UCA) Distance between teats before milking (DBTB), Distance between teats after milking (DBTA), Teat
diameter before milking (TDB), Teat diameter after milking (TDA), Teat length before milking (TLB), Teat
length after milking (TLA), Milk yield (MKY).
Phenotypic correlation between body measurement traits and udder measurement traits is
presented in Table 2. The body measurements showed a significant relationship with udder
measurements in Saanen goats. The highest significant correlation coefficient between body
measurement and udder measurements was recorded between BL and UWA, HG and UWB,
WH and ULB at (P<0.01). The lowest correlation coefficient was highlighted between BL
and DBTB, WH and UCB, SH and ULA, BW and ULA at (P<0.05). There was a negative
significant correlation between RH and TLB (P<0.05). The findings of this study suggest
that body length can be used to improve distance between teats and udder width whereas
Rump height can be used to improve udder width, distance between teats and teat length. The
findings are similar with the study conducted by others(7) who stated that there was a
significant correlation between body measurement traits and udder measurement traits such
as distance between teats, udder width, udder length, and udder circumference that had a
significant correlation with body measurement traits such as body length, rump height,
426
withers height, and sternum height respectively in ewes and does in Nigeria. Other studies
conducted in sheep indicated that body measurements such as body weight, withers height,
body length, heart girth, neck length, and neck circumference had a significant correlation
with udder circumference, udder width, teat length, and distance between teats in West
African Dwarf sheep(5).
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Table 2: Phenotypic correlation between body measurement traits and udder measurement traits
Traits BL RH WH SH HG BW ULB ULA UWB UWA UCB UCA DBTB DBTA TDB TDA TLB TLA
BL
RH 0.40*
WH 0.44* 0.23ns
SH 0.28* 0.29* -0.10ns
HG 0.57** 0.46* 0.45* 0.37*
BW 0.29* 0.15ns 0.05ns 0.22ns 0.23ns
ULB 0.13ns 0.06ns 0.50** -0.13ns 0.04ns 0.06ns
ULA 0.11ns 0.15ns 0.12ns 0.28* 0.10ns 0.27* 0.48*
UWB 0.16ns 0.25* 0.20ns 0.20ns 0.63** 0.21ns 0.48* 0.43*
UWA 0.72** 0.08ns 0.06ns 0.07ns -0.10ns 0.02ns 0.58** 0.71** 0.66**
UCB -0.11ns 0.12ns 0.35* -0.07ns 0.21ns 0.20ns 0.39* 0.25* 0.19ns 0.10ns
UCA 0.14ns 0.10ns 0.24ns 0.06ns 0.07ns 0.23ns 0.57** 0.61** 0.36* 0.46* 0.10ns
DBTB 0.38* 0.26* 0.17ns 0.23ns 0.24ns 0.18ns 0.12ns 0.25* 0.31* 0.32* 0.26* 0.05ns
DBTA 0.08ns 0.03ns -0.07ns -0.07ns -0.15ns 0.05ns -0.13ns 0.18ns 0.27* 0.38* 0.09ns 0.14ns 0.59**
TDB -0.18ns -0.05ns -0.07ns -0.02ns -0.21ns -0.12ns -0.03ns -0.14ns -0.13ns -0.39* 0.12ns 0.13ns -0.43* -0.34*
TDA 0.00ns 0.01ns 0.23ns -0.02ns -0.17ns 0.07ns 0.26* 0.04ns -0.03ns -0.14ns 0.11ns 0.33* -0.10ns -0.36* 0.61**
TLB -0.23ns -0.29* -0.08ns -0.03ns -0.03ns -0.17ns -0.09ns -0.29* -0.23ns -0.26* 0.10ns -0.16ns -0.18ns -0.30* 0.36* 0.44*
TLA -0.04ns -0.09ns 0.16ns 0.03ns 0.12ns -0.14ns -0.07ns -0.23ns -0.11ns -0.11ns 0.10ns -0.16ns 0.17ns -0.10ns 0.02ns 0.37* 0.81**
Body length (BL), Rump height (RH), Wither’s height (WH), Sternum height (SH), Heart girth (HG), Udder length before milking (ULB), Udder length
after milking (ULA), Udder width before milking (UWB), Udder width after milking (UWA), Udder circumference before milking (UCB), Udder
circumference after milking (UCA) Distance between teats before milking (DBTB), Distance between teats after milking (DBTA), Teat diameter before
milking (TDB), Teat diameter after milking (TDA), Teat length before milking (TLB), Teat length after milking (TLA), Non-significant (ns), Correlation is
significant at 0.05 level, (*), Correlation is significant at a 0.01 level (**).
428
Phenotypic correlation between udder measurement traits and milk yield is shown in Table
3. The udder measurements traits had no statistical significant correlation with MKY except
for DBTA that showed a positive correlation at (P<0.05). There was a highly negative
significant correlation between MKY and TDB (P<0.01) and a negative significant
correlation with TLB (P<0.05). Results indicated that all udder measurement traits had no
significance correlation with milk yield except for the DBTA, TLB, and TDB. The study is
in agreement with other study(11) who stated that the correlation coefficients between milk
yield and udder measurement traits were not significant in White Bornu and West African
Dwarf of Southern Nigeria. There current findings imply that udder measurements such as
distance between teats, teat length and teat diameter can be used in improving milk yield.
Similarly, Žujović et al(12) highlighted a relationship between breast width, breast depth, and
milk yield of domestic Balkan goat breed that is reared in the mountain range Sharplanina.
However, the study conducted in Pelibuey ewes(6) disagrees with the current study and stated
that there was no correlation between teat length and teat diameter in the relationship between
udder measurement and milk yield in Pelibuey Ewes. These results may differ due to
differences in the breed of animal used and environmental factors.
429
Table 3: Phenotypic correlation between udder measurement traits and milk yield
Traits ULB ULA UWB UWA UCB UCA DBTB DBT TDB TDA TLB TLA MKY
A
ULB
ULA 0.48*
UWB 0.48* 0.43*
UWA 0.58** 0.71* 0.66*

* *
UCB 0.39* 0.25ns 0.19ns 0.10ns
UCA 0.57** 0.61* 0.36* 0.46* 0.10ns

*
DBTB 0.12ns 0.25ns 0.31* 0.32 0.26* 0.05ns
DBTA -0.13ns 0.18ns 0.27ns 0.38* 0.09ns 0.14ns 0.59**
TDB -0.03ns -0.14ns -0.13ns -0.39* 0.12ns 0.13ns -0.43* -0.34*
TDA 0.26* 0.04ns -0.02ns -0.14ns 0.11ns 0.33* -0.10ns -0.36* 0.61**
TLB -0.09ns -0.29* -0.23ns -0.26ns -0.10ns -0.16ns -0.18ns -0.30* 0.36* 0.44*
TLA -0.07ns -0.23ns -0.11ns -0.11ns 0.10ns -0.16ns 0.17ns -0.10ns 0.02ns 0.37* 0.81*
*
MKY -0.18ns 0.07ns 0.25ns 0.19ns 0.04ns 0.12ns 0.28ns 0.45* -0.57** -0.29ns -0.48* -0.13ns
Udder length before milking (ULB), Udder length after milking (ULA), Udder width before milking (UWB), Udder width after milking
(UWA), Udder circumference before milking (UCB), Udder circumference after milking (UCA) Distance between teats before milking
(DBTB), Distance between teats after milking (DBTA), Teat diameter before milking (TDB), Teat diameter after milking (TDA), Teat
length before milking (TLB), Teat length after milking (TLA), Milk yield (MKY), Non-significant (ns), Correlation is significant at 0.05
level, (*), Correlation is significant at a 0.01 level (**).
430
Table 4 present the correlation between body weight and milk yield. The results of correlation
between milk yield and body measurements revealed that there was no significant correlation
between the five body measurements taken. Only BW showed a highly positive correlation
with MKY (P<0.01). On evaluation of relationship between body measurement traits and
milk yield, results recognized that all body measurement traits had no significance correlation
with milk yield except body weight which had highly positive statistical significance
correlation with milk yield. The study is in harmony with the findings of Kouri et al(3) who
stated that milk yield was positively correlated with body weight of Damascus and Zaraibi
goats. However, it disagrees with other study(2) who stated that body length had a positive
significance correlation with milk yield in Damascus and Zaraibi goats in Egypt. The
environmental factors and the breed morphological structure may factor for the difference in
the outcome of results obtained.
Table 4: Phenotypic correlation between body measurement traits and milk yield
Traits BL RH WH SH HG BW MKY
BL
RH 0.40*
WH 0.44* 0.23ns
SH 0.28ns 0.29* -0.10ns
HG 0.57** 0.46* 0.45* 0.37*
ns ns
BW 0.29* 0.15 0.05 0.22ns 0.23ns
MKY 0.06ns 0.24ns 0.09ns 0.15ns 0.18ns 0.54**
Body length (BL), Rump height (RH), Wither’s height (WH), Sternum height (SH), Heart girth (HG), Milk
yield (MKY),
Non-significant (ns), significant at 0.05 level, (*),significant at a 0.01 level (**).
The current study concludes that the following udder measurement traits had a relationship
with body measurement traits: udder width with body length, rump height and heart girth;
distance between teats with body length and rump height; teat length with rump height; udder
length with withers height and body weight; and udder circumference with withers height
and sternum height. Distance between teats, teat length, and teat diameter has a relationship
with milk yield. The body weight of Saanen goat showed a relationship with milk yield. This
finding can be used by communal farmers to improve the milk yield of the Saanen goats.
There is a need for more investigation on the relationship between body measurement traits
and udder measurement and milk yield to assist in the improvement of milk yield.
431
Acknowledgments
The authors would like to express their appreciation to the University of Limpopo for
providing the necessary resources that were needed to carry out the study and the Saanen
dairy farmer at the Sikline village in the Mankweng area, Capricorn district of Limpopo
Province, South Africa for allowing data collection.
Conflict of interests
The authors declare that they have no conflict of interest.
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Meriz goats. Res Opin Anim Vet Sci 2021;1(9):601-605.
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and milk production of goats in different lactations. Biotechnol Anim 2011;27(2):217-
225.
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Technical note
Miguel Ángel Domínguez Martínez a*
Víctor Hernández Núñez b
Araceli Mariscal Méndez a
Amparo Martínez Martínez c
Gisela Fuentes-Mascorro a
a
Universidad Autónoma Benito Juárez de Oaxaca. Facultad de Medicina Veterinaria y
Zootecnia. Cuerpo Académico Ciencias Veterinarias Aplicadas al Desarrollo Regional. Av.
Universidad S/N. Ex-Hacienda 5 Señores, 68120. Oaxaca de Juárez, Oaxaca, México.
b
Universidad Tecnológica de la Mixteca, Brigada de Promoción del Desarrollo. Oaxaca,
México.
c
Universidad de Córdoba, Facultad de Veterinaria. Córdoba, España.
*
Corresponding author: mdominguez.cat@uabjo.mx
Abstract:
The Mixteco Creole cattle is a little explored genetic resource, which, however, has great
value due to its potential to be used in production systems that are respectful of the
environment and adaptable to its conditions. The identification and characterization of this
local resource is essential for its conservation and improvement. For this reason, in the
present study it was carried out the analysis of the diversity and genetic relationships of the
Mixteco Creole cattle population of Oaxaca, using 19 microsatellite DNA markers and 32
reference cattle populations belonging to the BIOBOVIS consortium of the CONBIAND
Network. The mean number of alleles detected was 8.8 ± 2.1 and the estimated effective
number of alleles was 4.5 ± 1.2. The genetic diversity represented by the expected (0.7700 ±
0.0682) and observed (0.7170 ± 0.0998) heterozygosity values was within the range of
434
estimators obtained in previous studies with local cattle populations using microsatellite
markers. An analysis of the population structure revealed a predominant influence of Iberian
germplasm (Bos taurus). There is also a close relationship between the Mixteco Creole and
the rest of the Mexican Creole cattle populations, with the exception of the Tropical Dairy
Creole.
Keywords: Conservation, Creole cattle, Genetic characterization, Microsatellites.
Creole cattle represent a genetic resource of great importance for the supply of food and raw
materials in areas with extreme climatic conditions, scarce food resources and high incidence
of infectious and parasitic diseases(1), and potentially contribute to hunger and poverty
reduction, as well as to sustainable development(2). However, the inability to appreciate the
real biological, economic and cultural value of these animals has caused an aggressive
extension of highly selected breeds, causing a constant erosion that endangers the existence
of these resources and, thereby, an irreparable loss of genetic variability, which could be of
great value to face the effects of climate change.
In the state of Oaxaca, there is a population of Creole cattle located in the Mixteca region
(Figure 1A), known as Mixteco Creole. Phenotypically, they are medium-sized animals, with
an average height at the withers of 1.03 ± 0.16 m and an average weight of 176 ± 51.48 kg
(parameters reported for an age of 1 to 3 yr)(3); their coat can be uniform black or red, or
black- or red-spotted (Figure 1B). The origin of the Mixteco Creole cattle dates back to
colonial times, having probably been brought by the first Spaniards for the construction of
the convents along the Oaxacan territory, on what is currently known as the Dominican
Route(4).
435
Figure 1: A) Geographic location of the Mixteca region of Oaxaca. B) Mixteco Creole

bovine cattle
The image shows the coloration and morphostructural characteristics of the Mixteco Creole bovine cattle
Since its introduction into the region, the Mixteco Creole cattle have successfully adapted to
the geographical and environmental conditions prevailing in the area, which is characterized
by a complicated orography and fluctuations in the availability of food and water. However,
these cattle are capable of being productive under such conditions, which makes them
suitable for developing environmentally friendly, resilient production systems that are able
to cope with changes in the environment, especially with the current trend in Latin America
towards the development of more intensive and sustainable production systems(5).
According to the Food and Agriculture Organization of the United Nations (FAO), the
identification and genetic characterization of local livestock is an essential step in the
conservation and use of these genetic resources(6). In this regard, microsatellite molecular
markers have proven to be a highly effective tool to genetically characterize Creole cattle
populations in the Americas(7–10), Therefore, this study evaluated the genetic diversity of the
Mixteco Creole cattle population and their genetic relationships with other local and
specialized cattle populations, using 19 microsatellite markers and 32 reference populations
in order to generate information on the conservation status of this valuable local genetic
resource.
A total of 40 adult Mixteco Creole cattle (29 females and 11 males) were selected and
identified based on phenotypic characteristics of Creole cattle previously reported in the
literature, including color pattern, size, and zoometric parameters(3,11); those individuals that
exhibited phenotypic characteristics typical of Zebu breeds were discarded. In order to avoid
close kinship relationships among the selected individuals, only one specimen was included
for each one of the cattle production units sampled, geographically separated in different
436
communities of the Oaxacan Mixteca (17°48′00″ N, 97°46′00″ W), in addition to confirming,

through an interview, the absence of genetic connections (use of stallions) between the
production units. The sample size was defined taking as a reference the information published
for population genetic studies using microsatellite molecular markers(12), as well as the
sample size suggested by FAO for genetic characterization studies of local livestock
populations using microsatellites (n=25 to 40)(13).
The biological material consisted of whole blood samples with anticoagulant (EDTA)
obtained by aseptic puncture of the jugular vein, from which nucleic acids were extracted
using the ReliaPrepTM Blood gDNA Miniprep System kit (Promega), following the
manufacturer's instructions. A panel of 19 microsatellite markers recommended by FAO-
ISAG was used for the genetic analysis of the population(14) (Table 1), having been amplified
by PCR and processed in an ABI377XL capillary sequencer (Applied Biosystems), with
subsequent allelic typing, following the methodology established at the Laboratory for the
Improvement and Conservation of Animal Genetic Resources of the University of Cordoba,
Spain(7).
437
Table 1: Allele frequencies observed in the Mixteco Creole Cattle population

BM1818 BM1824 BM2113 CSRM60 CSSM66
Allele Frec. Allele Frec. Allele Frec. Allele Frec. Allele Frec.
260 0.0132 179 0.2750 126 0.0385 91 0.0125 179 0.0132
262 0.0395 181 0.1750 128 0.0513 93 0.1500 181 0.0921
264 0.3553 183 0.3875 130 0.0769 95 0.0125 183 0.1974
266 0.1842 185 0.0125 132 0.0256 97 0.1375 185 0.0789
268 0.3421 189 0.1500 134 0.0769 99 0.0250 187 0.0132
270 0.0526 136 0.2436 101 0.0125 189 0.2895
272 0.0132 138 0.3077 103 0.4125 191 0.0132
140 0.1026 105 0.1875 193 0.1842
142 0.0769 111 0.0375 197 0.1184
113 0.0125
ETH003 ETH010 ETH185 ETH225 HAUT27
103 0.0263 209 0.1667 220 0.0286 139 0.2000 128 0.0135
109 0.1053 211 0.0128 222 0.0571 141 0.0125 140 0.0270
115 0.0658 213 0.1026 226 0.0571 143 0.1125 142 0.0811
117 0.3421 215 0.1154 228 0.6000 145 0.0250 146 0.0135
119 0.1974 217 0.3333 230 0.0143 147 0.2625 148 0.5405
123 0.0658 219 0.1923 232 0.1000 149 0.2875 150 0.2297
125 0.1711 221 0.0256 234 0.1143 151 0.0125 152 0.0676
129 0.0263 223 0.0513 236 0.0143 153 0.0250 154 0.0270
240 0.0143 157 0.0625
HEL009 ILSTS006 INRA32 INRA63 MM12
149 0.0128 287 0.0556 168 0.0152 175 0.4250 105 0.0132
151 0.0128 289 0.0417 174 0.0152 177 0.3250 109 0.0132
153 0.3077 291 0.2361 176 0.1364 179 0.0125 117 0.1579
155 0.0641 293 0.2778 178 0.2879 183 0.2000 119 0.1184
157 0.0256 295 0.1111 180 0.3485 185 0.0375 121 0.2368
159 0.0256 297 0.2639 182 0.0303 123 0.1184
161 0.2308 299 0.0139 184 0.1212 125 0.0526
163 0.1154 186 0.0152 129 0.0132
165 0.0513 188 0.0303 131 0.0263
167 0.0513 133 0.1974
169 0.0128 135 0.0395
171 0.0897 139 0.0132
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SPS115 TGLA053 TGLA122 TGLA227

Allele Frec. Allele Frec. Allele Frec. Allele Frec.
242 0.0375 151 0.0658 134 0.0250 79 0.0769
244 0.4875 153 0.0395 140 0.0125 81 0.0256
246 0.0625 157 0.2105 142 0.0375 83 0.1282
248 0.1875 159 0.1711 144 0.0375 85 0.3077
250 0.0250 163 0.0526 146 0.0750 87 0.0128
252 0.0875 165 0.0921 148 0.0125 89 0.0641
254 0.0250 167 0.1842 150 0.3875 91 0.0641
256 0.0875 169 0.1447 152 0.2875 93 0.0128
175 0.0263 154 0.0125 95 0.0385
181 0.0132 156 0.0125 99 0.2692
160 0.0625
168 0.0125
174 0.0250
The information generated from allelic typing was used to calculate allele and genotypic
frequencies for each microsatellite marker using the MSTools add-in for Excel (Genetics
Dept, TCD, Ireland). The number of alleles per locus, effective number of alleles, observed
and expected heterozygosity, polymorphic information content (PIC), and FIS inbreeding
coefficient were estimated with Popgene v 1.32(15). The Hardy-Weinberg equilibrium test
was carried out with Arlequin v 3.1(16).
The analysis of the population structure and genetic relationships was carried out using allelic
information from 32 reference populations previously reported in the literature (Figure 2)(17),
belonging to the BIOBOVIS Consortium (https://BIOBOVIS.jimdofree.com/) of the
Network for the Conservation of Local Domestic Animal Biodiversity (CONBIAND). The
reference populations were divided into four groups according to their origin or specialization
(Mexican Creoles, Iberians, specialized Europeans, and Zebu). The genetic distance between
pairs of NEI populations (DST) was estimated with the Arlequin v3.1 software. Based on the
information generated in the genetic distance matrix, a graphical representation in the form
of a phylogenetic tree was created using the SplitsTree v.4.14.16 software, with the
Neighbor-Joining method. Finally, the Structure software was utilized(18) to infer the
population structure, using the following parameters: 100,000 warm-up iterations followed
by 1'000,000 Markov chain-based Monte Carlo (MCMC) iterations. A total of 32 different
runs (K2 to K33) were performed to estimate the most probable number of existing clusters.
The optimal K value was estimated by the modal value method of the Delta K distribution,
using the formula Delta K = mean (|L''(K)|) / sd (L(K)).
439
Figure 2: NEI's genetic distance (unbiased distance) tree using the Neighbor-Joining
method
The circle shows the location of the Creole populations within the genetic distance tree (except for the
Tropical Dairy Creole), revealing their intermediate position between the European Bos taurus breeds and the
Bos indicus breeds. Populations: MIX (Mixteco Creole Bovine), CRI (Tropical Dairy Creole), POB (Poblano
Creole), CBC (Baja California Creole), CHU (Chihuahua Creole), CNY (Nayarit Creole), CHI (Chiapas
Creole), TDL (Fighting), RET (Very dark), BCO (Red-spotted), BNE (Black-spotted), MAR (Marshland),
PAJ (Straw-colored), NAN (Andalusian Black), VCA (Canary Island cattle), PAL (Palmera), AAN
(Aberdeen Angus), BWC (British White Cattle), HER (Hereford), JER (Jersey), SHO (Shorthorn), DEX
(Dexter), BWS (Brown Swiss), CHA (Charolais), HOL (Holstein- Fresian), LIM (Limousin), SIM
(Simmental), GEB (Gelbvieh), GYR (Gyr), BRH (Brahman), SIN (Sindi), GUZ (Guzerat), NEL (Nelore).
Table 1 shows the results of the calculation of allele frequencies for each of the 19 loci
analyzed in the Mixteco Creole cattle population. Polymorphic variation was observed in all
the loci analyzed. In total, 168 alleles were detected distributed among the 19 microsatellites,
representing a mean number of alleles of 8.8 ± 2.1, with TGLA122 (Na= 13), MM12 (Na=
12) and HEL009 (Na= 12) markers having the highest number of alleles (Table 2). With
regard to the effective number of alleles, a mean of 4.5 ± 1.2 alleles was observed, while the
average polymorphic information content was 0.7286 ± 0.0748. The observed heterozygosity
value was 0.7170 ± 0.0998, and the expected heterozygosity was 0.7700 ± 0.0682. Table 2
shows the results of the main genetic diversity parameters for each of the microsatellite
440
markers evaluated. The HAUT27, ILSTS006, and TGLA227 markers were the only loci in
Hardy Weinberg disequilibrium (P<0.05). On the other hand, 15 of the 19 microsatellite
markers showed positive FIS values, and the remaining 4 showed negative values; however,
most of them were separated from the zero value, obtaining a mean FIS value of 0.058, with
TGLA227 and BM1818 markers showing the greatest deviation from the positive FIS value
and CSRM60 marker showing the greatest deviation from the negative FIS value.
Table 2: Genetic diversity parameters

Marker Na Ne PIC Ho He FIS P value
BM1818 7 3.5479 0.6695 0.5789 0.7277 0.1938 0.0640

BM1824 5 3.5834 0.6728 0.6500 0.7301 0.0984 0.7939
BM2113 9 5.3462 0.7907 0.7949 0.8235 0.0222 0.8494
CSRM60 10 4.0100 0.7195 0.8250 0.7601 -0.0991 0.6078
CSSM66 9 5.3780 0.7895 0.8684 0.8249 -0.0668 0.4087
ETH003 8 4.8456 0.7673 0.7632 0.8042 0.0384 0.8776
ETH010 8 4.9223 0.7704 0.7436 0.8072 0.0668 0.1559
ETH185 9 2.5574 0.5860 0.5143 0.6178 0.1555 0.2228
ETH225 9 4.7690 0.7597 0.7750 0.8003 0.0194 0.9550
HAUT27* 8 2.7939 0.6022 0.6216 0.6509 0.0319 0.0042
HEL009 12 5.5410 0.7990 0.8205 0.8302 -0.0012 0.8609
ILSTS006* 7 4.5474 0.7458 0.8056 0.7911 -0.0326 0.0136
INRA32 9 4.1644 0.7244 0.6667 0.7716 0.1227 0.4309
INRA63 5 3.0505 0.6101 0.6000 0.6807 0.1074 0.4384
MM12 12 6.5045 0.8283 0.8421 0.8575 0.0049 0.2746
SPS115 8 3.3934 0.6763 0.6750 0.7142 0.0430 0.7280
TGLA053 10 6.8274 0.8366 0.7368 0.8649 0.1367 0.1248
TGLA122 13 4.0455 0.7211 0.7000 0.7623 0.0702 0.5162
TGLA227* 10 4.9951 0.7743 0.6410 0.8102 0.1985 0.0017
Na= number of alleles; Ne= effective number of alleles; PIC= polymorphic information content; Ho=
observed heterozygosity; He= expected heterozygosity; FIS= coefficient of inbreeding; P value = Hardy
Weinberg equilibrium significance.
The genetic distance analysis revealed that the Mixteco Creole cattle clusters with Mexican
Creole populations (Figure 2), showing a smaller distance with regard to Creole cattle from
441
Chihuahua (D= 0.046), Baja California (D= 0.064), and Puebla (D= 0.078). As for the rest
of the population groups, the Mixteco Creole exhibited a smaller genetic distance in relation
to the Red-spotted population (D= 0.103), belonging to the Spanish landrace group, as well
as with respect to the Limousin breed (D= 0.190), which is one of the specialized European
breeds. The greatest genetic distance was observed with each of the Bos indicus breeds
(Nelore 0.588 – Gyr 0.734).
Finally, the results of the analysis of the population structure through the assignment test with
Structure software (Figure 3A) and the subsequent calculation of the optimal K value using
the modal value method of the Delta K distribution (Figure 3B), revealed that the optimal K
was 8. The proportions of assignment to each cluster for Mixteco Creole are shown in Figure
3A. According to the results of K8, it is observed that, with the exception of the Dairy Creole
population, the rest of the Mexican Creole populations, including the Mixteco Creole, exhibit
a higher percentage of assignment to cluster 1, which includes the local Black-spotted, Red-
spotted, Andalusian Black, and Straw-colored Spanish populations (Figure 3). The rest of the
Mixteco Creole cattle genome was distributed as follows: 14.2 % with the rest of Spanish
landraces, 13.8 % with specialized European breeds, and 9.9 % with the cluster of Zebu
populations.
Figure 3: A) Results of the Bayesian Structure analysis for the Mixteco Creole Cattle
population, using 32 reference populations and a total of 8 inferred clusters (K8). B)
Estimation of the optimal K using the modal value method of the Delta K distribution. C)
Individual of the Black-spotted breed
(Source of the photograph: National group of Black-spotted and Red-spotted cattle breeders’ associations).
442
Local populations play a key role in livestock breeding, as they have been the basis for the
development of specialized breeds and today constitute a reservoir of genetic diversity that
must be preserved(19). In this sense, the Mixteco Creole cattle represents a local livestock
resource of great value that must be preserved, for which purpose its genetic evaluation is
essential.
The results of allelic diversity, represented by the mean number of alleles observed, showed
a similarity with the data reported in previous studies in bovine populations, when compared
with the mean values per population [6.92 ± 0.99](7), [6.78 ± 1.88](8), [8.31 ± 2.10](20),
however, they are lower when compared with the mean number of alleles detected
considering the Creole populations as a group [14.21 ± 3.74](8), [15.5 ± 0.9](17). The
difference observed in the mean number of alleles when compared at the group level is due
to the heterogeneity of Creole populations in the Americas, which has been confirmed in
several studies using autosomal and mitochondrial polymorphisms(21-23). This heterogeneity
is probably the result of several factors such as differences in the origin of the populations(17),
differentiation by geographical location(18), as well as the process of genetic drift and the
contribution of animals of different origins, which have been mixed at some point with the
Creole populations, as has been described with the introgression of Zebu, African and British
breeds(21,23-24). This data should be taken with caution, since the heterogeneity observed could
also reflect the state of threat to the population, due to dilution as a result of intensive
interbreeding or as a consequence of isolation and abandonment(17).
The values of observed and expected heterozygosity represent a measure of genetic diversity
in a population; however, they are estimated using different data. On the one hand, expected
heterozygosity is estimated based on allele frequencies, while observed heterozygosity is
estimated from genotypic frequencies, so the differences observed between both estimates
can be an indicator of the level of inbreeding in the population(25). The present study showed
a difference between heterozygosity values; the observed heterozygosity was lower than the
expected heterozygosity, suggesting a tendency towards inbreeding, as could be subsequently
verified in the estimation of the FIS value. On the other hand, when comparing the expected
heterozygosity values and the effective number of alleles with the values reported for the
Creole breeds using a similar panel of microsatellites(10,17,20), a correspondence in the results
was observed. The effective number of alleles represents the number of alleles expected in a
population with the same heterozygosity but with equally distributed allele frequencies(26);
therefore, if the allele frequencies are highly unbalanced and only some alleles are in the
majority, the effective number of alleles will tend to be lower(27), as was observed in the
Mixtec Creole population, among which certain alleles are more frequent. The relevance of
estimating the number of alleles and effective number of alleles lies in the fact that these data
can be used as a conservation criterion, since allelic diversity can have important implications
in the response to selection for adaptation to changing environments(28), This is of great
importance when talking about Creole populations, as they are considered reservoirs of
443
genetic information to face potential environmental alterations due to climate change, so it is

of great importance to implement measures for the conservation of allelic diversity.
The polymorphic information content exceeded 0.5 for all markers, which, according to the
scale proposed by Botstein(29), indicates that the microsatellites evaluated are highly
informative, and could be used for further genetic diversity monitoring studies or parentage
testing. Regarding Wright's fixation index, the estimation of this parameter provides a
measure of the degree of inbreeding of individuals with respect to the population to which
they belong(30). Although generally positive, the FIS value can be negative if inbreeding is
systematically avoided within populations(31). In the present study, the estimated FIS values
were mostly positive, far from zero, suggesting a tendency for heterozygote deficiency
inbreeding. The result observed in the population, in which the deviation of FIS with respect
to the zero value is positive can be attributed to the condition of the domestic population,
where mating is not random and the proportion of males is lower compared to females, in
addition to being long production systems with few animals and whose replacements are
usually obtained within the same production units, which predisposes to inbreeding(32-33). On
the other hand, with respect to the Hardy-Weinberg equilibrium test, of the 19 markers
analyzed, only markers HAUT27, ILSTS006 and TGLA227 were found to be out of
equilibrium; this data increases the reliability of the results obtained in the study and also
suggests that the population is not being subjected to perturbing forces that cause significant
changes in their genotypic frequencies(34).
Genetic relationships between populations were analyzed using genotypic information from
32 breeds belonging to the BIOBOVIS consortium of the CONBIAND Network, which were
selected for their potential relationship with Mixteco Creole cattle. We used 6 Mexican
Creole populations, 9 Spanish local populations, which could be found among the founding
populations of Latin American cattle, 12 European specialized breeds, and 5 Zebu breeds.
As Figure 2 shows, the results of the genetic distance calculation show that the Mixteco
Creole groups with the Mexican Creole populations of Chihuahua, Nayarit, Baja California
and Puebla, separated from the European and Zebu cattle groups. This distribution has been
reported in previous work with Latin American Creole cattle(17,35) This shows that, like other
Creole populations in the Americas, the Mixteco Creole has an identity closely related to
other Creole cattle due to their shared origin; this identity has been preserved despite the
geographic distance between the populations. Interestingly, the smallest genetic distance to
a non-Creole population was observed with the Red-spotted population, which has been
described as one of the possible founding populations of American Creole cattle(20).
Currently, in the Mixtec region, the Zebu breeds have been replaced by Bos taurus cattle of
better temperament, as, according to producers, crossbreeding between Zebu and Creole
cattle generated individuals with a temperament that was difficult to manage, while European
cattle, having been subjected to more intensive selection for docility and ease of
management(36), do not exhibit this issue. This fact has probably prevented the introgression
444
of Zebu germplasm into the Creole population from increasing, maintaining the genetic
distance between these populations.
Regarding the population structure, Bayesian analysis was carried out with Structure
software, calculating different values of K (2 - 33), with the subsequent estimation of the
optimal K (K= 8). A model with K= 8 populations was suggested, because it was associated
with a higher probability (Figure 3B), suggesting that there are closely related races or groups
(Figure 3B)(7). For a K = 8, the results indicate that Mixteco Creole cattle maintain a relatively
uniform population structure, sharing 62 % of their genome with Mexican Creole
populations, except for the Tropical Dairy Cattle. Similarly to what was reported for
Ecuadorian local breeds, a contribution of Red-spotted and Straw-colored cattle and a low
relevance of Marshland cattle were observed(20), which suggests that the Mixteco Creole is
integrated in its origin with Latin American Creole populations and reinforces the theory that
these breeds are part of the founding populations of Creole cattle in Latin America, since, as
has been described in other works, most of the Spanish cattle that gave rise to the Creole
populations in America came from southern Spain(20). In addition, this cluster also includes
the Spanish Black-spotted (Figure 3C) and Andalusian Back landraces, which is interesting
because the characteristic coloration patterns of the Mixteco Creole are similar to the
coloration patterns of these Iberian populations, mainly in the Black-spotted population,
whose genome percentage assigned to this cluster was 87.4 %. The Mixteco Creole genome
composition for K= 8 exhibited genetic heterogeneity, which has been previously described
in other Creole populations using mitochondrial, autosomal and Y chromosome
markers(22,24,37). The results confirm that the influence of Iberian cattle is predominant in the
Mixteco Creole cattle, as is the case in the Creole populations of the Americas, which retain
genetic signatures of their Iberian ancestry(21). However, it is also possible to infer that there
may have been recent contributions of exotic germplasm belonging to cattle of different
origins, which, based on the percentages of allocation observed in lower proportions, would
be European and Zebu. The presence of Zebu germplasm may be the remnant of the
importation of bulls used as sires for females of Creole breeds, a practice of indiscriminate
crossbreeding widely used in several Latin American countries since the middle of the last
century(22,33), but which, as mentioned above, is not currently common among producers in
the Mixteca region. Another possible cause for the presence of Zebu germplasm may be
related to the flow of ancestral genes between African Zebu cattle and Iberian cattle before
they were brought to America, as has been suggested in previous studies using SNP markers
and mitochondrial DNA(24,38).
The Mixteco Creole cattle show a level of genetic variability similar to that reported in studies
of Creole cattle populations in the Americas. In addition, it is more genetically related to
other Mexican Creole cattle populations. However, there is evidence of the influence of
exotic germplasm, in smaller percentages, from specialized Bos taurus and Bos indicus
breeds.
445
Acknowledgments
The authors wish to express their gratitude to the BIOBOVIS consortium of the CONBIAND
Network for providing genotypic information on the reference cattle breeds used in this
study.
Conflict of interests
The authors declare that they have no conflict of interests.
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Technical note
Influence of the cut intervals on hay quality of Panicum maximum cv.

BRS Tamani in brazilian Cerrado
Eva Nara Oliveira Gomes a
Alexandre Menezes Dias a*
Luciana Junges a
Luís Carlos Vinhas Ítavo a
Gelson dos Santos Difante a
Juliana Oliveira Batistoti a
a
Federal University of Mato Grosso do Sul. Faculty of Veterinary Medicine and Animal
Science. Av. Senador Filinto Muller, 2443. Vila Ipiranga. CEP 79070-900 Campo Grande,
MS, Brazil.
*Corresponding author: alexandre.menezes@ufms.br
Abstract:
The grass Panicum maximum cv. BRS Tamani is a hybrid high-quality, small-sized forage
plant with intense tillering activity. This study was carried out to examine the potential of
the grass Panicum maximum cv. BRS Tamani at different regrowth ages (49, 63, 77, and
91 d) for hay production, in the rainy period. The experiment was conducted at the Farm
School of the Federal University of Mato Grosso do Sul-Brazil, between October 2015
and April 2016. The treatments corresponded to four regrowth ages, with four replicates,
distributed into 9 m2 plots. Regrowth ages influenced the morphogenetic characteristics
of the grass, except for leaf senescence and final leaf length. Green (9.6–17.6 t ha-1) and
dry-matter (2.6–5.9 t ha-1) yields, hay yield (3.4-6.9 t ha-1), and proportions of stem (91.6–
455.9 g kg-1) and senescent material (34.8–98.4 g kg-1) increased with the regrowth ages,
while the proportion of leaves (837.7-402.1 g kg-1) and the leaf: stem ratio (15.9–0.9)
decreased (P<0.05). The dry matter (881.7–852.8 g kg-1) and protein contents (81.2–47.6
g kg-1) of the hay decreased with the higher regrowth ages; however, the neutral detergent
fibre (746.5–759.2 g kg-1) acid detergent fibre (519.8–567.7 g kg-1) and lignin (74.3–86.4
450
g kg-1) contents rose as the regrowth ages did. Nutrient digestibility decreased with the
regrowth ages (P<0.05). Panicum maximum cv. BRS Tamani has the potential to produce
of hay ha-1, better nutritive value and a high proportion of leaves in the regrowth interval
of 49 to 63 d.
Key words: Dry matter production, Forage management, Nutritive value, Tropical grass.
Received: 29/04/02019
To ensure a year-round supply of high-quality feed in the livestock activity, producers

have sought techniques that make it possible to utilize the surplus herbage mass produced
during the rainy period. In this scenario, the production and use of hay from grasses of the
genus Panicum could be a very important alternative in the feeding of animals during the
dry period of the year.
The haymaking process consists of harvesting, drying, baling, and storing forage plants(1),
which are steps that can be performed manually or mechanically. To be considered ‘hay’,
the forage should have 10 to 15 % moisture, which allows for adequate storage conditions
and prevents the occurrence of deterioration processes and losses(2).
Hay can be made from any forage plant, but some characteristics render some plants more
suitable for hay making than others, how elevated forage yielding potential, adequate
nutritional quality, presence of thin stems, and high leaf percentage. Another interesting
feature of forage plant is tolerance to frequent harvests, since, the harvest interval can
influence their regrowth potential and persistence(3).
The BRS Tamani cultivar released by EMBRAPA-Brazil in 2015 with the aim of
improving the nutritional value of pasture and forage production in tropical regions(4), may
be a viable forage option for hay production. It has thin leaves, a high forage-yielding
potential, elevated ground cover ability, and high nutritional value, in addition to being
resistant to pests and diseases(5).
However, there is little scientific data on its use as hay, especially regarding its production
and nutritional potential. Therefore, research should be carried out with the objective of
obtaining data that identify the morphogenetic characteristics of the BRS Tamani cultivar,
relating its physiological behavior and productive and nutritional potential so that the
needs of the animals are met.
Besides that, this paper as a pilot study might not be statistically representative for the
reason that there was a short period of study, although, they will provide an interesting
insight into the morphogenetic characteristics and productive and nutritional potential of
451
the Panicum maximum cv. BRS Tamani grass. The present study objective was to evaluate
the potential of Panicum maximum cv. BRS Tamani at different ages of sprouting (49, 63,
77 and 91 d) for the production of hay during the rainy season.
The experiment took place in the Forage Crops Section of the Farm School, located in
Terenos - MS, Brazil (20°26’34.31’’S, 54°50’27.86’’ W, 530.7 m asl), and at the
Laboratory of Applied Animal Nutrition and Forage Crops, Faculty of Veterinary
Medicine and Animal Science, at the Federal University of Mato Grosso do Sul (UFMS).
The experimental period was October 2015 to April 2016. Monthly precipitation and
minimum, mean, and maximum temperature data during the experimental period were
collected at the Center for Weather, Climate, and Water Resource Monitoring of Mato
Grosso do Sul (CEMTEC) (Figure 1).
Figure 1: Average, minimum and maximum temperatures and rainfall from 2014-2018
Soil samples were harvested from the 0 to 20 cm layer to determine its fertility before the
experimental beds were implemented. The following results were obtained: pH (CaCl2):
5.31; P: 4.52 mg dm-3; organic matter: 35.34 g dm-3; K: 0.20 cmol dm-3; Ca: 7.35 cmol
dm-3; Mg: 1.20 cmol dm-3; Ca + Mg: 8.55 cmol dm-3; Al: 0.00 cmol dm-3; H + Al: 5.18
cmol dm-3; CEC: 13.93 cmol dm-3; base saturation: 628.1 g kg-1. Dolomitic limestone was
applied in the amount of 1.2 t ha-1 (PRNT: 800.0 g kg-1). Prior to sowing, 100 kg ha-1 of
P2O5 and 60 kg ha-1 of K2O were applied. After sowing, 100 kg ha-1 of N were applied in
the form of urea.
The grass Panicum maximum cv. BRS Tamani was sown in November 2015 and was
established in a total area of 36 m2 in October 2015. The sowing rate was 4 kg of viable
seeds for hectare and the area was divided into sixteen 9 m2-experimental plots. In
December 2015, a uniformity cut was made in all plots at 10 cm-stubble to start the study
this was following by application of 50 kg ha-1 of N in the form of urea.
452
Treatments consisted of four regrowth ages (49, 63, 77, and 91 d) of harvest evaluated at
rainy season. After every evaluation finished, were cut the plants in all the experimental
plots to a 10 cm of residual herbage. A randomized-complete-block experimental design
was adopted, with four replicates, with 16 experimental units.
The variance analysis was performed considering a randomized block design with four
replicates, and orthogonal decomposition of the sum of treatment squares into linear and
quadratic effects of different regrowth ages to probe the best fit of the model.
The model used was Yij = μ + Ri + eij, in which μ is the general average; Ri is the fixed
treatment i, i = 1… 4, and eij is the experimental error associated with each observation
Yij.
The significance of effects was analyzed by the Tukey test, at α=0.05, using PROC
MIXED (Statistical Analysis Systems – SAS, version 9.1, SAS Institute, Inc. Cary, NC,
USA).
To evaluate the morphogenetic and structural variables, five tillers representative of each
plot were chosen, identified with a colored thread, and evaluated during the entire
regrowth period of each age. The length of the marked tillers was measured every 7 d with
a centimeter-graduated ruler. The lengths of stem (from the soil to the last leaf with a fully
expanded ligule), leaf (measured from the expanded ligule to the extremity of the blade),
and leaf under elongation (measured from the ligule of the last expanded leaf until the end
of the blade) were measured in the tiller. Leaf appearance rate (LAR), leaf elongation rate
(LER), stem elongation rate (SER), leaf senescence rate (LSR), phyllochron, number of
live leaves per tiller (NLL), leaf lifespan (LL), and final leaf length (FLL) were calculated
as proposed by Lemaire and Chapman(6).
To quantify the herbage mass, forage samples were collected from each plot using a 1 m2
square frame and harvested at 10 cm from the soil surface, at random. After harvesting,
the sample collected from within each frame was taken to the laboratory to be manually
separated into the following morphological components: leaf (leaf blades), stem (stems +
leaf sheaths), and senescent material. These were then dried at 55 ºC in a forced-air oven
until reaching a constant weight for the determination of the dry weight and further
laboratory analyses.
After the herbage mass was collected, the hays were made for the evaluation of the
remaining forage from the entire experimental bed. The green (fresh) forage was chopped
and weighed and then spread on the floor of a covered shed for drying. Upon reaching the
haying point, the material was baled manually and weighed again. Four bales of hay were
made per regrowth age and stored for 30 d in an appropriate shed. Subsequently, a 0.5 kg
sample of each bale was collected, dried in an oven at 55 ºC, and then analysed in the
laboratory.
453
Samples were ground through to 1 mm particles for the analysis of chemical composition
of the morphological components of the plant (leaf and stem) and of the hay. The
concentrations of dry matter (DM), organic matter (OM), and crude protein (CP) were
determined as described in the AOAC(7). Neutral detergent fibre (NDF) content was
measured as proposed by Van Soest(8), without a heat stable amylase and not corrected for
ash. Acid detergent fibre (ADF) content was measured as proposed by Van Soest(9)
without corrected for ash. Lignin [lignin (sa)] was determined by solubilization of
cellulose with sulphuric acid. Lastly, the in vitro digestibility of DM and NDF was
evaluated according to the recommendations of Silva(10), using the technique described by
Tilley and Terry(11) adapted to an artificial rumen developed by ANKON®, as described
by Holden(12).
Leaf appearance rate (Figure 2A), NLL (Figure 2D) and LER (Figure 2E) decreased
linearly (P<0.05) as the regrowth age increased. The phyllochron increased linearly
(Figure 2B), though. Stem elongation rate (Figure 2F) and LL (Figure 2C) showed a
quadratic response (P<0.05), with maximum values of 0.80 cm tiller-1 day-1 (at 91 d) and
40.30 d (at 77 d), respectively. Despite the FLL (43.33 cm) and LSR (1.95 cm tiller-1 d-1)
were not influenced (P>0.05) by the regrowth ages. These results can be explained by as
a tiller ages, it gradually loses vigor, and this effect has great impacts on its morphogenetic
and structural characteristics(13). The higher LAR observed at the earlier ages can be
related to the higher photosynthetic efficiency of younger tillers at those ages in relation
to that observed in the older tillers found at the greater regrowth ages(13). In the same way,
LAR might have been reduced due to the changes occurring in LER and SER, because,
LAR is affected by two factors, when these vary in the tiller: leaf elongation rate and stem
length(14).
The regrowth age increased the elongation time of new leaves, then, this affect the
phyllochron positively, since the SER (Figure 2F) and the proportion of stem (Figure 3C)
rose with ages. Which can be explained by some researchers, which observed that
increasing phyllochrons as a plant ages are due to the longer time necessary for the leaf to
cover the distance between the apical meristem and the extremity of the stem, which is
longer at longer regrowth ages(15).
454
Figure 2: Leaf appearance rate (LAR); leaf lifespan (LL); number of live leaves per
tiller (NLL); leaf elongation rate (LER); stem elongation rate (SER) of Panicum
maximum cv. BRS Tamani at different regrowth ages (RA)
The increasing LL up to 77 d may have been a consequence of the adaptation of the forage
plant when it reduced its tiller renewal, as observed by the lower LAR and LER and higher
SER (Figure 2A, E, F), leaves already existing remained alive for a longer time, though.
In an experiment with Tanzania grass, the plant adapts when leaf-tissue renewal is low,
which allows the leaves to remain alive longer(16). Thus, older tillers are characterized by
lower LAR, which lead to lower NLL and longer LL as compared with younger tillers(17).
Number of live leaves is a genetically defined variable whose value is relatively constant.
However, this variable may be changed depending on climatic conditions and pasture
management(18), which might have caused the variations observed in this study with the
advancing regrowth ages. Another explanation is that the tiller entered in reproductive
stage after 63 d of regrowth and it carried nutrients in older leaves to panicle,
consequently, older leaves died and decreased the NLL with rose the regrowth ages.
Leaf elongation rate declines as a result of increased competition for photoassimilates as

the plant grows older, which leads to the appearance of new tillers or inflorescences(19). In
young tillers, in order to attain high growth rates from the capture of resources, the plant
increases its leaf elongation rate. Older tillers, in turn, depend on strategies to preserve
455
these resources(17) reducing these rates so as to minimize lesions caused by stressful

conditions(20).
The greater stem elongation demonstrated by SER is related to the increasing growth of
the plant that was observed with time, since the competition for quantity and quality of
light increases as the plant develops and recovers its leaf area. This culminates in greater
stem elongation as a plant’s attempt to place the leaf blades in the upper part of the canopy,
which receives a significant amount of light(16). Another likely explanation for the
increased stem elongation is the plant’s transition from vegetative to reproductive stage,
because during the experimental period, was observed that cv. BRS Tamani evolved to
the reproductive stage at 63 d of regrowth.
The proportion of leaves (Figure 3D) and the leaf: stem ratio (L:S; Figure 3F) declined
linearly (P<0.05) with the higher regrowth ages, while the opposite response was seen for
the proportions of stem (Figure 3E) and senescent material (Figure 3F). These responses
can be explained by the appearance of the panicle with the advance of maturity, which
was observed after 63 d of regrowth, since the grass reduces the leaf size and prioritizes
the growth of stems to expose the inflorescence. When plants enter the reproduction
period, the leaf blade percentage declines immediately, even in pastures under constant
management, which consequently elevates the proportion of stems(21).
At the higher regrowth ages the proportion of senescence material (Figure 3F) was also
higher, which is due to the gradual increase in competition for light and in stem elongation.
In this regard, the quantity and quality of light that reaches the canopy decline lead to
morphophysiological alterations in the plant(20). In this way, the leaves located near the
base and that are shaded accelerate the senescence process(18).
The decreasing L:S (Figure 3F) is a result of the increasing proportion of stem (Figure 3C)
and decreasing proportion of leaves (Figure 3D) observed with the progression of
regrowth days. Decreasing L:S are often related to the aging process of a forage plant (22).
The lower L:S found at 91 d of regrowth, it was 0.90, which characterizes this regrowth
age as inadequate for hay making for cv. BRS Tamani, because there was more
proportions of stem to leaves and greater senescent material than others regrowth ages.
Total green mass (TGM; Figure 3A), total dry mass (TDM; Figure 3B) and total hay (TH;
Figure 3C) rose linearly (P<0.05) with the regrowth age. Total green mass, TGM, and TH
increased by 0.18, 0.07, and 0.08 t ha-1 with the regrowth age, respectively. The increased
of all production can be explained by greater stem elongation and proportion of stem and
dead material, observed in Figure 2E, 2F and Figure 3F. While the leaf production
declined by 11.02 g kg-1 with every day of regrowth (Figure 3D).
456
Figure 3: Total green mass (TGM), total dry mass (TDM), and total hay (TH),
proportions of leaves, stems, and senescent material (Ῡ•) and leaf:stem ratio (Ῡ•; L:S) in
Panicum maximum cv. BRS Tamani at different regrowth ages (RA)
In the leaf, the DM, NDF, ADF, and lignin (sa) contents increased linearly (P<0.05) as
regrowth ages were greater (Table 1). Nonetheless, the opposite is true for CP content in
leaf. The DM, ADF and Lignin (sa) content of the stem and the hay increased linearly
(P<0.05) with the regrowth ages. Nevertheless, the CP in stem and hay decreased (P<0.05)
as regrowth ages were greater.
Raising DM contents in the leaf and in the stem as the regrowth age did are due to the
increasing amount of fibrous components in the cell wall (Table 1). However, the
decreasing DM content in the hays resulting from the progression of regrowth time may
be associated with the losses of leaf blades when the bales were made, besides the higher
losses of moisture content in the forage at earlier ages, when stems are younger(23). Despite
these decreases in DM values, they were within the acceptable limits, which correspond
to 10 to 15 % moisture, in which no losses or deterioration occur(2).
As a forage plant grows older, its fibrous fraction roses (Table 1) for the reason that the
development of supporting structures provided by the fibrous carbohydrates and lignin.
457
The cell wall then thickens and is lignified, due mainly to the increasing quantity and
thickness of stem(24). The opposite is true to crude protein concentrations declined as the
regrowth age of cv. BRS Tamani advanced. This result may be related to the thickening
of the cell wall observed at older ages, which may lead to a reduction of cell wall content,
which includes protein and soluble carbohydrates. Another explanation for the lower CP
content is that protein components complex to those of ADF, becoming the insoluble
fraction of the forage plant(25).
The lower CP observed in hays at older regrowth ages may be associated with a lower
L:S; i.e., a larger proportion of stems and a smaller proportion of leaves (Figure 3). Along
with this factor, the CP content of leaf and stem declined as the regrowth age advanced
(Table 1). The minimum CP level of the diet should be considered 70 g kg-1, and values
below that may compromise animal performance, since the development of rumen
microorganisms and digestibility would be negatively affected(26). Therefore, to prevent
the utilization of CP from being restricted, cv. BRS Tamani should be used from 49 to 55
d of regrowth, during which period the CP content of the material would be higher than
the minimum necessary (80.88 to 71.13 g kg-1).
The in vitro digestibility of dry matter (IVDMD) and neutral detergent fiber (IVNDFD)
decreased linearly (P<0.05) in the leaf, the stem and in the hay as the regrowth days were
greater (Table 2). The lowest values of these two components were in the stem. In the hay,
the lowest values of the respective components were 568.03 and 483.69 g kg-1, both
observed at 91 d of regrowth age.
The decreasing digestibility values (Table 2) detected with the advancing regrowth age
are due to the reduction of fiber quality, since the lignin contents of the cell wall increased
(Table 1). Athayde et al(23) stated that the unfavorable effects of lignin are more
pronounced in tropical grasses as their regrowth age progresses. This negative effect might
have generated a barrier that blocks the microorganisms from adhering and promoting
enzymatic hydrolysis(27). Another factor that can be lead to a reduction in digestibility is
an imbalance between nutrients. Vasconcelos(28) submitted that one of the reasons for
declines in rumen digestibility is nutritional imbalance, especially of energy
(carbohydrates) and protein. Thus, the increasing fiber and decreasing CP contents
resulting from the increasing regrowth period described in Table 1 reduce the fiber
utilization in the rumen.
In view of the present results, BRS Tamani has potential for hay production. Although the
later regrowth ages provided higher green matter, dry matter, and hay yields, they had a
negative impact on the morphogenetic characteristics, nutritional values, and digestibility
of the grass. Therefore, the regrowth ages of 49 and 63 d showed the best results for the
production of hay with the best nutritional value without compromising the
morphogenetic characteristics of the plant.
458
The Panicum maximum cv. BRS Tamani grass has the potential to produce 3.4 to 4.2 t
ha-1 of hay in the regrowth interval of 49 to 63 d. When defoliated in this range, BRS
Tamani hay presented better nutritive value and high proportion of leaves, which
characterizes a gramine suitable for use in the form of hay in larger cut intervals aiming
at higher productivity and nutritional quality. Above 63 d of regrowth, tillers from BRS
Tamani progressed from vegetative to reproductive stage, that resulted in greater stem
elongation rate and the nutrients decreased in the leaf and stem and come to seeds. Further
studies should be undertaken focusing on the regrowth ages with more than a single year,
so, it can evaluate the effect of the year under different environmental conditions.
Acknowledgements
The authors are extremely grateful to Universidade Federal de Mato Grosso do Sul
(UFMS), Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do
Estado de Mato Grosso do Sul (FUNDECT) and Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.
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463
Table 1: Chemical composition of leaf, stem, and hay of Panicum maximum cv. BRS Tamani at different regrowth ages
Regrowth ages (days) Regresion equations (r2) SEM P-Value
Ítem
49 63 77 91 L* C◊
Leaf
MS, g kg-1 280.1 303.5 299.1 349.5 Y=209.483+1.35917*ER (0.80) 0.907 0.0005 0.1827
MO, g kg-1 MS 930.7 918.0 919.3 921.8 Y=922.45 0.302 0.2888 0.1621
PC, g kg-1 MS 84.0 62.1 62.2 61.1 Y= 101.65-0.49*ER (0.64) 0.323 0.0002 0.0039
FDN, g kg-1 MS 740.9 745.5 750.0 723.0 Y=676.314+1.1145*ER (0.84) 0.833 0.0001 0.4212
FDA, g kg-1 MS 481.0 487.7 534.1 549.8 Y=434.156+0.9651*ER (0.68) 0.709 0.0002 0.1519
Lignin (sa), g kg-1 MS 49.4 52.6 78.8 80.9 Y=1.954+0.9107*ER (0.89) 0.256 0.0001 0.4267
Stem
MS, g kg-1 207.1 251.2 257.5 295.7 Y=121.281+1.81455*ER (0.94) 0.985 0.0001 0.6161
MO, g kg-1 MS 919.0 923.4 939.2 939.9 Y=930.38 0.503 0.0514 0.8268
PC, g kg-1 MS 42.6 44.0 25.5 21.9 Y=72.5572–0.538897*ER (0.83) 0.315 0.0001 0.2534
FDN, g kg-1 MS 798.9 803.8 801.9 804.0 Y=802.15 1.522 0.1777 0.2330
FDA, g kg-1 MS 566.5 590.3 669.8 651.9 Y=451.78+2.3978*ER (0.78) 1.320 0.0001 0.0124
Lignin (sa), g kg-1 MS 47.6 58.3 109.6 108.1 Y= -35.5+1.6629*ER (0.85) 0.853 0.0001 0.0431
463
464
Hay
MS, g kg-1 881.7 875.9 847.2 852.8 Y=920.197–0.7694*ER (0.77) 0.458 0.0001 0.0521
MO, g kg-1 MS 917.9 920.2 921.5 882.8 Y=910.60 1.149 0.2723 0.3296
PC, g kg-1 MS 81.2 79.5 63.6 47.6 Y=124.436–0.7789*ER (0.92) 0.456 0.0001 0.0982
FDN, g kg-1 MS 746.5 750.2 752.9 759.2 Y=752.2 0.293 0.0953 0.8046
FDA, g kg-1 MS 519.8 531.1 556.5 567.7 Y=462.039+1.1276*ER (0.97) 0.614 0.0001 0.9753
Lignin (sa), g kg-1 MS 74.3 73.2 82.8 86.4 Y=56.99335+0.3057*ER (0.85) 0.187 0.0001 0.1878
DM= dry matter; OM= organic matter; CP= crude protein; NDF0: neutral detergent fiber; ADF= acid detergent fiber; RA= regrowth ages. SEM= standard error of the means;
*Linear; ◊Quadratic.
464
465
Table 2: In vitro digestibility of leaf, stem, and hay of Panicum maximum cv. BRS Tamani at different regrowth ages
Regrowth ages (days) Regression equations (r2) SEM P-value
Item
49 63 77 91 L* Q◊
Leaf
IVDMD, g kg-1 DM 649.9 617.4 611.5 588.7 Y=708.383–1.2622*RA (0.94) 0.550 <0.0001 0.6026
IVNDFD, g kg-1 DM 610.2 565.0 539.0 515.8 Y=702.951-1.9743*RA (0.90) 1.034 <0.0001 0.2827
Stem
IVDMD, g kg-1 DM 554.1 485.0 445.1 431.8 Y= 694.551-2.9193*RA (0.89) 1.267 <0.001 0.4781
IVNDFD, g/kg DM 509.0 432.0 378.5 345.2 Y=746.255-4.6412*RA (0.91) 1.654 <0.001 0.3705
Hay
IVDMD, g kg-1 DM 638.2 601.8 586.4 555.0 Y=710.94-1.6959*RA (0.92) 0.985 <0.001 0.4135
IVNDFD, g kg-1 DM 598.8 557.1 520.7 489.8 Y=719.647-2.4983*RA (0.97) 1.103 <0.001 0.5073
IVDMD= in vitro dry matter digestibility; IVNDFD= in vitro neutral detergent fiber Digestibility; RA= regrowth ages. SEM= standard error of the means; *Linear;
◊Quadratic.
465
Technical note
Evaluation of sow seroconversion with the use of inoculum at different

doses and vehicles against porcine epidemic diarrhea
Nancy Paulina García Cano Rubí a
Francisco Ernesto Martínez-Castañeda b
Elein Hernández Trujillo c
Rosa Elena Sarmiento Silva a
Rolando Beltrán Figueroa a
Montserrat Elemi García-Hernández a
María Elena Trujillo-Ortega a*
a
Universidad Nacional Autónoma de México. Facultad de Medicina Veterinaria y Zootecnia.
Ciudad Universitaria, Av. Universidad #3000, Colonia, C.U., Coyoacán, 04510 Ciudad de
México, México.
b
Universidad Autónoma del Estado de México. Instituto de Ciencias Agropecuarias y
Rurales. México.
c
Universidad Nacional Autónoma de México. Facultad de Estudios Superiores Cuautitlán.
México.
*Corresponding author: elenam@unam.mx
Abstract:
Porcine epidemic diarrhea (PED) is a highly contagious enteric disease of pigs, which has
caused great economic losses to the swine industry worldwide. The known measure for PED
control prior to the development and launch of vaccines in 2017 in Mexico, was "feedback"
or "liquefaction". It was a widely used measure during the PED outbreak in 2013; however,
466
there is no homogeneity in its use among the various authors who recommend it. Currently,
several studies have experimented with other types of prophylaxis, such as oral immunization
with PED virus obtained from cell culture isolation, which allows quantification of the
infectious virus and ensures that only the virus, and no other agent, is being used as inoculum.
The objective of the present study was to compare the time of seroconversion in sows
inoculated with the quantified virus with four different vehicles (milk, wheat, direct, and
water) and different doses of vehicle (1 ml, 2 ml, and 3 ml) at different pregnancy stages and
with a different number of farrowings. The study was conducted at CEIEPP, a full-cycle farm
with 170 females. The present study showed that the vehicles with the best results were the
inoculum with water and the direct inoculum combined with the 1 ml dose, as the
combination of these vehicles and an inoculum dose resulted in seroconversion in more than
90 % of the sows from the second week post inoculation.
Key words: Inoculum, Porcine Epidemic Diarrhea, Seroconversion.
Porcine epidemic diarrhea is a highly contagious enteric disease in pigs caused by the Porcine
Epidemic Diarrhea (PED) virus(1), a single-stranded positive-sense enveloped RNA virus that
belongs to the genus Alphacoronavirus, family Coronaviridae(2). It infects mainly the
epithelial cells of the intestine of pigs, causing atrophy, necrosis, and detachment of the
intestinal villi, which affects nutrient absorption(3), causing problems such as watery diarrhea,
acute vomiting, anorexia, extensive dehydration, imbalanced blood electrolytes, and weight
loss in pigs of all ages(4). It is especially severe in seronegative piglets, among which the
morbidity and mortality rate is up to 100 %(5). The virus was identified in the 1970s in the
United Kingdom and Belgium. In 1976 a similar epidemic occurred in several European
countries, and was named EVD2; since then, the disease has been reported in many other
countries(6,7). In October 2010, a highly pathogenic variant of the PED virus strain was
identified in China, and later in May 2013, this same variant caused disease in the U.S.A,
from where it spread to Canada and other countries in Central and South America, including
Mexico(6,8). It was estimated that, in the U.S.A, the PED outbreak affected more than 8,400
farms(9), killing more than 7 million pigs equivalent to 10 % of their swine population(10),
with losses of $1.1 billion dollars for producers(11).
The known measure for control of the disease has been "feedback" or "liquefaction; however,
there is no homogeneity in the use of this technique(12,13). It consists in the ingestion of small
intestine, gastric contents, or diarrhea of pigs showing clinical signs of PED in the first 6 to
467
12 h after the onset of the disease. It can also be prepared from the intestinal scraping of
slaughtered piglets that had diarrhea in their last 4 h and can be mixed with evaporated milk,
trying to achieve a liquid (not pasty) consistency(14). Another type of prophylaxis is oral
immunization with PED virus obtained from cell culture isolation, which allows
quantification of the infectious virus and calculation of a protective dose, and ensures that
only the virus —and not any other agent— is being used as inoculum(15,16). Most of the
vaccines marketed in Mexico are live attenuated or inactivated vaccines that use strains
similar to CV777 and are administered orally(15), and they are recommended for use in
pregnant females in the 2nd and 3rd week prior to farrowing(16). Stress has been observed with
the use of the vaccine in pregnant sows(17), and its effectiveness is still under evaluation.
The objective of the present study was to compare the time of seroconversion in sows
inoculated with quantified viruses with four different vehicles (milk, wheat, direct, and water)
and different doses of vehicle (1 ml, 2 ml, and 3 ml), at different pregnancy stages and with
parity of the sows.
The study was conducted in a semi-technified full-cycle farm located in the northeast of the
State of Mexico, with an average of 170 Landrace x Yorkshire females in the inventory.
The method for animal handling was submitted to and approved by the Institutional
Subcommittee for the Care and Use of Experimental Animals (SICUAE) of the Faculty of
Veterinary Medicine and Zootechnics FMVZ CU-UNAM, with approval number MC-
2020/4-4.
The virus was obtained from the Virology Laboratory of the Faculty of Veterinary Medicine
and Zootechnics, Universidad Nacional Autónoma de México (UNAM), identified in the
Gen Bank with the accession number KM044335.1, which has a titer of 1x108
DICC50%/ml(18,19).
Sows were immunized with 12 different intervention protocols against PED on January 26,
2018. The variants of this protocol were to administer the quantified virus in four different
vehicles, which were: milk, wheat, water, and without a vehicle, i.e., direct (viral suspension
in a culture medium), with three different doses of each vehicle, 1 ml, 2 ml and 3 ml (Table
1). The inoculation protocol was performed on all sows on the farm in the form of a sheet.
468
Table 1: Summary of experimental design

Group Vehicle Dose Virus concentration No. of animals
(DICC50%/ml)
1 Milk 1 ml 1x108 8
2 Milk 2 ml 2x108 6
3 Milk 3 ml 3x108 10
4 Wheat 1 ml 1x108 8
5 Wheat 2 ml 2x108 8
6 Wheat 3 ml 3x108 10
7 Water 1 ml 1x108 8
8 Water 2 ml 2x108 8
9 Water 3 ml 3x108 10
10 Direct 1 ml 1x108 7
After the administration of the different intervention protocols, blood samples were taken
from the sows at wk 2, 4, 8, and 13, in tubes for blood sample collection without additive.
These samples were transferred in ice boxes at 2 to 8 °C to the Virology Laboratory of the
Faculty of Veterinary Medicine and Animal Husbandry of the UNAM. Samples were
processed using the ELISA technique of the ID Screen® PEDV Indirect Kit (ID-VET),
according to the supplier's specifications, ID Screen® PEDV Indirect - IDVet(18). The ELISA
test was used to monitor the different immunization protocols of the females and to identify
the females that exhibited seroconversion.
The seroconversion data were analyzed with descriptive statistics and with the Kaplan-Meier
survival curve and the Mantel-Cox log-rank test, respectively. A value of P<0.05 was
considered statistically significant. Table 2 shows intervention protocols.
Table 2: Comparison groups of different vehicles and doses using the Kaplan-Maier
survival curve test and the Mantel-Cox log-rank test
Groups Comparison
1 1 ml milk, 2 ml milk, and 3 ml milk
2 1 ml wheat, 2 ml wheat, and 3 ml wheat
3 1 ml direct, 2 ml direct, and 3 ml direct
4 1 ml water, 2 ml water, and 3 ml water
5 1 ml milk, 1 ml wheat, 1 ml direct, and 1 ml water
Each ml of vehicle contains 1x108 DICC50%/ml of the PED virus.
469
It was observed that two vehicles showed the best seroconversion response with the 1 ml
dose (direct vehicle and vehicle with water), while the vehicle with the lowest seroconversion
was wheat (Figure 1).
Figure 1: Percentage of seroconversion of the four different inoculum vehicles (water,

direct, milk, and wheat) at three different doses, at four different times after inoculum
administration
Differences in survival time were found only in groups 2 and 7: Mantel-Cox, χ2 = 12.56, 2
gl; P= 0.0019 and χ2 = 15.75, 3 gl; P= 0.0013 (Figure 2), respectively, The water and milk
vehicles showed a seroconversion above 45 % and 60 % at wk 2 and 6, respectively.
The use of different vehicles and doses has been implemented and described by various
authors(12,13,20), although the effectiveness of one over the other has not been proven. In this
work, the water and direct inoculum with 1 ml were the best. However, there was no
difference between the different vehicles and doses, with the exception of the wheat inoculum
and the three-milliliter doses with different vehicles (Figure 2).
470
Figure 2: Kaplan-Meier survival curve, A) curve of domestic pigs inoculated with the
wheat vehicle, days after inoculation. B) domestic pigs inoculated with 4 different vehicles
at 3 ml doses, days after inoculation
In serological assays, on average, antibodies are first detected in serum 6 to 14 d after contact
with the virus(21). The present work revealed that a small percentage of sows already exhibited
PED antibodies from the second week on, while most of the sows exposed to the PED virus
exhibited antibodies during the 10th and 14th wk. It was also found that the seroconversion
471
effect differed according to the vehicle or dose at which the sows were exposed to the PED
virus.
Antibody levels in sows naturally infected with the PED virus remain high for up to six
months, although the recovered fecal levels disappear within one to two months’ post-
infection(22).
Immunization of pregnant sows is reportedly important for controlling PED and reducing
piglet deaths(23,24). 100 % of piglets from sows immunized at 57 to 59 d of gestation have
been reported to survive(24), while those exposed at 19 to 22 d of gestation and those exposed
after 96 d of gestation showed piglet survival rates of 87 and 56 %, respectively(25).
The efficacy of the vaccine and of the intramuscular booster depends on the induction of IgA
antibody memory B-cells in sows previously exposed to the field virus or orally
immunized(26). This work utilized oral immunization protocols with different vehicles and
different doses.
Certain studies highlight the better performance of oral inoculation (including feedback)
versus intramuscular inoculation. However, both protocols can be inefficient as a
consequence of: 1) the lack of standardized feedback protocols; 2) the poor ability of current
intramuscular vaccines to induce lactogenic immunity; 3) the antigenic difference between
the vaccine and the epidemic strains, and 4) the potential and continuous re-infection with
PEDs due to the use of feedback(27).
The present study showed that the vehicles with the best results were the inoculum with water
and the direct inoculum combined with the 1 ml dose, as the combination of these vehicles
and doses with the inoculum resulted in seroconversion in more than 90 % of the sows from
the second-week post-inoculation.
Acknowledgments
This study was made possible thanks to the grant bestowed by the Support for Research and
Technological Innovation Projects Program (PAPIIT), to project No. IN221218,
"Differential analysis of proteins expressed in cell culture with different isolates of the
Porcine Epidemic Diarrhea virus obtained in Mexico and their relationship with changes in
antigenicity", for which Dr. María Elena Trujillo Ortega is responsible. The authors are
grateful to CONACYT for its support through the research scholarship program, Registration
No. 933765.
472
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Technical note
Effect of selenium source on productive behavior, serum and muscle

selenium content, and serum level of albumin, α-, β- and ∂-globulins in
Pelibuey sheep
Lino Rigoberto Cárdenas-Ramírez a
Carlos Sánchez del Real a
Agustín Ruíz-Flores a
Gabriela Pérez-Hernández a
Reyes López-Ordaz b
Claudio Vite-Cristóbal a
Rufino López-Ordaz a*
a
Universidad Autónoma Chapingo. Posgrado en Producción Animal. Departamento de
Zootecnia. Km 38.5. Carretera México-Texcoco. 56230. Chapingo, México.
b
Universidad Autónoma Metropolitana. UAM-Xochimilco. Departamento de Producción
Animal. Ciudad de México.
* Corresponding author: rlopezor@yahoo.com
Abstract:
The objective was to compare the effects of sodium selenite (SS) and Sel-Plex® (SP) on dry
matter intake (DMI), daily weight gain (DWG), feed conversion (FC), carcass yield, Se in
serum, muscle, albumin, and globulins in Pelibuey lambs. Fifty (50) animals (LW=23.0 ±
1.1 kg; 5 to 6 mo) were stratified and randomly assigned to one of five treatments (n=10): 1)
Basal diet, C); 2) C + 0.30 mg kg-1 DM of SS, 30SS; 3) C + 0.90 mg kg-1 DM of SS, 90SS;
4) C + 0.30 mg kg-1 DM of SP, 30SP; and 5) C + 0.90 mg kg-1 DM of SP, 90SP. There was
no effect (P>0.05) on DMI; while 90SP and 30SS showed higher DWGs (293 and 260 vs
476
245, 243, and 230 g d-1; P<0.05) compared to the other treatments. FC was better for 90SP
and C. Final LWs, carcass yields and dorsal fat were not affected (P>0.05). In the
Longissimus dorsi, 30SP increased (P<0.05) Se with respect to 90SP and C and was similar
with 30SS and 90SS. There was no effect (P>0.05) on the Gluteus maximus and Musculus
deltoideus. Albumin was higher with 30SP and 90SS; while α-globulin was higher with 30SS
and 90SP. In conclusion, 0.90 mg of SP improved DWG and FC. Selenite and SP increased
Se in serum up to 0.30, and it decreased with 0.90 mg per kilogram of SP. In the Longissimus
dorsi, Se was improved in 30SP with respect to 90SP and C and was not similar to 90SS and
30SS. The organic Se of 90SP improved the level of albumin and α-globulins.
Keywords: Selenium sources, Weight gain, Carcasses, Albumin, α-globulin, Longissimus

dorsi et lumborum, Growing sheep.
Selenium (Se) is essential in the antioxidant defense system in animals and humans. Among
its functions are to serve as part of the enzyme glutathione peroxidase (GSH-Px), which
destroys free radicals in the cytoplasm and protects tissues from oxidative stress(1). Se has
been studied for its functions in the immune system and DNA protection(2). On the other
hand, deficiency of the mineral is associated with diseases such as white muscle, and
repression of immunity in lambs. The negative effects of the deficiency are explained by the
relationship between the mineral and the hormones produced by the thyroid(3,4).
Sodium selenite (Na2SeO3) has been the preferred source of inorganic Se in ruminant feeding.
However, with the appearance of new sources of organic Se(5), the question as to which is
better arose. Most of the mineral is found as GSH-PX and selenoproteins that are produced
in the liver and distributed in serum. However, there are not many reports of the relationships
between dietary Se and serum concentrations of albumin, α-, ß- and ∂-globulins in sheep.
Selenite is mainly used for the formation of selenoenzymes and differs from organic forms
of greater availability than Se + yeasts(6,7,8). Others have shown that it is possible to increase
the Se content of meat with Sel-Plex®(9,10,11) compared to SS. Both forms increase in animal
tissues, improving the consumption of Se by humans who ingest meat from animals
supplemented with Se.
Based on the above, the objective was to study the effects of supplementation of the source
of Se on feed intake, changes in body weight, feed efficiency, carcass weights, selenium in
serum, muscles, albumin and globulins of growing Pelibuey lambs.
477
The study was carried out in the sheep module of the Experimental Farm of the Chapingo
Autonomous University, in Chapingo, Mexico (98° 29 ́23 ́ ́N and 98° 53 ́27 ́ ́ W); at 2,250 m
altitude with temperate subhumid climate. The temperature varies from 12 to 18 °C, with 645
mm of annual precipitation, distributed from July to September(12).
The study used 50 newly weaned Pelibuey lambs (LW=23 ± 1.1 kg; five to six months old),
which were stratified and randomly assigned to one of five treatments (n=10): 1) basal diet
(BD, C); 2) C+0.30 mg of Se kg-1 DM, of SS, 30SS; 3) C+0.90 mg of Se kg-1 DM, of SS,
90SS; 4) C+0.30 mg of Se kg-1 DM (Sel-plex™ OSe; Alltech, Inc., Nicholasville, KY), of
SP, 30SP; and 5) C+0.90 mg of Se kg-1 DM, of SP, 90SP. The diet was formulated according
to the recommendations of the NRC(10). In the final composition, diet C contained 0.1 mg of
Se per kg of DM, while 03SS and 03SP contained 0.4 mg. In the same sense, diets 09SS and
09SP contained 1.0 mg of Se per kg of DM (Table 1).
Table 1: Ingredients and nutritional composition of the experimental diet supplied to

fattening Pelibuey lambs that received 0.30 or 0.90 mg kg-1 of dry matter of sodium selenite
(SS) or Sel-Plex® (SP) during 56 d of confinement
Composition Percentage of inclusion, (g kg-1)
Rolled corn 300.0
Ground corn 290.0
Corn stover 140.0
Soybean hulls 80.0
Soybean meal 60.0
Molasses 50.0
Corn gluten 44.0
Mineral mixturea 15.0
Calcium carbonate 10.0
Urea 5.0
Common salt 5.0
Bypass fat 1.0
Chemical composition
478
Dry matter1, % 87.0
Metabolizable energy2, Mcal/kg DM 2.80
Crude protein1, % 16.00
Rumen undegradable protein2, % 6.00
Crude fiber1, % 10.00
Ether extract1, % 3.30
Ash1, % 5.80
Vitamin A1, IU/kg 1.50
Vitamin E1, IU/kg 16.70
Selenium1, mg/kg-1 DM 0.10
Determined in the laboratories of the Chapingo Autonomous University. 56230. Chapingo, State of Mexico.
1
NRC, (2007). aMineral mixture: Ca, 24%; Cl, 12%; Na, 8%; P, 3%; Mg, 2%; S, 0.5%; K, 0.5%; Zn, 5000 mg
2
kg-1; Mn, 4000 mg kg-1; Fe, 2000 mg kg-1; I, 100 mg kg-1; Co, 60 mg kg-1; Cr, 5 mg kg-1; Vitamin A, 500000
IU kg-1; Vitamin D, 300000 IU kg-1; Vitamin E, 1000 IU kg-1. Mezcla mineral-engorda® (Servicios
Especializados Profesionales; Chapingo, Mexico).
The lambs received feed twice a day. Fifty (50) percent of the feed offered was served at
0700 h and the rest at 1500 h. These animals were trained to eat in individual Calan door-
type feeders (American Calan, Inc.; Northwood, NH, US), equipped with a container of
approximately 15 kg. Feed was offered ad libitum (15 % more than the previous day’s
intake). The portion was weighed, recorded and deposited in the feeders. Uneaten feed was
removed, weighed and recorded. For each animal, a sample of the consumed feed and the
non-consumed feed was obtained weekly.
The total DM was determined in an oven at 100 ºC for 24 h and incinerated in a muffle at
500 ºC to quantify the OM and ash content. DM intake was estimated by multiplying daily
feed intake by its DM content.
The NDF and ADF contents of the diets were quantified following the procedures of Goering
and Van Soest(13); while the protein was obtained by Kjeldahl(14). Changes in live weight
(LW) were recorded weekly and used to calculate DWG. The lambs were slaughtered at the
end of the fattening period following official slaughter procedures(15).
The carcass yield, expressed as a percentage, was calculated as the proportion of the weight
of the hot carcass, divided by the LW and multiplied by 100. The weight of the cold carcass
479
was obtained 24-30 h after cold storage at approximately 2 ± 2 ºC. Carcass yield was
recalculated and reported as cold carcass yield.
The research protocols and management procedures were carried out following the Official
Mexican Standard (NOM-051-ZOO-195). During mobilization, the animals were treated in
accordance with the standard NOM-024-ZOO-195.
Every 14 d, a blood sample, approximately 10 ml, was taken by puncture of the jugular vein,
in vacutainer tubes without anticoagulant (Beckton-Dickinson, Franklin Lakes, NJ). The
samples were kept in the environment for 60 min in order for them to coagulate and then they
were refrigerated. The blood was centrifuged at 1,000 xg for 25 min at 4 ºC. The serum was
stored in vials at -20 ºC and later sent to the laboratories of the Faculty of Veterinary Medicine
and Zootechnics of UNAM, for the determination of albumin, α-, ß-, ∂-globulin and Se. The
globulins were determined by the procedures of Connell et al(16); while the Se in serum was
quantified with a spectrofluorometer (Perkin Elmer model LS30), following the procedures
of Tamari et al(17).
After 48 h of slaughter, the Longissimus dorsi, Gluteus maximus and Musculus deltoideus
muscles were removed from each carcass according to the procedures of Covenin(18). From
each muscle and carcass, three cuts approximately 2.54 cm thick were obtained and packed
individually. All cuts were frozen at -30 ºC and stored at -20 ºC until the corresponding
analyses. After thawing, the thickness of the dorsal fat layer between the 12th and 13th rib
was taken(18).
The meat samples were partially thawed at 4 ºC (to avoid fluid loss). Subsequently, the visible
adipose tissue was removed, mixed with a Black and Decker™ food processor (Model
HC3061, New Britain, CT, USA), packed in bags (Whirl-Pak Bags, Nasco, Fort Atkinson,
WI), and stored at -20 ºC until the final analyses. Se was quantified with an atomic absorption
spectrophotometer (SpectrAA 220®, New Britain, CT, USA), following the procedures of
the manufacturers.
Data were analyzed using the statistical package SAS(19) (version 9.2, SAS Institute Inc.,
Cary, NC, US). DMI, LW changes, and FC were analyzed with the Mixed procedure of SAS
in a completely randomized design with measures repeated over time(19). The model included
fixed effects of treatment, week and the treatment×week interaction. The random effect of
animal was nested in treatments and was taken as the repeated term. The statistical model is
described below, after removing the double or triple interactions that were not significant:
480
ijkl= μ + Ti+ Tj + T×Tij + Lk(i) + Eijkl
Where:
Yikjl is the response variable, μ is the overall mean;
Ti is the fixed effect of treatmenti (i = 1, 2, ..., 5);
Tj is the fixed effect of time (j = 1, 2, ..., 4);
T×Tij is the fixed effect of the interaction of i-th treatmenti and j-th timej;
Lk(i) is the random effect of the animal;
Eijkl is the random effect of experimental error.
A model similar to the previous one was used to study the levels of albumin, α-, ß- and ∂-
globulins. Se concentrations in muscles were analyzed with Proc Mixed in a completely
randomized design with a classification criterion(19). The results were declared significant
where it was observed that P<0.05. When differences between treatments were detected, the
means were compared with the Tukey procedure with α=0.05. The covariance structure that
produced the lowest Akaike(20) criteria was that of composite symmetry in all the variables
studied, except for Se levels in muscles, which better adapted to the autoregressive of order
(1).
Table 2 shows the results obtained for DMI, DWG and FC of growing lambs supplemented
with SS and SP for 56 d. The supplementation with Se did not influence (P>0.05) the DMI
of the sheep. This may be because the addition of Se increases the digestibility of OM, NDF
and N in the total tract and possibly facilitates the absorption of the mineral in the abomasum.
However, it was not enough to increase the DMI.
481
Table 2: Mean values of feed intake, daily weight gain, feed conversion, live weight,
carcass yield and dorsal fat of fattening Pelibuey sheep supplemented with 0.30 and 0.90
mg of selenium
Treatments (Selenium, mg kg-1

MS)
Variable Sodium selenite Sel-Plex™ EE1 P2
Treat. Tx
Control 0.30 0.90 0.30 0.90 Time
(T) Time
Feed intake, kg DM 1.10 1.28 1.29 1.26 1.29 0.10 0.99 0.01 0.44
d-1
Daily weight gain, kg 0.230b 0.243b 0.245b 0.260a 0.293a 0.04 0.01 0.02 0.33
d-1
Feed conversion, kg 4.78b 5.26a 5.26a 4.48b 4.40b 0.19 0.03 0.36 0.25
Final live weight, kg 42.5 38.50 39.10 39.00 39.50 1.40 0.81 0.41 0.32
Hot carcass yield, % 53.20 54.10 54.10 52.50 52.80 0.98 0.69 0.25 0.11
Cold carcass yield, % 52.20 53.10 53.00 51.50 51.40 0.99 0.32 0.50 0.20
Dorsal fat, mm 2.20 2.70 2.30 2.50 1.99 0.32 0.80 0.23 012
1
Standard error; 2Probability (P<0.05); abc Values in the same row with distinct literal are different (P<0.05).
The results obtained in this study agree with Alimohamady et al(4), who observed an
improvement in the digestibility of dietary components. Other studies reported different
results. Domínguez-Vara et al(21) observed no differences in DWG and feed conversion in
Rambouillet lambs fed 0.30 mg of Se per kg-1 of DM of SS compared to the control. The
non-difference was attributed to the state of Se and its low availability in the diets.
In the present study, supplementation with 0.30 and 0.90 mg kg-1 DM of SS or SP impacted
DWG. This may be due to an improvement in feed digestibility. On the contrary, the
superiority in feed conversion with 90SP, possibly, is explained because the Se from SP has
been shown to have a higher bioavailability with respect to SS(9,21).
Table 2 shows the final LWs, the weights of the hot and cold carcasses, and the thickness of
the dorsal fat layer of sheep fed SS and SP. There was no effect of the level and source of Se
(P>0.05) on the aforementioned variables. The lack of effect is explained by the similarity in
the DMI of the animals. As is known, the LW of animals depends on feed intake and in the
present case, this intake was similar between treatments, although the FC was different.
482
Vignola et al(6) indicated that supplementation with 0.3 and 0.9 mg of Se from SS or SP did
not affect the Longissimus dorsi area, dorsal fat thickness and the weights of hot and cold
carcasses of growing lambs. The loss of effect was attributed to the similar DMI among the
different sources of the mineral.
As shown in Table 3, the effect of the treatments influenced (P≤ 0.05) the content of Se in
the blood serum. As the Se in the diet increased, the concentration in blood serum increased
and then it showed a decreasing return with 0.9 mg of Se kg-1 DM.
Table 3: Mean values of serum concentrations of selenium, albumin, α-, β- and ∂-

globulins, Longissimus dorsi et lumborum, Gluteus maximus and Musculus deltoideus of
fattening Pelibuey sheep supplemented with 0.30 and 0.90 mg kg-1 DM of selenium from
sodium selenite or Sel-Plex™ for 56 days in confinement.
Treatments, mg kg-1
Control Sodium selenite Sel-Plex™ CI1 SEM2 P3
Variable
0.3 0.9 0.3 0.9
Blood serum concentrations, mg L-1
0.08-
Selenium 0.05c 0.08a 0.08a 0.09a 0.07b 0.001 0
0.504
24.0-
Albumin 45.91b 46.66b 49.91a 52.09a 48.09b 1.51 0
30.05
α-globulin 12.16c 14.44a 12.51c 12.62c 13.13b 1.14 0
β-globulin 13.45 14.98 13.65 13.61 18.83 2.03 0.7
∂-globulin 23.4 25.24 24.38 24.62 23.65 1.28 0.4
Muscle concentration, μ/100 g
Longissimus dorsi 9.0-
17.30b 20.50ab 20.22ab 23.82a 17.90b 2.8 0
et lumborum 40.004
Gluteus maximus 14.37 15.32 17.62 23.72 19.7 4.75 0.4

Musculus
15 24.57 29.82 31.32 20.3 4.77 0.3
deltoideus
1
Concentration interval. 2Standard error of the means. 3Probability, (P<0.05). 4Puls(22); 5Kaneko et al(24).
a,b,c
Values in the same row with different literal are different (P<0.05).
483
According to Puls(22), adequate levels of Se in blood serum of growing sheep are 0.08 to 0.50
mg L-1, in order to balance internal homeostasis. In the present study, all treatments were
within the indicated range, except for C and 30SS, which showed concentrations below 0.07
mg L-1.
As shown in Table 3, supplementation with different levels of SS or SP influenced (P<0.05)

the Se level in the Longissimus dorsi, with no apparent effects (P>0.05) in the Gluteus
maximus and Musculus deltoideus. The Se in skeletal muscle increases as the diet is richer in
the mineral. Based on the report by Puls(22), the observed values of Se in the muscles are
within the appropriate range for animals of the same characteristics as in the present study.
The highest response to supplementation was 30SP. Perhaps due to the greater availability
of the mineral to be incorporated into tissues, however, with the highest level, its response
tends to decrease.
The results obtained in the present study agree with others previously published. Juniper et
al(9) indicated that 0.35 mg of SS or SP increased the mineral in the muscles, in a manner
dependent on the diets of finishing bovines. The difference between sources was remarkable.
The Se of SS produced 0.31 mg of Se, while that of SP yielded 0.46 mg in the Longissimus
dorsi, which are similar to those observed in the present study.
The levels of albumin, α-, ß- and ∂-globulins in the blood serum of sheep are presented in
Table 3. The levels and sources of Se only increased (P<0.05) albumin and α-globulins. On
the contrary, they did not affect the levels of ß- and ∂-globulins. Serum albumin originates in
hepatocytes, from where it passes into the bloodstream (which represents approximately
13 % of the total protein produced by the liver)(23).
In the present study, the increased presence of albumin in lambs that consumed 90SS and
30SP was not due to the increase in the synthetic capacity of hepatic albumin. Nor was it
because the animals had a high capacity for synthesis. The difference is explained because
Se, as an antioxidant, improves the activity of hepatocytes, so perhaps it improved overall
protein production.
Based on the report by Kaneko et al(24), the albumin concentration obtained in the present
study was approximately twice the minimum recommended levels, and in all cases, it
exceeded the maximum indicated. The maximum level was reached with 30SP and
subsequently, it tended to decrease. The values observed in the present study are consistent
with those observed by De Paula Silva et al(25) with several sheep breeds created in tropical
conditions.
484
Immunoglobulins act as membrane receptors on β lymphocytes and are used by the immune
system to identify and neutralize viruses and bacteria(26). In the present study, the highest
concentration of α-globulins was found in lambs that consumed 30SS and 90SP. This
behavior was related to the antioxidant capacity of the mineral included in the diet.
In conclusion, the level of 0.90 mg of Sel-Plex™ improved DWG and FC. Selenite and SP
increased serum Se in 30SS, 90SS and 30SP and it decreased with 0.90 mg per kg of SP. In
the Longissimus dorsi, Se was improved in 30SP with respect to 90SP and C and was similar
to 90SS and 30SS. The organic Se of 90SP improved the level of albumin and α-globulins.
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Revista Mexicana de Ciencias Pecuarias
Edición Bilingüe
Rev. Mex. Cienc. Pecu. Vol. 14 Núm. 2, pp. 260-487, ABRIL-JUNIO-2023 Bilingual Edition
ISSN: 2448-6698
CONTENIDO
CONTENTS
ARTÍCULOS / ARTICLES Pags.

Producción de anticuerpos séricos en respuesta a la vacunación contra los virus de la rinotraqueitis
infecciosa bovina y la diarrea viral bovina con una vacuna comercial
Production of serum antibodies in response to vaccination against infectious bovine rhinotracheitis and bovine viral diarrhea viruses with a commercial vaccine
Jorge Víctor Rosete Fernández, Guadalupe Asunción Socci Escatell, Abraham Fragoso Islas, Sara Olazarán Jenkins, Ángel Ríos Utrera.....................……....…....…....…....…....…....….................…..........….........260
Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 14 Núm. 2, pp. 260-487, ABRIL-JUNIO-2023
Characterization of fetal bovine serum obtained from the meat industry for cell culture
Caracterización del suero bovino fetal proveniente de la industria cárnica mexicana en el cultivocelular
Francisco Javier Preciado-Gu�érrez, David Masuoka-Ito, José Luis Barrera-Bernal, Bryan Ivan Mar�n del Campo-Téllez, Vicente Esparza-Villalpando, Ricardo Ernesto Ramírez-Orozco...............................277

Acción ixodicida de productos naturales de plantas nativas mexicanas
Javier Sosa-Rueda, Fabiola Villarauz, Vanihamin Domínguez-Meléndez, Ida Soto-Rodríguez, Fernando C. López-Fentanes, David I. Mar�nez-Herrera,
Álvaro Peniche-Cardeña, FranciscoCen-Pacheco..............................................................................................................................................................................................................................................…….292
Efecto de Xoconostle (Opuntia matudae Scheinvar) sobre la concentración de metano y las variables ruminales
durante una fermentación in vitro de rastrojo de maíz
Effect of Xoconostle (Opuntia matudae Scheinvar) on methane concentration and ruminal variables during in vitro fermentation of corn stover
José Jesús Espino-García, Isaac Almaraz-Buendía, J. Jesús Germán Peralta-Or�z, Abigail Reyes- Munguía, Iridiam Hernández-Soto,
Lucio González-Mon�el, Rafael Germán Campos-Mon�el…..........…….….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....…….....….............…….....……...........309
Factores que afectan la tasa de preñez mediante transferencias de embriones por fertilización in vitro
en novillas multirraciales en condiciones de trópico colombiano
Factors affecting the rate of pregnancy by embryo transfers (ET) by in vitro fertilization in multibreed heifers under Colombian tropical conditions
Heli Fernando Valencia Ocampo, Nancy Rodríguez Colorado, Tatiana Mantilla…......…......…......…......….......….......….............…......…...............…......…......…......…......…......…......…......…......…..……..326
Estructura y variabilidad genética del bisonte americano (Bison bison) en México

Genetic structure and variability in American bison (Bison bison) in Mexico
Joel Domínguez-Viveros, Guadalupe Nelson Aguilar-Palma, Rafael Villa-Angulo, Nancy Hernández-Rodríguez, José Manuel Pérez-Cantú, Flora Moir, Pedro Calderón-Domínguez....…….......…….….............339
Composición química del rastrojo de tres cultivares de maíz esterilizados y colonizados por micelio de Ganoderma lucidum
Stover chemical composition in three corn cultivars after sterilization or colonization with Ganoderma lucidum mycelia
Liz Sarahy Pérez-Martell, Juan de Dios Guerrero-Rodríguez, Daniel Claudio Mar�nez-Carrera, Javier Francisco Enríquez-Quiroz, Efraín Pérez-Ramírez, Benito Ramírez-Valverde............……….............…......349
Nutrient concentrations, in vitro digestibility and rumen fermentation of agro-industrial residues

of Cannabis sativa L. as a potential forage source for ruminants
Concentraciones de nutrientes, digestibilidad in vitro y fermentación ruminal de residuos agroindustriales de
Cannabis sativa L. como fuente potencial de forraje para rumiantes
Elia Esther Araiza-Rosales, Esperanza Herrera-Torres, Francisco Óscar Carrete-Carreón, Rafael
Jiménez-Ocampo, Daniel Gómez-Sánchez, Gerardo Antonio Pámanes-Carrasco………………………...…………...…….………….........…...……...……...……...……...……...……...……...……...……...……...…......................…......366
REVISIONES DE LITERATURA / REVIEWS

Importancia de Haematobia irritans en la ganadería bovina de México: Situación actual y perspectivas. Revisión
Importance of Haematobia irritans in cattle in Mexico: Current situation and perspectives. Review
Roger Iván Rodríguez Vivas, Carlos Cruz Vázquez, Consuelo Almazán, Juan José Zárate Ramos..….....…….....…....….....……...……..…..……..…..……..…..……..…..……..…..……..…..……..…..……..…..……..…..……..…..….384
NOTAS DE INVESTIGACIÓN / TECHNICAL NOTES

Curvas de crecimiento en bovinos Limousin de raza pura y cruzados
Joel Domínguez-Viveros, Antonio Reyes-Cerón, Carlos Enrique Aguirre-Calderón, Ricardo Mar�nez-Rocha, Carlos Luna-Palomera, Nelson Aguilar-Palma….…….............................................................…….412
Relationship between body measurement traits, udder measurement traits and milk yield of Saanen goats in Capricorn district of South Africa
Relación entre rasgos de mediciones corporales, rasgos de mediciones de la ubre y producción de leche de cabras Saanen en el distrito de Capricorn de Sudáfrica
Thlarihani Cynthia Makamu, Molabe Kagisho Madikadike, Kwena Mokoena, Thobela Louis Tyasi…….....…….......…….....……........…….........…….........…….........…….........…….........…….........……..................…......423
Análisis genético del bovino Criollo Mixteco de Oaxaca

Miguel Ángel Domínguez Mar�nez, Víctor Hernández Núñez, Araceli Mariscal Méndez, Amparo Mar�nez Mar�nez, Gisela Fuentes-Mascorro......…......…......…......…......…......…......…..........….....…………...434
Influence of the cut intervals on hay quality of Panicum maximum cv. BRS Tamani in brazilian Cerrado
Influencia de los intervalos de corte en la calidad del heno de Panicum maximum cv. BRS Tamani en el Cerrado brasileño
Edgar Hernández Moreno, Joel Ventura Ríos, Claudia Yanet Wilson García, María de los Ángeles Maldonado Peralta,
Eva Nara Oliveira Gomes, Alexandre Menezes Dias, Luciana Junges, Luís Carlos Vinhas Ítavo, Gelson dos Santos Difante, Juliana Oliveira Ba�sto�..................……………………………………….……………...........…450
Evaluación de la seroconversión de cerdas con el uso de un inóculo a diferentes dosis y vehículos contra la diarrea epidémica porcina
Evaluation of sow seroconversion with the use of inoculum at different doses and vehicles against porcine epidemic diarrhea
Nancy Paulina García Cano Rubí, Francisco Ernesto Mar�nez-Castañeda, Elein Hernández Trujillo, Rosa Elena Sarmiento Silva,
Rolando Beltrán Figueroa, Montserrat Elemi García-Hernández, María Elena Trujillo-Ortega…....……....……....……....……....……....……....……....……....……....……....…...…...…...…...…...…...…...…......……....…….......466
Efecto de la fuente de selenio en el comportamiento productivo, contenido de selenioen suero y músculo,

y nivel sérico de albúmina, α-, β- y ∂-globulinas en ovinos Pelibuey
Effect of selenium source on productive behavior, serum and muscle selenium content, and serum level of albumin, α-, β- and ∂-globulins in Pelibuey sheep
Lino Rigoberto Cárdenas-Ramírez, Carlos Sánchez del Real, Agus�n Ruíz-Flores, Gabriela Pérez- Hernández, Reyes López-Ordaz, Claudio Vite-Cristóbal, Ruﬁno López-Ordaz………..……….......……....…..……476
Rev. Mex. Cienc. Pecu. Vol. 14 Núm. 2, pp. 260-487, ABRIL-JUNIO-2023

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RMCP Vol. 14 Num. 2 (2023) : April-June (English Version)

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Rev. Mex. Cienc. Pecu. Vol. 14 Núm. 2, pp. 260-487, ABRIL-JUNIO-2023

TIPOGRAFÍA Y FORMATO: Oscar L. Rodríguez Rivera

VISITE NUESTRA PÁGINA EN INTERNET

REV. MEX. CIENC. PECU. VOL. 14 No. 2 ABRIL-JUNIO-2023

Producción de anticuerpos séricos en respuesta a la vacunación contra los virus de

Ixodicide action of natural products from native Mexican plants

Efecto de Xoconostle (Opuntia matudae Scheinvar) sobre la concentración de metano

Factores que afectan la tasa de preñez mediante transferencias de embriones por

Composición química del rastrojo de tres cultivares de maíz esterilizados y colonizados

Nutrient concentrations, in vitro digestibility and rumen fermentation of agro-industrial

Importancia de Haematobia irritans en la ganadería bovina de México: Situación actual

Curvas de crecimiento en bovinos Limousin de raza pura y cruzados

Evaluación de la seroconversión de cerdas con el uso de un inóculo a diferentes dosis y

Efecto de la fuente de selenio en el comportamiento productivo, contenido de selenio

INSTRUCTIONS FOR AUTHORS

Revista Mexicana de Ciencias Pecuarias is a scientific

11. Literature cited. All references should be quoted in

Expresa sus más sinceras condolencias por el sentido fallecimiento del

Dr. Raymundo Martínez Peña

Nació el 20 de septiembre de 1939 en Nogales, Veracruz; se desempeñó

Falleció el 20 de marzo de 2023.

Production of serum antibodies in response to vaccination against

Jorge Víctor Rosete Fernández a

Guadalupe Asunción Socci Escatell b

Abraham Fragoso Islas a

Sara Olazarán Jenkins a

Ángel Ríos Utrera c*

*Corresponding author: ariosu@hotmail.com

vaccination (booster vaccine). To detect antibodies, serum samples were collected 30 d

Keywords: Infectious bovine rhinotracheitis, Bovine viral diarrhea, Polyvalent vaccine,

Infectious bovine rhinotracheitis (IBR), also known as infectious pustular vulvovaginitis, is

The objective was to determine the prevalence of serum antibodies in response to

Material and methods

Post-vaccination blood sampling and serum collection

Response variables and statistical analyses

Table 1: Statistical significance of treatment effect, by response variable and disease

BVD 0.4588 0.1769 0.1042

Table 2: Prevalences (%) of serum antibodies against infectious bovine rhinotracheitis

Table 4: Prevalences (%) of serum antibodies against infectious bovine rhinotracheitis

Infectious bovine rhinotracheitis

Outside Mexico, in dairy herds of Toca-Boyacá and Caquetá, Colombia, prevalences of

Bovine viral diarrhea

induced short-lived antibody production, as it does not promote immunological memory

Conclusions and implications

The commercial polyvalent vaccine induced the production of satisfactory levels of

2. Gu X, Kirkland PD. Infectious bovine rhinotracheitis. Australian and New Zealand

8. Betancur HC, Gonzales TM, Reza GL. Seroepidemiología de la rinotraqueitis infecciosa

16. De La Trinidad SH. Seroprevalencia y factores de riesgo de rinotraqueitis infecciosa

17. Abad-Zavaleta J, Ríos-Utrera A, Rosete-Fernández JV, García-Camacho A, Zárate-

19. Sánchez-Castilleja YM, Rodríguez DJG, Pedroso M, Cuello S. Simultaneidad

20. Ríos-Utrera Á, Rosete-Fernández JV, Zárate-Martínez JP, Fragoso-Islas A, Olazarán-

21. Ochoa X, Orbegozo M, Manrique AF, Pulido PM, Ospina J. Seroprevalencia de

28. OIE. Office International des Epizooties. Rinotraqueitis bovina

30. Pérez S, Inman M, Doster A, Jones C. Latency-related gene encoded by bovine

38. Milián-Suazo F, Hernández-Ortíz R, Hernández-Andrade L, Alvarado-Islas A, Díaz-

Characterization of fetal bovine serum obtained from the meat industry

Francisco Javier Preciado-Gutiérrez a

José Luis Barrera-Bernal b

Bryan Ivan Martín del Campo-Téllez b

Ricardo Ernesto Ramírez-Orozco c*

* Corresponding author: dcmrero@gmail.com

Material and methods

One lot of serum was collected from FREASA (Frigorífico y Empacadora de