RMCP Vol. 14 Num. 4 (2023) : October-December (English Version)

Edición Bilingüe
Bilingual Edition
ISSN: 2448-6698
Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 14 Núm. 4, pp. 745-930, OCTUBRE-DICIEMBRE-2023
Rev. Mex. Cienc. Pecu. Vol. 14 Núm. 4, pp. 745-930, OCTUBRE-DICIEMBRE-2023

REVISTA MEXICANA DE CIENCIAS PECUARIAS Volumen 14 Numero 4, Octubre-
Diciembre 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 octubre de 2023.
3° Concurso de Dibujo Infantil 2023
“Futuros Investigadores”
Categoría B, 3er. Lugar
Autor: Ari Enrique Mares Hernández
Edad: 9 años, Aguascalientes
DIRECTORIO
Título: El campo mexicano 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).
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II
I
REVISTA MEXICANA DE CIENCIAS PECUARIAS
REV. MEX. CIENC. PECU. VOL. 14 No. 4 OCTUBRE-DICIEMBRE-2023
CONTENIDO
Contents
ARTÍCULOS
Articles
Pág.
Estructura de la red de mercado de bovinos en México, 2017-2021
Structure of the cattle market network in Mexico, 2017-2021
Nicolás Callejas Juárez, José María Salas González...................................................................745
Prospectiva ambiental al 2030 en sistemas de producción de leche de vaca en México

Environmental outlook to 2030 in cow's milk production systems in Mexico
María del Rosario Villavicencio-Gutiérrez, Nicolás Callejas-Juárez, Nathaniel Alec Rogers-Montoya,
Vianey González-Hernández, Rodrigo González-López, Carlos Galdino Martínez-García, Francisco
Ernesto Martínez Castañeda……..............................................................................................760
Evaluación de resistencia a antibióticos en muestras de heces de terneros con diarrea

en la región Cajamarca, Perú
Assessment of antibiotic resistance in fecal samples from calves with diarrhea in the Cajamarca
region, Peru
Marco Antonio Cabrera González, Héctor Vladimir Vásquez Pérez, Carlos Quilcate-Pairazamán,
José Bazán-Arce, Medali Cueva-Rodríguez ..............................................................................782
Contaminación de alimento comercial seco para perro por Aspergillus flavus y

aflatoxinas en Aguascalientes, México
Contamination of commercial dry dog food by Aspergillus flavus and aflatoxins in Aguascalientes,
Mexico
Lizbeth Martínez-Martínez, Arturo Gerardo Valdivia-Flores, Teódulo Quezada-Tristán, Alma Lilián
Guerrero-Barrera, Erika Janet Rangel-Muñoz, Karla Isela Arroyo Zúñiga, Fernanda Álvarez-Días,
Marcelo Lisandro Signorini-Porchietto .....................................................................................796
Estimación del grado básico de calidad en canales bovinas conforme a madurez ósea,
marmoleo y predominancia fenotípica Bos indicus
Estimation of the basic quality grade of beef carcasses according to bone maturity, marbling, and
Bos indicus phenotypic predominance
Francisco Gerardo Ríos Rincón, Leslie Zelibeth González Rueda, Jesús José Portillo Loera, Beatriz
Isabel Castro Pérez, Alfredo Estrada Angulo, Jesús David Urías Estrada ....................................818
III
The effect of hesperidin added to quail diets on blood gas, serum biochemistry and
Hsp70 in heat stress
Efecto de la hesperidina añadida a las dietas de codorniz sobre los gases en sangre, la
bioquímica sérica y HSP 70 bajo estrés por calor
Abdullah Özbilgin, Aykut Özgür, Onur Başbuğ .........................................................................836
Evaluación antihelmíntica de cuatro extractos de árboles forrajeros contra el

nematodo Haemonchus contortus bajo condiciones in vitro
Anthelmintic evaluation of four fodder tree extracts against the nematode Haemonchus
contortus under in vitro conditions
Itzel Santiago-Figueroa, Alejandro Lara-Bueno, Roberto González-Garduño, Pedro Mendoza-de
Gives, Edgar Jesús Delgado-Núñez, Ema de Jesús Maldonado-Simán, Yagoob Garedaghi,
Agustín Olmedo-Juárez ...............................................................................................…………855
Efecto del uso de agua residual tratada sobre el suelo y cultivos forrajeros de
Chenopodium quinoa Willd y Zea mays L.
Effect of treated wastewater use on soil and forage crops of Chenopodium quinoa Willd and Zea
mays L.
Ana Lilia Velasco-Cruz, Vicente Arturo Velasco-Velasco, Judith Ruíz-Luna, José Raymundo
Enríquez-del Valle, Aarón Martínez-Gutiérrez, Karen del Carmen Guzmán-Sebastián ……………..…874
Correlación entre el comportamiento del toro de lidia en los corrales y el ruedo

Correlations between behavior in corrals and the bullring in Lidia breed bulls
Juan Manuel Lomillos, Eloy Marino, Enrique Recas, René Alonso, Marta Elena Alonso ................889
NOTAS DE INVESTIGACIÓN
Tehnical notes
Efecto ixodicida de los extractos vegetales de Cinnamomum zeylanicum y Tagetes

erecta sobre garrapatas Rhipicephalus microplus
Ixodicidal effect of plant extracts of Cinnamomum zeylanicum and Tagetes erecta on
Rhipicephalus microplus ticks
Perla Iris Miranda Reyes, Francisco Martínez Ibañez, Rodolfo Esteban Lagunes-Quintanilla,
América Ivette Barrera Molina ................................................................................................905
Detección de patógenos de importancia epidemiológica en cerdos ferales de Chihuahua

y Durango, México
Detection of pathogens of epidemiological importance in feral pigs from Chihuahua and Durango,
Mexico
Mario Enrique Haro Tirado, José Martín Fuentes Rodríguez, Claudia Chacón Zendejas, Alberto
Lafón Terrazas, Luis Lecuona Olivares, Rodolfo Pineda Pérez, Rosalba Carreón Nápoles ………..…915
Ovine pulmonary adenocarcinoma in Mexico

Adenocarcinoma pulmonar ovino en México
Johnatan Alberto Ruíz-Ramírez, Brayan Jossue Chávez-Ramírez, Jorge Luis García-Valle, Marcelo de
las Heras, Alfonso López-Mayagoitia, Luis Jorge García-Márquez ........... ...................................923
IV
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.
V
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
VI
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.
VII
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)
VIII
Updated: March, 2020
INSTRUCTIONS FOR AUTHORS
Revista Mexicana de Ciencias Pecuarias is a scientific Title page

journal published in a bilingual format (Spanish and Abstract
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IX
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Standard journal article (List the first six authors
mentioned in the text. Text, tables and figure
followed by et al.)
references should be identified by means of Arabic
numbers. Avoid, whenever possible, mentioning in the I) Basurto GR, Garza FJD. Efecto de la inclusión de grasa
text the name of the authors. Abstain from using o proteína de escape ruminal en el comportamiento
abstracts as references. Also, “unpublished de toretes Brahman en engorda. Téc Pecu Méx
observations” and “personal communications” should 1998;36(1):35-48.
not be used as references, although they can be
inserted in the text (inside brackets).
Issue with no volume
Key rules for references II) Stephano HA, Gay GM, Ramírez TC. Encephalomielitis,
reproductive failure and corneal opacity (blue eye) in
a. The names of the authors should be quoted
pigs associated with a paramyxovirus infection. Vet
beginning with the last name spelt with initial capitals,
Rec 1988;(122):6-10.
followed by the initials of the first and middle name(s).
In the presence of compound last names, add a dash III) Chupin D, Schuh H. Survey of present status of the
between both, i.e. Elias-Calles E. Do not use any use of artificial insemination in developing countries.
punctuation sign, nor separation between the initials World Anim Rev 1993;(74-75):26-35.
of an author; separate each author with a comma,
even after the last but one. No author given
b. The title of the paper should be written in full, IV) Cancer in South Africa [editorial]. S Afr Med J
followed by the abbreviated title of the journal without
1994;84:15.
any punctuation sign; then the year of the publication,
after that the number of the volume, followed by the
number (in brackets) of the journal and finally the 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).
Organization, as author
d. In the case of a single author’s book (or more
VI) The Cardiac Society of Australia and New Zealand.
than one, but all responsible for the book’s contents),
Clinical exercise stress testing. Safety and
the title of the book should be indicated after the
performance guidelines. Med J Aust 1996;(164):282-
names(s), the number of the edition, the country, the
printing house and the year. 284.
In press
X
VII) Scifres CJ, Kothmann MM. Differential grazing use of XVII) AOAC. Official methods of analysis. 15th ed.
herbicide-treated area by cattle. J Range Manage [in Arlington, VA, USA: Association of Official Analytical
press] 2000. Chemists. 1990.
Books and other monographs XVIII) SAS. SAS/STAT User’s Guide (Release 6.03). Cary
NC, USA: SAS Inst. Inc. 1988.
Author(s)
XIX) SAS. SAS User´s Guide: Statistics (version 5 ed.).
VIII) Steel RGD, Torrie JH. Principles and procedures of Cary NC, USA: SAS Inst. Inc. 1985.
statistics: A biometrical approach. 2nd ed. New
York, USA: McGraw-Hill Book Co.; 1980. Electronic publications
XX) Jun Y, Ellis M. Effect of group size and feeder type
Chapter in a book
on growth performance and feeding patterns in
IX) Roberts SJ. Equine abortion. In: Faulkner LLC editor. growing pigs. J Anim Sci 2001;79:803-813.
Abortion diseases of cattle. 1rst ed. Springfield, http://jas.fass.org/cgi/reprint/79/4/803.pdf.
Illinois, USA: Thomas Books; 1968:158-179. Accesed Jul 30, 2003.
XXI) Villalobos GC, González VE, Ortega SJA. Técnicas
Conference paper para estimar la degradación de proteína y materia
X) Loeza LR, Angeles MAA, Cisneros GF. Alimentación orgánica en el rumen y su importancia en rumiantes
de cerdos. En: Zúñiga GJL, Cruz BJA editores. en pastoreo. Téc Pecu Méx 2000;38(2): 119-134.
Tercera reunión anual del centro de investigaciones http://www.tecnicapecuaria.org/trabajos/20021217
forestales y agropecuarias del estado de Veracruz. 5725.pdf. Consultado 30 Jul, 2003.
Veracruz. 1990:51-56.
XXII) Sanh MV, Wiktorsson H, Ly LV. Effect of feeding
XI) Olea PR, Cuarón IJA, Ruiz LFJ, Villagómez AE. level on milk production, body weight change, feed
Concentración de insulina plasmática en cerdas conversion and postpartum oestrus of crossbred
alimentadas con melaza en la dieta durante la lactating cows in tropical conditions. Livest Prod Sci
inducción de estro lactacional [resumen]. Reunión 2002;27(2-3):331-338.
nacional de investigación pecuaria. Querétaro, Qro.
http://www.sciencedirect.com/science/journal/030
1998:13.
16226. Accesed Sep 12, 2003.
XII) Cunningham EP. Genetic diversity in domestic
animals: strategies for conservation and 12. Tables, Graphics and Illustrations. It is preferable
development. In: Miller RH et al. editors. Proc XX that they should be few, brief and having the
Beltsville Symposium: Biotechnology’s role in necessary data so they could be understood without
genetic improvement of farm animals. USDA. reading the text. Explanatory material should be
1996:13. placed in footnotes, using conventional symbols.
13. Final version. This is the document in which the

Thesis
authors have already integrated the corrections and
XIII) Alvarez MJA. Inmunidad humoral en la anaplasmosis modifications indicated by the Review Committee. The
y babesiosis bovinas en becerros mantenidos en una works will have to be elaborated with Microsoft Word.
zona endémica [tesis maestría]. México, DF: Photographs and images must be in jpg (or
Universidad Nacional Autónoma de México; 1989. compatible) format with at least 300 dpi resolution.
XIV) Cairns RB. Infrared spectroscopic studies of solid Photographs, images, graphs, charts or tables must
oxigen [doctoral thesis]. Berkeley, California, USA: be included in the same text file. The boxes should
University of California; 1965. not contain any vertical lines, and the horizontal ones
only those that delimit the column headings, and the
Organization as author
line at the end of the box.
XV) NRC. National Research Council. The nutrient
requirements of beef cattle. 6th ed. Washington, 14. Once accepted, the final version will be translated into
DC, USA: National Academy Press; 1984. Spanish or English, although authors should feel free
to send the final version in both languages. No
XVI) SAGAR. Secretaría de Agricultura, Ganadería y charges will be made for style or translation services.
Desarrollo Rural. Curso de actualización técnica para
la aprobación de médicos veterinarios zootecnistas 15. Thesis will be published as a Research Article or as a
responsables de establecimientos destinados al Technical Note, according to these guidelines.
sacrificio de animales. México. 1996.
16. Manuscripts not accepted for publication will be
returned to the author together with a note explaining
XI
the cause for rejection, or suggesting changes which ml milliliter (s)
should be made for re-assessment. mm millimeter (s)
min minute (s)
17. List of abbreviations:
ng nanogram (s)
cal calorie (s) P probability (statistic)
cm centimeter (s) p page
°C degree Celsius CP crude protein
DL50 lethal dose 50% PCR polymerase chain reaction
g gram (s) pp pages
ha hectare (s) ppm parts per million
h hour (s) % percent (with number)
i.m. intramuscular (..ly) rpm revolutions per minute
i.v. intravenous (..ly) sec second (s)
J joule (s) t metric ton (s)
kg kilogram (s) TDN total digestible nutrients
km kilometer (s) AU animal unit
L liter (s) IU international units
log decimal logarithm vs versus
Mcal mega calorie (s) xg gravidity
MJ mega joule (s)
The full term for which an abbreviation stands should
m meter (s) precede its first use in the text.
µl micro liter (s)
µm micro meter (s) 18. Scientific names and other Latin terms should be
written in italics.
mg milligram (s)
XII
https://doi.org/10.22319/rmcp.v14i4.6433
Article
Nicolás Callejas Juárez a*

José María Salas González b
a
Universidad Autónoma de Chihuahua. Facultad de Zootecnia y Ecología. Periférico
Francisco R. Almada Km. 1, CP 31453. Chihuahua, Chihuahua, México.
b
Universidad Autónoma Chapingo. Departamento de Sociología Rural. Estado de
México, México.
* Corresponding author: ncallejas@uach.mx
Abstract:
Cattle ranching, transportation, and utilization are issues associated with resource
endowment, distance traveled, and types of markets. The structure of the livestock
mobilization network by market at the municipal and state level in Mexico during the
2017-2021 period was investigated. The data were analyzed using measures of economic
structure and Social Network Analysis. During the period under analysis, an annual
average of 8.9 million heads of cattle were moved in Mexico: 57.9 % interstate and
42.1 % intrastate. The most important markets were for slaughter and fattening, the rest
corresponded to beef breeding, reproduction, fairs, and shows. The average market and
state specialization were low, with a higher specialization in the entertainment market.
The structure of the state network of all markets showed a high degree of average market
and density, but low centrality of output and input. These measures mean that, on average,
states can connect in 1.2 steps to the national network and in 1.7 steps to the network per
purpose. The authors conclude that the state structure of the livestock market in Mexico
is composed of 32 origins, 32 destinations, six markets, and major interstate mobilization
from the south to the north of the country.
Keywords: Regional localization, Regional specialization, Network analysis, Interstate
commerce, Intrastate trade.
Received: 22/03/2023
Accepted: 23/06/2023
745
Rev Mex Cienc Pecu 2023;14(4):745-759
Introduction
Research on cattle production and marketing has been carried out in Mexico, but other
important zootechnical objectives or market niches such as breeding, fattening, and
entertainment have not been addressed. Livestock mobility consists of moving live
animals from one place to another according to the laws of the market and the
government(1), while the social structure of markets is a refutation of the asocial market
conceptualizations that dominate the economic theory and policy(2).
Research of this type has been carried out in the U.S.(3), Argentina(4), Germany(5), Brazil(6),
France(7), Chile(8), Ecuador(9), Ireland(10), and Uruguay(11); they all agree on the
importance of the studies for resource allocation, improved market efficiency, and animal
health management.
Canada has an effective animal identification system, and its provinces are moving
towards a fully traceable system; however, the U.S. and Mexico have made little or no
progress in this sense(12). In the U.S., lack of traceability causes annual economic losses
of up to US$83 billion, and in the case of low- and middle-income countries, of up to
US$95 billion; 80% of these losses are related to food and water consumption(13). In
addition to the disruptions in the U.S. beef cattle supply chain and the drop in cattle prices
across the board caused by the COVID-19 pandemic(14), the epidemiological phenomenon
led to a historical increase in the difference between the price of cattle and the wholesale
price of meat(15), with losses estimated at US$ 13.6 billion(16).
In Mexico, the supply of cattle is important in terms of inventory, volume produced, value
of production, and spread throughout the national territory(17). The national inventory in
the year 2020 was 35.6 million heads of cattle: 92.7 % beef, and 7.3 % milk. 36.3 % were
concentrated in the states of Veracruz, Jalisco, Chiapas, and Chihuahua(18); the first three
states are characterized by the breeding of Zebu cattle, and the fourth, by raising European
breeds(19).
The development of information and communication technologies has given rise to

theories that form the basis of the current regional economic development. The network
theory is a tool for analyzing the structure of a market for any economic activity or
productive sector(20). The structural characteristics of social networks describe how actors
are connected to form a network or value chain(21); network measurements can be
calculated at node and network-wide level(22).
Faced with the problem of providing solutions for livestock production and distribution,
the objective of the research was to analyze the structure of the livestock mobilization
network in Mexico during the 2017-2021 period, through measures of economic location,
centrality and density of the networks by type of market motive.
746
Material and methods
The database used in the research considered 100 % of the daily records of all types of
cattle (milk, beef, rodeo, bullfight) legally moved by the quarantine stations of the
National Service for Agriculture and Food Health, Safety, and Quality (Servicio Nacional
de Sanidad, Inocuidad y Calidad Agroalimentaria, SENASICA) in Mexico for six
purposes or markets (slaughter, fattening, beef breeding, reproduction, fairs, and
entertainment) for the 2017-2021 period. The information was used with the express
authorization of SENASICA, and its analysis was performed in Microsoft Office® Excel.
A comprehensive data collection method(23) was used for the research. Municipal
mobilization records were grouped by state and by market. This grouping was done to
form municipal, state, and federal matrices in accordance with the method and techniques
utilized to estimate the indicators of the structure of the livestock market network in
Mexico. A total of 1,374 source municipalities (Ms), 1,842 municipalities of destination
(Md), and 44.7 million heads of cattle moved during the analysis period were analyzed.
By market, 951 municipalities moved cattle for slaughter, 900 for fattening, 652 for beef
breeding, 676 for reproduction, 391 for entertainment, and 484 for fairs; the
municipalities of destination were 853 for slaughter, 1,119 for fattening, 1,086 for beef
breeding, 1,355 for reproduction, 916 for entertainment, and 548 for fairs.
Municipal data were analyzed by state, with 𝑁𝑖 = 32 source municipalities (𝑋𝑖 ) and 𝑁𝑗 =
32 municipalities of destination (𝑋𝑗 ); this amounts to 1,024 exchange relationships. The
economic importance and networks were measured based on the number of cattle moved
across Mexican territory (𝑋𝑖𝑗 ). Two theories ―the spatial location theory(24) and social
network analysis― were used in the analysis of the network structure(25).
For the analysis of the regional economic structure, the livestock movement data were
arranged in two matrices, one for the sector-region of origin, and the other one, for the
sector-region of destination. The sectors were the six types of livestock movements (𝑉𝑖 ),
and the regions, the states of origin and destination of the livestock (𝑉𝑗 ). The variable of
analysis was the number of mobilized heads of cattle (𝑉𝑖𝑗 ).
The participation of the sector in the region of origin (𝑃𝑗𝑖1 ) and the sector in the region of
destination (𝑃𝑗𝑖2 ) represents interregional specialization; this data was obtained by
dividing the percentage of region j within the activity of sector i. The location coefficient
(Qij ) shows the proportion of each region within each sector and is a measure of
interregional sector distribution and absolute concentration; it is calculated based on the
share of sector i in region j and the share of the same sector in the national total. Finally,
the specialization coefficient (Qr) shows the degree of similarity of the regional economic
747
structure with the economic structure of the country and is used as a measure of regional
specialization(26).
𝑉𝑖𝑗 𝑉𝑖𝑗
𝑄𝑟 = 0.5 ∑ | −∑ | ; 0 ≥ 𝑄𝑟 ≤ 1
∑𝑖 𝑉𝑖𝑗 𝑗 ∑𝑖 ∑𝑗 𝑉𝑖𝑗
𝑖
For the analysis of the network, the information was organized in a matrix format (𝑋𝑖𝑗 ).
The rows correspond to the mobilized livestock by origin (Xi ), and the columns, to those
received by destination (Xj ). The main diagonal of the matrix was also considered because
it represents mobilization within a state, or intrastate (Xii ). The matrix elements were
transformed to binary form, assigning a value of 1 to livestock mobilization (Xij >0) and
of 0 to an absence of mobilization (Xij =0). A total of seven networks were analyzed, one
for all the purposes of livestock movement and one for each purpose. Likewise, all
analyses were performed for the period from 2017 to 2021.
The method used was Social Network Analysis (SNA)(25). The total structure and purpose
of livestock movement were analyzed using measures of density and centrality. Density
is a measure of cohesion among the elements of a network(27), and centrality measures the
importance of a particular element in the network(25).
The degree of centralization of the network measures the number of livestock movements
from origin to destination (𝐺𝐶𝑖𝑗 = ∑𝑖,𝑗 𝐺𝐶𝑖𝑗 = ∑𝑗,𝑖 𝑋𝑗𝑖 ); density measures the number of
livestock movements carried out divided by the number of possible movements (𝐷𝑖𝑗 =
𝑁𝑖𝑗 /𝑁); the outbound grade measures the number of connections between each source
and destination (Gi = ∑i Xij ), and the degree of entry measures the number of connections
between each destination and origin (Gj = ∑j Xji ). The eigenvector centralization measures
the qualitative aspect of a vertex's connections, based on the premise that connections to
more influential vertices are more important than connections to less influential vertices,
and it also considers the centrality of neighbors.
The eigenvector centrality measures the influence of a node on the network, assigning a
relative score to each node based on the principle that the links of important nodes
(measured by the degree of centrality) are worth more than the links of unimportant
nodes(8).
Homophily is an intrastate measure of livestock and is calculated based on the sum of the
elements of the main diagonal of the matrix (𝑇𝑟 = ∑ 𝑥𝑖𝑖 ). Homophily is the tendency of
states and municipalities to form groups for the purpose of selling or buying cattle.
Finally, social capital (SC) is a measure of social relationships and can represent an
advantage created by the location of a person with a relationship structure; it can take
three forms: 1) Obligations and expectations, 2) Information channels, and 3) Social
norms(28). Likewise, social capital consists of the information and reciprocity resources
that individuals can obtain from the structure of social networks(29). The social capital was
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estimated through the closure measure, which quantifies the preference of the origin for
a specific destination, that is, a particular origin always prefers a particular destination
and vice versa.
Results
The exploratory analysis made it possible to identify the dynamics of live cattle
movement in Mexico; mapping its geographical distribution was the basis for showing
the state structure during the 2017-2021 period. At the national level, an annual average
of 8.9 ± 0.3 million livestock heads were moved for all reasons, with an average annual
increase of 3.4 %.
The structure of the livestock moved consisted of six markets: slaughter, fattening, beef
breeding, reproduction, fairs, and entertainment. The two most important markets were
slaughter and fattening, with 53.5 % and 44.35 % of the cattle moved, respectively; the
remaining markets represented 1.1 % for slaughter, 0.5 % for beef breeding, 0.3 % for
fairs, and 0.2 % for entertainment. In addition, the structure by sex was higher in males
(65.8 %) than in females (34.2 %); however, during the period of analysis, the
mobilization of males decreased 16.9 % in that period (72.6 % in 2017 to 60.3 % in 2021),
and in females, it increased 44.9 % (27.4 % in 2017 to 39.7 % in 2021).
The proportion of cattle in intrastate markets was lower (42.2 %) than in interstate markets
(57.9 %). However, intrastate participation by market was 30.9 %; it was higher for the
slaughter market (71.8 %) and lower for the entertainment market (5.8 %). By state, the
most important intrastate markets were San Luis Potosí (7.2 %), Veracruz (5.7 %), and
Durango (5.2 %), and the interstate markets were Chiapas-San Luis Potosí (33 %),
Chiapas-Querétaro (2.3 %), and Chiapas-Veracruz (1.7 %).
Supply
The economic structure of livestock movement could be explained through measures of

interregional specialization. The average market specialization (0.39) was higher than for
the regions (0.33). The markets for entertainment and slaughter were the most and least
specialized, with values of 0.57 and 0.28, respectively; by state, Mexico City and
Aguascalientes were the most and least specialized, with 0.99 and 0.01, respectively. By
state, in one market 25.0 %, in two markets 18.8 %, in three markets 34.4 %, in four
markets 15.6 %, in five markets 6.3 % and none in the six markets. The specialization
rate by market was 43.8 % for the states specialized in slaughter, 46.9 % for those
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specialized in fattening, 43.8 % for beef breeding, 50.0 % for reproduction, 34.4 % for
fairs, and 40.6 % for entertainment (Figure 1).
Figure 1: Geographic specialization of bovine livestock supply in Mexico, 2017-2021
Demand
In the economic structure of demand, none of the states specialized in any of the six
purposes. The average specialization by purpose was higher than the specialization by
state, 0.40 and 0.27, respectively. The fair market was the most specialized (0.58), while
the market for slaughter was the least specialized (0.11). Specialization by state shows a
specialization rate of 15.6 % in one market, of 18.8 % in two markets, of 6.3 % in three
markets, of 43.8 % in five markets 43.8 %, and of 0 % in all six markets. Specialization
by market shows that 71.9 % of the states specialized in cattle for breeding, 65.6 % in
entertainment, 62.5 % in fairs, 62.5 % in beef breeding, 46.9 % in slaughter, and 43.8 %
in fattening (Figure 2).
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Figure 2: Geographic specialization of the cattle demand in Mexico, 2017-2021
Network analysis
The municipal cattle movement network in Mexico was composed of 1,374 source
municipalities (red points), 1,842 municipalities of destination (blue points), and 39,068
commercial links (edges). The measures of the structure of the entire network were low;
the density was 0.04, and the average grade, 52.4. However, the measures of
centralization were high: the degree of centralization was 0.34, for both outward
centralization and inward centrality. The average network density and degree measures
by market were lower than for the entire network; however, the centrality measures were
higher, amounting to twice as much in the market for shows (Figure 3).
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Figure 3: Mexico’s municipal network for the mobilization of live cattle, 2017-2021
The state cattle movement network in Mexico consisted of 32 states of origin (red nodes),
32 states of destination (blue nodes), as well as of 856 commercial relationships out of
1,024 potential ones. The average density and degree measures for the entire network
were high, 0.84 and 25.9, respectively; however, the centrality measures were low: the
degree of centrality was 0.02; the output centrality, 0.17, and the input centrality, 0.14
(Figure 4).
Figure 4: Mexico's state network for the mobilization of live cattle, 2017-2021
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The market nets had low average density and degree measures relative to the total net, of
0.50 and 15.0, respectively; the highest average density and degree values were for the
fattening and breeding nets (0.60 and 18.6), and the lowest one was for fairs (0.38 and
11.8). However, the degree of market centrality was 11.5 times higher than for the entire
network, and the origin and destination centralities were 2.5 times higher.
A measure of centrality that considers the relative importance of the network elements is
the eigenvector. The total network had an eigenvalue of 0.18, which is higher than that of
the markets (Figure 5). In the national network, the first eigenvector accounted for
80.8 % of the variation of all markets. In the total network, 28.1 % of the states had the
maximum eigenvalue (0.19). Jalisco was the most important state in market networks for
slaughter (0.24), fattening (0.21), beef breeding (0.24), reproduction (0.22), and fairs
(0.28), and the states of Michoacán and San Luis Potosí, for entertainment (0.28) (0.24).
Figure 5: Eigenvector by market network 2017-2021
Finally, measures of homophily and social capital (closure) robustly support the structure
of the cattle network in Mexico. The average homophily for the whole network was higher
(0.69) than per market (0.31), with the highest homophily for reproduction and the lowest
for beef breeding. Likewise, the average network social capital for all markets was higher
(0.90) than per market (0.71). The highest capital stock was in the fattening market
network (0.79), and the lowest, in the fairs (0.68).
Discussion
Mexico’s cattle market moved on average one third of the national inventory annually.
slaughter and feedlot markets accounted for the largest proportion of livestock moved,
breeding and finishing activities are carried out in the same production unit, but not for
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the fair, breeding and show markets (Table 1). Given that the slaughter and fattening
markets accounted for the largest proportion of livestock moved, beef breeding and
reproduction activities are carried out in the same production unit, unlike for the fair,
reproduction, and entertainment markets.
Table 1: Main cattle supply and demand states in Mexico, 2017-2021 (%)
State S1 D1a S2 D2a S3 D3a S4 D4a S5 D5a S6 D6a
Chiapas 0.9 0.7 28.6 1.3 20.1 17.4 10.8 10.1 24.2 24.4 1.5 1.5
Coahuila 4.3 4.4 3.6 3.1 7.0 18.4 2.6 2.7 0.8 0.4 0.5 1.3
Durango 13.7 10.2 1.8 15.7 8.6 11.9 1.9 3.3 1.1 0.8 0.3 2.0
Guerrero 0.1 0.1 3.4 0.0 0.3 0.3 1.7 2.1 0.7 0.3 22.5 2.5
Jalisco 4.5 1.3 6.5 3.0 4.4 6.5 9.5 9.0 10.3 9.5 7.8 9.4
State of Mexico 0.3 9.5 0.2 1.8 0.5 0.9 0.2 1.5 0.2 0.1 4.8 10.0
Michoacán 7.5 11.4 1.5 5.9 0.7 1.2 2.3 5.2 1.1 3.8 4.5 9.7
Nuevo León 7.9 8.8 2.1 8.6 1.9 3.3 10.2 4.1 12.3 8.9 0.7 1.8
San Luis Potosí 13.8 13.8 1.7 15.3 1.2 0.7 0.9 1.6 3.0 4.0 9.9 2.5
Tabasco 0.3 0.3 7.8 0.4 9.4 9.0 7.4 8.1 8.1 7.0 0.4 0.3
Tamaulipas 2.1 1.9 1.7 2.1 3.5 2.6 10.9 6.0 10.9 8.6 1.0 0.6
Tlaxcala 0.0 0.1 0.0 0.1 0.1 0.2 0.0 0.3 0.0 0.0 16.4 1.1
Veracruz 9.1 7.4 14.5 10.9 5.6 5.4 8.9 10.9 9.4 8.1 0.8 2.9
1= slaughtering supply 1ª= slaughtering demand, 2= fattening supply, 2a= fattening demand, 3= beef
breeding supply, 3a= beef breeding demand, 4= reproduction supply, 4a= reproduction demand, 5=
supply for fairs and shows, 5a= demand for fairs and shows, 6= entertainment supply, 6a= entertainment
demand.
Productive resources and fuel costs have allowed for greater market specialization; in
2021, the cost per kilometer traveled for land transportation was 0.52 US$ km-1 and
represented 43.8 % of the total cost(30). The southeastern states of the country have
specialized in the breeding and grazing markets due to the relative abundance of climate,
land, water and forage; Mexico's humid tropics are characterized by rainfall of up to 1,300
mm per year (Jaramillo, 1994, cited in Enríquez-Quiroz et al., 2021)(31), allowing a
maximum of 1.79 UA ha-1(32). The northern and central states specialize in fattening and
slaughtering to supply the large meat consumer markets of the central metropolis, and the
central markets, in fairs and entertainment events that are important for regional cultures.
The analysis of the regional economic structure and by livestock market in Mexico
indicates that, on average, livestock markets and regions in Mexico have a low level of
specialization, since both measures are less than 40 %. The most specialized markets
(fairs and entertainment) are related to the supply of fighting bulls and rodeo, while the
least specialized is related to the slaughter of cull animals (cows and bulls). The
specialization of cattle breeding in the northern states of the country is in the production
of calves for export; Chihuahua's cattle breeding activity is oriented to the export of
calves(33).
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The measure of average specialization by market and by state indicates that cattle farming
in Mexico has a low level of specialization, although it was higher by market than by
state. The average supply specialization for the states was not statistically different from
the average demand specialization for the states (P>005); the same was true for the
average market specialization (P>005). However, it was found that there are different
degrees of specialization in the markets; it was higher in the entertainment market and
lower in the slaughter market. Low specialization is explained by the diversity of
production scales, resource allocation, and reduced knowledge of the markets by the
suppliers (producers) and demanders (consumers).
Given the large number of municipalities and the dispersion and distance between these,
the average degree and centrality of the municipal livestock network in Mexico are
considered very low. A higher proportion of Mexico's municipalities participate in
livestock markets; 56.2 % of the municipalities participated in the markets of origin, and
73.3 %, in the markets of destination. 41.4 % of the municipalities participated in the
slaughter market of origin, and 58.9 %, in the destination market for breeding. The market
with the lowest participation of source municipalities was entertainment (16.4 %), and in
the destination municipalities, it was the fairs market (23.2 %).
The cost of transporting livestock between municipalities is high. The distance between
the two most important municipalities in the mobilization of livestock (Ezequiel Montes
Querétaro and La Paz Estado de México) is 224.9 km, but the distance to the second
market (Benemérito de las Américas, Chiapas, and Tamuín, San Luis Potosí) is 1,341.9
km, and the longest distance was from Matamoros, Coahuila, to Mexicali, Northern Baja
California, of 1,714 km. Livestock mobilization occurs in all Mexican states, but its
importance differs by market of origin and destination; the southeastern states were the
main origin of cattle for the reproduction, beef breeding, and fattening markets; the
northern markets are the main destination of cattle for fattening (Table 1).
Density is a measure of network connectedness and social capital. High density in the
national network is associated with the number of markets (six), the number of
slaughterhouses (1,175), and the availability of resources, while low centrality is
associated with low scales of production. The density by market is lower than for the
entire network due to the specialization of both origins and destinations, while the
centrality by market is higher because it is associated with the preference or social capital
of origins and destinations. The states obtain market information for an average of 26.8±6
states, but these represent only 3.1 % of the cattle moved.
The eigenvector is a measure of network centrality; the network pattern is represented by

80.8 % of the states. The eigenvalue of the states of origin and destination indicate that
the national cattle market in Mexico has a high stability. The degree of inequality of the
states is only 1.8 % of the potential maximum.
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The most important source states are related to the most important destination states, and
vice versa, while individual markets have 3.3 times more instability. The breeding and
fair markets had the highest and lowest stability, representing 2.3 and 4.2 times the
national stability. Special cases are weaned calves; stabilizing their replacement is the
basis for stabilizing the beef breeding, fattening, and slaughter markets. The instability of
the fairs is more associated with the economic stability of the country and events such as
the Covid-19 pandemic in 2021.
This research is the first in Mexico to analyze the structure of the six cattle markets at the
municipal level. Maps of specialization of supply and demand were constructed, the
economic index of specialization was estimated, the graph of the national livestock
movement network by market was presented, and measures of density and centrality of
the network were estimated. By knowing the origin, destination and quantity of livestock
moved in the country, it is possible to establish a system of sanitary surveillance and
registration of market information to improve the productivity of cattle production and
marketing systems in Mexico. The cattle market in Mexico is important because it
mobilizes on average more than one third of the national inventory, mainly for slaughter
and fattening. However, the markets present a low specialization due to variables such as
the large number of municipalities in the country. As a result, only a fourth of the states
of origin and a seventh of the states of destination specialize in the fattening and slaughter
markets. Likewise, the national mobilization structure presents a high degree of density,
with a low degree of centrality; whereas, by market, the density is lower, but with higher
centrality. Therefore, with homophily representing almost half of the market, the social
capital is high. These aspects allow an average of 1.2 states to connect to the entire
national network, and up to 1.7 states, to the network by purpose.
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Article
Environmental outlook to 2030 in cow's milk production systems in

Mexico
María del Rosario Villavicencio-Gutiérrez a
Nicolás Callejas-Juárez b
Nathaniel Alec Rogers-Montoya c
Vianey González-Hernández a
Rodrigo González-López d
Carlos Galdino Martínez-García a
Francisco Ernesto Martínez-Castañeda a*
a
Universidad Autónoma del Estado de México. Instituto de Ciencias Agropecuarias y
Rurales. Instituto Literario 100. Centro, 50000, Toluca, Estado de México. México.
b
Universidad Autónoma de Chihuahua. Facultad de Zootecnia y Ecología. Chihuahua,
México.
c
Colegio de Postgraduados. Ganadería. Estado de México, México.
d
Universidad Nacional Autónoma de México. Facultad de Medicina Veterinaria y
Zootecnia. Ciudad de México, México.
* Corresponding author: femartinezc@uaemex.mx
Abstract:
The objective of this study was to evaluate the environmental performance of cow milk
production in small and medium scale systems in Mexico, through life cycle analysis with a
cradle to farm gate approach, for the period 2021-2030. The established functional unit was
1 kg of milk corrected for fat and protein. The impact assessment was carried out with the
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OpenLCA 1.11.0 software, using the ReCiPe method, considering seven impact categories:
agricultural land occupation (ALO), marine ecotoxicity (ME), human toxicity (HT), climate
change (CC), fossil depletion (FD), soil acidification (SA), and water depletion (WD).
Among the main results of the research, the production of cattle feed was identified as the
chief contributor to environmental loads in most of the categories with percentages above
71 %, while on-farm emissions contribute to the environmental loads for the CC (28 %), FD
(26 %) and SA (59 %) categories. A comparison was made between pessimistic, base and
optimistic scenarios for the years 2021 and 2030, which confirmed an improvement in
environmental efficiency in the optimistic scenario, the increase in production volume
represents a decrease of 6 % and 5 %, respectively, in the assessed impact categories.
Keywords: Life-cycle analysis, Environmental impact, Sustainability.
Introduction
Worldwide milk production involves approximately 150 million households. In developing

countries, milk production by smallholder farmers is an important source of both nutrition
and income for millions of households(1). According to FAO, between 80 and 90 % of milk
production in developing countries is carried out in small-scale production systems(2).
Mexico is one of the developing countries with a long tradition of dairy production, ranking
15th in the world among milk-producing countries(3).
In Mexico, cow's milk is the third most important livestock product in terms of the economy;
in 2021, its output closed with a volume of 12,852 million liters and an economic value of
90,823 million pesos(3). 85 % of the dairy herd corresponds to the semi-intensive family
system(4). This type of system is characterized mainly by having a Holstein cattle herd whose
diet includes rainfed forage crops (corn, oats, wheat, triticale, barley, rye, rye grass, and
native and introduced grasses), legumes (alfalfa, vetch, and chickpeas), and residues from
agricultural plots(5).
Semi-intensive family livestock farming is recognized for its socioeconomic importance;
however, this activity faces different problems, including low yields in milk production,
derived from factors such as genetics, environment, diet(6) and climate change. The lack or
excess of rainfall and extreme temperatures(7) cause a decrease in agricultural production and,
therefore, insufficient conditions to maintain livestock production(8).
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On the other hand, Mexican cattle ranching is associated with the generation of 13.2 % of
GHG emissions in the country, which in 2019 amounted to 736.6 million t of CO₂
equivalent(9); it is also associated with the degradation of natural resources. The generation
of GHG emissions is attributed to low milk yields, inefficient management and feeding
practices, and an older age at the first calving(10). Environmental issues have increased the
interest in identifying mitigation alternatives in different scenarios and productive systems.
Baldini et al(11) highlight Life-Cycle Analysis as a method to identify and evaluate the
environmental burdens associated with milk production.
Environmental impact assessment of milk has been carried out for intensive(12-15) and semi-
intensive production systems(15-18). Some authors indicate that in order to reduce emissions
from milk production on small-scale farms it is necessary to produce milk at a larger
scale(17,19,20).
The objective of this study was to evaluate the environmental performance of cow's milk
production in a semi-intensive system in Mexico through life-cycle analysis with a cradle-
to-gate approach, for the period 2021-2030.
This study was conducted using the Life-Cycle Analysis (LCA) methodology, in compliance
with the principles established by ISO 14040 and 14044(21,22), which integrates four phases:
definition of objectives and scope; inventory analysis; impact assessment, and interpretation
of results.
Product system
The system under study corresponds to milk production in small and medium scale farms in
Mexico, considered as a semi-intensive system whose production inventory amounts to
85 % of the national total(23,24). It is a very heterogeneous production system with respect to
its technological, agroecological, and socioeconomic level(25). Small and medium-scale dairy
farming is characterized by a small number of animals in the production units(26). Out of a
total of 257 thousand small- and medium-scale producers, 47.30 % have 30 cows or less(27),
the milk-producing breeds are mainly Holstein, and the milking is done manually(28).
In 2021, the semi-intensive production system had an inventory of 2’579,223 heads of dairy
cattle and an output volume of 11’046,795.96 liters of fluid milk. The annual productivity
per cow was 4,017 liters, i.e., 13.17 liters per day(29).
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Definition of objectives and scope
The objective of this study was to evaluate the environmental performance of cow’s milk
production in a semi-intensive family system in Mexico in the year 2030. The functional unit
was 1 kg of milk adjusted for fat and protein (MAFP). According to the International Dairy
Federation (IDF), the use of the MAFP unit ensures a fair comparison with different breeds
or feeding regimes(30). The weight of raw milk was converted to MAFP using the following
equation:
MAPF (kg/year) = Output (kg/year) * [0.1226 fat% +0.0776 protein%+ 0.2534]

The fat and protein contents were established considering the average of the values
established in the book Bovine milk production in the family system in Mexico ("Producción
de leche de bovino en el sistema familiar en México"), being 4.5 % and 3.5 %, respectively(5).
System limits
System boundaries were established considering a cradle-to-gate approach (Figure 1), i.e.,
from the extraction of raw materials used in cattle feed until the milk is ready to leave the
farm. The system considered two main sub-systems: 1) Cow feed production: considers the
activities and processes related to the cultivation of fodder crops and legumes. 2) Milk
production: considers the activities of transporting feed to the farm and feeding the cows for
305 d corresponding to the milking period(31).
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Figure 1: Outline of the limits of the milk production system
Inventory analysis
The study considered data from secondary sources of information. The volume of production
and the inventory of heads at the national level were obtained from the database of the Agri-
Food Information System for Consultation (Sistema de Información Agroalimentaria de
Consulta)(29). Inputs used in livestock feed were obtained from scientific books of the
National Institute for Research on Forestry, Agriculture, and Livestock (Instituto Nacional
de Investigaciones Forestales, Agrícolas y Pecuarias)(5,6).
In order to determine the milk production per cow per year, the values corresponding to the
volume of national production and the national cattle head inventory were estimated(29). The
volume of fluid milk output (thousands of liters) in Mexico was forecast for the period 2021-
2030, using the univariate statistical method of series (ARIMA)(32). Statistical tests, model
estimation and forecasts were performed with the Simetar® software.
The probability distribution was used as a risk factor to determine the minimum and
maximum of the milk volume and price series. These confidence intervals were used to
construct the pessimistic (lower interval), baseline (mean), and optimistic (upper interval)
scenarios. Milk volumes were calculated by production system (barns with an average of 8
cows); the volume of production per cow per day and per year was calculated considering a
305-d lactation period (Table 1).
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Table 1: Production parameters for the semi-intensive system in the pessimistic, baseline
and optimistic scenarios
2021 2030
Pessimistic Baseline Optimistic Pessimistic Baseline Optimistic
Liters of cow’s
3,745 4,017 4,267 3,724 3,944 4,166
milk/305 d
Liters of cow’s
12.28 13.17 13.99 12.21 12.93 13.66
milk/d
Kg of cow’s
milk/305 d 4,033 4,325 4,594 4,010 4,246 4,486
(MAFP)
Kg of cow’s milk/d
13.22 14.18 15.06 13.16 13.92 14.71
(MAFP)
MAFP= milk adjusted for fat and protein.
Table 2 shows the various proportions of the dietary ingredients used in the feeding of the
cows.
Table 2: Main ingredients of the diet used for feeding cows in a semi-intensive system
Average ingredients per year
Inputs %
(kg)
Corn silage 65,450 25.81
Hay alfalfa 212 0.08
Corn stubble 34,277 13.52
Green alfalfa 133,057 52.47
Fodder corn 2,399 0.95
Other grains 1,938 0.76
Concentrate 01 5,021 1.98
Agricultural residues 8,445 3.33
Protein 10 2,780 1.10
Total 253,579 100.00
The inventory for the production of 1 kg of milk – MAFP was integrated according to the
above information. (Table 3).
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Table 3: Inventory per 1 kg of milk -MAFP produced in semi-intensive system

2021 2030
scenario scenario scenario scenario scenario scenario
S1
Inputs
Corn silage 1.96251 1.82989 1.72263 1.97376 1.86385 1.76424
Hay alfalfa 0.00636 0.00593 0.00558 0.00639 0.00604 0.00571
Corn stubble 1.02778 0.95832 0.90215 1.03367 0.97611 0.92395
Green alfalfa 3.98968 3.72007 3.50202 4.01255 3.78912 3.58662
Fodder corn 0.07194 0.06708 0.06315 0.07235 0.06832 0.06467
Other grains 0.05812 0.05419 0.05102 0.05846 0.05520 0.05225
Concentrate 01 0.15055 0.14038 0.13215 0.15142 0.14299 0.13534
Agricultural residues 0.25321 0.23610 0.22226 0.25466 0.24048 0.22763
Protein 10 0.08337 0.07773 0.07318 0.08385 0.07918 0.07495
Outputs
Total, feed 7.60352 7.08969 6.67414 7.64711 7.22129 6.83538
S2
Inputs
Land occupancy (stable) 0.00216 0.00201 0.00190 0.00217 0.00205 0.00194
Fuel 0.01126 0.01050 0.00989 0.01133 0.01070 0.01012
Electricity 0.00733 0.00683 0.00643 0.00737 0.00696 0.00659
Water 7.56322 7.05212 6.63877 7.60658 7.18301 6.79915
Feed 7.60352 7.08969 6.67414 7.64711 7.22129 6.83538
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Outputs
Ammonia 0.00991898 0.00924868 0.00870658 0.00997585 0.00942035 0.00891692
Methane from manure
management 0.01835012 0.01711005 0.01610718 0.01845532 0.01742764 0.01649630
Methane by enteric
fermentation 0.02454948 0.02289048 0.02154879 0.02469022 0.02331536 0.02206937
Nitrogen 0.00010911 0.00010174 0.00009577 0.00010973 0.00010362 0.00009809
Nitrous oxide 0.00000248 0.00000231 0.00000218 0.00000249 0.00000236 0.00000223
S1= food production subsystem, S2= milk production subsystem.
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The inventory for S1 was integrated considering the feed supplied to the cows: corn silage,
hay alfalfa, corn stubble, green alfalfa, fodder corn, concentrate 01, agricultural residues and
protein 10. The data were obtained from the Agricultural and Food Database
(AGRIBALYSE), Environment and Energy Management Agency (Agribusiness)(33).
The inventory for S2 was integrated considering:
Occupation of barn floor: bovine milk production in a semi-intensive system is regularly

carried out in individual cubicles with free access in a paved corral; according to the manual
of good livestock practices in bovine milk production units(34), under these conditions, dairy
cattle require a surface area of 9 m2/cow.
Fuel consumption: the number of liters of fuel per kg of MAFP produced was calculated
based on the type of vehicle required to transport the ingredients, the fuel efficiency
expressed in km/liter, and the load capacity, adjusted for the functional unit.
Electricity consumption: in small and medium-scale production systems, the milking is
done manually; therefore, only artificial lighting of the barn was considered(34), and
adjustment was made for the number of days (305) that the cows remain in lactation.
Water consumption: water consumption per cow was estimated for the lactation period (305
days); lactating cows consumed an average of 110 L of water per day(35).
Methane (CH4) emissions from manure fermentation and manure management, and nitrogen
(N) and nitrous oxide (N2O) emissions from manure management were estimated using the
emission factors established for Mexico by the National Institute of Ecology and Climate
Change (Instituto Nacional de Ecología y Cambio Climático)(36).
Environmental impact assessment
The impact assessment was performed in OpenLCA V.1.11.0 software, using the ReCiPe
2008 method. Seven midpoint categories were considered for this study: agricultural land
occupancy (ALO), climate change (CC), fossil depletion (FD), human toxicity (HT), marine
ecotoxicity (ME), soil acidification (SA), and water depletion (WD)(37). These categories
were selected for having the highest relative contribution of environmental impacts and for
the frequency of their use in the literature on similar researches.
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Results
Environmental impact assessment
The results of the characterization for the baseline scenario identified seven categories (ALO,
CC, FD, HT, ME, SA, and WD) as the main contributors to the environmental impacts in the
production of 1 kg of MAFP. Table 4 shows that the food production subsystem (S1) is
responsible for most of the total impact, with percentages of over 71 % in the following
categories: CC, FD, HT, WD, ME, and ALO. While the milk production subsystem (S2)
contributes to environmental loads in the categories SA (58.94 %), CC (28.16 %), and FD
(25.58 %).
Table 4: Midpoint impacts for 1 kg MAFP-Baseline Scenario 2021

Category S1 S2 Total Unit
2
ALO 6.13494 0.00219 6.13713 m *a
CC 0.65082 0.25508 0.90590 kg CO2 eq
FD 0.03501 0.01203 0.04704 kg oil eq
HT 1.59909 0.05472 1.65381 kg 1,4-DB eq
ME 1.72146 0.01833 1.73979 kg 1,4-DB eq
SA 0.01867 0.02680 0.04547 kg SO2 eq
WD 0.04327 0.00131 0.04458 m3
S1= food production subsystem, S2= milk production subsystem, ALO= agricultural land occupation, CC=
climate change, FD= fossil depletion, HT= human toxicity, ME= marine ecotoxicity, SA= soil acidification,
WD= water depletion.
The processes involved in the production of 1 kg MAFP involved in the generation of

environmental loads are presented in Figure 2. In subsystem 1, the main contributors are:
concentrate 01 production in categories HT (60.83 %), ME (44.12 %), FD (37.78 %) and CC
(17.35 %), corn silage production in categories SA (28.03 %), WD (26.74 %), ALO
(26.74 %), CC (17. 94 %) and ME (16.73 %), protein 10 production in categories HT
(37.63 %), ME (28.10 %) and FD (26.79 %), forage corn production in category WD
(67.79 %) and green alfalfa production in categories ALO (62.68 %) and CC (18.42 %).
In subsystem 2, the environmental load of the production of 1 kg MAFP, is derived from

livestock rearing mainly for categories TA (58.94 %) and CC (26.61 %) and from the
transport of inputs to the farm, specifically in category FD (25.58 %).
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Figure 2: Contribution of the processes involved to the different impact categories

100% 3.81%
4.26%
26.61% 25.58%
28.10%
Relative contribution (% )
80%
37.63% 58.94%
9.60%
26.79% 67.79%
60% 62.68%
17.35%
44.12%
40% 2.67%
3.78%
18.42% 60.83%
37.78%
20% 5.01% 4.18%
26.74% 28.84% 28.03%
17.94% 4.72% 16.73%
0% 4.66% 4.31%
-6.97%
-20%
ALO CC FD HT ME TA WD
Corn silage production Hay alfalfa production Corn stubble production

Green alfalfa production Fodder corn production Other grains production
Concentrate 01 production Agricultural residues production Protein 10 production
Transport Cattle breeding
Comparative analysis of scenarios for the production of 1 kg MAFP
Figure 3 shows the comparison of the environmental results in the baseline, optimistic and
pessimistic scenarios for the seven impact categories for the years 2021 and 2030.
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Figure 3: Comparative analysis of environmental loads between 2021 and 2030, for the
pessimistic, baseline, and optimistic scenarios.
Agricultural land occupancy (ALO) Climate change (CC)

6.800 0.990
6.600 0.960 0.972 0.977
6.582 6.620
6.400
0.930
kg CO₂ eq
6.200 6.251 0.923
m²*a
0.900 0.906
6.000 6.137
0.870
5.800 5.917 0.873
5.777 0.840 0.853
5.600
5.400 0.810
5.200 0.780
2021 2030 2021 2030 2021 2030 2021 2030 2021 2030 2021 2030
Fossil depletion (FD) Human toxicity (HT)

0.052 1.860
1.800
0.050 0.050 0.051
1.784
1.740 1.774
kg 1,4-DB eq
0.048 1.680
kg oil eq
0.048 1.685
0.046 0.047 1.620 1.654
0.045 1.560 1.594

0.044 1.557
0.044 1.500
0.042 1.440
1.380
0.040
2021 2030 2021 2030 2021 2030
2021 2030 2021 2030 2021 2030
Pessimistic Baseline Optimistic
Marine ecotoxicity (ME) Soil acidification (SA)

1.900 0.051
1.850 1.866
1.877 0.048 0.049 0.049
1.800
kg 1,4-DB eq
0.045 0.045
0.046
kg SO₂eq
1.750 1.772 0.044

1.740
0.042 0.043
1.700
0.039
1.650 1.677
1.600 1.638 0.036
1.550 0.033
1.500 0.030
2021 2030 2021 2030 2021 2030 2021 2030 2021 2030 2021 2030
Water depletion (WD

0.051
0.048
0.048 0.048
0.045
0.045
0.045
0.042 0.043
m³
0.042
0.039
0.036
0.033
0.030
2021 2030 2021 2030 2021 2030
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The comparative results between the pessimistic, baseline, and optimistic scenarios show
that, in 2021 and 2030, the optimistic scenario allows a reduction of 6 % and 5 %,
respectively, of emissions in all impact categories. This is due to the increase in production
volume and the improvement in production efficiency, which allows for a reduction in the
intensity of emissions(19).
For the years 2021 and 2030, in the ALO category, the optimistic scenario exhibited a
reduction of 0.36 and 0.33 m2 per kilo of MAFP, respectively. A pessimistic scenario implied
an increase of 0.44 and 0.37 m2 per kilo of MAGP.
In the CC category, the optimistic scenario allowed a decrease of 0.53 kg for 2021 and 0.49
kg CO2 eq per kg MAFP for 2030. On the other hand, the production of 1 kg of MAFP in a
pessimistic scenario implied an increase of 0.066 and 0.045 kg of CO2 eq per kg of milk,
respectively.
In the optimistic 2021 and 2030 scenarios, a decrease of 0.0027 and 0.0026 kg oil eq per kilo
of MAFP, respectively, was observed in the FD category. The pessimistic scenario showed
an increase of 0.0034 and 0.0028 kg oil eq per kilogram of MAFP.
For the year 2030, the HT and ME categories in the optimistic scenario predicted a reduction
of 0.059 and 0.095 kg 1,4-DB eq per kilo of MAFP, respectively, and in the pessimistic
scenario, an increase of 0.12 and 0.10 kg 1,4-DB eq per kilo of MAFP, respectively.
In the SA category, the optimistic scenario for the years 2021 and 2030 allowed a reduction
of 0.0027 and 0.0025 kg SO2 eq while the pessimistic scenario implied an increase of 0.0033
and 0.0027 kg SO2 eq per kg MAFP.
Finally, in the WD category for the years 2021 and 2030, there was a reduction of 0.0026
and 0.0024 m3 of water per kilo of MAFP, respectively, in the optimistic scenario, and an
increase of 0.0032 and 0.0027 m3 of water per kilo of MAFP, respectively, in the pessimistic
scenario.
Discussion
The characterization results of the baseline scenario for 1kg MAFP production in a semi-
intensive system show the highest environmental loads in the food production subsystem
with percentages of over 71 % in the categories agricultural land occupation (ALO), climate
change (CC), fossil depletion (FD), water depletion (WD), human toxicity (HT), and marine
ecotoxicity (ME). Similar results were found in the study by Carvalho et al(18), where crop
production for livestock feed was identified as one of the main contributors to the production
of 1 kg of MAFP in a semi-intensive system in Brazil, mainly for the categories of land
occupation, fossil resource depletion, water consumption, and soil acidification.
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Comparison with studies in intensive systems shows that food production also represents a
significant environmental impact(38).
Agricultural land occupancy
The environmental load of the production of 1 kg of MAFP in the ALO category was 6.14
m2 in its baseline 2021 scenario. The main contributors to this category are related to the
cultivation of green alfalfa (62.68 %), followed by corn silage (26.74 %). These results are
higher than the 1.89 m2/yr per kg of MAFP cited by Berton et al(16), where the land was
destined for agriculture that produces the inputs required for livestock feeding in traditional
small-scale systems in Italy, and also superior to the results presented by Xiaoquin et al(38),
where the production per kilo of MAFP requires occupancy of 1.16 m2 to 2.49 m2,
highlighting that 98 % corresponds to land occupation for feed production and 2 %
corresponds to stables. Rivera et al(39) reported an occupancy of 1.33 m2 per kilo of MAFP
in a conventional milk production system in Colombia.
However, the results of this study showed an occupancy below 8.8 to 11.2 m2 per kg of milk
in Ethiopia(40) and agree that fodder production on soils with low biomass yields is a
determinant for the contribution of impacts in the ALO category(18); thus, it is possible to use
2.25 m2 less soil in intensive systems than in less technified ones(41).
In the year 2021, the optimistic scenario presented a production of 4,594 kg of milk per cow
(15.06 kg/d), being the highest production of the compared scenarios, which meant the lowest
contribution in the ALO category with 5.78 m2, while in the pessimistic scenario milk
production decreased to 4,010 kg cow/yr (13.16 kg/d); this represented an increase in land
occupation of 6.62 m2 (Figure 3). An increase in milk production per area of agricultural land
is accompanied by an improvement in environmental efficiency(42).
Climate change
In the CC impact category, 0.85 kg CO2 eq was generated per 1 kg MAFP in the baseline
scenario. The main contributors for this category are related to cattle breeding (26 %),
followed by green alfalfa (18.32 %), corn silage (17.84 %), and concentrate 01 productions
(17.26 %) (Figure 2). Environmental burdens from livestock breeding are mainly attributed
to CH4 emissions from enteric fermentation and manure management, while environmental
burdens from forage and legume production are mainly related to N2O emissions generated
by the use of agrochemicals in agricultural practices.
The environmental impact of CC found in this study is below 1.42 kg CO2 eq per kilo of
MAFP in a semi-intensive system(18). Kim et al(43), compared small-scale (150 cows) and
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intensive (1,500 cows) systems and reported values of 1.22 and 0.98 kg CO2 eq, respectively,
demonstrating that feeding practices such as a reduction in the proportion of fodder to 50 %,
as well as the use of more digestible fodders, can help to improve the quality of the feed, and
that an increase in fat supplementation can reduce CC contributions by up to 7 %. With
respect to the production of concentrates(44), they point out that it is possible to relate this to
an increase in GHG generation by modeling a reduction in feed consumption, attributing the
increase to a diminution of the milk production volume per cow.
The values found in the different scenarios of the current study range from 0.853 kg CO2 eq
per kg MAFP for barns with a yield of 4,594 kg of milk per year (15.06 liters/d) in an
optimistic scenario, to 0.977 kg CO2 eq per kg MAFP for barns with a yield of 4,010 kg of
milk per year (13.16 liters/d), 0.788 kg CO2 eq per kg MAFP in a semi-stabled system in
Brazil with a yield of 6,335 kg milk(45), where lower CO2 eq. values may be associated with
higher levels of milk production per cow(18).
Fossil depletion
The environmental impact for the FD category was 0.48 kg oil eq. for 1 kg of MAFP; the
main contributions for the production of concentrate 01 (34.59 %) and protein 10 (24.52 %)
correspond to S1, while 25 % of the emissions are generated in the transportation of inputs
to the farm (Figure 2). This value is below the 4.82 kg oil eq(18), where the processes with the
greatest impact were corn silage production (45.7 %), pasture production (34.3 %), and
transportation of inputs to the farm (10 %). Ferreira et al(46) point out the importance of
knowing the origin of inputs in the supply chain in order to reduce transportation impacts.
The values found in the different scenarios considered in this study range from 0.044 kg oil
eq, in an optimistic 2021 scenario, to 0.051 kg oil eq, in a pessimistic 2030 scenario (Figure
3); these variations correspond to the increase or decrease in milk productivity per cow.
Soil acidification
For the SA category, 1 kg of MAFP generated a total of 0.043 kg SO2 eq in its 2021 baseline
scenario. The main generator of emissions for this category is livestock farming (58.75 %),
followed by corn silage production (28.83 %) and emissions generated by livestock farming
from the volatization of nitrogen in the form of ammonia (NH3) (Figure 2). Emissions from
corn silage production are NH3 and N2O.
The total emissions generated in this category are higher than the 0.001 kg SO2 eq.(18) and
the 0.020 kg SO2 eq. attributable mostly to emissions from manure management and nitrogen
fertilizer use reported(43). In both studies, corn silage was one of the main contributors to
emissions generation for this impact category. The specialized literature has shown how
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reliance on commercial concentrates can result in the contamination of soils and water bodies
by excess nutrients, in addition to competing directly with the production of other foods for
human consumption.
The optimistic 2021 scenario presents the lowest value in the SA category with 0.043 kg SO2
while the pessimistic 2021 and 2030 scenario presents the highest value with 0.049 kg SO2;
the increase in production volume allows the reduction of the environmental impact in the
SA category.
Water depletion
Water depletion per 1 kg MAFP was 0.04225m3 in its 2021 baseline scenario; the main
consumption was for the production of fodder corn (67.08 %) and corn silage (27.74 %).
Water consumption in this study is slightly above 0.00587 m3(18); similarly, in this study, the
highest water consumption was in corn crops. However, there is high variability in the WD
category with consumptions from 0.02800 m3 to 0.09900 m3, so that, as farm size increases,
water consumption decreases, because the largest water footprint of milk production
corresponds to the cultivation of fodder crops to sustain smaller scale production systems(41).
The values presented in the comparative scenarios (Figure 3) range from 0.042 m3 of water
for the optimistic scenario to 0.048 m3 for the pessimistic scenario. Water is an essential input
for the cleaning and consumption of animals(47), although there is no way to reduce the water
intake, as an animal’s physiological requirements and milk production influence its
consumption; proper water management is an adequate alternative to minimize losses of this
vital liquid.
Human toxicity and marine ecotoxicity
Toxicity-related categories presented values of 1.5 kg 1,4-DB eq for HT and 1.64 kg 1,4-DB
eq for ME. Although these values are not generally considered in the literature, since there
are no comparative reference data, in this study they represent an important relative
contribution to the environmental loads; the main contributors are the production of
concentrate 01 and protein 10 (Figure 2), with percentages of 60.83 % and 37.63 % for HT,
and 44.12 % and 28.10 for ME. The values presented in the comparative scenarios (Figure
3) are the lowest in the optimistic 2021 scenario (1.56 kg 1,4-DB eq for HT and 1.64 kg 1,4-
DB eq for ME), while the highest value is presented for the pessimistic 2030 scenario (1.78
kg 1,4-DB eq for HT and 1.88 kg 1,4-DB eq for ME).
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Environmental impact mitigation strategies
The results of the evaluated scenarios provide a great opportunity for action to position dairy
farming in a positive scenario; the increase in production volumes was observed to be
accompanied by a decrease in environmental loads. One strategy to improve the
environmental performance of semi-technified milk production systems is to improve the
productivity per lactating cow(18); this would allow the mitigation of the environmental
impacts without reducing the milk production. It is possible to increase the production
volume by increasing efficiency with fewer cows. This implies not only an environmental
benefit but also an economic and social benefit that allows progress towards the sustainability
of milk production systems.
This study identified the main processes that contribute to the generation of environmental
impacts, first of all, agricultural activities related to crop cultivation required for livestock
feeding and manure management. This gives way to the implementation of comprehensive
strategies such as the transition to a circular economy through regenerative processes to
eliminate losses and waste throughout the biological cycle. As an opportunity to close the
cycle, the feces and urine of livestock can be used as natural fertilizer; the use of good
management practices and with the corresponding monitoring, can contribute to soil health
and reduce CH4 emissions to the atmosphere(48).
The food production subsystem is the main contributor to the generation of environmental
loads in the ALO, CC, FD, HT, ME, and WD categories. For the ALO category, the input
that used the largest amount of soil was alfalfa. For the categories CC, FD, HT, and ME, the
inputs with the highest contribution to emissions generation were concentrate 01, protein 10,
and corn silage. In the WD category, the greatest impact is attributed to the forage corn crop.
Animal husbandry has its largest contribution to SA, CC, and FD categories; enteric
fermentation processes and manure management contribute to the generation of emissions
such as CH4 and NH3. The comparative scenarios confirm that the increase in production
volume represents a decrease of 5 % and 6 % for the years 2021 and 2030, respectively, in
the impact categories evaluated. Therefore, the improvement of productive efficiency per
lactating cow is one of the main goals to be established.
Acknowledgments
The authors wish to thank the National Council for Humanities, Science, and Technology of
Mexico (Consejo Nacional de Humanidades, Ciencia y Tecnología, CONAHCYT) for
project F003-320069, assigned to Francisco Ernesto Martínez Castañeda.
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Conflict of interest
The authors declare that they have no competing financial interests or known personal
relationships that might have influenced the work reported in this paper.
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Article
Assessment of antibiotic resistance in fecal samples from calves with

diarrhea in the Cajamarca region, Peru
Marco Antonio Cabrera González a
Héctor Vladimir Vásquez Pérez b
Carlos Quilcate-Pairazamán b
José Bazán-Arce a
Medali Cueva-Rodríguez a*
a
Instituto Nacional de Innovación Agraria (INIA). Estación Experimental Baños del Inca.
Dirección de Desarrollo Tecnológico Agrario, Jr. s/n Wiracocha, Baños del Inca,
Cajamarca 06004, Perú.
b
INIA. Dirección de Desarrollo Tecnológico Agrario, La Molina. Lima. Perú.
*Corresponding author: mcuevar@unc.edu.pe
Abstract:
Diarrhea is associated with infectious bacteria that cause mortality in calves, such as
Escherichia coli, representing a problem for milk and meat producers globally, causing
large economic losses. This study assessed the resistance to E. coli strains isolated from
diarrheal feces of newborn calves from the Cajamarca region. Fifty two (52) fecal samples
from calves from five provinces of the Cajamarca region were collected for the isolation
of E. coli on MacConkey agar with sorbitol. The molecular identification of E. coli was
performed by amplification of the uidA gene by conventional PCR and then antibiotic
susceptibility/resistance was assessed using the Kirby-Bauer methodology and antibiotic
discs with neomycin, tetracycline, sulfamethoxazole-trimethoprim and enrofloxacin. The
results were that 96.15 % of E. coli strains were resistant to tetracycline, 51.92 % to
sulfamethoprim, 26.92 % to neomycin and 9.61 % to enrofloxacin. It was also
demonstrated that 30.76 % had resistance to two drugs, 19.23 % to three drugs and
5.76 % to four drugs; a significant difference was found in resistance to tetracycline
(P<0.0001). It is concluded that newborn calves from the Cajamarca region that presented
782
diarrhea are carriers of antibiotic-resistant E. coli, representing a problem for cattle

farmers, since these strains can cause the death of animals and contribute to the spread of
antibiotic resistance.
Keywords: Resistance, Antibiotics, E. coli, Calves, Diarrhea.
Introduction
Diarrhea in calves has been linked to infectious pathogens, representing a challenge for
those engaged in milk and meat production globally(1). The most important infectious
bacteria that cause mortality associated with diarrhea in calves are E. coli and
Salmonella(2), generating large economic losses if the disease is not treated in time with
appropriate antimicrobials and supportive therapy(2,3). Antibiotics have been used in
animals for the treatment of diseases, prevention and control of common infections(4,5),
however, the inappropriate and excessive use of antibiotics contributes to antimicrobial
resistance, threatening the health of animals and humans(6). In relation to animals that are
destined for slaughter and finally for human consumption, they act as reservoirs of
antimicrobial-resistant strains(7).
For example, it has been reported that, in Egyptian dairy farms, strains of E. coli have
been isolated from diarrheal feces of calves, which were resistant to ampicillin(8). Calves
frequently eliminate microorganisms through their feces, generating the spread of
bacteria in the farm environment, which could cause infections in other animals. E. coli
isolates have been obtained from feces of dairy calves, which present resistance to
multiple antibiotics from the fluoroquinolone group and the iucD gene was determined
as the most prevalent(9). Likewise, other studies mention the aerobactin operon (iucD),
which produces four types of siderophores: enterobactin, salmochelin, aerobactin and
yersiniabactin. The genes involved in the biosynthesis of siderophores are found in
uropathogenic strains (UPEC) and commensal strains; nevertheless, salmochelin,
aerobactin and yersiniabactin are located in UPEC-associated pathogenicity islands, but
not in commensal strains, suggesting that iron uptake systems were acquired by horizontal
gene transfer. Aerobactin is a siderophore present in most UPEC strains, having a great
stability in the binding to Fe3+ and is one of the responsible for iron sequestration during
a urinary tract infection (UTI), the combination of adhesion/iron uptake/toxicity genes
shows the high virulence and the potential for damage that the strains have to cause a
UTI(9,10).
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On the other hand, the French surveillance network for antimicrobial resistance in sick
animals indicated that E. coli strains carrying most resistances have been isolated from
diarrheal feces of neonatal calves(10), with amoxicillin, tetracycline and streptomycin
being the main antibiotics to which resistance has been generated(7,11).
It is necessary to report the resistance that has been generated to multiple types of
antibiotics to consider their control and proper use in cattle, because it represents a danger
to public health. In addition, there are very few studies of antibiotic resistance in newborn
cattle in Peru, with this study being a contribution to have knowledge of the current
situation. The objective of the research was to assess the antibiotic resistance of E. coli
strains isolated from diarrheal feces of newborn calves from five provinces of Cajamarca.
Study location
Work was carried out in 18 dairy farms under a semi-intensive system, which are located
in the province of Chota, San Miguel, Celendín, San Marcos and Cajamarca, region of
Cajamarca, Peru. A total of 52 samples were collected, which were from calves with
diarrhea until the first month of life, of which 35 calves were males and 17 calves were
females, in the rainy season (November 2020 – May 2021) (Figure 1). The fecal samples
(approximately 3 g) were obtained directly from the rectum through the use of first-use
sterile polyethylene bags, these samples were identified and taken in an expanded
polystyrene box containing ice to the laboratory of Biotechnology in Animal Health| of
the Baños del Inca Agricultural Experimental Station, where the isolation of E. coli from
all the samples was carried out.
784
Figure 1: Geographical location of the farms that participated in the study, from the
provinces of Chota, San Miguel, Celendin, San Marcos and Cajamarca, region of
Cajamarca, Peru
Isolation and identification of Escherichia coli
With a sterile bacteriological loop, 300 μl of feces was seeded on MacConkey II agar with
sorbitol (Becton, Dickinson and Company® Loveton Circle Sparks, MD 21152, USA),
then incubated at 37 °C for 24 h in an oven (Universal oven Memmert UN-110/
Schwabach/Germany), colonies with typical morphology and development, such as
bright red colonies, were isolated, which were considered as E. coli.
Extraction of deoxyribonucleic acid (DNA) from Escherichia coli
Three selected E. coli colonies grown on MacConkey II agar with sorbitol were cultured
in liquid microbial growth and multiplication medium 2xYT medium (Sigma, REF.
Y2377) at 37 °C for 18 h; bacterial growth was determined by concentration of preset
values in the spectrophotometer (PCR MAX Lambda 64272, Bibby Scientific Ltd. United
Kingdom), the calculation of the Colony Forming Units (CFUs) was performed at 600
nm, determining the growth of viable bacteria in the growth medium. To obtain the E.
coli DNA template, the Wizard® Genomic DNA purification kit (Promega, REF. A1120)
was used, with the manufacturer’s indications, the DNA was stored in 1,500 μL
polypropylene microtubes (Eppendorf™) in refrigeration, using a Samsung refrigerator,
785
RT35K5930S8/PE, Samsung, Mexico of 4 °C, which is used for the different processes
of the Polymerase Chain Reaction (PCR).
Molecular identification of Escherichia coli
The molecular identification of E. coli was performed by PCR using “primers” (F5’-
TCAGCGCGAAGTCTTTATACC-3’, R5’-CGTCGGTAATCACCATTCCC-3’), for
the amplification of the uidA gene (248 bp)(11,12,13). In the PCR reaction, 1 μl (10 mM) of
F and R of each primer, 7.7 μl of molecular grade water and 12.5 μl of G2 Green Master
Mix (Promega, Madison, USA) were used, 2.8 μl of DNA at a concentration of 50 μg/ml
was used as template. The thermal profile of the PCR reaction was: denaturation 94 °C/2
min, 25 cycles denaturation 94 °C/30 sec, hybridization 55 °C/30 sec, extension 72 °C/45
sec; final extension 72 °C/2 min. The reference strain JM 109 of Escherichia coli
(Promega, REF L2004) was used as a positive control. Fragments of amplified DNA (248
bp) to identify E. coli strains were separated by their molecular weight by electrophoresis
– agarose 1 %. The fragments were analyzed by agarose gel staining with Sybr Green
(Thermo Fisher) and observed on a Labnet U1001 UV transilluminator, 302 nm, Taiwan.
Antimicrobial susceptibility test
For the evaluation of susceptibility/resistance of E. coli strains to antibiotics, the Kirby-

Bauer methodology was used, where parameters established for bacteria isolated from
animals by the Clinical and Laboratory Standards Institute (CLSI) were taken as a
reference(14) (Table 1). Before carrying out the susceptibility test, specific Mueller Hinton
agar (Merck KGaA 64271, Darmstact Germany) was prepared to determine the sensitivity
of clinically important pathogens according to the manufacturer’s instructions, and
sterilized in an autoclave (Automatic Digital Autoclave AVDA050 Liters, Biogenics Lab,
Peru); then it was poured into Petri dishes of 35 mm diameter / 10 mm high and two or
three isolated colonies were seeded with a bacteriological loop, incubated using an
incubator (Memmert CO2 ICO50 GmbH + Co. KG) under aerobic conditions for 18 h at
37 °C. Antimicrobial susceptibility of all isolated colonies was determined against
neomycin-N 30 μg, tetracycline-TE 30 μg, sulfamethoxazole-trimethoprim-SXT 25 μg
and enrofloxacin-ENR 5 μg (Discs - Thermo Scientific™); the sensitivity of the isolated
strains was classified as sensitive, intermediate or resistant by measuring the halo of
inhibition according to the parameters established by the CLSI(14).
786
Table 1: Interpretation of antibiotic sensitivity tests, by disc diffusion method (diameter

of inhibition zone)
Antibiotic Disc Sensitive Intermediate Resistant
concentration resistance
(μg)
Tetracycline 30 ≥19 15-18 ≤14

Sulfamethoprim 25 ≥16 11-15 ≤10
Neomycin 30 ≥17 13-16 ≤12
Enrofloxacin 5 ≥23 17-22 ≤16
Statistical analysis
Results were analyzed using the Graph Pad Prism 9.3.1 software (Prism Software, Irvine,
CA, USA). The normality of the data was determined by “Kolmogorov-Smirnoff”. The
analysis of variance (ANOVA), followed by Tukey’s multiple comparisons analysis for
evaluations between antibiotics (parameters related to sensitivity, resistance). The
information obtained was considered statistically significant at a P<0.05.
Results
From the total samples, a total of 52 with positive growth of E. coli on MacConkey II
Sorbitol agar were selected. The molecular identification of E. coli present in feces of
calves with diarrhea, the uidA gene encoding the β-glucoronidase enzyme(12,13,14) was
amplified. The processing of the PCR products was analyzed by 1 % agarose gel
electrophoresis, a simple and effective method to separate, identify and purify DNA
fragments with a molecular size of 0.5 to 25 kb. Electrophoretic bands of the expected
size were observed: 248 bp positive for the amplified region of the uidA gene. It was
detected in the 52 samples analyzed, evidencing the identification of E. coli, since this
gene is specific to the bacterium (Figure 2), of which 63.30 % (n= 35) corresponded to
male calves and 32.69 % (n= 17) came from female calves.
787
Figure 2: Amplification of the uidA gene from calf fecal samples by (1 %) agarose gel
electrophoresis
Lane 1 100 bp marker. Positive samples in all lanes n= 52.
The presentation of susceptibility/resistance to drugs of diarrheal fecal samples from

calves was analyzed, where different percentages of resistance could be observed; most
strains of E. coli showed resistance to tetracycline (96.15 %, 50/52), likewise, more than
50 % of the samples were resistant to sulfamethoxazole-trimethoprim (51.92 %, 27/52),
followed by a significant percentage of resistance to neomycin (26.92 %, 14/52), in
addition, less resistance to enrofloxacin was observed (9.61 %, 5/52) (Figure 3).
Figure 3: Percentage of E. coli strains with characteristics of resistance to four drugs
The presentation of antibiotic resistance was also determined in terms of the variation of
the strains isolated in each calf, most had resistance to one (42.30 %, 22/52) and two
(30.76 %, 16/52) drugs, the problem worsens in a significant number of calves, presenting
multiple resistance in the isolated strains of E. coli, observing resistance to three
788
(19.23 %, 10/52) and four (5.76 %, 3/52) drugs, with the resistance to tetracycline being
the most common in all; in addition, resistance to enrofloxacin was the one that occurred
in the lowest proportion in the isolated strains (Figure 4).
Figure 4: Percentage of multiple resistance of drugs used in the control of diarrhea in

calves
It was observed that both males and females presented resistance, with higher percentages
of resistance observed in males for tetracycline (68 %) and neomycin (64.28 %)
respectively; but curiously, regarding females, it could be observed that resistance to the
drugs sulfamethoprim (77.70 %), enrofloxacin (80 %) was higher compared to males
(Figure 5).
Figure 5: Percentage of drugs most commonly used in the control of colibacillosis in

calves
The statistical analysis revealed a significant difference (P<0.0001) regarding the

presentation of resistance of E. coli strains from samples of calves with diarrhea to
tetracycline (Figure 6).
789
Figure 6: Statistical difference in resistance of E. coli strains to tetracycline
(P<0.0001). The data are expressed in absolute values and percentage (%).
Discussion
E. coli is one of the main bacterial agents that cause urinary infections in animals,
septicemia and diarrhea in farm animals; the phenomenon of resistance expressed by E.
coli strains to the drugs used in their control shows therapeutic failure, in addition, many
cases of multiple resistance are being observed, which increases worldwide, with the
dissemination of resistance becoming a public health problem(15,16).
In this research, it was possible to determine different characteristics of resistance to

antibiotics and with different percentages, which has allowed determining that the
therapeutic failure to antibiotics expressed by the bacterium in calves raised in dairy cattle
farms in the region of Cajamarca, Peru, is widespread, being able to identify that all
isolated strains of E. coli show resistance; thus, it was possible to observe a higher
percentage of resistance to tetracycline and with a significant percentage of multiple
resistance with a higher prevalence to two drugs, followed by three and four antibiotics
evaluated.
The data obtained on antibiotic resistance in the Cajamarca region allows to mention that
it is due to the lack of sanitary control records in the herds, making it difficult to trace the
drugs used and, according to the version of the owners, some of these animals when
presenting the signs of the disease are treated with antibiotics and others are not, however,
all presented resistance to at least one of the antibiotics analyzed, which allows to mention
that resistance could be due to the misuse of antibiotics by farmers, being used
790
frequently(17), and it is advisable to monitor the calf by electrolyte reconstitution and, if

possible, not to administer antibiotics due to the presentation of resistance to these
drugs(18).
Another possible cause of this widespread resistance to antibiotics could be that the
majority of treated calves and mainly in untreated calves, possibly this phenomenon is
due to the fact that milk and colostrum from cows that have been treated with an antibiotic
facilitates the presence of antibiotic residues in milk, increasing the selection pressure of
E. coli strains, with which resistant strains are selected, a practice well established in the
regional livestock farming of Cajamarca(19,20,21), in addition, it can be assumed that there
is diffusion of genes between commensal and pathogenic strains of antibiotic resistance
among animals and herds, increasing resistance levels(22,23,24).
The pattern of resistance observed in isolated strains of E. coli has an order from highest
to lowest prevalence tetracycline, sulfamethoprim, neomycin and enrofloxacin in a
smaller proportion, common results that were also obtained by other researchers who
report a pattern of resistance to tetracyclines, sulfonamides, penicillins and
fluoroquinolones(25), cephalothin, tetracycline, trimethoprim-sulfadiazine, ampicillin(26),
the most common multiresistance pattern was ampicillin-kanamycin-streptomycin-
sulfamethoxazole-tetracycline(27).
The most frequent resistance phenotype of E. coli strains was to tetracycline 96.15 %
(50/52), this is possibly due to the fact that E. coli strains carry tetracycline-resistant
phenotypes due to the inappropriate use of the drug by producers, which has generated
greater selection pressure in strains carrying genes that confer resistance to tetracycline,
contributing to the transfer of antimicrobial resistance genes through strain diffusion(28);
in addition, these strains are possible sources that spread resistance to the environment
when manure is spread in grazing areas as fertilizer(29).
The presence of resistance of local strains of E. coli to tetracycline is widespread based

on its broad-spectrum use in animal health, as a reservoir of gram-negative bacteria with
genes of resistance to tetracycline as a source of infection and with a higher prevalence
in E. coli causing diarrhea in calves, a frequent problem that has also been reported in
other studies with similar results(30,31,32).
It is very important to determine the genes that are involved in these resistance processes
related to the use of these drugs, such as adhesion, iron transporters genes(33,34), as is the
case of the iucD gene(9,35), in addition, the processes of horizontal gene transfer, selection
pressure, caused by the indiscriminate use of antibiotics(36,37), all of the above is necessary
knowledge to evaluate the processes of treatment and control of diarrhea in calves in the
region of Cajamarca.
791
The strains of E. coli causing neonatal diarrhea in calves in the Cajamarca region present
a prevalence of multiple resistance to the drugs used by farmers in their control, observing
a profile of resistance to tetracycline, sulfamethoprim, neomycin and enrofloxacin
(TSNE), as a result of the misuse of drugs which increases the selection pressure on
strains that promote the expression of genes of virulence and resistance to antibiotics,
becoming foci of transmission of resistance to both animals and humans due to the
possibility of horizontal transmission between microorganisms. Considering that a
definitive diagnosis should be made, determining the etiological agent and susceptibility
to antibiotics, then correctly apply the selected antibiotic in correct dose and frequency.
In addition, based on the results obtained, it is necessary to determine the resistance genes
involved in multiple resistance to antibiotics.
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795
Article
Contamination of commercial dry dog food by Aspergillus flavus and

aflatoxins in Aguascalientes, Mexico
Lizbeth Martínez-Martínez a
Arturo Gerardo Valdivia-Flores a*
Teódulo Quezada-Tristán a
Alma Lilián Guerrero-Barrera b
Erika Janet Rangel-Muñoz a
Karla Isela Arroyo Zúñiga a
Fernanda Álvarez-Días a
Marcelo Lisandro Signorini-Porchietto c
a
Universidad Autónoma de Aguascalientes. Centro de Ciencias Agropecuarias. Av.
Universidad 940, Col Cd. Universitaria, 20131, Aguascalientes, Ags, México.
b
Universidad Autónoma de Aguascalientes. Centro de Ciencias Básicas. México.
c
Instituto Nacional de Tecnología Agropecuaria, Rafaela– Santa Fe, Argentina.
* Corresponding author: avaldiv@correo.uaa.mx; gerardo.valdivia@edu.uaa.mx
Abstract:
Commercial dry food (CDF) for dogs is a whole grain ration thoroughly mixed and die-cut
with heat and pressure to give it the shape of kibble. CDF is formulated with several agro-
industrial ingredients and by-products of agricultural and livestock origin. Contamination by
Aspergillus flavus and aflatoxins (AFs) in foods has been shown to be a global problem that
causes harm to human and animal health. The objective was to evaluate the presence of
fungal microbiota and contamination by AFs in CDF. A random sample (n= 77) of marketed
796
CDF was selected in Aguascalientes, Mexico. The samples were processed and cultured by
serial dilutions, obtaining monosporic isolates, which were characterized morphologically,
toxigenically (HPLC), and molecularly (PCR). The concentration of AFs in CDF was
quantified by HPLC. Fungal growth was observed in 53.2 % of CDF, and 7.8 % exceeded
the maximum permissible limit (MPL=106 CFU/g). The genera Aspergillus, Penicillium,
Cladosporium, Mucor, Alternaria, and Fusarium were found (69.4, 12.9, 9.4, 4.7, 1.7, and
1.1 %, respectively). All CDF samples showed contamination by AFs (14.8 ± 0.3 μg/kg), and
11.8 % exceeded the MPL (20.0 μg/kg) suggested by the regulations; contamination was
significantly associated (P<0.05) with some ingredients used, CDF moisture, and inclusion
of fungicides and sequestrants. The results obtained suggest that the CDF manufacturing
process does not wholly eliminate contamination by fungi or by the AFs present in the
ingredients used for its formulation; consequently, these remain in the finished product,
putting at risk the health of dogs and the efficacy of the food chain.
Keywords: Aspergillus flavus, Aflatoxins, Food chain, Kibble.
Introduction
Commercial dry food (CDF) for dogs is a whole grain ration thoroughly mixed and die-cut
by heat and pressure in the shape of kibble; it is composed of several agro-industrial products
and by-products of agricultural and livestock origin, so they are important as a frequent
output from agro-industrial supply chains(1). In Mexico, the use of CDF has become popular
to achieve the integration of dogs into the urban lifestyle; also, several brands of CDF have
proliferated to meet the variety of nutritional needs of these pets, according to their activity,
breed, age, and some special conditions(2). In Mexico, the National Council of Manufacturers
of Balanced Feed and Animal Nutrition registers 22 factories that produce 1.3 thousand
tonnes of CDF annually(3), which is complemented by an abundant offer of international
brands(4).
In the manufacture of CDF, agro-industrial products, and by-products of different

bromatological compositions are incorporated to comply with its nutritional design (5). The
nutritional and sanitary quality of these ingredients is transferred to the final formulation, so
it has been pointed out that CDF presents risks of contamination by various pathogens, such
797
as mycotoxigenic fungi(6-8). Fungal contamination occurs at multiple stages of the production

of plant ingredients, such as flowering, harvesting, processing, or storage of cereals; in
addition to the permanence of metabolic residues of mycotoxins in meat, dairy products, and
eggs(9-11).
The toxigenic fungal genera found in the ingredients for CDF formulation are Aspergillus
spp., Penicillium spp., and Fusarium spp.(12-14). Likewise, the mycotoxins frequently found
in food ingredients are aflatoxins (AFs)(12-13). The presence of AFs represents a risk factor
for animal health and economic losses for agribusiness because it reduces the nutritional
value of the food product(15).
The poisoning of dogs by AFs causes hemodynamic, digestive, and nervous alterations, as
well as changes in the biochemistry and clotting ability of the blood; which are especially
sensitive to AFs because they have a reduced activity of the enzyme glutathione S transferase
(GST), essential for the detoxification pathway of xenobiotics(16). Although there are no
specific maximum permissible limits (MPLs) for AFs in the CDF for dogs (4), the MPLs
established for foods intended for other domestic animals have been suggested to be used(17),
especially the guidelines indicated by the Codex Alimentarius or by the European
Community (20.0 or 5.0 μg/kg, respectively)(17,18).
Reports of outbreaks of clinical forms due to AFs poisoning in dogs are scarce, but their
geographical distribution is very diverse: North America, Latin America, Asia, and
Africa(19-21). This coincides with a worldwide distribution of toxigenic fungi both in CDF and
in the ingredients with which they are made(22,23). In addition, the way CDF is dispensed, in
generally large bags or sacks, allows the increase in the concentration of AFs, because the
dog must ingest all the content that is in each bag, but the fungal spores and toxins are
resistant to the manufacturing process(2). In summary, the presence of toxigenic fungi and
their toxins can be considered a severe problem for the dog to adequately perform its
zootechnical function as a companion, guardian, or sports animal. In addition, national and
international agribusiness must carry out better strategies to reduce fungal contamination and
its mycotoxins in CDFs. Therefore, this study aimed to evaluate the presence of fungal
microbiota and contamination by AFs in the CDF for dogs.
798
Study design
The study was conducted in Aguascalientes, Mexico (22°27'35 - 21°37'20" N; 101°50'07" -

102°52'27" W). The climate is semi-dry with an average annual temperature of 18 °C, with
an average rainfall of 526 mm, and the main rainy season in summer(24). A list of shopping
centers, pet stores, veterinarians, and grocery stores that sold CDF was obtained, and a visit
was made to get information on the brands and types in the establishments. A total of 145
types of CDF (Table 1) were found, which were considered as a sampling frame. The sample
size was calculated in 58 types of CDF using the following formula to estimate proportions
in a finite population(25):
𝑁𝑍 2 𝑝𝑞
𝑛=
𝑁𝑑 2 + 𝑍 2 𝑝𝑞
Where: n= sample size (58); N= population size (145 types of CDF); Z= standard normal
distribution value (1.96); p= prevalence or expected proportion of contamination with
Aspergillus spp. or with AFs in the CDF, a proportion value P= 0.5 was used, q= 1-p; d=
desired precision (maximum error= 0.10).
Table 1: Characteristics of commercial dry dog food marketed in central Mexico

Supply Sampling Protein Moisture Fiber Price
Type of
(N) (n) (n/N%) (min. (max. %) (max. *US$/kg±(SE)
food
%) %)
Origin
National 120 64 53.3 24.0 11.0 4.0 3.9b ±0.24
International 25 13 52.0 26.0 11.0 4.0 5.9a ± 0.92
Commercial classification
Standard 87 52 59.8 22.0 12.0 5.0 2.6b ± 0.17
Premium 58 25 43.1 27.0 11.0 4.0 7.7a ± 0.37
Prescription (Age)
Puppy 55 27 49.1 27.0 11.0 4.0 5.1a ± 0.45
Adult 90 50 55.6 22.0 11.0 4.0 3.7b ± 0.30
Prescription (Size)
General 72 43 59.7 22.0 12.0 5.0 2.2a ± 0.10
Specific 73 34 46.6 26.0 11.0 4.0 6.8a ± 0.39
Total 145 77 53.1
*Price in reference US dollars (www.banxico.org.mx: January 2020).
ab
Means with different literal show significant differences (P<0.05).
799
The selection of samples was performed using the snowball sampling technique(26), for which
the establishments were visited successively in alphabetical order, and samples of CDF sold
were acquired. The purchase of the CDF was suspended when the same types that had been
previously acquired were found in three successive stores. Finally, 77 different types of CDF
were purchased (Table 1).
The type of CDF was classified according to the prescription (age and size) and commercial
identification (standard and premium) declared by the manufacturer. The composition of the
CDFs was recorded from the nutritional information reported by the manufacturer to identify
the ingredients used. The CDFs were classified by the presence or absence of cereals,
oilseeds, vegetable oil, legumes, tubers, animal by-products, fungicides, and ingredients with
sequestering capacity and type of CDF.
Sample handling
The samples were dried in an oven with forced air circulation and pulverized (500-800 μm)
in a universal continuous mill and stored inside sealed bags in refrigeration (4-5 °C) until
processing (<2 wk).
The fungal isolation was performed using the direct plating technique with serial dilution for
the count of fungal colonies in the CDF. Samples were diluted (10-1, 10-2, 10-3, and 10-4) and
seedings were performed on rose bengal agar + chloramphenicol and Czapek. The incubation
period in the dark was 27-30 °C for 7 d(27). Preparations of fungal colonies were made with
cotton blue staining using lactophenol to observe microscopic characteristics(28). The isolates
were identified with macroscopic and microscopic morphological characteristics(29,30).
Molecular analysis
Genomic DNA was extracted from monosporic isolates consistent with the morphology of
A. flavus using previously standardized methods(31). The (1 %) agarose gel electrophoresis
technique was used to verify the quality of the DNA obtained. DNA samples were deposited
in the gel with loading buffer (Platinum II Green PCR Buffer 5X Thermo Fisher Scientific,
Waltham, MA, USA) to place them in the electrophoretic chamber with loading buffer (TAE
1X, 95 volts, 40 min). The resulting bands were observed in an image documenter (GEL
DOC XR, BIO-RAD Molecular Image CA, USA) with the Quantity One software (version
4.6.7.).
800
Polymerase chain reaction (PCR) was performed to amplify genomic DNA fragments in the
region of internal transcribed spacers (ITS1-5.8S-ITS2- rRNA), calmodulin gene (CaM), and
the primer gene of the aflatoxin biosynthetic pathway (aflR) following previously described
protocols(32,33). The following primers were used for ITS1: 5´-
TCCGTAGGTGAACCTGCGG-3´; ITS4: 5´-TCCTCCGCTTATTGATATG -3´; CMDA7-
F: 5´-GCCAAAATCTTCATCCGTAG-3´; CMDA8-R: 5´-
ATTTCGTTCAGAATGCCAGG-3´; aflR-F: 5´-
GGGATAGCTGTACGAGTTGTGCCAG-3´; aflR-R: 5´-
TGGKGCCGACTCGAGGAAYGGGT-3´ (Thermo Fisher Scientific, Waltham, MA, USA).
The enzyme Taq-polymerase (Platinum Green Hot Start PCR 2X Master Mix, Thermo Fisher
Scientific) was used for amplification, and amplification reactions were performed in a
thermal cycler (Labnet, Multigene, USA). The amplification protocol was introduced for the
ITS1-5.8S-ITS2 RNAr region using a denaturation period of 3 min at 94 °C, followed by 35
cycles (denaturation at 94 °C/1 min, annealing at 54 °C/1 min and extension at 72 °C/1 min)
and with a final extension of 9 min at 72 °C. The conditions for amplification of the CaM
gene were with a denaturation period, a cycle of 1 min/94 °C followed by 30 cycles (1
min/94 °C, for annealing 1 min/53 °C and for extension 1 min/72 °C) and a final extension
period of 10 min at 72 °C was added. Likewise, for the amplification of the aflR gene, a pre-
denaturation period of 1 min at 94 °C was used, followed by 35 cycles (denaturation at
94 °C/1 min, annealing 63 °C/1 min, and extension 72 °C/1 min) and with a final extension
of 10 min at 72 °C. The quality of the PCR products (ITS, CaM, and aflR) was verified by
the 1 % agarose gel electrophoresis technique. A ladder with a marker of molecular weight
(1.0 μL, 100 bp DNA ladder, 0.5 μg/μL. No. 15628019/15628050. Invitrogen DNA Ladder)
together with 1.0 μL of the buffer (BlueJuice Gel Loading Buffer 10X) were included. The
size in base pair (bp) of the amplicon for molecular identification was: ITS, 600-800; Calm,
468 and aflR, 796. The bands were visualized in the image documenter using the Quantity
One software (version 4.6.7.). PCR products were purified with the ExoSAP-IT PCR Product
Cleanup reagent (Affymetrix, Thermo Fisher Scientific Inc. Santa Clara, California, USA).
Mycotoxin quantification
The quantification of the concentration of AFs was performed in duplicate according to the
AOAC official method 990.33(34). The content of AFs was extracted using solid phase tubes
(SPE; SupelcleanTM LC-18 SPE tube, Sigma-Aldrich, USA), methanol:water, acetic acid,
tetrahydrofuran (THF), and hexane. Trifluoroacetic acid-derived extracts were injected into
an HPLC system with a fluorescence detector (Varian Pro Star binary pump; FP detector
2020, Varian Associates Inc., Victoria, Australia), C18 column and column protector (LC-
18 and LC-18; Thermo Fisher Scientific, Waltham, MA, USA). AFs estimates were obtained
801
with the help of a software (Galaxie Ver. 1.9.302.530), and concentrations were calculated
using standard curves of purified AFs (Sigma Aldrich, St. Louis, MO, USA).
The following mycotoxins were also quantified in the CDF: zearalenone (ZEA), ochratoxin
(OTA), fumonisins (FUM), and deoxynivalenol (DON) by indirect ELISA analysis(4)
(Ridascreen Fast: Zearalenon R5502, Fumonisin R5602, Ochratoxin A R5402,
Deoxynivalenol R5902, R-Biopharm, Germany).
Data were analyzed using a normality test with the Kolmogorov-Smirnov method at a 95 %
confidence level. The comparison of the sample means for each variable was performed by
means of the Tukey test (HSD) with a statistical software (Statgraphics Centurion, version
16.1.03). To identify the risk of exceeding the MPL established for the concentration of AFs,
the Chi-square test (χ2) of the probability ratio or odds ratio (OR) was performed, calculating
the portion of CDF that exceeded the MPL for the concentration of AFs and that was exposed
to a specific factor (formulation with the inclusion of cereals, oilseeds, vegetable oil,
legumes, tubers, by-products of animal origin, fungicides and ingredients with sequestering
capacity and type of CDF) divided by the portion of CDF that exceeded the MPL for the
concentration of AFs but was not exposed to that specific factor. A probability level of
P<0.05 was considered in all analyses.
Results
Most CDF acquired (82.8 %) were manufactured by national manufacturers, while 17.2 %
were made by international commercial brands (Table 1). More than half of the CDF samples
(41/77= 53.2 %) had fungal contamination, while 7.8 % (6/77) contained a fungal
concentration above the maximum recommended levels (106 CFU/g). Eighty-five (85)
purified fungal isolates were obtained, which showed morphological characteristics
corresponding to the following main toxigenic genera (Figure 1): Aspergillus spp. (69.4 %),
Fusarium spp. (1.1 %) and Penicillium spp. (12.9 %). Isolates with morphology
corresponding to the genera Cladosporium spp., Mucor spp., and Alternaria spp. (9.4, 4.7,
and 1.7 %, respectively) were also identified. Of the isolates of Aspergillus spp., 40.7 %
(24/59) corresponded to the morphology of A. flavus(30); 75.0 % (18/24) of the A. flavus
isolates demonstrated in vitro the capacity to produce aflatoxins (9.8 ± 0.64 μg/kg in 7 d) and
also expressed the CaM and aflR genes and the ITS region (Figure 2) by PCR analysis.
802
Figure 1: Macroscopic and microscopic morphological structure (40x) of monosporic

isolates. Panels: A) Aspergillus spp., B) Fusarium spp., C) Penicillium spp., D) Aspergillus
spp., E) Fusarium spp., and F) Penicillium spp.
Figure 2: 1 % agarose gel electrophoresis of PCR products
Panel A: amplification of the internal transcribed spacer (ITS) region. Panel B: amplification of Calmodulin
(Calm). Panel C: amplification of the primer gene of the aflatoxin biosynthetic pathway (aflR). First lane:
100-2000 base pair molecular weight marker; C1-C18: isolates of Aspergillus flavus.
All CDF samples showed detectable concentrations of AFs (Figure 3). The frequency of AFs
concentration showed a normal approximation (P=0.14); the minimum concentration was 8.6
μg/kg and the maximum concentration was 22.2 μg/kg; a mean concentration of 14.8 ± 0.3
μg/kg was estimated, with a 95.0 % confidence interval of 14.2-15.4 μg/kg. It was also
detected that approximately one in ten (11.8 %) of the CDF analyzed exceeded the MPL of
AFs recommended by most legislations of American countries for the use of cereals (20.0
μg/kg)(35), while all the CDF exceeded the European recommendations (5.0 μg/kg)(18).
803
Concentrations of OTA, FUM, and DON were below detection limits; while the estimated
concentrations of ZEA (228 ± 13.8 μg/kg) in no case exceeded the MPL (400 μg/kg)
suggested to regulate this mycotoxin(35).
Figure 3: Frequency of the concentration of aflatoxins (AFs) in commercial dry dog food
in central Mexico
MPL= maximum permissible limit (MPL): Americas (20 μg/kg); European Union (5.0 μg/kg).
In this study, no significant difference (P>0.05) was observed between the concentration of
AFs and the general characteristics of the CDF, such as origin, commercial classification
(standard or premium), or prescription by age or size of the dog; nor was any statistical
association detected that would allow these characteristics to be identified as risk factors that
generate concentrations above the MPL (Table 2).
804
Table 2: Association between commercial dry dog food characteristics and aflatoxin
concentration
Mean ±SE >MPL P value
Type of food (n) OR
(µg/kg) (%) (χ2)
Origin
National 64 15.0a ±0.32 12.5 0.49 1.71
a
International 13 13.9 ±0.61 7.7
Commercial classification
Standard 52 15.1a ±0.34 11.5 0.93 0.95
a
Premium 25 14.2 ±0.50 12.0
Prescription (age)
Puppy 27 15.0a ±0.48 18.5 0.05 2.6
a
Adult 50 14.7 ±0.35 8.2
Prescription (size)
General 43 15.3a ±0.38 14.0 0.32 1.7
a
Specific 34 14.2 ±0.42 8.8
SE= standard error; MPL= maximum permissible limit (20 μg/kg); P(χ 2)= Chi-square; OR= odds ratio.
ab
The average concentration of AFs in the CDF presented significant differences associated
with the characteristics of the CDF and with the ingredients used. The CDF with moisture
higher than 10 % showed an estimated concentration of AFs significantly higher (P<0.05)
than that in CDF containing lower moisture. It was also detected that there was a significant
three times higher risk (OR χ2 P<0.05) of finding concentrations above the MPL in those
CDF that registered moisture greater than 10 % (Table 3) in relation to the CDF that had
moisture less than 10 %. Although it was also detected that there was a higher risk for CDF
that contained a higher concentration of protein, fat, and ash (>22, >12, and >7 %,
respectively) in their formulation, the statistical association was not significant (P>0.05).
805
Table 3: Association between bromatological analysis of commercial dry dog food and
aflatoxin concentration
Mean ±SE >MPL P value
Characteristic (n) OR
(µg/kg) (%) (χ2)
Relative moisture
>10% 31 17.4a ± 0.36 19.4 0.01 3.4
≤10% 46 13.0 b
± 0.29 6.5
Protein
>22% 47 14.9a ± 0.36 14.9 0.12 2.5
≤22% 30 14.7 a
± 0.46 6.7
Fat
>12% 22 14.9a ± 0.53 18.2 0.11 2.2
≤12% 55 14.8 a
± 0.34 9.1
NFE
Present 47 14.4a ± 0.52 12.8 0.78 1.1
a
Absent 107 15.0 ± 0.34 11.2
Fiber
>4% 30 15.2a ± 0.46 13.3 0.61 1.3
≤4% 47 14.5 a
± 0.36 10.6
Ash
>7% 38 15.5a ± 0.40 15.8 0.11 2.3
≤7% 39 15.1 a
± 0.39 7.7
SE= standard error; MPL= maximum permissible limit (20 μg/kg); P(χ 2)= Chi-square; OR= odds ratio; NFE=
nitrogen-free extract.
ab
The average concentration of AFs in the CDF that contained wheat was significantly higher
(P<0.05) compared to the estimated concentration of AFs in the CDF that did not use this
ingredient (Table 4). Nevertheless, when calculating the risk of exceeding the MPL, there
was no significant association (P>0.05) between the proportion of CDF that contained wheat
and those that did not include it in their formulation. No significant difference (P>0.05) was
observed between the AFs concentration means in the presence or absence of any by-product
of animal origin in the CDF. However, a significant association (P<0.05, χ2) was detected in
the proportion of CDF that exceeded the MPL among the CDF that presented fishmeal and
fish oil in their formulation, compared to those that did not; therefore, the risk (OR) of finding
concentrations above the MPL was three times higher than in the CDF that registered absence
of the ingredients. All the samples purchased used meat and bone meal in the formulation of
CDF, so no association with these ingredients could be established.
806
Table 4: Association between aflatoxin concentration and the inclusion of agro-industrial

foods and by-products in commercial dry dog food
Mean ± SE >MPL P value
Ingredient (n) OR
(µg/kg) (%) (χ2)
Wheat
Present 47 15.5a ± 0.35 12.8 0.60 1.3
b
Absent 30 13.8 ± 0.44 10.0
Barley
Present 22 15.5a ± 0.53 18.2 0.11 2.2
a
Absent 55 14.5 ± 0.33 9.1
Corn
Present 55 15.0a ± 0.34 9.1 0.11 0.45
a
Absent 22 14.3 ± 0.53 18.2
Rice
Present 38 15.2a ± 0.40 13.2 0.57 1.4
a
Absent 39 14.4 ± 0.40 10.3
Oilseeds
Present 27 15.1a ±0.48 14.8 0.37 1.6
a
Absent 50 14.6 ±0.35 10.0
Vegetable oil
Present 38 14.8a ±0.41 13.2 0.57 1.3
a
Absent 39 14.8 ±0.40 10.3
Legumes
Present 49 14.8a ±0.36 12.2 0.77 1.1
a
Absent 28 14.8 ±0.47 10.7
Tubers
Present 45 14.9a ±0.37 13.3 0.45 1.5
a
Absent 32 14.7 ±0.44 9.4
Egg and milk
Present 27 15.1a ±0.48 18.5 0.05 2.6
a
Absent 50 14.7 ±0.35 8.0
Fishmeal and fish oil
Present 31 15.3a ±0.45 19.4 0.01 3.4
a
Absent 46 14.5 ±0.37 6.5
SE= standard error; MPL= maximum permissible limit (20 μg/kg); P(χ 2)= Chi-square; OR= odds ratio.
ab
CDF that contained fungicides or mycotoxin mineral sequestering agents showed

significantly lower mean AFs concentration (P<0.05) compared to the estimated
concentration of AFs in CDFs where these additives were not included (Table 5). In addition,
a significant protective association (P<0.05, χ2) was detected when comparing the proportion
807
of CDF that exceeded the MPL but did not include these components and those that did add
them to their formulation, so the risk (OR) of presenting concentrations above the MPL was
lower than in the CDF that included fungicides or sequestering agents.
Table 5: Association between the inclusion of fungicides and sequestering agents with the
concentration of aflatoxins in commercial dry dog food
Mean ± SE >MPL P value
Ingredient (n) OR
(µg/kg) (%) (χ2)
Fungicides
Present 46 13.5a ± 0.33 6.5 0.01 0.29
b
Absent 31 16.7 ± 0.40 19.4
Organic adsorbents
Present 43 14.7a ± 0.38 9.3 0.30 0.59
a
Absent 34 14.9 ± 0.43 14.7
Mineral sequestrants
Present 43 13.9a ± 0.36 7.0 0.04 0.35
b
Absent 34 15.9 ± 0.41 17.7
SE= standard error; MPL= maximum permissible limit; P(χ 2)= Chi-square; OR= odds ratio.
ab
Discussion
Commercial dry food or kibble has represented an important market for various industries
that produce food for dogs incorporated into the urban lifestyle(3). As in other products of
agricultural and animal origin, contamination by fungal microbiota and mycotoxins is
virtually inevitable(11). The present study detected contamination by toxigenic Aspergillus
flavus in one third (18/77= 31.2 %) of a random sample of CDF, as well as a detectable
concentration of aflatoxins in all samples; in addition, 11.8 % of the CDF exceeded the
maximum permissible limit of AFs (20.0 μg/kg) suggested by the regulations(35). This finding
has not been previously reported in Mexico and contamination by AFs puts at risk the health
of dogs and the proper performance of their zootechnical function (company, guard, work,
etc.) for which they are raised(36). Likewise, it economically affects the agro-industrial
branches that provide the ingredients by altering the safety of the product and deteriorating
its economic and nutritional value(15).
In this study, it was found that CDF had low to moderate concentrations of other mycotoxins.
The levels of OTA, FUM and DON were estimated to be below the detection limits. The
concentration of ZEA reached concentrations close to half (57.0 %) the maximum
permissible level used in European countries that regulate this mycotoxin (400 μg/kg)(35);
808
however, this finding of absence of significant concentrations of mycotoxins other than AFs
does not guarantee that these contaminants could not be present in other circumstances,
because mycotoxins are common contaminants in cereals that are used as common
ingredients in the manufacture of dog food(37); this suggests that CDF manufacturing should
be properly managed due to the severity of mycotoxin contamination(38).
Although the information on the presence of A. flavus and AFs in various ingredients in
human food is extensive, studies of contamination in CDF are scarce, despite being
formulated with similar ingredients(2). In this study, Aspergillus spp. was the genus detected
most frequently (69.4 %) in CDF, which agrees with several authors(12,14,39) who identified
the same fungal genera contaminating CDF for dogs in other countries. In the present study,
fungal microbiota was found in 53.2 % of the samples, and 7.8 % exceeded the maximum
concentration of fungi (106 CFU/g) suggested as the maximum permissible(40). The
confirmation of the identity of isolates with the morphology of A. flavus was achieved by
amplification of genes and gene regions (ITS, CaM, and aflR), which has been proposed as
a default barcode for the identification of these fungi with the capacity to produce AFs(41,42).
These findings suggest that the persistence of active forms of fungi with toxigenic capacity
means an additional risk since if the usual processes of kibble production are not able to
destroy the fungal microbiota, when the environmental conditions (water activity and
temperature) change due to the opening of the bags where the finished product is stored, the
spores and sclerotia of A. flavus can give rise to new vegetative forms capable of using food
substrates, producing aflatoxins and increasing the pre-existing concentration in CDF(43). In
addition, the usual amount of CDF contained in the bag is sufficient for a duration of several
days or weeks in which the dog has to consume all the material, regardless of its quality and
safety(44).
In this study, a significant association was found between some characteristics of CDF and
the detected concentration of AFs, which was reinforced by the estimation of the increase in
the risk of exceeding the MPL. Especially relative moisture above 10 % showed three times
more risk of presenting concentrations above the MPL compared to foods with a lower
relative moisture (Table 3). This finding coincides with other studies that report that the
activity of water present in the food matrix is a relevant factor for the expression of genes
regulating the AFs biosynthesis pathway(45). Therefore, if the substrate contains more
moisture or is rehydrated during storage, AFs concentrations may increase(46). This result
could be attributed to the fact that the extruded raw material for the formulation of CDF
presents in the initial stage of the process an excess of relative moisture content (20-25 %),
and although it is reduced by drying to low levels (8-12 %), only the growth of the vegetative
forms of the fungal microbiota is inhibited, but its spores and mycotoxins produced within
the processed material remain stable(45).
809
The results of this study also showed that there was greater contamination by AFs in the
presence of some ingredients used in the manufacture of the food. The CDFs that contained
wheat or fishmeal and fish oil had higher concentrations of AFs or a higher risk of exceeding
the MPL. In the case of cereals, contamination has been attributed to crop exposure at various
stages of production (flowering, harvesting, transport, processing, or storage)(47). These
ingredients are widely used as a source of carbohydrates, fiber, proteins, fats, minerals, and
vitamins(48); on the other hand, fishmeal and fish oil are products resulting from the
processing of whole fish or by-products (cooking, pressing, dehydration and milling) and
constitute a source of protein that is rich in fatty acids of high nutritional value
(eicosapentaenoic acid, docosahexaenoic acid, and omega-3 acid)(49). These ingredients are
included in formulas because of their low cost and because they maintain an acceptable
nutritional value for the dog’s physiology, in addition, their inclusion does not affect the
palatability and digestibility of nutrients(38). This suggests that AFs contamination may be
common in CDF with the presence of cereals or fish by-products(50,51). Therefore, the quality
of these ingredients should be guaranteed, and proper handling and effective process
management of the finished product should be carried out to ensure protection against
contamination by AFs(52).
The results of this study showed that CDF that included fungicides or mineral sequestrants
in their formulation had both a lower mean concentration of AFs and a lower percentage of
AFs above the MPL (P<0.05) compared to those that did not, which suggest a protective
association of these agents against the risk of AFs contamination higher than the MPL. This
finding suggests that the use of fungicidal and sequestering agents is a helpful method to
reduce the toxic effects of AFs, since fungicides have an inhibitory effect on fungal growth
by acidifying their cytoplasmic content(53); while mineral sequestrants exert their protective
association through β-dicarbonyl chemisorption of AFs, which reduces their bioavailability
through gastrointestinal absorption(54,55).
Surprisingly, in this study, no association (P>0.05) was found between the concentration of
AFs or the proportion that exceeded the MPL suggested by the regulations (Table 2) against
some characteristics considered as evidence of quality by users (premium foods, international
origin, or higher price). This suggests that consumer confidence is based on criteria other
than the safety of CDF, such as marketing diffusion, supposed association between quality
and price, palatability, or appearance(2).
810
In the present study, considerable contamination by toxigenic Aspergillus flavus was

detected, as well as a significant concentration of aflatoxins in all samples collected in a
random and representative sampling of commercial dry dog food. These findings suggest that
the health of dogs and the proper development of their zootechnical function are at risk, and
it could also affect the agro-industrial branches that provide this food since the safety of the
product is altered, and its economic and nutritional value deteriorates. The results of the study
indicated that some bromatological characteristics and the formulation used in the
preparation of CDF generated a greater risk of contamination by fungi and mycotoxins; it
follows that there is a need to design and implement more effective strategies to verify the
safety of ingredients and processes used in the manufacture of CDF. In addition, the
establishment of maximum permissible levels of AFs specific for CDF and research on
prolonged exposure of dogs to low concentrations of mycotoxins should be encouraged.
Acknowledgements
The authors wish to thank the funding from the National Council of Science and Technology
of Mexico (CONACYT No. 738906) and the Autonomous University of Aguascalientes
(project No: PIP/SA 22-2).
Conflicts of interest
The authors declare that there is no potential conflict of interest concerning the present
research, authorship, or publication of this paper.
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Article
Estimation of the basic quality grade of beef carcasses according to bone

maturity, marbling, and Bos indicus phenotypic predominance
Francisco Gerardo Ríos Rincón a*
Leslie Zelibeth González Rueda a
Jesús José Portillo Loera a
Beatriz Isabel Castro Pérez a
Alfredo Estrada Angulo a
Jesús David Urías Estrada a
a
Universidad Autónoma de Sinaloa. Facultad de Medicina Veterinaria y Zootecnia.
Boulevard San Ángel # 3886, Fraccionamiento San Benito, 80246, Culiacán, Sinaloa,
México.
*Corresponding author: fgrios@uas.edu.mx
Abstract:
In order to estimate the Basic Quality Grade of beef carcasses according to bone maturity,
marbling and Bos indicus racial predominance, data from 1,417 carcasses processed in four
Federal Inspection Type establishments were analyzed. The following variables were
recorded: cavitary fat, rib eye area, dorsal fat thickness, hump length and height, marbling,
and bone maturity. Using the variables marbling and bone maturity, the Basic Grade of
Quality was estimated in accordance with the norm NOM-004-SAGARPA-2018. The hump
height was used as a criterion to determine racial predominance, and four groups were
generated from this information. Based on the recorded values, descriptive statistics, analysis
of variance, comparison of means, and Tukey's test were determined. The hump height in
each group was 7.19, 10.54, 14.38, and 20.11 cm (P<0.01), respectively. 82 % of the
carcasses were predominantly Bos indicus. The hot carcass weight was 310.05 kg for group
818
1 vs 326.99 kg for group 4 (P<0.01). The rib eye area was 85.59 cm2 for group 1 vs 89.14
cm2 for group 2 (P<0.05). Of the total number of carcasses evaluated, 60 were classified as
Supreme quality (4.23 %), 655 as Select quality (46.22 %), 621 as Standard quality
(43.82 %), and 81 as Commercial quality (5.72 %). The beef carcasses in this study have
mainly a Bos indicus breed component, and their Basic Quality Grade corresponded primarily
to a greater number of carcasses with grade A bone maturity, but with less marbling.
Keywords: Bovine carcass, Bos indicus, Marbling, Bone maturity.
Introduction
Mexican beef production in 2021 was 2'128,590 t, which represents 2.3 % more than in 2020,
when beef production was 2'078,158 t(1); this is indicative of a very dynamic agricultural and
livestock sector. The interest in adding value to beef production in Mexico has its origin in
the establishment of the Livestock and Beef Classification Service in Mexico, implemented
for the first time in 1969 by the Government of the State of Sonora(2). Years later, in order to
identify differences in carcass quality and yield, on September 18, 1991, the norm NMX-FF-
078-1991(3) was published in the Official Gazette of the Federation (Diario Oficial de la
Federación). This norm is based on the classification system of the United States of America
and adapts the concept of carcass evaluation to emphasize the existing differences between
production systems. This norm recognized and awarded the following classification grades:
Prime, Choice, Select, Standard, and Commercial; it also awarded performance grades
identified as 1, 2, 3, 3, 4, and 5. It was subsequently repealed and gave rise to the norm NMX-
FF-078-2002(4), also of a voluntary nature, whose purpose was to support cattle breeders and
other agents involved in the beef production, processing, marketing and consumption chain,
by defining the quality characteristics that carcasses must meet for marketing purposes; with
the following quality grades: Prime, Choice, Standard, Commercial, and Unclassified,
considering the levels of marbling and texture or firmness; however, unlike its predecessor
NMX norm, it does not specify or take into account the performance grades.
Through all these years, the issue of the evaluation of Mexican bovine carcasses has always
been controversial; for some, it is an incentive for livestock production activities; however,
for others it is an uncomfortable method to punish the product(5). With the recent approval of
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the Mexican Official Norm NOM-004-SAGARPA-2018, "Bovine Meat-Classification of

carcasses according to their physiological maturity and marbling characteristics"(6), it is
established that one of the accepted ways to provide certainty and, therefore, order to the beef
carcass supply sector is to establish a quality classification that will provide information on
product attributes, preventing confusion in the domestic and export markets, as well as the
arbitrary establishment of qualities that are not officially recognized. In this regard, beef
carcass classification or grading seeks to evaluate the final merit of an animal by assessing
parameters of economic importance for the carcass(7), since the variables that help classify
bovine carcasses seek to define parameters that can be accurately identified, either in absolute
terms (weight) or in relative terms (scores), which will converge in a fair commercialization
of the carcasses(8).
Mexican beef production comes from different production systems; therefore, its quality
must be assessed through the evaluation and classification of carcasses, considering the racial
factor, age, and type of cattle, in addition to the specifications of the norm NOM-004-
SAGARPA-2018. Based on the above, the objective of this study was to estimate the Basic
Quality Grade of bovine carcasses according to bone maturity, marbling, and Bos indicus
breed predominance.
Study area
Data from 1,417 beef carcasses processed in four Federal Inspection Type (TIF)
establishments located in the state of Sinaloa, Mexico, were analyzed. The information was
obtained from the primary cuts’ production line. All cattle came from an intensive beef
production system in finishing pens located within a radius of 50 km from the TIF
establishments.
Post mortem procedure
After stunning and bleeding of the bovines, their chronological age was estimated based on
the appearance and wear of the teeth, according to the guidelines established in the
820
Operational Manual, which serves as a tool for the identification, separation, and elimination
of Specific Risk Material(9). The animals were thus classified into two groups: cattle under
30 mo of age and cattle aged 30 mo or more. During the process, the bovines were
decapitated; their forelimbs and hind limbs were removed, and they were skinned and
eviscerated so that they could be cut along the midline and divided into two half-carcasses.
Once the carcasses were washed and sanitized, they entered the refrigeration chamber, after
weighing the hot carcass (HCW). Carcasses of male and female animals less than 30 mo old,
as well as from male and female animals 30 mo old or older, were included in this study.
Recording variables of interest
After 48 h after slaughter and kept refrigerated at a temperature of 2 to 4 °C, the following
variables were evaluated in the bovine carcasses(10).
Estimation of renal, pelvic, and cardiac fat. The amount of cavitary fat was determined
subjectively and expressed as a percentage of the weight of the cold carcass; normally the
weight of these fat accumulations represents between 1 and 5 % of the weight of the cold
carcass. The weight of the kidneys was excluded from this measurement.
Determination of the rib eye area. This was measured with a template marked with small
squares, where each square was added up to measure the complete area of the Longissimus
dorsi muscle, the muscle area was perfectly delineated with a permanent marker; in this
measurement, the adjacent fat and other surrounding tissues were not included.
Dorsal fat thickness. This variable was determined at the height of the 12th rib, three-quarters
of the distance from the long axis of the Longissimus dorsi muscle, starting from the midline.
The thickness was measured and recorded in millimeters with the help of a caliper.
Hump length and hump height. The dimensions of the hump indicate the approximate degree
of Bos indicus ancestry of the animals; the height of the hump was measured at the middle
of its base, taking as a reference the ligament of the nape of the neck; while the length of the
hump was measured in a straight line, from the beginning of the base to the end of the hump.
Marbling. The amount and distribution of intramuscular fat (marbling) in the M. longissimus
dorsi was evaluated after the half carcass was presented with a deep cross-section, with one
of the following categories being awarded: devoid, trace, slight, small, modest, moderate and
slightly abundant, stated in the norm NOM-004-SAGARPA-2018.
821
Determination of bone maturity. This variable was estimated visually on the carcass based
on the degree of ossification of the cartilages of the first three spinous processes below the
cut line with reference to the 12th and 13th ribs. The maturity values were determined
according to the bone maturity criteria established in NOM-004-SAGARPA-2018, which
refers to the Average Percentage of Ossification. It establishes maturity criteria A for cattle
from 9 to 30 mo of age; maturity B, for cattle from 30 to 42 mo of age, and maturity C, for
cattle over 42 mo of age.
Based on the variables of marbling and bone maturity, the Basic Grade of Bovine Carcass
Quality was estimated according to the following categories: Prime, Choice, Select, Standard
and Commercial, in accordance with the provisions of the norm NOM-004-SAGARPA-
2018. This standard establishes that once the physiological maturity and the degree of
marbling have been determined; these two factors must be considered in order to classify the
beef carcass, under the following integral classification system shown in Figure 1.
Figure 1: Integral classification of bovine carcasses based on the norm NOM-004-

SAGARPA-2018
Maturity
Degree of marbling Group A Group B Group C
(9 to 30 months) (30 to 42 months) (over 42 months)
Slightly abundant Prime
Moderate
Modest Choice
Low
Slight Select
Traces Commercial
Practically Standar
devoid
The hump height was used as a criterion to determine breed predominance, based on the
information shown in Table 1.
822
Table 1: Breed predominance based on hump height recorded in cattle processed in

processing plants in Mexico
Predominance of zebu cattle Hump height, cm (mean ± SE)
≤ ¼ Bos indicus 7.92 ± 0.95
½ Bos indicus 9.44 ± 0.17
¾ Bos indicus 13.13 ± 0.18
4/4 Bos indicus 14.07 ± 0.34
Source: Rubio et al(10); SE= standard error.
Carcass data were entered into a Microsoft Excel® spreadsheet, based on the hump height
described in Table 1, and each carcass was assigned to a group according to Bos indicus
predominance: group 1= ≥ zebu; 2= ½ zebu; 3= ¾ zebu; 4= zebu. Descriptive statistics were
obtained for the following variables: hot carcass weight (HCW), rib eye area (REA), back fat
thickness (BFT), renal pelvic fat (RPF), hump height (HH), hump length (HL), marbling, and
bone maturity. Next, the normality analysis of the values was performed with the
Kolmogorov-Smirnov test corrected by Lilliefors(11), with the R software(12). Analysis of
variance between groups was performed for the variables HH, HCW, and REA, and the
means were compared using Tukey's test. The variables marbling and bone maturity did not
have a normal distribution, so they are presented with descriptive statistics of the median and
interquartile range. In order to determine the distribution of basic quality grades and
marbling, the results are shown as absolute frequency (n) and percentage. The association
between groups with the degrees of marbling was carried out with the Chi-square test for a 5
x 4 contingency table (5 degrees x 4 groups). Because there was a statistical association, Chi-
square tests were then performed for permutations of 4 groups, taking 2 at a time (4P2) in
each degree of marbling. In the case of groups 3 and 4 in the commercial grade, Fisher's exact
test was used. In all statistical analyses, the maximum alpha level for statistical significance
was 0.05.
Results and discussion
Table 2 shows the results of the characteristics of beef carcasses from intensive finishing
pens and processed in Federal Inspection Type establishments.
823
Table 2: Characteristics of beef carcasses from intensive finishing and processed in Federal
Inspection Type establishments (n= 1,417)
Variable Mean SD Minimum Maximum CV (%)
HCW, kg 318.16 36.43 201.80 451.80 11.45
2
REA, cm 87.87 11.36 49.03 141.93 12.93
BFT, mm 6.70 3.73 1.0 33.0 55.67
CF, % 2.10 0.65 1.0 4.0 30.95
HL, cm 27.86 5.25 8.0 49.0 18.84
HH, cm 12.58 4.40 4.0 30.0 34.98
Marbling 300.00* 100** 100.0 500.0 25***
Maturity 100.00* 0** 100.0 400.0 0***
HCW= hot carcass weight; REA= rib eye area; BFT= back fat thickness; CF= cavitary fat; HL= hump length;
HH= hump height; SD= standard deviation; CV= coefficient of variation. *Medians; ** Interquartile range
(IQR). ***(IQR/Range)x100.
According to the information presented above, the characteristics that show the highest
coefficient of variation are dorsal fat thickness (55.67 %), hump height (34.97 %), and
cavitary fat (31.39 %). Back fat or subcutaneous fat thickness is related to body condition, as
well as to the energy reserves of the bovines(13). In the present study, the wide variation in
this value may be mainly due to the number of days that the bovines remain in the finishing
pens which, given the heterogeneity in terms of breed type, starting weight, body condition
and gender, determines the duration of their stay in intensive production units before being
processed in the slaughterhouses. In Mexico, Vázquez-Mendoza et al(14), observed
significant differences in the characteristics of the carcasses of bovines of different genotypes
finished in feedlots; precisely this variable is included in the USDA carcass classification
system, as is the percentage of renal, pelvic, and cardiac fat(15). The accumulation of cavitary
fat is influenced by a variety of factors, including the level of feed intake during fattening,
the energy concentration of the diet, the finishing time of the bovines in the feedlot, and the
use of muscle growth promoters(16).
The marbling and bone maturity variables used in the norm NOM-004-SAGARPA-2018 to
assign a quality grade to Mexican beef carcasses that were evaluated in the present study
showed disparate values; marbling exhibited a coefficient of variation of 35.18 %, while bone
maturity exhibited a coefficient of variation of 18.0 %. This indicates that the bone maturity
of the carcasses favorably impacts the assignment of a higher Basic Quality Grade according
to the integral classification of the beef carcass, but, to a certain extent, prevents them from
being classified with a higher quality grade, given the low level of marbling in the rib eye
area.
824
Table 3 shows the hump height values by group according to racial predominance. The mean
values between the four groups showed a statistical difference (P<0.01). It is worth noting
that the coefficient of variation for this variable in groups 1, 2, and 3 was less than 10 %,
indicating little dispersion of these values.
Table 3: Hump height by group as an indicator of Bos indicus breed predominance in

bovines from intensive finishing units and processed in Federal Inspection Type
establishments (n= 1,417)
Group n Mean (cm) SD (cm) CV (%)
≤ ¼ zebu 252 7.19 a 0.89 12.37
½ zebu 536 10.54 b 1.10 10.44
¾ zebu 399 14.38 c 1.13 7.89
zebu 230 20.11 d 3.07 15.28
SD= standard deviation; CV= coefficient of variation.
abcd
Means with different letters are significantly different (P<0.01).
According to Boleman et al(17), hump length indicates the approximate degree of Bos indicus
ancestry; bovines with a hump height above 10.2 cm will have phenotypic characteristics of
cattle with this racial predominance. To reaffirm the above, a study(18) determined that the
hump height in Bos indicus (Brahman) cattle ranged between 15 and 18 cm. In another
research(19), it was observed that, based on hump height, approximately 90 % of the beef
cattle population in Mexico have a strong Bos indicus genetic background. Thus, based on
the fact that groups 2, 3, and 4 have a mean hump height of over 10 cm, it can be deduced
that the genetic background of the zebu carcasses included in this study is 82 %.
Table 4 shows the values for hot carcass weight and rib eye area according to breed
predominance in cattle from intensive finishing units and processed in Federal Inspection
Type establishments. The results show that the mean HCW of group 1 is lower than that
recorded in groups 3 and 4 (P<0.01), but similar to the mean of group 2 (P<0.01).
825
Table 4: Hot carcass weight and rib eye area by group according to the racial
predominance of cattle from intensive finishing and processed in Federal Inspection Type
establishments (n= 1,417)
HCW REA
Group n Mean SD CV Mean SD CV
(kg) (kg) (%) 2
(cm ) 2
(cm ) (%)
a c
1 252 310.05 37.79 12.19 85.59 11.32 13.23
ab a
2 536 316.89 34.33 10.83 89.14 11.73 13.17
bc ab
3 399 319.91 35.73 11.17 88.31 11.11 12.59
c bc
4 230 326.99 38.83 11.88 86.68 10.49 12.11
HCW= hot carcass weight; REA= rib eye area; SD= standard deviation; CV= coefficient of variation. Group
1= ≤zebu; 2= ½ zebu; 3= ¾ zebu; 4= zebu.
abcd
Means with different letters are significantly different (P<0.01).
The HCW values of group 2 are similar to those of group 3, but the mean values of group 3
are equal to those of group 4 (P<0.01). In one experiment, hot carcass weights between 354
and 412 kg were recorded in Hereford x Angus bovines that received different levels of
zilpaterol during finishing(20). Cancian et al(21) recorded 292 and 321 kg of HCW in young
Nelore oxen and bulls, respectively. On the other hand, Huerta et al(8), recorded 272 kg of
HCW in predominantly zebu bovines. In another study(22), the carcass performance of
Brahman bulls and F1 crossbreeds fattened in tropical pastures was evaluated. Also, the
HCW in the Brahman breed was observed to be 242 kg, while it was 255 kg in F1 Angus 249
kg in F1 Chianina, 272 kg in F1 Romosinuano, and 252 kg in F1 Simmental bovines. These
values indicate that European-type racial dominance favorably influences the HCW;
however, the predominance of the zebu breed in the present study resulted in a better HCW.
In the values of rib eye area per group according to their racial dominance, a significant
difference (P<0.01) of 3.55 cm2 was observed when comparing groups 1 and 2, which
indicates that the presence of Bos indicus half-blood bovines improved the REA compared
to animals containing only ≤¼ Zebu blood. When comparing the animals with less zebu racial
predominance (group 1) against those with more racial predominance (group 4), no
significant differences were observed; this suggests that for the regions where this study was
carried out, crosses between Bos indicus and Bos taurus cattle are better for the REA variable
than purebred animals. The REA in cattle is an indicator of muscularity and an important
factor in determining yield grade; therefore, as the REA increases, retail product yield
increases. In this regard, Torrescano-Urrutia et al(23), carried out a study to characterize cattle
carcasses in the center of the state of Sonora, and found that the REA registered a range of
80.66 to 82.15 cm2; subsequently, in another study(24), values ranging from 69.2 to 89 cm2
were observed. The results of both experiments are for cattle finished in feedlots, so they are
similar to those recorded in the present study. However, the expression of the value of the
bovine HCW is influenced by the production system, as shown in the results of another study
previously referred to(22), which was carried out in a grazing system and shows the following:
826
the Brahman’s REA was 54.84 cm2; that of F1 Angus, 63.80 cm2; that of F1 Chianina, 61.06
cm2; that of F1 Romosinuano, 76.39 cm2, and that of F1 Simmental, 60.05 cm2. The values
shown therein are lower than those recorded in the present research, probably due to the type
of production system used, although the study indicates that crossbred animals improve the
REA, in agreement with the results of the present work.
Table 5 shows the distribution of beef carcasses by bone maturity and marbling. It was
observed that 1,191 carcasses (84.05 %) registered maturity A, i.e. it is estimated that they
belong to animals under 30 mo of age, while 226 carcasses (15.95 %) correspond to cattle
over 30 mo of age. With regard to the marbling grades, 1,339 bovine carcasses (94.48 %)
were described as Virtually Devoid, Traces and Slight. According to the integral
classification of the bovine carcass indicated in the norm NOM-004-SAGARPA-2018, this
may have contributed to the assignation of the Select and Standard basic quality grades to
the carcasses. However, their Basic Quality Grade may be lower when the Marbling indicator
is associated to the Bone Maturity factor.
Table 5: Distribution of bovine carcasses according to bone maturity and degree of

marbling
Bone maturity Marbling
Carcasses % Carcasses %
A 1,191 84.05 Virtually devoid 254 17.92
B 140 9.88 Traces 388 27.38
C 75 5.29 Slight 697 49.18
D 11 0.77 Small 74 5.22
Total 1,417 100 Modest 4 0.28
According to Lee et al(25), age is a fundamental factor in beef carcass grading systems when
combined with other factors, such as nutrition and genetics. One of the main factors affecting
carcass quality is marbling, which translates as stored body energy; therefore, this fat deposit
will augment as the age of the cattle and the energy density of the diet increase.
The presence of marbling in the Longissimus dorsi muscle depends on the genetic potential
of the bovine and the amount of energy consumed. Thus, young cattle that are processed at
15 mo of age have been shown to have equal or higher marbling scores than genetically
similar bulls processed between 18 and 24 mo of age when fed diets with a sufficient energy
density to allow marbling(26). The presence of marbling in the REA is of particular importance
in the grading system of the United States of America and northern Mexico(27) and is given a
unique value as it is related to the tenderness and palatability of the meat.
827
Table 6 shows the distribution of the degree of marbling in bovine carcasses by group
according to the predominance of marbling Bos indicus. The marbling of the REA in meat
producing animals is related to intramuscular fat content and plays an important role in
several aspects of meat quality.
The results of this study indicate that in 17.9 % of the bovine carcasses there was no marbling,
in 27.4 % there were traces of marbling, and in 49.2 % there was slight marbling. These
values show that the marbling of 94.49 % of beef carcasses has a low intramuscular fat
content, which has a direct impact on the classification of these carcasses. According to the
degree of marbling, it is observed that the nil marbling classification corresponds to ½ and ¾
carcasses of Bos indicus, occurring less frequently (P<0.05) than in ¼ carcasses or in over ¾
carcasses; as to the trace degree, the frequency increases for ½ and ¾ carcasses of Bos indicus
and decreases significantly in carcasses under ¼ and above ¾. Slight marbling occurred more
frequently in under ¼ carcasses of Bos indicus, being similarly frequent in ½ carcasses of
Bos indicus (P>0.05). There were no significant differences in the degree of "little" marbling,
which was found in similar proportions in all the groups of carcasses.
Table 6: Distribution of the degree of marbling in bovine carcasses by group according to

the predominance of Bos indicus
Degree of marbling, n (%)

Group Nil Traces Slight Small Modest*
1 58 (23.02) a 38 (15.08) b
140 (55.56) a
16 (6.35) a 0 (0)
2 69 (12.87) b 165) a 271 (50.56) ab 29 (5.41) a 2 (0.36)
3 63 (15.79) b 135 (33.83) a 184 (46.12) b 16 (4.01) a 1 (0.25)
4 64 (27.83) a 50 (21.74) b 102 (44.35) b 13 (5.65) a 1 (0.43)
Total 254 (17.9) 388 (27.4) 697 (49.2) 74 (5.2) 4 (0.3)
Group 1= ≤¼ zebu; 2= ½ zebu; 3= ¾ zebu; 4= zebu
ab
Different letters in the frequencies within the degree of marbling indicate significant difference (Chi-square
test 2 x 2; P<0.05)
* No analysis was performed due to expected frequencies under 5 in each cell.
Intramuscular fat content has been reported to vary between species, between breeds and
between muscle types within the same breed. Although there are other factors involved in
the variation of marbling in animals, including sex, age and diet, it has been indicated that
the variability in intramuscular fat content is linked mainly to the number and size of
intramuscular adipocytes, so the rate of intramuscular fat accumulation depends on the rate
of muscle growth. In animals that have a higher muscle content with high glycolytic activity,
they show a reduced intramuscular fat content(28). In this regard, the intramuscular fat content
analyzed by solvent extraction is stated to show a variation from 1.0 to 8.9 %; therefore, meat
is considered to be lean when it has less than 3.6 % of intramuscular fat(29). This agrees with
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the findings recorded in carcasses with little marbling and lean carcasses produced in tropical
region(19,30). Given the importance of the marbling score in the global beef market, studies
have been conducted to better understand the low marbling score in Bos indicus-influenced
cattle compared to Bos taurus cattle. In a review of these studies(31), it was observed that there
is no strong relationship between the ability to synthesize fatty acids de novo and the
marbling score or the adipocyte volume, concluding that the low marbling scores typically
observed in cattle with Bos indicus influence are mainly attributed to lower intramuscular
adipocyte volume compared to Bos taurus breeds.
Table 7 shows the distribution of Basic Grades of carcass quality, grouped according to cattle
breed predominance.
Table 7: Distribution of carcasses by basic quality grade by Bos indicus predominance
Basic quality grades

Group Prime Select Standard Commercial
n % n % n % n %
1 9 (3.57) a 117 (46.43) a 85 (33.73) c 41 (16.27) a
2 21 (3.92) a 256 (47.76) a 221 (41.23) b 38 (7.09) b
3 16 (4.01) a 181 (45.36) a 200 (50.13) a 2 (0.50) c*
4 14 (6.09) a 101 (43.91) a 115 (50.0) a 0 (0.0) c
*
Total 60 (4.23) 655 (46.22) 621 (43.82) 81 (5.72)
Probability 0.49 0.77 0.01 0.01
Group 1= ≤zebu; Group 2= ½ zebu; Group 3= ¾ zebu; Group 4= zebu.
abc
Different letters in the frequency percentages within the same basic quality grade indicate statistical
difference (2 x 2 Chi-square test); P<0.05); * Fisher's exact test (P = 0.5354).
In general, and regardless of breed predominance, there were 60 Prime quality carcasses
(4.23 %), 655 Select quality carcasses (46.22 %), 621 Standard quality carcasses (43.82 %),
and 81 Commercial carcasses (5.72 %). Of Group 1, with a lower predominance of the Bos
indicus breed, 3.57 % of the carcasses were identified as Prime, 46.43 % as Select, 33.73 %
as Standard, and 16.27 % as Commercial. A similar percentage distribution is observed
among the rest of the groups from lesser to greater Bos indicus predominance; as the number
of carcasses in each quality grade decreases or increases, the predominance of fattening is
greater or lesser from group 2 onward. Thus, for example, in the Select Basic Grade, there
are 256 carcasses in group 2,117 in group 1,181 in group 3, and 101 in group 4.
829
There are no previous studies with which these results can be compared, since they take as
reference the Mexican Official Norm that recently came into force in Mexico. However,
based on the now repealed Norm NMX-FF-078-2002, Zorrilla-Ríos et al(32) classified beef
carcasses produced in a tropical region of Mexico according to five criteria: maturity, age,
conformation, lean color, fat color, and distribution of subcutaneous fat cover. Carcasses
were classified as 13.4 % Select, 45.8 % Standard, 27.4 % Commercial, and 10.6 %
Unclassified. Based on this classification, no Prime category carcasses were recorded; in this
regard, the authors describe that 79 % of the carcasses attained the Prime classification grade
in the first instance, but when conformation was evaluated, only 0.5 % of the carcasses
attained the definitive grade of Prime.
Table 8 shows the distribution of Basic Quality Grades of beef carcasses categorized by sex,
age, and racial predominance. According to sex and age, in the group of females younger
than 30 mo and males younger and older than 30 mo, there was a greater distribution of
carcasses in the Select and Standard grades, and in the group of females aged more than 30
mo, there was a larger number of Commercial grade carcasses. In relation to the
predominance of the zebu breed, it may be observed that, as this breed component increases,
the number of carcasses in each category gradually decreases. This is attributed to the poor
development of intramuscular fat in zebu cattle compared to European cattle(19,33).
830
Table 8: Distribution of Basic Quality Grades of carcasses categorized by sex, age, and
group, according to racial predominance of cattle from intensive finishing units and
processed in Federal Inspection Type establishments (n= 1,417)
Basic Quality Grades
Sex Age Group Prime Select Standard Commercial Totals
n % n % n % n % n
1 2 4.55 33 75 9 20.45 0 0.00 44
<30 2 0 0.00 18 85.71 3 14.29 0 0.00 21
3 0 0.00 1 50.00 1 50.00 0 0.00 2
F 4 0 0.00 0 0.00 0 0.00 0 0.00 0
2 2.98 52 77.61 13 19.40 0 0.00 67
1 2 1.77 43 38.05 27 23.89 41 36.28 113
>30 2 2 2.82 20 28.17 11 15.49 38 53.52 71
3 0 0.00 5 41.67 5 41.67 2 16.67 12
4 0 0.00 0 0.00 0 0.00 0 0.00 0
4 2.04 68 34.69 43 21.94 81 41.32 196
1 5 5.38 39 41.94 49 52.69 0 0.00 93
<30 2 18 4.16 211 48.73 204 47.11 0 0.00 433
3 15 4.12 167 45.88 182 50.00 0 0.00 364
M 4 14 6.28 97 43.50 112 50.22 0 0.00 223
52 4.67 514 46.18 547 49.14 0 0.00 1113
1 0 0.00 2 100.0 0 0.00 0 0.00 2
>30 2 1 9.09 7 63.64 3 27.27 0 0.00 11
3 1 4.76 8 38.10 12 57.14 0 0.00 21
4 0 0.00 4 57.14 3 42.86 0 0.00 7
2 4.87 21 51.21 18 43.90 0 0.00 41
Totals 60 4.23 655 46.22 621 43.82 81 5.72 1417
F= females; M= males; <30: aged less than 30 months; >30: aged more than 30 months; Group 1=≤ zebu; 2=
½ zebu; 3= ¾ zebu; 4= zebu.
The cattle carcasses in this study had a mainly zebu breed component. Most of the carcasses
corresponded were classified as belonging to the "Select and Standard" basic grades, a large
831
number of them having a grade A bone maturity but low scores in the marbling grades; this
fact limited their classification to a better basic quality grade in accordance with the
provisions of the norm NOM-004-SAG/ZOO-2018, as an effect of the racial predominance
of Bos indicus.
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835
Article
The effect of hesperidin added to quail diets on blood gas, serum

biochemistry and Hsp70 in heat stress
Abdullah Özbilgina*
Aykut Özgürb
Onur Başbuğc
a
Sivas Cumhuriyet University Veterinary Faculty, Department of Animal Nutrition and
Nutritional Disorders. Sivas, Turkey.
b
Gaziosmanpaşa University. Artova Vocational School. Laboratory and Veterinary
Health Program. Tokat, Turkey.
c
Sivas Cumhuriyet University. Department of Veterinary Internal Medicine. Veterinary
Medicine Faculty. Sivas, Turkey.
*Corresponding author: abdullahozbilgin@gmail.com
Abstract:
The aim of this study was to determine the effects of flavonoid, which is a product of
citrus production, on blood parameters and HSP 70 concentration in quails applied at
thermoneutral and heat stress. In this study, 160 quails (Coturnix coturnix japonica,
male), 6 wk old and 150-200 g live weight, were housed in cages for 1 wk of exercise and
5 wk of trial period. The study design consists of 4 groups of 40 animals and 4 subgroups
with 10 animals in each group. Thermoneutral (24 ± 0.1 o C) groups are NC (0 g
hesperidin/kg basal feed) and NHES3 (3 g hesperidin /kg basal feed) and heat stress (34
± 0.1 oC) groups are HC (0 g hesperidin/kg basal feed) and HHES3 (3 g hesperidin /kg
basal feed) were randomly generated. In the case of heat stress, pO2, pH, HCO3, Cl
concentrations decreased in the HHES3 group compared to the HC group (P<0.05). ALP
enzyme concentration showed a significant decrease in the HHES3 group compared to
the HC group in the heat stress condition. Heat shock protein (HSP70) protein level
increased in blood serum, kidney, liver and thigh tissues in HC group with cellular stress
during heat stress; however, HSP70 concentration decreased significantly in the HHES3
836
group. As a result, positive effects of hesperidin supplementation in the diet were found
in both heat stress and thermoneutral conditions.
Keywords: Flavonoid, Quail, Thermoneutral, Heat shock protein, Hesperidin.
Introduction
Different environmental factors can cause stress in poultry farming. Environmental

temperature is an important factor in poultry production since it affects the performance
of the animal and causes economic problems(1-4). In general, the thermoneutral
temperature has been reported as 16-25 ˚C in poultry(5). It has been reported that
physiologically stress occurs if the ambient temperature remains above the thermoneutral
temperature(6). When exposed to stress, adrenocorticotropic hormone (ACTH) is secreted
depending on CRH that secretes from the hypothalamus. ACTH provides the secretion of
corticosteroids and adrenaline. Thus; glucose, lipid and protein metabolisms are regulated
by secreting high amounts of corticosteroids into the environment as metabolic adaptation
during heat stress(7-9). Metabolism, nutrition and environmental conditions are effective
on the acid-base balance of the body. The most important parameters that indicate the
acid-base state of the blood are blood pH, bicarbonate (HCO3-), and the concentrations of
sodium (Na+), potassium (K+) and chlorine (Cl−) ions. Monovalent minerals play an
important role for the acid-base balance(10-12). Animals maintain homeostasis under heat
stress conditions through vasodilatation, convection, and evaporation(13). Initially,
environmental stress factors alter metabolic functioning in poultry and causes the
production of glucose to maintain homeostasis during the presence of stressors. At heat
stress, air sacs play an important role in gas exchange, as they increase air circulation to
the surface which results in evaporation that causes heat to spread(14).
Due to stress, oxidation occurs in the structure of proteins and DNA in the blood and the
tissues. As a result of heat stress, an increase in heat shock proteins is observed(15). Heat
shock proteins (HSP) are a family of proteins produced by cells in response to stressors
that are or are not related to temperature(16). HSPs are an important family of proteins that
have been preserved throughout evolution and are expressed in all living things from
prokaryotes to eukaryotes. HSPs have performed tasks such as folding newly synthesized
proteins in the cell, preventing protein aggregation, stabilizing proteins, and eliminating
misfolded proteins. HSPs are divided into five main classes according to their molecular
837
mass: small HSPs (<40 kDa), HSP60 (60 kDa), HSP70 (70 kDa), HSP90 (90 kDa) and
HSP100 (100 kDa). Each HSP has different isoforms, and they are localized in different
parts of the cell. The Hsp70 molecular chaperone plays a central role in protein quality
control. By binding to Hsp70 protein substrates, they help them fold, break down,
transfer, regulate, and prevent clustering. Hsp70 substrate binds to hydrophobic regions
in proteins and helps the newly synthesized proteins and partially folded proteins to fold
correctly(17-21).
Previous studies reported that heat stress causes poor performance in the animal and
suppresses the immune system(22). Following the heat stress; decrease in live weight,
paleness in the color of meat(23), low immunity, fluid-electrolyte balance and irregularity
in blood pH(24), even cases such as sudden death can be observed in broilers. When heat
stress occurs in broilers, acid-base balance disturbance and respiratory alkalosis may
occur(25).
Hesperidin is an effective antioxidant that reduces oxidative stress. It also inhibits lipid
peroxidation(26,27). It has been reported that the concentration of lactate dehydrogenase
and heat shock protein (Hsp70), which are markers of heat stress, decreases with the
addition of hesperidin to poultry rations(28). It has been reported that in order to overcome
the negative effects of heat stress on Japanese quails, a good nutrition strategy should be
administered(29). Diets supplemented with hesperidin provide an alternative to the use of
synthetic additives, can improve the lipid profile of chicken meat, and ensure higher
quality poultry meat production(30,31). Additionally, recent studies have reported that the
contribution of hesperidin to the ration has positive effects on meat quality, egg quality
and intestinal micro flora in quails(32-34).
Heat stress has been shown to have adverse effects on broilers, including increased feed
consumption as well as reduced growth rate and vitality of broilers(35). In addition, it may
decrease the quality of the products obtained from broilers by increasing their abdominal
fat(36). In current study, the effects of hesperidin, a citrus by-product, on blood parameters
and HSP 70 levels will be determined.
In the study, 160 quails (Coturnix coturnix japonica, male) at the age of 6 wk with a live
weight of 150-200 g were housed in cages for 1 week of exercise and for 5 wk of
experimental period with 10 quails per cage, a total of 42 d. Quails (45cm width X 20cm
height X 90cm length) were housed in cages. The study design consists of 4 groups with
40 animals and 4 subgroups within each group. Thermoneutral (24 ± 0.1 ˚C) groups are
NC (0 g hesperidin/kg basal feed) and NHES3 (3 g hesperidin /kg basal feed) and heat
838
stress (34 ± 0.1 ˚C) groups are HC (0 g hesperidin/kg basal feed) and HHES3 (3 g
hesperidin /kg basal feed) were randomly generated. The hesperidin (C28H34015, cas no:
520-26-13, 91 % purity, Chem-Impex International Company, USA) used in the study
was commercially available. The rations used in the experiment were formulated
according to the recommendations of the NRC(37) (Table 1).
Table 1: Diet compositions used in the experiment

Diets****
Thermoneutral Heat stress
Ingredients, % NC NHES3 HC HHES3
Wheat 52.03 52.03 52.03 52.03
Maize 10.42 10.42 10.42 10.42
Vegetable oil 2.76 2.76 2.76 2.76
Soybean meal, %48 27.52 27.52 27.52 27.52
Limestone* 5.55 5.25 5.55 5.25
Dicalcium phosphate 1.17 1.17 1.17 1.17
Salt 0.26 0.26 0.26 0.26
Vitamin-mineral premix** 0.25 0.25 0.25 0.25
L threonine 0.03 0.03 0.03 0.03
Hesperidin*** - 0.30 - 0.30
Calculated values
Dry matter, % 90.30 90.30 90.30 90.30
Crude protein, % 19.96 19.96 19.96 19.96
Crude ash, % 9.80 9.50 9.80 9.50
Crude cellulose, % 2.86 2.86 2.86 2.86
Ether extract, % 4.56 4.56 4.56 4.56
Metabolic energy, kcal/kg 2900 2900 2900 2900
Calcium, % 2.50 2.38 2.50 2.38
Available phosphorus, % 0.35 0.35 0.35 0.35
Methionine +cystine, % 0.64 0.64 0.64 0.64
Lysine, % 1.00 1.00 1.00 1.00
Threonine, % 0.74 0.74 0.74 0.74
Tryptophan, % 0.27 0.27 0.27 0.27
* *Limestone was reduced and added instead of hesperidin in the experimenting groups.
**Vitamin-Mineral premix contained per kg: mg: retinol (vit A) 3, tocopherol (vit E) 30, menadione (vit
K3) 5, thiamine (vit B1) 1, riboflavin (vit B2) 5, pyridoxin (vit B6) 3, nicotinic acid 30, pantothenic acid
10, folic acid 0.8, ascorbic acid (vit C) 10, choline chloride 450, Co 0.2, I 0.5, Se 0.3, Fe 25, Mn 120, Cu
10, Zn 100; μg: cholecalciferol (vit D3) 62.5, cobalamin (vit B12) 20, biotin 100 μg.
*** Hesperidin obtained from Chem-Impex Int. company, molecule formula (C28H34O15), cas no (520-
26-13), purity grade 91% (Chem-Impex, Wood Dale, IL, USA).
****NC= Control (0g hesperidin/kg feed), (24 ± 0.1 ˚C); NHES3: thermoneutral temperature (24 ± 0.1
˚C), (3g hesperidin/kg feed); HC= heat stress temperature (34 ± 0.1 ˚C); HHES3= heat stress temperature
(34 ± 0.1 ˚C), (3g hesperidin/kg feed).
In the study, granular feed and water were given ad libitum to animals. During the study
period, there was a relative humidity of 50-60 % in the cages. Fluorescent lamps were
used for the lighting of the trial room, and a timer (Cata CT 9181, China) was used during
the trial to provide a 16-h light 8-h darkness along with sunlight. In order to create heat
stress in the cages, electric heaters were used for heating up the compartments. The trial
839
cage was kept at the room temperature, while the group subjected to stress using the
electrical thermostat control heaters during the trial period was kept at 34 ± 0.1 ˚C, and
the quails in the thermoneutral group were kept at 24 ± 0.1 ˚C. During the experimentation
period, the relative humidity of the trial room was constantly measured with a hygrometer
and kept under control. Electric fans were used to regulate air circulation and get rid of
the accumulated dust and harmful gases in the cages.
Ethical approval
This study has been conducted with the permission of Tokat Gaziosmanpaşa University,
Animal Experiments Local Ethics Committee dated 20.05.2021 and numbered 51879863-
36.
Biochemical analysis of blood gas and serum
At the end of the trial, 3 animals were randomly selected from each subgroup, which
equals to 12 from each group and a total of 48 overall. Blood samples from the Vena
saphena brachialis were taken before slaughter, and the blood gas values were
determined by photometric method using a commercial kit (epoc BGEM blood test,
Germany). Immediately after the blood samples were taken, they were centrifuged for 10
min at 3,000 rpm, and then the serum collected at the top was transferred to 2 ml
Eppendorf tubes. The serums were frozen and stored for analysis in a freezer at -80 °C.
Biochemical values were detected in blood serum samples using an autoanalyzer device
(Mindray BS200, China).
Hsp70 gene expression analysis
At the end of the study, tissue samples of 2-3 g were taken from liver, kidney, and breast
muscles from each animal under hygienic conditions. The tissue and blood samples were
then stored at -80 °C for Hsp70 gene analysis. After 0.9 ml of physiological saline was
added to the 0.1 g tissue sample weighed, the tissue samples (0.1 g) were homogenized
in a homogenization buffer (0.15 M NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1
mM PMSF, 0.1 M). E-46, 0.08 µM of aprotinin, 0.1 µM of leupeptin and 0.1% NP-40(38)
and homogenates were centrifuged at 4 °C for 20 min at 12,000 ×g using an Ultra-turrax
840
homogenizer on ice. The supernatant was collected and stored at -20 °C until protein
determination. The amount of protein was determined using ELISA (BT-LAB,
E0124Ch). The standard curve and immunological detection of proteins have been carried
out mainly according to the manufacturer's instructions.
The data were expressed as mean ± standard error and the significance level was tested
with Oneway ANOVA. The difference between the groups was determined by the
Bonferroni and Tamhane’s T2 multiple comparison test with a significance confidence
interval of P<0.05.
Results
Within the scope of the experiment, there is a statistically significant difference between
thermoneutral and heat stress groups in terms of pCO2, pO2, pH, HCO3, Na, K, Cl
concentrations among the blood parameters (P<0.05); however, all groups yielded the
same results in terms of hematocrit and hemoglobin (P>0.05). In blood gas parameters,
pCO2 was the lowest in heat stress group HC; it was highest in the HHES3 group
(P<0.05). pO2 was highest in the HC group, which is the heat stress group, and lowest in
the HHES3 group (P<0.05). Blood pH was the lowest in the HHES3 group; it was highest
in the HC group (P<0.05). The blood Na and Cl concentrations were the lowest in the
heat stress groups; was highest in thermoneutral groups (P<0.05). The K concentration
was the lowest in the HC group and the highest in the HHES3 group (P<0.05) (Table 2).
841
Table 2: The effect of adding hesperidin to quail diets at thermoneutral and heat stress
on blood gas parameters
Thermoneutral (24 ˚C) Heat stress (34˚C)
NC NHES3 HC HHES3 p
Hgb, g/dL 12.34±0.53 12.40±0.50 10.80±0.07 11.20±0.58 0.06
b b c a
PCO2, mmHg 37.99±0.18 38.13±0.14 31.24±0.06 48.85±2.20 0.001*
c ab a d
PO2, mmHg 46.81±0.69 50.16±0.50 52.36±0.02 40.09±1.16 0.001*
Hct, % 36.53±1.62 36.26±1.45 31.81±0.30 32.70±1.66 0.05

pH 7.42±0.01b 7.41±0.001b 7.53±0.01a 7.31±0.02c 0.001*
HCO3, mmol/L 23.84±0.01b 24.13±0.06b 25.92±0.44a 24.60±0.03b 0.001*
Na, mmol/L 162.97±0.74a 159.23±0.56b 144.41±0.15d 147.85±0.83c 0.001*

K, mmol/L 4.55±0.09b 4.73±0.05b 3.94±0.01c 5.93±0.17a 0.001*
a b c c
CI, mmol/L 129.35±1.33 118.81±1.79 109.00±0.001 108.48±0.14 0.001*
Hgb= hemoglobin, PCO2:= partial carbon dioxide pressure, PO2= partial oxygen pressure, Hct=
hematocrit, pH= potential of hydrogen, HCO3= hydrogencarbonate, Na= natrium, K= potassium, Cl=
chloride.
*There is a statistically significant difference between the experimental groups (P<0.05).
In addition, serum alkaline phosphatase (ALP) enzyme concentration in the blood serum
parameters was the lowest in the HHES3 group; was highest in the HC group (P<0.05);
however, all groups are similar in terms of other parameters (Table 3).
Table 3: Effects of adding hesperidin to quail diets at thermoneutral and heat stresss on
blood serum parameters
Thermoneutral (24 ˚C) Heat stress (34 ˚C)

NC NHES3 HC HHES3 P
Glucose, mg/dl 169.62±30.94 144.50±32.89 204.44±35.20 165.39±23.14 0.59
Triglyseride, mg/dl 1156.98±69.06 983.02±176.00 1225.63±3.60 1022.87±141.10 0.48
HDL, mg/dl 59.40±20.54 26.83±13.27 81.90±17.21 43.98±11.74 0.20
Total cholestrol,
313.03±43.29 262.43±15.81 268.06±18.46 256.43±10.26 0.33
mg/dl
Total protein, mg/dl 4.52±0.24 4.69±0.27 5.32±0.51 4.72±0.16 0.31
Albumin, g/dl 1.90±0.11 1.87±0.06 1.79±0.06 1.79±0.08 0.69
Globulin, g/dl 3.40±0.41 2.82±0.21 2.94±0.13 2.73±0.16 0.23
ALT, u/l 6.50±0.92 5.67±0.21 8.44±1.09 6.14±1.03 0.17
AST, u/l 194.00±22.52 181.83±10.64 216.33±17.64 199.86±26.28 0.67
ALP, u/l 845.93±161.17ab 622.83±154.0b 1272.91±146.42a 507.16±67.60b 0.001*
Ca, mg/dl 26.21±3.11 22.17±3.18 21.36±1.14 22.40±2.67 0.60
Mg, mg/dl 7.11±0.44 6.91±0.51 6.77±0.27 6.88±0.30 0.94
P, mg/dl 11.60±1.11 12.03±1.46 12.27±0.81 12.68±0.89 0.90
HDL= high density lipoprotein, ALT= alanine transaminase: AST= aspartate transaminase: ALP=
alkaline phosphatase, LDH= lactate dehydrogenase, Ca= calcium, Mg= magnesium, P= phosphor.
*There is a statistically significant difference between the experimental groups (P<0.05).
842
In terms of HSP 70 parameter, it was the lowest in thigh tissue in thermoneutral groups
(P>0.05). Concentration was similar in all tissues in thermoneutral groups (P>0.05). In
the heat stress groups, the serum concentration was highest in the HC group, but lower in
the HHES3 group (P<0.05). Concentration in liver and kidney tissues was high in the HC
group under heat stress, while it was significantly lower in the HHES3 group. In addition,
in the heat stress groups, the Hsp70 concentration in the liver tissue was the lowest in the
HHES3 group (P<0.05) (Figure 1).
Figure 1: Protein expression level of the Hsp70 in experimental groups
Discussion
There are many studies on the effect of heat stress on the addition of vitamins, amino
acids and minerals in poultry feed(3,5). Current study was conducted to observe the effects
of hesperidin, a flavonoid included in the diet, on blood biochemistry and expression of
Hsp70 in quails exposed to heat stress.
Depending on the increase in environment temperature, some changes occur in the blood
and metabolism. In case of rapid breathing, high loss of carbon dioxide, a decrease in
partial CO2 pressure (pCO2) in the blood and an increase in blood pH occur.
Hyperventilation alters the acid-base balance in poultry through the development of
respiratory alkalosis(39). In the current study, pCO2 pressure was lowest in the HC group
while pO2 pressure was highest. While the highest pCO2 pressure was seen in the HHES3
group, the pO2 pressure was the lowest. While pCO2 and pO2 pressures, which were
similar in the thermoneutral group, increased in the heat stress groups, pCO2 increased
as expected, the pO2 decreased. Attia et al(3) reported that the addition of amino acids to
the ration in broilers at heat stress was slightly higher blood pH in the heat stress control
group. Depending on the ambient temperature, the highest pH in the HC group and the
843
decrease in pH in the HHES3 group may be associated with hesperidin supplementation.

In current study, a decrease in blood pH level below the neutral pH level (7.35), an
increase in pCO2 to 48 mmHg, and a decrease in PO2 to 40 mmHg in the HHES3 group
at heat stress is seen as a table of respiratory acidosis. Additionally, blood HCO3
concentration is highest in the HC group, while it is at the level of thermoneutral groups
in the HHES3 group. No compensation effect on blood pH due to the addition of
hesperidin has been observed.
While the blood hemoglobin concentration was lowest in the HC group, it approached
that in the thermoneutral groups in the HHES3 group. In previous studies, it has also been
reported that the hemoglobin concentration in the blood that occurs at normal temperature
tends to decrease due to an increase in heat stress(40,41).
In general, Na, K and Cl concentrations are important for blood acid base balance in terms
of pH. The blood pH rises with the formation of respiratory alkalosis. In the current study,
blood pH was highest in the HC group from the heat stress groups, as expected, depending
on the alkalosis status. However, blood pH shifted to neutral pH in the HHES3 group,
which was thought to be related to the contribution of hesperidin. Contrary to the
thermoneutral groups, the Na concentration, which is an important cation, is not expected
to be the lowest in the HC group. Likewise, the K concentration in the HC group has also
showed a decrease. Although Na and K concentrations showed a general similarity in the
normal and heat stress groups in current study, higher blood K and lower blood Cl
concentration have been observed in the HHES3 group with a low pH. Similarly, in a
study conducted on the effects of heat stress on dairy cattle, it has been reported that a
decrease in Na and K concentrations in the rumen fluid causes urinary excretion of Na
and loss of K in the skin(42). The normal interval for the concentration of chlorine in the
blood is between 97 and 107 mEq/L. It has been reported that, when stress occurs in the
body, electrolyte levels may become irregular; hence, an increase in the chlorine
concentration in the blood occurs(43). However, in current study, while the blood chlorine
concentration is above the normal levels at thermoneutral groups, it is thought that the
blood chlorine concentration of an animal under heat stress had hit the upper limit as a
result of compensation.
It has been reported that gluconeogenesis is stimulated by increasing the number of free
radicals in the environment due to heat stress, secreting the ACTH and cortisol hormones,
and preventing insulin release from β-cells in the pancreas; thus, increasing the serum
glucose levels(44). Rudich et al(45) have reported in their study that oxidative stress
conditions negatively affect insulin secretion. In current study, it has also been determined
that the blood glucose level is lower at thermoneutral groups than it is at HC group. As a
result, as in previous studies(46,47), blood glucose concentration increased in the HC group
under heat stress; and hesperidin in the NHES3 group and the lowest concentration in the
HHES3 group in heat stress compared to the HC group. It has been reported that stress
caused by the administration of adrenocorticotropic hormone (ACTH), one of the stress
hormones, increases the blood glucose, cholesterol, and high-density lipoprotein (HDL)
844
levels, but reduces the triglyceride level(48). Moeni et al(49) reported that chromium
contribution to broiler rations reduces blood triglyceride, cholesterol and LDL levels but
increases cholesterol and HDL concentrations.
As reported in previous studies, it was observed that the blood triglyceride concentration
was highest in the HC group under heat stress and close in the HHES3 group, while it
was close in the NHES3 group. That means that the concentration of triglycerides in the
blood has increased due to the heat stress. According to Rashidi et al(47), this increase in
the level of lipids in the blood is due to heat stress, a decrease in feed consumption, and
the provision of energy needs by mobilization of lipid resources. On the other hand, in
current study, they reported that the addition of organic chromium and selenium to the
ration decreased serum lipid content, similar to the decrease in serum total cholesterol
and triglyceride levels in the NHES3 and HHES3 groups compared to the HC group. It
was observed that the blood HDL concentration in thermoneutral groups was lower in the
NHES3 and HHES3 groups compared to the NC group under heat stress, compared to the
HC group. As a result, it is believed that the HDL ratio decreased with the addition of
hesperidin.
Additionally, in support of the current work, Moeini et al(49) reported that, when stress
was created at heat stress (33 ± 3 ˚C), the total cholesterol level decreased in the trial
groups where organic chromium was added compared to the control group, depending on
the increase in the dose. Similarly, in the current study, the total cholesterol concentration
in NHES3 group decreased without dependence on heat stress. The group with the
additional hesperidin, which is at a heat stress, has the lowest cholesterol level. It is
believed that the decrease in total cholesterol concentration in the HHES3 group
compared to both the normal and the heat stress control group occurred due to hesperidin
addition of 3 g/kg and the dosage.
Oxidative stress caused by heat stress increases the production of free radicals, which
leads to oxidation of the cell membrane, lipid peroxidation that leads to hepatocellular
damage, increase in the intracellular enzyme levels, which include aspartate
aminotransferase (AST) and Lactate dehydrogenase (LDH). There is a statistically
significant difference between normal and heat stress groups in terms of blood serum ALP
enzyme levels (P<0.05). In the current study, the blood serum concentration of alkaline
phosphatase (ALP) enzyme in the HC group under heat stress increased due to heat stress.
However, a significant decrease observed in both the NHES3 group in the thermoneutral
groups and the HHES3 group, which is the heat stress group, may be due to hesperidin
supplementation. In general, the concentrations of ALT, AST, ALP and LDH enzyme
have been observed to change at both thermoneutral and heat stress groups due to the
contribution of hesperidin. Mehaisen et al(50) observed a similar increase in ALT, AST
enzyme concentration in the heat stress control group due to the addition of propolis to
the ration on heat stress. In the same study, it was reported that the ALT and AST enzyme
levels were decreased with the addition of propolis in the trial groups in a similar way
they were in the current study. The results obtained in current study are consistent with
845
previous studies(51,52). In current study, the AST level has been observed to be slightly
higher at thermoneutral groups than the heat stress groups. However, AST data obtained
in the heat stress study conducted by Abdelhady et al(53) have reported a lower
concentration than current study.
In Figure 2, it was observed that the enzyme level of LDH was lower in heat stress groups
compared to thermoneutral groups, but both NHES3 and HHES3 groups were lower than
NC and HC groups. Similarly to current study, Al-Mashhadini et al(54) have reported that
the use of sesame oil on animals exposed to heat stress has reduced the blood LDH
enzyme concentration in the group fed with additional sesame oil compared to the control
group at normal temperature, that the LDH enzyme concentration increased due to the
stress effect in the control group at heat stress, and that the LDH enzyme concentration
was found to be lower in the group fed with additional sesame oil than in the control
group. Additionally, there are multiple studies reporting that the blood LDH
concentration increases due to heat stress in poultry exposed to 41-42 ˚C
temperature(55,56).
Figure 2: Lactate dehydrogenase enzyme level at thermoneutral and heat stress
In current study, total protein levels have been observed to have a little bit higher
concentration at thermoneutral than at heat stress (Table 3). The addition of hesperidin to
the ration in HHES3 group increased the protein level. The total protein level increased
under HC groups in heat stress, but a decrease has been observed in the HHES3 group
with the additional hesperidin. A high level of total protein at heat stress is associated
with an increase in the concentration of heat shock proteins (Hsp70)(57,58).
The albumin concentration in the blood serum were a similar concentration at

thermoneutral groups as the heat stress groups. As a result, except for a slight decrease in
the albumin level from thermoneutral to heat stress, it is believed that the hesperidin
addition does not have a positive effect on the albumin concentration in the blood. Effect
846
of vitamin E on heat stress, Şahin(59) has reported that heat stress inhibited the total, there
is a similar situation in terms of globulin. A lower level of globulin was found at heat
stress than normal temperature.
Among the recent studies on heat stress, Al-Mashhadani et al(54) have determined the
effect of sesame oil on heat stress and found that the concentration of albumin in the blood
increased towards heat stress group; however, the group with heat stress and sesame oil
has presented a decrease, like current study. Known as a molecular chaperone, Hsp70 is
a protein that has been preserved throughout evolution and it is produced by the cells of
all living things in response to stress stimuli. Hsp70 levels are quite high at the first times
when cellular stress commences. Hsp70 is vital in all stages of cell metabolism, including
growth, differentiation, division, and even cell death. In particular, heat stress and the
amount of ROS that increases accordingly disrupt the 3-dimensional structures and
stability of proteins in cells, leading to their denaturation. Cellular stress factors in the
cytosol complicate the protein folding process. Therefore, protein quality control is
necessary for the cell to maintain its viability. Hsp70 has functions such as correct folding
of newly synthesized protein chains, inter-membrane protein translocation, inhibition of
protein aggregation, and targeting decayed proteins for degradation. Thus, Hsp70 has
been recognized as an important biomarker for increasing Hsp70 expression levels to
maintain cellular integrity in cases of increased stress in the cell and for monitoring heat
stress and ROS that increases accordingly(17-19). In previous studies, it has been reported
that quercetin and several other flavonoids inhibit the induction of Hsp70 caused by
cellular level heat shock at the level of mRNA accumulation(60). Budagova et al(61)
reported that quercetin, one of the natural flavonoids of the in vitro cell response to heat
stress-induced stress, completely inhibits the synthesis and intracellular accumulation of
heat shock protein (Hsp70) in response to hyperthermia. Kim et al(62) has reported that
fisetin, a dietary flavonoid, can inhibit HSP activity, interact with cancer cell
proliferation, and induce apoptosis in their study. Xu et al(63) has reported in their study
that quercetin may have a cytoprotective role that can act through a mitochondrial
pathway during heat stress exposure. In the current study, there was a decrease in Hsp70
levels in quail blood serum, kidney, liver and thigh tissues in the HHES3 group compared
to the HC group in the heat stress groups. However, Hsp70 level in liver, kidney and thigh
tissues in thermoneutral groups was similar to that of the NC group. According to these
results, it is thought that hesperidin supplementation in cases of heat stress has a great
potential as an important contribution to reducing the increased stress in tissues due to
heat increase, similar to previous studies.
As a result, prevention and treatment of diseases using phytochemicals, in particular

flavonoids, are well known. Fruits and vegetables are natural sources of flavonoids.
847
Various flavonoids found in nature have their own physical, chemical and physiological
properties. These substances are more widely used in developing countries. As a result of
the study, when hesperidin, a flavonoid, was added to the food, compared to the HC group
in the HHES3 group, it caused an improvement in hemoglobin, pO2, pH, HCO3, Cl
concentrations in case of heat stress. In addition, in the case of heat stress; blood glucose,
triglyceride, HDL and total cholesterol concentrations decreased in the HHES3 group
compared to the HC group. ALP enzyme concentration showed a significant decrease in
the HHES3 group compared to the HC group in the heat stress condition. Hsp70 protein
level increased in blood serum, kidney, liver and thigh tissues in HC group with cellular
stress during heat stress; however, Hsp70 concentration decreased significantly in the
HHES3 group. It is thought that the use of Hesperidin, which is a supplement added to
the feed in heat stress, may offer a potential nutritional strategy to overcome the harmful
effects of stressors in poultry farming.
Acknowledgments and conflict of interest
This study received no funding. The authors have no relevant financial or non-financial
interests to disclose. All authors contributed to the study conception and design. Material
preparation, data collection and analysis were performed by [Abdullah Özbilgin], [Onur
Başbuğ] and [Aykut Özgür]. The first draft of the manuscript was written by [Abdullah
Özbilgin] and all authors commented on previous versions of the manuscript. All authors
read and approved the final manuscript.
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854
Article
Anthelmintic evaluation of four fodder tree extracts against the nematode

Haemonchus contortus under in vitro conditions
Itzel Santiago-Figueroa a
Alejandro Lara-Bueno b
Roberto González-Garduño c
Pedro Mendoza-de Gives d
Edgar Jesús Delgado-Núñez e
Ema de Jesús Maldonado-Simán b
Yagoob Garedaghi f
Agustín Olmedo-Juárez d*
a
Universidad Nacional Autónoma de México. Facultad de Estudios Superiores Cuautitlán.
Cuautitlán Izcalli, Estado de México, México.
b
Universidad Autónoma Chapingo. Posgrado en Producción Animal. Chapingo, Estado de
México, México.
c
Universidad Autónoma Chapingo. Unidad Regional Universitaria Sur Sureste. Teapa,
Tabasco, México.
d
Instituto Nacional de Investigaciones Agrícolas, Forestales y Pecuarias. Centro Nacional de
Investigación Disciplinaria en Salud Animal e Inocuidad. Carr. Fed. Cuernavaca-Cuautla
8534, Jiutepec 62574, Morelos, México.
e
Universidad Autónoma de Guerrero. Facultad de Ciencias Agropecuarias y Ambientales.
Iguala, Guerrero, México.
f
Islamic Azad University. Faculty of Veterinary Medicine, Department of Parasitology.
Tabriz, Iran.
*Corresponding author: olmedo.agustin@inifap.gob.mx, aolmedoj@gmail.com
855
Abstract:
The objective was to evaluate the nematocidal effect of four hydroalcoholic extracts (HAE)
of Brosimum alicastrum (HAE-Ba), Guazuma ulmifolia (HAE-Gu), Erythrina americana
(HAE-Ea) and Leucaena leucocephala (HAE-Ll) against Haemonchus contortus. The tests
of egg hatching inhibition (EHI) and larval (infective larvae) mortality were used. The
treatments were HAEs at concentrations of 6.25-50 mg/mL for EHI and 25-100 mg/mL for
larval mortality, ivermectin (5 mg/mL, positive control) and distilled water (negative
control). Data were analyzed using an ANOVA and treatments with a concentration-
dependent effect were subjected to a regression analysis to determine lethal concentrations
(LC50 and LC90). In addition, a phytochemical analysis was performed on the extracts to
identify the presence of the main secondary metabolites. The best ovicidal and larvicidal
activity was observed in HAE-Gu with 96.78 % EHI at 6.25 mg/mL and 57.2 % larval
mortality at 75 mg/mL. Followed by HAE-Ba showing 90 % EHI at 6.25 mg/mL and 58.0 %
larval mortality at 75 mg/mL. The LC50 and LC90 of HAE-Gu on EHI were 2.7 and 4.4
mg/mL, respectively. While the LCs of this same extract on larvae were LC50= 64 and
LC90= 125 mg/mL. The phytochemical analysis indicates that all extracts contain tannins,
coumarins, flavonoids and terpenes. The fodder species G. ulmifolia and E. americana could
be candidate plants for the control of H. contortus.
Keywords: Fodder trees, Secondary metabolites, Haemonchus contortus, Larval mortality,

Egg hatching inhibition.
Introduction
In tropical regions, gastrointestinal nematodes (GIN) represent a serious problem in small

ruminants; and to reduce the impact that these organisms have on animals, it is necessary to
perform some type of treatment(1). Haemonchus contortus is a hematophagous nematode with
the highest prevalence worldwide in sheep and goats, which affects their health(2,3). This
parasite causes different alterations in its host, including reduced growth rate, anemias and
can cause sudden death(3). The main method for the control of GIN in small ruminants,
including H. contortus, is through the use of broad-spectrum anthelmintics such as
benzimidazoles, macrocyclic lactones, imidazothiazoles and more recently the amino-
856
acetonitrile derivative. The inappropriate and excessive use of these antiparasitics has
triggered a problem of multiple anthelmintic resistance worldwide(4).
In small ruminant production systems under grazing conditions, the use of tree species with
fodder potential represents a viable option for their feeding, because they contain a rich
source of energy and protein(5). It has been determined that these tree species contain
secondary metabolites, so they could have anthelmintic activity(6). Among the best-known
tree species are: Brosimum alicastrum, which contains 14 to 17 % crude protein (CP)(7,8);
Guazuma ulmifolia, which contains 17 % CP(9,10); Erythrina americana, which provides 14
to 18.9 % CP(11,12); and Leucaena leucocephala, which provides 23.4 to 33.2 % of CP,
depending on the age of regrowth and the season of the year(13,14). Some secondary
metabolites have been identified in these tree species; for example, in the foliage of B.
alicastrum, phenols such as gallic acid are reported(15), G. ulmifolia presents phenols such as
caffeic acid, chlorogenic acid and flavonoids such as catechin, quercetin and luteolin(16).
Erythrina americana contains alkaloids (erysotrine) in seeds, flowers and foliage(17) and
phenols such as hydrolysable tannins(12). For its part, L. leucocephala contains flavonoids
such as quercetin, kaempferol, luteolin, among others(18).
Due to the presence of these secondary metabolites in the foliage of these plants and their
availability in tropical regions, it is interesting to know their effect on the GINs of small
ruminants; however, there is limited information on some of these plants. In sheep fed G.
ulmifolia for 30 days, a highly significant decreasing trend (P<0.001) was found in the count
of eggs per gram of feces(19), while the methanolic extract of the seed of E. americana exerts
a nematocidal and insecticidal effect on Panagrellus redivivus and Anopheles sp.,
respectively(20-22). On the other hand, aqueous extracts of L. leucocephala and G. ulmifolia
showed inhibitory effect of egg hatching of 50 % at 1.25 mg/ml on GINs from sheep(23). In
order to know the effect of B. alicastrum, G. ulmifolia, E. americana and L. leucocephala,
on the nematode H. contortus, hydroalcoholic extracts were evaluated on eggs and infective
larvae of the parasite H. contortus under in vitro conditions.
Fodder samples
The collection of plant material was carried out in the Huasteca Potosina region, located in
the state of San Luis Potosí. This region has a subhumid climate with rains in summer(24).
Leaves and stems of mature trees aged 3, 12, 20 and 30 yr for L. leucocephala, G. ulmifolia,
857
B. alicastrum and E. americana, respectively, were collected. It should be noted that the
material collected were non-senescent leaves and stems. The collection was carried out
during the months of June to October 2017. The material was then dried in a forced air oven
and ground to a particle size of 0.5 cm.
Hydroalcoholic extract
Each tree species was macerated with a hydroalcoholic solution, placing 300 g of the dried
and ground plant material in a solution of 70 % water and 30 % methanol and it was left to
macerate for 24 h. Each extract was then filtered to remove the plant material. After obtaining
the liquid part, the solvents were removed by distillation under reduced pressure using an R-
300 rotary evaporator (BUCHI, Switzerland) until semisolid extracts were obtained. Then
each extract was frozen at -80 ° C for 24 h and finally they were brought to total dryness by
lyophilization processes and stored at -40 ° C until further use.
Qualitative analysis of secondary compounds of extracts
The chemical profile of the hydroalcoholic extracts was determined by following different
phytochemical procedures using reference compounds(25). The identification of alkaloids was
performed using the technique by Dragendorff, Mayer and Wagner(25). The presence of
coumarins was determined with the Bornträger test, while the flavonoid content was
determined with the Mg2+ and HCl test(26,27). The ferric chloride, saline and gelatin test was
used to identify tannins(28,29). The identification of terpenes was determined using the
Liebermann-Burchard and Salkowski tests and foam formation was the indicator used to
identify the presence of saponins(27).
Biological material
Eggs and larvae of Haemonchus contortus were obtained from a donor sheep free of
gastrointestinal nematodes, of three and a half months of age and 22 kg of live weight,
previously artificially infected with a monospecific strain of the parasite under study (strain
INIFAP-HcIVMr-SAI). The sheep was housed in an elevated individual cage provided with
alfalfa, commercial feed and freely accessible water. The lamb was cared for following health
and welfare care according to the standard NOM-062-ZOO-1999.
858
Collection of H. contortus eggs
Feces were collected directly from the rectum of the infected animal. Subsequently, they
were washed with clean water through sieves of different diameters (240, 150, 120 and 30
μm) and the suspension of the last sieve was collected in 15 mL Falcon tubes containing the
parasites. Then the tubes were centrifuged at 3,500 rpm for 5 min (three times) in order to
obtain eggs free of fecal residues. Finally, they were quantified by aliquots to verify a
concentration of 100 ± 15 eggs in an aqueous suspension of 50 μL(30).
Obtaining of infective larvae (L3) of H. contortus
The L3 were obtained by stool cultures of the donor animal. The feces collected from the
animal were kept moist at room temperature for 7 d. After the required time, the larvae were
recovered using the Baermann technique(31). The L3 obtained were stored in culture dishes at
4 °C. Prior to performing the bioassays, the L3 were suspended in hypochlorite (187 μL
chlorine and 4,813 mL of distilled water) for 5 min so that they unsheathed. Then the L3 were
washed with distilled water three times by centrifugation (3,500 rpm for 5 min).
Subsequently, different dilutions were made until obtaining 100 ± 15 L3 contained in 50 μL
of an aqueous suspension.
Egg hatching inhibition (EHI)
Bioassays were performed on 96-well microtiter plates. Each extract was evaluated
individually in triplicate considering four repetitions per replication (n= 12). The HA-Es of
the four tree species were evaluated at concentrations of 50, 25, 12.5 and 6.25 mg/mL. In
addition, each bioassay included distilled water was as a negative control and ivermectin (5
mg/mL) as a positive control. Fifty microliters of an aqueous suspension containing 100 ±
15 eggs were added to each well and then 50 μL of extract at the required concentration or
controls were added as appropriate. The plates were incubated in a wet chamber at 25-30 °C
for 48 h. After this time, the number of eggs and larvae in each well was counted (Motic®
10x microscope). The percentage of egg hatching inhibition (%EHI) was determined by the
following formula:
%EHI = [(number of eggs)/(number of larvae + number of eggs)] x 100
859
Larval mortality
Bioassays were performed on 96-well microtiter plates (n=12). Each extract was evaluated
individually in triplicate considering four repetitions per replication (n=12). The treatments
were the extracts at different concentrations (100, 75, 50 and 25 mg/mL). Ivermectin (5
mg/mL) and distilled water were used as positive and negative controls, respectively. An
aqueous suspension of 50 μL containing 100 ± 15 L3 was added to each well and then 50 μL
of the treatments was added as appropriate. The plates were incubated in a wet chamber at
25-30 °C for 48 h. Subsequently, the live and dead larvae contained in each well were
quantified based on the criteria described by Olmedo-Juárez et al(32). The percentage of larval
mortality (LM) was determined by the following equation:
(number of dead larvae)

%LM = [ ] x 100
number of live larvae + number of dead larvae
The percentages of EHI and LM were previously normalized using the square root and
analyzed by ANOVA under a completely randomized design with the general linear model
(PROC GLM) of the SAS statistical package version 9.0(33). The comparison of means was
performed using the Tukey test at a significance level of 0.05. Treatments with concentration-
dependent effect were subjected to a regression analysis to determine lethal concentrations
50 and 90 (LC50 and LC90) using the PROC PROBIT system of the SAS statistical
package(33).
Results
Egg hatching inhibition and larval mortality
Table 1 shows the results of the ovicidal and larvicidal activity of the HAE of B. alicastrum
on the nematode H. contortus. This activity was different (P<0.05) in each concentration
evaluated, obtaining the greatest inhibitory effect of egg hatching at 50 mg/mL. On the other
hand, in the larval mortality test, only a mortality percentage of 29 % was achieved at 100
mg/mL.
860
Table 1: Percentage of egg hatching inhibition (%EHI) and mortality of infective larvae
(L3) of Haemonchus contortus caused by a hydroalcoholic extract of Brosimum alicastrum
Average eggs and Average live and
%EHI % Mortality
Treatments larvae dead larvae
± SD ± SD
Eggs Larvae Dead Live
Distilled water 2.9 138.2 2.07 ± 1.0f 2.8 74.5 4.6 ± 4.3c
a
Ivermectin (5 127.2 0.8 99.9 ± 0.2 139.3 0 100a
mg/ml)
HAE-Ba
(mg/ml)
100.0 --- --- --- 34.8 87.7 29.0 ± 11.1b
75.0 --- --- --- 32.3 94.1 26.1 ± 7.6b
50.0 92.8 21.1 81.4 ± 3.5b 14.3 103.6 14.4± 14.4b
c
25.0 82.0 36.6 69.1 ± 5.1 11.3 121.6 8.5 ± 5.2c
12.5 75.5 43 63.8±2.4d --- --- ---
6.25 70.0 56.1 55.5 ± 1.7e --- --- ---
Coefficient of variation 0.62 23.1
R2 0.99 0.95
Standard error of the mean (SEM) 0.04 0.15
P value <0.001 <0.0001
HAE-Ba= Hydroalcoholic extract of Brosimum alicastrum. ---= not evaluated. SD= standard deviation.
a-f
Means with different literal within the same column indicate a difference (P<0.05).
The HAE of G. ulmifolia exhibited an ovicidal effect close to 100 % from the concentration
6.25 mg/mL, being statistically equal to that obtained with ivermectin up to the concentration
of 12.5 mg/mL (Table 2). A similar effect was observed using the HAE of E. americana at
concentrations of 50, 25, and 12.5 mg/mL (Table 3).
861
Table 2: Percentage of egg hatching inhibition and mortality of infective larvae (L3) of
Haemonchus contortus caused by a hydroalcoholic extract of Guazuma ulmifolia
Average eggs Average live
%EHI % Mortality
Treatments and larvae and dead larvae
± SD ± SD
Distilled water 5.5 135.3 2.07 ± 1.0f 1.9 153.8 1.1 ±1.8e
Ivermectin (5 127.3 0 99.9 ± 0.2a 158.1 0 100a
mg/ml)
HAE-Gu (mg/ml)
100.0 --- --- --- 109.7 18.5 85.9 ± 7.4b
75.0 --- --- --- 85.3 60.5 57.2 ± 15.5c
50.0 118.8 0.4 99.5 ± 0.7ab 43.5 103.8 26.8± 15.0d
25.0 112.5 2.3 97.8 ± 2.8ab 11.3 138 7.7 ± 4.9e
12.5 120.8 0.75 99.4± 0.9ab --- --- ---
b
6.25 113.4 3.9 96.78± 5.3 --- --- ---
2
R 0.99 0.95
P value <0.001 <0.0001
SD= standard deviation; HAE-Ba= Hydroalcoholic extract of Guazuma ulmifolia. ---= not evaluated.
a-f
862
Haemonchus contortus caused by a hydroalcoholic extract of Erythrina americana
Average eggs Average live and %
%EHI
Treatments and larvae dead larvae Mortality
± SD
Eggs Larvae Dead Live ± SD
c
Distilled water 5.9 134.0 4.1 ± 2.0 3.0 96.3 3.6 ±2.9d
Ivermectin (5 mg/mL) 111.5 0.2 99.7± 0.6a 145.4 0 100a
HAE-Ea (mg/mL)
100.0 --- --- --- 93.4 49.1 60.0±
13.5b
75.0 --- --- --- 102.7 50.1 58.0 ± 24.8b
50.0 111.4 2.6 97.0 ± 7.5ab 86.8 49.2 62.6± 10.2b
25.0 86.4 0.5 99.5 ± 0.7a 50.3 93.3 35.8± 7.3c
12.5 91.3 2.0 97.7 ± 2.6a --- --- ---
6.25 94.0 9.3 88.8 ± --- --- ---
ab
19.0
2
R 0.94 0.89
P value <0.0001 <0.0001
HAE-Ba= Hydroalcoholic extract of Erythrina americana. ---= not evaluated. SD= standard deviation.
a-d
The highest larvicidal activity (85 % LM) of the extract of G. ulmifolia was achieved using
the highest concentration (100 mg/ml). While the HAE of E. americana only caused 60 %
mortality at the same concentration. On the other hand, the results obtained with the HAE
from L. leucocephala showed the highest percentage of EHI (83.2 %) when the concentration
of 50 mg/mL was used. And for LM, only 63 % was achieved using 100 mg/ml of the HAE
(Table 4).
863
Haemonchus contortus caused by a hydroalcoholic extract of Leucaena leucocephala
Average live
Average eggs
%EHI and dead % Mortality
Treatments and larvae
± SD larvae ± SD
c
Distilled water 7.7 131.3 5.6 ± 3.5 4.7 122.0 5.2 ±2.9d
Ivermectin (5 112.5 0.1 99.9 ± 0.2a 145.4 0 100a
mg/mL)
HAE-Ll (mg/mL)
100.0 --- --- --- 75.7 40.0 63.0± 22.9b
75.0 --- --- --- 27.6 99.5 21.7 ± 8.4c
50.0 97.0 20.4 83.2 ± 12.4a 13.0 95.2 12.0± 2.1cd
25.0 53.9 66.8 48.9 ± 31.7b 7.5 114.2 6.2± 2.9d
12.5 50.5 65.9 48.4 ± 35.3b --- --- ---
b
6.25 44.4 65.9 45.9 ± 38.6 --- --- ---
2
R 0.59 0.93
P value <0.0001 <0.0001
HAE-Ba= Hydroalcoholic extract of Leucaena leucocephala. ---= not evaluated. SD= standard deviation.
a-d
Lethal concentrations (LCs)
The Cs 50 and 90 required to cause EHI and larval mortality are shown in Table 5. The
regression analysis indicated that the extracts with the best inhibitory effect on egg hatching
were HAE-Ea (LC50=0.16 mg/mL and LC90=4.41 mg/mL) and HAE-Gu (LC50=2.7
mg/mL and LC90=4.4 mg/mL). Regarding larval mortality, the best treatment was observed
in HAE-Gu with LC50 and LC90 of 64.0 and 125.2 mg/mL, respectively.
Identification of secondary metabolites
The phytochemical analysis showed the presence of secondary metabolites in the four plant
extracts, such as tannins, coumarins, saponins, alkaloids and flavonoids (Table 6).
864
Table 5: Lethal concentrations (LC50 and LC90) of hydroalcoholic extracts of four fodder tree species required to inhibit egg hatching
and kill infective larvae (L3) of Haemonchus contortus at 48 hours
% Egg hatching inhibition % Mortality of infective larvae (L3)
Plant LC50 CI 95% limits LC90 CI 95% limits LC50 CI 95% limits LC90 CI 95% limits
(lower-upper) (lower-upper) (lower-upper) (lower-upper)
HAE-Ba 4.8 (3.88-5.70) 197 (145.6-293.1) 187.8 (156.67-2.70.6) 608.7 (376.7- ..)
HAE-Gu 2.7 (2.6-2.8) 4.4 (2.62-2.80) 64.0 (62.45-65.66) 125.2 (119.6-132.0)
HAE-EA 0.16 (0.04-0.38) 4.1 (2.8-5.4) NA --- NA ---
HAE-LL 17.9 (16.8-19.1) 201.9 (167.6-251.0) 93.12 (91.61-94.71) 124.5 (119.6-131.36)
CI= confidence interval. NA= not active. HAE-Ba= Brosimum alicastrum, HAE-Gu= Guazuma ulmifolia, HAE-Ea= Erythrina americana.
865
Table 6: Results of the qualitative phytochemical analysis of the hydroalcoholic extracts

Hydroalcoholic extract (HA-E)
Colorimetric
Metabolite Brosimum Guazuma Erythrina Leucaena
reaction
alicastrum ulmifolia americana leucocephala
Dragendorff - - - +
Alkaloids Mayer - - - +
Wagner - - - ++
Coumarins Borntraeguen - + + +
2+
Flavonoids Mg and
- - + +
HCL
Ferric
+++ +++ +++ +++
chloride
Gelatin
- - - -
Tannins solution
Gelatin and
- - - -
saline
Saline +++ +++ +++ +++
Liebermann-
Triterpenes/ - + - +
Burchard
Steroids
Salkowski + + + +
Saponins Foam
+ - + ++
formation
(-) Not detected (+) positive light reaction (++) positive reaction (+++) strong positive reaction.
Discussion
Natural products obtained from plants rich in secondary metabolites have been evaluated for
different medicinal purposes, such as antioxidants, antimicrobials and antiparasitics(34-36).
The four hydroalcoholic extracts evaluated in the present study exhibit nematocidal activity
against Haemonchus contortus, a hematophagous parasite of greater prevalence in sheep and
goats, which affects their health. There are few studies on the use of Brosimum alicastrum as
an anthelmintic, although it is an abundant resource in tropical regions; the extract of
acetone:water (70:30) on H. contortus larvae has been observed to inhibit 95 % of the ability
to unsheathe at a concentration of 1.2 mg/mL(37). While in the present study, using extract
based on methanol:water, 187.8 mg/mL was required to cause 50 % mortality. On the other
hand, an acetonic extract of G. ulmifolia has been shown to exhibit ovicidal activity on
Cooperia punctata, another parasitic nematode of cattle, inhibiting up to 70 % of hatching at
a concentration of 9.6 mg/mL(38). Likewise, an ethanolic extract (100 mg/mL) of this plant
866
species has shown nematocidal effect on Pheritima posthuma(39). In a recent study, a

hydroalcoholic extract of G. ulmifolia has been shown to exhibit significant ovicidal effect
(90 % EHI) at a concentration of 0.50 mg/mL(40). The ovicidal activity reported in the present
study with the hydroalcoholic extract of G. ulmifolia indicates that a higher concentration
(LC50=4.4 mg/mL) than reported by the previous work is required. This could be explained
by the fact that a plant species collected in a different region was used and probably the
content of bioactive compounds could be different between both plant species. Although in
the present work it has been reported that G. ulmifolia contains some secondary compounds
such as tannins, flavonoids, coumarins and terpenes, it is very important to know the content
of each of these compounds to relate them to anthelmintic activity. On the other hand, in vivo
studies have also been conducted in kids artificially infected with infective larvae of H.
contortus, which were fed with 10 % of G. ulmifolia foliage and no differences were obtained
in the count of eggs per gram of feces (EPG) compared to the control group(41). The same
results were observed in Pelibuey ewes fed with 30 % of G. ulmifolia, however, a highly
significant trend (P<0.001) towards the decrease of EPG was observed in these ewes(19).
It is known that species of the genus Erythrina have a wide variety of alkaloids that have
been identified and are attributed a neuromuscular blocking effect(20), in addition, the use of
methanolic extract on Daphnia magna turned out to be highly toxic(21), so the nematocidal
effect found in the present study could be attributed to those compounds. A methanolic
extract of E. variegate has been evaluated against crustaceans of the genus Artemia, as well
as earthworms (Eisenia foetida) and parasitic helminths of birds such as Ascardi galli and
Raillietina spiralis and mortality was reported in these biological models using
concentrations of 10 mg/mL(42,43). On the other hand, in a study conducted in Pelibuey sheep
fed with E. americana foliage, no changes in egg count were observed during the
experimental phase(12).
The LCs 50 and 90 for B. alicastrum in gastrointestinal nematode larvae reported in another
study were 291.6 and 666.6 mg/mL, respectively(44), which were similar to those reported in
the present study (187.8 and 608.7 mg/mL). Regarding G. ulmifolia, the results of the present
study indicate that, to inhibit 50 % of the hatching of H. contortus eggs, 2.2 mg/mL of the
hydroalcoholic extract is required, while in another study with an extract of acetone:water
(70:30) of G. ulmifolia against C. punctata, it was 8.84 mg/mL(38). In the same study, the
authors report a LC50 of 11.77 mg/mL of the extract of acetone:water 70:30 of L.
leucocephala(38). In the present research work, the LCs calculated for the HAE of the leaves
of this tree species were higher (LC50=52.8 and LC90=308 mg/mL) respectively (Table
5)(45). The LC of E. americana on H. contortus has not been previously reported, however,
for the species E. variegata, on crustaceans of the genus Artemia, the LC50 was 3.99
mg/mL(43), a value higher than that of the present study (0.19 mg/ml).
867
Some secondary metabolites such as tannins, saponins and coumarins have been identified
in the bark and leaves of B. alicastrum(46,47). In the present study, the chemical profile in the
extract of B. alicastrum indicated the presence of tannins and saponins. On the other hand,
saponins, cyanogenic glycosides, phenols and steroids, which were also found in the present
study, have been reported qualitatively in G. ulmifolia(48). In other species of the genus
Erythrina, they have been reported to contain secondary metabolites similar to those found
in the hydroalcoholic extract of E. americana. E. variegate has been reported to contain
alkaloids, saponins and flavonoids(43). In another study in E. americana from Tabasco,
Mexico, high levels of tannins have been identified(12). The secondary metabolites reported
in L. leucocephala depend on the type of extract; for instance, saponins, phenols, tannins,
terpenes, among others, have been identified in aqueous and ethanolic extracts, similar to the
profile found in this study(45,49-51).
It is concluded that the hydroalcoholic extract of the four trees studied may be an option for
the control of Haemonchus contortus in small ruminants, especially G. ulmifolia and E.
americana. It is recommended to continue with their study to identify the active compounds
in each case.
Acknowledgements
The authors thank the National Council of Humanities, Sciences and Technologies for the
financing during the period of Doctoral Studies of the main author (grant number: 429558).
The authors declare that they have no conflict of interest.
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Article
Effect of treated wastewater use on soil and forage crops of Chenopodium

quinoa Willd and Zea mays L.
Ana Lilia Velasco-Cruz a
Vicente Arturo Velasco-Velasco a*
Judith Ruíz-Luna a
José Raymundo Enríquez-del Valle a
Aarón Martínez-Gutiérrez a
Karen del Carmen Guzmán-Sebastián a
a
Tecnológico Nacional de México. Instituto Tecnológico del Valle de Oaxaca. Nazareno,
71230, Xoxocotlán, Oaxaca, México.
*
Corresponding author: vicente.vv@voaxaca.tecnm.mx
Abstract:
Given the scarcity of water resources for agricultural use, it is necessary to promote the use
of wastewater for agriculture. The towns of Capulálpam de Méndez and Ixtlán de Juárez in
the State of Oaxaca have anaerobic wastewater treatment plants (WWTP). The
morphological growth, biomass production and N and P content were evaluated in two forage
species —Chenopodium quinoa Willd and Zea mays— irrigated with treated wastewater
(TWW). A complete randomized design (CRD) was established in each municipality, given
the homogeneity of the soil, using a 2 x 2 factorial arrangement, i.e., two forage crops
(Quinoa and corn) and two types of irrigation (fresh water and treated wastewater), with 4
replicates per treatment. Analyses of variance and Tukey mean tests (P≤0.05) were
performed for the studied variables. In the soils, the pH level was "moderately acid" to
"neutral" (5.1 to 7.3); the EC indicated "negligible effects of salinity"; organic matter was
found at intervals of "medium to high", and the texture was sandy clay loam in Ixtlán and
874
clay loam in Capulálpam. Growth variables (plant height, stem diameter, and number of
leaves) and biomass were significantly higher in plants irrigated with treated wastewater in
both forage crops. Nitrogen and phosphorus contents were significantly higher in quinoa and
corn plants receiving TWW. TWW could be an alternative that would help reduce the use of
chemical fertilizers, as it is an important source of nutrients in forage crops.
Keywords: Biomass, Fresh weight, Dry weight, Water treatment.
Introduction
In the world, more than 70 % of fresh or potable water withdrawals are related to the
agricultural sector(1), and in Mexico, 76 %. Part of this, approximately 29 %, is used for
growing fodder crops(2). One of the most widely cultivated species is fodder corn, due to the
high energy value it provides to livestock(3), whose function is to generate proteins for human
consumption. Protein content can be considered a valuable unit of measurement for
comparing foods(4). The Food and Agriculture Organization of the United Nations (FAO) has
estimated that, in order to overcome the problem of water scarcity or lack of water, at least
20 million hectares of agricultural land are irrigated with untreated or partially treated
wastewater(5). Thus, land irrigated with wastewater amounts to 10 % of the total distributed
in fifty countries around the world. The World Health Organization (WHO) and FAO attach
importance to the use of treated wastewater (TWW) in agricultural irrigation, as well as to
the switch from freshwater to treated or reused wastewater. TWW contains essential nutrients
for crops and, for irrigation purposes, counteracts environmental and health risks(5,6,7).
Forages are highly water-demanding and are an indirect human consumption product. The
use of TWW is a way of guaranteeing water for the future; it is a process of sustainability
and a small step towards the productivity of local agroecosystems. For this reason, it is
advisable to establish fodder crops close to the sites where the wastewater treatment plants
are located. Quinoa (Chenopodium quinoa Willd) as forage has the advantage of being
cultivated at altitudes ranging from sea level to 4,000 m asl; it tolerates frost and drought and
adapts to different regions with acidic and alkaline soils (pH 4 to 9), and its nutritional value
lies in the ideal balance of amino acids in its protein, which makes it an ideal component in
diets(8).
875
Corn is the world's most widely grown agricultural product(9). By 2025, it is estimated that
60 % of the global corn consumption will be destined for animal feed, and that this percentage
will grow at an average annual rate of 1.8 %, driven by the expansion of livestock in
developing countries(10). In Mexico, corn is utilized as fodder, grain, stubble, silage, and for
industrial uses (tamale leaves), and it is one of the main irrigated crops with untreated
wastewater(11).
In order to ensure that TWW does not pose a risk to soil, crops, and human health, it is
recommended to use wastewater that has passed through a treatment plant(12,13). The use of
TWW in crops will save costs, protect the aquifers, and make fresh water available to the
population. Therefore, the purpose of this study was to evaluate the morphological growth,
biomass production and N and P content in two forage species —Chenopodium quinoa Willd
and Zea Mays— irrigated with treated wastewater (TWW) in Ixtlán de Juárez and
Capulálpam de Méndez, Oaxaca, Mexico.
Study area
The study was conducted in the "Sierra Norte" of Oaxaca, Mexico, located in the sub-
province of the Southern Sierra Madre of Mexico, and in the hydrological region RH28,
"Papaloapan", the second largest watershed in the country(14). The research was carried out
in the towns of Ixtlán de Juárez and Capulálpam de Méndez. The municipality of Ixtlán
de Juárez (17° 20' N, 96° 29' W), is located at an altitude of 2,030 m asl; the climate is C
(w) temperate sub-humid, and the area has a rugged orography; the average rainfall is 900
to 1,100 mm per year; the average annual temperature is 20 oC(15), and the soil type is
humic Acrisol (HA). The municipality of Capulálpam de Méndez (17° 18' N, 96° 27' W)
is located at an altitude of 2,040 m asl; the climate is classified as C (w2) (w) b (i ') g
temperate sub-humid; the average precipitation is 1,115 mm per year and occurs between
June and October; the average annual temperature is 15.2 oC(16); the soil type is cambisol.
Experimental design and sowing
A complete randomized design (CRD) was used in each municipality (Ixtlán de Juárez and
Capulálpam de Méndez). The treatments consisted of a 2 x 2 factorial, i.e. the two forage
876
crops (Chenopodium quinoa Willd and Zea Mays L) and two types of irrigation: fresh water
(DW) and treated wastewater (TWW), with four replicates per treatment. The study was
established in March 2017. The cultivated plot in Ixtlán was established in an area of 300 m2
(20 x 15 m) divided into four sections of 60 m2 each (15 x 4 m), with a slope of 3 %. The
cultivated area in Capulálpam was 400 m2 (20 x 20 m) divided into four subplots of 80 m2,
with a slope of 1 %. The cultivated area of both Ixtlán and Calpulálpam was subdivided into
two parts: in the first subplot, sown with quinoa, was irrigated with freshwater and treated
wastewater; in the second section, corn was grown and also irrigated with treated wastewater
and freshwater (FW). The subplots were divided by five unsown furrows for the application
of the types of irrigation. The soil was prepared with a tractor, the distance between furrows
of both crops was 80 cm, drawn parallel to the slope. The quinoa variety Ontifor was sown
by hand continuously in the bottom of the furrow, at a depth of less than 3.0 cm
approximately, with an approximate density of 450,000 plants ha-1(8,17). Corn (creole Zea
mays) was planted by hand continuously at the bottom of the furrow for forage, at a depth of
approximately 4.0 cm, with a density of 60,000 plants ha-1.
Obtainment of treated wastewater (TWW)
The wastewater treatment plants (WWTPs) receive all the domestic water from the same
localities; Ixtlán receives a flow rate of 3.3 L s-1 and Capulálpam receives a flow rate of 1.0
L s-1. The WWTPs have a pretreatment system based on grids of different diameters, which
are located at the inlet of the reception channel to retain solid wastes, such as soda caps, hair,
wood, PET, etc. The water passes through the sand trap (3.0 m long channel), where the first
process of sedimentation of solids takes place. The wastewater enters the biodigesters by
gravity; each drop has a free fall of 3.10 m (this is where most of the sludge settles) and, by
laminar flow, the water reaches the tubular sedimentation area (inner tank) and gradually
rises until it overflows into the biodigester area (outer tank). The biodigester area is composed
of polyethylene fabrics to facilitate the accommodation of anaerobic bacteria, which is why
they are called hosts. Bacteria generally form granules at the apexes of the hosts, and they
take care of the biodigestion of the wastewater, transforming it into treated (biodigested)
wastewater. The process takes 14 h.
Obtainment of fresh water (FW)
In Capulálpam, fresh or clean water was extracted directly from a well and piped to the plot.
In Ixtlán de Juárez, fresh water was obtained from the potable water system. Neither synthetic
877
fertilizers nor pest or disease control substances were applied. Crops were established during
the dry season following the World Health Organization's Guidelines and Recommendations
for the Safe Use of Wastewater in Agriculture(6). Irrigations with fresh water and with treated
wastewater were carried out at the same time, for five days, using 2" diameter hoses to flood
the furrows.
Soil analysis
Soil samples were collected from each study site for analysis(18). Four soil samples were
obtained at the beginning stage, four at the intermediate stage, and four at the end of the
cultivation process, adding up to a total of 12 samples in the cultivated area of Ixtlán.
Likewise, a total of 12 soil samples were extracted from the cultivated area of Capulálpam.
The initial samples were taken before planting and irrigation, and the intermediate and final
samples, 45 and 90 d later, respectively. Two kg of each sample were collected at a depth of
30 cm from each site. The pH and electrical conductivity (EC) were determined with a
potentiometer (Conductronic PC45), organic matter (Walkley and Black method), texture
(Bouyoucos method), nitrogen (by micro Kjeldahl), and phosphorus (Bray and Kurtz 1
method), the latter using a UV-Vis spectrophotometer (GBC CITRA10).
Measurement of plant growth variables
For each treatment, ten Quinoa plants and six corn plants of an easy-to-handle size were
randomly selected from each crop, discarding the plants on the edges. Their height was
measured with a flexometer from ground level to the last main leaf; their stem diameter was
measured with a vernier at 3.0 cm from the ground, and the number of leaves on each plant
was counted. Variables were read at 60 and 90 d in quinoa and corn, respectively. The plants
were harvested after 90 d to obtain forage because more than 50 % of the plants no longer
showed increased growth in height and stem diameter. To account for biomass, four plants
in each treatment were destructively sampled and leaves, stems and roots were separated 90
d after planting. The fresh material was placed in forced air circulation ovens at 65 oC until
a constant weight was reached. The fresh and dry weight of each plant was obtained. The
nitrogen content was measured with an organic elemental analyzer (PerkinElmer, series II,
model 2400), and the phosphorus content, by the vanadomolybdic method in a UV-Vis
spectrophotometer (GBC, model CITRA10)(19).
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Analyses of variance and Tukey's mean tests (P≤0.05) were performed on the data obtained
in the field for the variables studied in the quinoa and corn crops. The effect of the factors
and treatments, i.e., the type of irrigation on both crops established at each site, was
evaluated.
Results and discussion
Physical and chemical properties of soil
The pH of the soils before crop establishment was "moderately acidic", i.e. pH 5.1 - 6.5(19);
at 45 and 90 d after planting. In some cases, the pH increased to a "neutral" level (pH 6.6 -
7.3); such pH increase could be due to nitrates being transformed to atmospheric nitrogen
(N2)(20). This condition is contrary to that of other studies where soils irrigated with
secondary treatment water show decreases in pH of 8.2 to 7.6(21) and 8.0 to 7.7(22). The
electrical conductivity (EC) in the soils before planting was 0.31 to 0.44 dS m-1, which,
according to the norm NOM-021-2000, is considered as "negligible salinity effects" (< 1.0
dS m-1) in both the Ixtlán and Capulálpam soils(23,24). 45 and 90 d after planting, the EC
increased mainly when irrigated with TWW, still within the "negligible effects of salinity";
only two soil samples increased to 1.05 and 1.27 dS m-1, the latter value being categorized as
"very slightly saline" (1.1 – 2.0 dS m-1). The cause of this could be the fact that soils retain
cations, expressed as cation exchange capacity (CEC), due to the increase in clay and organic
matter(25). For the same reason, an increase of a few tenths was observed in soils irrigated
with TWW. This confirmed the findings of other researchers in the sense that soil EC
increased when TWW irrigation was applied: 0.34 – 0.42 dS m-1(21) and 2.73 – 4.70 dS m-
1(22)
.
A similar trend was obtained with soil organic matter. In the initial sampling, the values
indicated a "medium" (1.6 - 3.5 %) to "high" (3.6 - 6.0 %) content; after sowing and irrigation
(45 d) the percentages increased in both crops and remained in the same "medium" and "high"
ranges. In this regard, some researchers indicate that wastewater contributes organic matter
to the soil, helping to maintain soil fertility(24,26,27). However, in the final stage (90 d), the
organic matter content decreased. In general, TWW improves soil fertility by providing
nutrients and other benefits to crops, thereby reducing the use of chemical fertilizers(28). The
soils have average fertility, average erosion capacity, and average organic matter
879
mineralization capacity, all easy to manage for the farmer. The region where these localities
are located has a rugged orography.
Table 1: Quantification of pH, electrical conductivity, and organic matter in soils as a

function of locality, crop, irrigation water type, and sampling time
Soils of Ixtlán Soils of Capulálpam
Quinoa Corn Quinoa Corn
Parameter Sampling time FW TWW FW TWW FW TWW FW TWW
pH Initial 5.39 5.38 5.33 5.43 5.40 5.65 5.23 5.35
Intermediate 6.62 6.26 6.92 6.08 5.93 5.57 5.47 5.73
Final 6.75 6.25 6.57 6.65 6.21 5.31 5.69 5.78
EC, dS m⁻¹ Initial 0.31 0.32 0.33 0.31 0.39 0.36 0.44 0.37
Intermediate 0.57 0.71 0.68 0.74 0.49 1.05 0.47 0.58
Final 0.56 0.77 0.84 0.7 0.46 1.27 0.39 0.51
Organic Initial 2.44 1.89 2.99 2.68 4.23 3.40 5.23 4.07
matter, % Intermediate 2.79 2.85 4.31 4.52 3.65 2.94 4.70 3.59
Final 2.22 3.52 2.41 3.00 3.42 1.90 3.30 3.93
Texture Loamy clayey sandy Loamy clayey
FW= fresh water; TWW= treated wastewater; EC= electrical conductivity; Initial=samples before planting;
Intermediate= 45 d after planting; Final= 90 d after planting.
Plant growth
Quinoa, a domesticated Andean crop, belongs to the Amaranthaceae family and is also
considered a forage crop(29). Corn belongs to the Poaceae family, a crop with different uses
such as fodder for livestock(30). The type of growth and vegetative development differs
between the two forage species. An analysis of variance showed significant differences
(P≤0.01) in the factor "type of water" for the variables height, stem diameter, and number of
leaves in quinoa plants, at 60 and 90 d after planting; the same occurred in corn plants except
for the variable number of leaves.
Plant height, stem diameter, and number of quinoa leaves were significantly higher (P≤0.05)
when irrigation was with treated wastewater at 60 and 90 days after planting (Table 2). The
results for corn were similar (Table 3), except for the number of leaves, which showed no
significant difference, which means that the number of leaves was similar when the plants
were irrigated with treated wastewater and with freshwater. Plant height was significantly
greater (P≤0.05) in plants that received treated wastewater. The results show that treated
880
wastewater should no longer be seen as a waste product, but as a water resource that can be
treated and reused productively(12).
Table 2: Quinoa growth at 60 and 90 days after planting in two locations irrigated with
freshwater and treated wastewater
Water Height SD No. of Height SD No. of
Site type (cm) (cm) leaves (cm) (cm) leaves
----------- 60 days --------- ---------- 90 days ------------
b b b
Ixtlán FW 73.52 0.44 59 136.92 b 0.60 b 80 b
TWW 125.73 a 0.75 a 92 a 175.5 a 0.94 a 113 a
Capulálpam FW 29.15 c 0.35 b 48 b 57.61 c 0.44 c 62 b
TWW 117.42 a 0.87 a 113 a 143.75 b 0.95 a 123 a
x̅ FW 51.34 b 0.40 b 54 b 97.27 b 0.77 b 71 b
x̅ TWW 121.58 a 0.81 a 103 a 159.63 a 0.95 a 118 a
FW= fresh water; TWW=treated wastewater. SD= stem diameter.
acb
Values with the same letter within each column and within each crop are not different (P≤0.05).
Table 3: Corn growth at 60 and 90 days after planting in two locations irrigated with
freshwater and treated wastewater
Water Height SD No. of Height SD No. of
Site type (cm) (cm) leaves (cm) (cm) leaves
---------- 60 days -------- --------- 90 days ----------
b b b
Ixtlán FW 111.70 2.11 11 255.91 b 2.31 b 14 a
TWW 174.08 a 2.40 a 13 a 340.22 a 2.57 a 14 a
Capulálpam FW 74.37 c 1.91 b 9c 157.72 c 1.92 c 11 b
TWW 124.29 b 2.58 a 11 b 248.91 b 2.66 a 12 b
x̅ FW 93.04 b 2.01 b 10 b 206.81 b 2.12 b 13 a
x̅ TWW 149.19 a 2.49 a 12 a 294.57 a 2.62 a 13 a
FW= fresh water; TWW=treated wastewater. SD= stem diameter;
abc
Values with the same letter within each column and within each crop are not different (P≤0.05).
Biomass
An analysis of variance showed significant differences (P≤0.05) in root, leaf, and stem fresh
and dry weight of the studied quinoa and corn plants when irrigated with fresh water versus
treated wastewater. The fresh and dry weights of the quinoa and forage corn crops were
significantly higher (P≤0.05) in plants irrigated with treated wastewater (Tables 4 and 5).
The fresh and dry weight of the organs of both crops was root < leaves < stems. In quinoa
plants irrigated with treated wastewater, the total fresh weight of the leaves + stems were 3.4
881
times higher, and the dry weight was 3.2 higher. Likewise, the fresh weight of corn was 2.3
higher, and the dry weight 2.6 times higher, when irrigated with treated wastewater. This
may be due to the higher nutrient content provided by the TWW, as other research has
shown(26,31,32). In a similar study to the present one, the N content in the leaves of corn
irrigated with TWW was shown to increase from 1 to 3 % with respect to the crops irrigated
with aquifer water(33), and the corn plot irrigated with TWW had a yield of 2.58 t ha-1, while
the yield of the control plot was 1.61 t ha-1(34).
Table 4: Fresh and dry weight of Quinoa irrigated with fresh water and treated wastewater
in two locations
Root Leaves Stem
Site-Water
Fresh Dry Fresh Dry Fresh Dry
type
------------------------------------------- g ------------------------------------
I-FW 4.60 b 0.58 b 15.77 c 2.10 b 25.80 c 6.35 b
I-TWW 6.98 a 1.91 a 52.91 a 6.05 a 106.91a 18.74 a
C-FW 1.77 c 0.73 b 12.80 c 1.92 b 15.30 c 3.04 b
a a b a b
C-TWW 6.05 1.93 28.15 5.52 53.25 13.19 a
x̅ FW 3.19 b 0.66 b 14.29 b 2.01 b 20.55 b 4.70 b
x̅ TWW 6.52 a 1.92 a 40.53 a 5.79 a 80.08 a 15.97 a
I=Ixtlán, C=Capulálpam, FW= fresh water, TWW=treated wastewater. 𝑥̅ = average.
abc
Values with the same letter within each column are not significantly different (P≤0.05).
Table 5: Fresh and dry weight of corn irrigated with fresh water and treated wastewater in
two locations
Root Leaves Stem
Site –Water
type Fresh Dry Fresh Dry Fresh Dry
------------------------------------------- g --------------------------------------
I-FW 28.92 a 4.25 b 133.75 b 25.75 b 406.30 b 42.25 b
I-TWW 73.25 a 11.56 a 266.00 a 56.00 a 895.50 a 99.00 a
C-FW 32.85 a 6.82 b 119.00 b 22.75 b 381.50 b 29.75 b
C-TWW 83.50 a 15.50 a 261.50 a 54.25 a 1056.30 a 111.00 a
x̅ FW 30.89 b 5.54 b 126.38 b 24.25 b 393.90 b 36.00 b
79.31% 82.06% 85.95% 80.81% 77.17% 90.86%
a a a a a
x̅ TWW 78.38 13.53 263.75 55.13 975.90 105.00 a
70.55% 82.73% 78.33% 79.10% 80.06% 89.24%
I=Ixtlán, C=Capulálpam, FW= Fresh water, TWW=Treated wastewater. 𝑥̅ = average.
ab
Values with the same letter within each column are not different (P≤0.05).
The water content was significantly higher in quinoa and forage corn plants that were
irrigated with TWW than in plants that were irrigated with fresh water (Table 6); the leaves
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showed lower percentage values of water and a significantly higher water content. In corn
plants, the water content ranged between 80 and 90 %; in quinoa, it was 70 to 85 %,
depending on the organ concerned. These results express the influence of TWW on the water
content.
Table 6: Water content in quinoa and corn plants irrigated with freshwater and treated
wastewater in two locations
Root Leaves Stem
Site –
Quinoa Corn Quinoa Corn Quinoa Corn
Water type
------------------------------------- g --------------------------------------
I-FW 4.02 b 24.67 b 13.67 c 108.00 b 19.45 c 364.05 b
I -TWW 5.07 a 61.69 a 46.86 a 210.00 a 88.17 a 796.50 a
C-FW 1.07 c 26.03 b 10.88 c 96.25 b 12.26 c 451.75 b
C-TWW 4.12 b 68.00 a 22.63 b 207.25 a 40.06 b 945.30 a
x̅ FW 2.53 b 25.35 b 12.28 b 102.13 b 15.86 b 357.90 b
x̅ TWW 4.60 a 64.85 a 31.75 a 208.63 a 64.12 a 870.90 a
abc
Values with the same letter within each column are not different (P≤0.05).
Plant nitrogen and phosphorus content
In general, the nitrogen and phosphorus content were significantly higher in the organs of
quinoa and feed corn plants irrigated with TWW, with the exception of some organs in both
crops (Table 7). Research on citrus fruit trees(35) and vegetables(36) reports a higher N
concentration in leaves and a higher crop growth, due to the higher amount of nutrients and
organic matter in wastewater(12). The concentration of N and P in the organs of the plants
irrigated with TWW does not represent a danger for the plants themselves, and, therefore,
neither for the human or animal that consumes it(12). In Almeria, Spain, wastewater is reused
because of its moderate salt content and its high content of nutrients for plants, particularly
of N, P, and K(37). These results indicate that the plants were able to grow more easily when
they obtained more N and P. The highest N and P content in quinoa plants was found in the
leaves, followed by the stem and nutrient the root, respectively. In forage corn plants, P
content was second to that of quinoa, and the N content was higher in the stem, followed by
the leaves and the root (Table 7). Khaskhoussy et al(22), found higher N content in corn
irrigated with TWW: leaves 1.2 % and root 0.6 %, as compared to corn irrigated with FW:
leaves 1.0 % and root 0.45 %, Munir et al(38) showed that the N content of TWW-irrigated
corn plants was significantly higher than that of TWW-irrigated corn plants —of 1.08 %—,
compared to plants irrigated with FW (0.66 %). A similar tendency was reflected in the P
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content of TWW-irrigated plants: 0.19 %, and FW-irrigated plants: 18 %. Therefore, the use
of treated wastewater could be considered as an alternative that will help reduce the use of
chemical fertilizers on forage crops in the region and replicate them in another site.
Table 7: Nitrogen and phosphorus content in organs of quinoa and forage corn plants when
irrigated with freshwater and with treated wastewater
Site – Water Root Leaves Stem Root Leaves Stem
type
-------- Quinoa -------- -------- Corn --------
Nitrogen (%)
I-FW 2.15 a 1.70 ab 4.07 b 0.67 c 1.64 c 0.76 a
I- TWW 2.07 a 2.48 a 6.03 a 1.35 b 2.97 ab 3.32 a
C- FW 1.32 a 0.09 b 2.36 c 1.33 b 2.73 b 1.57 a
C-TWW 1.91 a 1.95 ab 5.30 a 2.18 a 3.52 a 2.30 a
x̅ FW 1.73 a 3.21b 1.33 b 1.00 b 1.16 b 2.18 a
x̅ TWW 1.99 a 5.66 a 2.21 a 1.76 a 2.81 a 3.24 a
Phosphorus (mg kg-1)
I- FW 0.12 b 0.17 b 0.25 b 0.10 b 0.28 c 0.24 b
I- TWW 0.22 a 0.20 a 0.34 a 0.16 a 0.45 a 0.43 a
C- FW 0.08 b 0.13 c 0.24 b 0.17 a 0.38 b 0.27 b
C-TWW 0.10 b 0.11 c 0.39 a 0.08 b 0.30 c 0.10 c
x̅ FW 0.10 b 0.24 b 0.15 a 0.13 a 0.33 b 0.25 a
x̅ TWW 0.16 a 0.37 a 0.16 a 0.14 a 0.38 a 0.27 a
acb
Values with the same letter within each column, crop, and element are not different (P≤0.05).
In the soil pH, neither the electrical conductivity nor the organic matter content represented
any risk when irrigation with treated wastewater was applied. The plant height, stem
diameter, and number of leaves of quinoa plants were significantly higher when these were
irrigated with treated wastewater; the same applies for corn, except for the variable number
of leaves, which did not show a significant difference. The biomass of both forage crops was
significantly higher in those plots where treated wastewater was utilized, compared to
irrigation with freshwater. The nitrogen and phosphorus content were significantly higher in
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both quinoa and corn plants that were irrigated with treated wastewater. Treated wastewater
is an important source of nutrients for forage crops and represents an alternative for
significantly reducing the use of chemical fertilizers.
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Article
Correlations between behavior in corrals and the bullring in Lidia

breed bulls
Juan Manuel Lomillos a*
Eloy Marino b
Enrique Recas b
René Alonso b
Marta Elena Alonso c
a
Universidad Cardenal Herrera-CEU. Facultad de Veterinaria. Departamento de
Producción y Sanidad Animal, Salud Pública Veterinaria y Ciencia y Tecnología de los
Alimentos C/ Tirant lo Blanc, 7. 46115 Alfara del Patriarca. Valencia, España.
b
Equipo Veterinario. Plaza de Toros de las Ventas. Madrid, España.
c
Universidad de León. Facultad de Veterinaria. Departamento de Producción Animal.
León, España.
* Corresponding author: juan.lomillos@uchceu.es
Abstract:
The value of fighting bulls (Lidia breed) is quantified based on their behavior in the
bullring. Predicting this behavior is challenging because the heritability of behavior
patterns is unknown and their interpretation subjective. An analysis was done of the
possible relationship between bull behavior during pre-bullfight handling (unloading, first
and second veterinary examinations) and during the bullfight. Behavioral parameter data
was recorded for 200 adult bulls during pre-bullfight handling and the bullfight. Among
the six genetic lines in the sample, the Santa Coloma and Albaserrada lines exhibited the
highest values for mobility, aggressiveness, respiratory rate, and fight rate. Correlations
were identified between some behaviors in pre-bullfight handling and others during the
bullfight. Mobility during unloading and the first examination was positively correlated
with Exit speed in the opening, Focus on banderillero (lancer on foot) in the second
period of the bullfight and Determination in the third period. In contrast, aggressiveness
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during unloading was negatively correlated with mobility parameters during the second
and third periods. No differences between animals were observed during the second
examination, indicating that bulls quickly adapted to the corrals. The results suggest that
some aspects of bull behavior prior to the bullfight can provide valuable information to
bullfighters and breeders.
Keywords: Behavior, Lidia breed, Ethology.
Introduction
The breed of cattle bred for bullfighting is known as Lidia. It is the only breed exploited
for its ethological performance. Since the 18th Century, individuals have been selected
based on behavioral traits observed during what is known as a tienta, a test of
aggressiveness(1). Because it has been a genetic improvement process carried out at the
herd level, it has produced multiple genetic lines characterized by phenotypic(2) and
ethological(3) traits that are both stable and defined(4). The resulting bulls are raised for
four to five years to fight in the bullring for approximately fifteen minutes(5). The goal of
selection is to produce bulls deemed apt for bullfights, which exhibit behavior broadly
termed bravura (implying both ferocity and bravery). However, defining ideal behavior
for a fighting bull is extremely difficult and highly subjective(6-7).
To produce bulls with the desired bravura, breeders keep exhaustive records of behavior
in breeding animals during the tienta, and of bulls during bullfights(8). This is a complex
task that does not necessarily produce high heritability of the desired traits(9). One
challenge in attaining trait heritability is defining what the desired behavioral traits are.
Many authors have tried to objectively describe the desired behavioral traits in fighting
bulls by defining the ethological patterns, positive or negative, defining the opposing
qualities of bravura and meekness(10-14). Some have devised ethological assessment
methods based on the frequency of behavioral patterns. For example, there is a fighting
bull rating table(15), a bull aptitude test(16), and bull evaluation sheets(17,18). In addition, a
computer program and ethological assessment methodology(19) have been
implemented(20-22). These studies focus on quantifying or evaluating bull behavior, be it
during the tienta or a bullfight. The intent is to select breeding animals or verify bull
performance during bullfights.
890
A bull’s behavior during pre-bullfight handling (transport, unloading and stabling) may
reflect the ethological traits it will exhibit during the bullfight. If this hypothesis holds
true, observing bull behavior prior to a bullfight could provide valuable information on
its potential behavior in the ring. This would be merely predictive since the performance
of the bullfighters and the variables in each bullfight also strongly influence on a bull’s
behavior. This connection has been suggested previously(16), but without scientific
analysis to support the argument. The present study objective was to analyze bull behavior
during pre-bullfight handling in an effort to begin defining ethological guidelines that
could predict bull behavior in the bullring.
Data were collected for two hundred animals which fought at bullrings in the cities of
Valencia and Madrid, Spain. All animals were between three and five years of age. They
came from 17 herds, and belonged to 6 different lines of the Lidia breed.
Pre-bullfight handling did not differ, following a standard procedure. This begins with
transport to the bullring three days before the bullfight where the bulls are unloaded
individually into a small pen (approx. 150 m2), and cooled with water until they have
calmed down. They are then moved through a narrow corridor to a scale, where they are
weighed individually, and into a larger corral (approx. 300 m2). Here they have an initial
veterinary examination. The bulls remain together in the corral for 48 h. On the day of
the bullfight, they are moved individually into another (approx. 300 m2) corral where they
have a second, and final, veterinary examination.
Bull behavior during the pre-bullfight handling process was documented on a form by
three veterinarians who agreed on the evaluation before recording it. Behavior was
assessed at four stages in the process: unloading (1); first veterinary examination (2);
second veterinary examination (3); and during time in corrals (4).
At stages 1, 2 and 3, mobility, aggressiveness and respiratory rate were assessed using a
three-point scale (1= low, 2= intermediate, and 3= high). At stage 4, once all the animals
were in the same corral, two types of collective behavior were assessed: nervousness, (1-
calm, 2-vigilant, 3-nervous) and fight rate (1-infrequent, 2-moderate, 3-frequent).
Video recordings were taken of each animal’s bullfight and later evaluated by the same
person, with experience in ethological assessment. The software and methodology
described by Sánchez et al(19), and validated by Alonso et al(23-25), were used. This allows
evaluation of an animal’s behavior during each of the bullfight’s three periods (known as
tercios). During each tercio, a total of 21 behavioral variables (see below) were graded
891
on a five-point scale and recorded in an independent spreadsheet file, along with the
duration of each tercio.
1. Exit speed (rapisal). The speed at which the bull runs through the door into the ring. 0=
animal exits walking and stops in corridor. 5= animal exits at a run.
2. Stops at door (parapu). If the bull stops when it steps onto the sand. 0= animal maintains
exit speed when stepping onto sand. 5 = animal stops.
3. Ring circuit (recorre). If the bull moves around the ring before being stopped by the
first flourishes of a cape. 0= animal remains standing at some point in the bullring. 5=
animal makes one or more circuits.
4. Distance at start of charge (acudlar). Distance at which the bull begins to charge when
first provoked. 0= animal only charges when a bullfighter is very close. 5= animal charges
at any distance, no matter distance to the bullfighter.
5. Attacks shields (remata). If bull gores or hits shield behind which bullfighter shelters.
0= under no circumstances does animal attack shield. 5= animal attacks shields every
time it gets near them during initial lancing.
6. Head height vis-a-vis horse (humillacab). Estimated height at which bull places horns
on horse’s body. 0= animal raises horns towards lancer. 5= animal places horns on lower
portion of protective armor or on horse’s abdomen.
7. Push. Extent to which, once it has made contact, the bull uses its dorsal muscles and
hind quarters to push the horse. 0= animal does not push at all, remains static or leans
slightly into horse. 5= animal uses dorsals and hind legs in attempt to displace horse.
8. Goring. If bull gores horse’s protective armor and to what extent. 0= animal firmly
pushes against armor, without changing from point of initial contact. 5= animal insistently
gores armor, and even tries to detach the lance.
9. Release (suelto). If animal releases or disengages from horse after feeling pain from
the lance, and runs away from horse without bullfighters challenging it. 0= animal
remains with horse and does not escape, requires challenge. 5= animal disengages and
quickly runs away after feeling pain from lance.
10. Response to pain (crecedol). Extent to which the bull, upon feeling pain, increases its
force and aggressivity against the horse. 0= animal decreases aggressivity in response to
pain. 5= animal attacks more decisively after lancer’s aggression.
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11. Distance to banderillero (largoban). The distance from which the bull charges the
banderillero (lancer on foot) when called. 0= animal waits until banderillero is very
close. 5= animal charges before being called on all three attempts.
12. Focus on banderillero (fijoban). Extent to which animal focuses on banderillero or is

distracted. 0= animal is constantly distracted by the crowd and other bullfighters. 5=
animal focuses on banderillero from the first call until the lancing ends.
13. Follows banderillero (sigueban). The tenacity with which the bull follows the
banderillero once the darts have been anchored in its back. 0= animal stands still after
encounter. 5= animal insistently follows banderillero, normally until he takes cover
behind a shield.
14. Gallop. Frequency with which bull gallops. 0= animal never gallops. 5= animal is
constantly galloping.
15. Distance of charge at muleta (largomu). Distance from which bull begins charge at
muleta (cape supported with stave). 0= animal does not charge until muleta is very close.
5= animal begins charge from large distance when given the option.
16. Head height vis-a-vis muleta (humillamu). Head height of bull when charging muleta.
0= animal keeps head high from beginning to end of charge. 5= animal lowers head when
beginning charge and keeps it low until finishing.
17. Determination (codicia). The passes in each “set” of charges at the muleta can
continue without pause after each one. This is frequently the case at the beginning of the
third tercio, but can decrease as it progresses. 0= animal stops after each set. 5= animal
does not pause between passes in all sets.
18. Delay (tardea). Number of calls or challenges required for bull to charge. 0= animal
charges the moment cape is shown. 5= animal requires repeated calls, on each pass, before
charging.
19. Charge area (embiste). The third tercio occurs in an area chosen by the bullfighter or
one preferred by the bull. 0= encounters must happen in area preferred by animal, usually
near the shields or the exit to the corrals. 5= animal exhibits no preference.
20. Focus on muleta (fijomul). Extent to which bull remains focused on muleta. 0= animal
looks at the bullfighter or the crowd, is constantly distracted. 5= animal remains focused
on muleta at all times.
21. Runs away from muleta (huyemul). Extent to which bull runs away from muleta, in
search of an exit, after first charges. 0= animal exhibits no intent to avoid muleta. 5=
animal constantly runs away, making it impossible to complete passes.
893
Results for all the variables described above were expressed as a mean with standard
deviation. A one-way ANOVA was applied to identify any effect of genetic line or
moment of observation (unloading, first examination, second examination) on the
ethological variables documented pre-bullfight. If a factor of variation had two levels and
was statistically significant (P≤0.05), a Student-Newman-Keuls test (P≤0.05) was run a
posteriori to compare groups of homogeneous means. Any possible linear relationships
between the ethological variables documented in the corrals and those documented during
the bullfight were identified with a Pearson’s linear correlation test. All statistical
analyses were run with the IBM® SPSS® ver. 19.0 software program(26).
Results
Analysis (ANOVA, Student-Newman-Keuls) of the ethological values collected during

pre-bullfight handling indicated that animal attitude, represented by mobility,
aggressiveness and respiratory rate, exhibited no changes during unloading and the first
examination, which take place on the same day and consecutively. Aggressiveness and
respiratory rate decreased significantly in the second examination (Figure 1).
Figure 1: Ethological values (means) during unloading and two veterinary

examinations
ab
Different letters over columns in the same stages indicate significant difference (P<0.05).
Analysis of bull behavior by genetic origin (line) identified differences between them
during pre-bullfight handling (Table 1), but not during the bullfight. In the corrals, the
Santa Coloma and Albaserrada lines exhibited the highest values for mobility,
894
aggressiveness and respiratory rate, especially at unloading and first examination. Values
for the nervousness variable were highest in Santa Coloma in the corrals, and highest in
Albaserrada and Santa Coloma animals during the bullfight (P<0.05).
The Pearson’s linear correlation analysis identified multiple significant correlations

(Tables 2, 3, 4, 5 and 6). Results have been simplified by grouping the results from the
unloading and first examination stages since their means did not differ (Figure 1).
895
Table 1: Ethological parameter values (mean) by genetic line during pre-bullfight handling
Unloading 1st Exam 2nd Exam Corrals
Line n Mobi Agre Resp Mobi Agre Resp Mobi Agre Resp Nerv Fight
Murube 18 1.55a 1.13a 1.23a 1.76a 1.02a 1.21a 1.46a 1.09a 1.24a 1.24a 1.08a
Núñez 24 2.27b 1.26a 1.58a 2.17a 1.41a 1.59ab 2.25b 1.53a 1.01a 1.27a 1.02a
Domecq 100 2.35b 1.44a 1.30a 1.96a 2.02a 1.80b 1.84ab 1.48a 1.53a 1.28a 1.35a
Atanasio 22 1.92ab 1.87a 1.83a 1.55a 1.70a 1.83b 2.20b 1.23a 1.25a 1.22a 1.38a
Albaserrada 18 2.64c 2.91b 2.83b 2.52b 2.53b 2.58c 2.3b 2.88b 1.53a 1.51a 2.20b
Santa Coloma 18 2.55c 2.88b 2.54b 2.53b 2.01a 2.77c 2.74c 2.29ab 1.81a 2.43b 2.76b
Total 200 2.20 2.01 1.82 2.41 2.14 1.94 2.34 1.74 1.39 1.59 1.79
Mobi= mobility; Agre= aggressiveness; Resp= respiration rate; Nerv= nervousness; Fight= fight rate.
ab
Different letter superscripts in the same column indicate significant difference (P<0.05).
896
Table 2: Pearson’s linear correlation analysis between bull ethological parameters

recorded during pre-bullfight handling and during the opening of the bullfight
OPENING
Rapisal Parapu Recorre Acudlar Remata
Unloading Mobi 0.34* 0.12 0.09 0.12 -0.03
Agre 0.13 -0.07 0.15 0.01 0.27*
Resp 0.08 0.04 -0.07 -0.02 0.12
Examination Mobi 0.03 0.03 -0.06 0.11 0.05
Agre 0.11 0.02 0.09 0.06 0.00
Resp 0.17 0.08 0.03 0.11 0.05
Corrals Nerv 0.14 -0.07 0.12 0.19 0.04
Fight 0.04 0.08 0.03 -0.18 0.03
Rapisal= Exit speed; Parapu= Stops at door; Recorre= Ring circuit; Acudlar= Distance start of charge;
Remata= Attacks shield; Mobi= Mobility; Agre= Aggressiveness; Resp= Respiration Rate; Nerv=
Nervousness; Figh = Fight rate.
* (P<0.05).

recorded during pre-bullfight handling and during the first tercio (varas) of the bullfight
VARAS
Humillacab Empuja Cabecea Suelto Crecedol
Unloading Mobi 0.14 0.05 -0.09 0.06 0.00
Agre 0.12 -0.04 0.03 -0.16 0.10
Resp -0.07 0.16 0.10 0.08 0.01
Examination Mobi 0.08 0.12 -0.05 0.12 0.07
Agre 0.03 -0.06 0.15 0.08 0.12
Resp 0.09 0.19 0.01 0.16 0.18
Corrals Nerv 0.17 0.13 0.06 0.11 -0.11
Fight 0.08 -0.17 0.12 -0.03 -0.18
Humillacab= Head height vis-a-vis horse; Empuja= Push; Cabacea= Goring; Suelto= Release; Crecedol=
Response to pain; Mobi= Mobility; Agre= Aggressiveness; Resp= Respiration Rate; Nerv= Nervousness;
Fight= Fight rate.
* (P<0.05).
897

recorded during pre-bullfight handling and during the second tercio (banderillas) of the
bullfight
BANDERILLAS
Largoban Fijoban Sigueban Galopa
Unloading Mobi 0.58* 0.05 0.12 0.09
Agre -0.22* 0.01 -0.21* -0.41*
Resp 0.12 0.06 0.17 0.10
Examination Mobi 0.18 0.02 -0.01 0.21*
Agre -0.03 0.11 0.11 0.05
Resp 0.1 -0.07 -0.02 0.01
Corrals Nerv 0.17 -0.11 0.16 0.17
Fight -0.05 0.05 0.09 -0.07
Largoban = Distance to banderillero; Fijoban = Focus on banderillero; Sigueban = Follows banderillero;
Galopa = Gallops; Mobi = Mobility; Agre = Aggressiveness; Resp = Respiration Rate; Nerv =
Nervousness; Fight = Fight rate.
* (P<0.05).

recorded during pre-bullfight handling and during the third tercio (muleta) of the
bullfight
MULETA
Largomu Humillamul Codicia Tardea Embiste Fijomul Huyemul
UL Mobi 0.11 0.19 0.11 -0.19 0.29* -0.04 -0.15
Agre -0.31* 0.08 0.17 -0.13 0.16 0.13 0.02
Resp -0.03 0.06 -0.08 0.14 0.07 0.17 0.19
EX Mobi 0.03 -0.07 0.16 -0.17 0.12 -0.00 -0.11
Agre 0.07 0.11 -0.10 0.45* 0.08 0.16 0.02
Resp 0.00 -0.19 -0.18 0.07 0.18 0.04 0.17
COR Nerv 0.11 0.14 -0.27* 0.04 0.07 0.13 -0.12
Fight 0.08 -0.14 -0.12 0.01 -0.09 0.15 0.04
Largomu = Distance of charge at muleta; Humillamul = Head height vis-à-vis muleta; Codicia =
Determination; Tardea = Delay; Embiste = Charge area; Fijomul = Focus on muleta; Huyemul = Runs
from muleta; UL = Unloading; EX = Examination; COR = Corrals; Mobility; Agre = Aggressiveness;
Resp = Respiration Rate; Nerv = Nervousness; Fight = Fight rate.
* (P<0.05).
In the pre-bullfight handling results, the most valuable information in terms of predicting
animal behavior during the bullfight is that for unloading and first examination. At this
time, the animal is in a state of stress after transport, which brings out its intrinsic
characteristics of mobility and aggressiveness. In the opening of the bullfight, mobility
during unloading and first examination positively correlated to the parameter “Exit
speed”, as well as to “Distance to banderillero” in the second tercio (banderillas) and
“Charge area” during the third tercio (muleta). In contrast, the ethological parameter
values for the second examination were not strongly predictive of animal behavior during
898
the bullfight. However, the mobility parameter in this stage positively correlated with the
“Gallops” parameter in the second tercio, a reflection of good physical condition in the
corrals and the bullring. Also, the aggressiveness parameter in the second examination
positively correlated with the “Delay” parameter in the third tercio.
Discussion
Research done in production animal management facilities to assess animal stress in

response to transport to the slaughterhouse indicates that animals adapt to their new
surroundings within a few hours(27-29). This agrees with the present results in which bull
aggressiveness and respiratory rate values were significantly lower at the second
examination after they had adapted to the corrals for two days, which coincides with
previous reports(30).
In the pre-bullfight stages, bulls of the Santa Coloma and Albaserrada lines exhibited the
highest mobility, aggressiveness and respiratory rate values during unloading and first
examination. This coincides with genetic studies of the Lidia breed which state that this
difference in behavior has resulted from selection focused on more temperamental or
fierce behavior(17,31), which has raised consanguinity rates in these lines(32). The literature
addressing the characteristics of the Lidia lines also indicates that the Santa Coloma and
Albaserrada lines have a different capacity to adapt to the stress of transport and
corralling(12,33). Bulls from these lines also exhibit distinct behavior during the
bullfight(27).
Aggressive behavior during unloading and the first veterinary examination is common in
domestic animals, and even more so in those raised in extensive systems. In the Lidia
breed, the lack of space in the corrals generates greater aggressiveness between
individuals(34). The present results showed a positive correlation between aggressiveness
in the pre-bullfight stages and the ethological parameter “Attacks shield” during the
bullfight. However, it exhibited a negative correlation with the parameters “Distance to
banderillero”, “Follows banderillero” and “Gallops” in the second tercio (banderillas),
and with “Distance of charge at muleta” in the third tercio (muleta). A bull’s
aggressiveness in the corrals was apparently linked to its entrance into the ring and goring
of the shields, which, in principle, would result from breed line and bravura. But the
multiple negative correlations suggest that the most aggressive individuals during pre-
bullfight handling did not perform well in the bullfight; indeed, they tended to be meeker
as shown in their lack of mobility and zeal during both the second and third tercios.
899
As suggested in a previous study, bulls exhibiting greater aggressiveness during pre-

bullfight handling may be experiencing greater stress, preventing them from resting
adequately and resulting in their being less aggressive during the bullfight(35). Perhaps the
animals included in the present study that were more aggressive in the corrals were more
stressed. They expended energy during pre-bullfight handling and were therefore more
tired during the bullfight itself. This is supported by the negative correlation between
nervousness in the corrals and lower “Determination” values during the third tercio.
Animals exhibiting nervousness and aggressiveness in the corrals may be badly adapted
to handling, undermining their physical condition and causing them to perform below
expectations during the bullfight. Considering this, a bull’s pre-bullfight behavior may
affect its physical performance, pushing any correlation between ethologies into the
background.
Respiratory frequency showed no correlation with the analyzed ethological parameters,

although its relationship to animal physical condition during the bullfight, indicated by
time in motion in the ring(35-36), is worth further study.
No previous data, be it published or personal testimony from breeders, is available on bull

behavior in the field or during pre-bullfight handling(37-38). The present results suggest
some correlation between certain ethological patterns during pre-bullfight handling and
behavior during the bullfight. Broader studies including analyses of bull behavior in the
field and prior to the bullfight could continue to improve predictability of behavior in the
ring.
Acknowledgements
The authors wish to thank the teams of veterinarians at Valencia and Las Ventas de
Madrid, the companies Plaza 1 and Nautalia, the Comunidad de Madrid and the
Diputación de Valencia.
900
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904
Technical note
Ixodicidal effect of plant extracts of Cinnamomum zeylanicum and Tagetes

erecta on Rhipicephalus microplus ticks
Perla Iris Miranda Reyes a
Francisco Martínez Ibañez b
Rodolfo Esteban Lagunes-Quintanilla c*
América Ivette Barrera Molina a*
a
Universidad Autónoma del Estado de Morelos. C. Ixtaccíhuatl 100, Vista Hermosa, 62350,
Cuernavaca, Mor. México.
b
Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria. Centro Nacional de
Servicios en Constatación en Salud Animal. Jiutepec 62550, Morelos, México.
c
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Centro Nacional de
Investigación Disciplinaria en Salud Animal e Inocuidad. Jiutepec 62550, Morelos, México.
*Corresponding author: america.barrera@uaem.mx
Abstract:
One of the main problems in cattle farming is infestations caused by Rhipicephalus microplus
ticks, the most important parasitic species in the cattle industry. Its control is based mainly
on the application of ixodicides. However, the excessive and inappropriate use of these
products has generated resistant strains. As an alternative, biological control has been
proposed as a promising method, as it prevents environmental contamination, promotes the
safety of animal products, and contributes to sustainability. For this reason, the objective of
the present study was to perform an in vitro evaluation of the ixodicidal effect of two plant
extracts of Cinnamomum zeylanicum and Tagetes erecta on R. microplus ticks. The analysis
was carried out using the larval immersion technique (LIT) and the adult female immersion
technique (AIT), and the morphological damage to the cuticular structure of the ticks was
905
then determined by stereo microscopy. The most significant result was 100 % larval mortality
(P<0.05) for the C. zeylanicum extract at a concentration of 6%, presenting evident
morphological damage in the cuticular structure. In contrast, T. erecta extract showed no
ixodicidal activity. Finally, it is concluded that the plant extract of C. zeylanicum shows
efficacy in in vitro tests against R. microplus larvae and may prove useful as an economical
and sustainable alternative for tick control.
Keywords: Cinnamomum zeylanicum, Biological control, Plant extracts, Rhipicephalus

microplus, Tagetes erecta.
Ticks are one of the most important ectoparasites in tropical and subtropical areas(1). In
Mexico, 82 species of ticks have been recorded in both wild and domestic animals,
Rhipicephalus microplus being the most predominant one in livestock. This tick generates
economic losses related to reduced production levels, disease transmission, mortality, and
high control costs(1,2). In recent years, the most widely used strategy for their control has been
the application of chemicals known as ixodicides such as organophosphates, carbamates,
formamides, synthetic pyrethroids, macrocyclic lactones, phenylpyrazolones, etc., which
work adequately at recommended doses. However, the irrational use of these products has
led to the selection of resistant tick populations, making control increasingly complex(3,4).
For this reason, the availability of alternative methods has become a necessity for the
producers, as well as for the consumers who demand pesticide-free products made with
environmentally safe technology, such as plant extracts with active ingredients that exhibit
insecticidal and pest control effects, especially in ecological and organic production
systems(5,6,7).
In view of this situation, the control of ticks with extracts such plants as garlic and Mexican
oregano has been widely studied in some species of livestock importance, using various
methods of obtainment and application(8-11). On the other hand, commercial extracts from
plants like cinnamon and marigold tend to have low toxicity to human health, slow
development of resistance, and instability in the environment, and their active ingredients are
rapidly metabolized by solar radiation and microclimatic humidity(12). For this reason, the
objective of this research was to evaluate the ixodicidal effect of the extracts of two plants
—cinnamon (Cinnamomum zeylanicum) and marigold (Tagetes erecta)— on the mortality
of R. microplus ticks using the larval package technique (LPT) and the adult female
906
immersion technique (AIT), and to determine the morphological damage caused at the
microscopic level.
The present research utilized 7- to 15-d-old R. microplus tick larvae and adult ticks 21 to 23
d collected from bovines infested artificially. The susceptible reference strain from Moyahua,
Zacatecas, Mexico, was used, having been provided by the ectoparasites and diptera
laboratory of the National Center for Animal Health Testing Services (Centro Nacional de
Referencia en Parasitología Animal y Tecnología Analítica, CENAPA) of the National
Service for Agri-Food Health, Safety and Quality (Servicio Nacional de Sanidad, Inocuidad
y Calidad Agroalimentaria, SENASICA).
Two plant extracts formulated by oil-in-water emulsion of C. zeylanicum 15 % (equivalent

to 151.80 g a.i./L) and T. erecta 90 % (equivalent to 831.60 g a.i./L) in weight of active
compound were assessed and used mainly to control botanical pests such as red spider mites
(Tetranychus urticae, Oligonychus punicae) or whiteflies (Bemisia tabaci). In order to
evaluate the ixodicidal effect of each of the extracts, solutions were prepared using the
commercial concentration recommended as a reference (1.5 %).
In order to evaluate the ixodicidal effect of the extracts of C. zeylanicum and T. erecta 90 %,
a Probit analysis was performed, using five different concentrations —6 %, 3 %, 1.5 %,
0.75 % and 0.375 %— and a control group of 0.375 %. The bioassay was performed under
the larval immersion methodology (LIT)(13). A solution was prepared in distilled water
(according to the manufacturer's instructions) with the highest concentration of the extracts
(6 %); serial double dilutions were then made with a dilution factor of 0.5, established by the
technique up to the lowest concentration (0.375 %). In glass Petri dishes of 15 cm in diameter,
a 12.5 cm Whatman No. 1 paper filter was placed, and 10 ml of each solution were added to
the respective dish. Subsequently, ~ 400 larvae distributed on the paper were placed with a
brush and covered with another paper filter simulating a larval immersion for 10 min. After
the exposure time had elapsed, ~100 larvae were taken and transferred to 7.5 x 8.5 cm filter
paper packets, which were secured with a BACO™ Bulldog-type clip. Each package
containing the treated larvae was identified and kept in an incubator at a temperature of 28 ±
2 ºC and at a relative humidity of 80-90 % for 24 h. Three replicates were performed for each
concentration. Finally, package readings were taken by counting the number of live and dead
larvae, following the methodology described by FAO in 2004(14).
On the other hand, the immersion test of R. microplus adult females (AIT) was performed
using the methodology described above(15). Only the commercial concentration
recommended for C. zeylanicum and T. erecta according to the norm NOM-006-ZOO-1993
was used to evaluate their toxicity on female weight and inhibition of oviposition and
hatching. Ticks were placed in beakers with 30 ml of 1.5 % solution of both extracts and kept
in immersion for one minute while being subjected to circular movements. Subsequently,
907
they were removed in order to eliminate the excess product and placed in Petri dishes to be
incubated at 28 ± 2 ºC and 80-90 % relative humidity. On d 14, the eggs were separated and
weighed to determine the percentage of oviposition inhibition between the treated and control
groups. Finally, 1 g vials of duly identified eggs were placed and incubated for 26 d until
hatching. The shells and eggs within 3x3 quadrants were counted in order to determine the
percentage of hatching.
At the end of the immersion test, a morphological analysis was performed using R. microplus
tick larvae to determine potential morphological damage to the cuticular structure after
exposure to C. zeylanicum and T. erecta extracts. Ten (10) larvae per treatment were selected
from 6 %, 3 %, 1.5 % and 0.75 % concentrations and analyzed by stereo microscopy (Leica
microscope) at the helminthology laboratory of the National Center for Disciplinary
Research on Animal Health and Safety (Centro de Nacional de Investigación Disciplinaria
en Salud Animal e Inocuidad, CENID-SAI, INIFAP).
Larval mortality parameters were obtained (% mortality = number of dead larvae x

100/number of total larvae)(16). Data were subjected to analysis of variance and to the
Kruskal-Wallis test in order to determine significant differences between treatments and
controls. Additionally, the data generated in the bioassays from the Probit analyses were
submitted to the Polo Plus program (LeOra Software, Petaluma, CA), with the purpose of
calculating the lethal concentration 50 (LC50) with a 95% confidence interval.
Probit analysis of the ixodicidal effect of the plant extracts applied using the larval immersion
technique made it possible to determine a gradual mortality from the highest concentration
(6 %) to the lowest (0.375 %), as shown in Table 1. The extract of C. zeylanicum produced a
significant mortality (P<0.05) at concentrations of 6 % and 3 %, obtaining results of 100 and
97.8 %, respectively. On the other hand, the mortality resulting from the 1.5 % concentration
was 64.2 %, which was not statistically significant, similarly to the lowest concentrations that
exhibited 0 % mortality. In contrast, the extract of T. erecta showed no biological activity in
any of the concentrations applied through the Probit methodology, yielding mortality
percentages of 0 %, indicative of a null toxic effect on R. microplus ticks larvae.
Table 1: Mortality of R. microplus tick larvae treated with C. zeylanicum and T. erecta
extracts by Probit analysis with five different concentrations
Extract concentrations (%)
Treatment 6 3 1.5 0.75 0.375
C. zeylanicum 100* 97.8* 64.2* 0 0
T. erecta 0 0 0 0 0
Control 0 0 0 0 0
* Statistical difference with respect to the Control group, P<0.05.
908
Table 2 shows the values obtained when analyzing the mortalities of each of the
concentrations used in the Probit analysis. With the results of each concentration, an analysis
was carried out using the Polo PC program to calculate the LC50 for each of the replicates.
The total number of R. microplus tick larvae used in the first repetition was 1,413; it was
1,556 in the second repetition, and 1,529 in the third repetition. The LC50 ranges obtained in
each repetition were a minimum of 1.37 and a maximum of 2.41, with a 95% confidence
interval.
Table 2: Values obtained through the Polo PC program with the mortalities obtained from
C. zeylanicum extract on R. microplus tick larvae
n Slope LC50 CI (95 %)
1413 3.862 ± 0.185 2.41 1.92 - 3.09
1556 6.326 ± 0.338 1.43 1.29 - 1.58
1529 9.316 ± 0.766 1.37 1.24 - 1.48
n= number of R. microplus larvae; LC50= lethal concentration; CI= confidence interval.
The results for the inhibition of oviposition and hatching indicate that there was no
statistically significant difference for any of the extracts evaluated with respect to the control
group (Table 3). The percentage of oviposition inhibition was 3.88 % and 0 % for C.
zeylanicum and T. erecta, respectively. The percentage of hatching inhibition was 0 % for
both extracts.
Table 3: Oviposition and hatching inhibition percentages of C. zeylanicum and T. erecta

extracts on R. microplus ticks
Average Average
Extract Concentration female egg % O. I. % H. I.
weight (g) weight (g)
C. zeylanicum 1.5 % 3.69 2.02 3.88 0
T. erecta 1.5 % 3.75 2.10 0 0
Control ---- 3.75 2.12 ---- ----
O. I.= oviposition inhibition; H. I.= hatching inhibition.
Morphological analysis by stereo microscopy showed remarkable effects on R. microplus

larvae after treatment with C. zeylanicum extract. It was observed that, as the concentration
of the extract increased, the structural damage became more noticeable. In panel A of Figure
1, the larva exhibits evident morphological alterations in the gnathosome (mouthparts),
characterized by a reduction in the size of the pedipalps and hypostome, a diminished amount
of cuticle and a change in the coloration of the legs. In addition, the shield present in the
gnathosome shows an orange coloration that differs from panel D or the control group,
suggesting inadequate development and an affected gnathosome. Panel B and C show that
the idiosome (body) is whiter and more transparent, indicating an adverse effect on the
909
intestinal caecum that is not adequately appreciated, possibly due to the toxicity of the
extracts at the concentrations used. No morphological damage in panel D or the control
group.
Figure 1: Morphological damage identified in R. microplus larvae treated with different

concentrations with C. zeylanicum extract
A= 6 % concentration (the black arrows show structural damage). B= 3 % concentration. C= 1.5 %

concentration; D= control larva (water).
In the present study, C. zeylanicum extract was observed to exhibit ixodicidal activity on R.
microplus larvae. In particular, it was determined that the use of this extract at a 6 %
concentration produced 100 % larval mortality. These findings are in agreement with
previous studies where the efficacy of a chemotype derived from C. verum against R.
microplus ticks was evaluated by LPT and AIT, observing that the use of essential oils with
benzyl benzoate showed efficacy against R. microplus in its larval stage(17). Likewise,
previous findings(18), were similar to those obtained in the present investigation, where they
evaluated the compounds (E)-cinnamaldehyde and α-bisabolol derived from cinnamon
essential oils and obtained a 100 % larval mortality at concentrations of 2.5, 5 and 10 mg/ml,
concluding that the essential oils and their compounds have high acaricidal activity; however,
they report low toxicity in adult ticks filled with R. microplus.
On the other hand, it has been reported that the essential oils of C. zeylanicum act on adult
ticks filled with R. microplus in pure form, reaching mortalities of 100 %, 97 %, and 62 % at
concentrations of 10, 5, and 1 mg/ml, respectively(19). However, in the present study, when
evaluating the commercial concentration recommended of 1.5 %, no effect was observed on
910
the inhibition of oviposition and hatching of adult ticks. It is important to note that
evaluations above the recommended concentration were not performed, since the norm
NOM-006-ZOO-1993 states that, if this concentration does not have a 98 % mortality rate in
adult ticks, the efficacy is considered negative.
However, it should be noted that the use of the commercial concentration of 1.5 % is for the
control of phytophagous insect pests. In this work, a strategy of concentrations was
standardized, considering that these arthropods are hematophagous and are at different stages
of development. The 6 % concentration of C. zeylanicum, which is lethal to R. microplus tick
larvae, proves that a higher amount is required than for the target species. In addition, no
effectiveness was observed on adult ticks, which could indicate that this product does not
have the potential to biologically affect the ectoparasite in the adult stage. However, previous
studies assessed the combination of three essential oils, demonstrating that increasing the
concentration of C. zeylanicum resulted in greater effectiveness in controlling adult stage
ticks. These findings suggest that the use of higher concentrations of C. zeylanicum may be
an effective strategy to combat R. microplus ticks at all life stages. However, further research
is needed to determine the optimal concentrations and to evaluate their potential side effects,
environmental effects, and economic feasibility(20).
Further studies using different concentrations on adult ticks are needed, as, in this case, only
the commercial concentration recommended was used. These additional studies will make it
possible to evaluate the efficacy of the product at different doses and to determine whether
there are more effective concentrations for the control of adult ticks. Thus, it will be possible
to obtain a better understanding of the response of ticks to different concentrations and to
establish more precise guidelines for their control.
Furthermore, in the present study, no distillation or chromatography was performed to

separate the compounds from the evaluated extract. However, it has been reported that the
main biocomponent of C. zeylanicum is eugenol, which could be involved in its toxicity
against R. microplus tick larvae(21). In addition, the plant C. zeylanicum is known to possess
certain properties, notably its insecticidal and acaricidal activity(6,18,20), which suggests that
it is an alternative method for the biological control of ticks. In contrast, assessment of the
T. erecta extract showed no toxicity on R. microplus larvae or adults. In this regard, no
acaricidal activity of T. erecta extract against ticks has been reported. To date, there are only
studies where the ixodicidal effect of T. minuta essential oil against R. microplus tick
infestation was evaluated, and statistically significant results were obtained in the productive
parameters (number and weight of the ticks, oviposition, and viability of larvae), showing an
efficacy of 99.9 % at a concentration of 20 %(22). Therefore, future research is needed to
isolate, identify and characterize the compounds of T. erecta extract in order to determine
whether it has ixodicidal properties against R. microplus ticks.
911
The efficacy shown by the plant extract of C. zeylanicum for the control of R. microplus tick
larvae highlights the potential use of this natural product as a biological control method and
as an economical and sustainable alternative for tick control. However, additional studies are
needed to evaluate different methods of plant extraction; in vivo tests and toxicity studies
must yet be performed in order to elucidate its effect on adult R. microplus ticks and the
possible risk of using it on domestic animals.
The authors declare that they have no conflict of interest in relation to the preparation,
revision, and publication of this work.
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1. Tabor AE, Ali A, Rehman G, Rocha Garcia G, Zangirolamo AF, Malardo T, et al. Cattle
tick Rhipicephalus microplus-host interface: a review of resistant and susceptible host
responses. Front Cell Infect Microbiol 2017;(7):506.
2. Estrada-Peña A, Mallón AR, Bermúdez S, de la Fuente J, Domingos A, García MPE, et

al. One health approach to identify research needs on Rhipicephalus microplus ticks in
the Americas. Pathogens 2022;(11):1180.
3. Yessinou RE, Akpo Y, Adoligbe C, Adinci J, Assogba MN, Koutinhouin B. et al.

Resistance of tick Rhipicephalus microplus to acaricides and control strategies. J
Entomol Zool Stud 2016;(4):408-414.
4. Rojas MC, Loza RE, Rodríguez CSD, Figueroa MJV, Aguilar RF, Lagunes QRE, et al.
Antecedentes y perspectivas de algunas enfermedades prioritarias que afectan a la
ganadería bovina en México. Rev Mex Cienc Pecu 2021;(12):111-148.
5. Soto GA. Manejo alternativo de plagas de ácaros. Rev Cie Agr 2013;(2):34-44.
6. Chungsamarnyart N, Jiwajinda S. Rattanagrithaku C, Jansawan W. Practical extraction

of sugar apple seeds against tropical cattle ticks. Kasetsart J 1991;(25):101-105.
7. Williams LAD. Adverse effects of extracts of Artocarpus altilis Park, and Azadirachta
indica (A. Juss) on the reproductive physiology of the adult female tick, Boophilus
microplus (Canest). Invertebr Reprod Dev 1993;(23):159-164.
8. Panella NA, Karchesy J, Maupin GO, Malan J, Piesman J. Susceptibility of immature

Ixodes scapularis (Acaris: Ixocidae) to plants derived acaricides. J Med Entomol
1997;(34):340-345.
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9. Vatsya S, Yadav C, Kumar R, Banerjee P. In vitro acaricidal effect of some medical

plants against Boophilus microplus. J Vet Parasitol 2006;(20):141-143.
10. Álvarez V, Loaiza J, Bonilla R, Barrios M. Control in vitro de garrapatas (Boophilus

microplus, Acari: Ixodidae) mediante extractos vegetales. Rev Biol Trop 2008;(56):291-
302.
11. Bravo M, Coronado A, Henríquez H. Eficacia in vitro del amitraz sobre poblaciones de
Boophilus microplus provenientes de explotaciones lecheras del estado de Lara,
Venezuela. Zoot Trop 2008;(26):35-40.
12. Broglio-Micheletti SMF, Neves-Valente EC, Alves de Souza L, Silva-Dias ND, Girón-
Pérez K, Prédes-Trindade RC. Control de Rhipicephalus (Boophilus) microplus (Acari:
Ixodidae) con extractos vegetales. Rev Colomb Entomol 2009;(35):145-149.
13. Shaw RD. Culture of an organophosphorus-resistant strain of Boophilus microplus

(Can.) and an assessment of its resistance spectrum. Bull Entomol Res 1966;(56):389-
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14. FAO. Acaricide resistance: diagnosis, management and prevention. guidelines

resistance management and integrated parasite control in ruminants, animal production
and health division, agriculture department. Food and Agriculture Organization of the
United Nations. Rome. 2004.
15. Drummond RO, Ernst SE, Trevino JL, Gladney WJ, Graham OH. Boophilus annulatus
and B. microplus: laboratory tests of insecticides. J Econ Entomol 1973;(66):130-133.
16. Reyes-Domínguez IJ, Arieta-Román RJ, Fernández-Figueroa JA, Romero-Figueroa

MZ, Peniche-Cardeña AJE. Resistencia de Rhipicephalus (Boophilus) microplus a
ixodicidas en ranchos bovinos del municipio Evangelista, Veracruz, México. Rev
Electron de Vet 2013;(14):1-6.
17. Monteiro IN, Monteiro ODS, Costa-Junior LM, da Silva Lima A, Andrade EHA, Maia
JGS, et al. Chemical composition and acaricide activity of an essential oil from a rare
chemotype of Cinnamomum verum Presl on Rhipicephalus microplus (Acari: Ixodidae).
Vet Parasitol 2017;(238):54-57.
18. Dos Santos DS, Boito JP, Santos RCV, Quatrin PM, Ourique AF, Dos Reis JH, et al.
Nanostructured cinnamon oil has the potential to control Rhipicephalus microplus ticks
on cattle. Exp Appl Acarol 2017;(73):129-138.
913
19. Marchesini P, Oliveira DR, Gomes GA, Rodrigues THS, Maturano R, Fidelis QC, et al.
Acaricidal activity of essential oils of Cinnamomum zeylanicum and Eremanthus
erythropappus, major compounds and cinnamyl acetate in Rhipicephalus microplus.
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formulations to control the cattle tick Rhipicephalus microplus using a mixture desing
approach. Exp Paratitol 2019;(30):26-33.
21. Gende LB, Floris I, Fritz R, Eguaras, MJ. Antimicrobial activity of cinnamon
(Cinnamomum zeylanicum) essential oil and its main components against Paenibacillus
larvae from Argentine. Bull Insectology 2008;(61):1-4.
22. Andreotti R, Garcia MV, Cunha RC, Barros JC. Protective action of Tagetes minuta
(Asteraceae) essential oil in the control of Rhipicephalus microplus (Canestrini, 1887)
(Acari: Ixodidae) in a cattle pen trial. Vet Parasitol 2013;197(1-2):341-345.
914
Technical note
Detection of pathogens of epidemiological importance in feral pigs from

Chihuahua and Durango, Mexico
Mario Enrique Haro Tirado a

José Martín Fuentes Rodríguez b
Claudia Chacón Zendejas c
Alberto Lafón Terrazas c
Luis Lecuona Olivares d
Rodolfo Pineda Pérez e
Rosalba Carreón Nápoles a*
a
Universidad Nacional Autónoma de México. Facultad de Medicina Veterinaria y Zootecnia.
Departamento de Medicina y Zootecnia de Cerdos. Avenida Universidad 3000, 04510
Coyoacán Ciudad de México, México.
b
Asesor privado. México.
c
Protección de la Fauna Mexicana, A.C. México.
d
USDA APHIS/México.
e
Comisión Nacional de Áreas Naturales Protegidas, Reserva de la Biósfera La Michilía.
México.
*Corresponding author: rcn@unam.mx
Abstract:
This study aimed to evaluate in feral pigs the presence of Salmonella spp (Spp), porcine
reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2),
swine influenza virus (SIV), porcine epidemic diarrhea virus (PEDV), Mycoplasma
hyopneumoniae (Mhyo) and Actinobacillus pleuropneumoniae (App). The samples were
obtained from pigs in the states of Chihuahua and Durango, Mexico. The tests analyzed for
915
the animals from Chihuahua were nasal swabs for SIV, rectal swabs for Spp and PEDV,
serum for PRRSV and PCV2, lung, liver, and lymph nodes for Spp, SIV, PRRSV, and PCV2,
as well as serum for serological tests. From animals in the state of Durango, the following
was collected: nasal swabs for SIV, rectal swabs for Spp and PEDV, and serum for PCV2 for
molecular and serological studies. The molecular results in both states showed samples
positive for PCV2, 73.3 % in Chihuahua and 91.3 % in Durango; likewise, two positive
samples were obtained for Spp in the state of Chihuahua (13.3 %) and one in Durango
(6.6 %), for SIV there were two positive (8.7 %) in Durango. For PRRSV and PEDV, samples
were negative in both states. Serological results in pigs from the two states showed positivity
for PCV2, Spp, and App. Samples were negative for PRRSV, PEDV, and Mhyo in both
states. It is important to highlight the molecular and serological detection of feral pigs
positive for various infectious agents important in pig production with zoosanitary
repercussions on public health, which implies an epidemiological relevance of these animals
in the context of “one health”.
Keywords: Feral pigs, Salmonellosis, Influenza, Porcine Circovirus type 2, PRRS, Porcine
epidemic diarrhea.
Feral pigs are animals that live in the wild and are widely distributed worldwide. They are
considered invasive species due to the damage they cause to agriculture, livestock farming,
and natural resources and because they interfere with other species(1-2). In Mexico, the
National Commission for the Knowledge and Use of Biodiversity (CONABIO, for its
acronym in Spanish) has reported the presence of these animals mainly in the northern states,
particularly in the Santa Elena Canyon in Chihuahua and in the La Michilía Reserve located
in the state of Durango, also in the Laguna de Términos in Campeche and some areas of
Central Mexico.
Several studies have shown that feral pigs can be an important source of bacterial diseases,
such as brucellosis, viral diseases, such as Aujeszky, and parasitic diseases, such as
trichinellosis, which represents a risk to public and animal health(3-6). Currently, in Mexico,
there is little information on the sanitary aspect of these animals; the available information
indicates that the presence of swine influenza, leptospirosis, salmonellosis, and brucellosis
has been detected in the states of Baja California Sur and Durango(7-8). To expand the health
information available so far, the present study determined the presence and frequency of
Salmonella spp (Spp), porcine reproductive and respiratory syndrome virus (PRRSV),
porcine circovirus type 2 (PCV2), swine influenza virus (SIV), porcine epidemic diarrhea
916
virus (PEDV), Mycoplasma hyopneumoniae (Mhyo) and Actinobacillus pleuropneumoniae

(App) in samples collected from feral pigs in the states of Chihuahua and Durango, Mexico.
During the period from 2019 to 2020, with the support of the feral pig control program in the
Santa Elena Canyon, Chihuahua, and in the La Michilía Biosphere Reserve, Durango, 15 and
23 animals, respectively, of both sexes and different sizes were opportunistically captured.
After capture, the animals were sacrificed at the trapping site according to the guidelines of
NOM-033-SAG/ZOO-2014. Immediately after slaughter, biological samples were collected
from the animals: nasal and rectal swabs, which were stored in minimum essential medium
(MEM) as a means of transport until their analysis. It was determined the presence of SIV in
the nasal swabs and the presence of Spp and PEDV in the rectal swabs by PCR.
Blood samples were taken from all pigs from the anterior vena cava using a 16G x 4-inch
needle and placed in a tube with separator gel, then centrifuged at 1,500 rpm for 10 min, and
the serum was stored in refrigeration until analysis for PRRSV and PCV2 by PCR and
antibodies of PRRSV, PCV2, App, Mhyo, Spp, and PEDV. In the case of the animals
captured in Chihuahua, it was also possible to obtain organs such as lung, liver, and lymph
nodes, collecting a fragment of approximately 5 cm of each one, which were collected in new
plastic bags that were stored in freezing at -20 °C until the analysis of Spp, SIV, PRRSV, and
PCV2. All determinations were carried out in the diagnostic laboratory of the Department of
Medicine and Zootechnics of Pigs of the Faculty of Veterinary Medicine and Zootechnics of
the National Autonomous University of Mexico.
For molecular studies, nucleic acids were extracted from nasal, rectal, serum, and tissue
samples using the commercial kit (QIAamp cador Pathogen Mini Kit QIAGEN) following
the manufacturer’s instructions. Subsequently, the real-time polymerase chain reaction
(PCR) was performed, for which a commercial kit (GeneReach Pockit) was used for each of
the agents mentioned above; these kits were used under the protocol for each of the agents
with their respective positive and negative controls; the interpretation as positive was with a
CT equal to or less than 35 according to the supplier. Serological tests were performed using
commercial kits for Spp (Idexx Laboratories Inc), PCV2 (BioNote Inc.), PRRSV (Civtest
Suis PPRS A/S Hipra), PEDV (ID Screen PEDV indirect), Mhyo (Civtest Suis MHYO Hipra)
and App (ID Screen App Screening indirect); the methodology and interpretation were
performed according to the supplier’s instructions.
For the information analysis, a descriptive statistic was made due to the non-homogeneity
between the samples, their small number, and the absence of other information on the
sampled animals, obtaining the percentages of positive samples for each etiological agent in
each state.
917
In the two states analyzed, 11 positive samples for PCV2 (73.3 %) in Chihuahua and 21
(91.3 %) in Durango were detected by molecular means; likewise, two samples positive for
Spp in the Chihuahua group (13.4 %) and one for Durango (6.6 %) were obtained. In the case
of SIV, there were two positive samples (8.7 %) in Durango. In the case of PRRSV and
PEDV, the samples were negative in both states (Table 1).
Table 1: Number of samples positive and negative for different pathogens in feral pigs
from the states of Chihuahua and Durango: 2019-2020
Agent Chihuahua Durango

(+) (-) (+) (-)
SIV 0 15 2 21
Salmonella spp 2 13 1 -
PEDV 0 15 0 23
PRRSV 0 15 0 23
PCV2 11 4 21 2
SIV= swine influenza virus; PEDV= porcine epidemic diarrhea virus; PRRSV= porcine reproductive and
respiratory syndrome virus; PCV2= porcine circovirus type 2.
Serology results showed 12 (80 %) samples positive for PCV2 and 13 for Salmonella spp
(56.5 %), respectively; 2 (13.3 %) samples positive for Spp in Chihuahua and 23 (100 %) for
Durango were also identified. For App, there were two positive samples (2.6 %) for
Chihuahua and 23 (100 %) for animals from Durango. Samples were negative for PRRSV,
PEDV, and Mhyo in all samples, Table 2.
Table 2: Number of positive and negative samples by serology for different pathogens in
feral pigs from the states of Chihuahua and Durango: 2019-202
Agent Chihuahua Durango

(+) (-) (+) (-)
Salmonella spp 3 12 23 0
PCV2 12 3 13 10
PRRSV 0 15 0 23
PEDV 0 15 0 23
Mhyo 0 15 0 23
App 2 13 23 0
PCV2= porcine circovirus type 2; PRRSV= porcine reproductive and respiratory syndrome virus; PEDV=
porcine epidemic diarrhea virus; Mhyo= Mycoplasma hyopneumoniae. App= Actinobacillus
pleuropneumoniae.
918
The high number of positive samples detected in serum and organs for PCV2 in both states
suggests that this virus actively circulates in the sampled feral pig population, which is
consistent with what has been reported in other similar reports(9-11). The above may suggest
that feral pigs could be a reservoir for domestic pigs or vice versa. Regarding SIV, despite
being a widely distributed disease in the world, only two positive samples were found in
Durango; nevertheless, success in antigen detection is complicated because the virus has a
very short excretion period, and sampling must be performed when the pig is in a febrile
period(12).
The results of this study coincide with previous studies(13-14) in which the detection of this
virus is also affected by external factors such as interaction with other species(15). In the case
of Spp, it is known that the bacteria can be eliminated intermittently in feces for long periods,
so possibly, when the animals were captured, they were not eliminating it. It must be
considered that large populations are also required to maintain the infection in the
environment, so there is a possibility that the bacteria is not present in feral pigs. The results
showed an uneven behavior as a high frequency was detected in pigs from Durango, but a
low frequency in Chihuahua, possibly due to the type of habitat that influences the
availability of water and food, and this is a stressor that causes the pig to eliminate the
pathogen and have more frequent contact with the bacteria, feces, and water(1).
In the case of PRRS and PED, the negative results suggest that these agents have little or no
circulation in feral pigs, which coincides with what has been reported by several
authors(14,16,17), since they require specific epidemiological conditions for their spread within
a herd, such as overpopulation, which happens constantly in technified pig farms; feral pigs
are low-density populations, which reduces the possibility of contact with these viruses, so
transmission would not be possible. Regarding Mhyo, not finding positive samples may
indicate that this agent is probably not present, but it should be considered that there may be
subclinical infections detectable only with histopathology(3,18). There are studies with data
positive for Mhyo, but it may be due to geography, time of collection, and number of
samples(19). In the case of App, the results show the presence of this agent, which is consistent
with other studies(9,14) where a high frequency of this disease is reported, and if it is added
that this kit has high sensitivity and detects the 12 relevant serotypes in domestic pigs, it can
be inferred that this agent circulates in these populations(20-21).
This work demonstrated the presence of animals positive for several important agents in
commercial pig production and public health, as had already been reported in a previous
study in Baja California Sur. It is relevant to comment on the implication of the above
because these animals move long distances and in herds searching for food and water, which
leads to their interaction with other wild and domestic species and humans, representing a
risk as it is a reservoir of various infectious agents. Although CONABIO indicates the
presence of this species throughout Mexico, the total population is unknown, so it would be
919
good to conduct more research to know exactly where they are present, estimate the
population in each of those places to be able to implement control programs such as the one
carried out by La Michilía, and at the same time carry out studies with more targeted designs
and with a larger sample size to continue with the detection of infectious agents present in
these animals. The above will allow to know a more accurate situation in Mexico, where the
information is virtually zero.
Acknowledgments
Candelario Cárdenas Figueroa, operational technician of the La Michilía Biosphere Reserve;

Pedro Roldan Morales, park ranger of the La Michilía Biosphere Reserve; Rogelio Martínez
González, community watchman of the Ejido El Alemán Nuevo, Suchil, Dgo.; Biol. María
Elena Rodarte García, CONANP Regional Director. Dr. Gabriela Gómez Verduzco from the
Secretariat of Postgraduate and Research and Dr. Roberto Martínez Gamba, both from the
FMVZ, UNAM.
Funding
Project funded by USDA under the Memorandum of Understanding between the National
Autonomous University of Mexico and the Animal and Plant Health Inspection Service of
the United States Department of Agriculture. Registration number 41991-1701-3-VII-15.
The authors state that they have no conflict of interest in this study.
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922
Technical note
Johnatan Alberto Ruíz-Ramírez a,b
Brayan Jossue Chávez-Ramírez a
Jorge Luis García-Valle a
Marcelo de las Heras c
Alfonso López-Mayagoitia d
Luis Jorge García-Márquez a*
a
Universidad de Colima. Facultad de Medicina Veterinaria y Zootecnia. Colima, México.
Carretera Colima-Manzanillo Km 40, Col. La Estación. 2810 Tecomán, Colima, México.
b
Universidad de Colima. Facultad de Medicina. Colima, México. Programa de Doctorado en
Ciencias Médicas
c
Universidad de Zaragoza. Facultad de Veterinaria. Zaragoza, España. Departamento de
Patología Animal
d
University of Prince Edward Island. Department of Pathology and Microbiology, Atlantic
Veterinary College. Charlottetown, PEI. Canada.
*
Corresponding author: ljgm_cmv@hotmail.com
Abstract:
Ovine pulmonary adenocarcinoma is a transmissible pulmonary malignancy of sheep caused

by a beta-retrovirus currently named Jaagsiekte sheep retrovirus (JSRV). Club cells (formerly
Clara cells) in the bronchiole and type II pneumonocytes in the alveoli are the target
oncogenic cells for this virus. Characterized clinically by intermittent cough, abundant nasal
discharge, and progressive weight loss, the tumors randomly involve all lung lobes or have a
923
cranioventral distribution mimicking bronchopneumonia. The definitive diagnosis of ovine

pulmonary adenocarcinoma requires identifying Jaagsiekte sheep retrovirus or associated
specific proteins in neoplastic cells such as JSRV-Env oncogenic protein. A two-year-old
Male Pelibuey sheep with a history of chronic cough and progressive weight loss was treated
unsuccessfully with antibiotics and died a few days later. Postmortem examination revealed
lung edema and several nodular to locally extensive masses in the lungs. Microscopically the
tumoral tissues were composed of clusters of neoplastic epithelial cells exhibiting a lepidic
growth pattern typical of pulmonary carcinoma. Tumoral cells were immunopositive for
Jaagsiekte sheep retrovirus-Env oncogenic protein. Based on these findings, the final
diagnosis of ovine pulmonary adenocarcinoma was made.
Keywords: Ovine pulmonary adenocarcinoma, Ovine pulmonary adenomatosis, Jaagsiekte,

retrovirus, JSRV, Immunohistochemistry, Pelibuey.
Ovine pulmonary adenocarcinoma (OPA), also called Jaagsiekte in South Africa, and ovine
pulmonary carcinoma (OPC) or pulmonary adenomatosis, is a transmissible malignant lung
tumor of sheep(1,2). First reported in South Africa in 1855, OPA occurs in many parts of the
world and is considered by some researchers the most common pulmonary neoplasia in
sheep(3). In the Americas, it has been reported from Argentina(4), Peru(5), Brazil(6) and
Mexico(7). In some countries, OPA is endemic and causes a significant economic impact on
sheep production, and in some geographic regions can reach an annual mortality rate of
2 %(3).
OPA is caused by a retrovirus belonging to the genus Betaretrovirus, family Retroviridae(1,2)

and is referred to as Jaagsiekte sheep retrovirus (JSRV). Its genomic RNA is formed
approximately by 7,460 nucleotides(8,9) and the viral genome possesses the gag, pro, pol and
env genes characteristic of retroviruses; JSRV also contains an envelope glycoprotein that
plays a fundamental role in the oncogenic transformation of cells(10,11). Like some other
retroviral infections, JSRV has a long incubation period of up to two years, and the neoplasia
can be reproduced experimentally by inoculating susceptible sheep(10,12).
JSRV infection has been reported in various breeds of domestic sheep (Ovis aries), only
infrequently in goats and mouflons (wild sheep Ovis gmelini), and never in any other animal
species(2,6). The primary target cells for JSRV in the lung are the bronchiolar Club cells
(formerly Clara cells) and the pneumonocytes Type II in the alveolar wall(1,9,13). This virus
924
also infects B-lymphocytes, T-lymphocytes (CD4+ and CD8+), macrophages and has been
detected in peripheral blood monocytes(9,11,14).
Sheep with OPA typically show progressive weight loss, exercise intolerance, coughing, and
overflowing fluid nasal secretions. The lungs are notably heavy on necropsy because of
severe pulmonary edema, and the pulmonary parenchyma exhibits multiple, firm grey
tumoral nodules that are firm in texture. Histologic features are those of a well-differentiated
lepidic or papillary carcinoma(3,12,15). OPA requires laboratory confirmation by identifying
JSRV or associated proteins in affected tissues by immunolabelling or PCR(4,14,16). It was
described the gross, microscopic and immunohistochemical changes in the lungs of a sheep
infected with JSRV, which evolved to OPA in Colima, Mexico.
A two-year-old male Pelibuey (Ovis aries) farmed in Colima, Mexico was presented to the
local veterinarian with a two-month history of nasal discharge, chronic cough, respiratory
distress, and progressive weight loss. The animal was separated from the flock and treated
for 7 d with broad-spectrum antibiotics and expectorants. The sheep deteriorated, became
prostrated and finally died. The carcass was submitted for postmortem examination to the
Pathology Laboratory, Faculty of Veterinary Medicine of the University of Colima.
At postmortem examination, the sheep appeared emaciated and exhibited mark corneal
opacity of the right eye due to traumatic injury to the periorbital tissue. Internally, there were
some fibrous adhesions on the right caudal lobes between the visceral and parietal pleura.
Overall, the lungs appeared pale and distended with rounded margins and were heavy and
edematous (Figure 1A). There were two distinct types of tumoral infiltrations in the lung: the
first types consisted of well-delineated firm tumoral masses (1-7 cm) protruding from the
pleural surface (Figures 1A and 1B); the second type consisted of a locally extensive
neoplastic infiltration grossly resembling broncho pneumonic consolidation (Figure 1B). The
trachea and bronchi contained large quantities of frothy fluid (Figure 1C), the heart showed
mark right-sided dilation, and the liver was congested. No other significant lesions were
observed on postmortem examination. Tissues were fixed in 10 % in buffered formalin and
routinely processed for histopathological examination.
925
Figure 1: Ovine pulmonary adenocarcinoma; lung
A. Lateral view of the right lung showing partially distended and pale pulmonary parenchyma. The dorsal
caudal lobe shows a focal, dark grey discoloration (arrows) with a firm texture on palpation. B. Ventral view
of the lungs and the pericardial sac (p). Note a locally extensive area of dark consolidation in the left caudal
lobe resembling bronchopneumonia (arrowheads) and a prominent raised tumoral nodule (arrow). C.
Abundant foamy fluid (asterisk) oozing from the trachea (t). D. Microscopic view of tumor showing alveoli-
like structures composed of thin bands of stromal tissue lined by cuboidal and columnar neoplastic cells.
Hematoxylin-eosin. E. Alveolar-like structures are composed of a thin layer of connective tissue lined by
cuboidal or columnar cells. Hematoxylin-eosin. Inset: Neoplastic cells show strong immunolabeling for
JSRV-Env oncogenic protein in the cytoplasm and cell membrane. IHC; Monoclonal antibody. F.
Microscopic view of the junction between the tumor and normal lung (dotted circle). Note lack of
encapsulation or junctional fibrosis between tumor and adjacent lung tissue. Hematoxylin-eosin. Inset:
Cluster of neoplastic cells showing strong immunolabeling for JSRV-Env oncogenic protein. IHC;
Monoclonal antibody.
Microscopically, the tumoral masses were poorly delineated, unencapsulated and composed
of focal to coalescing clusters of epithelial cells growing in lepidic and papillary patterns on
thin bands of connective tissue and forming alveoli-like structures (Figure 1D). Neoplastic
cells were cuboidal or columnar, had abundant eosinophilic cytoplasm with round nuclei and
subtle chromatin patterns containing one or two nucleoli (Figures 1D and 1E). There was
926
mild anisokaryosis and 1-2 mitotic figures per high power field (40x). The surrounding
alveoli were normal except for the presence of some edema and scattered alveolar
macrophages, but without evidence of encapsulation (Figure 1F). Based on the gross and
microscopic findings, a tentative diagnosis of OPA was made. Paraffin-embedded samples
of the affected lung were shipped away for immune-detection of JSRV-Env oncogenic
protein using monoclonal antibodies(11,16), and the results were positive (Figures 1E and 1F).
Infection with JSRV is distributed globally in all continents, with the notable exception of
Australia and New Zealand(9,13,14). In some countries, JSRV infection is endemic and affects
80 % of the ovine population, although only 4-30 % of the infected sheep go onto developing
OPA(8,17). The first confirmed case of OPA reported in Mexico was in 2019 in a flock of
sheep in the state of Tabasco in southern Mexico(7). Until 2016 OPA was an official notifiable
exotic disease in Mexico, but starting in 2018, the Official Mexican Sanitary Norm no longer
recognizes OPA as a reportable disease(18,19), perhaps conforming to the revised OIE
recommendations. The true prevalence of JSRV infection and OPA incidence in Mexico is
unknown mainly because the overall postmortem and laboratory investigations for farm
animals are seldom performed in this country.
The clinical history, gross, microscopic and immunohistochemical findings for the 2-yr-old
sheep reported here were classical of OPA. According to the veterinary literature, respiratory
signs and weight loss usually manifest between 2-4 yr of age, but initial infections likely
occur during colostrum ingestion and early life(2,5). The abundant liquid secretion from the
nostrils in a sheep with chronic cough and weight loss is a piece of relevant information that
OPA needs to be investigated by the practicing veterinarian. Holding the affected sheep
upside-down by the hind limbs (“wheelbarrow” test) results in clear whitish liquid's runoff
from the nostrils(8,12,17). Unfortunately, this hallmark sign was not adequately investigated by
the local veterinarian. It should be noted that OPA's exuded nasal fluid is not edema or
exudate "senso strictu", but rather excessive fluid oversecreted by neoplastic bronchiolar
Club cells and alveolar type II pneumonocytes(2,15). Secondary bacterial infections are
common in OPA lungs, but this was not the case in these sheep.
OPA clinical signs are nonspecific and can be seen in other chronic respiratory infections of
sheep(12,15). Also caused by a retrovirus (genus lentivirus), Maedi induces a severe
progressive lymphocytic interstitial pneumonia in sheep, and the clinical signs are
indistinguishable from OPA(3,15). Unfortunately, no routine laboratory assays are currently
available to distinguish these two retroviral diseases in everyday ovine practice. Electron
microscopy would be a rather unpractical and costly method to detect JRSV in OPA
neoplastic cells(8,13). Therefore, necropsy, histopathology and virus detection by
immunolabelling or PCR are indispensable for conventional diagnostic work. Besides OPA
and Maedi, other non-viral sheep diseases also manifest clinically with chronic cough and
weight loss such, as chronic bacterial bronchopneumonia, verminous pneumonia, caseous
927
lymphadenitis, and other lung neoplasms(2,3,12). There was no evidence of any of these lung
diseases on postmortem and microscopic examination in our sheep.
One unique feature of OPA is that tumors often have cranioventral lung distribution,
mimicking bronchopneumonia's classical distribution. In some other cases like ours, the
tumors involve all pulmonary lobes indistinctively, as it happens with most pulmonary
neoplasia in domestic animals(2,3,15). Tumor distribution in this case was dorsal, not
cranioventral, as reported in the literature. The pulmonary changes in OPA can be grouped
into two morphologic patterns, namely "classical" and "atypical"(20). The classical OPA tends
to be locally extensive, has a cranioventral distribution and on the cut-surface oozes clear
fluid. In contrast, the atypical OPA tends to be focal and sometimes solitary with no fluid
evidence on the cut-surface. It seems that this case deviated slightly from the classical
presentation, also showing the atypical pattern. Regardless of the l type of presentation, the
microscopic findings are the same(12).
In this sheep, there was no evidence of metastasis to the mediastinal or bronchial lymph nodes
or distant tissues, which was not surprising since only 10 % of OPA metastasize to these
nodes(3,17), and metastasis to other distant organs is extremely rare(8). The OPA microscopic
features are classic of a bronchioloalveolar carcinoma; however, this same type of pulmonary
neoplasia can also develop spontaneously in sheep's lungs in the absence of JSRV
infection(3,12). The classification of lung carcinomas in human and veterinary pathology has
changed continuously in the last three decades. Current classification distinguishes
microscopically five main types of lung adenocarcinomas based on the predominant growth
pattern of neoplastic cells: lepidic, papillary, acinar, squamous and adenosquamous(3).
Lepidic growth relates to proliferating neoplastic cells along the intact surface of alveolar
walls without vascular stromal or vascular invasion(12,15). In this case, the prevailing growth
patterns were lepidic and papillary types, which are the most commonly seen in OPA(3,20).
Retroviral-induced neoplasia of small ruminants such as OPA and also enzootic (ethmoidal)
nasal carcinoma are of immense interest to researchers in human medicine worldwide. OPA
has been used extensively as an animal model for human carcinogenesis studies(9,10,16). There
is still much to learn about sheep retroviral oncogenesis, and the current scientific advances
in molecular pathology will likely lead to important discoveries soon.
The authors declare that they have no conflicts of interest.
928
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Revista Mexicana de Ciencias Pecuarias
Edición Bilingüe
Rev. Mex. Cienc. Pecu. Vol. 14 Núm. 4, pp. 745-930, OCTUBRE-DICIEMBRE-2023 Bilingual Edition
ISSN: 2448-6698
CONTENIDO
CONTENTS
Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 14 Núm. 4, pp. 745-930, OCTUBRE-DICIEMBRE-2023
ARTÍCULOS / ARTICLES Pags.
Estructura de la red de mercado de bovinos en México, 2017-2021
Nicolás Callejas Juárez, José María Salas González...................................................................…...........................…..........…..........…..........…..........…..........…..........…..........…........….................…..........….........745
Prospectiva ambiental al 2030 en sistemas de producción de leche de vaca en México

Environmental outlook to 2030 in cow´s milk production systems in Mexico
María del Rosario Villavicencio-Gu�érrez, Nicolás Callejas-Juárez, Nathaniel Alec Rogers-Montoya, Vianey González-Hernández,
Rodrigo González-López, Carlos Galdino Mar�nez-García, Francisco Ernesto Mar�nez Castañeda…….................................................................................................................................................................760
Evaluación de resistencia a antibióticos en muestras de heces de terneros con diarrea en la región Cajamarca, Perú
Assessment of antibiotic resistance in fecal samples from calves with diarrhea in the Cajamarca region, Peru
Marco Antonio Cabrera González, Héctor Vladimir Vásquez Pérez, Carlos Quilcate-Pairazamán, José Bazán-Arce, Medali Cueva-Rodríguez..............................................................................................…….782
Contaminación de alimento comercial seco para perro por Aspergillus flavus y aflatoxinas en Aguascalientes, México
Contamination of commercial dry dog food by Aspergillus flavus and aflatoxins in Aguascalientes, Mexico
Lizbeth Mar�nez-Mar�nez, Arturo Gerardo Valdivia-Flores, Teódulo Quezada-Tristán, Alma Lilián Guerrero-Barrera,
Erika Janet Rangel-Muñoz, Karla Isela Arroyo Zúñiga, Fernanda Álvarez-Días, Marcelo Lisandro Signorini-Porchie�o....…….....…….....…….....……...................…….....…….....…….....…….....…...............……...........796
Estimación del grado básico de calidad en canales bovinas conforme a madurez ósea, marmoleo y predominancia fenotípica Bos indicus
Estimation of the basic quality grade of beef carcasses according to bone maturity, marbling, and Bos indicus phenotypic predominance
Francisco Gerardo Ríos Rincón, Leslie Zelibeth González Rueda, Jesús José Portillo Loera, Beatriz Isabel Castro Pérez, Alfredo Estrada Angulo, Jesús David Urías Estrada...........….…..818
The effect of hesperidin added to quail diets on blood gas, serum biochemistry and Hsp70 in heat stress
Efecto de la hesperidina añadida a las dietas de codorniz sobre los gases en sangre, la bioquímica sérica y HSP 70 bajo estrés por calor
Abdullah Özbilgin, Aykut Özgür, Onur Başbuğ…………………………………………………………………………………………………………………………….…….……………….…………….…………….…………….…………....................................836
Evaluación antihelmíntica de cuatro extractos de árboles forrajeros contra el nematodo Haemonchus contortus bajo condiciones in vitro
Anthelmintic evaluation of four fodder tree extracts against the nematode Haemonchus contortus under in vitro conditions
Itzel San�ago-Figueroa, Alejandro Lara-Bueno, Roberto González-Garduño, Pedro Mendoza-de Gives, Edgar Jesús Delgado-Núñez,
Ema de Jesús Maldonado-Simán, Yagoob Garedaghi, Agus�n Olmedo-Juárez..............................................................................................................................................................................................…...... 855
Efecto del uso de agua residual tratada sobre el suelo y cultivos forrajeros de Chenopodium quinoa Willd y Zea mays L.
Effect of treated wastewater use on soil and forage crops of Chenopodium quinoa Willd and Zea mays L.
Ana Lilia Velasco-Cruz, Vicente Arturo Velasco-Velasco, Judith Ruíz-Luna, José Raymundo Enríquez-del Valle, Aarón Mar�nez-Gu�érrez, Karen del Carmen Guzmán-Sebas�án.............…..................…......874
Correlación entre el comportamiento del toro de lidia en los corrales y el ruedo

Correlations between behavior in corrals and the bullring in Lidia breed bulls
Juan Manuel Lomillos, Eloy Marino, Enrique Recas, René Alonso, Marta Elena Alonso……..…..…….…….…….…….…….…….…….…….…….…….…….…….…….…….…….…….…...…..................……..…..……..…..……..….....889
NOTAS DE INVESTIGACIÓN / TECHNICAL NOTES
Efecto ixodicida de los extractos vegetales de Cinnamomum zeylanicum y Tagetes erecta sobre garrapatas Rhipicephalus microplus
Ixodicidal effect of plant extracts of Cinnamomum zeylanicum and Tagetes erecta on Rhipicephalus microplus ticks
Perla Iris Miranda Reyes, Francisco Mar�nez Ibañez, Rodolfo Esteban Lagunes-Quintanilla, América Ive�e Barrera Molina......…......…......…......…......…..........…..........….........…..........…..................….………..905
Detección de patógenos de importancia epidemiológica en cerdos ferales de Chihuahua y Durango, México

Detection of pathogens of epidemiological importance in feral pigs from Chihuahua and Durango, Mexico
Mario Enrique Haro Tirado, José Mar�n Fuentes Rodríguez, Claudia Chacón Zendejas, Alberto Lafón Terrazas, Luis Lecuona Olivares, Rodolfo Pineda Pérez, Rosalba Carreón Nápoles………………...........…915

Adenocarcinoma pulmonar ovino en México
Johnatan Alberto Ruíz-Ramírez, Brayan Jossue Chávez-Ramírez, Jorge Luis García-Valle, Marcelo de las Heras, Alfonso López-Mayagoi�a, Luis Jorge García-Márquez...…...…...…....................….....…….......923
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REV. MEX. CIENC. PECU. VOL. 14 No. 4 OCTUBRE-DICIEMBRE-2023

Prospectiva ambiental al 2030 en sistemas de producción de leche de vaca en México

Evaluación de resistencia a antibióticos en muestras de heces de terneros con diarrea

Contaminación de alimento comercial seco para perro por Aspergillus flavus y

Evaluación antihelmíntica de cuatro extractos de árboles forrajeros contra el

Correlación entre el comportamiento del toro de lidia en los corrales y el ruedo

Efecto ixodicida de los extractos vegetales de Cinnamomum zeylanicum y Tagetes

Detección de patógenos de importancia epidemiológica en cerdos ferales de Chihuahua

Ovine pulmonary adenocarcinoma in Mexico

INSTRUCTIONS FOR AUTHORS

Revista Mexicana de Ciencias Pecuarias is a scientific Title page

13. Final version. This is the document in which the

Structure of the cattle market network in Mexico, 2017-2021

Nicolás Callejas Juárez a*

* Corresponding author: ncallejas@uach.mx

The development of information and communication technologies has given rise to

Material and methods

The economic structure of livestock movement could be explained through measures of

Figure 1: Geographic specialization of bovine livestock supply in Mexico, 2017-2021

Figure 2: Geographic specialization of the cattle demand in Mexico, 2017-2021

Figure 5: Eigenvector by market network 2017-2021

The eigenvector is a measure of network centrality; the network pattern is represented by

Conclusions and implications

3. Fike K, Spire MF. Transportation of cattle. Veterinary Clinics of North America -

5. Brzoska L, Fischer M, Lentz HHK. Hierarchical structures in livestock trade

7. Hoscheit P, Anthony É, Vergu E. Dynamic centrality measures for cattle trade

8. Alocilla O, Monti G. Network analysis of cattle movements in Chile: Implications

9. Vinueza RL, Durand B, Zanella G. Network analysis of cattle movements in

12. Knutson RD. Discussion: animal identification systems in North America:

18. SIAP. Sistema de información Agropecuaria y Pesquera. Producción ganadera.

.24. Bosier S. Planning a system of regions: methods and techniques of interregional

26. Lira L. Técnicas de análisis regional. Instituto Latinoamericano y del Caribe de

30. Barrones-Sanz, LD. Costos operativos en el transporte de mercancía por carretera:

31. Enríquez-Quiroz JF, Esqueda-Esquivel VA, Martínez-Méndez D. Rehabilitation of

32. Camacho-Vera JH, Vargas-Canales JM, Quintero-Salazar L, Apan-Salcedo GW.

Environmental outlook to 2030 in cow's milk production systems in

María del Rosario Villavicencio-Gutiérrez a

Nathaniel Alec Rogers-Montoya c

Carlos Galdino Martínez-García a

Francisco Ernesto Martínez-Castañeda a*

* Corresponding author: femartinezc@uaemex.mx

Keywords: Life-cycle analysis, Environmental impact, Sustainability.

Worldwide milk production involves approximately 150 million households. In developing

Material and methods

Definition of objectives and scope

MAPF (kg/year) = Output (kg/year) * [0.1226 fat% +0.0776 protein%+ 0.2534]

Figure 1: Outline of the limits of the milk production system

Table 3: Inventory per 1 kg of milk -MAFP produced in semi-intensive system

The inventory for S2 was integrated considering:

Occupation of barn floor: bovine milk production in a semi-intensive system is regularly

Environmental impact assessment

Environmental impact assessment

Table 4: Midpoint impacts for 1 kg MAFP-Baseline Scenario 2021

The processes involved in the production of 1 kg MAFP involved in the generation of

In subsystem 2, the environmental load of the production of 1 kg MAFP, is derived from

Figure 2: Contribution of the processes involved to the different impact categories

Corn silage production Hay alfalfa production Corn stubble production

Comparative analysis of scenarios for the production of 1 kg MAFP

Agricultural land occupancy (ALO) Climate change (CC)

Fossil depletion (FD) Human toxicity (HT)