RMCP Vol. 15 Num. 2 (2024) : April-June (English Version)

Edición Bilingüe
Bilingual Edition
ISSN: 2448-6698
Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 15 Núm. 2, pp. 249-482, ABRIL-JUNIO-2024
Rev. Mex. Cienc. Pecu. Vol. 15 Núm. 2, pp. 249-482, ABRIL-JUNIO-2024

REVISTA MEXICANA DE CIENCIAS PECUARIAS Volumen 15 Numero 2, Abril-Junio
2024. 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 abril de 2024.
Establo lechero San Cristóbal en Tepatitlán,
Jalisco; con promedio de producción de 31
lts/vaca/dia bajo sistema familiar.
Autor: Adriana García Ruíz DIRECTORIO
FUNDADOR
John A. Pino
EDITOR EN JEFE EDITORES ADJUNTOS
Arturo García Fraustro Oscar L. Rodríguez Rivera
Alfonso Arias Medina
EDITORES POR DISCIPLINA
Dra. Yolanda Beatriz Moguel Ordóñez, INIFAP, México Dr. Juan Ku Vera, Universidad Autónoma de Yucatán, México
Dr. Ramón Molina Barrios, Instituto Tecnológico de Sonora, Dr. Ricardo Basurto Gutiérrez, INIFAP, México
Dr. Alfonso Juventino Chay Canul, Universidad Autónoma de Dr. Luis Corona Gochi, Facultad de Medicina Veterinaria y
Tabasco, México Zootecnia, UNAM, México
Dra. Maria Cristina Schneider, Universidad de Georgetown, Dr. Juan Manuel Pinos Rodríguez, Facultad de Medicina
Estados Unidos Veterinaria y Zootecnia, Universidad Veracruzana, México
Dr. Feliciano Milian Suazo, Universidad Autónoma de Dr. Carlos López Coello, Facultad de Medicina Veterinaria y
Querétaro, México Zootecnia, UNAM, México
Dr. Javier F. Enríquez Quiroz, INIFAP, México Dr. Arturo Francisco Castellanos Ruelas, Facultad de
Dra. Martha Hortencia Martín Rivera, Universidad de Sonora Química. UADY
URN, México Dra. Guillermina Ávila Ramírez, UNAM, México
Dr. Fernando Arturo Ibarra Flores, Universidad de Sonora Dr. Emmanuel Camuus, CIRAD, Francia.
URN, México Dr. Héctor Jiménez Severiano, INIFAP., México
Dr. James A. Pfister, USDA, Estados Unidos Dr. Juan Hebert Hernández Medrano, UNAM, México
Dr. Eduardo Daniel Bolaños Aguilar, INIFAP, México Dr. Adrian Guzmán Sánchez, Universidad Autónoma
Dr. Sergio Iván Román-Ponce, INIFAP, México Metropolitana-Xochimilco, México
Dr. Jesús Fernández Martín, INIA, España Dr. Eugenio Villagómez Amezcua Manjarrez, INIFAP, CENID
Dr. Maurcio A. Elzo, Universidad de Florida Salud Animal e Inocuidad, México
Dr. Sergio D. Rodríguez Camarillo, INIFAP, México Dr. José Juan Hernández Ledezma, Consultor privado
Dra. Nydia Edith Reyes Rodríguez, Universidad Autónoma del Dr. Fernando Cervantes Escoto, Universidad Autónoma
Estado de Hidalgo, México Chapingo, México
Dra. Maria Salud Rubio Lozano, Facultad de Medicina Dr. Adolfo Guadalupe Álvarez Macías, Universidad Autónoma
Veterinaria y Zootecnia, UNAM, México Metropolitana Xochimilco, México
Dra. Elizabeth Loza-Rubio, INIFAP, México Dr. Alfredo Cesín Vargas, UNAM, México
Dr. Juan Carlos Saiz Calahorra, Instituto Nacional de Dra. Marisela Leal Hernández, INIFAP, México
Investigaciones Agrícolas, España Dr. Efrén Ramírez Bribiesca, Colegio de Postgraduados,
Dr. José Armando Partida de la Peña, INIFAP, México México
Dr. José Luis Romano Muñoz, INIFAP, México Dra. Itzel Amaro Estrada, INIFAP, México
Dr. Jorge Alberto López García, INIFAP, México
Dr. Alejandro Plascencia Jorquera, Universidad Autónoma de
Baja California, México
TIPOGRAFÍA Y FORMATO: Oscar L. Rodríguez Rivera
Indizada en el “Journal Citation Report” Science Edition del ISI . Inscrita en el Sistema de Clasificación de Revistas Científicas y
Tecnológicas de CONACyT; en EBSCO Host y la Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
(RedALyC) (www.redalyc.org); en la Red Iberoamericana de Revistas Científicas de Veterinaria de Libre Acceso
(www.veterinaria.org/revistas/ revivec); en los Índices SCOPUS y EMBASE de Elsevier (www.elsevier. com).
I
REVISTA MEXICANA DE CIENCIAS PECUARIAS
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II
REVISTA MEXICANA DE CIENCIAS PECUARIAS
REV. MEX. CIENC. PECU. VOL. 15 No. 2 ABRIL-JUNIO-2024
CONTENIDO
Contents
ARTÍCULOS
Articles
Pág.
Estudio de la Estructura y Diversidad genética de ganado Holstein del sistema familiar

en México
Study of the Genetic Structure and Diversity of Holstein cattle in the small holder system in Mexico
Felipe de Jesús Ruiz-López, José G. Cortés-Hernández, José Luis Romano-Muñoz, Fernando
Villaseñor-González, Adriana García-Ruiz ................................................................................249
Efecto de diferentes protocolos de castración en indicadores productivos de cerdos:

meta-análisis
Effect of various castration protocols on production indicators in pigs: meta-analysis
Humberto Rafael Silva-Santos, Francisco Ernesto Martínez-Castañeda, Gregorio Álvarez-Fuentes,
María de la Salud Rubio-Lozano, María Elena Trujillo-Ortega ...................……............................267
Acumulación de materia seca, rendimiento y calidad nutricional del forraje de híbridos

de maíz cosechados a diferentes días después de la siembra
Dry matter accumulation, yield, and nutritional quality of forage of corn hybrids harvested at
different days after sowing
Diego Eduardo Ramírez Gutiérrez, José de Jesús Olmos Colmenero, Alfonso Peña Ramos, Juan
Isidro Sánchez Duarte, Ernesto Medina Núñez, Silviano Gallardo Ramírez, Omar Iván Santana …287
Análisis por microscopía electrónica y difracción de rayos X de enterolitos de equinos en

el valle de Aburrá, Antioquia, Colombia
Electron microscopy and X-ray diffraction analysis of equine enteroliths from the Aburrá Valley in
Antioquia, Colombia
Sergio Andrés Vélez Gil, Juan José Patiño Marulanda, José Ramón Martínez Aranzales................302
Prevalencia y factores de riesgo asociados a Cryptosporidium spp. en bovinos de leche

de Chiquinquirá (Colombia)
Prevalence and risk factors associated with Cryptosporidium spp. in dairy cattle in Chiquinquirá
(Colombia)
Diana M. Bulla-Castañeda, Deisy J. Lancheros Buitrago, Leneth B. Castañeda Sedano, Rosa I.
Higuera Piedrahita, Martin O. Pulido-Medellin ..........................................................................310
III
Influence of the type of container and traditional methods on the long-term storage of
honey produced by stingless Scaptotrigona mexicana: bioactive compounds and
antioxidant properties
Influencia del tipo de recipiente y de los métodos tradicionales en el almacenamiento a largo plazo
de la miel producida por Scaptotrigona mexicana sin aguijón: compuestos bioactivos y propiedades
antioxidantes
Naida Juárez-Trujillo, Simón Carrouché, María Remedios Mendoza-López, Juan L. Monribot-
Villanueva, José A. Guerrero-Analco, Maribel Jiménez-Fernández……………………………………………323
Un efecto novedoso del extracto acuoso de semillas de Pimpinella anisum sobre

garrapatas de perros domésticos (Canis lupus familiaris)
A novel effect of aqueous extract of Pimpinella anisum seeds on ticks of domestic dogs (Canis lupus
familiaris)
William Fernando Várguez-Tec, Sara Luz Nahuat-Dzib, Julia Cano-Sosa, Lorena Reyes-Vaquero, Edgar
E. Lara-Ramirez, Benjamín Abraham Ayil-Gutiérrez, Angel Virgilio Domínguez-May.......................344
Conocimiento socio-ecológico de la actividad apícola en la Costa Chica de Guerrero,

México
Socio-ecological knowledge of the beekeeping activity in the Costa Chica region of Guerrero, Mexico
José Cámara-Romero, William Cetzal-Ix, Luis Alaniz-Gutiérrez, Agustín Rojas-Herrera, José Aparicio-
López, Columba Rodríguez-Alviso .....................................………..............................................360
Prevalencia de Fasciola hepatica y Calicophoron spp. en vacunos de crianza extensiva

del distrito Florida (Amazonas), Perú
Prevalence of Fasciola hepatica and Calicophoron spp. in extensively reared cattle in the Florida
district (Amazonas), Peru
Medali Cueva-Rodríguez, Teófilo Torrel, Cristian Hobán, Wuesley Alvarez-García, Flor Mejía, Luis
Vargas-Rocha........................................................................................................................376
Influence of feedlot living space on production variables, carcass and meat quality
traits in Holstein steers
Influencia del espacio vital del corral de engorda en las variables de producción, rasgos de calidad
de la canal y la carne en novillos Holstein
Ana Mireya Romo-Valdez, Cristina Pérez-Linares, Francisco Gerardo Ríos-Rincón, Fernando
Figueroa-Saavedra, Alberto Barreras-Serrano, Beatriz Isabel Castro-Pérez, Eduardo Sánchez-López,
Georgina Valentina Cervantes Cazarez ....................................................................................393
REVISIONES DE LITERATURA
Reviews
Lenteja de agua (Lemna minor): potencial alimentario y ambiental. Revisión

Common duckweed (Lemna minor): food and environmental potential. Review
Olga Jaimes Prada, Olga Lora Díaz, Katherine Tache Rocha......................................................404
IV
Implicación de las Fusariotoxinas en la producción avícola. Revisión
Implication of Fusariotoxins in poultry production. Review
Gabriela Guadalupe Gómez Verduzco, Ernesto Ávila González, Guillermo Téllez Isaías, Juan Carlos
Del Río García, Jacqueline Uribe Rivera....................................................................................425
Contribución de gramíneas forrajeras a la fijación biológica de nitrógeno y su respuesta

a la inoculación de diazótrofas. Revisión
Contribution of forage grasses to biological nitrogen fixation and their response to diazotroph
inoculation. Review
Dania Fonseca López, Nelson Vivas Quila, Raúl Cuervo Mulet, Carlos Eduardo Rodríguez Molano.446
NOTAS DE INVESTIGACIÓN
Technical notes
Frecuencia de seropositividad contra el circovirus porcino tipo 2 (PCV2) en el área

metropolitana de Monterrey, Nuevo León y su área periférica
Frequency of seropositivity against porcine circovirus type 2 (PCV2) in the metropolitan area of
Monterrey, Nuevo León, and its peripheral area
José Pablo Villarreal-Villarreal, César Dávila-Martínez, Heidi Giselle Rodríguez-Ramírez ..............462
Prevalencia e intensidad de virosis de abejas melíferas (Apis mellifera) en seis regiones

del estado de Jalisco, México
Prevalence and infection intensity of honey bee (Apis mellifera) viral diseases in six regions of the
state of Jalisco, Mexico
Ana Karen Ramos-Cuellar, Álvaro De la Mora, Francisca Contreras-Escareño, Nuria Morfin, José
María Tapia-González, José Octavio Macías-Macías, Tatiana Petukhova, Adriana Correa-Benítez,
Ernesto Guzman-Novoa .........................................................................................................471
V
Actualización: octubre, 2023
NOTAS AL AUTOR
La Revista Mexicana de Ciencias Pecuarias se edita bibliográficas una extensión máxima de 30 cuartillas y
completa en dos idiomas (español e inglés) y publica tres 5 cuadros.
categorías de trabajos: Artículos científicos, Notas de
6. Los manuscritos de las tres categorías de trabajos que
investigación y Revisiones bibliográficas.
se publican en la Rev. Mex. Cienc. Pecu. deberán
Los autores interesados en publicar en esta revista contener los componentes que a continuación se
deberán ajustarse a los lineamientos que más adelante se indican, empezando cada uno de ellos en página
indican, los cuales, en términos generales, están de aparte.
acuerdo con los elaborados por el Comité Internacional de Página del título
Editores de Revistas Médicas (CIERM) Bol Oficina Sanit Resumen en español
Panam 1989;107:422-437. Resumen en inglés
Texto
1. Sólo se aceptarán trabajos inéditos. No se admitirán
Agradecimientos y conflicto de interés
si están basados en pruebas de rutina, ni datos
experimentales sin estudio estadístico cuando éste Literatura citada
sea indispensable. Tampoco se aceptarán trabajos
que previamente hayan sido publicados condensados 7. Página del Título. Solamente debe contener el título
o in extenso en Memorias o Simposio de Reuniones o del trabajo, que debe ser conciso pero informativo; así
Congresos (a excepción de Resúmenes). como el título traducido al idioma inglés. En el
manuscrito no se debe incluir información como
2. Todos los trabajos estarán sujetos a revisión de un
nombres de autores, departamentos, instituciones,
Comité Científico Editorial, conformado por Pares de
direcciones de correspondencia, etc., ya que estos
la Disciplina en cuestión, quienes desconocerán el
datos tendrán que ser registrados durante el proceso
nombre e Institución de los autores proponentes. El
de captura de la solicitud en la plataforma del OJS
Editor notificará al autor la fecha de recepción de su
(revisar el Instrucctivo para envío de artículos en la
trabajo.
dirección: http://ciencias pecuarias.inifap.gob.mx.
3. El manuscrito deberá someterse a través del portal de
8. Resumen en español. En la segunda página se debe
la Revista en la dirección electrónica:
incluir un resumen que no pase de 250 palabras. En
http://cienciaspecuarias.inifap.gob.mx, consultando
él se indicarán los propósitos del estudio o
el “Instructivo para envío de artículos en la
investigación; los procedimientos básicos y la
página dela Revista Mexicana de Ciencias Pecuarias”.
metodología empleada; los resultados más
Para su elaboración se utilizará el procesador de
importantes encontrados, y de ser posible, su
Microsoft Word, con letra Times New Roman a 12
significación estadística y las conclusiones principales.
puntos, a doble espacio. Asimismo, se deberán llenar
A continuación del resumen, en punto y aparte,
los formatos de postulación, carta de originalidad y no
agregue debidamente rotuladas, de 3 a 8 palabras o
duplicidad y disponibles en el propio sitio oficial de la
frases cortas clave que ayuden a los indizadores a
revista.
clasificar el trabajo, las cuales se publicarán junto con
4. Por ser una revista con arbitraje, y para facilitar el el resumen.
trabajo de los revisores, todos los renglones de cada
9. Resumen en inglés. Anotar el título del trabajo en
página deben estar numerados de manera continua a
inglés y a continuación redactar el “abstract” con las
lo largo de todo el documento; asimismo cada página
mismas instrucciones que se señalaron para el
debe estar numerada, inclusive cuadros, ilustraciones
resumen en español. Al final en punto y aparte, se
y gráficas.
deberán escribir las correspondientes palabras clave
5. Los artículos tendrán una extensión máxima de 20 (“keywords”).
cuartillas a doble espacio, sin incluir páginas de Título,
10. Texto. Las tres categorías de trabajos que se publican
y cuadros o figuras (los cuales no deberán exceder de
en la Rev. Mex. Cienc. Pecu. consisten en lo
ocho y ser incluidos en el texto). Las Notas de
siguiente:
investigación tendrán una extensión máxima de 15
cuartillas y 6 cuadros o figuras. Las Revisiones
VI
texto, en los cuadros y en las ilustraciones se deben
a) Artículos científicos. Deben ser informes de trabajos
identificar mediante números arábigos entre
originales derivados de resultados parciales o finales
paréntesis, sin señalar el año de la referencia. Evite
de investigaciones. El texto del Artículo científico se
hasta donde sea posible, el tener que mencionar en el
divide en secciones que llevan estos
texto el nombre de los autores de las referencias.
encabezamientos:
Procure abstenerse de utilizar los resúmenes como
Introducción Material referencias; las “observaciones inéditas” y las
y MétodosResultados “comunicaciones personales” no deben usarse como
Discusión referencias, aunque pueden insertarse en el texto
Conclusiones e implicaciones (entre paréntesis).
Literatura citada
Reglas básicas para la Literatura citada
En los artículos largos puede ser necesario agregar Nombre de los autores, con mayúsculas sólo las
subtítulos dentro de estas divisiones a fin de hacer iniciales, empezando por el apellido paterno, luego
más claro el contenido, tanto en Material y métodos como iniciales del materno y nombre(s). En caso de
en las secciones de Resultados y de Discusión, las apellidos compuestos se debe poner un guión entre
cuales también pueden presentarse como una sola ambos, ejemplo: Elías-Calles E. Entre las iniciales de
sección. un autor no se debe poner ningún signo de
b) Notas de investigación. Consisten en puntuación, ni separación; después de cada autor sólo
modificaciones a técnicas, informes de casos clínicos se debe poner una coma, después del último autor se
de interés especial, preliminares de trabajos o debe poner unpunto.
investigaciones limitadas, descripción de nuevas El título del trabajo se debe escribir completo (en su
variedades de pastos; así como resultados de idioma original) luego el título abreviado de la revista
investigación que a juicio de los editores deban así ser donde se publicó, sin ningún signo de puntuación;
publicados. El texto contendrá la misma información inmediatamente después el año de la publicación,
del método experimental señalado en el inciso a), luego el número del volumen, seguido del número
pero su redacción será corrida del principio al final del (entre paréntesis) de la revista y finalmente el número
trabajo; esto no quiere decir que sólo se supriman los de páginas (esto en caso de artículo ordinario de
subtítulos, sino que se redacte en forma continua y revista).
coherente.
Puede incluir en la lista de referencias, los artículos
c) Revisiones bibliográficas. Consisten en el
aceptados, aunque todavía no se publiquen; indique la
tratamiento y exposición de un tema o tópico de
revista y agregue “en prensa” (entre corchetes).
relevante actualidad e importancia; su finalidad es la
de resumir, analizar y discutir, así como poner a En el caso de libros de un solo autor (o más de uno,
disposición del lector información ya publicada sobre pero todos responsables del contenido total del libro),
un tema específico. El texto se divide en: después del o los nombres, se debe indicar el título
Introducción, y las secciones que correspondan al del libro, el número de la edición, el país, la casa
desarrollo del tema en cuestión. editorial y el año.
11. Agradecimientos y conflicto de interés. Siempre Cuando se trate del capítulo de un libro de varios
que corresponda, se deben especificar las autores, se debe poner el nombre del autor del
colaboraciones que necesitan ser reconocidas, tales
capítulo, luego el título del capítulo, después el
como a) la ayuda técnica recibida; b) el
nombre de los editores y el título del libro, seguido del
agradecimiento por el apoyo financiero y material,
país, la casa editorial, año y las páginas que abarca el
especificando la índole del mismo; c) las relaciones
capítulo.
financieras que pudieran suscitar un conflicto de
intereses. Las personas que colaboraron pueden ser En el caso de tesis, se debe indicar el nombre del
citadas por su nombre, añadiendo su función o tipo de autor, el título del trabajo, luego entre corchetes el
colaboración; por ejemplo: “asesor científico”, grado (licenciatura, maestría, doctorado), luego el
“revisión crítica de la propuesta para el estudio”, nombre de la ciudad, estado y en su caso país,
“recolección de datos”, etc. Siempre que corresponda, seguidamente el nombre de la Universidad (no el de
los autores deberán mencionar si existe algún la escuela), y finalmente el año.
conflicto de interés.
12. Literatura citada. Numere las referencias
consecutivamente en el orden en que se mencionan
por primera vez en el texto. Las referencias en el
VII
Autor de capítulo.
Emplee el estilo de los ejemplos que aparecen a
continuación: IX) Roberts SJ. Equine abortion. In: Faulkner LLC editor.
Abortion diseases of cattle. 1rst ed. Springfield,
Illinois, USA: Thomas Books; 1968:158-179.
Revistas
Artículo ordinario, con volumen y número. (Incluya el Memorias de reuniones.
nombre de todos los autores cuando sean seis o X) Loeza LR, Angeles MAA, Cisneros GF. Alimentación
menos; si son siete o más, anote sólo el nombre de de cerdos. En: Zúñiga GJL, Cruz BJA editores.
los seis primeros y agregue “et al.”). Tercera reunión anual del centro de investigaciones
I) Basurto GR, Garza FJD. Efecto de la inclusión de grasa forestales y agropecuarias del estado de Veracruz.
o proteína de escape ruminal en el comportamiento Veracruz. 1990:51-56.
de toretes Brahman en engorda. Téc Pecu Méx XI) Olea PR, Cuarón IJA, Ruiz LFJ, Villagómez AE.
1998;36(1):35-48. Concentración de insulina plasmática en cerdas
Sólo número sin indicar volumen. alimentadas con melaza en la dieta durante la
inducción de estro lactacional [resumen]. Reunión
II) Stephano HA, Gay GM, Ramírez TC. Encephalomielitis, nacional de investigación pecuaria. Querétaro, Qro.
reproductive failure and corneal opacity (blue eye) in 1998:13.
pigs associated with a paramyxovirus infection. Vet
Rec 1988;(122):6-10. XII) Cunningham EP. Genetic diversity in domestic
animals: strategies for conservation and
III) Chupin D, Schuh H. Survey of present status of the use development. In: Miller RH et al. editors. Proc XX
of artificial insemination in developing countries. VI eltsville
B Symposium: Biotechnology’s role in genetic
World Anim Rev 1993;(74-75):26-35. improvement of farm animals. USDA. 996:13.
1
No se indica el autor. Tesis.
IV) Cancer in South Africa [editorial]. S Afr Med J XIII) Alvarez MJA. Inmunidad humoral en la anaplasmosis
1994;84:15. y babesiosis bovinas en becerros mantenidos en una
zona endémica [tesis maestría]. México, DF:
Suplemento de revista. Universidad Nacional Autónoma de México; 1989.
V) Hall JB, Staigmiller RB, Short RE, Bellows RA, Bartlett XIV) Cairns RB. Infrared spectroscopic studies of solid
SE. Body composition at puberty in beef heifers as oxigen [doctoral thesis]. Berkeley, California, USA:
influenced by nutrition and breed [abstract]. J Anim University of California; 1965.
Sci 1998;71(Suppl 1):205.
Organización como autor.
Organización, como autor.
XV) NRC. National Research Council. The nutrient
VI) The Cardiac Society of Australia and New Zealand.
requirements of beef cattle. 6th ed. Washington,
Clinical exercise stress testing. Safety and performance
DC, USA: National Academy Press; 1984.
guidelines. Med J Aust 1996;(164):282-284.
XVI) SAGAR. Secretaría de Agricultura, Ganadería y
En proceso de publicación. Desarrollo Rural. Curso de actualización técnica para
la aprobación de médicos veterinarios zootecnistas
VII) Scifres CJ, Kothmann MM. Differential grazing use of
responsables de establecimientos destinados al
herbicide treated area by cattle. J Range Manage [in
press] 2000. sacrificio de animales. México. 1996.
XVII) AOAC. Oficial methods of analysis. 15th ed.
Arlington, VA, USA: Association of Official Analytical
Libros y otras monografías Chemists. 1990.
Autor total. XVIII) SAS. SAS/STAT User’s Guide (Release 6.03). Cary
VIII) Steel RGD, Torrie JH. Principles and procedures of NC, USA: SAS Inst. Inc. 1988.
statistics: A biometrical approach. 2nd ed. New XIX) SAS. SAS User´s Guide: Statistics (version 5 ed.).
York, USA: McGraw-Hill Book Co.; 1980.
Cary NC, USA: SAS Inst. Inc. 1985.
VIII
Publicaciones electrónicas Abreviaturas de uso frecuente:
XX) Jun Y, Ellis M. Effect of group size and feeder type cal caloría (s)
on growth performance and feeding patterns in cm centímetro (s)
growing pigs. J Anim Sci 2001;79:803-813. °C grado centígrado (s)
http://jas.fass.org/cgi/reprint/79/4/803.pdf. DL50 dosis letal 50%
Accessed Jul 30, 2003. g gramo (s)
XXI) Villalobos GC, González VE, Ortega SJA. Técnicas ha hectárea (s)
para estimar la degradación de proteína y materia h hora (s)
orgánica en el rumen y su importancia en rumiantes i.m. intramuscular (mente)
en pastoreo. Téc Pecu Méx 2000;38(2): 119-134. i.v. intravenosa (mente)
http://www.tecnicapecuaria.org/trabajos/20021217 J joule (s)
5725.pdf. Consultado 30 Ago, 2003. kg kilogramo (s)
XXII) Sanh MV, Wiktorsson H, Ly LV. Effect of feeding level km kilómetro (s)
on milk production, body weight change, feed L litro (s)
conversion and postpartum oestrus of crossbred log logaritmo decimal
lactating cows in tropical conditions. Livest Prod Sci Mcal megacaloría (s)
2002;27(2-3):331-338. http://www.sciencedirect. MJ megajoule (s)
com/science/journal/03016226. Accessed Sep 12, m metro (s)
2003. msnm metros sobre el nivel del mar
13. Cuadros, Gráficas e Ilustraciones. Es preferible µg microgramo (s)
que sean pocos, concisos, contando con los datos µl microlitro (s)
necesarios para que sean autosuficientes, que se µm micrómetro (s)(micra(s))
entiendan por sí mismos sin necesidad de leer el texto. mg miligramo (s)
Para las notas al pie se deberán utilizar los símbolos ml mililitro (s)
convencionales. mm milímetro (s)
14 Versión final. Es el documento en el cual los autores min minuto (s)
ya integraron las correcciones y modificaciones ng nanogramo (s)
indicadas por el Comité Revisor. Se les enviará a los P probabilidad (estadística)
autores un instructivo que contendrá los puntos p página
esenciales para su correcta elaboración. Las PC proteína cruda
fotografías e imágenes deberán estar en formato jpg
PCR reacción en cadena de la polimerasa
(o compatible) con al menos 300 dpi de resolución.
Tanto las fotografías, imágenes, gráficas, cuadros o pp páginas
tablas deberán incluirse en el mismo archivo del texto. ppm partes por millón
Los cuadros no deberán contener ninguna línea % por ciento (con número)
vertical, y las horizontales solamente las que delimitan rpm revoluciones por minuto
los encabezados de columna, y la línea al final del seg segundo (s)
cuadro. t tonelada (s)
15. Una vez recibida la versión final, ésta se mandará para TND total de nutrientes digestibles
su traducción al idioma inglés o español, según UA unidad animal
corresponda. Si los autores lo consideran conveniente UI unidades internacionales
podrán enviar su manuscrito final en ambos idiomas. vs versus
16. Tesis. Se publicarán como Artículo o Nota de xg gravedades
Investigación, siempre y cuando se ajusten a las Cualquier otra abreviatura se pondrá entre paréntesis
normas de esta revista. inmediatamente después de la(s) palabra(s)
17. Los trabajos no aceptados para su publicación se completa(s).
regresarán al autor, con un anexo en el que se
19. Los nombres científicos y otras locuciones latinas se
explicarán los motivos por los que se rechaza o las
deben escribir en cursivas.
modificaciones que deberán hacerse para ser
reevaluados.
18.
IX
Updated: October, 2023
INSTRUCTIONS FOR AUTHORS
Revista Mexicana de Ciencias Pecuarias is a scientific Title page

journal published in a bilingual format (Spanish and Abstract
English) which carries three types of papers: Research Text
Articles, Technical Notes, and Reviews. Authors interested Acknowledgments and conflict of interest
in publishing in this journal, should follow the below- Literature cited
mentioned directives which are based on those set down
by the International Committee of Medical Journal Editors
(ICMJE) Bol Oficina Sanit Panam 1989;107:422-437. 7. Title page. It should only contain the title of the
work, which should be concise but informative; as well
1. Only original unpublished works will be accepted. as the title translated into English language. In the
Manuscripts based on routine tests, will not be manuscript is not necessary information as names of
accepted. All experimental data must be subjected to authors, departments, institutions and
statistical analysis. Papers previously published correspondence addresses, etc.; as these data will
condensed or in extenso in a Congress or any other have to be registered during the capture of the
type of Meeting will not be accepted (except for application process on the OJS platform
Abstracts). (http://cienciaspecuarias.inifap.gob.mx).
2. All contributions will be peer reviewed by a scientific 8. Abstract. On the second page a summary of no more
editorial committee, composed of experts who ignore than 250 words should be included. This abstract
the name of the authors. The Editor will notify the should start with a clear statement of the objectives
and must include basic procedures and methodology.
author the date of manuscript receipt.
The more significant results and their statistical value
3. Papers will be submitted in the Web site and the main conclusions should be elaborated briefly.
http://cienciaspecuarias.inifap.gob.mx, according the At the end of the abstract, and on a separate line, a
“Guide for submit articles in the Web site of the list of up to 10 key words or short phrases that best
Revista Mexicana de Ciencias Pecuarias”. Manuscripts describe the nature of the research should be stated.
should be prepared, typed in a 12 points font at 9. Text. The three categories of articles which are
double space (including the abstract and tables), At published in Revista Mexicana de Ciencias
the time of submission a signed agreement co-author Pecuarias are the following:
letter should enclosed as complementary file; co-
authors at different institutions can mail this form a) Research Articles. They should originate in primary
independently. The corresponding author should be works and may show partial or final results of
research. The text of the article must include the
indicated together with his address (a post office box
following parts:
will not be accepted), telephone and Email.
Introduction
4. To facilitate peer review all pages should be numbered
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consecutively, including tables, illustrations and
Results
graphics, and the lines of each page should be
Discussion
numbered as well.
Conclusions and implications
5. Research articles will not exceed 20 double spaced Literature cited
pages, without including Title page and Tables and In lengthy articles, it may be necessary to add other
Figures (8 maximum and be included in the text). sections to make the content clearer. Results and
Technical notes will have a maximum extension of 15 Discussion can be shown as a single section if
pages and 6 Tables and Figures. Reviews should not considered appropriate.
exceed 30 pages and 5 Tables and Figures.
b) Technical Notes. They should be brief and be
6. Manuscripts of all three type of articles published in evidence for technical changes, reports of clinical
Revista Mexicana de Ciencias Pecuarias should cases of special interest, complete description of a
contain the following sections, and each one should limited investigation, or research results which
begin on a separate page. should be published as a note in the opinion
of the editors. The text will contain the same
X
information presented in the sections of t he e. When a reference is made of a chapter of book
research article but without section titles. written by several authors; the name of the author(s)
of the chapter should be quoted, followed by the title
c) Reviews. The purpose of these papers is to
summarize, analyze and discuss an outstanding topic. of the chapter, the editors and the title of the book,
The text of these articles should include the following the country, the printing house, the year, and the
sections: Introduction, and as many sections as initial and final pages.
needed that relate to the description of the topic in f. In the case of a thesis, references should be
question. made of the author’s name, the title of the research,
10. Acknowledgements. Whenever appropriate, the degree obtained, followed by the name of the City,
collaborations that need recognition should be State, and Country, the University (not the school),
specified: a) Acknowledgement of technical support; and finally the year.
b) Financial and material support, specifying its
nature; and c) Financial relationships that could be the Examples
source of a conflict of interest.
The style of the following examples, which are partly
People which collaborated in the article may be based on the format the National Library of Medicine
named, adding their function or contribution; for of the United States employs in its Index Medicus,
example: “scientific advisor”, “critical review”, “data should be taken as a model.
collection”, etc.
11. Literature cited. All references should be quoted in
their original language. They should be numbered Journals
consecutively in the order in which they are first
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.
XI
In press XVII) AOAC. Official methods of analysis. 15th ed.
Arlington, VA, USA: Association of Official Analytical
VII) Scifres CJ, Kothmann MM. Differential grazing use of
Chemists. 1990.
herbicide-treated area by cattle. J Range Manage [in
press] 2000. XVIII) SAS. SAS/STAT User’s Guide (Release 6.03). Cary
NC, USA: SAS Inst. Inc. 1988.
Books and other monographs
XIX) SAS. SAS User´s Guide: Statistics (version 5 ed.).
Author(s) Cary NC, USA: SAS Inst. Inc. 1985.
VIII) Steel RGD, Torrie JH. Principles and procedures of

Electronic publications
statistics: A biometrical approach. 2nd ed. New
York, USA: McGraw-Hill Book Co.; 1980. XX) Jun Y, Ellis M. Effect of group size and feeder type
on growth performance and feeding patterns in
Chapter in a book growing pigs. J Anim Sci 2001;79:803-813.
http://jas.fass.org/cgi/reprint/79/4/803.pdf.
IX) Roberts SJ. Equine abortion. In: Faulkner LLC editor. Accesed Jul 30, 2003.
Abortion diseases of cattle. 1rst ed. Springfield,
Illinois, USA: Thomas Books; 1968:158-179. XXI) Villalobos GC, González VE, Ortega SJA. Técnicas
para estimar la degradación de proteína y materia
Conference paper orgánica en el rumen y su importancia en rumiantes
en pastoreo. Téc Pecu Méx 2000;38(2): 119-134.
X) Loeza LR, Angeles MAA, Cisneros GF. Alimentación
http://www.tecnicapecuaria.org/trabajos/20021217
de cerdos. En: Zúñiga GJL, Cruz BJA editores.
5725.pdf. Consultado 30 Jul, 2003.
Tercera reunión anual del centro de investigaciones
forestales y agropecuarias del estado de Veracruz. XXII) Sanh MV, Wiktorsson H, Ly LV. Effect of feeding
Veracruz. 1990:51-56. level on milk production, body weight change, feed
XI) Olea PR, Cuarón IJA, Ruiz LFJ, Villagómez AE. conversion and postpartum oestrus of crossbred
Concentración de insulina plasmática en cerdas lactating cows in tropical conditions. Livest Prod Sci
alimentadas con melaza en la dieta durante la 2002;27(2-3):331-338.
inducción de estro lactacional [resumen]. Reunión http://www.sciencedirect.com/science/journal/030
nacional de investigación pecuaria. Querétaro, Qro. 16226. Accesed Sep 12, 2003.
1998:13.
12. Tables, Graphics and Illustrations. It is preferable
XII) Cunningham EP. Genetic diversity in domestic that they should be few, brief and having the
animals: strategies for conservation and necessary data so they could be understood without
development. In: Miller RH et al. editors. Proc XX reading the text. Explanatory material should be
Beltsville Symposium: Biotechnology’s role in placed in footnotes, using conventional symbols.
genetic improvement of farm animals. USDA.
1996:13. 13. Final version. This is the document in which the
authors have already integrated the corrections and
Thesis modifications indicated by the Review Committee. The
works will have to be elaborated with Microsoft Word.
XIII) Alvarez MJA. Inmunidad humoral en la anaplasmosis
y babesiosis bovinas en becerros mantenidos en una Photographs and images must be in jpg (or
zona endémica [tesis maestría]. México, DF: compatible) format with at least 300 dpi resolution.
Universidad Nacional Autónoma de México; 1989. Photographs, images, graphs, charts or tables must
be included in the same text file. The boxes should
XIV) Cairns RB. Infrared spectroscopic studies of solid not contain any vertical lines, and the horizontal ones
oxigen [doctoral thesis]. Berkeley, California, USA: only those that delimit the column headings, and the
University of California; 1965.
line at the end of the box.
Organization as author
14. Once accepted, the final version will be translated into
XV) NRC. National Research Council. The nutrient Spanish or English, although authors should feel free
requirements of beef cattle. 6th ed. Washington, to send the final version in both languages. No
DC, USA: National Academy Press; 1984. charges will be made for style or translation services.
XVI) SAGAR. Secretaría de Agricultura, Ganadería y 15. Thesis will be published as a Research Article or as a
Desarrollo Rural. Curso de actualización técnica para Technical Note, according to these guidelines.
la aprobación de médicos veterinarios zootecnistas
responsables de establecimientos destinados al 16. Manuscripts not accepted for publication will be
sacrificio de animales. México. 1996. returned to the author together with a note explaining
XII
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)
XIII
https://doi.org/10.22319/rmcp.v15i2.6366
Article
Study of the Genetic Structure and Diversity of Holstein cattle in the

small holder system in Mexico
Felipe de Jesús Ruiz-López a
José G. Cortés-Hernández b
José Luis Romano-Muñoz a
Fernando Villaseñor-González c
Adriana García-Ruiz a*
a
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Centro
Nacional de Investigación Disciplinaria en Fisiología y Mejoramiento Animal. Ajuchitlán
Colón, 76280 Querétaro, México.
b
Universidad Nacional Autónoma de México. Facultad de Medicina Veterinaria y Zootecnia.
Ciudad Universitaria, Ciudad de México. México.
c
INIFAP. Campo Experimental Centro-Altos de Jalisco. México.
*Corresponding author: garcia.adriana@inifap.gob.mx
Abstract:
The objective was to know the population structure of Holstein animals in the family dairy
system, to identify possible origins of the genetic material, to know the degree of inbreeding
and to identify possible traces of selection in the genome, which allow glimpses of the traits
that have been improved over the years. The study included 270 animals genotyped with the
GGP-50K® chip. After genotype quality control, 43,548 autosomal SNPs were included. To
know the population structure, analyses of mixtures and principal components (PCs) were
performed. To know genomic inbreeding and detect traces of selection, information on runs
of homozygosity (ROH) was used. Mixture analysis was performed with the Admixture
software, and PC, ROH and inbreeding analyses were performed with SVS-v7.6.8. Mixture
249
Rev Mex Cienc Pecu 2024;15(2):249-266
analysis showed evidence of six components, all linked to Holstein bulls families with
different country of origin. The PCs did not show stratification of the population by herd.
The mean inbreeding coefficient was 0.59 ± 0.53 %. In the regions of the genome with ROHs
most frequent in the population (≥20 animals), numerous associations, QTLs and genes
related to milk production and composition, fertility parameters, susceptibility to diseases,
body conformation, feed efficiency and some characteristics of carcass composition have
been reported. The results reflect the existence of a wide genetic diversity in this population
and the possibility of carrying out genetic improvement work through selection without
affecting inbreeding levels.
Keywords: Genetic diversity, Traces of selection, Inbreeding, Family dairy system.
Received: 17/12/2022
Accepted: 09/05/2023
Introduction
The cattle dairy industry in Mexico produced around 11.489 billion liters of milk nationwide
in 2020 (SIAP, 2020)(1), of which more than 30 % of the volume was produced in the family
dairy system (FDS), which includes approximately 78 % of the farms(2). In the FDS farms,
Holstein animals predominate, although Brown Swiss animals and their crosses can be
found(3). Currently, in this system there is little information on production records by animal
and on rare occasions genealogical information can be collected, which makes it unfeasible
to carry out genetic evaluations of the animals in this system. The genetic improvement of
these animals has been carried out by the selection performed by the cattle farmer within
their herd, or by the introduction of genetic material, but there is no evidence of directed
mating for a specific genetic purpose.
The use of genomic information has made it possible to describe the structure of populations
that do not have genealogical information or records. The study of these populations or
animals has been carried out from the study of single nucleotide polymorphism (SNP)
markers or their clustering pattern, such as for example, runs of homozygosity (ROHs),
which are homozygous segments in the genome, identical by descent, which can be used to
study population structure, demographic history and to decipher the genetic structure of
complex diseases(4). ROHs are the result of crossbreeding between related individuals(5) from
populations with a high level of selection intensity, influenced by the availability of
250
replacements and the adoption of technological and reproductive tools(6), or low rates of
recombination(7). Their distribution and length depend on the intensity of selection, being
more frequent and more extensive when it is higher(8), when mating between close relatives
is frequent or when the size of populations is small(4).
The potential of ROHs to help the genetic improvement of production animals is big due to
the fact that they contain a large number of genes that encode traits of interest(9). In addition,
the identification of ROHs can help to visualize and recognize haplotype patterns
characteristic of breeds or species(7), allowing the identification of genomic regions with
possible traces of selection for the breed(10) and the calculation of individual inbreeding
levels. The latter is done by evaluating the portion of the genome covered by ROH segments,
especially since there is a high probability of detecting genomic information from ancient
relationships(11). This is a useful tool for populations that do not have genealogical
information(12).
Traces of selection are regions of the genome that have been conserved for generations in
populations due to natural or artificial selection. These sequences of genetic material are
related to functionally important traits(13) and their detection helps to identify candidate genes
that have been favored in the selection processes to which populations have been exposed,
and to identify beneficial mutations. In addition, they help to understand the molecular
pathways related to phenotypic traits(14,15).
With SNP marker analyses, it is also possible to know the population structure through
mixture analysis and to know the most influential origins in a population. In addition, through
information-reductive methods, such as principal component analyses, it is possible to
determine patterns of population structure, important information for establishing the basis
for a genetic improvement program.
The objective of this study was to know the population structure, identify possible origins of
the genetic material, know the degree of inbreeding, and identify possible traces of selection
in the genome that allow us to glimpse the characteristics that have been improved over the
years by the decisions of cattle farmers in family production systems in Mexico.
Material and methods
A total of 270 Holstein cow genotypes were used, randomly chosen from the population
present in three FDS herds located in the region of Tepatitlán, Jalisco, Mexico. The animals
were genotyped with the GeneSeek Genomic Profiler Bovine GGP 50K® chip. Quality
251
control of genomic information consisted of excluding animals with a call rate <0.90,
excluding SNPs with minor allele frequency (MAF) < 0.02, or with a call rate < 0.95, or with
a Hardy Weinberg P-value < 0.0001(16,17). After quality control, 43,548 autosomal SNPs were
included.
In order to know the structure and main population origins, a mixture analysis was carried
out through estimation based on likelihood models that define the structure of ancestry in
unrelated individuals, a methodology implemented in the Admixture V 1.3.0(18) software. On
the other hand, Principal Component (PC) analyses were performed to identify possible
population groupings by herd. To estimate inbreeding with genomic information and traces
of selection in the population, ROHs were searched in the genome. To define ROHs, runs
with a minimum length of 500 kb and a minimum number of 25 SNPs, with a minimum
density of 1 marker every 50 kb and a maximum gap between contiguous homozygous
markers of 500 Kb were included. With the aforementioned parameters, the risk of including
very short ROHs was avoided, a common case due to linkage disequilibrium (LD)(17). LD is
associated with the presence of linked genes, so when they are inherited from parents to
children, they do so jointly, affecting the frequency of recombination (less than 50 %) and
the presence of ROHs in the genome(19). In addition, 1 heterozygous SNP and 5 genotypes
lost per run were allowed(20,21).
ROH analysis was performed using the bioinformatics platform called SNP & Variation Suite
v7.6.8 Win64 (Golden Helix, Bozeman, MT, USA)(22), while the analyses of the data
obtained were carried out with SAS Institute 9.3.(23). To analyze the distribution of 𝐿𝑅𝑂𝐻 , six
classes were defined according to their length, which were 0.5 to 4, >4 to 8, >8 to 12, >12 to
16, >16 to 20, and >20 Mb(24).
Traces of selection were detected through the loss of genetic variation, using the ROHs
identified in the genome, using the most frequent in the population (in at least 10 % of the
animals). According to their physical position in the genome, annotations, or regions
previously related to genes, QTLs or traits that have been made in other populations and that
are reported in the Animal QTLdb Release 43 database(25) were identified. In addition, traces
of selection previously reported in the Bovine Genome Variation database (BGVD) were
searched in order to find genomic information that helps to know possible characteristics that
have been selected in the population of the Family Dairy System (FDS) in Mexico.
For the calculation of the coefficient of inbreeding by runs (FROH), the methodology
i
proposed by Mcquillan et al(27) was used, who defined it as FROH = ΣLiROH / Lauto , where
i
FROH is the endogamy coefficient of individual i calculated by ROH, ΣLiROH is the total sum
of the ROH segments of an individual i above a specified minimum length, in this case > 500
kb, and Lauto is the length of the autosomal genome covered by SNPs including centromeres.
252
As the year of birth of the animals is unknown, the inbreeding trend by lactation number of
the animals at the time of sampling was calculated.
Additionally, the calculation of the inbreeding coefficient was carried out through observed
and expected homozygous markers (FHOE) for all animals, which has been reported to have
a correlation of 0.96 ± 0.001 with the genomic relationship matrix in Holstein cattle(28), the
values of FHOE can range from −1 to +1. The negative numbers refer to the exogamy present
in mating between individuals from different populations and the positive values indicate the
level of endogamy of individuals from the same population; the calculation was performed
through the method referenced by Ferenčaković et al(11) with the program called SNP &
Variation Suite v7.6.8 Win64 (Golden Helix, Bozeman, MT, USA)(22).
Results and discussion
In the analysis of mixtures, the value that best defined the number of ancestral populations
(K) was six and according to the information collected from some cow parents, six large
families were identified, defined mainly by the country of origin of the bulls. Figure 1-a
shows the population structure linked to the six main groups by country of origin of the bulls,
although some of these families share the same country of origin. Therefore, the groups that
shared the same origin were combined, leaving only three large groups represented, two
representing the United States of America and one representing the United Kingdom (USA,
840 and GBR, respectively) (Figure 1-b). The origins USA and 840 correspond to the United
States of America, only that the 840 is assigned to animals that use radio frequency
identifications (RFID), devices issued by the International Committee for Animal Recording
(ICAR), while animals registered with the USA country of origin do not carry an RFID and
the use of genetic material is locally or more limited than those of the 840(29). The results of
this study show the genetic dependence of the family system in Mexico on foreign material,
mainly from the United States, since more than 80 % of the origins were linked to families
with origins from this country, either from local trade or from those registered internationally.
253
Figure 1: Population structure of Holstein cattle in the family system a) including the six
origins attributed to bull families and b) grouped by country of origin
Although another study(29) had reported the influence of other breeds of dairy cattle on the
family system, the present study found no evidence of the use or crossbreeding with other
breeds. These results could suggest that cattle farmers have followed a more directed mating
system, and that they limit themselves to using animals of the same breed in services.
In the PC analyses (Figure 2), no stratification was found by country of origin of the bull,
and when the herd of origin was evaluated, a homogeneous herd (purple) was observed in
the population, and a difference was observed between the animals of the other herds (red
and blue). The percentage of variability associated with each component was 2.9, 2.0, and
1.8 for components or eigenvalues (EV) 1, 2, and 3, respectively.
254
Figure 2: Principal component analysis of the family system population with Holstein
phenotype, defined by herd of origin
The total number of ROHs (𝑁𝑅𝑂𝐻 ) found in the studied population was 15,695, with an
average length (𝐿𝑅𝑂𝐻 ) of 4.79 Mb, a minimum and maximum length of 0.5 and 91.49 Mb,
respectively. The average length of the genome covered by ROH was 278.76 Mb, with a
minimum and maximum of 13.28 and 535.83 Mb, respectively. According to the frequency
of ROHs in the population, they were identified as unique (in a single animal) or repeated,
the latter with the same length (identical) or of variable length. Thirty-five point eight six
(35.86) percent of the ROHs were unique (Table 1), while 64.14 % (10,067) were repeated.
Table 1: Number and percentage of unique runs of homozygosity (ROHs) and repeated
ROHs with the same start and end position, as well as variable positions
Type of ROH Length Number of ROHs Percentage
Unique - 5,628 35.86

Identical 5,663 36.08
Repeated Variable 4,404 28.06
The 𝑁𝑅𝑂𝐻 was lower compared to that reported in animals that come from specialized
production systems, which could be attributed to a lower intensity of selection since the loss
of genetic variation or the formation of ROHs in the genome is influenced, among other
factors, by the level of intensity of selection in the populations, which in turn is determined
255
by the availability of replacements and adoption of technological and reproductive tools, such
as artificial insemination (AI) and embryo transfer (ET). In dairy cattle, the intensity of
selection is very high and the selection of genetic material is influenced by a limited number
of parent families, so the mating of related individuals may be common(30). In Holstein cattle
from the specialized production system in Mexico, the 𝑁𝑅𝑂𝐻 was 88,529, with a larger
population size (~4,500 animals) and 𝐿𝑅𝑂𝐻 was greater than 8.95 Mb(24). In other studies in
Holstein cattle from the specialized system, the 𝐿𝑅𝑂𝐻 reported are even higher; for example,
in the US it is 299.6 Mb(31) and in Italy it is 297 Mb(32).
The average number of ROHs per animal was 58.13 ± 11.89, with a maximum and minimum
of 92 and 10, which is a high value compared to the results of Holstein cattle from the
specialized system in Mexico(24), reported on average at 20.07 ROHs per animal, with a
maximum of 283 and a minimum of 1. Studies in other Holstein populations of intensive
production have reported around 82.3 ± 9.83 ROHs per animal in Holstein cattle from the
US(31) and 81.7 ± 9.7 runs per animal in Holstein cattle from Italy(32). These differences may
be due to the high degree of selection in the populations of specialized production systems
in both the US and Italy, as well as the availability of genetic material from highly selected
bulls compared to the FDS that was analyzed, where the selection objectives are not so
defined.
According to the classification of 𝐿𝑅𝑂𝐻 , the most frequent runs were the shortest (0.5 to 4
Mb) with 64.42 %, followed by those from 4 to 8 Mb with 20.45 %, and the least frequent
were the longest runs (>16 to 20 and >20; Table 2). The lengths of the ROHs provide
information on the number of generations in which the common ancestor is shared, with the
longest being those formed in recent generations(21), so the length of the ROHs found in this
population reflects recent and low inbreeding.
Table 2: Frequency of runs of homozygosity (ROH) at different lengths
Length (Mb) Number Percentage

0.5 to 4 10,110 64.42
>4 to 8 3,209 20.45
>8 to 12 1,131 7.21
>12 to 16 539 3.43
>16 to 20 296 1.89
>20 410 2.61
Mb= Megabases.
The average inbreeding coefficient (FROH) in the population was 0.59 ± 0.53 %, with a
maximum of 3.35 % and a minimum of 0.034 %. The results are consistent with the small
256
number of ROHs found and the short average length. These values were well below what
was reported in other highly selected Holstein cattle populations; for example, 4.2 % in the
US(33). Although the inbreeding found in this population was insignificant, a value that is
confirmed by the values calculated for FHOE, which were -0.02 ± 0.08, when reviewing the
averages by number of births, a slight increase in FROH was found in recent generations,
which could indicate the beginning of an unfavorable trend for the group studied (Figure 3).
Figure 3: Genomic inbreeding (FROH) percentages by lactation number at the time of

sampling
To identify traces of selection throughout the genome, it was searched for specific
chromosomes and regions in the location and distribution of ROHs. The presence of ROHs
occurred to a greater extent in the long chromosomes than in the short ones, although the
latter presented a higher proportion of the genome covered by homozygous regions, as was
the case of chromosomes 10 and 20, which presented 10 and 20 with 16.98 % and 17.76 %
(Figure 4), behavior similar to what was reported by Szmatola et al(34) in Holstein cows from
Poland, suggesting that these regions have been subject to greater selection, due to
association with traits of economic interest. The percentages of homozygosity per
chromosome are higher than the average FROH value because the length of the chromosome
is taken as one hundred percent and not the total length of the genome covered by the SNPs;
this gives a better perception of the length of the chromosome covered by ROHs.
257
Figure 4: Percentage of chromosome covered by runs of homozygosity (ROH)
Throughout the genome, a positive relationship was observed between chromosome size and
the number of ROHs detected on that chromosome, but this was not the case for the
percentage of chromosome length covered by ROHs since short chromosomes showed a
higher proportion covered by ROHs (Figure 5), this is because the average length of ROHs
was greater in short chromosomes than in long chromosomes, because long chromosomes
have more recombination than short chromosomes(8). Of the total ROHs determined in the
population, chromosomes 1 and 6 were the ones with the highest number of ROHs (5.98 and
5.39 %) and the chromosomes with the lowest number were chromosomes 28 and 27 (1.31
and 1.50 %), results that are similar to those of Purfield et al(17), who also reported a higher
number of ROHs on the long chromosomes than on the short ones.
Figure 5: Percentage of runs of homozygosity (ROH) on each chromosome
258
According to the frequency of repeated ROHs in the population, only 35 were found in 10 or
more animals and were distributed on chromosomes 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15,
17, 19, 21, 22, 23, 26, and 29. The most frequent ROHs were found on chromosomes 2 and
22, in 27 and 23 animals, respectively, with the length in these runs being 1.82 Mb and 1.61
Mb. In the same position as the runs found on chromosome 2 (83.84-85.66 Mb), Cole et al(35)
reported QTL (Quantitative Trait Loci) related to hip width and height in Holstein cattle from
the US; Cai et al(36) reported QTLs associated with milk fat production in Holstein cattle from
Nordic countries, which may indicate traces of selection on these chromosomes(37).
Of the total ROHs of variable length, only 37 were found in 10 or more animals, distributed
throughout the genome, except on chromosome 8. In the region where the most frequent
ROHs in the population were found (≥20 animals), numerous associations, QTLs and genes
related to milk production and composition, fertility parameters, susceptibility to diseases,
body conformation, feed efficiency and some characteristics of carcass composition (Table
3) have been reported. The results show that, although the ROH lengths in this population
(~4.79 Mb) suggest crossbreeding of animals related approximately 16 generations ago(11),
the conserved regions could indicate that selection in this population is aimed at improving
milk production, composition, fertility, and health, as could be expected in milk production
systems. On chromosomes 1 and 2, in addition to associations with characteristics of interest
in dairy cattle, associations with carcass characteristics are observed, findings that could
suggest possible crosses with other breeds.
To search for ROH annotations with variable length, the shortest ROH with respect to the
final position was used as a reference (Table 3), to avoid providing information outside the
region common to all animals with a specific ROH.
259
Table 3: Genome annotations found in regions where the most frequent runs of
homozygosity (ROH) in the family dairy population were detected
Start Length Associations /QTL Genes
BTA NoA Traces of selection
position (pb) reported NCBI
13 389,736 1,593,318 38 CALEASE, PTAT, FY, Associated: TMX4, PLCB1,
MY, NM, PY, UHT, 287026. MIR2285M-1.
SB, STA, FANG,
FTLEG, UA, RLEGR,
RLEGS, RTPL, SCS,
MRCT, FSC,
CONCEPT, MBCASP,
MPFRAT, DYF,
DYST, TPL, TLGTH,
UDPTH, PP, FP,
HTINT.
1 761,316 1,116,706 32 PP, MKCASP, Associated: ATP5O, ITSN1,
CONCRATE, 506426. CRYZL1, DONSON,
MUGKCASP, BTBS, Candidate: SON, GART,
SCS, FATTH, PY, RFI. 282257. DNAJC28,
TMEM50B,
IFNGR2, IFNAR1,
LOC104970778,
IL10RB, IFNAR2,
LOC526226, OLIG1,
OLIG2.
17 67,686 1,209,677 32 FY, PY, CONCRATE, TMEM192, KLHL2,
CONCEPT. MSMO1, CPE,
LOC101903170.
2 83,841,602 1,794,112 31 FSC, NRR, Candidate: SLC39A10, DNAH7,
CONCRATE, 526800 STK17B,
CONCEPT, Candidate: LOC531691.
MUGKCASP, MSPD, 19122
BTBS, FY, BD, Gene:
CALEASE, PTAT, 521004.
FTPL, UA, NM, PL,
RTPL, UHT, RUMWD,
SCS, SB, STA, STR,
UC, UDPTH, LMY,
EY, BW, BVDV.
7 153,780 811,014 31 CALEASE, SB, FANG, Gene: LOC107131408,
FTLEG, PTAT, FTPL, 338031. LOC100125913,
UA, NM, PL, RLEGR, LOC101902704,
RLEGS, UHT, SCS, FLT4, CNOT6,
STA, UC, UDPTH, GFPT2, MAPK9,
MBCASP. RASGEF1C.
BTA= chromosome, NoA= number of animals.
Associations /QTLs reported. CALEASE= calving ease, PTAT= conformation final score, FY= milk fat
yield, MY= milk yield, NM= net merit, PY= milk protein yield, UHT=udder attachment height,
260
SB=stillbirths, STA= stature, FANG= foot angle, FTLEG= foot and leg conformation, UA=udder attachment,
RLEGR= rear leg placement - rear view, RLEGS= rear legs- side view, RTPL= rear teat placement, SCS=
somatic cell score, MRCT= milk rennet coagulation time, FSC= first service conception, CONCEPT= number
of inseminations per conception, MBCASP= milk B-casein percentage, MPFRAT= milk protein-to-fat ratio,
DYF= dairy character, DYST= dystocia, TPL= teat placement, TLGTH= teat length, UDPTH=udder depth,
PP= milk protein percentage, FP= milk fat percentage, HTINT= heat intensity, MKCASP= milk Kappa casein
percentage, CONCRATE= conception rate, MUGKCASP= milk non-glycosylated kappa casein percentage,
BTBS= bovine tuberculosis susceptibility, FATTH= fat thickness in the 12th rib, RFI= residual feed intake,
NRR= non-return rate, MSPD=milking speed, BD= Body depth, FTPL=front teat placement, PL= productive
life span, RUMWD= rump width, STR=milk strength, UC=udder cleft, LMY=lean meat yield, EY= energy of
milk yield, BW= birth body weight, BVDV= bovine viral diarrhea susceptibility.
NCBI genes. 287026= phospholipase C beta 1, 506426= crystallin zeta protein encoder, 282257= subunit 1 of
interferon alpha and beta receptor, 526800= ankyrin repeat domain 44, 19122= prion protein, 521004= solute
carrier family 39 member 10, 338031= FMS-related receptor tyrosine kinase 4.
Traces of selection (protein-coding genes). TMX4= thioredoxin-related transmembrane protein 4, PLCB1=
phospholipase C beta 1, MIR2285M-1= microRNAs involved in post-transcriptional regulation of gene
expression, ATP5O= ATP synthase peripheral stalk subunit, OSCP= ATP synthase peripheral stalk subunit,
ITSN1= intersection 1, CRYZL1= crystallin zeta protein encoder, DONSON= DNA replication fork
stabilization factor, SON= DNA and RNA-binding protein, GART=phosphoribosylglycinamide
formyltransferase synthetase, Phosphoribosylaminoimidazole synthetase, DNAJC28= heat shock protein
family, TMEM50B= transmembrane protein 50B, IFNGR2=interferon gamma receptor 2, IFNAR1=
interferon alpha and beta receptor subunit 1, LOC104970778= uncharacterized RNA gene, IL10RB=
interleukin receptor subunit beta, IFNAR2= interferon alpha and beta receptor subunit 2, LOC526226=
histone H4, OLIG1= oligodendrocyte transcription factor 1, OLIG2= oligodendrocyte transcription factor 2,
TMEM192= transmembrane protein 192, KLHL2= kelch of family 2, MSMO1= methylsterol
monooxygenase 1, CPE= carboxypeptidase E, LOC101903170= uncharacterized RNA gene, SLC39A10=
solute carrier family 39, DNAH7= dynein axonemal heavy chain 7, STK17B=serine/threonine kinase 17b,
LOC531691= HECT, C2 and WW domain containing E3 ubiquitin protein ligase 2, LOC107131408=
olfactory receptor family 5 subfamily W member 39, LOC100125913= uncharacterized gene,
LOC101902704= C-type lectin domain family, 7, A, FLT4= fms-related receptor tyrosine kinase 4, CNOT6=
CCR4-NOT transcription complex subunit 6, GFPT2= glutamine-fructose-6-phosphate transaminase 2,
MAPK9= mitogen-activated protein kinase 9, RASGEF1C= RasGEF domain family member 1C.
In the study population, ROHs that have been maintained as a result of the selection process
of the family system population were identified. In these conserved regions, associations of
SNP markers, QTL and gene are found, which are mostly related to characteristics of
economic interest in the dairy industry, such as milk production and composition, fertility
parameters, susceptibility to diseases, body conformation, feed efficiency and some other
characteristics such as carcass composition, which could be taken as traces of selection
(Table 3). These results show the traits that have been included in the selection processes in
the population, either intentionally because of the selection made by cattle farmers or
unintentionally because of the availability of genetic material in the market, since, by using
AI, the choice of bulls guides the cattle farmer to modify the genetics of their animals in the
way that AI companies do.
261
The dairy cattle from the FDS have ancestral origins from countries that are suppliers of
genetic material internationally, such as the US and GBR, showing no evidence of recent
crossbreeding with other dairy breeds. Within the studied population, it can be observed
genetically homogeneous, with a lower number and length of ROHs than animals in
specialized production systems, reflecting a wide genetic variation caused by a low intensity
of selection. In this work, ROHs that have been maintained as a result of the selection process
were identified, which are mostly related to characteristics of economic interest in the dairy
industry. The results of this study reflect the existence of a low level of inbreeding in the
population and a greater genetic diversity in this population compared to those found in
specialized systems, so it is possible to carry out genetic improvement work aimed at the
characteristics of interest of the producers through selection, without inbreeding
compromising the productivity and health of the population.
Acknowledgments and funding source
Project funded by INIFAP-CENIDFyMA with the name “Development of a sustainable

comprehensive strategy to increase the availability of good quality Holstein replacements in
the family milk production system in Mexico” with SIGI Number: 15352034772.
Conflict of interest
The authors declare that there are no conflicts of interest.
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Article
Effect of various castration protocols on production indicators in pigs:

meta-analysis
Humberto Rafael Silva-Santos a
Francisco Ernesto Martínez-Castañeda b
Gregorio Álvarez-Fuentes c
María de la Salud Rubio-Lozano d
María Elena Trujillo-Ortega e*
a
Universidad Nacional Autónoma de México. Posgrado en Ciencias de la Producción y de
la Salud Animal. Ciudad de México, México.
b
Universidad Autónoma del Estado de México. Instituto de Ciencias Agropecuarias y
Rurales. Toluca, México.
c
Universidad Autónoma de San Luis Potosí. Instituto de Investigación en Zonas Desérticas.
San Luis Potosí, México.
d
Universidad Nacional Autónoma de México. Centro de Enseñanza Práctica e Investigación
en Producción y Salud Animal. Ciudad de México, México.
e
Universidad Nacional Autónoma de México. Programa Universitario de Alimentación
Sostenible. Circuito de la investigación científica s/n, Ciudad universitaria. 04510. Ciudad
de México, México.
* Corresponding author: elenam@unam.mx
Abstract:
The objective was to evaluate the effect of various castration protocols through a meta-
analysis of indicators of daily feed intake, feed conversion, daily weight gain, slaughter
267
weight, hot carcass weight, and carcass yield. 179 publications from three electronic sources
(Scopus, PubMed, and Web of Science) were reviewed over a 24-year period, out of which
the 26 studies that met the inclusion criteria were selected. The effect was analyzed with six
comparisons: C1=surgical castration vs whole; C2=standard immunocastration vs whole;
C3= standard immunocastration vs surgical castration; C4= alternative immunocastration vs
whole; C5= alternative immunocastration vs surgical castration, and C6= alternative
immunocastration vs standard immunocastration. Averages were estimated for feed intake
(kg) (0.23, 0.23, -0.05, 0.32, 0.11, -0.09), feed conversion (kg:kg) (0.27, 0.05, -0.16, 0.11,
0.11, 0.11, -0.19), daily weight gain (g) (-9. 54, 39.08, 40.70, 107.63, -53.0, 69.14), carcass
weight (kg) (-9.54, 39.08, 40.70, 107.63, -53.0, 69.14), and hot carcass weight (kg) (1.23,
0.85, 0.46, 1.03, 1.02, -0.42) respectively. The indicators of feed consumption, feed
conversion, daily weight gain, slaughter weight, and hot carcass weight proved different
(P<0.05); only the carcass yield variable exhibited no difference (P>0.05). The conclusion is
that immunocastration improves performance in production and carcass indicators; surgical
castration improves carcass yield; whole pigs have better feed conversion, and standard and
alternative immunocastration differ in their response in terms of production and carcass
measurement indicators.
Keywords: Immunocastration, GnRH, Meta-analysis, Carcass, Stockbreeding.
Introduction
In whole pigs for slaughter, the sexual taste and odor in the meat is perceptible(1). Therefore,
at early ages, it is preferable to castrate pigs surgically; this technique is the most commonly
used, while it is also invasive(2). Various methods of castration were addressed in the 1990s(3),
among which the results of the application of immunization against GnRH on sexual odor,
the response in production and carcass indicators were most prominent(4,5).
The standard immunization protocol consists of two subcutaneous doses at 12 and 16 wk of

age(6). The former allows for immune recognition, antibody production, and anchoring to
gonadotrophs. The second dose increases the immune response, causing gonadal atrophy and
eliminating androstenone as a sexual odor precursor(7).
268
An increase in daily weight gain, higher carcass weight at slaughter and a reduction in back
fat thickness have been reported to occur with immunocastration(8,9); however, other authors
report the opposite, observing a lower carcass yield and a lower daily weight gain(10,11).
Studies have been published in which different GnRH immunization protocols involving age,
interval, and number of doses have been applied. Some of the results observed in these studies
in the protocols that had 4- and 6-wk intervals between doses were improvements in feed
conversion and carcass weight(12) and improved growth performance indicators in late
immunocastration and prepubertal protocols(13), while the standard and delayed
immunization protocols resulted in better feed efficiency(14).
The results of the different studies on castration methods have been analyzed with the
statistical tool of meta-analysis. An example of this can be seen in the 2012 paper by Batorek
et al(15), where the comparison between immunocastration versus surgical castration and
whole pigs showed that immunocastrated pigs had longer carcasses compared to surgically
castrated and whole pigs, as well as a faster growth rate and a better feed conversion than
whole pigs.
In another 2018 study(16), immunocastration in pigs produced a higher daily weight gain and
a lower feed conversion rate than surgical castration. On the other hand, higher daily weight
gain, feed intake, trace weight and back fat were observed in immunocastrated sows
compared to whole sows(17).
The objective of the present meta-analysis was to evaluate the effect of the different
castration protocols used until 2020, with an emphasis on the analysis of the castration
technique, as well as the effect on the age of application of the immunocastration protocol,
and to compare between standard and alternative immunization application, through the
production indicators daily weight gain, daily feed consumption, feed conversion, and
carcass evaluation based on live slaughter weight, hot carcass weight, and carcass yield.
The development of the present work included a literature review of the publications related
to the different castration protocols (surgical castration, standard GnRH immunization, and
alternative immunocastration) and also considered whole pigs. The methodological
procedure was: information search; systematic review; quantitative synthesis; categories of
analysis, and statistical analysis.
269
Information search
The search began with the approach and the research question of this work, which brought
the focus of the literature review to those publications that studied the effect of castration
protocols, either surgical or immunological, on the production process of pigs for slaughter
during breeding and the subsequent output of finished pigs to the slaughterhouse, as well as
the collection and measurement of the carcass. The search for information began in 1994,
when the first results on this subject were reported, up to 2020. In the systematic review, the
electronic meta-search engines Scopus, PubMed, and Web of Science were used to search
for papers.
The primary search was performed by mixing keywords related to the topic (Figure 1). The
available publications in the fields of biological sciences, animal production sciences, and
meat science and technology were segregated, and the corresponding results, mean values,
and dispersion measures were published.
Figure 1: Keyword combinations for article searches
Systematic review
The selection of the papers was applied to 179 articles by two team members, who
determined the criteria for selection and exclusion of the articles based on the initial research
270
question and approach (Figure 2). Once these criteria were established, the selection process
resulted in 26 articles for analysis (Figure 3).
Figure 2: Inclusion and exclusion criteria for article selection

Inclusion Exclusion
 GnRH immunization, surgical  To have any other treatment in

castration and use of whole pigs. addition to surgical and immunological
 Use of pigs for slaughter and castration protocols.
consumption as research subjects.  Subjects of research not intended for
 Pigs with a terminal genetic basis in consumption, slaughter or production
the conformation of different local processes.
genetic lines.  Work directed to a social field to
 Work with results on growth, which no corresponding results were
development and finishing indicators. reported for pork production,
 Papers with results on indicators of procurement, or consumption
interest in meat science and processes.
technology.  Papers that reported nonparametric
 All with results in the chemical and results, as well as those whose results
nutritional composition of the meat. were ranges or subjective values.
 Works by various nationalities.  Observational studies with data
 Works by different institutes and reported by qualitative indicators.
educational or governmental entities.
Figure 3: Information selection flow
271
Quantitative summary
Table 1 shows the characteristics of the works included in the analysis in terms of technique
and method of castration.
Table 1: Details of publications selected for statistical analysis

Experimental
Author Gentics Sex
groups
(19)
Andersson et al Yorkshire x Landrace m, h Q, Ie, aI, W
(3)
Bonneau et al Large white x Pietrain h Q, Ie, W
(20)
Channon et al Large White x (Landrace x Duroc x m sI, W
Largewhite)
(11)
Daza et al Duroc x (Landrace x Large white) m, h Q, sI, W
(21) Terminal h sI, W
Di Martino et al
(8)
Dunshea et al Large white x Landrace m Q, sI, sI, W
(22)
Font et al Terminal m, h Q, sI, W
(23)
Galleos et al Terminal m sI, Ia
(24)
Gamero et al Ibérico x Duroc h Q, sI, W
(25)
Gogic et al Swallow-bellied Mangalitsa m sI, W
(9)
Grela et al Polish Zloynika m Q, sI, aI, W
(12)
Laeliifano et al Large White x Landrace m sI, aI, W
(26)
Morales et al Terminal m Q, sI, W
(27)
Oliviero et al Landrace m Q, W,
(28)
Pauly et al Large white m Q, sI, W
(29)
Rikard-Bell et al Terminal m sI, W
(30)
Rodriguez et al Terminal h sI, W
(31)
Skrelp et al Cerdos gordos eslovacos x Duroc m Q, sI, W
(32)
Skrelp et al Large white x Landrace x Duroc m Q, sI, W
(33)
Stupka et al Duroc x (Large White x Landrace) m, h Q, sI, W
(10)
Turkstra et al (Deutch Landrace x Finnish Landrace) m Q, sI, aI, W
x Large White
Van den Broeke et Terminal m, h Q, sI, aI, W
al(34)
Weiler et al(35) Terminal m, h Q, sI, W
(36)
Yuan et al Duroc·(Landrace·x Large White) m Q, sI
(14)
Zoels et al Piétrain × Large White x Landrace m Q, sI, aI, W
*sex: f=females, m=males; Experimental group: Q= surgical castration, sI= standard immunocastration, aI=
alternative immunocastration; W=whole pigs.
272
The groups for analysis were the following:
Whole: pigs that remained without any type of intervention during the production period.
Surgical castration: pigs that had their testicles surgically removed before 7 d of age.
Standard immunocastration: pigs that underwent the immunization protocol as indicated by
the manufacturer, two doses subcutaneously, at approximately 12 and 16 wk of age.
Alternative immunocastration: pigs that received the immunization protocol at ages other
than the standard or with a longer interval between doses.
Categories of analysis
The analysis of the information was divided into two stages:

a) Production period: described by indicators of feed intake, feed conversion ratio, and daily
weight gain.
b) Carcass measurement process: described by the indicators slaughter weight, hot carcass
weight, and carcass yield.
From the identification and description of the categories of analysis, the following
comparisons were established for the analysis of the information in each of the
aforementioned indicators:
C1: surgical castration

C2: standard immunocastration vs whole.
C3: standard immunocastration vs surgical castration.
C4: alternative immunocastration vs whole.
C5: alternative immunocastration vs surgical castration.
C6: alternative immunocastration vs standard immunocastration.
Statistical analysis
The statistical analysis used to synthesize the results of various studies on the effect of
castration protocols on the indicators of the productive period and the carcass measurement
process was carried out with the NCSS software (NCSS Statistical System for Windows,
Kaysville, UT: Number Cruncher Statistical Systems, 2021). Dispersion averages were
estimated based on the values of the mean, standard deviation, and number of observations
for each indicator under study. A random effects model was utilized to test the hypothesis of
273
heterogeneity, the average standard difference of the effect, and its confidence interval (α=
0.05); this decision was supported by Chi-square tests(18).
Results
Table 2 shows the comparisons, the result of the differences between the means points to the
first analysis protocol for each of them.
Production period indicators
For the feed intake indicator, the alternate immunocastration (C4 and C5), surgical castration
(C1 and C3) and standard immunization (C2 and C6) protocols obtained the highest feed
intake (Table 2). Animals within the two immunization protocols had a higher intake than
castrated animals (C3 and C5). Finally, among the immunization protocols, the standard
protocol was the one that had the highest intake.
For feed conversion (Table 2), the whole pig protocol obtained the best value (C1, C2, and
C4), followed by the standard immunocastration (C3 and C6) and surgical castration
protocols (C5).
For daily weight gain (Table 2), the standard immunization protocol exhibited the best results
(C2, C3 and C6), followed by the alternate immunization (C4 and C5) and whole pigs (C1)
protocols.
Indicators of the carcass measurement process
274
Table 2: Results of the analysis of the indicators for each comparison

C1 C4
Surgical castration vs whole Alternative immunocastration vs whole
Difference Difference
Coefficient Coefficient
Variable n between means ± P Variable n between P
limits (95%) limits (95%)
SW means ± SW
Feed intake, kg 16 0.23 ± 0.03 (0.16; 0.29) 0.01 Feed intake, kg 8 0.32 ± 0.09 (0.13; 0.51) 0.01
Feed conversion, Feed conversion,
kg:kg 13 0.27 ± 0.04 (0.20; 0.34) 0.01 kg:kg 5 0.11 ± 0.15 (-0.18; 0.40) 0.01
Daily weight Daily weight gain,
gain, g 15 -9.54 ± 16.62 (-42.12; 23.03) 0.01 g 8 107.63 ± 58.75 (-7.52; 222.78) 0.01
Slaughter weight, Slaughter weight
kg 14 4.11 ± 3.35 (-2.45; 10.68) 0.01 kg 5 0.09 ± 1.36 (-2.56; 2.76) 0.01
Hot carcass Hot carcass weight,
weight, kg 17 1.23 ± 0.51 (0.22; 2.24) 0.01 kg 6 1.03 ± 0.86 (-0.66; 2.72) 0.01
Carcass yield, % 9 0.33 ± 0.52 (-0.69; 1.35) 0.97 Carcass yield % 6 -0.22 ± 0.63 (-1.46; 1.01) 0.94
C2 C5
Standard immunocastration vs whole Alternative immunocastration vs surgical castration
Feed intake, kg 17 0.23 ± 0.08 (0.08; 0.38) 0.01 Feed intake, kg 5 0.11 ± 0.12 (-0.13; 0.34) 0.01
kg:kg 15 0.05 ± 0.04 (-0.0; 0.13) 0.01 kg:kg 5 -0.19 ± 0.14 (-0.56; 0.08) 0.01
Daily weight Daily weight gain,
gain, g 21 39.08 ± 11.37 (16.79; 61.34) 0.01 g 6 69.14 ± 32.67 (5.10; 133.18) 0.01
Slaughter weight, Slaughter weight,
kg 21 1.24 ± 0.59 (0.07; 2.42) 0.01 kg 7 1.28 ± 0.50 (0.29; 2.27) 0.01
weight, kg 25 0.85 ± 0.47 (-0.06; 1.76) 0.01 kg 7 -0.42 ± 0.44 (-1.28; 0.44) 0.01
Carcass yield, % 12 -0.68 ± 0.46 (-1.58; 0.22) 0.99 Carcass yield, % 4 -0.10 ± 0.75 (-1.58, 1.37) 0.89
C3 C6
Standard immunocastration vs surgical castration Alternative immunocastration vs standard immunocastration
Feed intake, kg 12 -0.05 ± 0.07 (-0.19; 0.09) 0.01 Feed intake, kg 6 - 0.09 ± 0.11 (-0.31; 0.12) 0.01
kg:kg 10 -0.16 ± 0.03 (-0.23; -0.09) 0.01 kg:kg 7 0.11 ± 0.07 (-0.03; 0.24) 0.01
275
Daily weight Daily weight gain, 1 -53.00 ± (-100.66; - 0.01

gain, g 14 40.70 ± 12.18 (17.03; 64.77) 0.01 g 2 24.32 5.34)
Slaughter weight, Slaughter weight,
kg 14 1.21 ± 0.53 (0.16; 2.25) 0.01 kg 9 0.47 ± 2.07 (-3.58; 4.53) 0.01
weight, kg 17 0.46 ± 0.69 (-0.90; 1.81) 0.01 kg 9 1.02 ± 1.67 (-2.25; 4.30) 0.01
Carcass yield, % 9 -0.66 ± 0.52 (-1.68; 0.35) 0.45 Carcass yield, % 9 0.29 ± 0.52 (-0.72; 1.31) 0.76
276
Final weight showed favorable indicators for alternative immunization at C4, C5, and C6
(Table 2), followed by standard immunization at C2 and C3, and surgical castration
(C1).When analyzing the hot carcass weight, the highest value was obtained with the standard
immunization protocol (C2 and C3), followed by the alternative immunization protocol (C4
and C6), and surgical castration (C1 and C5).
Contrary to the final weight and the hot carcass weight, the response observed in the carcass
yield analysis showed that the surgical castration protocol obtained higher percentages in C1,
C3, and C5. On the other hand, whole pigs show higher performance at C2 and C4, leaving
only the alternative immunization protocol with a favorable indicator at C6.
Figures 4 and 5 represent the heterogeneous behavior (P<0.05) in the analysis of the
alternative immunocastration protocol at C4, C5, and C6. In both cases, an increase in
favorable results was observed with the application of the alternative immunization.
Discussion
The response of the analyzed indicators is explained on the basis of the results of a whole
animal, considered as the ideal productive animal for its metabolic qualities and their effect
on the carcass(37).
Production-period indicators
Feed intake
The amount of food consumed is regulated by the satiety center, which responds to serum
concentrations of leptins, produced by adipocytes. Several factors can modify feed intake
such as: the presentation and formulation of the food, the physical and physiological state of
the individual, and the physical and social activity within the group(38).
The physical and physiological state of the animal modifies the feed intake; therefore,
surgical castration or immunocastration are protocols that alter this indicator. Surgical
castration increases feed intake due to the increase in the serum concentration of leptins, 2.97
ng/ml — as a consequence of the redistribution and increase of adipose tissue after the
removal of the gonads(39,40)—, saturating and inhibiting the satiety center located in the
central nervous system(41). In immunocastrated pigs, serum leptin concentrations remain
similar to those of whole pigs (2.68 ng/ml); therefore, satiety is not altered(15).
277
Likewise, the interruption of the hypothalamic-pituitary-gonadal axis when the gonads are
removed affects the consumption and satiety habits, also influenced by sex hormones,
especially by estrogens, which when the testes are removed are not aromatized; this causes
the estrogen to be produced by the adipose tissue in low concentrations, of 0.34 pg/ml, and,
therefore, the pig exhibits resistance to glucose and a higher feed intake(42). Estradiol
concentration decreases in immunized pigs, 0.37 pg/ml; however, its effect may vary
depending on the age at which the second dose is applied(43).
Another element that modifies the intake is the physical and social activity within the group.
While whole pigs show greater sexual activity, dominance and competitiveness for food,
whereby their feed intake diminishes(37), immunocastration and surgical castration reduce the
presence of androgens and, therefore, the pigs’ sexual activity and dominance, increasing
their feed intake(15).
With respect to immunization protocols, studies report no differences, contrary to the

findings of this study, where variability in the timing of alternative immunizations may alter
the feed intake response(23).
Feed conversion
The best feed conversion is established as the one with the lowest feed intake in relation to
the production of one kilogram of meat; among the factors that can modify it are the physical
and physiological state of the pig.
Somatotropin is one of the elements required for muscle development, which is altered if the
physical state changes(41) as a consequence of the reduction of the blood IGF-1 concentration
in castrated pigs to 256 ng/ml, related to the reduction of estradiol, whereby the mechanisms
of muscle growth and development become altered(43). In immunized pigs the concentration
of IGF-1 reaches 332 ng/ml, compared to its value in whole pigs, which is 459 ng/ml(43).
In late alternative immunization protocol, the feed conversion value increases with the second
dose; therefore, the age of application is a relevant factor in allowing the physiological
mechanisms to remain for a longer period of time(15,44).
Daily weight gain

The physiological mechanisms and state, and the physical and social activity of the pigs are
some of the factors that influence weight gain. The utilization of nutrients in the diet promotes
the growth and development of tissues. An example of this is the response of the muscle
masses to the presence of lysine, which causes a retention of nitrogen in muscle of 31.24
g/d(45,46). Surgical castration allows a retention of 25.51 g/d of nitrogen, while
immunocastrated pigs retain only 22.95 g/d(45,47,48).
278
The physical activity of whole pigs is more dynamic due to the hierarchy within groups, as
well as the competition in feed intake, also considering that, as the age of puberty approaches,
the incidence of aggressions increases(37).
In the case of surgically castrated pigs, physical activity and energy demand are lower;
however, competition for feed intake prevails and, in some cases, increases because of
alterations in the satiety centers. In immunized pigs, the increase in this indicator responds
to the reduction of aggressions and dominance attitudes among pigs, which allows a
homogeneous feed consumption and reduces the need to heal wounds and injuries(41).
Indicators of the carcass measurement process
Slaughter weight
At the end of the production cycle, the final weight of the animals will depend on the physical
condition and age at slaughter; it should be noted that the result is related to the feed
conversion rate and daily weight gain, as well as to the metabolic mechanisms of these
indicators. The production cycle of pigs for slaughter usually lasts approximately 20 to 22
wk, during which time the whole pig reaches an average weight of 110 kg, attributed to its
metabolic efficiency, considering that a large part of this value corresponds to muscle
mass(38).
Surgically castrated pigs eventually gain weight due to the redistribution and growth of
adipose tissue, especially subcutaneous fat(49). The weight of immunocastrated pigs involves
a similar muscle development to that of whole pigs, a thicker fat cover —without reaching
those of the surgical castration protocol—, homogeneous feed consumption, metabolic
utilization, and a higher weight gain due to docile behavior(41).
Hot carcass weight

After slaughtering and the process of removing the head and viscera, the carcass is obtained;
its weight is determined by the morphology of the pig, as well as the amount of fat it contains
and its relation to the muscles. The carcass of whole pigs is leaner because it has little fat
cover and intermuscular fat, in addition to the 1.4 % reduction in weight corresponding to
the testicles(50), which coincides with the results of the meta-analysis performed in 2012(15).
In surgically castrated pig carcasses, the amount of fat cover and intermuscular fat increases
the weight, and the difference in final weight is not affected by the removal of the
reproductive tract(41).
279
In immunized pigs, the fat cover is thicker than that of whole pigs and thinner than that of
surgically castrated pigs; they have a better metabolic response after the second dose and
allow similar muscle growth and development to those of a whole pig(51). Alternative
immunization protocols have a better response, especially in late immunization or with a
longer interval between the first and second dose(19).
Carcass yield
The ratio between live weight and carcass weight, as well as the number of cuts that can be
obtained and their weight, determine the carcass yield, which is influenced by the amount of
inter- and intramuscular fat, as well as the ratio between the volume of the meat pieces and
the covering fat(52). It is important to consider that certain external factors may influence the
percentage expression of yield, such as fasting prior to slaughter, transportation, and travel
time.
In the case of surgically castrated pigs, there is a greater content of intermuscular adipose
tissue, giving more weight to the cuts, despite the fact that the number of cuts in whole pigs
is larger due to the length of the carcass(53).
The volume of meat of pigs immunized with either one of the application protocols is lower
than that of whole pigs, as well as of the meat and intermuscular fat developed by surgically
castrated pigs, and, therefore, their performance is lower(15,16).
In conclusion, the meta-analysis of various castration protocols in pigs under experimental

conditions shows that immunocastrated pigs, with both the standard and alternative
application protocols, are more efficient in terms of the indicators of consumption and weight
gain, as well as of live weight and hot carcass weight. A higher carcass yield is observed in
surgically castrated pigs. Whole pigs have better feed conversion. There are differences in
production indicators and carcass measurement between the standard and the alternative
immunization protocols.
It is important to consider that the age of immunocastration modifies the results in the
production indicators. Castration protocols exhibit different effects on pig production,
depending on the type and age at which they are applied. Absence of gonadotropin-releasing
hormone and androgen concentration modifies the physiological response to productive
performance. Standard immunocastration enhances the response of pig production process
280
traits. Alternative immunocastration enhances carcass trait response; further research on the
age of application of this protocol and its potential effects is required.
Acknowledgments and conflict of interest
This study is part of the doctoral thesis project of Humberto R. Silva Santos, as part of the
PhD program in Animal Production and Health Sciences of the National Autonomous
University of Mexico (Universidad Nacional Autónoma de México). Funding was provided
by the PAPIIT project. IN216921. There is no conflict of interest on the part of the authors.
The authors are grateful to CONAHCyT for the scholarship awarded for their studies.
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Article
Dry matter accumulation, yield, and nutritional quality of forage of corn

hybrids harvested at different days after sowing
Diego Eduardo Ramírez Gutiérrez a
José de Jesús Olmos Colmenero a
Alfonso Peña Ramos b
Juan Isidro Sánchez Duarte c
Ernesto Medina Núñez d
Silviano Gallardo Ramírez e
Omar Iván Santana b*
a
Centro Universitario de los Altos – Universidad de Guadalajara. Jalisco, México.
b
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP) – Campo
Experimental Pabellón. Aguascalientes, México.
c
INIFAP – Campo Experimental La Laguna. Coahuila, México.
d
Escuela Nacional de Lechería Sustentable SPR de RL. Jalisco, México.
e
Proteína Animal S. A. de C.V. San Juan de los Lagos, Jalisco, México.
* Corresponding author: santana.omar@inifap.gob.mx
Abstract:
The objective was to evaluate the dry matter (DM) accumulation by component, yield, and
nutritional composition of forage of four corn hybrids harvested at 121, 128, 135 and 142 d
after sowing. In each harvest, five plants were randomly taken and separated into their
components (stem, leaves, grain, cob, bracts, and tassel) and DM was determined; chemical
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composition and in-situ digestibility were analyzed in a composite sample of a whole plant.
The accumulation of grain in the total DM increased from 35.8 to 43.9 % from 121 to 142 d
to harvest, respectively, and diluted the other components, especially the proportion of stem
and leaves, which decreased inversely proportional to the accumulation of grain. Total DM
content differed between hybrids, from 3.8 and up to 8.3 percentage units on the same days
to harvest. Nonetheless, the hybrid did not affect DM yield or grain production, increasing
by 2.1 and 1.4 t ha-1 between harvests, respectively. NDF content decreased and starch
increased (both linearly), affecting net energy for lactation, which increased from 1.49 to
1.56 Mcal kg-1 from 121 to 142 d to harvest, respectively. The interaction between days to
harvest and hybrid affected starch content, which was 5.2 units higher in a hybrid with similar
NFC and NDF content than its counterparts. DM, NDF and starch digestibilities were
affected by the hybrid, but not by the days to harvest.
Keywords: Starch, Corn silage, Dairy cow.
Introduction
In northern and central Mexico, corn forage is widely used as silage in dairy cow diets(1),
where it represents between 40 and 60 % of the dry basis of the diet(2). The level of inclusion
of corn silage in the ration is a function of the yield and nutrient quality of the forage(3). In
Mexico, in the last 10 yr, the yield per hectare of irrigated forage corn (fresh) has remained
relatively unchanged(4). This is mainly associated with inadequate selection of hybrids and
early harvests(2,5), which reduces dry matter (DM) yield and grain content, which is where
the highest energy value of forage is concentrated(6).
In central Mexico, the dairy basins of Aguascalientes and Altos-Norte de Jalisco share similar
agroclimatic characteristics, contribute 9 and 19 % of national milk production, and cultivate
around 15,000 and 45,000 ha of irrigated forage corn, respectively(4). In this region, water
scarcity, growing demand for corn silage and high costs of grains and concentrates make it
necessary to make corn production more efficient per unit area and per m3 of water used(2).
Therefore, increasing forage yield and quality is essential to achieve more sustainable milk
production(1,2,7).
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The use of outstanding hybrids is the first step to high yield and nutrient quality of forage(8).
Selecting the hybrid and harvesting it at an optimal stage of maturity is essential to achieve
maximum accumulation of DM in the grain in a reasonable time(8,9). Corn silage with better
nutritional quality and locally produced can displace imported corn kernel from the diet and
reduce feed costs. As the days to harvest are delayed, there is a greater accumulation of grain
in the plant, increasing the energy value of the as the proportion of other components in the
plant with less digestibility dissolve(9). Nevertheless, increasing the number of days to harvest
to favor grain accumulation decreases the digestibility of neutral detergent fiber (NDF),
which negatively affects feed consumption in dairy cattle(6,9). Thus, the hypothesis of the
present study was that delaying the days to harvest increases the yield of DM and grain
accumulation without affecting the digestibility of DM and NDF, which are mostly
influenced by the hybrid effect. Therefore, the objective was to determine the response of
four hybrids harvested at 121, 128, 135 or 142 d in DM accumulation by component, total
DM yield, bromatological composition and digestibility of DM, NDF and starch.
Area of study and experimental design
The study was carried out under surface drip irrigation conditions in the SS-2019 cycle in a
farm located in San Juan de los Lagos, Jalisco (21º17’40” N and 102º18’01” W) at 1,838 m
asl; where the climate is semi-dry temperate with an average rainfall of 600 mm. The soil is
alkaline (pH 7.8) with 1.9 % organic matter content and 71 mg kg-1 inorganic N. A
randomized block design was used with an arrangement in split plots with four replications,
where the large plot was the hybrid and the small plots the days to harvest. The hybrids used
were DK-4018 (H1, Dekalb®), Noble (H2, Aspros®), Antílope (H3, Asgrow®) and XR-49
(H4, Ceres®), which were selected for having a yield above the average from a local
assessment in the previous year. All hybrids were intermediate cycle and semi-toothed white
grain. Harvesting was carried out at 121, 128, 135 and 142 d after sowing, which
corresponded approximately to a grain maturity stage of approximately R2, R3, R4 and R5,
respectively. The experimental plot consisted of four furrows 5.0 m long and 0.75 m wide,
and the useful plot consisted of the two central furrows. Sowing was carried out on May 30
in moist soil, depositing the seed manually at a distance of 15 cm at the bottom of the furrow;
the population density to harvest averaged 93,211 ± 2,090 plants ha-1.
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Agronomic management, data collection and sampling
Prior to sowing, between the first and second harrow pass, 4 t ha-1 of cattle compost and
poultry manure with a concentration of 1.1 % N and 0.8 % P were applied; additionally, 200
kg of ammonium nitrate was applied between vegetative stages V3 and V6. The total N
available in the soil for the crop was estimated at 340 kg ha-1. There were no diseases and
only one application for fall armyworm (Spodoptera frugiperda) in stage V3 was necessary,
which was controlled with an application of chlorantraniliprole (Coragen, FMC®, Mobile,
AL). Precipitation and minimum and maximum temperatures were recorded at a weather
station (Em50, Meter Group Inc., Pullman, WA) located 80 m from the experimental site.
Growing degree-days (GDDs) were calculated as the difference between the average
temperature and the base temperature of corn (10 °C). Flowering was recorded when 50 %
of the experimental plot exhibited male inflorescence (tassels releasing pollen) and female
inflorescence (stigmas on young ears).
At 121, 128, 135 and 142 d after sowing (DAS), all the plants in the useful plot were
harvested at 15 cm above ground level and the total fresh weight was recorded. A random
sample of five whole plants was separated into five components: ears of corn, stem, leaves,
tassel, and bracts. Each component was weighed fresh and placed in paper bags to dry at
55 °C to constant weight to determine DM. After drying, the ear of corn was separated into
cob and grain, and samples of each component were ground (SR300 Retsch®, Staufen,
Germany) to pass a 1 mm sieve; subsequently, a composite sample of 100 g (dry weight) of
whole plant was made in proportion of each component to the total dry weight. The whole
sample was used to perform bromatological and in-situ digestibility analyses.
Bromatological and digestibility analyses
Chemical analyses were carried out in the forage laboratory of Unión de Cooperativas de
Consumo Alteñas S.C. de R.L. (UCCA, San Juan de los Lagos, Jal.). Ash content was
determined by introducing 1.0 g of sample into a crucible and incinerating at 550 °C for 6 h
in a muffle furnace. The content of NDF and ADF was determined sequentially in 0.5 g of
sample introduced into a F-57 bag in the fiber analyzer (A200, Ankom Tech., Macedonia,
NY); first, the determination of NDF was performed using alpha-amylase and sodium sulfite,
followed by the determination of ADF in CTAB and H2SO4 solution. The total nitrogen (N)
concentration was determined using the Dumas dry combustion procedure (Leco FP-528, St.
Joseph, MO) and the crude protein (CP) content was calculated as % N × 6.25. The starch
content was determined using the enzymatic-colorimetric procedure(10). Initially, glucose was
290
released by incubating 1.0 g of sample at 100 ºC for 1 h in 30 ml of 100 mM acetate buffer

solution at pH 5.0 and 100 μL of alpha-amylase (Megazyme Ltd., Wicklow, Ireland) was
added, then the reaction was incubated for 2 h at 50 ºC in 3 ml of GOPOD solution
(Megazyme Ltd., Wicklow, Ireland) and then the absorbance was determined at 505 nm in a
visible-light spectrophotometer (Genesys 10S, Thermo Sci., Madison, WI). Finally, the
content of ethereal extract (EE) was analyzed with the gravimetric method in the Golfish
equipment (Novatech GF-6, Tlaquepaque, Jal.) using hexane as a solvent.
Digestibility was determined in situ using two rumen-fistulated cows between 70 and 95 d in
milk (ENLS, Zapotlanejo, Jal.) fed a fully mixed ration composed of 50 % corn silage, 25 %
ground corn grain and 25 % protein-mineral core. First, 4.5 g of sample was placed in 10 ×
20 cm dacron bags (R1020, Ankom Tech., Macedonia, NY) and they were secured with a
strap. Duplicate samples were introduced into the ventral sac of the rumen to determine DM
digestibility, NDF digestibility (NDFD) at 48 h, non-digestible fraction of NDF (uNDF) at
120 h, and starch digestibility at 12 and 24 h. All samples were removed simultaneously and
rinsed in a 12 min cycle in a washing machine until clear water was obtained. Subsequently,
the bags were dried at 55 ºC to constant weight to calculate the digestibility of the DM by
initial vs final weight difference; NDFD, uNDF and starch digestibility were calculated by
analyzing the bag residue with the procedures already described for NDF and starch.
All data were analyzed in the R statistical program (R Studio Inc., Boston, MA) using the
agricolae package and the aov instruction for analysis of variance (ANOVA) with the
following model:
Y = µ + Ai + Hj + δij + Dk + (H × D)jk + Eijk
Where:
Y is the response variable,
μ is the overall average,
A is the random effect of replication i (i= 1 to 4),
H is the fixed effect of the j-th hybrid (j= 1 to 4),
δ is the experimental error associated with the large plot (hybrid),
D is the fixed effect of the k-th day to harvest (k= 1 to 4),
(D × H) is the interaction between hybrid and days to harvest
Eijk is the residual error. For digestibility data, the random effect of the cow (l= 1 to 2) was
included using the model described above.
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All data are least squares means and statistical significance was stated at P≤0.05. Hybrid and
day-to-harvest means, when a linear or quadratic effect was detected, were separated using
Tukey’s HSD test.
Flowering, days to harvest, and GDD accumulation
The number of days to flowering was 73 for hybrids H1, H2 and H3 and 71 for hybrid H4.
Days to harvest were September 29 (121 d), October 6 (128 d), October 13 (135 d) and
October 20 (142 d); for each date, 1,266, 1,329, 1,397 and 1,470 GDDs accumulated,
respectively. In the first 34 d of the crop, the mean temperature averaged 25 °C and then
fluctuated between 19 and 23 °C.
DM accumulation by component
As shown in Table 1, the analysis of variance did not detect significant interactions between
DAS and hybrid in five plant components, except for percentage of cob (P=0.01), where the
differences were minimal. The components with the lowest proportion were tassel, cob, and
bracts, which remained relatively similar in proportion in the four harvests and together
accounted for about 14.5 % of the total DM. In contrast, the components with the highest
proportion were stem, leaves, and grain, with the percentage of the latter increasing as the
days to harvest progressed.
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Table 1: Dry matter (DM) accumulation by plant component, DM content, and whole plant
yield of four hybrids harvested at different days after sowing (DAS)
Component, % of total DM Whole plant
Stem Leaves Tassel Grain Cob Bracts DM, % DM, t/ha
DAS1
121 20.4A 28.3A 0.8 35.8C 6.7 8.0A 25.8D 24.9C
128 18.6B 26.2B 0.8 40.2B 6.6 7.6B 29.5C 25.7C
135 18.7B 25.6B 0.8 41.7B 6.4 6.8BC 34.6B 30.3B
142 19.3B 23.3C 0.6 43.8A 6.4 6.6C 37.8A 33.3A
Hybrid2
H1 19.7b 25.8 0.7b 39.9b 6.2 7.7a 32.3b 29.0
H2 19.2b 26.7 0.6b 39.7b 7.0 6.8b 29.0c 28.1
H3 17.3c 25.3 0.7b 42.0a 6.7 8.0a 35.2a 28.9
H4 20.7a 25.6 1.0a 40.0b 6.1 6.6b 31.2b 28.3
SEM 0.354 0.451 0.025 0.481 0.074 0.163 0.885 0.791
DAS Q** L** NS Q* NS L** L** L**
Hybrid < 0.01 0.184 < 0.01 < 0.01 NS < 0.01 < 0.01 0.805
D×H 0.662 0.089 0.468 0.226 0.013 0.061 0.014 0.865
1
Sowing date: May 30, 2019.
2
Hybrid: (H1: DK-4018; H2: Noble; H3: Antílope; H4: XR-49).
SEM= standard error of the mean; DAS= response of linear (L) or quadratic (Q) days to harvest denoted by:
*0.01 < P≤0.05 and **(P<0.01), D × H= interaction between DAS and hybrid, NS= not significant.
ABC
Means with different uppercase literals differ in DAS (P≤0.05)
abc
Means with different lowercase literals differ between hybrids (P≤0.05).
The percentage of stem showed a quadratic response (P<0.01), decreasing from 121 to 128
d to harvest and from which it remained without significant changes. In addition, it was
observed that the hybrid affected the stem percentage (P<0.01), H4 surpassed H1, H2 and
H3 by 1.1, 1.6 and 3.5 units, respectively. The percentage of leaves decreased linearly
(P<0.01), decreasing 1.2 percentage units between harvests, but no hybrid effects were
detected in this component. The percentage of grain increased quadratically (P=0.02) with
the days to harvest, increasing by 4.5 percentage units from 121 to 128 d, and between 0.6
and 2.2 units from 135 and 142 d, respectively. Hybrid H3 outperformed hybrids H1, H2 and
H4 in grain percentage by 2.1, 2.3 and 2.1 units, respectively. The largest increase in grain
ratio from 121 to 128 corresponded to a decrease in stem percentage over the same period.
The proportion of tassel was not affected by the days to harvest, but differences between
hybrids (P<0.01) were detected, which may not be important in the total composition of the
plant due to its low proportion to the total DM. The percentage of cob was not affected by
the days to harvest or hybrid and remained relatively constant, averaging 6.5 ± 0.3 % of the
total DM. The proportion of bracts exhibited a linear response (P<0.01), decreasing at a rate
293
of 0.3 percentage units between harvests; a difference was also detected between hybrids
(P<0.01) and it was higher in H1 and H3 compared to hybrids H2 and H4 (7.7 and 8.1 vs 6.8
and 66 %, respectively). Hybrids H1 and H3, with the highest proportion of bracts, also had
higher grain accumulation.
Whole plant DM content and yield
The interaction between hybrid and days to harvest affected DM content (P<0.01), as shown
in Figure 1. Hybrids H3 and H2 had the highest and lowest DM content in the four harvests,
respectively; on the other hand, hybrid H1 exhibited a DM accumulation almost linear and
intermediate between H3 and H2. In contrast, hybrid H4 showed higher variation in DM
accumulation between harvests. These discrepancies may be mostly associated with grain
accumulation, but it could also be that the stay-green trait of each hybrid to conserve moisture
(mainly in the stems) affects the DM content(11). The accumulation of DM increased linearly
(P<0.01) at a rate of 3 weekly percentage units, equivalent to 0.4 % per day (Table 1). DM
accumulation has been reported to be around 0.7 to 1.0 % per day under temperate
conditions(12,13). In the present study, drip irrigation and the regular distribution of rainfall
recorded in the cycle could have helped to keep soil moisture constant and reduce plant
moisture loss.
Figure 1: Dry matter (DM, %) content in forage of four corn hybrids (H1=DK-4018,
H2=Noble, H3=Antilope, and H4=XR-49) grown under irrigated conditions and harvested
at 121, 128, 135 and 142 days after sowing
abcdfghi
Means with different literals differ statistically (P≤ 0.05).
294
DM production increased linearly (P<0.01) at a rate of 2.1 t ha-1 per week, but there was no
hybrid effect despite the interaction detected in DM accumulation. The increase in DM
production was mainly due to grain accumulation as it was the only component that increased
in proportion to the total DM with days to harvest. Grain production (t ha-1) increased linearly
with days to harvest (P<0.01) and was 8.9, 10.3, 12.6 and 14.6 t ha-1 at 121, 128, 135 and
142 d to harvest, respectively, but no hybrid effect or its interaction with days to harvest was
detected.
Chemical composition
Except for CP and EE contents, the other bromatological variables were affected by the days
to harvest and hybrid (Table 2). CP and EE contents remained within normal and relatively
stable ranges, with significant differences between hybrids, but these were minimal. In
contrast, the values of NDF, ADF, NFC and starch differed to a greater degree between days
to harvest and between hybrids. NDF content decreased linearly (P<0.01) at a rate of 1.6
percentage units between harvests, while the proportion of ADF increased linearly (P<0.01)
at a rate of 0.9 percentage units per week. Differences were also detected between hybrids
for NDF and ADF contents (both P<0.01), where hybrids H3 and H4 accumulated lower
percentages of NDF and ADF than H1 and H2 (Table 2). These findings differ from those
reported in a local study in which NDF and ADF decreased by about 3.1 and 1.0 percentage
units, respectively, over a 10-d period(14). In another study in which four harvests were carried
out at similar DM content, a reduction in NDF and ADF was also reported; this was attributed
to the dilution of these components due to the increase in grain percentage(9). In the present
work, it is speculated that the low cut height (15 cm) at which the harvest was carried out
may have caused more cellulose at the expense of hemi-cellulose; this has been documented
in other studies in which a shorter stem accumulates more ADF and lignin(15,16).
295
Table 2: Dry matter (DM) chemical composition in whole corn plant of four hybrids
harvested at different days after sowing (DAS)
% DM1
NEL
CP NDF ADF STA NFC EE ASH
Mcal kg-1
DAS2
121 8.0 52.1A 22.5D 19.8D 30.9D 2.4 6.6A 1.49D
128 7.8 49.9B 24.2C 20.9C 33.4C 2.6 6.3A 1.51C
135 7.5 48.3C 24.8B 23.2B 35.1B 2.8 6.3A 1.52B
142 7.9 45.6D 25.9A 25.4A 38.1A 2.8 5.6B 1.56A
Hybrid3
H1 7.6c 49.5a 25.1a 20.6d 33.6b 2.8b 6.5a 1.51b
H2 7.3d 49.4a 25.0a 21.5c 34.5ab 2.2d 6.6a 1.50c
H3 7.9b 48.7b 23.7b 25.2a 34.5a 3.0a 5.9b 1.54a
H4 8.5a 48.2b 23.7b 22.0b 34.7a 2.7c 5.9b 1.53a
SEM 0.020 0.246 0.062 0.034 0.286 0.022 0.064 0.006
DAS NS L** L** L** L** NS Q* L*
Hybrid < 0.01 < 0.01 < 0.01 < 0.01 0.042 < 0.01 < 0.01 < 0.01
D×H 0.610 0.054 0.201 < 0.01 0.072 < 0.01 < 0.01 0.060
1
Expressed as % of total dry matter (DM) of whole plant, unless otherwise indicated.
CP= crude protein, NDF= neutral detergent fiber, ADF= acid detergent fiber, STA= starch, NFC= non-fibrous
carbohydrates, EE= ethereal extract, ASH= ashes; NEL= net energy for lactation calculated with the chemical
composition presented here and digestibility of NDF at 48 h (Eq. 2.11; NRC, 2001).
2
3
Hybrid: (H1= DK-4018; H2= Noble; H3= Antílope; H4= XR-49).
SEM= standard error of the mean; DAS= response of linear (L) or quadratic (Q) days to harvest denoted by:
*0.01 < P≤0.05 and **(P<0.01), D × H= interaction between DAS and hybrid, NS= not significant.
ABC
Means with different uppercase literals differ in DAS (P≤0.05)
abc
Means with different lowercase literals differ between hybrids (P≤0.05).
NFC content increased linearly (P<0.01) at a rate of 1.8 percentage units per week. The
increase in NFC was inversely proportional to the decrease in NDF. Although there were
differences in NFC between hybrids (P<0.01), these were minimal, from 0.9 to 1.1 %, and
only hybrid H1 differed with the lowest NFC content. In the present study, the values
obtained for NFC at 135 and/or 142 d to harvest were lower than those reported in other
studies at similar days to harvest(9,14). Starch accumulation was affected by the interaction
between days to harvest and hybrid (P<0.01); hybrid H3 consistently outperformed the other
materials at 128, 135 and 142 d to harvest, except at 121 d to harvest, when starch content
differed slightly between hybrids (Figure 2). This could be related to the variability observed
in DM and grain accumulation, which affects starch synthesis in grain(17). Regarding days to
296
harvest, a linear effect (P<0.01) was detected in starch accumulation, which increased at a
rate of 1.4 units between harvests.
Figure 2: Starch content in the dry matter (DM) of forage of four corn hybrids (H1= DK-
4018, H2= Noble, H3= Antílope, and H4= XR-49) grown under irrigated conditions and
harvested at 121, 128, 135 and 142 days after sowing
abcdfg
Means with different literals differ statistically (P≤0.05).
Digestibility
Table 3 shows the digestibility parameters evaluated at different incubation times in situ. No
interactions between hybrid and days to harvest were detected for any of the parameters
evaluated. Contrary to expectations, the days to harvest did not affect DM digestibility, NDF
digestibility (NDFD) or starch digestibility. These findings differ from those reported in other
studies in which NDFD at 36 or 48 h decreases by delaying the days to harvest and the
maturity of the plant(14,18). On the other hand, the NDFD values reported here are lower than
those reported in other local studies using the same in-situ method and incubation time(14,19).
On the other hand, DM digestibility and NDFD were affected by the hybrid (P=0.02 and
P=0.01, respectively). Hybrid H1, which had the highest DM digestibility, also obtained the
superior NDFD. The increase in DMD is associated with grain accumulation, while the
decrease is attributed to a lower NDFD(9,20). However, in the present study, the higher grain
accumulation of hybrid H3 did not compensate for its lower NDF digestibility.
297
Table 3: In-situ digestibility of dry matter, neutral detergent fiber and starch of four corn
hybrids harvested at different days after sowing (DAS)
In-situ digestibility
DMD48 NDFD48 uNDF120 STD12 STD24
% DM % NDF % NDF % starch % starch
DAS1
121 59.6 32.2 47.3 46.9 96.7
128 59.0 30.1 47.6 47.0 95.6
135 60.2 30.2 48.8 46.6 94.5
142 60.7 30.0 49.3 44.6 94.4
Hybrid2
H1 62.0a 34.2a 47.1 43.2c 96.9
H2 57.9c 30.5ab 49.6 48.6a 95.3
H3 60.0b 29.6b 49.2 46.8b 93.8
H4 59.5b 28.2b 48.2 46.5b 95.1
SEM 1.600 1.130 1.530 1.230 1.620
DAS NS NS NS NS NS
Hybrid 0.021 0.041 0.257 0.014 0.265
D×H 0.140 0.072 0.128 0.124 0.202
1
2
Hybrid: (H1: DK-4018; H2: Noble; H3: Antílope; H4: XR-49).
DMD48= dry matter (DM) digestibility at 48 h of incubation, NDFD48= neutral detergent fiber digestibility
(NDF) at 48 h of incubation, uNDF120= non-digestible NDF at 120 h of incubation, STD12= starch
digestibility at 12 h of incubation, STD24= starch digestibility at 24 h of incubation.
SEM= standard error of the mean; DAS= linear (L) or quadratic (Q) effects of days to harvest denoted by:
*0.01 < P≤0.05 and **(P<0.01), D × H= interaction between DAS and hybrid.
abc
Means with different literals differs (P≤0.05).
The fraction of non-digestible NDF (uNDF) at 120 h did not differ between days to harvest
or between hybrids, and the means were 48.2 ± 0.9 and 48.2 ±1.4 %, respectively. In the
present study, uNDF values were up to 10 units higher than those reported in other
studies(18,19). A high value of uNDF is associated with lignified fiber fractions, mainly from
the base of the stem, where more lignin accumulates with plant senescence and increase in
DM content(19,20,21). Thus, it is possible that the low values of uNDF found in this study are
associated with the low cut height used in the present study, compared to the aforementioned
studies (15 vs 25 cm, respectively) and another local study using up to 40 cm of cut height(22).
Finally, starch digestibility at 12 or 24 h was not affected by the advance of days to harvest,
and only a hybrid effect (P=0.01) was detected on starch digestibility at 12 h. Although all
hybrids used were semi-toothed grains, it is possible that the vitreosity gradient at grain
maturation affected starch digestibility at 12 h and this was without effect at 24 h(23).
298
In the present study, grain accumulation and DM content increased by delaying the days to
harvest and were influenced by the hybrid effect. Nonetheless, DM yield was not affected by
the hybrid and only increased with days to harvest. The NDF content decreased and the starch
content increased as the days to harvest progressed, but this factor did not affect the
digestibility parameters evaluated. In general, DM yield and grain accumulation can be
maximized by delaying harvest by up to 142 d without affecting NDF digestibility, but some
agronomic strategies to harvest need to be explored to reduce the value of uNDF.
Acknowledgements
To CONAHCyT for the support granted to the first author to carry out his postgraduate
studies. Thanks to Proteína Animal S.A. de C.V (PROAN), Unión de Cooperativas de
Consumo Alteñas S.C. de R.L. (UCCA) and the National Institute of Forestry, Agricultural
and Livestock Research (INIFAP, for its acronym in Spanish), who provided the financial,
technical, and scientific means to carry out the study. Likewise, thanks are given to Escuela
Nacional de Lechería Sustentable S.P.R. de R.L. (ENLS) for the care of the rumen-fistulated
cows used in the digestibility study.
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Article
Electron microscopy and X-ray diffraction analysis of equine

enteroliths from the Aburrá Valley in Antioquia, Colombia
Sergio Andrés Vélez Gil a

Juan José Patiño Marulanda a
José Ramón Martínez Aranzales a*
a
Universidad de Antioquia, Medellín, Colombia. Facultad de Ciencias Agrarias.
Escuela de Medicina Veterinaria. Línea de Investigación en Medicina y Cirugía Equina
– LIMCE, Grupo de Investigación Centauro. Medellín, Colombia.
* Corresponding author: jose.martinez@udea.edu.co
Abstract:
The objective of this study was to determine the mineralogical composition of equine
enteroliths from the Aburrá Valley in Antioquia, Colombia. Samples of eight enteroliths
from eight horses were subjected to semi-quantitative X-ray diffraction (XRD) and
transmission and scanning electron microscopy (TEM/SEM) analysis. The TEM/SEM
of the analyzed enteroliths reported the presence of carbon, oxygen, phosphorus,
magnesium, calcium, silicon, potassium, bromine, iron, sulfur, and aluminum. The
XRD identified struvite, newberyte, kyanite, low quartz, actinolite, nitratine, cordierite,
and vivianite. Both techniques used in the analysis of the enteroliths were correlated by
matching the mineral compounds with the detected chemical elements. The main
mineral components of the enteroliths were magnesium phosphates, struvite and
newberyte being the most common.
Keywords: Colic, Enterolithiasis, Equine, Struvite.
302
Introduction
Enteroliths are concretions derived from mineral precipitations around a nucleus or

nidus of organic or inorganic material, located in the gastrointestinal tract(1,2). These
foreign bodies have different shapes, among which the most common are those of
spherical or tetrahedral and irregular conformation, with different sizes and weights(3).
Geographic regions with a high predisposition to enterolith formation due to specific
mineral components of the soil, water, and plant species have been identified(2-8).
Risk factors such as water sources and high consumption of alfalfa hay with high levels
of magnesium, nitrogen, and phosphorus in the diet may contribute to the formation of
enteroliths, as the struvite formed by these minerals predisposes to their formation(9).
Alfalfa facilitates the formation of magnesium oxide by promoting an alkaline pH,
which favors conditions for the deposition and formation of enteroliths; hence, this
legume in the diet of horses is described as a potential risk factor. Among other factors
involved, the environment, intestinal pH, hypomotility, and the presence of nuclei are
reported to make the formation of these foreign bodies possible(1,8,10).
In addition to exogenous predisposing factors, endogenous factors such as breed, sex,

age, and physiological particularities of horses are described for the presentation of
enteroliths and phytobezoars(4,9). For example, 15-yr-old horses have been found to have
enterolithiasis in the major colon, and 13-yr-old horses, in the minor colon(3); however,
this condition is also reported in animals of all ages(2,4), beings less common in young
animals because of the time required for its development(7).
The speed of enterolith formation in the intestinal tract is variable, as it is related to

particularities of the luminal microenvironment of the colon, type of feed —mainly
concentrate—, and management in confinement(8,9,10), growth form from the nucleus,
and presence of minerals and trace elements(11). Alterations in intestinal pH can
contribute to both the formation and dissolution of enteroliths, thus affecting the time of
formation(1). In these situations, studies are needed to identify and determine the
involvement of predisposing factors in order to establish appropriate preventive
measures and avoid surgical solution as a last resort(12). Therefore, the objective of this
study was to evaluate the mineralogical composition by electron microscopy (chemical
elements) and X-ray diffraction (chemical compounds) of enteroliths obtained from
horses in Colombia.
It was used enteroliths collected (by surgical extraction and spontaneous excretion)
from horses (Colombian Criollo and Argentine Silla) aged 12 to 16 yr, fed with
commercial concentrate, Angleton hay (Dichantium aristatum), salt, and water at will,
in the Aburrá Valley, in Antioquia, Colombia. Once photographically registered, the
enteroliths were weighed and classified by appearance, shape, and size, and later
fragmented with an electric saw, allowing the identification of their nidus or central
nucleus. The slices facilitated the evaluation of color and internal architectural features
such as texture and porosity. Eight samples of enteroliths from an equal number of
303
horses were analyzed in laboratories specialized in mineralogy, crystallography, or

characterization of materials; by X-ray diffraction and transmission and scanning
electron microscopy (TEM/SEM).
Fragments of enteroliths were pulverized and subsequently placed on the quartz crystal
for mineralogical composition analysis, through the semi quantitative X-ray diffraction
(XRD) technique (Empyrean® Series II - Alpha 1, Model 2012, Madrid, Spain). The
analysis of crystalline phases and quantification was performed with the HighScore Plus
software and the ICDD/PDF-4-2012 database for phase identification, with standard
reflection configuration, angle 2ᶿ - 5-80°, step: 0.0263°, time: 46.359 sec. Mineral
identification was obtained by comparing the diffraction patterns of the enterolith
samples with the standard patterns.
On the other hand, samples of the enteroliths were cut into slices that were polished on
both sides, subsequently dehydrated on a hot plate, and prepared according to the
routine procedure for examination with TEM/SEM (FEI Tecnai® G2 F20), along with a
dispersive X-ray spectroscopy for the scanning of the study material.
The data were tabulated and systematized in MS Excel spreadsheets, analyzed with
descriptive statistics, and presented in frequency tables with reports in percentages of
the elements and mineral compounds in the composition of each of the enterolith
samples. This study was approved by the Ethics Committee for Animal Experimentation
(CEEA, Spanish acronym) of the University of Antioquia, Medellin - Colombia
(protocol No. 1062016).
Results
Figure 1 shows the size, shape and texture of the collected enteroliths. Spherical,
polyhedral and irregular shapes with smooth, rough, and porous surfaces were
predominant. The figure also shows the macroscopic and electron microscopic texture
of some of the enteroliths. The enteroliths ranged in size from 5 to 15 cm, and weighed
664.14 ± 385.01 g (maximum 1,157 g; minimum 127 g). On the other hand, material of
plant origin (fiber and seeds) was identified in all the cores of the enteroliths studied,
when they were fragmented with the saw.
304
Figure 1: Enteroliths obtained from equines
a) Shape, size and texture of the enteroliths. b) Electron microscopy image of enteroliths by texture and
conformation of struvite crystals.
Table 1 shows the chemical elements in compositional percentages reported during the
analysis of each enterolith by TEM/SEM. The elements with the highest percentage
were carbon (C), 46.06 %; oxygen (O), 26.85 %; phosphorus (P), 11.55 %; magnesium
(Mg), 5.97 %, and calcium (Ca), 3.71 %, with minerals such as silicon (Si), 2.74 %;
potassium (K), 1.24 %; bromine (Br), 0.35 %; iron (Fe), 0.71 %; sulfur (S), 0.61 %, and
aluminum (Al), 0.17 %. The presence of these elements —especially those considered
as trace elements— varied among the enteroliths.
Table 1: Compositional percentages of mineral elements in enteroliths from eight

horses from Valle de Aburrá in Antioquia, Colombia, analyzed by transmission and
scanning electron microscopy (TEM/SEM)
Enterolith Element (%)
C O P Mg Ca Si K Br Fe S Al
1 31.86 21.07 32.46 12.32 0 0 2.29 0 0 0 0
2 38.07 30.61 11.32 2.56 13.96 2.88 0.60 0 0 0 0
3 57.46 24.00 3.27 1.27 4.12 4.37 1.35 0 1.98 1.48 0.71
4 27.16 27.22 30.18 13.29 0 0 2.15 0 0 0 0
5 61.81 23.79 1.18 0 1.18 9.37 0 2.66 0 0 0
6 32.56 30,45 7.13 11.23 5.21 3,56 2.34 0 3.45 3.40 0.67
7 63.45 27.04 2.34 4.23 1.56 1.23 0 0.15 0 0 0
8 56.12 30.67 4.56 2.89 3.67 0.56 1.23 0 0.30 0 0
Table 2 shows the chemical compounds detected in each enterolith by XRD analysis.
The mineral compounds with the highest concentration were struvite (magnesium
ammonium phosphate hexahydrate [MgNH4PO4-6H20]), 78.68 %; newberyte
(magnesium acid phosphate), 11.23 %; kyanite (aluminum silicate), 3. 18 %; low quartz
(silicon oxide), 2.36 %; actinolite (inosilicate), 2.15 %; nitratine (sodium nitrate),
1.45 %; cordierite, (magnesium cyclocyclicate), 0.46 %, and vivianite, (hydrated iron
phosphate) 0.45 %. None of the samples contained more than five of these compounds.
305
Table 2: Concentration percentages of mineral compounds in horse enteroliths from the

Aburrá Valley in Antioquia, Colombia, analyzed by X-ray diffraction (XRD)
Compound (%)
Enterolith Low
Struvite Nitratin Newberyte Cordierite Actinolite Vivianite Kyanite
quartz
1 83.8 7.8 1.1 6.9 0.3 0 0 0
2 99.6 0 0.4 0 0 0 0 0
3 81.9 3.3 3.1 4.0 0 7.8 0 0
4 81.5 0 18.1 0.1 0 0.3 0 0
5 96.1 0 0.5 0 3.4 0 0 0
6 96.4 0 0 0 0 0 3.6 0
7 55.7 0 33.3 7.9 0 3.1 0 0
8 34.5 0.5 33.4 0 0 6.0 0 25.5
Discussion
The literature reports that equine enteroliths are formed mainly by the precipitation of
struvite, with increased presence of Mg, nitrates, phosphates, and high concentrations of
cations within an alkaline environment in the colon(5,7,13). In addition, high Mg
concentrations in the equine colon have been associated with alfalfa-based diets (>
50 %) and are considered to predispose the formation of enteroliths. However, not all
horses fed alfalfa develop enterolithiasis; this indicates the existence of other factors
that may induce the formation of these concretions, such as individual issues,
hypomotility, bacterial flora, diets, buffering capacity, and water quality, which may
influence the intestinal pH and the colonic mineral content(6,9,10).
This study did not analyze the predisposing factors of enterolith formation or the
evolution of the clinical pictures of horses diagnosed with enterolithiasis. This is a
recognized limitation of this work, as it does not allow to infer the participation of these
factors in the formation of enteroliths; only the composition of these factors is
described. However, the enteroliths come from a geographical area of the department of
Antioquia, Colombia, where it is unusual to feed horses with alfalfa and there is no
desert context, in contrast with previous reports where regions of the world with a high
alfalfa supply and sandy soils have been reported to have the highest frequencies of
occurrence of enterolithiasis(1-4,6,7), indicating that the genesis of enteroliths may be
multifactorial.
The variety of shape, size and texture, and configuration of the nidi were similar to
those of other reports(13). However, unlike in other studies, all nidi were identified,
being the predominant plant material, in contrast with other studies that have described
materials other than plant material(1,9). It was not possible to verify the single or multiple
presence of enteroliths; spherical enteroliths are interpreted as single presence of foreign
bodies, and polyhedral enteroliths, as multiple presence(14,15), as complete information
on the medical history of the equines was not available.
Struvite is identified as the predominant mineral compound in enteroliths as in other

studies(6,7,13). Likewise, the presence of vivianite, although in a lower proportion in the
composition, was similar to that reported by Hassel et al(13). Conversely, the presence of
306
newberyte, kyanite, low quartz, actinolite, nitratine, and cordierite —mineral elements
and trace minerals determined by TEM/SEM (Table 1)— has not been reported;
however, it cannot be inferred that this is a characteristic of enteroliths obtained from
animals from this geographic region, given the low number of samples. Particularly
noteworthy is the finding of more than three compounds in most of the enteroliths, with
the exception of the one constituted by struvite and vivianite.
Although the presence of eight compounds was determined in the group of selected
enteroliths, the presence of apatite (Ca phosphate) was not found, in consonance with
the study by Hassel et al(13), although a larger number of samples are required to
confirm this finding. However, in canines and felines, struvite urinary stones may be
accompanied by apatite stones(16,17), indicating special conditions and interaction or
substitution of ions that can influence the crystallization of apatite, as is the case of K
and Mg(18).
As for the major elements, the concentrations of P, Mg, K, Ca and organic C and trace
elements such as Fe, were similar to those reported in the petrographic and
mineralogical studies carried out by Rouff et al. (11) in samples of enteroliths from
different geographic regions. On the other hand, the present study reported
concentrations of S, Si, Br, and Al, but did not detect the presence of Zn or Mn. In
addition, copper (Cu) was not detected in any of the studies, despite being found in the
nutritional analysis of equine feed carried out by the same authors(11). Therefore, it is
possible that the precipitation and crystallization of mineral compounds depends not
only on saturation but also on the interaction of ions and pH conditions in the colonic
fluid(18). Based on the above, it is possible to hypothesize that the difference in contexts
and feeding systems of the equines may affect the ionic saturation in the colonic fluid,
which could partly explain the amount of major and trace elements determined in this
work.
Despite the type of food supplied to the horses and the presence of certain minerals in
their colonic fluid, these do not form compounds, or such compounds are not detected in
the composition of the enteroliths, a fact that reinforces the hypothesis of the existence
of other predisposing factors involved in their formation and growth(11,13,18). However, it
is interesting that struvite is the major component of the enteroliths analyzed in several
parts of the world, which might suggest the existence of a potential analogy with the
formation of struvite urinary calculi, in which there is evidence of microbial metabolism
rather than mineral saturation(19,20). However, this process is complex, and there is still
no evidence that it occurs in the equine colon(9,21).
The recognition of trace elements and organic impurities in the composition of struvite
is important, as the higher the concentration of these elements, the greater the
susceptibility to decomposition(11). In addition, canine and feline apatite stones are more
resistant than struvite stones(16,17); however, they are absent in equine enterolithiasis.
Therefore, it is possible to consider medical treatments to dissolve the enteroliths and
diet manipulation strategies to prevent their formation, given that the mineralogical
analyses showed high impurities of organic material and trace elements that make them
susceptible to disintegration and, depending on the composition, solvents like
carbonated beverages such as Coca-Cola® may be utilized for this purpose (12).
307
Both techniques (TEM/SEM and XRD) used in the analysis of the enteroliths were
correlated by matching the mineral compounds with the detected chemical elements. In
sum, the main mineral components of the analyzed enteroliths were Mg phosphates, the
most common of which are struvite and newberyte, unlike vivianite which was also
detected, but in a lower proportion than previously reported(13). Other compounds were
also reported to be distributed in all the analyzed samples; however, studies with a
larger number of samples and with relevant information on the management, feeding,
and clinical condition associated with enterolithiasis of the animals are required to
determine the association with the mineralogical composition of the enteroliths.
Acknowledgments
This work was financed with resources from the Research Development Committee
(CODI) of the Research Vice-Rectory of the University of Antioquia, Research Center
of the Faculty of Agricultural Sciences (CIAG) and Sustainability Resources 2019 -
2020 of the Centauro Group.
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309
Article
Prevalence and risk factors associated with Cryptosporidium spp. in dairy

cattle in Chiquinquirá (Colombia)
Diana M. Bulla-Castañeda a
Deisy J. Lancheros Buitrago a
Leneth B. Castañeda Sedano a
Rosa I. Higuera Piedrahita b
Martin O. Pulido-Medellin a*
a
Universidad Pedagógica y Tecnológica de Colombia – UPTC. Grupo de Investigación en
Medicina Veterinaria y Zootecnia – GIDIMEVETZ. Tunja, Colombia.
b
Universidad Nacional Autónoma de México. Facultad de Estudios Superiores Cuautitlán,
Cuautitlán, México.
*Corresponding author: martin.pulido@uptc.edu.co
Abstract:
Cryptosporidiosis is a disease characterized by episodes of diarrhea in cattle worldwide,

caused by a protozoan parasite of the genus Cryptosporidium spp. of the phylum
Apicomplexa and Family Cryptosporiidae. It is responsible for important economic losses,
and, in addition to this, it generates an impact on human health, as it can parasitize humans.
The objective of the study was to determine the prevalence of and risk factors associated with
Cryptosporidium spp. in cattle in Chiquinquirá (Colombia). A descriptive cross-sectional
study with simple random sampling was carried out, with a sample size of 1,044 head of
cattle, including males and females of different breeds and age groups, using the WinEpi
statistical software. Fecal samples were taken directly from the rectum and processed with
the modified Ziehl-Neelsen (ZN) technique for the identification of parasite oocysts using a
100X objective. The data were processed with the Epi Info® statistical software. An overall
310
prevalence of 7.3 % (73/1000) was found; females, 2 to 4-yr-old bovines, and crossbred cattle
were the most prevalent. No significant statistical association was found between breed, age,
and sex of the individuals evaluated, and protozoan positivity (P≥0.05). The purchase of
animals and larger productions were considered risk factors for parasitosis. Protozoan
prevention and control plans should be designed and implemented based on sanitary practices
to prevent the dissemination of oocysts found in fecal matter.
Keywords: Crypstoporidium spp., Cryptosporidiosis, Cattle.
Introduction
Cryptosporidium spp. is a protozoan, coccidian, zoonotic, obligate intracellular parasite that

is part of the phylum Apicomplexa and the family Cryptosporiidae; it is distributed across
the world(1-5). The parasite affects the gastrointestinal tract of vertebrate species such as cattle,
birds, small ruminants, rodents, canines, felines, rabbits, squirrels, and even humans(6-9).
Recent reports indicate that more than 40 species of Cryptosporidium spp. have been
described, among which C. parvum, C. bovis, C. ryanae, and C. andersoni are routinely
found in cattle(5).
Parasites of this genus cause a serious gastrointestinal disease known as cryptosporidiosis(7,8)

that impacts both human health and animal health(1,5,6). Cattle, especially calves, have been
identified as one of the most common reservoirs of this protist(1,4), which is one of the main
causes of morbidity and mortality in calves aged 1 mo or less worldwide(10). However, there
is a wide variety of hosts that can act as reservoirs of the parasite, favoring the persistence of
Cryptosporidium spp. in the environment for long periods of time as oocysts and, therefore,
increasing the risk of their transmission to susceptible hosts(6,7).
Cryptosporidium spp. infections constitute a substantial public health burden and are
responsible for economic losses in livestock herds worldwide(11). Therefore, the reduction of
disease and shedding of Cryptosporidium spp. oocysts is considered an important objective
in livestock productions, by inhibiting the transmission of the protist through direct contact
with infected animals, or ingestion of feed and water contaminated with animal feces(9). The
diagnosis of the protozoan is based on the identification of oocysts at the laboratory, where
311
it is a common practice to carry out a microscopic observation of the oocysts applying a

Ziehl-Neelsen (ZN) stain with an acid alcohol solution, auramine with phenol, or
immunofluorescent stain to fecal smears(12).
The therapeutic options available to treat cryptosporidiosis are limited(11). Despite the
substantial interest in this type of parasite, progress in terms of treatment development and
understanding of most of the life cycle of this unusual organism is scarce(7). So far, in the
Department of Boyacá there are no recent studies on the identification of parasite oocysts in
fecal material by microscopy, nor the analysis of different variables(13). Therefore, the
objective of this research was to determine the prevalence and risk factors associated with
Cryptosporidium spp. in cattle in Chiquinquirá (Colombia).
Geographical location
Boyacá has four municipalities specialized in milk production (Chiquinquirá, Caldas, San
Miguel de Sema, and Saboyá), reaching a volume of 70,000 L per day derived from
approximately 50,000 cows destined for milk production(14). According to national
government data, livestock farming in Chiquinquirá represents an important part of the
economy of the municipality, which is located at 5°36'48" N and of 0°15'21" W of the
meridian of Bogotá, at an altitude of 2,000 to 3,200 meters above sea level, having an average
temperature of 15 ºC(15).
Sample size
In the year 2022, Chiquinquirá reported 33 398 head of cattle, according to the National
Livestock Census of the Colombian Agricultural Institute (ICA)(16). Based on the reported
data and following the formula obtained from the WinEpi statistical program, a sample size
of 947 female and 47 male cattle of various age groups and breeds with dairy potential was
determined. In addition, a confidence interval of 95%, an accepted error of 5%, a sampling
fraction of 1.15% and an expected prevalence of 50% were considered.
312
𝑍 𝑎
2√𝑝(1−𝑝) 𝑍 2 𝛼/2 ∙ 𝑝(1 − 𝑝)
𝑛=( )=
𝐸 𝐸2
Where: n= sample size; E= accepted error; p= expected value of the ratio; α= queuing
probability.
Sample collection and processing
A total of 2 to 5 g of fecal material were taken directly from the rectum by rectal palpation.
The samples were labeled and stored in refrigeration coolers to be transported to the
Veterinary Parasitology Laboratory of the Pedagogic and Technological University of
Colombia (Universidad Pedagógica y Tecnológica de Colombia, UPTC) for processing. For
the identification of Cyptosporidium spp. oocysts in bovine feces, the modified Ziehl-
Neelsen (ZN) or Kinyoun cold staining technique was utilized. A thin smear of fecal material
was made on the slide and allowed to air dry. The slides were then placed in staining racks
where they were stained for 10 min with ZN fuchsin. The slides were then placed in staining
racks where they were stained for 10 min with ZN fuchsin. The slides were examined
microscopically using a 100x objective with immersion oil. In these samples,
Cryptosporidium spp. oocysts stained bright red were considered positive(17).
The identification of Cryptosporidium spp. oocysts in bovine fecal material and the data
obtained in the epidemiological survey were consolidated and filtered. Among the evaluated
factors, it is important to mention that reference is made to the absence or presence of
management practices; large herds were those with more than 10 animals in production,
while small herds were those with 10 animals or less. In terms of the water sources, the only
one that provided potable and treated water was the aqueduct. The results were analyzed with
the Epi Info® statistical software, version 7.2.4.0.
The proportion of individuals affected by Cryptosporidium spp. and exposed to the factors
evaluated in the study were compared with the same proportion of a population not exposed
to that factor to estimate prevalence ratios (PR). The PR was employed to measure the
association between cryptosporidiosis and the hypothesized causal factors, as well as the
significance of these associations using Fisher's exact test(18).
313
PR values above 1 (lower 95% confidence interval < 1) and with P<0.05 were considered
risk factors, whereas PR values below 1 (upper 95% confidence interval < 1) and with P<0.05
were regarded as protective factors. The dependent variable included the modified ZN
results, while the independent variables were all the determinant variables established in the
epidemiological survey applied during sampling. Once these factors were established, a
logistic regression was performed(19).
Ethical considerations
The study was conducted per Resolution 8430 of the Colombian Ministry of Health and
Social Protection and the 1989 Law No. 84. These set out the standards that are appropriate
for the welfare of animals during research. In addition, before blood sampling, an informed
consent form was signed by the owners of the cattle.
Results
An overall prevalence of 7.3 % (73/1000) was determined in the municipality of

Chiquinquirá. Females were more prevalent than males, with a prevalence rate of 7.39
(70/947) and 6.98 % (3/43), respectively. Cattle aged 2 to 4 yr and crossbred cattle had a
higher presence of Cryptosporidium spp. oocysts (Table 1). No significant statistical
association was found between the breed, age, or gender of the individuals evaluated and
protozoan’s positivity (P≥0.05).
Table 1: Prevalence of Cryptosporidium spp. by age group and breed in cattle in the
municipality of Chiquinquirá, Boyacá
Variable N Positive Cryptosporidium spp. Prevalence (%)
Age groups
< 2 years 304 20 6.58
2-4 years 84 10 11.90
> 4 years 612 43 7.03
Breeds
Ayrshire 138 11 7.97
Crossbreed 95 9 9.47
Holstein 767 53 6.91
314
Regarding the assessed variables, management practices such as the presence of cattle
belonging to other owners (P=0.0018), pasture leasing (P=0.0010), and the purchase of
animals (P=0.0062) were statistically significantly associated with the occurrence of the
parasite in the evaluated cattle (Table 2).
Table 2: Analysis of management practices as potential risk factors associated with

Cryptosporidium spp. infections
Confidence
Variable Category PR P-value
interval (95%)
Pen 0.9769 0.9416 - 1.0135 0.1234
Other owners’ livestock 0.9427 0.9136 - 1.0729 0.0018
Other species 0.9352 0.8351 - 1.0474 0.1113
Management
Lease of pastures 0.9455 0.9138 - 1.0783 0.0013
practices
Purchase of animals 1.0472 1.0118 - 1.0839 0.0062
Damaged fences 1.0056 0.9696 - 1.0430 0.4254
Deworming 0.9352 0.8351 - 1.0474 0.1113
The results are presented as prevalence ratio (PR) and 95% confidence interval (CI).
The association between diarrhea and the presence of Cryptosporidium spp. oocysts in the
analyzed fecal samples was statistically significant. Herd size too was statistically significant:
large herds were considered as a potential risk factor, while small herds were established as
a preventive factor against the occurrence of the parasite. On the other hand, when analyzing
the source of drinking water, the aqueduct and the stream exhibited a statistically significant
association with the positivity of the protozoan, and the stream was established as a potential
risk factor, while the aqueduct was a protective factor (Table 3).
Table 3: Analysis of clinical manifestations, herd size, and drinking water source as
potential risk factors associated with Cryptosporidium spp. infections
Confidence interval
Variable Category PR P-value
(95%)
Clinical Diarrhea 0.9552 0.9208 - 1.0909 0.0078
manifestations Fever 0.9699 0.9366 - 1.0044 0.0545
Herd size Large herd 1.051 1.0169 - 1.0863 0.0081
Small herd 0.9515 0.9206 - 0.9834 0.0072
Water source Aqueduct 0.9496 0.9175 - 0.9829 0.0023
Cistern 0.9694 0.9364 - 1.0035 0.0657
Gully 1.0589 1.0122 - 1.1078 0.0041
The results are presented as prevalence ratio (PR) and 95% confidence interval (CI).
315
The analysis of the variables that were determined as potential risk factors through logistic
regression allowed determining that the purchase of animals and production units with more
than 10 animals as risk factors for the occurrence of Cryptosporidium spp. oocysts in the
evaluated cattle (Table 4).
Table 4: Analysis of variables as potential risk factors associated with infections by

Cryptosporidium spp.
Variable Odds ratio LCI UCS P-value
Purchase of animals 2.252 1.3358 3.7965 0.0023
Large herd 2.6677 1.2593 5.651 0.0104
Gully 1.5773 0.9484 2.6232 0.0791
LCI= lower confidence interval; UCS= upper confidence interval.
Discussion
Enteric protozoan infection in cattle can pose a threat to the productivity and survival of the
animals, resulting in negative impacts on the livestock industry(20). Within this group of
pathogens affecting animal health, Cryptosporidium spp. is an obligate intracellular parasite
transmitted by the fecal-oral route after ingestion of oocysts that can contaminate, persist,
and resist disinfection in the water and food(21). The published literature on the parasite is
extensive, providing details of its distribution in most regions of the world(22). Prevalence
rates of 52.2 % in Algeria(10), 16.2 % in Ethiopia(4), 53 % in Latvia(23), and 64 % in cattle
sampled in the Lagoon region of Mexico have been reported(24).
Similarly, at the national level, there are prevalence rates of 22 % in the Central Savannah
province (Cundinamarca)(25), 22 % and 7 % in Chiquinquirá(26,27), and 48 % in bovines in
Boyacá(28); microscopic diagnosis revealed that 115 calves (26.6 %) from 44 farms (59.5 %)
in a central area of Colombia (Antioquia, Boyacá, Cundinamarca, and Meta) tested
positive(29), these rates being higher than those found in the present study. The reported
variations may be caused by different environmental conditions, management practices, and
the number of animals in the farms; therefore, the role of the environment in direct and
indirect contamination should be considered, mainly the accumulation of oocysts having
occurred previously in animals of the herd, which facilitates the fecal-oral transmission route
among the cattle and can thus modify the prevalence of infection by the parasite(30).
In the present study, cattle aged 2 to 4 yr had the highest prevalence of the parasite, unlike in
Africa(6,10), Asia(20), and South America(29), where a higher infection rate was detected in
young cattle compared to adult animals. No significant statistical association was found
316
between cattle age (P≥0.05) and the prevalence of Cryptosporidium infection in cattle from
central Ethiopia(31); however, in cattle from dairy farms in Colombia(28), United States(32), and
India(2), reports have detected an association between the age of the bovines and the excretion
of oocysts in fecal matter. Although age was not considered a risk factor for the occurrence
of the protozoan in the present study, bovines aged <12 mo were associated with the excretion
of Cryptosporidium spp. oocysts in Colombia(28). In this regard, it should be taken into
account that nursing calves are more predisposed to acquire infection by the parasite; in
addition, the clinical disease may be influenced by the immune status of the host(33).
Das et al(2) report that there is statistical significance between positivity to Cryptosporidium
spp. and the sex of the cattle, which was not evident in the present study; however, similarly
to our results, in Ethiopia(31) and Nigeria(6) no significant statistical differences in the
prevalence of Cryptosporidium infection were found between males and females. On the
other hand, crossbreeds had the highest oocyst excretion of the protozoan, with no statistical
association between cattle breed and parasite occurrence (P≥0.05). Likewise, in Addis Ababa
and its surroundings(31), the prevalence of infection is likely due to the potential occurrence
of the coccidian in beef and dairy cattle(2) regardless of the breed of the animals.
The risk factors for Cryptosporidium spp. are mainly associated with the handling and
sanitary condition of the animals(31). The presence in the herds of bovines belonging to other
owners, the leasing of pastures, and the purchase of animals whose health and deworming
history was unknown were associated with the presence of oocysts in the evaluated samples
(P≤0.05). This is because the transmission of cryptosporidiosis is mainly due to management
practices that allow the dissemination that oocysts found in the environment or in diseased
animals or susceptible hosts. Similarly, the purchase of animals was identified as a risk factor
for the presence of the parasite, possibly because Cryptosporidium spp. is not specific to the
host. Thus, an environment contaminated with oocysts during an outbreak in cattle can lead
to the infection of other species that subsequently use the same grazing area; the unknown
health history of individuals can also increase the potential transmission of the protozoan(2).
Herd size was associated with Cryptosporidium spp. oocyst excretion, where smaller herd
sizes were considered a protective factor, and larger herds were established as a risk factor
for infection, consistently with the positive association between higher cattle population
density and fecal excretion of Cryptosporidium spp. in Africa(31), Asia(20), Europe(34), and
North America(32). Likewise, individual calf rearing reduces the potentiality of infection by
the protozoan by approximately 2.5 times compared to group calf rearing(31), as the rate of
oocyst shedding differs between housing systems, exhibiting a higher prevalence in calves
kept as a group compared to the individual system. However, this will depend on the age of
the animals(34) which demonstrates the importance of the facilities used in intensive farms
with higher animal densities(29).
317
The aqueduct differed significantly (P≤0.05) from the stream as a source of drinking water,
the aqueduct having been established as a protective factor against parasite positivity. Farms
with drinking water sources such as wells, rivers, or streams acquired 2.4 and 2.9 times more
Cryptosporidium than farms using tap water to provide water to cattle(31). Likewise, herds
that dispose of wastewater in the field compared to farms that discharge wastewater into
nearby wells may also be more likely to be infected with the protozoan(31). Infection by
Cryptosporidium spp. is also significantly associated with the symptoms of the infected
animals(2), as in the case of the presence of diarrhea in the evaluated individuals (P≤0.05).
However, previous studies showed no association between the presence of diarrhea and
oocyst shedding(6,34,35), as well as only a slightly higher prevalence in diarrheic cattle
compared to non-diarrheic cattle(6). Despite this, the rate of infection by Cryptosporidium
spp. in Colombia(27,28) and Algeria(10) is higher in animals with diarrhea compared to those
that do not have it, consistently with the findings in Chiquinquirá, which highlight that the
risk of occurrence of this symptom in bovines may decrease as they reach adulthood(23).
There is currently no vaccine or drug in the market for the treatment and control of
cryptosporidiosis in ruminants, which makes its prevention difficult. In this sense, it is
necessary to implement strategies to reduce the spread of infection in herds, including good
disease management practices, such as the separation of cattle with diarrhea; cleaning and
disinfecting the facilities before introducing animals; the removal and disposal of fecal matter
or wet garbage; good hygiene of the feed and water troughs, and adequate supply of
colostrum to newborns, as well as the development of strategies to reduce humidity in the
herds(36).
A moderate prevalence of infection by Cryptoporidium spp. was found in cattle in

Chiquinquirá, where females, cattle aged 2 to 4 yr, and crossbreeds were the most prevalent.
Although infection by the protozoan occurs more frequently in calves, adults can become a
source of dissemination of the parasite; therefore, its prevention and control in herds should
be paramount. The purchase of animals and larger productions were considered as risk factors
for parasitosis; in this sense, sanitary and management practices should be adjusted to
minimize the excretion of oocysts in fecal matter in extensive systems and in those where
animals whose sanitary history is unknown are allowed to enter.
318
The authors of this article declare that they have neither conflict of interest nor any economic,
personal, political, financial, or academic relationship that might influence their judgment.
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Article
Influence of the type of container and traditional methods on the long-

term storage of honey produced by stingless Scaptotrigona mexicana:
bioactive compounds and antioxidant properties
Naida Juárez-Trujillo a
Simón Carrouché b,c
María Remedios Mendoza-López d
Juan L. Monribot-Villanueva e
José A. Guerrero-Analco e
Maribel Jiménez-Fernández a*
a
Universidad Veracruzana. Centro de Investigación y Desarrollo en Alimentos. Av. Dr. Luis
Castelazo, s/n. 91000. Xalapa, Veracruz, México.
b
Istom. Ecole-Supérieure Dágro-Développment International, Francia.
c
Chasseurs De Saveurs S.L. de R.L. Company, Veracruz, México.
d
Instituto de Química Aplicada, Universidad Veracruzana, Veracruz, México.
e
Instituto de Ecología, A.C. Red de Estudios Moleculares Avanzados. Veracruz, México.
*Corresponding author: maribjimenez@uv.mx
Abstract:
Scaptotrigona mexicana honey is characterized by its nutritional and antioxidant properties,

but it has a high moisture content that affects its stability during storage. The objective of this
work was to evaluate the physicochemical and antioxidant properties by UV-Visible
spectroscopy, profile of phenolic compounds by ultra-high performance liquid
chromatography coupled to mass spectrometry and fatty acids and volatile compounds by
323
gas chromatography coupled to mass spectrometry, minerals by microwave plasma atomic

emission spectroscopy, from honey stored in different containers that, along with traditional
methods, are commonly used to increase its stability. Most physicochemical and antioxidant
properties were not significantly different from those of freshly harvested honey. The results
suggest that the packaging with an exhaust check valve has a significant effect on the
decrease in moisture content and water activity, but not on the physicochemical and
antioxidant properties for at least 2 yr of storage. These results suggest that the type of
container should be considered when storing honey as it significantly (P<0.05) affects its
properties and quality.
Keywords: Antioxidant activity; Container; Honey; Scaptotrigona mexicana; Meliponine

stingless bees.
Received. 08/05/2023
Introduction
Stingless bee honey is highly demanded by consumers, due to its healing properties and
quality(1). However, this type of honey is characterized by a higher moisture content, which
increases the probability of its deterioration. In addition, it has been reported that excess
water can be a negative quality attribute since it creates a high risk of inducing fermentation
processes and consequently altering the organoleptic properties of honey(2). Various
methodologies have been reported to maintain the nutritional and sensory properties of
honey, increase its stability, and improve its handling and marketing conditions to obtain a
safe product for the consumer(3), such as the use of dehydration in plastic trays using
controlled-temperature ovens(4). However, exposure to high temperatures produces an
increase in the content of hydroxymethylfurfural(5). It has been reported that heating to
boiling temperature eliminates yeasts and reduces moisture content and that certain
containers, such as unglazed clay pots, produce a reduction in moisture content of up to
20 %, increasing the shelf life of honey(6). However, clay containers have the disadvantage
of being fragile, of low capacity and not functional for transport. In a traditional method used
in the Totonacapan region, Veracruz, Mexico, beans are added to honey since, according to
the inhabitants, these seeds absorb moisture from the honey, making it more stable. Another
traditional technique is the use of vacuum packing to avoid the entry of air. Therefore, the
objective of this research was to investigate the effect of storage in different plastic containers
on the physicochemical properties, antioxidants, phenolic compounds and fatty acids profile,
minerals and volatile compounds in honey during storage.
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Chemicals
2,2´-Diphenyl-1-picrylhydrazyl (DPPH), Trolox (6-hydroxy- gallic acid, quercetin, Folin–

Ciocalteu reagent and 2,4,6-Tris(2-pyridyl)-1,3,5-triazine (TPTZ) were purchased from
Sigma (St. Louis, MO, USA).
Sample collection
Twenty-four (24) liters of honey were provided by the company Chasseurs De Saveurs S.A.
C.V. They were collected from the Zozocolco region, Veracruz, Mexico. Samples were
collected from hives in rustic wooden boxes that meliponicultors keep in their homes. The
extraction was carried out during the month of May 2018 using a 20 mL syringe. The 24-L
batch of honey collected was divided into four batches (6 L per batch). The honey from each
batch was divided into three 2-L containers (Figure 1). T0 (control) was immediately used
for the analysis of the evaluated parameters and collected from rustic wooden boxes. Batches
T1 to T4 were placed in plastic containers commonly used for the commercialization of
honey. The description of the treatments is presented below: T1: honey stored in an opaque
plastic tray (high density polyethylene), T2: honey stored in an opaque plastic tray added
with five bean seeds, T3: honey stored in an opaque plastic tray with an exhaust check valve
(ZAZOLYNE, China) used in the fermentation of wines, T4: honey stored in a transparent
plastic container (polyethylene terephthalate, 1). The honey samples were placed in a room
with a temperature of 25 ºC and were analyzed at the beginning and after 2 yr of storage. All
the determinations were made by triplicate.
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Figure 1: Packaging treatments for honey Scaptotrigona mexicana
T0= control; T1= honey stored in an opaque plastic tray; T2= honey stored in an opaque plastic tray
added with five bean seeds; T3= honey stored in an opaque plastic tray with an exhaust check
valve; T4= honey stored in a transparent plastic container.
Physicochemical properties
The moisture, electrical conductivity, pH, and titratable acidity of the honey samples were
determined, using the appropriate analytical standard procedures(7). Electrical conductivity
was determined using a conductivity meter (Mettler Toledo, ME 226 model, Pittsburgh,
USA) and water activity was measured using a water activity meter (AquaLab, Model 4TE,
Meter group, Inc, USA). The color was measured with a colorimeter (ColorFlex V1–72
SNHCX 1115, Hunter Lab, USA) using parameters CIE L*,a*, b* and total color change,
browning index and Chroma were calculated.
Total phenolic compounds content, vitamin C and antioxidant activity
The content of total phenolic compounds and the antioxidant activity: DPPH (2,2-Diphenyl-
1-picrylhydrazyl), FRAP (Ferric reducing ability of plasma) and ABTS (2,2'-azino-bis-(3-
ethylbenzothiazoline-6-sulphonic acid) assays were determined using methanolic honey
extract with a 1:100 dilution(8).
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The total phenolic compounds were determined by the Folin-Ciocalteu method with some
modifications(9). Thirty microliters of each sample and 30 μL of Folin-Ciocalteu were mixed
and incubated for 2 min (40 °C). After 240 μL of Na2CO3 (5%) were added, they were
incubated for 20 min (40 °C). After that time, the absorbance was read (λ= 765 nm). Vitamin
C content was determined using a standard curve made with L-ascorbic acid (99% purity;
Sigma Rec. 84272, St. Louis, Missouri, USA) at a concentration of 0–50 mg and the results
were expressed as mg equivalents of ascorbic acid (AAE) per gram.
The percentage of inhibition of the DPPH radical was determined by mixing 30 µL of each
sample with 270 µL of DPPH reagent, then incubated for 30 min, subsequently the
absorbance was read (λ= 517 nm, Multiskan FC, model IVD, Finland). ABTS was
determined, 30 μL of extract and 270 μL of ABTS reagent were mixed and then incubated
for 30 min (25 °C). Then the absorbance (λ= 734 nm, Multiskan FC, model IVD, Finland)
was measured. Finally, FRAP was determined, 30 μL of extract and 270 μL of FRAP reagent
were mixed, then incubated for 30 min (37 °C) and absorbance was measured (λ= 593 nm,
Multiskan FC, model IVD, Finland). For the two technics 0.1–1 mg/mL Trolox calibration
curve was performed(9). The results are expressed in miligrams of Trolox equivalents per
gram of dry weight of each sample.
UPLC-MS analysis
For the honey extracts, one gram of honey was weighed, 10 mL of methanol was added, and
it was subjected to ultrasonication (Sonics Materials VCX 750 ultrasonic microprocessor,
Connecticut, USA) for 10 min, this process was repeated until exhaustion. Subsequently, the
solvent was evaporated to dryness in a rotary evaporator (Rotavapor R-100, Büchi, Flawil,
Switzerland). Then, the honey extract (10 mg) was re-dissolved in 1.0 mL of MeOH with
0.1% of formic acid (Both MS grade, Sigma-Aldrich), filtered and placed in a 1.5 mL Ultra
High-Performance Liquid Chromatography (UPLC) vial. The identification and quantitation
of individual phenolic compounds was performed with an UPLC coupled to a triple
quadrupole mass spectrometer (Agilent Technologies 1290-6460, Santa Clara, California,
USA). Chromatographic conditions were: flow 0.3 mL/min, injection volume 2 µL, and
column temperature 40 °C. The gradient started at 1% B, then changed to 50% B in 30 min,
then 99 % B in 4 min followed by an isocratic step to 99 % B for 4 min. Subsequently, a
gradient to 1% B in 1 min followed by an isocratic step for 5 min. The mass spectrometry
conditions were electrospray ionization in positive and negative modes, temperature (T) of
the gas 300 °C and T of the sheath gas 250 °C with flows of 5 and 11 L/min, respectively.
The nebulizer pressure was 45 Psi and the capillary and nozzle voltages were 3,500 and 500
V, respectively. Forty-eight (48) compounds were searched: shikimic acid, gallic acid, L-
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phenylalanine, protocatechuic acid, 4-hydroxybenzoic acid, gentisic acid, 4-

hydroxyphenylacetic acid, (-)-epigallocatechin, (+)-catechin, vanillic acid, scopoline,
chlorogenic acid, caffeic acid, malvin, kuromanin, procyanidin B2, vanillin, keracyanin, (-)-
epicatechin, 4-coumaric acid, mangiferin, umbelliferone, (-)-gallocatechin gallate,
scopoletin, ferulic acid, quercetin 3,4-di-O-glucoside, 3-coumaric acid, salicylic acid, sinapic
acid, epicatechin gallate, ellagic acid, myricitrin, pelargonidin, quercetin 3-D-galactoside,
rutin, p-anisic acid, quercetin 3-glucoside, luteolin 7-O-glucoside, malvidin, 2,4-dimethoxy-
6-methylbenzoic acid, penta-O-galloyl-B-D-glucose, kaempferol 3-O-glucoside, quercitrin,
naringin, rosmarinic acid, trans-cinnamic acid, luteolin, and kaempferide. Each compound
was identified using a dynamic multiple reaction monitoring method and quantified using
calibration curves from 0.25 to 19 µM, with a coefficient of determination greater than
0.99(9).
GC-MS compounds
Volatile compounds (esters, aldehydes, ketones and terpenes that are characteristic of this
type of sample) were determined in 3.0 g of honey stored. The honey was placed in a vial
sealed with a PTFE / Teflon cap and heated to 100 °C, then the sample was injected using an
Agilent Technologies Head-space model 7694E and a gas chromatograph (Agilent
Technologies™, model 6890 N, Net Work GC system, Santa Clara, California, USA)
equipped with a DB-5 capillary column (60 m × 0.25mm id × 0.25 µm film thickness) was
used. The GC conditions were initial temperature: 45 °C for 5 min, heating ramp: 15 °C/min
up to 280 °C, for 1 min. Helium at a flow of 1 mL/min, injector temperature of 250 °C.
Identification of volatile compounds was performed by mass spectrometry using the Agilent
Technologies™ Model 5975 inert XL mass spectrometer, mass spectra were obtained by
electron impact ionization at 70 eV. For identification, the mass spectra obtained for each
compound were compared with a database (HP Chemstation-NIST 05 Mass Spectral search
program, version 2.0d).
Fatty acid profile
Oily material was extracted from honey using a Soxhlet extractor with hexane (60–80 °C)
for 6 h. The oily extract was filtered and concentrated under vacuum (Büchi, Flawil,
Switzerland) to get crude extracts. Methyl Esters of Fatty Acids (FAMEs) were obtained
through an esterification process and analyzed by gas chromatography coupled to mass
spectrometry (GC-MS)(10). A gas chromatograph (Agilent Technologies™, model 6890 N,
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Net Work GC system, Santa Clara, California, USA) equipped with a DB-5 column (5%
methylpolysiloxane, cat-1225082, J&W Scientific, USA) was used. The GC conditions were:
initial temperature: 150 °C for 5 min, heating ramp: 30 °C/min to a temperature of 210 °C,
1 °C/min to 213 °C for 40 min, finally 20 °C/min up to 280 °C for 40 min. Helium at a flow
of 1 mL/min, injector temperature of 250 °C. Identification of fatty acids was performed by
mass spectrometry using the Agilent Technologies™ Model 5975 XL Inert Mass
Spectrometer. The identity of each fatty acid was assigned using an external standard
(FAMEs mix, C8:C22, cat no. 18920-1AMP, Sigma-Aldrich) which contained: octanoic
acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid,
pentadecanoic acid, 9-hexadecenoic acid, hexadecanoic acid, cis-9,12, heptadecanoic acid,
octadecadienoic acid, cis-9-octadecaenoic acid, heptadecanoic acid, eicosanoic acid, 11-
eicosenoic acid.
Minerals analysis
For mineral extraction, honey (1 g) was digested in digester tubes with a nitric acid solution
(5%) in a 1:10 (p:v) ratio. The tubes were placed in a Kjeldahl digester (Speed Digester K-
439, Büchi, Flawil, Switzerland) and digested at 170 °C for 2 h until an almost clear solution
was obtained. This solution was filtered and later transferred to a 50 mL volumetric flask and
diluted with 5% HNO3 to finally be injected. The determination was performed with an
Agilent MP 4100 MP-AES (Santa Clara, California, USA) consisting of a One Neb inert
nebulizer, a double-pass glass cyclonic spray chamber, and a charge-coupled detector (CCD)
of solid state. The plasma gas flow was 20 L/min and the makeup gas flow 1.5 L/min. A
calibration curve was made from a mixture of 27 elements (Ag, Al, As, B, Ba, Be, Ca, Cd,
Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, Pb, Sb, Se, Si, Sr, Ti, Tl, V, Zn) with eight points
(concentrations: 100, 75, 50, 25, 10, 7.5, 5.0 and 1 ppm). The coefficient of determination
was greater than 0.98 for each element. The conditions of the equipment for the analysis
were: capture time 13 sec, plasma stabilization time with sample aspiration 15 sec, reading
time 3 sec (reading in triplicate) and washing time 20 sec.
Microbiological analysis
The count of total mesophilic aerobic bacteria and molds and yeasts was carried out by
weighing 1 g of honey that was mixed with 9 mL of PBS buffer. Subsequently, serial
dilutions were made until the 10-9 dilution was obtained. Finally, 1 mL of each dilution was
seeded in Petri dishes and plate count agar (Difco™, BD Detroit US) and PDA agar (potato
329
dextrose agar, BD™ Difco™ plate count agar) for aerobics were poured bacteria and molds
and yeasts, respectively. Finally, they were incubated for 48 h and 5 d to 35 °C, and the
colonies were counted.
Treatment and analysis were performed in triplicate, and the values were expressed as mean
± SD (standard deviation). All data were analyzed using one way of variance (ANOVA),
followed by a Tukey’s test with a significance level of 5% (P<0.05) using Minitab 16
statistical software (Minitab Inc. State College, PA, USA)(6).
Results
Physicochemical property analysis
Table 1 shows the physicochemical properties of honey stored in different packages. The
moisture content of the analysed samples varied from 20.60 to 23.40 %, while the water
activity varied from 0.663 to 0.675 after 2 yr of storage in the different treatments. Honey
stored in a container with an anaerobic valve showed the greatest decrease in moisture
content (20.60 %) and in water activity (0.667) compared to the control treatment, followed
by honey stored in a transparent plastic container (moisture content: 22.80 %, aw= 0.675).
High moisture content is related to the environment in which the flowers from which the bees
collect nectar are found. In addition, it must be considered that honey obtained from stingless
bees contains a greater amount of water, so the effect that the type of container and its
permeability has a greater effect on its stability compared to commercial honey obtained from
Apis mellifera(3,5).
The color parameters of the honey exhibited slight changes during storage; these changes
were reflected in the chroma values and in the total color change. The sample stored in
transparent containers (T4) exhibited a greater total color change (8.08).
The pH of the samples varied slightly from 3.23 to 3.66. The values of pH obtained in the
samples were in the range reported for this type of honey(11). The total acidity of the samples
stored in different containers (T1-T4) ranged from 85.66 to 87.33 meq/kg. The samples
stored in the different types of containers (T2–T4) were not significantly different from each
330
other but were different from the control sample (73.66 meq/kg). The acidity values for
treatments T1–T4 were similar to those reported for stingless bee honey of 85 meq/100 g(12).
The hydroxymethylfurfural (HMF) values of the samples stored in different containers (4.00–
4.78 mg/kg) were not significantly different from those for the control treatment (4.09
mg/kg).
Antioxidant activity analysis
Table 2 shows that the content of total phenolic compounds, vitamin C and antioxidant
compounds of honey stored in different containers were not significantly different (P>0.05)
from that of the control treatment, except for vitamin C and DPPH radical inhibition. The
content of total phenolic compounds varied from 12.55 to 14.31 mg GAE/100 g honey and
that of vitamin C from 86.86 to 114.17 mg AA/g. Consistent with these values, the
antioxidant activity determined by the DPPH radical scavenging activity presented high
inhibition values (75.57–94.70 %). Similarly, the range of values determined by the FRAP
(2.23-3.70 mg TE/g) and ABTS (0.61-1.13 mg TE/g) tests for the different types of
containers show that honey contains compounds with a high capacity to reduce ferric ions
and that they are stable during storage. These results are consistent with those reported for
other types of honey(13) and the opposite to those reported for honey subjected to a
temperature of 22–40 °C after 90 d of storage(14).
Table 2: Antioxidant properties of the honey recently harvested (T0-control), stored after
two years in different containers (T1-T4)
Property T0 T1 T2 T3 T4
91.40 ±
Vitamin C, mg AAE/g 87.50±8.26a 86.86±1.02a 114.17±2.16d 106.64±1.35c
5.24a,b
Total phenolic compounds, 12.55 ±
13.46±3.19a 14.06±3.70a 11.82±3.50a 14.31± 2.63a
mg GAE /g 3.24a
87.25 ±
DPPH inhibition, % 94.05±2.69c 75.57±5.65a 94.70±1.88c 84.71±4.65b
2.65b
2.97 ±
FRAP, mg TE/g 3.70±1.87a 2.94±1.05a 2.23±0.98a 2.94±1.60a
0.95a
0.68 ±
ABTS, mg TE/g 0.61±0.22a 0.72±0.31a 0.69±0.24a 1.13±0.76a
0.18a
The values are shown as the mean ± SD (n=3). AAE: Ascorbic acid equivalents. GAE: Gallic acid
equivalents, TE: Trolox equivalents.
Different letters in each row indicate significant differences (P<0.05).
331
Phenolics identification and quantification by UPLC-MS
Table 3 shows the analysis of phenolic compounds present in freshly harvested honey and
honey stored in different containers. A total of 17 phenolics plus two precursors (shikimic
acid and L-phenylalanine) were identified in the honey stored in the different containers.
Shikimic acid (35511–38504.90 µg/g dry extract), 4-hydroxybenzoic acid (2781.36–2996.87
µg/g dry extract), 4-hydroxyphenylacetic acid (1685.49–2294.62 µg/g dry extract) and L-
phenylalanine (2917.68–3004.45 µg/g dry extract) were the major compounds in the samples.
No significant differences (P>0.05) were found in most of the phenolics and precursors of
the samples stored in the different containers after 2 yr of storage. The phenolics gentisic
acid, 4-hydroxyphenylacetic acid, p-anisic acid and the precursor shikimic acid exhibited
significant differences (P<0.05) in the samples stored in different containers, mainly in T4
(honey stored in a transparent plastic container). The profile of phenolic compounds found
was similar to that reported for stingless honey by other authors(8), however, variations were
found in relation to concentration, these differences in concentration have been attributed to
floral and geographical variation and a collection time(8).
Volatile compounds
Table 4 shows that 18 volatile compounds were found in honey in the different treatments,
ethyl acetate (20.20–30.24 %), cis-linalool oxide (30.05–34.73 %), trans-linalool oxide
(12.97–15.75 %), and 1,5,7-octatrien-3-ol, 3,7-dimethyl (12.55-14.67 %) were the major
compounds, representing approximately 50 % of the volatile compounds present in the
samples. Alcohol derivatives were the predominant ones found in honey during storage.
Table 4: Volatile compounds (%) determined in the honey recently harvested (T0-control),
stored after two years in different containers (T1-T4)
N Compound name RT (min) T0 T1 T2 T3 T4
1 Ethyl acetate 5.46 - 20.20b 22.25b 30.24c 28.67c
2 3-Methyl butanal 5.66 - - - - -
3 Hexane, 3 methyl 7.46 - 1.79a 1.77a 2.18b 1.91b
4 2-Hexene, 3 methyl 8.64 - 0.240b 0.236b 0.272c 0.16a
Propanoic acid, 2-hydroxy-,
5 ethyl ester 8.83 8.85c 1.49a 1.54a 1.78b 1.66b
6 Furfural 9.60 - 1.71b 1.87b 0.96a 0.68a
7 D-Limonene 10.90 0.37 - - - -
8 2-Heptanal acetate 10.93 1.38 - - - -
9 Lilac alcohol B 11.05 - 0.14b 0.15b 0.110a 0.112a
10 Lilac alcohol C 11.19 0.09a 0.11a 0.10a 0.09a 0.09a
332
11 Benzaldehyde 11.68 0.87a 1.76b 1.98b 1.99b 2.47c

12 trans-γ-Caryophyllene 11.89 18.72 - - - -
13 Benzeneacetaldehyde 12.53 - 6.93b 6.19b 3.24a 3.70a
14 cis-Linalool oxide 12.71 48.07c 34.09b 34.73b 30.05b 31.51b
15 trans-Linalool oxide 12.90 21.648b 15.75a 14.81a 12.97a 13.44a
16 1,5,7-Octatrien-3-ol, 3,7-dimethyl 13.11 - 13.67a 12.55a 14.67a 13.44a
17 Nerol oxide 13.67 - 1.52c 1.25b 0.85a 1.51c
18 Linalool oxide 14.00 - 0.61a 0.62a 0.69a 0.69a
Results are expressed as the mean ± SD (n=3).
RT= retention time.
ab
Different letters in the same row are significantly different (P<0.05). --Not present.
Fatty acids present in the honey
Analysis of the hexane extract of the honey samples revealed the presence of eight fatty acids
(Table 5). Hexadecanoic acid (31.12–49.65 %), octadecanoic acid (21.48–26.86 %) and cis-
9-octadecadienoic acid (14.31–40.04 %) were the major compounds found in the different
stored samples.
Table 5: Relative area (%) of fatty acids in the hexane extract in the honey recently
harvested (T0-control) and stored after two years in different containers (T1-T4)
Compound name RT (min) T0 T1 T2 T3 T4
Decanoic acid 6.42 - 0.47d 0.25b 0.22b 0.19a

Dodecanoic acid 8.34 29.72c 1.96a 7.02b 2.27a 1.90a
Tetradecanoic acid 10.49 1.91c 2.07b 1.15a 2.48b 1.19a
9-Hexadecenoic acid 12.21 - 0.65a 0.75a 0.55a 0.68a
Hexadecanoic acid 12.41 22.27a 43.08c 34.33b 49.65c 31.12b
cis-9,12, Octadecadienoic acid 13.79 1.41a 2.96b 3.24b 3.71b 3.41b
cis-9-Octadecaenoid acid 13.83 15.90a 22.36b 32.23c 14.31a 40.04d
Octadecanoic acid 13.98 28.80b 26.47b 21.44a 26.86b 21.48a
RT= retention time.
abcd
Different letters in the same row are significantly different (P<0.05). -Not present.
Mineral content analysis
The mineral content remained constant during storage, and it descended in the following
order: K > Mg > Ca > Na > Si, for honey stored in the different containers (Table 6). The
333
concentration of As, Be, Cd, Mo, Ni, Pb, Sb, Ti, Tl and V was similar to that reported by
Villacrés-Granda et al(2). K (109.36–125.68 mg/100 g DW) and Mg (31.60–100.49 mg/100
g DW) were found in higher concentrations compared to the other minerals present.
Potassium was the majority mineral, representing a third of the total content and exceeding
that of other minerals by approximately 10 times. No statistically significant differences were
found for the minerals investigated in most samples evaluated during storage.
Table 6: Mineral and trace elements (mg / 100 g DW) in the honey recently harvested (T0-
control) and stored after two years in different containers (T1-T4)
Mineral T0 T1 T2 T3 T4
Al 1.378±0.01a 1.352±0.04a 1.451±0.10a 1.221±0.07a 1.235±0.00a
As - - - - -
B 1.670±0.02b 1.208±0.03a 1.456±0.10ª,b 1.765±0.09b 2.089±0.00c
Ba - - - - -
Be - - - - -
Ca 76.090±0.10b 43.172±0.5a 95.441±0.90c 96.662±0.95c 61.210±1.00b
Cd - - - - -
Co - - - - -
Cr - - - - -
Cu 0.578±0.07b 0.476±0.03b 0.554±0.03b 0.753±0.02c 0.159±0.01a
Fe 0.970±0.08b 0.740±0.02b 1.179±0.09c 0.815±0.03b 0.613±0.06a
K 115.90±3.00a 125.686±2.76b 109.97±2.00a 109.361±3.09a 122.840±2.67b
Mg 84.32±1.00b 100.490±1.98b 31.608±1.65a 96.608±1.49b 87.096±1.34b
Mn 0.11±0.00a 0.123±0.00a 0.14±0.00a 0.113±0.00a 0.189±0.00b
Mo - - - - -
Na 28.65±1.02b 35.794±1.17c 20.562±1.07a 26.655±0.45b 21.470±0.98a
Ni - - - - -
Pb - - - - -
Sb - - - - -
Se 4.870±0.98a 4.589±0.76a 4.892±0.49a 4.891±0.29a 5.494±0.57a
Si 45.88±1.00a 46.798±1.06a 40.195±0.30a 53.352±0.69a 51.485±0.70a
Sr - - - - -
Ti - - - - -
Tl - - - - -
V - - - - -
Zn 0.678±0.05a 0.814±0.06b 0.972±0.05b 0.349±0.06a 0.995±0.04b
These values are the average of three determinations.
ab
Different letters in the same row are significantly different (P<0.05). -: no detectable.
334
Microbiological analysis
The results showed a higher number of microorganisms in the initial samples for total aerobic
mesophilic bacteria (1.50 x 102 CFU/g) and molds, and yeast (2.30 x 102 CFU/g), compared
to the samples in different containers stored for two years (Table 7). The results obtained for
the analysis of microorganisms for the samples at the beginning of storage ranged from 0.78
x 102 - 0.98 x 102 CFU/g of sample for aerobic mesophiles and 0.03 x 102- 0.32 x 102 CFU/g
of sample for molds, and yeasts.
Table 7: Total aerobic mesophilic bacteria, and molds, and yeast present in samples at the
beginning and after two years of storage in different containers
Treatment code Total Aerobic mesophilic count Total molds and yeast
(CFU/g) count (CFU/g)
T0 (initial storage) 1.50 x 102a 2.30 x 102a
T1 0.89 x 102b 0.32 x 102b
T2 0.98 x 102b 0.15 x 102b
T3 0.95 x 102b 0.08 x 102b
T4 0.78 x 102b 0.03 x 102b
These values are the average of five determinations (n=5).
ab
Different letters in the same column are significantly different (P<0.05).
Discussion
Determination of the physicochemical properties, such as moisture, pH, acidity, and Brix
degrees allow the evaluation of honey quality. The moisture of honey favors the growth of
bacteria and fungi present in honey. The range of moisture values obtained for the different
treatments was consistent with those reported for the honey from Ecuadorian stingless bees(2).
It was found that the sample stored in the container with an escape check valve showed a
significant reduction in moisture content compared to the initial treatment, which it is
possibly due to the gases produced by fermentation dragging the moisture present into the
headspace of the container, preventing the moisture from returning. At the same time, color
changes in honey are related to its botanical origin, mineral content, the content of phenolic
compounds, antioxidant properties, room temperature, and storage time. The change in honey
stored in the transparent container is possibly because light could affect the components of
the honey such as carotenoids and flavonoids(15)..
The values of pH and the total acidity, play an important role in the quality of the honey(11).
The acidity values show that this honey stored for 2 yr has a higher acidity. The increase in
335
acidity value during storage may be related to the fermentation of honey and to its
antimicrobial properties, but it may also result in an undesirable vinegar taste because of
acetic acid production. The acidity of honey is related to the glucose content. Glucose is
converted by the action of the enzyme D-glucose oxidase into gluconic acid. This process
produces hydrogen peroxide which is a component of the antimicrobial action of honey(16).
The production of acids occurs not only by enzymatic action but also by fermentation of the
microorganisms present in the matrix. Increased acidity may also be associated with the
transformation of sugars from honey into alcohols and then into organic acids by osmophilic
yeasts. It is also necessary to consider that when the moisture content is high, the bacteria
grow and ferment the sugars, producing compounds such as acetic acid that can affect the
taste of honey(17).
A very important quality factor in honey is the HMF concentration, since it is an indicator of
the quality, freshness, and aging of honey. Under conditions such as processing or aging,
mainly influenced by temperature fluctuation, pH, storage conditions, and floral origin, may
help bring about its presence(18).
The antioxidant activity of honey depends on its floral origin and the processing conditions
and is closely related to the chemical compounds it possesses. The components of honey –
phenolic compounds, flavonoids, and phenolic acids, as well as chlorophyll, carotenoids, and
vitamin C – contribute to its antioxidant activity(15), coupled with the fact that the antioxidant
properties are related to its color and the moisture content.
The antioxidant activity of honey is due, among other factors, to the presence of phenolic
compounds, which are produced in plants as a protection system and are entrained in the
nectar extracted by bees. UPLC analysis revealed the presence of shikimic acid, 4-
hydroxybenzoic acid, 4-hydroxyphenylacetic acid and L-phenylalanine, among others. These
compounds confer antioxidant activity to honey since they possess delocalized electrons,
which cause free radical scavenging activity(19). The radical scavenging activity of phenolics
mainly depends on the number and position of hydroxyl groups in the molecules. The
presence of these compounds is explained by the fact that shikimic acid is a precursor of
aromatic metabolic intermediates, within which are flavonoids such as luteolin(20). The
presence of these phenolic compounds may suggest possible anti-inflammatory and
antimicrobial activity, among other properties. Consistent with the analysis of total phenolic
compounds, the concentration of most of the quantified phenolic compounds decreased in
the sample subjected to heat treatment, compared to the control. This helps to explain the
decrease in antioxidant activity.
Identifying volatile compounds plays a crucial role in assessing the quality of honey. These
compounds, linked to flower nectar, geographical origin, and overall stability, offer insights
into the honey's unique characteristics(21). When honey is stored in various containers, there
336
is an increase in volatile compounds over the initial storage day. Notably, esters, aldehydes,
ketones, and alcohols become predominant after 2 yr, contributing significantly to the honey's
odor and flavor. The appearance and rise of specific volatile compounds may be linked to the
fermentation process, with packaging type influencing oxygen availability and anaerobic
respiration enhancement. Moisture content further affects fermentation, favoring the
production of alcohol, carbon dioxide, and acetic acid, all influencing the concentration of
volatile compounds in honey(17).
Free fatty acids, akin to volatile compounds, serve as lipid markers reflecting the floral origin
of honey and can be crucial authenticity indicators(22). Changes in the concentration of certain
volatile compounds in honey stored in containers are likely due to the container material's
permeability. Plastics, in particular, may retain some volatile compounds, facilitating their
transfer between honey and the container material. This involves the adsorption or retention
of volatile compounds in honey, causing shifts in their concentration. Additionally, some
volatile compounds might be lost or absorbed, affecting the honey's aromatic profile and
consequently altering its taste and aroma. Fatty acids such as hexadecanoic acid increased in
proportion, while the proportion of dodecanoic acid decreased, and others like decanoic acid
and 9-hexadecenoic acid emerged during storage. These changes may be related to variations
in water activity and the permeability of different containers used for storage. Another factor
is that honey crystallization can impact the mobility and availability of fatty acids,
influencing their proportion.
Mineral content is another quality factor for honey. The analysis shows that the type of
minerals and their concentration does not vary during storage in the evaluated containers,
and it was similar to those reported by other authors regarding other types of honey;
Consistent with other reports, potassium was the most abundant mineral, this is considered
the most quantitatively important mineral in the honey, accounting for around 50 % of the
total mineral content. The presence of Al, Ba, Si, and Co is mainly since these minerals are
naturally present in the environment, demonstrated that the honey is a very good
environmental indicator so reflects the content of toxic elements in the surrounding water,
soil, and air(23). Honey can contribute to the diet with elements such as Mg, Ca, and K. Mg
and K are important micronutrients for the human body since they are involved in many
physiological processes and are essential for the maintenance of the normal function of cells
and organs, by which they make an important contribution to health(24). These results are also
consistent with those reported in a study on honey from stingless bees from Brazil, where it
was found that these minerals are the most important quantitatively(8).
Honey can contain microorganisms from different resources, such as pollen digestive tracts
dust, air soil, and nectar, or due to handling and processing. The presence of these
microorganisms can affect the quality of honey during storage, so an analysis of the total
count of aerobic microorganisms and molds, and yeasts were performed at the beginning and
337
end of storage in different containers. These values were below the limit reported by other
authors and that established for Apis mellifera honey, which may be due to proper handling
in harvesting and the presence of phenolic compounds, organic acids, and other bioactive
compounds present in the honey that has an inhibitory effect on this type of microorganism.
The concentration of total aerobic microorganisms and molds and yeasts decreased in honey
during storage, which is consistent with the reduction in water activity and moisture. This
could be attributed to various factors such as sugar crystallization or water evaporation due
to plastic permeability. The decrease in microorganism concentration is a positive factor that
ensures the quality of honey during its storage.
In this study, a comparison was made between plastic containers used commercially, since
the use of other types of containers, such as glass or metal, are more expensive for the
producer. The study demonstrated that storing honey in traditional plastic containers (high-
density polyethylene and polyethylene terephthalate) and using certain traditional
methodologies provide significant differences in the moisture content of honey during
storage, with the moisture content being minor in honey stored in the container with an escape
check valve (T3). It was also found that, in general, storage for 2 yr does not produce major
changes in the physicochemical properties and in the content of phenolic compounds, which
are associated with a decrease in antioxidant properties and volatile compounds that together
can affect the honey quality. Furthermore, storage had a positive effect on the microbiological
analysis of the honey. Finally, the evaluation of these parameters suggests that treatment T3
would be the most suitable for storing honey since it presented a total color change of less
than 3, an important quality parameter for consumers.
Acknowledgments
The Authors would like to thank Company Chasseurs De Saveurs S.A. C.V. from Coatepec
Veracruz for providing the honey. Also, we are grateful to the Mexican Association of Edible
Forests.
338
Disclosure statement
No potential conflict of interest was reported by the authors.
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Table 1: Physicochemical properties of the honey recently harvested (T0-control), stored after two years in different containers (T1-
T4)
Properties
T0 T1 T2 T3 T4
b ab b a
Moisture, gH20/ 100 g d.m. 25.70 ± 0.47 23.23 ± 0.28 23.40 ± 0.09 20.60 ± 1.33 22.80 ± 0.39a
Water activity (25 °C) 0.733 ± 0.006b 0.663 ± 0.001a 0.671 ± 0.007a 0.667 ± 0.006a 0.6759 ± 0.003a
L* 18.33 ± 0.37a 20.80 ± 1.85a 21.67 ± 3.59a 18.27 ± 0.43a 25.64 ± 2.55b
a* 2.84 ± 0.75a 6.66 ± 0.64b,c 6.27 ± 0.87b,c 5.40 ± 0.64b 8.91 ± 1.82c
b* 5.39 ± 0.23a 7.69 ± 2.34b 8.41 ± 4.58b 4.63 ± 0.08a 7.26 ± 3.45b
Chroma 6.11 ± 1.53a 10.17 ± 2.21a 10.49 ± 4.22a 7.11 ± 0.51a 10.02 ± 3.54a
Total color change - 4.81 ± 0.54b 5.32 ± 0.38b 2.69 ± 0.45a 8.08 ± 0.37c
Browning index - 0.38 ± 0.09a 0.42 ± 0.05a 0.42 ± 0.03a 0.39 ± 0.02a
pH 3.66 ± 0.05a 3.23 ± 0.05a 3.26 ± 0.05a 3.40 ± 0.06a 3.43 ± 0.05a
a a a a
Brix (º) 71.66 ± 0.57 71.90 ± 0.55 72.03 ± 0.55 72.10 ± 0.26 72.76 ± 0.37a
Electric conductivity, mS/cm 293.33 ± 11.54b 320.25 ± 20.00 a 293.33 ± 15.27b 303.33 ± 5.77b 296.00 ± 15.16b
Density, g/mL 1.40 ± 0.02b 1.36 ± 0.01a 1.36 ± 0.00a 1.36 ± 0.02a 1.36 ± 0.01a
Hydroxymethylfurfural, mg /kg 4.09 ± 0.53a 4.33 ± 0.20a 4.00 ± 0.39a 4.23 ± 0.22a 4.78 ± 0.52a
Titratable acidity, meq/kg d.m. 73.66 ± 0.57a 87.33 ± 6.65b 87.33 ± 0.57b 87.33 ± 2.08b 85.66 ± 1.15b
Data represent the average of three replicates or measurements ± standard deviation.
abcd
Different letters in the same row indicate significant differences (P<0.05).
-- Not present.
342
Table 3: Phenolic compounds (µg/g dry extract) of the honey recently harvested (T0-control), stored after two years in different
containers (T1-T4)
T0
Phenolic compound T1 T2 T3 T4
Gallic acid 126.78 ± 10.00a 136.17 ± 16.20a 137.88 ± 4.91a 143.32 ± 5.97ª 135.25 ± 8.16a
4-Hydroxybenzoic acid 2996.87 ± 135.76a 2847.66 ± 207.18a 2906.04 ± 118.78a 2934.22 ± 105.08a 2781.36 ± 146.05a
Protocatechuic acid 637.87 ± 21.79a 660.19 ± 22.55a 666.90 ± 13.40a 648.85 ± 24.07a 634.87 ± 34.48a
Vanillic acid 654.89 ± 38.33a 612.57 ± 59.27a 645.12 ± 39.07a 677.20 ± 29.77a 633.24 ± 27.82a
Gentisic acid 312.90 ± 89.87a 312.82 ± 36.07a 280.94 ± 29.34a 294.31 ± 10.82a 541.69 ± 48.62b
4-Hydroxyphenylacetic acid 2198 ± 129.88a 2294.62 ± 115.55b 1702.48 ± 195.65a 2036.27 ± 133.77b 1685.49 ± 103.27a
Sinapic acid 1677.33 ± 87.99a 1677.70 ± 81.08a 1636.52 ± 23.01a 1663.18 ± 57.32a 1565.35 ± 80.53a
Salicylic acid 1244.11 ± 199.55a 1240.45 ± 409.54a 1053.87 ± 54.18a 1074.40 ± 30.10a 1098.68 ± 32.97a
p-Anisic acid 399.97 ± 29.73b 317.57 ± 26.29ª 455.27 ± 39.95c 384.97 ± 38.77b 456.20 ± 52.69c
Rosmarinic acid 297.45 ± 12.95a 275.61 ± 20.76a 220.68 ± 58.40a 248.44 ± 16.14a 276.69 ± 67.38a
4-Coumaric acid 301.86± 38.26a 291.65 ± 37.60a 320.81 ± 13.12a 323.82 ± 7.57a 271.41 ± 53.92a
Trans-cinnamic acid 139.87 ± 9.41a 124.02 ± 7.43a 123.18 ± 4.77a 122.89 ± 3.74a 127.99 ± 3.83a
Luteolin 289.56 ± 29.44a 273.83 ± 38.24a 325.69 ± 104.80a 315.66 ± 45.43a 320.41 ± 62.01a
Scopoletin 689.85 ± 58.33ª 673.76 ±72.47ª 720.55 ± 26.69ª 740.75 ± 17.46ª 619.54 ± 120.49ª
Ferulic acid 132.63 ± 27.82a 127.52 ± 21.48a 136.69 ± 20.29a 150.77 ± 40.86a 111.18 ± 27.34a
Caffeic acid 478.86 ± 96.43a 410.22 ± 122.14a 570.36 ± 24.02a 590.65 ± 27.56a 452.59 ± 120.71a
Shikimic acid 36986.87±3999.20a 38200.95±3974.40a 37735.48±2688.39a 38504.90±2817.73a 35511.01±5741.62a
Vanillin 97.45 ± 6.98a 96.17±5.16a 97.36 ± 16.74a 85.01 ± 12.63a 98.26 ± 12.83a
L-phenylalanine 2930.99 ± 120.89a 2934.82±100.09a 2886.78 ±115.55a 3004.45 ± 130.22a 2917.68 ± 118.89a
ab
Different letters in the same row are significantly different (P<0.05).
343
Article
A novel effect of aqueous extract of Pimpinella anisum seeds on ticks of

domestic dogs (Canis lupus familiaris)
William Fernando Várguez-Tec a,c
Sara Luz Nahuat-Dzib b
Julia Cano-Sosa c
Lorena Reyes-Vaquero d
Edgar E. Lara-Ramirez e
Benjamín Abraham Ayil-Gutiérrez f
Angel Virgilio Domínguez-May a*
a
TecNM. Instituto Tecnológico Superior del Sur del Estado de Yucatán (ITSSY). Carretera
Muna-Felipe Carrillo Puerto, tramo Oxkutzcab-Akil Km 41+400, Oxkutzcab, 97880,
Yucatán, México.
b
TecNM, Campus Mérida. Departamento de Ingeniería Química y Bioquímica. Yucatán,
México.
c
Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C.
Subsede Sureste. Yucatán, México.
d
CONAHCYT-Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de
Jalisco A.C. Subsede Sureste. Yucatán, México.
e
Instituto Politécnico Nacional. Centro de Biotecnología Genómica, Laboratorio de
Biotecnología Farmacéutica. Tamaulipas, México.
f
CONAHCYT, Instituto Politécnico Nacional, Centro de Biotecnología Genómica,
Biotecnología Vegetal. Tamaulipas, México.
*Corresponding author: adominguez@suryucatan.tecnm.mx
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Abstract:
Synthetic pesticides used to combat ticks lose their effectiveness against certain species and
can affect human health. The present study evaluated in vitro and in vivo the effect of the
aqueous extract of Pimpinella anisum (P. anisum) seeds against Rhipicephalus sanguineus
and Ixodes affinis, domestic dog ticks. In the in vitro evaluations, concentrations of 1.25, 2.5,
5, 10, 25, 50, 75 and 100 % of the aqueous extract of P. anisum were applied directly to ticks.
The concentrations that had the highest effectiveness in immobilization were 50 %, 75 % and
100 %, but the latter caused immobilization for a longer time (55.89 ± 0.16 min). In the in
vivo evaluation, the concentrated aqueous extract was applied to ticks attached to the skin of
domestic dogs. Amitraz, a commercial tickicide, was used as a positive control. Both the
concentrated aqueous extract and Amitraz caused 100 % of tick detaching. Nonetheless,
concentrated aqueous extract of P. anisum seeds was more effective in reducing the average
time of tick detaching (60.81 ± 3.17 min) compared to the commercial tickicide Amitraz
(145.12 ± 15.97 min). This research suggests that the p-anisaldehyde identified in the
aqueous extract could be linked to the immobilization and detaching of R. sanguineus and I.
affinis from domestic dogs, suggesting that this extract could be used as a biopesticide to
control ticks in domestic dogs.
Keywords: Biopesticide, Ticks, Immobilization, Domestic dogs, In vitro, In vivo.
Introduction
Ticks of the family Ixodidae are hematophagous arthropods of worldwide distribution, which
parasitize various species of mammals, birds, and reptiles. When these ectoparasites feed on
their host (various domestic and wild animals, including humans), they can transmit
pathogenic microorganisms such as bacteria, viruses, protozoa, and helminths(1,2).
Ticks are pests that are considered economically harmful in livestock and other animal
species as they can cause severe anemia and weight loss(3). Dogs that inhabit rural and urban
areas are hosts of the species R. sanguineus. These ticks can be found in tropical and
subtropical regions, adapting to indoor conditions(4). Ixodes affinis is a species that is also
common in dogs and cats worldwide(5).
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The use of synthetic pesticides causes damage to the environment and is a health hazard(6,7);
however, in recent years, alternatives have been sought to control pests without polluting the
environment, one of them is the use of plants with insect or mite control capabilities.
The use of medicinal plants is a practice that has carried out since ancient times and has
contributed to the origin of modern medicine(8). Numerous medicinal plants contain
secondary metabolites and pigments, among other components that are toxic against various
microorganisms. It has been reported that phytochemicals isolated from medicinal plants are
key to the generation of biopesticides; they are also considered less toxic and easily
degraded(9,10). In recent years, the applications of extracts and essential oils from plant species
have become new alternatives as environmentally friendly pesticides, with the purpose of
limiting the use of synthetic pesticides in the agricultural sector(7,11).
Among the plant species used to combat pests is Pimpinella anisum(12), commonly known as
anise, green anise or badian(13), it belongs to the family Umbelliferae, currently called
Apiaceae, and has been used in traditional medicine as a carminative, aromatic, disinfectant
and galactagogue. P. anisum seeds have antimicrobial, antifungal, antiviral, antioxidant and
anticonvulsant activities and muscle relaxing, analgesic, and hypoglycemic effects(14), and
have also been reported to have insecticidal activity(15).The essential oil of the seeds of this
plant species has been shown to have a lethal effect against Tribolium castaneum(12), repellent
activity against adults of Culex pipiens(16) and is toxic against Daphnia magna(17). Recent
studies have shown that P. anisum seed oil has acaricidal activity against Tetranychus
urticae(18), while aqueous and methanolic extracts have antimicrobial activity against
Candida albicans(19) and Escherichia coli(20), respectively. Compounds such as trans-
anethole, methyl chavicol, anisaldehyde, estragole, and γ-hymachalen(14) have been
identified in the seeds of P. anisum, and p-anisaldehyde(14) has been identified in the essential
oil, which is a compound that causes immobilization, repellency, and mortality effects in
insects such as Haematobia irritans irritans (L.) and Musca domestica L., and the response
of its application depends on the stage of development in these insects(21,22,23). According to
the background and effects caused by the species P. anisum against other insects, this study
aimed to evaluate the effect of the aqueous extract of P. anisum seeds against ticks that
commonly attack domestic dogs, in order to reduce the use of synthetic insecticides that are
not friendly to the environment.
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Biological material
The seeds of P. anisum were obtained from the commercial house Granos y semillas Yael,
located at Calle 52 No. 540 and 67, Centro, Mérida, Yucatan, which were dehydrated.
Preparation of aqueous extract of P. anisum seeds
The concentrated aqueous extract of P. anisum seeds was obtained from 50 g of seeds, which
were crushed in a manual mill (Del Rey brand) to obtain particles of 1.5 mm in diameter on
average. The seeds were decocted using 12.5 g in 1 L of purified H2O (Bonafont® brand) at
90 °C. Cooking time was 20 min. Finally, the aqueous extract obtained was kept in amber
bottles and preserved under refrigerated conditions at 4 °C until use. Subsequently, the
concentrated aqueous extract was diluted with purified H2O (Bonafont®) to prepare
concentrations of 1.25, 2.5, 5, 10, 25, 50, 75 % and the 100 % concentrated aqueous extract.
Purified H2O was used as a negative control and the commercial compound Combatick®
(12.5 % Amitraz), an insecticidal and acaricide solution, was used as positive control, which
was prepared and applied according to the indications on the product label (2 ml of the
solution per 1 L of H2O).
Toxicity bioassay
To determine the toxicity of the aqueous extract of P. anisum seeds, a toxicity test was
performed on Artemia salina (White Mountain, Great Salt Lake, Utah, USA); the cysts were
incubated in filtered seawater for 24 h, at a temperature of 29 ± 4 °C, with constant
aeration(24).
Bioassays were performed on 24-well plates. Four concentrations (0.0005, 0.05, 5, 500
mg/ml) of the aqueous extract of P. anisum seeds were prepared by serial dilution. Ten
nauplii of A. salina were placed in each well. Filtered sea H2O without extract was used as a
negative control. All treatments were analyzed fivefold. The plates were incubated at 29 ±
4 °C for 24 h; after this time, they were observed under a stereo microscope (SMZ800, Nikon)
and the number of live nauplii was counted. Mortality was considered when no movement
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was observed after 10 sec. Mortality percentage and median lethal dose (LC50) were
calculated. To consider whether the plant extract is toxic, the toxicity criteria proposed by
Clarkson et al(25) were followed: non-toxic when LC50 ˃1,000 μg/mL, low toxicity 500 <
LC50 <1,000 μg/mL, moderate toxicity 100 < LC50 <500 μg/mL, and highly toxic 0 < LC50
<100 μg/mL.
Tick collection on domestic dogs
A total of 270 adult ticks were collected from 10 naturally infested domestic dogs of different
breeds, ages, and sexes from the municipalities of Ticul (20°23′43″N, 89°32′02″W) and
Oxkutzcab (20°18′10″N 89°25′06″W), Yucatan, Mexico; these animals received no previous
treatment. The ticks were placed in glass bottles with perforated lids and stored in the
laboratory at 29 ± 4 °C for 24 h.
In vitro evaluation in ticks
For the in vitro toxicity evaluation, was used the concentrated aqueous extract of P. anisum
seeds and seven dilutions of this same extract (1.25, 2.5, 5, 10, 25, 50, 75 %) and purified
H2O as a negative control. For each of the treatments, 30 ticks and 0.5 ml of the solution were
used; the solution was applied by spraying it on the ticks. After 30 min, the percentage of
immobilized ticks (% I) was calculated using the formula:
% I= (Ni/NT) x 100
Where: % I= percentage of immobilized ticks; Ni= number of ticks immobilized; NT= total
number of ticks treated.
In vivo evaluation in domestic dogs
The concentrated extract was used to evaluate the effect of the aqueous extract of P. anisum
seeds on ticks in domestic dogs. For this test, the positive control was Amitraz, which is a
commercial miticide and insecticide, and purified water of the Bonafont® brand was used as
a negative control. For the evaluation, 12 domestic dogs of different breeds, sexes and ages
that presented problems with the presence of ticks on their body were included and they were
divided into three groups of four canine specimens each for the application of the product to
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be evaluated and the count of the number of ticks present in each individual (Tables 1, 2 and
3). For each dog, a volume of 0.5 ml of concentrated extract of P. anisum was used and
applied to the left ear, tail, armpits and on the back of the animal where the ticks were.
Amitraz was applied in accordance with the commercial producer’s instructions. To
determine the time it took for each treatment to detach the ticks, we waited until the last tick
became detached by application area.
Table 1: Breeds of dogs infested with ticks in different areas, treated with the negative
control (purified H2O)
Number of ticks by area
Total number
Left ear Tail Armpits Back of ticks
Breed
Chihuahua 6 12 5 12 35
Maltese dog 5 15 11 11 42
German
5 15 11 11 42
shepherd
Mixed breed 13 7 12 3 35
Table 2: Breeds of dogs infested with ticks in different areas, treated with concentrated
aqueous extract of P. anisum
Number of
Left ear Tail Armpits Back ticks by area
Breed
German
7 7 8 9 31
shepherd
Table 3: Breeds of dogs infested with ticks in different areas, treated with the positive
control (Amitraz)
Number of
Left ear Tail Armpits Back ticks by area
Breed
German
7 7 8 9 31
shepherd
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Identification of tick species in domestic dogs
The identification of the 100 randomly selected ticks, treated in the laboratory, as well as
those that became detached from domestic dogs after the application of the aqueous extract
and Amitraz, was by morphological characteristics, the shape of the hypostome, capitulum,
and pedipalp of each of the ectoparasites. The ticks were placed in a 70 % ethanol solution
for 8 min, during which time the ticks remained motionless. They were then observed with a
stereo microscope (Stemi 305, Zeiss). The identification of the species was carried out using
the images of ticks published by Lord CC(26) and Solís Hernández(27) as a reference.
Identification of p-anisaldehyde in the aqueous extract of P. anisum
To determine the presence of p-anisaldehyde, 5 mL of the concentrated extract was used,

which was filtered through 0.22 μM nylon membranes (Thermo Scientific Cat. No. 726-
2520). Subsequently, the solution was frozen in a deep freezer for 3 h (Thermo Fisher
Scientific Inc., Model-TSX400D, No. 144DT0B01A) and lyophilized (LABCONCO-No. cat
77540-00) for 24 h. The lyophilized product was resuspended in 5 mL MeOH (TEDIA-
MS1922-001), stirred (Vortex-genie-Serial No G-560) and centrifuged again for 5 min
(Galaxy mini centrifuge – Serial No. 1204), obtaining the supernatant. For sample analysis,
2 μL of the supernatant was injected into a gas chromatograph coupled to mass spectrometry
(Agilent 7890A, Wilmington, Delaware USA), equipped with a hydrogen flame ionization
detector for compound identification. Compound separation was performed with an HP5MS
column (Agilent Technologies, 30 m × 0.250 mm, 0.25 μm, Cat. No. 190915-435, USA).
The injector temperature was set to 250 °C and the initial oven temperature was 70 °C for 3
min, increasing by 5 °C/min to 250 °C.
The data obtained in the in vitro and in vivo treatments were analyzed with one-way ANOVA
with the Holm-Sidak test using the Sigma Plot version 12 program.
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Results
Toxicity bioassay
The toxicity results of the aqueous extract of P. anisum seeds showed that at a concentration
of 500 mg/mL, the highest mortality percentage (14.3 %) was obtained, while with the other
concentrations no mortality was observed (Table 4). The median lethal dose (LC50) was 4645
μg/mL. With the toxicity and LC50 results obtained and, according to the toxicity criteria
proposed by Clarkson et al(25), the aqueous extract of P. anisum seeds is considered to be
non-toxic for use in canines.
Table 4: Percentage of mortality of Artemia salina in the presence of the aqueous extract of
P. anisum seeds
Concentration (mg/mL) Mortality (%)
Control 0.0
0.0005 2.0
0.05 0.0
5 0.0
500 14.3
Immobilizing effect of aqueous extract of P. anisum seeds in vitro
In the in vitro treatment, the aqueous extract of P. anisum seeds was sprayed on the ticks,
and after 30 min it was observed that the concentration of 1.25 % of the aqueous extract had
no effect on tick mobility. At the concentration of 2.5 %, an immobilization percentage of
16.7 ± 5.8 was observed. In the higher concentrations, the percentage of immobilization
increased significantly; with 25 %, more than 96 % of the total number of ticks evaluated
were immobilized, and with concentrations of 50, 75 and 100 % of extract, 100 % of the
treated ticks were immobilized, with no significant difference observed in the last four
treatments (Table 5).
The average time of tick immobilization differed depending on each concentration of the
aqueous extract of P. anisum seeds. It was observed that the 2.5 % aqueous extract caused
the ticks to remain motionless for 3.00 ± 0.04 min; with 10 % of the aqueous extract the time
was 14.9 ± 0.21 min; with 50 %, the immobilization time was 45.14 ± 0.07 min. The 100 %
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concentrated extract caused the ticks to remain motionless for more than 50 min (55.89 ±
0.16 min). Immobilization time was statistically different between each treatment (Table 5).
Table 5: Percentage of immobilized ticks and immobilization time caused by the effect of
the aqueous extract of P. anisum seeds under in vitro conditions
Aqueous extract concentration Immobilized ticks Immobilization time
(%) (%) (min)
0 0a 0a
1.25 0a 0a
2.5 16.7±5.8b 3±0.04b
5 43.3±5.8c 10.07±0.13c
10 60±00d 14.9±0.21d
25 96.7±5.8e 28.07±0.07e
50 100±00e 45.14±0.07f
75 100±00e 50.14±0.07g
100 100±00e 55.89±0.16h
abcd
Different letters, placed as a superscript, indicate significant differences. One-way ANOVA (P<0.005).
In vivo effect of aqueous extract of P. anisum seeds on ticks attached to

domestic dogs
In order to demonstrate the effect of P. anisum seeds on ticks attached to domestic dogs, the
concentrated aqueous extract was evaluated, which, under in vitro conditions, caused ticks to
be immobilized for longer than dilutions (1.25, 2.5, 5, 10, 25, 50, and 75 %).
In the comparison of the effects of the negative control of purified H20 (Bonafont®), the
aqueous extract of P. anisum seeds and Amitraz as a positive control, it was shown that the
purified water did not detach ticks from the skin of domestic dogs; in contrast, the aqueous
extract of P. anisum seeds caused all ticks to detach in an average time of 60.81 ± 3.17 min,
while Amitraz required 145.12 ± 15.97 min, observing that the average time of tick
immobilization of these treatments was statistically different (Table 6). After the detachment
of the ticks from the domestic dogs, the concentrated aqueous extract maintained the effect
of immobilizing the ticks in the ground for 14.625 ± 1.36 min. On the other hand, Amitraz
did so for 44.93 ± 2.38 min (Table 7), with a significant difference in the time of
immobilization of ticks on the ground.
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Table 6: Effect of aqueous extract of P. anisum seeds on the detaching time of ticks
attached to domestic dogs
Total time of tick detaching
Treatments Detached ticks (%) (min)
Purified water 0 0
a
Aqueous extract (100%) 100+00 60.813 ± 3.17
b
Amitraz 100±00 145.125 ± 15.97
ab
Table 7: Immobilization time of ticks after detachment in domestic dogs, due to the effect
of the aqueous extract of P. anisum seeds
Average immobilization time of the total
Treatments number of ticks (min)
Purified water 0
Aqueous extract (100%) 14.625 ± 1.36a
Amitraz 44.938 ± 2.38b
ab
Morphology of ticks evaluated
Morphological analyses suggest that ticks that were immobilized in vitro, as well as those
that became detached from domestic dogs when the aqueous extract of P. anisum and
Amitraz were applied, vary in shape and size of the hypostome, pedipalp, capitulum shape,
and color. In all the ticks that were evaluated, four pairs of legs were counted, observing
80 % R. sanguineus and 20 % I. affinis.
Identification of compounds of the aqueous extract of P. anisum seeds
Nine compounds were identified in the aqueous extract of P. anisum seeds: p-anisaldehyde
(4-methoxybenzaldehyde), butanoic acid, benzyl alcohol, and falcarinol (compounds with
antimicrobial activity), phenol (antioxidant property), 2-myristynoyl pantetheine (aromatic
compound), paromomycin (antileishmaniasis property), 10-heptadecen-8-ynoic acid, methyl
ester, (E)- and d-mannose (anti-inflammatory activity) (Table 8).
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Table 8: Compounds identified in the aqueous extract of P. anisum seeds by gas

chromatography coupled to mass spectrometry
N Area
Compound name Formula Reported biological activity
(%)
Immobilizing and repellent
1 4-methoxybenzaldehyde C8H8O2 0.55 effect(21,22,23)
Antifungal activity(28)
2 Butanoic acid C4H8O2 6.31 Antibacterial activity(29)
3 Phenol C6H6O 0.96 Antioxidant property(30)
4 Leishmania amazonensis
Paromomycin C23H45N5O14 0.04
treatment(31)
5 2-myristynoyl
C25H44N2O5S 0.02 Sensory property(32)
pantetheine
6 It inhibits the reproduction of β-
Benzyl alcohol C8H10O2 2.62 hemolytic Streptococcus and
Proteus spp(33)
7 Falcarinol C17H24O 0.49 Antimycobacterial activity(34)
8 10-heptadecen-8-ynoic
C18H30O2 0.03 Anti-inflammatory(35)
acid, methyl ester, (E)-
9 d-Mannose C6H12O6 0.02 Anti-inflammatory(36)
Discussion
In the evaluation of the aqueous extract of P. anisum seeds under in vitro conditions, the main
effect was observed to be the immobilization of ticks in domestic dogs. The percentage of
ticks that were immobilized and the duration of the effect depended on the increase in the
concentrations of the aqueous extract of P. anisum seeds. The effect of tick detachment and
immobilization when P. anisum extract concentrate is applied could be due to the compound
identified as p-anisaldehyde (4-methoxybenzaldehyde).
Likewise, it was shown that the correlation between the aqueous extract and its effect in this
study agrees with published results, Showler and Harlien(21), where they evaluated the
activity of p-anisaldehyde powder at 98 % purity of the sigma brand, observing that by
increasing the concentration of this product from 0.125 to 2.5 %, the number of immobilized
adults of Haematobia irritans irritans (L) increased. Showler and Harlien(22,23) reported that
p-anisaldehyde has lethal and repellent effects on Musca domestica. It has also been shown
that the increase in the concentration of p-anisaldehyde powder causes greater mortality of
354
Amblyomma americanum larvae; nevertheless, the effect it generated was in accordance with
the application technique(37). Considering this background, the present study suggests that
the effect of tick immobilization could be due to the presence of p-anisaldehyde in the
aqueous extract of P. anisum seeds, which the less concentrated it is, the shorter the duration
of its immobilization effect.
When the aqueous extract was applied to ticks attached to domestic dogs, it caused them to
detach, suggesting that the aqueous extract of P. anisum seeds generates immobilization; this
effect causes ticks to detach from the skin of domestic dogs, as does Amitraz (positive control
in this study). Amitraz is a widely used product for the treatment of ticks in domestic animals;
unfortunately, the extensive use of this product has caused certain species of ticks such as
Rhipicephalus microplus to become resistant(38); it is also a product that can cause poisoning
by inhalation and dermal contact(39).
In this research, it was found that, under the applied conditions, the aqueous extract of P.
anisum seeds detaches adult ticks from the skin of domestic dogs by 100 %, having similar
effects to the commercial product Amitraz. This means that the effect generated by this
extract could be related to the concentration used, or the application technique.
Likewise, it was found that the aqueous extract of P. anisum seeds formulated with 50 g of
seeds per liter of purified water is not toxic to the person who applies it, according to the
studies carried out with A. salina and with the criteria of Clarkson et al(25); in addition, the
aqueous extract of seeds did not cause irritation or redness of the skin of domestic dogs.
According to morphological analyses, ticks with an elongated, brown body, with short
hypostome and pedipalp, and a hexagonal shape of their capitulum belong to the species of
R. sanguineus, which are characteristics that coincide with data published by Lord CC(26). On
the other hand, ticks with a round body, with long hypostome and pedipalp, and a triangular
shape of their capitulum and dorsal shield indicate that they belong to the genus Ixodes or to
the species of I. affinis, characteristic data that coincide with specimens of I. affinis published
by Solís-Hernández et al(27).
Although the oily extract has been used in some publications, this does not limit the
evaluation of the use of the aqueous extract of P. anisum seeds as a sustainable and
economical alternative. The purpose of this research was to demonstrate that the aqueous
extract of P. anisum seeds can also work for the treatment of ticks, in addition to being
prepared in a simple manner and at a lower cost than extracting the essential oil from the
seeds.
In this work, it was observed that the aqueous extract of P. anisum seeds has potential as a
commercial use for tick control and is also affordable for domestic consumers. This study is
355
one of the first to be published in which the aqueous extract of Pimpinella anisum seeds is
evaluated.
Aqueous extract of P. anisum seeds can be a sustainable alternative for the treatment of ticks
in domestic dogs; this extract has been shown to have a more effective detaching time than
Amitraz, in addition to having an effect of 100 % ectoparasite detaching from the skin of
domestic dogs and keeping them immobilized for a certain amount of time, although shorter
than Amitraz. It was shown that the number of seeds used per liter of water for the production
of the aqueous extract makes it non-toxic. In addition, this extract did not cause irritation in
the area of application. It is expected that the results of this research will provide the basis
for future research on the aqueous extract made from the seeds of Pimpinella anisum and that
it will be applied to other pests that afflict living beings.
Acknowledgements
To the Higher Technological Institute of the South of the State of Yucatan for allowing the
use of materials and facilities to carry out this research, and the support provided by the
parents who allowed their canines to be evaluated.
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Article
Socio-ecological knowledge of the beekeeping activity in the Costa Chica

region of Guerrero, Mexico
José Cámara-Romero a
William Cetzal-Ix b*
Luis Alaniz-Gutiérrez c
Agustín Rojas-Herrera d
José Aparicio-López a
Columba Rodríguez-Alviso a
1
Universidad Autónoma de Guerrero. Centro de Ciencias de Desarrollo Regional. Acapulco,
Guerrero, México.
2
Tecnológico Nacional de México. Instituto Tecnológico de Chiná, Campeche, México.
3
Universidad Autónoma de Guerrero. Facultad de Medicina Veterinaria y Zootecnia No 2,
Cuajinicuilapa, Guerrero, México.
4
Universidad Autónoma de Guerrero. Facultad de Ecología Marina. Acapulco, Guerrero,
México.
*Corresponding author: rolito22@hotmail.com
Abstract:
Beekeepers need to identify melliferous flora (MF) in those areas where apiaries are
established because bees (Apis mellifera) depend on these floral resources for food and honey
production. The objective of the study was to analyze the socio-ecological aspects of
beekeeping, considering the knowledge of the melliferous flora by the producers in the Costa
Chica region of Guerrero (GCC), Mexico. A non-probabilistic convenience sampling was
360
carried out. The final sample consisted of 75 surveyed beekeepers. Descriptive statistics and
cross-tabulations were used for data analysis; botanical collections were made to identify the
species cited. Beekeeping is traditional (5-50 hives), the average age was 48 yr, with 10 years
of schooling and 12 yr of experience. Producers mentioned 33 MF species (26 native and
seven cultivated) belonging to 16 botanical families. In addition, they classified them by their
use as nectapolliniferous (14 species), polliniferous (10), and nectariferous (9). It was
recorded that native species flower during the winter (herbaceous) and spring (trees),
coinciding with the honey harvest season, while cultivated species flower during the rainy
season (summer) and are an important resource during the post-harvest season. GCC
beekeepers registered low knowledge of the vegetation surrounding their apiaries, but have
a high knowledge of the main MF species, finding that the older they are, the more knowledge
they have about the MF species that bees use in their food (nectar or pollen).
Keywords: Beekeeper, Floral resources, Traditional knowledge, Vegetation.
Introduction
Beekeeping is an activity that is directly linked to the sustainable management of natural

resources, as beekeepers depend on areas with native or introduced flora to install their hives
and thus provide bees with food sources (nectar or pollen) for the production of honey(1,2).
This activity is compatible with biodiversity conservation and with the surrounding
traditional crops, as they contribute to pollination and food sovereignty(3). Beekeeping
requires little investment and provides an important income for the economic stability of the
producers in the rural communities where it is practiced(4).
In Mexico, beekeeping is a livestock activity that influences socioeconomic and ecological

aspects because it generates a significant foreign exchange(5). The country ranks ninth in the
world in terms of production volume and eleventh in terms of number of beehives; at the
continental level, it ranks third in both areas(6). Despite being among the main honey
producers in the world, Mexico has shown a downward trend in honey volume and hive
inventories for the last two decades(7). This decline in production is due to multiple factors,
such as pests and diseases (varroasis, foulbrood, small hive beetle), technical-social issues
(lack of training and organization, middlemen, and competition in the international market),
361
and ecological issues (variations in phenology and floral synchronization)(8). These obstacles
have caused instability in the Mexican beekeeping sector, mainly in the beekeeping regions
of the country (North, Central Highlands, Pacific, Gulf, and Yucatan Peninsula)(5).
Beekeeping in Guerrero is favored by its geographic location and its diversity of climates
and natural resources. The state is located in two beekeeping regions (Central Highlands and
Pacific), which results in a high honey-production potential for the seven regions of the state
(Acapulco, Costa Chica, Costa Grande, Centro, La Montaña, Norte, and Tierra Caliente)(10).
In 2021, Guerrero ranked as the tenth largest honey producer in the country, with 2,081 t;
there has been a decline in this state’s production in recent years(7) due to deforestation, land
use change, and insecurity, which limits the development of this activity(10). Costa Chica in
Guerrero is the main honey-producing region at the state level, as it has a more extensive
coverage of vegetation in low and medium tropical forests, where the supply of floral
resources is more constant than in other regions; therefore, it is still necessary to expand the
knowledge related to the melliferous flora (MF). This information can be useful to learn about
the most important plant species for beekeeping, as well as to maintain established colonies
and increase their development(11).
In order to assess the experience and knowledge generated by beekeepers in a specific area
in regard to the vegetation and the diversity of MF species, with their flowering periods and
their food utility (pollen and nectar) for bees, it is necessary to have the observations of the
producers, information that is collected and validated through interviews, questionnaires or
field studies, and which allows to know the flora of interest for honey production(12). For this
reason, the objective of the study was to analyze the socio-ecological aspects of the
beekeeping activity, based on the knowledge of the MF of the producers of the Costa Chica
de Guerrero (GCC), Mexico, in order to have updated information on the panorama of the
beekeeping activity in this region.
The GCC region is made up of 15 municipalities: Ayutla de los Libres, Azoyú, Copala,
Cuautepec, Cuajinicuilapa, Florencio Villarreal, Igualapa, Juchitán, Marquelia, Ometepec,
San Luis Acatlán, San Marcos, Tecoanapa, Tlacoachistlahuaca, and Xochistlahuaca; the
GCC is bordered to the north by the La Montaña and Central regions; to the south, by the
Pacific Ocean; to the east, by the state of Oaxaca (Costa Chica region of Oaxaca), and to the
west, by the Acapulco region(13) (Figure 1).
362
Figure 1: Municipalities where interviews with beekeepers were conducted in the Costa
Chica region of Guerrero, Mexico
1= Ayutla de los Libres, 2= Azoyú, 3= Copala, 4= Cuajinicuilapa, 5= Cuautepec, 6= Florencio Villarreal, 7=

Igualapa, 8= Juchitán, 9= Marquelia, 10= Ometepec, 11= San Luis Acatlán, 12= San Marcos, 13= Tecoanapa,
14= Tlacoachistlahuaca, 15= Xochistlahuaca.
The GCC has a predominantly warm-sub-humid climate, with temperatures ranging between
20 and 29 °C and rainfall of 1,100 to 2,200 mm from June to October. The topography varies
from hilly terrain, in the municipalities of San Luis Acatlán and Ometepec, to flat or semi-
flat, in the municipality of Marquelia. The vegetation is composed of a third of low and
medium deciduous forests, and pine and oak forests in the areas near the Montain region(13).
A non-probabilistic convenience sampling(14) was carried out, where individuals were

selected for their willingness to provide detailed information on beekeepers' knowledge and
perception of MF in the GCC. A questionnaire was designed to collect the information, and
a survey was administered during meetings of beekeepers' cooperatives and associations. The
final sample consisted of 75 beekeepers surveyed during the period January to December
2021.
The questionnaire consisted of two sections: I) General data on the beekeeper (age, schooling,
time in this activity, and main occupation) and on the beekeeping unit (land tenure,
transhumance). II) Ecological knowledge of the flora of the region (acquisition of knowledge
of the MF, reforestation, types of vegetation in the area surrounding the apiaries, main species
close to their apiary and the contribution of nectar and pollen of the MF of the region); in
363
order to determine the beekeepers’ knowledge of the flora, they were asked, of the total
(100 %) of the plants in their region, what percentage they consider that they know.
For the purpose of identifying the MF cited by beekeepers, 10 random walks were conducted
in low and medium tropical rainforests in the study area, guided by a key beekeeper. Species
identification was based on a joint analysis between researchers and beekeepers and
documentary research available for the area(9,15,16). Species that could not be identified in the
field were collected according to the described technique(17) and were sent to the María
Agustina Batalla Herbarium at the Faculty of Sciences (FCME) of the National Autonomous
University of Mexico (Universidad Nacional Autónoma de México, UNAM), for
identification.
Data analysis
Descriptive statistics were used for data analysis, and the information was processed using
the SPSS statistical software, version 19. Frequency analysis and cross-table analysis(18) were
performed to compare the means of the indicators between the two sections of the survey and
to determine whether or not there is a relationship between the social variables and the
ecological variables. The Chi-square test was used to detect the association of the variable
age and knowledge of vegetation, experience, and land tenure with reforestation. Beekeepers
were classified into three categories, according to the number of hives they own: 1)
traditional, from 10 to 50 hives, 2) semi-technified, from 51 to 200 hives, and 3) technified,
> 200 hives.
Results
General characteristics of beekeepers
The average age of the beekeepers was 48 yr, and their average experience in beekeeping
was 12 yr (Table 1); their average schooling was 10 yr (Table 2). According to the
classification, traditional beekeeping engaged the largest number of beekeepers (58.7 %);
semi-technified beekeeping employed 45.3 %, and technified beekeeping, a mere 4 %), while
the latter have more experience (17 yr) and age (59 yr).
364
Table 1: Main socio-demographic characteristics of beekeepers

Type of Experience Age Education level
beekeeper (years) (years)
Basic High- Higher Total
school
Traditional 9 ± 1.02 43 ± 1.1 18 13 7 38
Semitechnified 15 ± 1.44 53 ± 1.31 20 9 5 34
Technified 17 ± 2.01 59 ± 6.1 2 0 1 3
Average 12 48 - - - -
Number of - - 40 22 13 75
beekeepers
Total % 53.4 29.3 17.3 100
Fifty-six percent of the beekeepers said that their apiaries are located on land that they legally
own, and the remaining 44 % indicated that their apiaries are located on borrowed or rented
land (Table 2). Technified beekeepers (4 %) have more than 200 hives and need to rent land
to establish their apiaries; therefore, most of them practice transhumant beekeeping, which is
not very deeply rooted in this region: only 12 % of the beekeepers practice it, generally
moving to areas in the “La Montaña” region.
Table 2: Economic activities of beekeepers and apiary ownership

Type of
Land tenure Main activity
beekeeper
Rented Private Beekeeper Farmer Salaried
employee
Traditional 19 19 10 11 17
Semitechnified 13 21 6 20 8
Technified 1 2 0 2 1
Number of 33 42 16 33 26
beekeepers
Total % 44.0 56.0 21.3 44.0 34.7
With respect to the main activity of the beekeepers, it was observed that 21.3 % are
exclusively dedicated to beekeeping, which means that beekeeping is a complementary
activity to other agricultural and livestock farming activities. However, technified beekeepers
were considered entrepreneurs, as they add value to beekeeping and diversify their economic
activities.
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Melliferous flora
Beekeepers identified 31 MF species, composed of 16 botanical families, of which Fabaceae

had the highest number of species(14), followed by Boraginaceae and Malpighiaceae, with
two, respectively, while the other 13 families had only one species; two species could not be
identified (Table 3).
According to the knowledge of beekeepers, 14 species are considered nectariferous, 11

produce nectar and pollen, and 8, pollen; of all these species, 26 are wild and 6 are cultivated,
the most prominent being Mangifera indica L., Citrus × aurantiaca (L.) Swingle and Cocos
nucifera L., as they are widely cultivated in the region (Figure 2). Although sesame
(Sesamum indicum L) is another important crop in the region, only two beekeepers mentioned
it.
Figure 2: Vegetation and melliferous flora
A) Secondary vegetation, Vista Hermosa, Ometepec. B) Ceiba pentandra (L.) Gaertn. C) Enterolobium
cyclocarpum (Jacq.) Griseb. D) Pithecellobium unguis-cati (L.) Benth. E) Bauhinia pauletia Pers. F) Vista
panorámica, Selva mediana subcaducifolia. G) Senna mollissima (Humb. & Bonpl. ex Willd.) H.S. Irwin &
Barneby.
366
In order to know the perception of the MF of the region, cross tabulations were generated
with age and knowledge of the vegetation, finding that 24 % of the beekeepers identify or
know 40 % of the vegetation, and 12 % of the beekeepers know between 80 % and 100 % of
the vegetation (Figure 3). Knowledge of the flora increases with age; beekeepers in the 26-
50 age group know 60 % of the region's flora, while the 15-25 age group knows only 30 %
of the flora.
Figure 3: Cross-tablulation. A. Between age and knowledge of the flora of the region. B.
Experience and reforestation. C. Land tenure and reforestation
* The Chi-square test showed that there was no correlation (P>0.05) between these three cross-tabulations
As for the beekeepers’ years of experience and practice on reforestation, 40 % were shown
to have 1 to 5 yr of experience, while only 33 % had between 6 and 9 yr of experience in
reforesting. Likewise, it was observed that 45 % of the beekeepers with legal ownership of
the land reforested, and 76 % who rented land did not do so.
367
Discussion
According to the historical national average number of hives per beekeeper(19), beekeeping
in Mexico can be classified as traditional (1-50), semi-technified (51-200), or technified
(more than 201).
The average age of beekeepers in the region was 48 yr, with an interval of 23 to 75 yr, similar
to that recorded in other entities such as Yucatán(20); the central-southern region of Jalisco(21),
with 49 yr, and Campeche(22), with 57 yr. Consequently, the average age of beekeepers in the
study region suggests that a generational changeover is occurring in the beekeeping activity;
this is an advantage because older and more experienced beekeepers are less willing to
change their traditional forms of production and to learn new techniques, compared to
younger beekeepers(23).
The experience of beekeepers at the national level varies between 21 and 23 yr; these data
are similar to those reported in the State of Campeche(20), where a value of 21 yr in the activity
was reported; an average of 22 yr of experience was found in the Sierra Centro-Norte region
of Veracruz(24), while a value identical to that of this study, of 23 yr of experience, was found
in the south-central region of Jalisco(23). The similarity of these results indicates that, at the
national level, beekeepers have acquired knowledge, skills, and competencies for the practice
of this activity.
The average schooling of the surveyed beekeepers was 10 yr, which is equivalent to the first
year of high school, similarly to the average schooling of beekeepers in Jalisco(21), of 9 yr,
but higher than the average schooling (5 yr) registered in Yucatán(25). The low level of
education is one of the main factors why field records or logs are not kept, a fact that limits
the possibility of managing information, maintaining traditional practices, and applying new
technologies(21).
Land ownership conditions are very different in the study region, since 44 % of the
beekeepers rent the land where the apiary is established —compared, for example, to the state
of Yucatán(22), where 74 % are privately owned and only 26 % are on rented land. This
reflects regional and intergenerational contrasts in social land ownership and is related to the
changes brought about by the 1992 agrarian reform, which encouraged the fragmentation of
communal lands and led to disruptions among deeply rooted indigenous and peasant
cultures(26). This phenomenon of forest fragmentation forces beekeepers to move their hives
to places with preserved vegetation in search of suitable species.
368
In this regard, transhumant beekeeping was carried out by only 12 % of beekeepers in the
GCC. These results are similar to those recorded in the Central and Northern Region of
Veracruz, where beekeepers with more than 150 hives (20 %) are the ones who practice
transhumance(24). On the other hand, the inadequate location of the apiary causes a lower
honey yield per hive; therefore, transhumance implies maximizing the productive efficiency
in function of the MF density, reducing the bee's foraging route and counteracting the
investment of economic resources(27).
Beekeeping in the GCC is a complementary activity for traditional and semi-technified

beekeepers. In the state of Yucatán, beekeeping is the main economic activity for 19 % of
beekeepers; the percentage rises to 25 % if the apiary has between 50 and 100 hives(25). The
greater the number of apiaries, the more beekeepers perceive beekeeping as their main
economic activity.
Community participation is a way to obtain reliable and useful results to solve issues and
improve situations or the collective knowledge of their region(28). This knowledge of the
region's flora, especially that which has melliferous potential, serves as a tool for the
beekeepers themselves, allowing them to better manage their apiaries, decide when to
supplement the bees' nutrition or change their apiaries to places with adequate MF for the
bees to forage for pollen and nectar, which contributes to the production of quality honey(1).
Mention by the surveyed beekeepers of certain species that are not important for the
beekeeping activity (Tamarindus indica L., Ehretia tinifolia L., and Persea americana Mill.)
confirms that beekeepers' perceptions are biased in favor of culturally influenced landscape
species, both cultivated and wild(29). The 72 % of beekeepers are familiar with 80 % of the
vegetation in their surroundings; such familiarity with these landscapes makes it difficult to
identify other types of species present in the vegetation of the forests.
Beekeepers in the municipality of Hopelchén, Campeche, recorded 50 species, three

subspecies, and three varieties of MF, distributed in 26 botanical families(12) ―a higher
number than that found in this study, where 33 species of MF were recorded.
In regions with a strong change in land use and where agricultural landscapes predominate,
the remnants of natural vegetation are mainly dominated by tree species that become
important for beekeeping(30). Similar values to those of this study were registered in a tropical
dry forest in Ecuador(31), where 28 MF species were identified by the beekeepers. However,
these questions addressed only those species they consider important for the bees, and not all
vegetation in general. Similarly, another study carried out in Nicaragua(32) identified 89
species, but without specifying whether or not all of them correspond to MF.
369
Also, of the 33 species recorded in this study, seven are cultivated; however, the flowering
period for bees is concentrated from November to May, while during the rest of the year,
according to certain authors, nectar and pollen resources are harvested from monoculture
plots(33). In the study region, plantations such as sesame, citrus, hibiscus, coconut, mango,
etc., play an important role as floral resources for beekeeping, due to their established surface
area and to flowering periods that are not simultaneous with those of the wild vegetation. A
noteworthy fact is that the beekeepers did not mention any introduced grass species, despite
the fact that these are abundant in the region's tropical dry forests. This is because beekeepers
associate grasslands with pollen production and do not consider them as an important floral
resource for bees. However, in other parts of the Caribbean, such as the Dominican
Republic(29), beekeepers have identified such invasive species as Leucaena leucocephala
(Lam.) de Wit, Syzygium jambos (L.) Alston and Prosopis juliflora (Sw.) DC. as plants of
beekeeping interest.
Finally, when asked about the level of knowledge of the flora around the apiaries, only 12 %
of the beekeepers considered that they knew between 80 % and 100 % of the MF, a lower
value than that registered in Campeche(12), where they found that 60 % are familiar with the
vegetation of their apiaries as a result of the transmission of knowledge through generations.
GCC beekeepers find it easier to adopt new technologies and diversify hive products due to
their average age, which is lower than the national average. Transhumant beekeeping is
carried out by technified beekeepers. Younger people have little knowledge of the
environment and MF, but identify specific flora that provide nectar or pollen for bees,
acknowledge that agricultural crops are important for bee activity, and recognize the
importance of the environment for bee activity. On the other hand, the tenure of the land
where the apiaries are located influences their reforestation, and because a high percentage
of the land is rented, this practice is not carried out. Beekeepers' perception of the resources
utilized by bees is an important source of knowledge about the flora of beekeeping interest,
which increases with age and is an invaluable source of information.
Acknowledgments
The authors are grateful to the National Council for Science, Humanities, and Technology
(Consejo Nacional de Humanidades, Ciencias y Tecnologías) for the financing of project
319942 Plants of "minor importance" for the survival of bees and the development of
370
beekeeping in the Costa Chica region of Guerrero), within the framework of the "2022 Call
for Proposals on Basic Science or Frontier Science, Modality: Paradigms and Controversies
of Science".
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Table 3: Species cited by beekeepers in the Costa Chica region of Guerrero

Family Taxa FR QFR SC F M A M J J A S O N D RSDH NMSB
Anacardiaceae Mangifera indica L. P Re X X - - - - - - - - - - --- 17
N-
Arecaceae Cocos nucifera L. Re - - - - - - X X X X X - --- 2
P
Tabebuia rosea (Bertol.) N- L. Alaniz et al.
Bignoniaceae Ab - - X X - - - - - - - - 3
DC. P 1008 (FCME)
N- L. Alaniz et al.
Boraginaceae Cordia dentata Poir. Re X X X - - - - - - - - - 4
P 289 (FCME)
Combretum fruticosum N- L. Alaniz et al.
Combretaceae Re - - X X X - - - - - - - 5
(Loefl.) Stuntz P 715 (FCME)
Ipomoea trifida (Kunth) G. L. Alaniz et al.
Convolvulaceae N Ab X - - - - - - - - - - X 30
Don 611 (FCME)
L. Alaniz et al.
Dilleniaceae Curatella americana L. N Ab X - - - - - - - - - - X 11
805 (FCME)
Andira inermis (W. Wright) L. Alaniz et al.
Fabaceae N Ab - X X X - - - - - - - - 36
Kunth ex DC. 1000 (FCME)
Enterolobium cyclocarpum L. Alaniz et al.
Fabaceae N Ab - - - X X - - - - - - - 2
(Jacq.) Griseb. 1002 (FCME)
Gliricidia sepium (Jacq.) L. Alaniz et al.
Fabaceae N Ab X X - - - - - - - - - X 29
Kunth ex Walp. 1003 (FCME)
L. Alaniz et al.
Fabaceae Hymenaea courbaril L. N Ab - - X X X - - - - - - - 46
1004 (FCME)
N- L. Alaniz et al.
Fabaceae Pterocarpus orbiculatus DC. Ab X - - - - - - - - - - X 6
P 771 (FCME)
L. Alaniz et al.
Fabaceae Bauhinia pauletia Pers. P Re - - - X X X - - - - - - 3
730 (FCME)
374
Senna mollissima (Humb. &

L. Alaniz et al.
Fabaceae Bonpl. ex Willd.) H.S. Irwin P Re X X - - - - - - - - - - 3
538 (FCME)
& Barneby.
Fabaceae Tamarindus indica L. P Re - - - X X - - - - - - - --- 2
Vachellia farnesiana (L.) L. Alaniz et al.
Fabaceae P Re - - - X X - - - - - - - 2
Wight & Arn. 547 (FCME)
Lauraceae Persea americana Mill. N Ab - - - - X X X - - - - - --- 2
Byrsonima crassifolia (L.) L. Alaniz et al.
Malpighiaceae N Ab - - - X X X - - - - - - 15
Kunth 1001 (FCME)
N- L. Alaniz et al.
Malpighiaceae Malpighia ovata Rose Ab - - - - X X X - - - - - 10
P 1007 (FCME)
ND Gusanillo (common name) N Ab X X - - - - - - - - - - --- 3
ND Tanalocote (common name) N Ab - X X - - - - - - - - - --- 7

Pedaliaceae Sesamum indicum L. N Ab - - - - - - X X - - - - --- 2
Coccoloba barbadensis L. Alaniz et al.
Polygonaceae N Ab - X X - X X - - - - - - 21
Jacq. 520 (FCME)
Citrus × aurantiaca (L.)
Rutaceae N Ab - - - - - - X X X X - - --- 7
Swingle
FR= Floral resource: N= nectar, P= pollen, N-P= nectar-pollen. QFR= quantity of the floral resource: AB= abundant, SC= scarce, RE= regular. Floral
calendar with months of the year (January-December). RSDH= representative specimen deposited in the FCME herbarium (based on Alaniz et al.,
collection number). NMSB= number of mentions of the species by beekeepers.
375
Article
Prevalence of Fasciola hepatica and Calicophoron spp. in extensively

reared cattle in the Florida district (Amazonas), Peru
Medali Cueva-Rodríguez a,b*
Teófilo Torrel c
Cristian Hobán d
Wuesley Alvarez-García b
Flor Mejía e
Luis Vargas-Rocha c
a
Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas. Laboratorio de
Enfermedades Infecciosas y Parasitarias de Animales Domésticos - LABISAN, Calle Higos
Urco N° 342-350-356 - Calle Universitaria N° 304. Ciudad de Chachapoyas (Amazonas)
Perú.
b
Instituto Nacional de Innovación Agraria. Dirección de Desarrollo Tecnológico Agrario.
Estación Experimental Baños del Inca. Ciudad de Los Baños del Inca (Cajamarca), Perú.
c
Universidad Nacional de Cajamarca. Facultad de Ciencias Veterinarias. Laboratorio de
Parasitología Veterinaria y Enfermedades Parasitarias. Ciudad de Cajamarca, Perú.
d
Universidad Nacional de Cajamarca. Facultad de Ciencias Veterinarias. Laboratorio de
Inmunología. Ciudad de Cajamarca, Perú.
e
Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas. Instituto de
Investigación de Ganadería y Biotecnología – IGBI. Ciudad de Chachapoyas (Amazonas),
Perú.
* Corresponding author: mcuevar@unc.edu.pe
376
Abstract:
The present study determines the prevalence of eggs of Fasciola hepatica and Calicophoron
spp. and of mixed infection in grazing cattle from six cattle ranches in the district of Florida,
Department of Amazonas (Peru). Using the natural sedimentation technique, 358 fecal
samples were examined. The prevalence of F. hepatica was 69.83 % (95% CI 65.08 - 74.59),
followed by Calicophoron spp. 60.34 % (95% CI 55.27 - 65.40) and a prevalence of mixed
infection 41.62 % (95% CI 36.51 - 46.73). The presence of F. hepatica eggs did not differ
among farms, breeds, and age groups (P>0.05). The presence of Calicophoron spp. and
mixed infection with F. hepatica showed differences between towns and breeds (P<0.05),
unlike the age groups, which were statistically similar (P>0.05). A high prevalence of fecal
eggs of F. hepatica and spp. was found, a situation that could be due to the environmental
conditions that allow the optimal development of the intermediate host and the cattle grazing
system.
Keywords: Prevalence, Coprology, Extensive breeding, Liver fluke, Rumen fluke.
Introduction
Parasitic infections are considered one of the most frequent and important health issues in
grazing animals. Parasites are an obstacle to profitable livestock farming, causing reduced
production and economic losses due to the costs of control, treatment, and mortality(1,2).
Fasciolosis is a disease of veterinary and public health importance that develops from the
ingestion of Fasciola hepatica metacercariae in feed or drinking water(3,4). The parasite is
located in the bile ducts and gallbladder, causes severe traumatic hepatitis during the
migratory and biliary stages, and can lead to loss of liver function as a result of damage to
liver parenchyma and bile ducts triggering liver fibrosis(5,6). In various regions worldwide, it
is considered a reemerging disease and a growing threat, mainly due to the rapid evolution
of human activities(7-9).
On the other hand, paramphistomosis, a disease caused by rumen trematodes of the

Paramphistomidae family, has been associated with significant morbidity and severe
377
pathological disorders such as enteritis and anemia, caused especially by the activity of
juvenile trematodes in the intestine of the definitive host, the ruminant(10,11). In acute
infections, the immature forms can cause the death of the animal(12). Adult parasites cause
rumenitis, acute catarrhal diarrhea, hemorrhage, detachment of rumen papillae, and fibrosis,
as well as the occurrence of areas with reticulum acanthosis, edema, ulceration, etc.(13-15). As
in the case of F. hepatica, ruminants become infected by ingesting metacercariae encysted in
forage or in water(16).
Both parasitizes are distributed across the world, mainly in tropical and subtropical
regions(17,18). Because they share the same intermediate host (snails of the family
Lymnaeidae), co-infections are possible in both the intermediate host and the definitive
host(19). The presence of these parasites is exacerbated under favorable conditions such as
wet soils, high rainfall, extensive farming systems, and fresh water bodies that host
snails(20,21). On the other hand, they cause great negative economic impact on the livestock
industry, affecting growth rate, feed conversion efficiency, reproductive performance,
carcasses in poor condition, animals experience reductions in milk production and
quality(22-25).
The lack of knowledge about the proper control of animal health issues and the low level of
education of farmers, particularly in small production systems, could partly explain the high
prevalence of bovine fasciolosis in certain scenarios(26). Given the frequent reports of rumen
and liver parasites found in cattle slaughtered for human consumption, the importance for
public health and the high economic costs involved in pharmacological treatments, the
present study determines the prevalence of F. hepatica and Calicophoron spp. in grazing
cattle in six annexes of the Florida district of the Department of Amazonas (in Peru). In this
way, it was seek to understand and achieve a more accurate picture of the presence of both
trematodes in cattle in the study area, with the consequent adoption of preventive and
prophylactic measures.
Study area
The study covered six villages in the district of Florida (Figure 1), located in the province of
Bongará, Department of Amazonas, in the northeastern Peruvian Amazon. The study area
has a humid tropical climate with frequent rainfall throughout the year and an average annual
temperature of 16 °C.
378
Figure 1: Location map of the sectors in the study area. The towns are located at an
altitude ranging between 2,280 and 2,750 m asl and have a relative humidity of 70 to 95
%
Animal selection and feces sampling
The sample size (n= 358) was estimated based on a population of 5,200 cattle (previous
census), an expected proportion of 0.5, a 95 % confidence level, and a precision level of 5%.
A stratified sampling with allocation proportional to the number of cattle determined the
number of samples to be considered for each sector. Female cattle over 2 yr of age and of
any breed were considered. Identification and age were taken from the ear tags. The animals
were raised in extensive rearing systems, fed rye grass (Lolium multiflorum), clover
(Trifolium repens), Kikuyu (Pennisetum clandestinum), and other native grasses (Figure 2).
379
Figure 2: Evaluated Brown Swiss cattle raised in open fields and fed green forage
Fecal samples (approximately 100 g) were collected directly from the rectum of the animals
using sterile obstetric gloves. Each animal was restrained by the owner with the help of a
rope, trying to cause the less pain as possible, with hands covered with latex gloves and the
perianal region was washed with soap and water. The samples were transported to the
Immunology Laboratory of the Faculty of Veterinary Sciences, National University of
Cajamarca (Universidad Nacional de Cajamarca) in an expanded polystyrene box with
cooling gels (2 to 4 °C). The transfer time lasted between 8 and 10 h. In the laboratory, they
were kept refrigerated at 4 °C until processing after 24 h. Clean and labeled materials were
used to avoid cross-contamination.
Analysis of the samples
Samples were processed by natural sedimentation(27). Eggs were observed under a

stereoscope with halogenated light at 5X (Nikon SMZ 745 - USA), and identification was
based on the morphological characteristics of the egg of each parasite(28-31).
Cattle breeders' attitude towards parasites in cattle
According to the observations made during the fecal sample collection process, it was found
that farmers in the evaluated areas lacked knowledge about mechanisms for the prevention
380
and control of trematodes in their animals. No control or prevention measures were identified,
such as the proper management of excreta, the management of drinking troughs, the
implementation of drainage systems on farms, the adoption of technified irrigation practices,
or the implementation of strategies aimed at controlling the intermediate host, among others.
In addition, no consistent information was obtained regarding the existence of deworming
programs; in fact, farmers were unaware of the presence of rumen trematodes in their
animals.
According to cattle ranchers, they sometimes perform deworming with albendazole-based

chemicals to control F. hepatica, known locally as Fasciola, Liver fluke, Alicuya, Lunguash,
Liver slug, Dystoma, and Coca Leaf. This process was carried out during the rainy season
(December to April) or when the animals presented persistent diarrhea or showed signs of
decay, without the supervision of a livestock professional, and in the absence of a
parasitological laboratory diagnosis by observation of fecal eggs or by any other method.
Data were processed using descriptive statistics. Positive cases were expressed as
percentages with 95% confidence intervals(32). The degree of association between prevalence
by sector, race, and age group was determined using the nonparametric Kruskal-Wallis test.
The Phi correlation function was used to determine the degree of the linear relationship
between the mixed prevalence of trematodes.
Results
Brown Swiss and Fleckvieh cows raised on pasture, ranging from 2 to more than 6 yr of age,
were found in the six farms of the Florida district. Eggs of Fasciola hepatica and
Calicophoron spp. were observed in all sectors (Figure 3). F. hepatica eggs were observed
in 69.83 % (95% CI: 65.08-74.59) of the animals, followed by Calicophoron spp. 60.34 %
(95% CI: 55.27-65.40) and a prevalence of mixed infection of 41.62 % (95% CI: 36.51-
46.73) (Table 1).
381
Figure 3: Calicophoron spp. eggs (a) and Fasciola hepatica (b). Stereomicroscopic view
(top) and microscopic view (bottom)
Discussion
The trematode Fasciola hepatica outnumbered Calicophoron spp. by less than a tenth
(9.12 %), with an overall mixed infection prevalence of 41.16 % (95% CI 36.09 - 46.23).
The presence of F. hepatica eggs did not differ between sectors, breeds, or age groups
(P>0.05). The prevalence of Calicophoron spp. and mixed infection with F. hepatica showed
differences between sectors and breeds (P<0.05), unlike the age groups, among which they
were statistically similar (P>0.05). The Phi correlation showed variable results of the
associated occurrence of both parasites in the animals.
The high presence of parasite eggs may be due to the good development of the intermediate
host in optimal environmental conditions, humid areas, and a variety of temperatures and
altitudinal levels, for example. The locations evaluated range from 11 to 20 ºC and have a
relative humidity of 60 to 95 %. Since both trematodes share the same intermediate host,
freshwater pulmonate mollusks of the family Lymnaeidae(33,34), this condition would
facilitate co-infection in both the intermediate and definitive host.
The areas where the sampling was carried out are located between 1,300 and 2,750 m asl,
ideal ranges for the development of parasitoses. Parasitic forms of F. hepatica have been
382
reported even in intermediate hosts below 400 m asl(35) and up to 4,500 m(36). Calicophoron
microbothrioides can be found below 200 m and also in mountainous areas above 3,000 m,
where there is stagnant water available for the cycle of the intermediate hosts, in areas used
for livestock farming, and in the areas without water for the cycle of the intermediate hosts(37).
Climate influences the rate of parasitic infection in livestock(38,39). As shown in Figure 1, the
study area includes large vegetation and bodies of water, ―favorable conditions for the
development of the intermediate host. In general, the province has a humid tropical climate
with frequent rainfall throughout the year and an average annual temperature of 16 °C. In an
area close to the study area, researchers found that water sources, mainly streams, irrigation
ditches and rivers, are risk factors for F. hepatica(40).
The breed of cattle has been reported as a risk factor in several studies. Purebreds are more
susceptible to infection than crossbreds(9). In a study conducted in Amazonas (Peru), it was
determined that the Brown Swiss breed is more susceptible to infection by F. hepatica and
other parasites(40). However, it should be noted that, in their study, the sample size for this
breed was larger than for other breeds. In the present research, a higher prevalence of
trematodes was found in the Fleckvieh breed. Despite an evident higher number of Brown
Swiss (n= 203) versus Fleckvieh (n= 155) cattle, the prevalence was statistically equal;
therefore, the results do not consolidate the breed as a risk factor.
Similarly, another study reported that the Simmental breed was a risk factor for F. hepatica
infection compared to Brown Swiss and other breeds(41). Although the sample size of the
Simmental breed was larger (as in the present study), the results were statistically similar to
those of Brown Swiss, only differing with the Jersey, Holstein and crossbred breeds, although
the sample size of these breeds was very low. Similar to the present study, several authors
have not reported conclusive results in which breed is a risk factor, but rather that the
presence of parasites is influenced by a higher population of a certain race in a certain
place(14,42,43).
The age of the definitive host is also closely related to infection(15). As in other reports, the
presence of parasites was higher in older animals. Most authors show that the prevalence of
trematodes is higher in animals older than 2.5 yr(21,43,44). No association has been found
between age and rumen trematode infection(14). Infected animals have an age limit for
becoming infected, since the life cycle of Paramphistomidae lasts at least 6 to 8 mo(9);
therefore, animals 12 to 24 mo of age may be at higher risk of infection. In addition, the
animals are raised under grazing conditions from birth until they leave the herd.
Several studies have indicated that extensive animal husbandry is a factor in parasite
infection(40,43). Cattle managed in extensive or semi-extensive regimes where access to
383
pasture occurs almost year-round throughout the animal's life are more predisposed to
grazing than those managed in an intensive or semi-intensive system(45).
The high prevalence of F. hepatica in cattle has been reported within the same Amazonas
region, with a prevalence of 45.6 %(40), and 59.5 %(41). The presence of trematodes in cattle
has also been described in other regions of Peru. In three districts of the province of
Oxapampa (Pasco - Peru), by rapid sedimentation of 408 samples of dairy cattle, a prevalence
of 10.0 ± 2.9 % of F. hepatica and 28.4 ± 4.4 % of a digenean of the Paramphistomidae
family were found(46).
Although studies on rumen trematodes in cattle in Peru are scarce, C. microbothrioides has
been reported in Amazonas(31). Trematode eggs of the Paramphistomidae family were
identified in the Loreto region (Peruvian jungle)(47). In San Martin (Peruvian jungle region)
Cotylophoron sp. has been reported in cattle(48). Both parasites have been identified in
different parts of the world. In South America, Cotylophoron cotylophorum has been
described in Colombia(49), Cotylophoron marajoensis n. sp. in Brazil(49) and C.
microbothrioides in Chile(37). As well as in the Americas, the presence of both trematodes
has been reported in European(50,51,52), African(34,26,38), Asiatic countries(53), etc. These regions
have similar conditions to those of the present study ―extensive breeding, climatic
conditions, age, breed, etc.―, as risk factors. Climate change and globalization contribute to
the distribution of parasites in a territory where the intermediate host has adapted(19).
Due to the high prevalence of trematode eggs identified in the sampled areas, it is suggested
that this situation may be attributed to the conditions in which cattle are raised, where no
formal programs for the control and prevention of parasitoses are available. Despite the
possible use of albendazole for the management of F. hepatica, no local studies of chemical-
based antiparasitic efficacy have been reported.
A high prevalence of fecal eggs of Fasciola hepatica and Calicophoron spp. was detected.
Climatic and geographic conditions, in addition to the grazing system and the absence of
control and prevention programs, predispose to the high presence of both trematodes.
However, further studies are needed to evaluate drainage systems, pasture, and watering
trough management practices in the control and prevention of trematodes, as well as
evaluations of antiparasitic resistance and comprehensive studies from a One Health
approach.
384
Sources of financing
This work was financed by the scholarship and co-financing program of the CONCYTEC,
CIENCIACTIVA of the Ministry of Education of Peru (Conv-191-2015-Fondecyt). M.C.-R.
is grateful to CONCYTEC for the financing. The authors are grateful to Dr. Rodrigo
Sanabria, VD, researcher of the Faculty of Veterinary Sciences, UNLP – Argentina, for the
guidance and direction of the research.
The authors declare that they have no conflict of interest that could have interfered with the
results of this research.
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Table 1: Prevalence (%) of trematodes found in grazing cattle in the district of Florida, Amazon (Peru)
Fasciola hepatica Calicophoron spp. Association

Variable Category No Phi Coefficient
Positive % (95% CI) Positive % (95% CI) Positive % (95% CI)
El Chido 41 26 63.41 (48.67 - 78.16)a 19 46.34 (31.08 - 61.61)a 11 26.83 (13.27 - 40.39)a -0.106
Florida 145 96 66.21 (58.51 - 73.91)a 100 68.97 (61.44 - 76.50)b 67 46.21 (38.09 - 54.32)ab 0.025
Miraflores de
41 30 73.17 (59.61 - 86.73)a 27 65.85 (51.34 - 80.37)ab 18 43.90 (28.71 - 59.09)ab -0.204
Levanto
Hamlet Nuevo Gualulo 58 45 77.59 (66.85 - 88.32)a 42 72.41 (60.91 - 83.92)b 32 55.17 (42.37 - 67.97)b -0.054
San José 15 13 86.67 (69.46 - 100)a 5 33.33 (9.48 - 57.19)a 5 33.33 (9.48 - 57.19)ab 0.277
San Lorenzo 58 40 68.97 (57.06 - 80.87)a 23 39.66 (27.07 - 52.24)a 16 27.59 (16.08 - 39.09)a 0.011
P value 0.347 <0.001 0.013
Brown Swiss 203 141 69.46 (63.12 - 75.79)a 142 69.95 (63.64 - 76.26)b 99 48.77 (41.89 - 55.64)b 0.009
Race Fleckvieh 155 109 70.32 (63.13 - 77.51)a 74 47.74 (39.88 - 55.61)a 50 32.26 (24.90 - 39.62)a -0.058
P value 0.860 <0.001 0.002
2 - 4 years 244 167 68.44 (62.61 - 74.27)a 148 60.66 (54.53 - 66.79)a 101 41.39 (35.21 - 47.57)a 0.007
Age > 4 - 6 years 72 52 72.22 (61.88 - 82.57)a 41 56.94 (45.51 - 68.38)a 27 37.50 (26.32 - 48.68) a -0.073
group > 6 years 42 31 73.81 (60.51 - 87.11)a 27 64.29 (49.79 - 78.78)a 21 -0.137
50.00 (34.88 - 65.12)a
P value 0.693 0.192 0.424
Total 358 250 69.83% (65.08 - 74.59) 216 60.34 (55.27 - 65.40) 149 41.62 (36.51 - 46.73)
ab
For each variable, the different letters between their levels are significant differences in each factor (Kruskall-Wallis, P<0.05).
392
Article
Influence of feedlot living space on production variables, carcass and

meat quality traits in Holstein steers
Ana Mireya Romo-Valdez a
Cristina Pérez-Linares a*
Francisco Gerardo Ríos-Rincón b
Fernando Figueroa-Saavedra a
Alberto Barreras-Serrano a
Beatriz Isabel Castro-Pérez b
Eduardo Sánchez-López a
Georgina Valentina Cervantes Cazarez a
a
Universidad Autónoma de Baja California. Instituto de Investigaciones en Ciencias
Veterinarias, Domicilio Conocido, Km 3.5 Carretera a San Felipe, Fraccionamiento
Campestre, 21386, Mexicali, B.C., México.
b
Universidad Autónoma de Sinaloa. Facultad de Medicina Veterinaria y Zootecnia.
Culiacán, Sinaloa, México.
*Corresponding author: cristina.perez@uabc.edu.mx
Abstract:
A determination of how the amount of allotted feedlot living space influences both
production indicators as well as carcass and meat quality traits obtained from Holstein
steers was performed by forming two treatment groups, T14: 65 steers/pen (14 m2/head
of space allowances) and T16: 57 steers/pen (16 m2/head of space allowances), with five
replications each treatment. The average arrival weight 238 ± 0.74 kg. During the
fattening period the cattle was feed twice a day with commercial diets. The steers were
slaughtered after a 261-d period. At the moment of the first reimplant a greater average
body weight was found in T16 vs T14 (384.25 vs 378.38 kg; P<0.05) and the difference
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continued until day 261 (612.35 vs 595.54 kg; P<0.05); regarding ADG, hot carcass
weight and cold carcass weight the result were: 1.50 vs 1.46 kg (P<0.05), of ADG kg/d;
367.34 vs 360.35 kg (P<0.05) and 366.68 vs 358.78 kg (P<0.05). No difference between
treatments were found in dorsal fat, marbling, pH and meat color. The results suggest that
an increase from 14 m2/animal to 16 m2/animal improves the production results as well
as the hot and cold carcass weight, with no effect on the quality traits of the carcass and
beef.
Keywords: Living space, Holstein steers, Feedlot, Carcasses, Meat quality.
Introduction
During their stay in the pen beef cattle require enough space to express its natural
behavior(1). According to Lagos et al(2) it is necessary to provide at least 18.5 m2/head to
ensure the ideal conditions of space for each animal however in case that during the
fattening period increases it is recommended that additional space is provided based on
the increase in body weight, for cattle with a weight up to 300 kg, the recommended space
is 15 m2/head, for cattle with weights higher than 400 kg a 20 m2 area is suggested. In
Mexico, the manual of good practices for intensive beef cattle production published by
the Agriculture Secretary (SAGARPA)3 estimates that a space between 12 and 12.5
m2/animal is enough for cattle to display its natural behavior.
Holstein calves have become an important input for feedlot beef production(4), so that it
accounts for 20 % of the total amount of cattle fatten in the United States of America(5),
a similar situation is now being observed in northern Mexico. Holstein steers offer certain
advantages since show desirable carcass traits like a superior distribution of intramuscular
fat and better dorsal fat width(6). It has been reported that adult Holstein cattle fatten in
feedlots exhibit an unpredictable and aggressive behavior(7), and for this reason this race
of cattle requires a larger amount of space than the beef producing races. Another fact to
take into consideration is that Holstein cattle more and more often so that the ground
condition in the pens is not good(8,9). Taking into consideration what has been above stated
an increase in the feedlot vital space per animal would have a positive impact the cattle’s
welfare and thus better beef production results(10).
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The objective of this study was to evaluate the effect that pen space had on the production
variables, as well as on the quality traits of carcass and meat obtained from Holstein
steers.
This study was review and approved by Veterinary Sciences Research Institute ethics
committee, with the project number 201/2399.
Geographical location
This study was carried out in Mexicali, Mexico, which is found at 32° 32´00 N, 115°
12’41 W. The region is characterized by a dry desert climate with an average temperature
of 34.7 °C (-5 °C winter and 50 °C summer), with an annual rainfall of 37 mm, and a
relative humidity above 50 %(11).
Animals and design of the study
The study was performed using castrated Holstein calves between the ages of 7 and 8 mo,
with an average weight of 238 ± 0.74 kg. Twenty four hours after the cattle arrived to the
feedlot they were vaccinated, dewormed and implanted with a product that contained
trembolone acetate, estradiol and tilosine. On arrival during spring (April-June) the
animals were assigned to one of two groups so that two treatments may be established.
Each treatment included five pens. The first treatment included 65 Holstein steers, in this
case each animal had a space allowance of 14 m2/per animal (T14), in the second
treatment a 16 m2/animal (T16) was allocated to each of 57 Holstein steers. The cattle
were fed twice a day using a feeding program that included three different diets given
during the fattening and finalization periods. In different proportions the ingredients of
all diets were: sudangrass, wheat hay, tallow, dried distillers grains (DDGs) and a premix
minerals.
After a 261 fattening period the steers were slaughtered, the average weight of the group
was 604 ± 5.67 kg. On the day the steers were slaughter they were transported 36 km by
truck to the slaughter house where they were put in waiting pens for 3.5 h, during this
time only water was provided. The steers were slaughter in a Federal Inspection Type
slaughter house (FIT) following the procedure described in the Mexican Official Norm
NOM-033-SAG/ZOO-2014, “Slaughter methods to be used in domestic and wild animal”
395
Production behavior
The following production result: initial weight, weight after first reimplant, weight after
second reimplant, final weight, average daily gain (ADG) and food conversion, were
obtained from the company´s records. Each of the animals slaughter weight was obtained
in the stunning box.
Carcass and meat evaluation
Carcasses from both treatments were chilled at 2 °C for 24 h and ribbed between the 12th
and 13th ribs to collect additional carcass data. A total of 178 carcasses from T14 and 176
carcasses from the T16 were available by the slaughterhouse to be considered for the
study of all the variables. The measurements of hot carcass weight (HCW) and cold
carcass weight (CCW), dorsal fat , marbling, ribeye area, pH and color of each carcass
were taken. Dorsal fat was measured in mm using a metric ruler. The ribeye area was
evaluated using a plastic grid method suggested by Iowa State University and the
marbling score (scale of slight; small; modest; moderate; slight abundant; moderately
abundant), were both evaluated following the methodology described by AMSA(12). The
pH was determined using a potenciometer (HANNAH INSTRUMENTS Inc. pH 101),
the color values (L*, a*, b*, C*, H*) were measured on the surface of the cut from the
Longissimus dorsi muscle between the twelfth and thirteenth intercostal space using a
MINOLTA CM-2002 spectrophotometer (Minolta camera, Co., Ltd., Japan) with a
specular component included (SCI), a D65 illuminant, and a 10° observer, where L* is
the index of luminosity, a* is the red color intensity and b* is the yellow color intensity
and C* measure color saturation.
Productive data was analyzed using the following statistical linear model: 𝑌𝑖𝑗 = 𝜇 + 𝜏𝑖 +
𝛽𝑗 + 𝜀𝑖𝑗 where 𝑌𝑖𝑗 is the response variable, 𝜇 is the true mean effect, 𝜏𝑖 is the fixed
treatment effect, 𝛽𝑗 is the fixed pen effect and 𝜀𝑖𝑗 is the random residual error iid N (0,
𝜎𝑒2 ). The hypothesis that treatment effects do not differ, was performed by F test statistic
in the ANOVA. Differences between treatments were declared when P≤0.05.
Carcass and meat quality data were analyzed as a randomized complete block design with
sampling, with pen as the experimental unit and carcass as the observational unit. The
statistical linear model was as follows: 𝑌𝑖𝑗𝑘 = 𝜇 + 𝜏𝑖 + 𝛽𝑗 + 𝜀𝑖𝑗 + 𝛿𝑖𝑗𝑘 , where 𝑌𝑖𝑗𝑘 is the
response variable, 𝜇 is the true mean effect, 𝜏𝑖 is the fixed treatment effect, 𝛽𝑗 is the fixed
396
pen effect, 𝜀𝑖𝑗 is the random residual error iid N (0, 𝜎𝑒2 ) and 𝛿𝑖𝑗𝑘 is the random sampling
error iid N (0, 𝜎𝑑2 ). The hypothesis that treatment effects do not differ, was performed
using an F test statistic in the ANOVA. Differences between treatments were declared
when P≤0.05.
The hypothesis that treatment effects do not differ for proportions within each marbling
class was done using a Chi-square test statistic in one frequency table. Differences
between treatments were declared when P≤0.05. The analysis was made using the
MIXED and FREQ procedures of the SAS 9.4 (TS1M7) statistical package.
Production results
A relevant finding of this study was that steers with a larger pen space had a higher weight
during all the fattening period; these results are presented in Table 1 and show that after
receiving the first reimplant (day 94 after arrival to the feedlot), the steers from T16
showed an average higher weight when compared to the animals in T14 (P<0.05); this
same result was observed after the second reimplant and through all the fattening period
(P<0.05); the observed weight difference between the groups was 16 %. Similar results
regarding weight differences have been reported by other authors(13), who found a higher
final weight in Hanwoo steers when they were provided with a larger pen space.
Table 1: Holstein steers Median weight values ± SEM per treatment

Treatment
Variable SEM Pr>F
14 m2 16 m2
Initial weight, kg 238.57 237.62 0.74 0.2000
st b a
Weight at 1 reimplant, kg 378.38 384.25 1.65 0.0004
d b a
Weight at 2 reimplant, kg 506.73 515.21 2.52 0.0008
b a
Final weight, kg 595.54 612.35 5.67 0.0032
SEM= standard error of the mean.
a,b
Different letter indicates differences between treatments (P<0.05).
Table 2 shows the production results for both groups of steers. It was found that weight
gain was higher for the steers in T16, however no difference was found in feed conversion
and feed intake. Similarly, to this study Kim et al(14), observed that Holstein steers 20 mo
of age that were provided with 16 m2/animal, reached a 750.39 kg final weight and daily
weight gain of 1.36 kg. A study in Holstein steers that did not considered the amount of
living space per animal as a variable have reported a final weight between 613.3 a 631.4
kg, a 1.41 to 1.46 kg/d of ADG(15), while a study carried out in Mexico found that Holstein
steers reached a final weight of 604.9 kg with a daily gain of 1.46 kg and a feed
397
consumption of 8.41 kg per day(16), another study performed by Carvalho et al(17) found
that Holstein steers gained daily 1.73 kg/d with a final weight of 598 kg. Although in
Mexico the federal norm(3) establishes that pen space for an animal under 400 kg should
be 12 m2 and for one above 400 kg 20 m2 (2). It may be expected that the world trend to
reduce the space allowance per animal in cattle feedlot(18) is impacting Mexico, so it is
likely that welfare and production variables will be affected because of smaller allowed
space for feedlot cattle.
Carcass and meat evaluation
Table 2: Median production results ± SEM per treatment

Treatment
Variable SEM Pr>F
14 m2 16 m2
Daily weight gain, kg 1.46b 1.50a 0.01 0.0327
Feed conversion 7.51 7.17 0.17 0.1260
Feed consumption, kg 10.80 10.62 0.15 0.2967
a,b
The group of steers that was provided with the largest living space showed a difference
of 7 kg both in the hot and cold carcass weight (P<0.05), these results are shown in Table
3 and correspond with it was reported by Ha et al(13) who provided a greater living space
to steers that were in the finalization period. A similar study(19) reported a larger hot
carcass weight for feedlot steers which were provided with 16 m2/animal, when compared
with two other groups of animals that had a living space of 10.6 and 8 m2/animal.
Table 3: Carcass median production results ± SEM per treatment

Treatment
Variable SEM Pr>F
14 m2 16 m2
Hot carcass weight, kg 360.35b 367.34a 2.98 0.0196
b a
Cold carcass weight, kg 358.78 366.68 2.96 0.0079
Dorsal fat, mm 9.1 9.3 0.83 0.1939
2
Ribeye area, cm 96.14 98.66 2.31 0.9277
a,b
In the present study dorsal fat and ribeye space showed no statistical difference between
groups (P>0.05), this result corresponds to what is reported in Hanwoo cattle
carcasses(19). In contrast with this study, researchers(20) found no differences (P>0.05)
between Hanwoo carcasses obtained from animals that were provided with different
living spaces. Other authors have reported lower dorsal fat numbers, 5.15 mm(14); 5.8
mm(17,21); while Carvalho et al(15) reported a dorsal fat measurement between 8.6 and 9.3
398
mm, Torrentera et al(16) observed a dorsal fat depth of 10.9 mm results that are similar to
what was observed in the present study.
Authors have found that dairy cattle tend to deposit greater amounts of fat in the
abdominal cavity and to accumulate less subcutaneous fat(22), in this context bovine races
that are bigger and take more time to mature have a larger proportion of inter and
intramuscular fat when compared with smaller races which mature earlier(23).
In the case of ribeye area, the present study found that they were larger than the ones
reported by Ha et al(13) for Hanwoo steers (91.0 and 94.6 cm2 for 10 and 16.7 m2 of living
space) likewise other studies in Holstein steers reported ribeye areas of 72.36 cm2 (17);
73.7 cm2 (21); 74.9-82.5 cm2 (15); 77.21 cm2 (14); 81.22 cm2 (16).
Regarding the amount of intramuscular fat in the meat (Table 4) the results indicate that
there is no difference between the groups, however the findings support the reports from
other researchers that in the case of Holstein steers choice beef is the grade that is
observed(16,17,21). In this study, 130 of the steer’s carcasses produce beef that was classified
as small while a second group of 159 carcasses yielded modest beef.
Table 4: Marbling score per treatment

Treatment
Variable 14 m2 16 m2 Pr>χ2
n = 178 n = 177
Slight 10 14 0.4142
Small 57 73 0.1605
Modest 87 72 0.2342
Moderate 23 17 0.3428
Lightly abundant 1 1 ---
Table 5 show both groups physicochemical results, it was found that in the case of pH,
L*, a* y C* no differences were observed (P>0.05), and although the values for b* y H*
showed differences (P<0.05), this dissimilarity does not result in noticeable differences
in color between treatments.
Table 5: Meat physicochemical median results ± SEM per treatment

Treatment
Variable SEM Pr>F
14 m2 16 m2
pH 5.67 5.60 0.06 pH
L* 29.97 31.96 0.85 L*
a* 17.08 16.79 1.24 a*
a b
b* 15.45 15.02 0.83 b*
C* 23.08 22.59 1.47 C*
a,b
399
In regard to pH, values between 5.5 and 5.8 are considered as normal for bovine meat(24);
so, the results obtained by the present study may be viewed as typical. Similar pH values
and have been reported in studies done with Holstein by other authors(6,25). In the case of
meat color, based in what has been reported by others authors(24), the meat obtained from
both groups is considered as dark cutting, another research have reported similar results
(L* =37.50, a*=14.69 y b*=12.39)(26) and (L*= 38.02, a*=19.86, b*=8.19, C*=21.49)(14);
the reason for this may be explained by the pre slaughter stress that the animals were
submitted to, which depleted blood glycogen and affected the beef´s color(27). Authors
have informed that the way animals are handled, the novelty of environment and fatigue,
are factors that contribute to stress(28).
It is very important that feedlot cattle is provided by sufficient living space during the
whole fattening period and considering that there is a trend to reduce the space allowance
per animal, it is very important to better understand the negative impact that a reduce pen
space has on the animal welfare and how this impacts beef production. As suggested by
the results of the present study a relatively small increase of living space has a positive
impact on carcass weight which at the end will translate into an increase of income.
Acknowledgments
We are very thankful to the employees and management from Ganadera Mexicali S.A.
for all the assistance and support provided to this study.
Conflict of interests
The authors declare that they have no conflict of interest.
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403
Review
Common duckweed (Lemna minor): food and environmental potential.

Review
Olga Jaimes Prada a
Olga Lora Díaz a*
Katherine Tache Rocha a
a
Universidad del Sinú Elías Bechara Zainúm. Facultad de Ciencias de la Salud. Cartagena,
Colombia.
*
Corresponding author: olora@unisinucartagena.edu.co
Abstract:
Common duckweeds are flowering plants of the family Araceae, comprising the smallest
angiosperms of the plant kingdom, a species of aquatic algae of universal distribution, found
on the surface of freshwater bodies, mainly in puddles, swamps, lakes, and calm rivers.
Recently, different research has been carried out on its potential and usefulness. Due to its
nutritional composition, protein contribution, high fiber content and low fat and carbohydrate
content, it would be an adequate input to generate products of high nutritional value,
characteristics that make it interesting compared to other species. It is used as a complement
to commercial diets in a wide variety of animals such as birds, ruminants, non-ruminants,
crustaceans, and fish, reducing feed costs by up to 50 %. Likewise, used in remediation
processes of a wide range of chemical contaminants with a high elimination rate, they can
absorb some dissolved substances and provide oxygen through photosynthesis. It has been
indicated that they are low cost of construction, maintenance, easy to operate, have a wide
tolerance to growing conditions, are generally easy to harvest, and do not compete with
farmland. In the environmental field, it is important to find alternative and innovative raw
materials, even without the need to use growth media or fertilizers, however, their acceptance
as a food source needs extensive research regarding their nutritional value, large-scale yield,
economic market supply and analysis of antinutritive components for human food.
404
Keywords: Common duckweed, Lemna minor, Nutritional profile, Environmental

remediation, Human and animal food.
Introduction
In recent decades, the rapid growth of the world’s population and the climate crisis have
become a serious problem that threatens the food and feed supply, generating dietary patterns
deficient in proteins and vitamins, the development of malnutrition due to excessive
consumption of simple sugars and stigmatization of nutrients(1). In this sense, common
duckweeds have taken center stage in recent research in the search for new foods that provide
healthy alternatives, pharmaceutical products that are sustainable and profitable on a large
scale of production.
These small aquatic plants comprise a group that float on the surface of bodies of water with
little movement, with a great capacity for reproduction and accelerated growth. Traditionally,
they have been used as remediation agents for water body pollution due to their ability to
absorb minerals, salts, nitrogenous substances, and heavy metals in water bodies(2).
From an ecological point of view, it can be seen that, given its interactions with other species,
it can be considered as a keystone species in its habitat, although it has a small size, due to
its rapid growth, high tolerance to pollution and capacity to absorb nitrogen and phosphorus,
Lemna minor has previously been used for wastewater treatment(3,4).
In Asian countries and recently in Western countries, they are being included in plant
mixtures for the raising of farm animals and fish cultures, showing favorable results in the
development and growth of these animals, reducing feeding costs(2).
Common duckweeds are known to contain essential nutrients such as protein, carbohydrates,
and fats. They also contain a variety of secondary metabolites that are beneficial to humans.
Therefore, consideration of common duckweed cultivation methods is vital for their best
utilization in various industrial applications. A number of reports have been generated on
common duckweed utilization, metabolites, and cultivation; these should be reviewed and
summarized as fundamental information to improve the application of common
405
duckweeds(5,6). It is important to note that if they are used as pollution remediation agents,
their use in human and animal food should be evaluated.
This paper aims to provide a global overview of common duckweeds, especially the species
Lemna minor, through a bibliographic review from taxonomic description to their use as an
alternative for inclusion in animal and human diet, considering their nutritional composition
and current lifestyle patterns. In addition, environmental impact as biomarkers,
environmental remediators, agricultural amendments in crops, biofuel sources and
pathogenesis models is studied.
Common duckweed (Lemna minor)
Common duckweed (Lemna minor) is the world’s fastest-growing, free-floating aquatic

angiosperm plant that grows in immobile waters, usually in freshwater or wetlands in most
parts of the world and is characterized by its small size and great reproductive capacity, which
allows it to occupy large aquatic spaces in a truly short time(7,8). They are generally described
as aquatic or floating grasses of quite simple structure, lacking stem, or leaves, occasionally
with small thread-like roots on their underside. It is an aquatic plant that can often be seen
floating or just below the surface or moving very slowly(9).
Lemna minor has a thalloid-shaped vegetative body, characteristic of some plants in which it
is not possible to differentiate the leaves from the stems, small in size with a flat structure,
green coloration in its leaves and a single thin white root. According to the description of
some authors, this characteristic is associated with a modified leaf that fulfills the functions
of the stem, leaf, and axis to support flowers, as shown in Figure 1(10,11).
Figure 1: Illustration of the vegetative body and roots of common duckweed (Lemna
minor)
Source: Landolt, E(11).
406
On the other hand, roots, generally related to the nutrient uptake aspect of the plant, seem to
have a slightly different function in these species. Some researchers have reported that the
consumption of nutrients via roots is little or non-existent, functioning as a stabilizing organ
in the body of water; however, the common duckweed, Lemna minor, has been shown to
acquire significant amounts of inorganic nitrogen through the root. The plant grows at
different temperatures varying between 5 and 30 °C, growing optimally between 15 and
18 °C. It adjusts favorably to any lighting conditions. It grows rapidly in calm, nutrient-rich
locations with high levels of nitrogen and phosphates. Iron often limits the proper
development of this species. It is also tolerant to a wide pH range, between 4.5 and 7(12,13).
Cultivation of common duckweed (Lemna minor)
Common duckweed crops can be produced quite easily and inexpensively, even without the
need to use growth media or fertilizers, as they are characterized by a high relative growth
rate (RGR). This means that they are able to produce large amounts of biomass in a short
time and in relatively small ponds, filled with a few centimeters of natural water (30 to 50
cm deep). The control and monitoring of the aquatic medium in which the plants grow is
particularly important. The productivity of common duckweed increases more if the optimal
ecological conditions for growth are respected, however, they are generally broad. These,
although varying slightly from species to species, generally consist of moderately warm,
sunny, nutrient-rich waters, as documented in ecological studies on some common duckweed
species of the genus Lemna.
In recent decades, it has become common to grow them outdoors, but it can be difficult to
optimize and control operationally. However, common duckweeds also represent a suitable
crop for indoor farming, with most species, due to their flat structure particularly suitable for
cultivation in multi-level (stacked) systems that use indoor floor space efficiently. Indoor
cultivation also expands the scope of crop managing and allows for pest-free conditions and
even sterile conditions. However, the technical and operational parameters required for large-
scale effective interior have received little attention in the literature(14).
It is important to note that sporadic common duckweed growth causes serious damage to
aquatic resources, with several economic implications. The dense and extensive blanket
created by the plant in the block of surface water and water channels makes activities such
as water flow, sailing, canoeing, swimming, and fishing impossible. It also affects irrigation,
floods canals, can clog hydroelectric turbines and disturbs rice fields. A dense layer of
common duckweeds shuts outs and inhibits competing aquatic plants, including algae that
407
require sunlight. For this reason, it is important to have integrated control or strategies that
allow their exploitation.
According to various objectives and targets, optimal cultivation of common duckweeds will
be necessary from an economic and industrial point of view. Various cultivation methods
using various types of bioreactors and conditions for their use as food resources,
pharmaceuticals, phytoremediators, and biofuels must be employed. Aquaponics combining
aquaculture and hydroponics could be a sustainable production system for plants(15).
Currently, the technology enables the mass production of good quality by controlling
environmental conditions, such as irrigation irradiation, atmospheric pressure, wind, speed,
temperature, and humidity.
Nutritional composition
In recent decades, several scientific studies have highlighted the nutritional value of common
duckweeds, which, due to their supply, could improve the quality of food in the future.
However, according to some studies, the metabolism of the plant and, therefore, its nutritional
composition depends a lot on the nutrients found on the surface of the body of water in which
it is found. These extremely important factors capable of influencing the nutritional
composition of the plant are reflected in the different results obtained in each study(16).
Proteins
The high-quality protein content reaches 20 to 35 % in dry matter, when grown under optimal
conditions, higher than the protein present in cereals(6). This means that common duckweed
biomass can be considered as an ingredient for animal or human food and can contribute to
improving food security through the development of sustainable methods of producing food
with high nutritional value(17). It has been documented that the protein production of common
duckweeds per harvested area was higher than that of soybeans, rice, and corn; therefore, it
could solve the problem of the scarcity of arable land to produce food(5).
The amino acid profile of common duckweeds stands out from some plant-based protein
sources currently known in the human diet, an aspect that some authors have come to relate
to an amino acid profile more similar to animal protein(18,19). Recent studies analyzing the
nutritional composition of several common duckweed crops showed, unlike previous
analyses, a content of amino acids, such as isoleucine, leucine, cysteine, methionine,
408
threonine, and valine, similar to the recommendations for consumption for the population
(Table 1); it can also be noted that the amounts were not lower than those recommended by
the WHO, 2007(20,21). Jahreis et al(20) found that the amino acid composition of common
duckweeds is comparable to that of legume meals, such as chickpeas, lupins, or peas. Clinical
nutrition studies have shown that the essential amino acids and vitamin B12 content of
common duckweeds are comparable to peas and cheese(5).
In addition, their source of proteins, which could replace soybean meal, is expected to be
used as a substitute to reduce environmental pollution created by the expansion of soybean
cultivation(21). Consumption of plant protein instead of animal protein could reduce energy
use and greenhouse gases and alleviate the negative aspects of feed production(5).
Table 1: Amino acid content of common duckweed (Lemna minor)

Amino acids G / 100 g
protein
Cysteine CYS 0.9
Methionine MET 1.6
Asparagine ASP 8.2
Threonine THR 4.0
Serine SER 4.1
Glutamine GLU 9.8
Glycine GLY 4.6
Alanine ALA 5.1
Valine VAL 4.6
Isoleucine ILEU 3.7
Leucine LEU 7.3
Tyrosine TYR 3.1
Phenylalanine PHE 4.4
Lysine LYS 5.0
Histidine HIS 1.5
Arginine ARG 4.8
Proline PRO 3.8
Source: Appenroth, et al(7).
Fats
Fats play an important role in the human diet; despite being stigmatized as nutrients harmful
to health, in the last decade some studies have shown the great impact they have by
contributing as protective factors against degenerative diseases such as Alzheimer’s,
409
reducing the risk of cardiovascular accidents and even, mortality. Authors report a content of
4 to 6 % fat content per dry weight(6).
An approximate content of 30 % saturated fatty acids, specifically high levels of palmitic

acid, is highlighted. Recently, in terms of fatty acid contribution by common duckweeds, it
has been shown that it adapts very well to the requirements of the human population, with a
low fat intake and a good ratio of polyunsaturated to monounsaturated fatty acids, generally
close to or more than half of the total fatty acid content, ranging from 55 to 63 %(22). It is also
important to highlight the presence of n-3 class polyunsaturated fatty acids (alpha-linolenic,
eicosapentaenoic, and docosahexapentaenoic acids), which are important in human
metabolism and act as anti-inflammatories(23). Other authors have described that 48 to 71 %
of fats are polyunsaturated fatty acids and the ratio of omega-6 to omega-3 fatty acids is 0.5
or less(6).
Carbohydrates and fiber
Several studies with common duckweeds have concluded that starches or carbohydrates are
fairly underrepresented in the analysis of the nutritional composition of these plants; in the
best of cases, carbohydrates represent approximately 10 % of the total nutritional value of
common duckweeds(24).
Some authors have shown that the growth environment, genetics of the species, nutrients of
the medium, temperature range, time, and intensity of sunlight cause differences in the
biochemical components (crude protein, ash, cellulose, water, fats, and minerals). It has been
documented that the protein content of common duckweed depends primarily on the nutrient
content in the water body, while the accumulation of minerals in common duckweed tissue
depends primarily on the water conditions in the growing environment. During artificial
cultivation, the starch, lipid, and protein content in common duckweeds can be controlled by
changing factors affecting the common duckweed growing environment, such as pH value,
temperature, medium structure, etc.(25).
On the other hand, the fiber content, unlike carbohydrates, is considerably high; common
duckweeds can contain up to 25 % of the total nutritional value in fiber(26). This high fiber
content represents an excellent option for inclusion in the human diet, which, together with
a large contribution of protein and a small contribution of fats and carbohydrates, would be,
according to several authors, completely beneficial in healthy lifestyles(27).
410
Minerals and trace elements
The nutritional composition in terms of minerals in common duckweeds is characterized by

being plants rich in potassium and iron, and poor in sodium; in contrast, in terms of trace
elements, the content of manganese, zinc, copper, among others, stands out (Table 2). On the
other hand, the total ash content is moderately high, with ranges of up to 18 % in some
studies, however, these values may vary according to the composition of the medium(6,26).
Table 2: Contribution of trace elements from common duckweeds in mg per kg of total

edible part
Trace elements mg / kg
Magnesium Mg 2850  710
Iron Fe 230  90
Manganese Mn 230  98
Iodine I 0.39  0.19
Cadmium Cd 0.076  0.145
Source: Ziegler P, et al(26).
Vitamins
Regarding vitamins, there are few studies on common duckweeds that have focused on
assessing the nutritional composition and presence of these micronutrients. The vitamins
found in the highest amount in common duckweeds are carotenoids, precursors of vitamin
A; the dominant carotenoid in this plant is lutein, followed by β-carotene. Other carotenoids
are found in much lower amounts, such as α-tocopherol and zeaxanthin(28,29) (Table 3).
Table 3: Carotenoid content in common duckweeds

Nutrients
Contribution
carotenoids
Lutein mg/100 g 40 – 80
-carotene mg/100 g 10 – 30
-tocopherol mg/100 g 0.5 – 13
Zeaxanthin mg/100 g 0.8 - 10
Source: Sree K, et al(28).
411
Human nutrition
In a study carried out by the Panel on Nutrition, Novel Foods, and Food Allergens (NDA) on
the safety of the complete plant material of Lemna minor and Lemna gibba as a novel food
in accordance with Regulation (EU) 2015/2283 in 2022(17) for consumption as a vegetable,
toxicological, nutritional, microbiological analyses of powder from common duckweeds
grown in greenhouses under controlled conditions were carried. Based on the proposed uses,
expected intake and compositional data, the intake of heavy metals, microcystins and
micronutrients, except manganese, it does not pose safety concerns for consumption as a
novel food. However, the findings on neurotoxicity and the possible higher susceptibility of
some subgroups of the general population, oral exposure to manganese beyond that normally
present in foods and beverages could pose a risk of adverse health effects without evidence
of any health benefit. On the other hand, the likelihood that the product may trigger allergic
reactions in humans is similar to that of other leafy vegetables, and therefore the level of risk
is considered low.
In this research, there were two human trials: a randomized crossover trial and a parallel
controlled trial with healthy subjects. The commission’s panel noted that the human studies
provided were primarily designed to research putative beneficial effects and addressed only
a limited number of safety-relevant evaluation criteria. The Panel considers that no adverse
events related to consumption were reported, however, it is noted that no conclusions can be
drawn from these studies about the safety of the product.
In some parts of Southeast Asia, such as Laos, Thailand and Myanmar, its consumption is
normal in preparations such as salads, soups, curries or omelets as a source of vegetable
protein, however, it has not been included as part of the diet in Western countries(16).
Common duckweed has been shown to have an amino acid profile that favors the diet of
aquatic and terrestrial animals, a contribution of vitamins and minerals that contribute to its
palatability, a concentration of fats (4 to 7 %) and starches (4 to 10 %) adequate when
compared to other plant-based foods such as dried legumes; in addition, knowing the poverty
rates in some populations of the world, and the high need for nutritional supplementation in
populations with limited access to healthy food, it could be of great relevance in human
nutrition(16,30).
In the case of Thailand, common duckweeds, in their variety of species, are marketed in
vegetable markets and their acceptance in the population such as Khai Nam, Khai Pum, and
Khai Phae (generally translated as “water eggs”) stands out in the preparation of traditional
local dishes such as salads, curried vegetables, and omelets(31).
412
Israeli studies seeking to compare the postprandial and nocturnal glycemic response by using
dairy shakes from common duckweeds of the species Wolffia globosa observe in these plants
a great opportunity in human nutrition, especially in population groups with difficulties in
carbohydrate metabolism. According to these studies, this species of algae could serve as an
emerging alternative plant protein source with potential beneficial postprandial glycemic
effects, however, no scientific information has been found with the species Lemna minor(29).
Considering the nutritional contribution of common duckweeds, especially their outstanding

contribution of proteins, fats, beta-carotenes, minerals, and low carbohydrate contribution, in
addition to the absence of antinutritional substances, common duckweeds (Lemna minor)
could represent an excellent option for consumption as a supplement in the dietary pattern of
needy communities throughout the world(32). However, in cases of uncontrolled growing
conditions, and particularly when fertilizers, pesticides and other organic contaminants are
present in large quantities at cultivation sites or in cases of water pollution by algae or
microbes, the high concentration of contaminants or toxins in those plants may pose a
potential risk to human health that should be considered(17). On the other hand, Appenroth et
al(6) indicate that Wolffiella hyalina and Wolffia microscopica are suitable for human
nutrition, even compared to other common duckweed species, regarding amino acid
composition and fatty acid distribution.
Animal nutrition
The high cost of feed for animal raising has led to the constant search for alternatives to
improve animal production. At present, soybean meal is one of the most widely used
alternative ingredients to replace fishmeal in animal feed due to its high protein content and
relatively well-balanced amino acid profile, which can generally meet the requirements of
many fish species. However, soybean meal is already in high demand in the human food
chain, both directly and indirectly in feed for farmed terrestrial animals. This competition
means that soybean meal is an expensive ingredient and this may limit its use as an ingredient
to meet future fish feed demands. Therefore, there is a constant need to find other ingredients
(insects, vegetables, algae, byproducts of aquatic organisms) to replace both fishmeal and its
main substitute, soybean meal, in farmed fish feed. Ideally, such ingredients should be
unconventional in order to avoid or minimize competition with other animal feed sectors(33).
Among these alternatives, common duckweed represents a great opportunity given its
accelerated growth and great ability to adapt to the environment in which it grows; this,
without leaving aside the source of raw vegetable protein that it represents, its contributions
413
of minerals, xanthophylls and amino acids such as lysine, threonine, and valine(18,34). Hence
the great opportunity from an economic point of view that it can represent.
Under experimental conditions, the production rate can be close to 183 extrapolated metric
tonnes/ha/year of dry matter, although yields are closer to 10-20 tonnes DM/ha/year under
real conditions. Its use has mostly occurred in a wide variety of animals of social interest
such as farmed birds, ruminants, non-ruminants, and farmed fish(35). In which, through
different inclusion models, it has been shown that it can be a good complement to the food
diet of livestock and fish(36,37).
Models implemented in small farms in the Asian continent focused on the recovery of
nutrient flow from animal waste have used the resulting common duckweed biomass as a
fresh feed for ducks, farmed fish and pigs; all this evidencing the contribution to the correct
nutrition of these animals and reducing feed costs(7,38,39).
Common duckweed has similar or superior characteristics to plant-based proteins, such as

legumes, however, it is rich in some essential amino acids. In Asian and Latin American
countries, there are reports of the use of common duckweed in the diet of farmed pigs, with
an inclusion of up to 10 % of the total feed consumption, showing excellent results in their
reproductive response(37).
In Latin American countries such as Mexico and Venezuela, common duckweed is used to
feed pregnant sows and piglets, replacing 80 % of protein from soybean cake or fishmeal as
a whole, with exceptionally good results in production(8). According to some authors,
common duckweed reaches protein levels of up to 38 % of its biomass. This protein
contribution and its ease of cultivation has allowed trials as feed for domestic ducks,
obtaining results in weight gain and egg production comparable to the usual protein
supplement, with the advantage of a 25 % decrease in feed costs in Asian countries(33).
Other agricultural models have implemented common duckweed as a fodder crop for
livestock raising, considering that common duckweed biomass has a protein content of more
than 30 % of dry weight, representing an excellent complement in the feed of farmed animals,
environmental sustainability, and cost reduction(34). When it is used as the only source of
nutrition, at a rate that should not exceed 6 % of body weight (dry basis), the results are much
lower than those obtained with conventional diets, at which point they cease to be potentially
beneficial; however, experiences in polycultures have shown that common duckweed
supplementation increases production per hectare(8). In this way, for more than 50 years,
science has studied the different alternatives that common duckweed represents in the
nutrition of different species of animals for consumption, yielding promising results as it is
a rich and sustainable source of protein.
414
Now, some research on feeding dry common duckweed, Lemna minor, as a protein source in
the diet of common carp fry, has shown that there are no significant differences in the growth
and development of fish that are fed diets supplemented with up to 20 % common duckweed
versus commonly used fish protein fodder. Showing that a diet consisting of up to 20 %
common duckweed content could be used as a complete replacement for commercial feed in
the formulation of the diet for common carp fry, allowing cost reduction(40).
According to research carried out by Goswani et al(41), when evaluating the impact of protein
from dry common duckweeds (L. minor) compared to the impact of the standard and
commercial diets of the fry of rohu Labeo rohita (carp native to the rivers of India and Asian
regions), slight modifications in the digestive enzymatic activity of fish were identified. The
diet with protein from common duckweed stimulates amylase, trypsin, and chymotrypsin
activities, which were significantly higher compared to other diets, but without altering or
modifying the growth rate of the fish. In this sense, the inclusion of raw common duckweed
in the feed, replacing amounts of up to 30 % of fishmeal in the diet, can be well tolerated by
farmed fish without affecting growth(42).
In the case of farmed fish for human consumption, recent studies have researched the transfer
of toxic heavy metals, such as cadmium, from common duckweed (Lemna minor) to
freshwater tilapia (Oreochromis mossambicus). Through regression analysis, significantly
positive correlations were found between the concentration of cadmium in common
duckweed and freshwater tilapia meat, concentrations that were especially found in greater
quantity in the tissues of intestine, edible muscle and remains. From this perspective, the
analyses suggest the assessment of toxicity risks(40). Other studies have researched the
potential of common duckweed as an animal feed through the fermentation process with the
addition of two probiotic strains, Bacillus strains, and B. subtilis, which have demonstrated
health benefits for poultry, demonstrating that common duckweed is a promising alternative
resource and has the opportunity to become a valuable resource in multiple industries such
as that of foods, biofuels, pharmaceuticals, and wastewater phytoremediation. With the
potential to increase sustainability, food security and reduce environmental impact(43).
Environmental impact of common duckweeds
With industrialization and the increase in the production of needs on a large scale by society,
the pollution of both surface and underground water bodies has become a major problem
with environmental and social impact(44). The accumulation of numerous toxic substances in
waters has led to the search for inexpensive and reachable options that allow to identify the
level of toxicity that these natural spaces may have. Thus, the recent use of common
415
duckweed (Lemna minor and Lemna gibba) crops has made it widely possible to analyze the
toxicity transmitted by water to organisms higher in the biological chain (animals and
humans)(45).
Other studies where growth parameters and assessment criteria, such as pigment content,
peroxidase activity, lipid peroxidation and alkaline comet assay, were used to detect the toxic
and genotoxic effects of surface water samples in common duckweed plants were able to
indicate the ability of selected biomarkers to predict the phytotoxic and genotoxic effects of
complex water mixtures on living organisms, as well as the relevance of common duckweed
as a sensitive indicator of water quality(46).
The inhibition of growth and reduction in the photosynthetic pigment of this plant when
growing in polluted water environments have allowed its use as an effective biomarker in the
non-specific detection of toxic components in water bodies. However, it should be
recognized that although it is a good indicator of water pollution, common duckweed does
not allow the nature of the agents or substances responsible for such toxicity to be determined
by itself(47).
Recent studies, despite the impossibility of identifying these toxic agents from common
duckweed, have managed to document the adaptive capacity they have to metabolize some
of these substances, such as nickel and ammonia, bringing the quality of water bodies to
acceptable levels in a prudent time, a process known as phytoremediation of water bodies(48).
Aquatic plants have been shown to be highly efficient in removing organic and inorganic
pollutants(49). Lemna minor has been widely applied for the remediation of various chemical
contaminants. The plant is used separately or in combination with other aquatic macrophytes
as an ecologically based pollution treatment technology(50). L. minor has been reported as a
floating microphyte highly successful for the phytoremediation of organic pollutants; it was
the most effective plant in the treatment of wastewater for the remediation of municipal
effluents. There was 98.8 % removal for total nitrogen and phosphorus, with a higher level
of oxygen dissolution due to an improvement in nutrient loading by common duckweed(51).
Common duckweed has shown great potential for phytoremediation of organic pollutants,
heavy metals, agrochemicals, pharmaceuticals, personal care products, radioactive waste,
nanomaterials, petroleum hydrocarbons, dyes, toxins, and related contaminants(50).
Substances that pose a serious risk to the environment and all forms of life because they can
be persistent, are easily transported through the media, and can cause poisoning of tissues
and organs(52,53,54). Tufaner(55) reported more than 90 % removal of heavy metalloids
(chromium, zinc, aluminum, arsenic, cadmium, cobalt, copper, lead, and nickel), while 83 %
for mercury in a mixture of wetland with L. minor. On the other hand, Lemna minor shows
an increase in chromium absorption percentage of 6.1, 26.5, 20.5, and 20.2 % at a different
exposure concentration of chromium stress(56,57). In addition, research conducted in relation
416
to agricultural chemicals such as fertilizers, pesticides, herbicides, and fungicides shows that
common duckweeds can accumulate and degrade these agrochemicals(58,59,60). Common
duckweed has the ability to conserve nature by acting as a hyperaccumulator. The wide
application of the plant is due to its ubiquitous nature, invasive mechanism, sporadic
reproductive capacity, bioaccumulation potentials and resilience in polluted environments(61).
Toxicity and antinutritional substances
Studies have highlighted the presence of antinutritional components, substances, or factors

in common duckweed(3). After several toxicity tests, it was discovered that common
duckweeds are extremely sensitive to triazines, sulfonylureas and pyridines, compounds
currently classified as toxic with a great polluting impact on the environment and that, given
the way of nutrition of this plant, they can absorb them. However, many authors point out
that the amounts of these compounds in this plant are small, and that they could be susceptible
to denaturation when subjected to heat treatments(62).
Antinutritional factors are substances or compounds that have the ability to interfere with the
biological use or exploitation of a food or nutrient, affecting a person’s health and some or
more of the physiological processes of the body. Some authors have reported the presence of
tannins and phytic acid in common duckweeds in concentrations of 0.02 and 0.09 %,
respectively(63).
Likewise, other studies showed concentrations of trypsin inhibitors at 1.47 %, calcium

oxalates at 3.5 % and tannins in concentrations much higher than previously cited studies,
0.9 %(64). However, recent research has highlighted low concentrations of cyanide at 0.15 %,
phytic acid 0.58 %, and tannins 0.48 % when analyzing a wide variety of common duckweed
strains; likewise, these samples were subjected to heat treatments where the deactivation or
inhibition of these substances was evidenced, thus eliminating the toxicity that could be
implied by the consumption of the plant(65).
According to research carried out by Sree et al(66) to determine the cytotoxic effects and
antiproliferative activity in human cell lines of several common duckweed species, including
L. minor, it was found that whole plant extracts do not have any detectable adverse effect in
human cell lines, which is a step towards ensuring the global use of common duckweed as a
component of human nutrition.
417
Conclusions
In recent years, common duckweed has taken on a prominent role in biotechnology and
agricultural applications. It could potentially be an important resource as an alternative source
of food for humans and animals. It has been used as raw or processed feed for meal
production, making it interesting in the animal feed industry, aquaculture, health
supplements, biofertilizers, biofuels, and emerging human food products.
It has demonstrated its strong potential for phytoremediation of organic pollutants, heavy
metals, agrochemicals, pharmaceuticals, personal care products, radioactive wastes,
nanomaterials, petroleum hydrocarbons, dyes, toxins, and related contaminants. The wide
application of the plant is due to its ubiquitous nature, invasive mechanism, sporadic
reproductive capacity, bioaccumulation potentials, and resilience in polluted environments.
The nutrients in the water in which it is grown critically affect its nutritional value, so it will
likely need to be decontaminated before feeding the animals if there are heavy metals in the
water, since common duckweed concentrates them. In this sense, it is important to highlight
the scarcity of studies on the use of these plants in human nutrition; therefore, it is necessary
to continue research to determine the role they could take and be included in the human diet
and the safety associated with their continuous consumption, large-scale yield, economic
market supply and sustainability.
Despite the challenges and knowledge gaps, there are realistic opportunities to develop and
operate controlled, autonomous, high-capacity common duckweed crops under indoor
conditions, for a wide range of purposes that ensure the characteristics of the final product.
It should be noted that accelerated growth, the impact of climate change, the decrease in
arable land, the depletion of soil, nutrients and water supply make it increasingly difficult to
obtain quality food in the quantities required.
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424
Review
Gabriela Guadalupe Gómez Verduzco a
Ernesto Ávila González a
Guillermo Téllez Isaías b
Juan Carlos Del Río García c
Jacqueline Uribe Rivera c*
a
Universidad Nacional Autónoma de México. Facultad de Medicina Veterinaria y Zootecnia.
Ciudad de México, México.
b
Universidad de Arkansas. Departamento de Ciencia Avícola. Arkansas, Estados Unidos de
América.
c
Universidad Nacional Autónoma de México. Facultad de Estudios Superiores Cuautitlán.
Carretera Cuautitlán-Teoloyucan Km. 2.5, San Sebastián Xhala, CP 54714 Cuautitlán Izcalli,
Estado de México, México.
* Corresponding author: juribe_mvz@hotmail.com
Abstract:
Mycotoxins are secondary metabolites produced by fungi of various genera. Among the most
important mycotoxins are those produced by fungi of the genus Fusarium sp., which can be
divided into several groups for study, which are the groups of trichothecenes (and T-2 toxin),
fumonisins, mainly fumonisin B1 (B1, B2, B3, B4, A1, and A2), and zearalenone with
estrogenic effects. Although fusariotoxins cause similar effects because they share the same
mechanism of action, by altering protein synthesis in intoxicated poultry, it is important to
mention the incidence as well as the characteristics between each of them. Therefore, the
characteristics of each group mentioned are described in each section.
425
Keywords: Mycotoxins, Fusariotoxins, Poultry, Fungi.
Introduction
Within livestock production, feed accounts for 65 to 70 % of the total cost of production;
however, despite constant efforts to achieve the safety of food intended for animal
production, there are still factors that diminish its quality, such as biological pathogens (such
as Salmonella spp., E. coli, Listeria spp., Campylobacter spp.), chemical substances
(fungicides, herbicides, and insecticides), and presence of fungi (Fusarium, Penicillium,
Mucor etc.)(1). Fungal contamination can occur under various conditions associated with both
environmental factors and factors associated with the fungus, such as size and species(2).
Mycotoxins are secondary metabolites produced by filamentous fungi(3), considered toxic

substances that present themselves as organic compounds of low molecular weight, which is
why they do not have immunogenicity characteristics. The production of mycotoxins
depends on a series of environmental factors such as humidity, temperature, ventilation,
constitution of the substrate in which the fungus develops, damage to the integrity of the
grains, and interaction between the various fungi present in the substrates(4). It is mentioned
that 25 % of crops worldwide are contaminated with mycotoxins(5).
Fusariotoxins
Among the toxins reported with the highest incidence within animal production are those
produced by different species of fungi of the genus Fusarium sp, which are capable of
inducing acute effects, as well as chronic effects, depending on the type of mycotoxin, the
level and duration of exposure, and the species and age of the animal.
Avian species are considered resistant to the occurrence of fusariotoxin intoxication, which
has been explained either by low sensitivity to toxicity mechanisms or by differences in
toxicokinetic properties(6). These fungal species have been classified within the group of field
fungi, with grains requiring a high percentage of moisture (approximately 20 to 22 %) for the
production of mycotoxins. The main substrate where the production of fusariotoxins is
observed is corn; nevertheless, their growth has also been reported in other substrates such
as sorghum, wheat, oats, barley, and soybeans. Fusariotoxins, in turn, are classified into three
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groups for study, among which are the trichothecenes group (which in turn is classified
according to the presence or absence of a macrocyclic ring in their chemical structure), the
zearalenone group and the fumonisin group (Table 1).
Table 1: Mycotoxins produced by fungi of the genus Fusarium sp.

Mycotoxins produced Species of producing fungi
 Trichothecenes Fusarium tricinctum, F. sporotrichioides, F.

graminearum, F. culmorum, F. poae, Fusarium
cephalosporium, F. myrothecium, F. acuminatum,
F. nivale, F. oxysporum, F. solani
 Zearalenone Fusarium graminearum, F. culmorum, F. poae, F.
roseum, F. moniliforme, F. avenaceum, F.
equiseti, F. nivale
 Fumonisins Fusarium proliferatum, F. verticillioides, F.

oxysporum
Adapted from(11,94,95).
Experimental studies have agreed that the main effect of the toxins produced by Fusarium
sp. has a direct impact on intestinal integrity (increased intestinal permeability), an effect
correlated in turn with a decrease in the animal’s immune response (alteration in the
production of cells involved in the immune response)(7,8). Both effects have their origin in
the alteration of protein synthesis that takes place in the different metabolic processes in the
animal(9,10). It is important to mention that regardless of the mycotoxin or mycotoxins that
are present in the feed, the cells of the digestive tract are the first to be in contact with the
mycotoxins; this means that, possibly, the entire intestinal epithelium can be compromised
even before the absorption of the feed begins, and in turn, it can also be affected by
mycotoxins that have a low absorption rate, such as fumonisins and deoxynivalenol.
Although fusariotoxins may have similar mechanisms of action among them, it is important
to mention the individual characteristics of each(11).
Trichothecenes generalities
More than 150 different types of trichothecenes have been identified, and it has been
established that, based on their occurrence in food, the most prevalent are T-2 toxin,
deoxynivalenol (DON or vomitoxin), and diacetoxyscirpenol (anguidine), which in their
chemical structure consist of a tetracyclic sesquiterpene skeleton, an oxane ring, and a stable
epoxide group (12,13-epoxytrichothecene) that confers its toxicity (Table 2)(7). The main
substrates where can be find the presence of trichothecenes are mainly corn, wheat, barley,
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oats, rice, and soybeans. Broadly speaking, trichothecenes are considered to be cytotoxic,
immunosuppressive and inhibitors of protein synthesis(8). Although trichothecenes can
generally cause gastrointestinal, dermatotoxic, immunotoxic, and genotoxic effects, it has
been reported that, under acute intoxication, they can be identified by evident skin
inflammation, diarrhea, edema, dermal necrosis, hemorrhages in the mucosa of the
gastrointestinal tract, and negative alterations in productive parameters(12,13,14).
Table 2: Structural differences in trichothecenes present in poultry production
Trichothecenes R1 R2 R3 R4 R5
Type A
HT-2 toxin OH OH OAc H OCOCH2CH(CH3)2
T-2 toxin OH OAc OAc H OCOCH2CH(CH3)2
Diacetoxyscirpentriol OH OAc OAc H H
Type B
Deoxynivalenol OH H OH OH O
3-acetyl-deoxynivalenol OAc H OH OH O
15-acetyl-deoxynivalenol OH H OAc OH O
Nivalenol OH OH OH OH O
Fusarenon X OH OAc OH OH O
Adapted from(18).
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T-2 toxin
Main characteristics and chemical structure
The T-2 toxin, belonging to group “A” of trichothecenes, contains in its tetracyclic chemical
structure a sesquiterpenoid system and an epoxy-trichothecene group in C-12 and C-13. It
can mainly be found in corn, wheat, oats, and barley(15,16).
It has been determined that the main fungus producing this mycotoxin is Fusarium
tricinctum, however, it can be produced in turn by Fusarium acuminatum, nivale, oxysporum,
poae, solani and sporotrichioides. In relation to this, the environmental conditions required
for the production of the fungus are an average environmental temperature of 18 to 30° C, as
well as a relative humidity of around 95 % and a water activity (AW) value greater than 0.88
(which must be 0.91 for the fungus to produce the mycotoxin)(17). The chemical structure of
this mycotoxin makes it a non-volatile compound soluble in acetone, chloroform and ethyl
alcohol; it is of low molecular weight (approximately 466.52 g/mol) and is described as a
mycotoxin highly resistant to heat (200-210 °C) and UV radiation, therefore, it is not easily
inactivated in stages during feed processing; nevertheless, it has been reported that the
addition of sodium hypochlorite or hydroxide for a period of 4 h can work as an inactivation
method(18).
It has been reported that the maximum levels allowed within the European regulation in
finished feed for T2 toxin range from 0.2 to 2 mg/kg of finished feed(19,20) and a concentration
of 4.97 mg/kg of feed has been established as LD501 and 10 mg/kg as LDPV2(18). It should be
considered that T-2 toxin can generate interactions in the presence of other mycotoxins,
which will be additive in the presence of deoxynivalenol (DON), ochratoxin A (OTA) and
fumonisin B1 and synergistic in the presence of nivalenol and aflatoxins(21-25).
Metabolism
The main pathways by which T-2 toxin will be metabolized in poultry are mainly de-
epoxidation and de-acetylation(26). In the case of de-epoxidation, which is the most common
pathway, the result will be the loss of the “epoxy” group, which confers toxicity to the
mycotoxin, while during the loss of the “acetyl” group, the result will be the obtaining of
secondary metabolites of the toxin, such as HT-2 and T2-tetraol(6).
Mechanism of action and main effects in poultry
T-2 toxin and its derivatives base their toxicity on the negative alteration it will cause on
protein, DNA and RNA synthesis, as well as on the cell cycle in different types of cells and
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its ability to induce apoptosis and necrosis, as well as lipid peroxidation, mainly in labile or
actively producing cells. It interacts with the peptidyl transferase of the ribosomal 60s
subunit, inhibiting the formation of new peptide bonds(27). Apoptosis caused by T-2 toxin
was evidenced in cell lines such as Vero and human hepatocarcinogenic(28). In addition, it
has been reported that T2 toxin reduces production parameters, with oral exposure to the
toxin being the main route of access to the body(18).
Effects on the immune system
In general, T-2 toxin has a time/dose-dependent immunosuppressive effect, either high

concentrations for a short period of time or low concentrations continuously(29). The presence
of leukopenia has been reported, leading to an increased susceptibility to secondary infections
(Listeria monocytogenes and Salmonella sp). T-2 toxin has also been associated with a
decrease in the amount of antibodies against Newcastle disease and infection of the bursa of
Fabricius(18). In addition, it has been described that it can act as an immunostimulant by
increasing IgA levels; this is related to the abnormal and transient activation of genes
involved in the inflammatory response(26,27). It is also mentioned that it can alter the
maturation of antigen-presenting cells by modifying antibody levels by lymphocyte
proliferation, leading to an increase in susceptibility to infectious agents(30).
Effects on the digestive system
Although T-2 toxin has a rapid absorption in the gastrointestinal tract and an elimination of
approximately 80 to 90 %(19), its toxicity is mainly effected during the enterohepatic
circulation, and the negative effect it has on the liver is the decrease of the enzymatic activity
necessary for the metabolism of toxic substances and the induction of lipoperoxidation,
which will consequently result in the formation of free radicals(18,31,32). It is important to
mention that the alteration on mucous membranes will be an evident characteristic related to
the diagnosis of mycotoxicosis caused by trichothecenes, especially by T-2 toxin, this related
to the caustic effect of the toxin, and it will be visible particularly in the oral cavity, being
observed as dermonecrotic lesions that will go from a whitish coloration at the beginning of
exposure to a black coloration in chronic exposures. Necrotic lesions may also be observed
in gizzards, intestinal mucosa, proventriculus and liver(33,34).
Effect on the nervous system and productive performance
The effect on the nervous system occurs due to the inhibition of protein synthesis, which
leads to an increase in the concentration of the amino acid tryptophan, a precursor of
serotonin, which in turn increases the concentration of serotonin, thus causing the activation
of serotonergic neurons(18,35). The synergistic presence of DON with T-2 toxin can increase
this effect, and anorexia, locomotor problems and vomiting can be observed(36,37); lesions
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present in the oral cavity should also be considered, which will contribute to the decrease in
consumption, thus affecting body weight at the end of the cycle, also observing a deficient
uniformity in the flock(19,38,39). In the case of laying hens, a decrease in egg production, as
well as in shell quality and hatchability, can be observed(38).
Deoxynivalenol
Deoxynivalenol, also known as DON or vomitoxin, is a common fusariotoxin in production

poultry, which may be more resistant than other consumption species(24,21,40). Deoxynivalenol
can be found in finished products such as pasta, bread, cookies, and beer. Although it is
considered a teratogenic mycotoxin, it has not been classified as carcinogenic, mutagenic, or
genotoxic(41).
Characteristics and metabolism
DON is a stable organic polar compound with high resistance to acidic pH media and high
temperatures (up to 180 °C). Its structure contains three free hydroxyl groups associated with
its toxicity(42). The lower susceptibility of poultry has been related to their low percentage of
intestinal absorption, which is approximately 5 to 20 %, while their excretion is 78.6 to
98.5 %, mainly through the bile duct, in a period of 24 to 72 h(43,44). In the case of poultry,
the main metabolic pathways of DON are sulfation, glucuronidation, and de-epoxidation(45).
When DON is metabolized through acetylation processes, compounds such as 3-acetyl-DON
and 15-acetyl-DON will be obtained, which can also be found in feeds, and their importance
lies in the fact that they can easily reverse DON and recover its toxicity(46). Other compounds
derived from deoxynivalenol are DON-3-glucoside, as well as masked compounds that have
gained importance for their ability to recirculate in the body, recovering their toxicity
characteristic; in addition, they have been recognized as having the capacity to produce
mixed toxic effects, causing adverse effects on animal welfare and productivity(15,47,48).
Inhibition of protein synthesis
Inhibition in protein synthesis occurs when DON forms a bond through the binding of its
epoxide group with the ribosomal 60s fraction, causing an alteration in the binding of the
latter with the 40s fraction, preventing the translation of messenger RNA and preventing the
binding of amino acids to the polypeptide chain, either in elongation or during the termination
of protein synthesis, also generating an increase in the amount of polyribosomes (80s), since
the uncoupling of messenger RNA and the release of the peptide chain are inhibited(46). It has
also been reported that it can alter the activity of the enzyme ribosomal peptidyl transferase
by forming peptide bonds between amino acids(46,49). It is worth mentioning that changes in
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the conformation of ribosomes can induce a stress response, thus leading to the activation of
mitogen-activated protein kinases (MAPKs)(50).
Effect on the immune system
An increase in serum IgA concentration has been observed, which has been related to the
inhibition of protein synthesis of protein components necessary for the transport of IgA to
the hepatobiliary system(11). Alterations in humoral immunity due to DON intoxication have
been determined by evaluating the response to vaccination, mainly against Newcastle disease
virus and infectious bronchitis virus, and a decrease in antibody titers has been observed. In
the case of cellular immunity, induction of apoptosis in leukocyte cells such as T and B
lymphocytes and macrophages, alteration of the activation of CD4 and CD8 T lymphocytes
to CD4+ and CD8+, and a reduction in the serum concentration of TNF-α(51) have been
observed. DON has the ability to alter the expression of genes that code for the production
of pro-inflammatory cytokines and chemokines(52). This has also been related to the
activation of genes that also code for the activation of cyclo-oxygenase-2 (COX-2) and
nuclear factor Kappa of activated B cells (NF-ΚB)(26).
Effect on intestinal health
Deoxynivalenol is known to be a potent anorexic and emetic compound due to the alteration
in the regulation of several signaling pathways. These alterations can affect the secretion of
anorexigenic or orexigenic hormones, such as serotonin, released by enterochromaffin or
Kulchitsky cells. It has also been observed that DON, by causing severe suppression of
cytokine signaling processes, generates the activation of pro-inflammatory cytokines,
affecting growth hormone signaling by suppression of two proteins, which are the hepatic
acid-labile subunit as insulin-like growth factor and insulin-like growth factor I(52). In
addition, an increase in transepithelial electrical resistance (TEER) in jejunum is mentioned,
mainly attributed to the decrease in space between the tight junctions and a decrease in
paracellular permeability to ions(53).
Effect of DON on productivity
In the case of laying hens, it has been reported that under a concentration of 2 to 3 mg
DON/kg of feed, a slight decrease in egg production can be observed; however, fertility and
hatchability can be maintained unchanged at this same concentration(45,46). It is important to
mention that the effects may vary according to the dose ingested, and with the consumption
of high concentrations (5 to 10 mg/kg of feed) of deoxynivalenol, diarrhea may be observed,
preventing the animal from correctly assimilating the nutrients, delaying its growth, and
causing unevenness in the flock(54-56). In the case of consumption of low concentrations,
anorexia and stunted growth have been observed(6,46).
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Fumonisins
Fumonisins are a group of toxins produced by several species of fungi (Table 1), within
which the main producers of fumonisins are: Fusarium verticillioides and F. proliferatum(57).
These fungi will be found as common contaminants of substrates such as corn, rice, sorghum,
barley, peanuts, and cotton, where their optimal growth will be in temperatures between 22.2
and 27.5 °C with an AW of 0.97-0.98 for the production of the toxin(58-60).
Main features and metabolism
There are six types of fumonisins, B1, B2, B3, B4, A1 and A2 (Table 3); nevertheless, the
most important due to their level of incidence and toxicity are B1 and B2. The basic structure
of fumonisins consists of a 20-carbon alkylamine, with one or two hydroxyl groups and one
or more methyl groups or esterified tricarballylic acid(61). Fumonisins are polar compounds
that are soluble in water and organic compounds such as methanol and acetonitrile, but are
insoluble in non-polar compounds, which in turn facilitates their elimination from the body(6).
In the case of poultry, it is now known that, like pigs, acetylated or hydrolyzed derivatives
(HFB1) are also generated as a product of acetylation after hydrolysis, as part of the toxicity
mechanisms associated with their metabolism in the body(62-65).
Table 3: Basic chemical structure of fumonisins B1, B2, B3, and B4
Fumonisin B1 R1=OH; R2=OH; R3=OH

Fumonisin B2 R1=OH; R2=OH; R3=H
Fumonisin B3 R1=H; R2=OH; R3=OH
Fumonisin B4 R1=H; R2=OH; R3=H
Adapted from (66,96-98).
Mechanism of action of fumonisins
The mechanism of action of fumonisins is based on the interference in the metabolism of

sphingolipids by competitive inhibition with sphinganine from de novo synthesis and
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sphingosine from sphingolipid turnover, generating an accumulation of sphingoid bases,

blocking the synthesis of complex sphingolipids through the inhibition of the enzyme
ceramide synthase(15,66,67). In addition, sphingosine and sphinganine have pro-apoptotic,
cytotoxic, and growth-inhibiting effects(68). Sphingolipids can act as first and second
messengers in a variety of signaling pathways and play a vital role in the formation of
membrane microdomains called lipid rafts(69). Ceramide is involved in the processes of cell
differentiation, senescence, and death. Sphingosine 1-phosphate (S1-P), on the other hand,
promotes cell survival and proliferation(66,67). The immediate consequence of the inhibition
of ceramide synthase is the accumulation of the sphingoid base that functions as a substrate,
(Sa) sphinganine and, to a lesser extent, (So) sphingosine(66). The liver and kidneys are the
main target organs, although variations have been observed depending on the species, dose,
and sex(70-74). The gastrointestinal tract can also be a target organ for fumonisins since
glycosphingolipids bind to sites for microbial pathogens and their toxins, through the
inhibition of ceramide synthase in the digestive tract, it can alter the expression of
glycosphingolipid binding sites or the transport of microbial toxins, and consequently the
sensitivity of animals to infectious agents(17).
The different effects of fumonisins on different productive species have been reported, and
it has been observed that they can vary from alterations in productive variables to changes in
biochemical parameters and immune response.
Effect on production parameters
Although the negative effect on productivity has generally been observed in high
concentrations, it has also been reported that, in concentrations around 5 ppm, it can cause
low uniformity in production parameters, mainly on body weight at the end of the cycle in
broilers(75).
Morphological and blood biochemistry alterations
The morphological alterations that have been observed in the case of production poultry are
a decrease or increase in relative weight in organs (heart, liver, spleen, bursa of Fabricius,
proventriculus)(76-78), hydropericardium, fatty liver or friable liver, hyperplasia of bile ducts,
cardiac degeneration and necrosis, and loss of tonicity in gizzard and proventriculus(77,79). In
addition, an increase in serum calcium and cholesterol values and a decrease in liver enzyme
values (aspartate amino transferase, alanine amino transferase, lactate dehydrogenase, γ
glutamyl transferase) have been observed, suggesting an injury to hepatic metabolism(80,81).
434
Effect on immune response
Among the lesions that have been observed as part of the damage of fumonisins to the
immune system in poultry, hemorrhages, leukocyte infiltrations, fat infiltration, necrotic
lesions, fibrosis in the liver, kidneys, lung, heart, intestine, gizzard, bursa of Fabricius and
pancreas, as well as edema and hemorrhages in the brain are described. Cortical atrophy in
the thymus, multifocal hepatic necrosis, and biliary hyperplasia have also been observed,
leading to lymphoid depletion(82). On the other hand, a reduction in the size of the spleen may
occur along with depletion of white pulp, thinning of cardiac myocytes, lymphoid cell
depletion in the bursa follicles, and renal tubular nephrosis at dosages exceeding 150 mg/kg
of feed(83-85). Studies on the effect of fumonisins have been conducted using high
concentrations of fumonisin; however, it has been reported that, on average, a concentration
of between 3 and 5 mg/kg of fumonisin B1 has been found in the main components of diets
intended for animal feed, such as corn(86).
Zearalenone
Zearalenone (previously known as F-2 toxin) is a non-steroidal estrogenic (mainly in pigs),

hematotoxic and genotoxic (rodents) fusariotoxin. The main fungi that produce zearalenone
are F. graminearum, F. oxysporum, F. roseum, which require temperatures of between 21
and 25 °C and an AW of approximately 0.87 for the production of the toxin. In particular, it
can be found contaminating corn, barley, rice, and soybeans, as well as finished products
such as meals and beer(87). In the case of poultry, it is considered that the consumption of a
high concentration is required to observe negative effects on production; in any case, it has
been reported that turkeys and ducks are the most susceptible species in poultry, as is the
case with other mycotoxins(88).
Metabolism and chemical structure
The characteristics of zearalenone allow it to be a compound of rapid biotransformation and

excretion. This means that it is not easy to find zearalenone in poultry products(89).
Zearalenone has a percentage of absorption by the digestive tract of approximately 10 % in
poultry, and a high percentage of elimination of both zearalenone and its conjugation
metabolites (approximately 65 %)(90,91). The structure of zearalenone is composed of a
resorcyclic lactone ring like its main derivative, zearalenol (α-zearalenol), which will be a
derivative with a higher degree of toxicity than zearalenone. In the case of poultry, the main
pathways involved in zearalenone metabolism are sulfation and glucuronidation(6).
435
Mechanism of action
The main mechanism of action used by this mycotoxin is the disruption of endocrine
metabolisms, this is done through interaction with nuclear estrogen receptors (ERs), with a
direct effect on transcription (which is estrogen dependent) in the nucleus (mechanisms of
competition with the same estrogens); although the structure of zearalenone is not similar to
that of estrogens, it can place an OH group belonging to its lactone ring in an advantageous
position for its interaction with estrogen receptors(92).
Alterations caused by zearalenone in poultry
In the case of roosters, a reduction in the size of the testicles can be observed, which can
microscopically show fatty degeneration and atrophy of the germinal epithelium. Finally,
production parameters can also be observed to be decreased, related to a decrease in feed
consumption(93).
Conclusions
It is important to correlate the effects produced experimentally, which are generally based on
the use of concentrations much higher than those found naturally in the feed, and generally
continuously for long periods, and not to underestimate the presence of more than one
mycotoxin that can enhance the individual effect of each of them. It should also be considered
that, in the case of poultry, pathways of absorption, distribution and metabolism of toxins
may be different from other species. As already described, fusariotoxins can lead to large
economic losses in production, either due to the alteration of production parameters or their
alternate effect on intestinal integrity and immune response, therefore, it is necessary to make
a correct diagnosis if the presence of mycotoxins in the feed is suspected.
The authors declare no conflict of interest.
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Review
Contribution of forage grasses to biological nitrogen fixation and their

response to diazotroph inoculation. Review
Dania Fonseca López a
Nelson Vivas Quila b
Raúl Cuervo Mulet c
Carlos Eduardo Rodríguez Molano d*
a
Universidad Santo Tomás. Centro de Investigación en Ciencia y Tecnología para el
Desarrollo Sostenible. Tunja, Colombia.
b
Universidad del Cauca. Facultad de Ciencias Agrarias. Grupo de Investigación
NUTRIFACA. Popayán, Colombia.
c
Universidad de San Buenaventura. Facultad de Ingeniería. Cali, Colombia.
d
Universidad Pedagógica y Tecnológica de Colombia UPTC. Facultad de Ciencias
Agropecuarias. Tunja, Colombia.
* Corresponding author: carlos.rodriguez@uptc.edu.co
Abstract:
The use of chemical inputs has led to the loss of microbial diversity involved in the N cycle,
such as diazotrophic bacteria, which are inhibited by saturation of the receptors responsible
for activating nitrogenase. Biological nitrogen fixation (BNF) in forage grasses can be used
as an ecosystem service. The aim of this review was to analyze the contribution of forage
grasses to BNF and their response to inoculation of non-symbiotic diazotrophs in order to
find study opportunities. The analysis of the information was carried out using the prisma
methodology of systematic reviews and meta-analyses. It should be noted that the main
forage species that contribute to BNF are Brachiaria sp. and Pennisetum sp. The inoculation
of Azospirillum sp. has generated a growth-promoting effect in grasses, but the response of
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the inoculated forage depends mainly on the synergy between plant and bacteria, showing
neutral, antagonistic, and positive effects.
Keywords: Fertilization, Nitrogen fixation, Forage, Nitrogenase, Pastures.
Introduction
In livestock systems, animal feed is economically viable when the ration is mainly made up
of forage. Nonetheless, it is necessary to produce grass in an eco-efficient scenario to
compensate for the environmental footprint caused by livestock farming, considering that in
Colombia it occupies 80 % of agricultural land(1). The proposed strategies include the use of
improved forage species, diversification of the system(1) and the utilization of natural
phenomena such as biological nitrogen fixation (BNF)(2). This is a process in which
diazotrophs transform atmospheric nitrogen (N) into ammonium from the nitrogenase
enzyme complex, and contributes about 62 %, which is equivalent to 11.29 million tonnes
(Mt) of nitrogen per year, which enters the Latin American agricultural ecosystem, while
chemical fertilization contributes approximately 6.81 Mt N per year(3). BNF is a resource that
can be used as a technological tool to reduce the application of nitrogen fertilizers of synthetic
origin that have low efficiency (approximately 40-50 %) and contribute to the emission of
greenhouse gases (ammonium, ammonia, and nitrous oxide)(4) and soil salinization(5).
Nevertheless, little is known about the contribution of forage grasses to BNF and the bacterial
species with the best productive effect. Therefore, this paper aimed to analyze the
contribution of forage grasses to BNF and their response to the application of biofertilizers
constituted by non-symbiotic diazotrophic bacteria, pure and in consortium, based on a
systematic review of literature to find study opportunities.
The prisma methodology of systematic reviews(6) was used; the databases consulted were
Scopus and Web of Science; for the search for information, the following criteria were
established: a) specificity, based on the use of Boolean operators, b) sensitivity, with CAB
descriptors; c) comprehensiveness, through the verification of descriptors of interest. The
search strategy was based on the following routes: TITLE-ABS-KEY (“Biofertilizer”) and
TITLE-ABS-KEY (“Biofertilizer and Grass”). With the general search, a total of 6,813
records were found between the Scopus (n= 4,621) and Web of Science (n= 2,192) databases.
The search was limited to the Boolean connectors “Biofertilizer and Grass” from which 128
records were found (Scopus: 84 records and Web of Science: 44 records), which were
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imported into the Mendeley software and grouped by years; the analysis was limited to the
period 2012-2022 (n= 80 records), then duplicate documents were removed (n= 2 records).
Articles evaluating the effect of the application of biofertilizers on forages or the contribution
of nitrogen fixed by these plants were included in the analysis. Publications with a title
outside the search of interest (n= 5) and with only descriptive information that did not meet
the inclusion criteria (n= 13 records) were excluded. Each record was independently
reviewed by all authors for a total of 50 studies included within the review. The results of the
analysis were defined as: a) Nitrogen fixed by forage grasses, b) Biofertilizers applied and
their effect on forage grasses. The data of interest in the study (fixed nitrogen and plant effect)
were tabulated and grouped by topic to measure their effect. A nonlinear regression analysis
was performed with the number of records obtained from the sigmoidal models 3,4,
Gompertz 3, and Hill 3. The models with the highest fit were selected based on the
significance value and fit of the coefficient of determination to establish the overall trend of
the area of interest.
Biological nitrogen fixation in forage grasses
In this review, it was identified that the test of choice for determining nitrogen fixed by forage
grasses is natural abundance of 15N(7). In the main studies reporting N fixed by forage, it is
highlighted that the rate of N fixation differs between species (Table 1). This has a direct
relationship with the populations of diazotrophic bacteria that interact with each type of
forage, in Brachiaria sp., approximately 102 to 108 CFU g-1 soil are estimated(8). On the other
hand, in Pennisetum sp., the diazotrophic bacterial population is reported to be 102 to 106
CFU g-1 soil(9).
Table 1: Some reports of forage species contributing to biological nitrogen fixation

according to the review analysis
Crop N fixed (%) Source
Aristida laevis 36 Marques AC, et al(2)
Pennisetum purpureum 18-70 De-Morais RF, et al(10)
Megathyrsus maximus sp. 16 - 39 De-Carvalho EX, et al(11)
Brachiaria sp. 5.1 – 45 Leite RDC, et al(12)
Miscanthus giganteus 16 Leite RC, et al(13)
Source: prepared based on the indicated citations.
It was found that the main bacterial genera that persist in the rhizosphere and plant tissue of
Brachiaria sp., Pennisetum sp., Megathyrsus sp., and Panicum sp., correspond to
Enterobacter sp. (6 %)(10), Azospirillum sp. (25 %)(12,13,14), Azotobacter sp., Bacillus sp.
(14 %)(2,15), Herbaspirillum sp. (11 %), Burkholderia sp. (8 %)(14), Bradyrhizobium sp. (6 %),
Klebsiella sp. (5 %)(11,16), Sphingomonas sp. (4 %)(17), other (2 %). However, their
distribution in roots, leaves and stems varies by forage species, locality, and soil type(18).
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These microorganisms do not cause structural modifications in the plant and are encoded by
the nifH(4) gene. The BNF process is carried out in sites with lower oxygen saturation to avoid
nitrogenase inactivation, such as in clays, or through a reduction in intracellular oxygen
concentration through an increase in cellular respiration(19). During the reaction, eight
electrons are pumped at high speed from a donor agent (ferredoxin or flavodoxin) to the
nitrogenase enzyme complex consisting of the metalloenzymes dinitrogenase reductase or
protein Fe encoded by the nifH gene and the dinitrogenase metalloenzyme encoded by the
nifD and nifK genes(20). Dinitrogenase reductase transfers each electron to dinitrogenase and
they are stored in the FeMo cofactor, the binding site of N until it is reduced to NH3, thus
consuming 16 ATP, and producing 2 mol of ammonium and 1 mol of H2 for each fixed N
molecule(21). As a result of the review, it was found that the differences in fixed nitrogen
ranges between forages and species of the same genus are mainly determined by the factors:
plant, soil, anthropogenic activities, and climate (Figure 1).
Figure 1: Factors influencing biological N fixation in forage grasses
Source: prepared based on citations(2,5,11,18,21,22).
Effect of climate on forage BNF
Although there are few studies analyzing the effect of climate on the BNF process, it is
highlighted that cloudiness has a negative influence on this process due to the lower
availability of photoassimilates that are produced in the leaves and distributed to the roots
for the formation of rhizo-exudates(23). The increased production of photoassimilates seems
to have a direct relationship with the persistence of inoculated diazotrophs, which favors their
449
effect; for example, with the application of Azospirillum brasilense in Urochloa brizantha, it
has been observed that at the beginning of the dry season in which solar radiation increases,
the mass of the roots of inoculated plants was 27 % higher than in non-inoculated plants, and
although during the transition period the production of grass decreased, in inoculated plants,
it decreased by only 7 % and its height increased by 16 % compared to non-inoculated plants,
due to the greater absorption of nutrients(12). Similar responses are reported with the
application of Bacillus sp. on Megathyrsus maximus(24).
Ensuring the persistence of diazotrophic communities can reduce dependence on nitrogen

fertilization(13); however, in the rainy season, N fixation due to bacterial effect may decrease
perhaps due to the entrainment of microorganisms(1,9). This may explain why it is reported
that at the end of the wet season, root biomass decreases by 15 % in inoculated plants and
there is a lower N content in the leaves compared to plants fertilized with N(12).
In N-deficient environments, BNF increases as a control response when there are low
mineralization rates(4,25). Thus, higher accumulated N is reported in autumn than in spring
due to the effect of a lower temperature in the forages Axonopus affinis (37.6 kg N ha-1.),
Paspalum notatum (27.7 kg N ha-1.) and Andropogon lateralis (1.6 kg N ha-1), estimating
that on average, the percentage of N from BNF is 33 %, 22 %, and 25 % respectively(2).
Soil effect on forage BNF
Soil characteristics also influence BNF(22), a greater diversity of diazotrophic populations in

soils with high organic matter is highlighted. The persistence of these microorganisms is
modulated by the type and quality of nutrients in the soil(22), it is explained that diazotrophs
increase their activity with the presence of iron (Fe), molybdenum (Mo), and vanadium (V)
because these elements can be exchanged to be part of the nitrogenase structure. This
enzyme, when inactivated by oxygen, requires anaerobic microsites to catabolize nitrogen
fixation, which is why it seems that in clay soils there is greater chemical and mineral
mobilization, and eventually greater BNF(21).
Effect of anthropogenic activities on forage BNF
Soil is a system that is naturally self-regulating, but abrupt changes in its characteristics due
to anthropogenic management activities (tillage, fertilization) and use (permanent pastures
with and without intervention, livestock) cause imbalance in bacterial communities since
they alter the structure of the pores, the availability of elements, the content of organic carbon
and the pH, factors that determine the richness, uniformity, and diversity of
microorganisms(2,22).
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Excessive application of Ca, nitrate and N during fertilization has a negative effect on
diazotrophic populations(17). The main cause is related to soil pH(12,22); variations of 1.5 in
the soil pH value can reduce the growth of microorganisms by up to 50 % in soils with a pH
between 5 and 7(12,22). There are reports of the inhibition of the growth of some microbial
populations, such as Azotobacter, Azospirillum, Herbaspirillum and Gluconacetobacter
diazotrophicus, with high fertilization doses of N(2,10,26), for example, with the application of
430 kg N ha-1 in B. brizantha and B. ruziziensis(27). Nevertheless, the type and amount of
fertilizer applied influences the abundance and diversity of microbial populations; an
increase in methanotrophs with inputs greater than 200 μg N g-1 of ammonia has been
observed when the active site of ammonia monooxygenase is exceeded(28). In general, the
structural modification of the bacterial community is a natural mechanism for controlling the
nitrogen status in the soil(2).
Effect of the plant factor on forage BNF
The morphophysiological characteristics of grasses generate dissimilar microenvironments

in leaves, stems, and roots, which promote the selective growth of members of the bacterial
population during the growth phase(4). In the early phase, the activity of rhizospheric
diazotrophic populations is greater due to an increase in rhizo-depositions as a mechanism
for plant recovery after grazing(13). The interaction between diazotrophic bacteria and plants
occurs through rhizo-depositions that include several molecules such as sugars,
polysaccharides, inorganic organic acids, amino acids, vitamins, flavonoids, siderophores,
peptides, proteins, and fatty acids(29). These chemical signals control the interactions that take
place in the soil and are responsible for promoting the selective growth of members of the
rhizospheric community and allow the movement of bacteria to the plant root and root
hairs(2,4). The diverse functional capacity of diazotrophic bacteria allows them to modulate
the growth response of forage and generate positive, negative, or neutral interactions. The
main findings in relation to forage response with diazotroph inoculation are discussed below.
Biofertilizers made up of diazotrophs that have been used in grasses
From 1985 onwards, the first scientific studies in the area of biofertilizers applied to forage
were reported, although historically it is a practice that dates back to 500 B.C., originating in
India, a country that continues to lead scientific advances with a 30 % global share, followed
by Brazil (10 %) and China (8.8 %). In the area of biofertilizers applied to forages, authors
such as Gupta et al(4), Li H et al(15) and De Sousa et al(30) stand out. Rapid growth is estimated
in the area with an inflection point by 2034 (Table 2), a projection that shows the existence
of study opportunities that are linked to the phenomenon of climate change and the challenge
of using sustainable fertilization strategies that reduce the application of chemicals obtained
by burning fossil fuels such as urea.
451
Table 2: Nonlinear regression models obtained for the searches “Biofertilizer” and
“Biofertilizer and grass”
Boolean Model Inflection Durbin a b R2 P-
code year Watson value
Sigmoidal 3 2034 1.07 10988 5.7 0.99 0.01
Biofertilizer Parameter
Sigmoidal, 3 2029 1.87 21.42 4.34 0.96 0.01
Parameter
Sigmoidal, 4 2018 2.89 26.96 5.4 0.90 0.01
Parameter
Gompertz, 3 2018 2.93 33.68 10.42 0.90 0.01
Biofertilizer Parameter
and grass Hill, 3 2016 0.82 21.34 92.85 0.58 0.01
Parameter
Source: Authors’ own preparation.
The trend of biofertilizer use in forages is sigmoidal with an inflection point towards the year
2029, as observed in the logistic model with the highest fit that obtained a Durbin Watson
value close to 2(7), although the prediction by the Gompertz and Hill models is earlier, they
have a lower fit (R2), therefore, they do not predict reliable behavior (Table 2). The inflection
point is associated with the rapid growth phase of the technology and corresponds to the
maximum value of the curve from which biofertilizer-related publications are expected to
begin to decline. These predictions with high variation are related to areas of application in
increasing development, and organic fertilization is beginning to gain importance in the
livestock sector due to the rise in the cost of chemical fertilizers.
From the review analysis, it was found that biofertilizers used in pastures have been applied
by seed inoculation in the product for 30 min to 24 h, followed by a drying time prior to
sowing(2,31) or by spraying in dosages ranging from 200 – 500 ml of inoculant ha-1 diluted in
water at 0.1 - 1.3 % in a minimum concentration of 106 CFU ml-1 or 106 CFU g-1(32-36).
The inoculation of microorganisms can modify the development of forage with high
variability between genera and strains applied or even cause no effect or generate a negative
response(35) (Table 3). When biofertilizers have been applied together with a synthetic N
source, responses greater than or equivalent to the application of 100 % of the N requirement
have been achieved due to more efficient absorption, reducing N losses caused by leaching
by up to 95 %(36). The best results in terms of production and economy have been observed
with the combined application of the inoculant and N(36-40).
452
Table 3: Some studies of the effect of diazotroph application on forage grasses

Forage Inoculant Percentage increase in Source
biological parameters
compared to non-inoculated
plants
(1)
Brachiaria Herbaspirillum 12 % in crude protein
decumbens rubrisubalbicans and H.
seropedicae
(15)
Megathyrsus Bacillus sp. and Bacillus 7.32 %, 25.3 % , 3.32 %,
maximus megaterium 20.3 %, 2.43 % in height, root
biomass, digestibility, protein
and neutral detergent fiber,
respectively
(16)
Avena saliva Klebsiella sp. 20 % in biomass
L.
(19)
Panicum Burkholderia 27 % in height
virgatum L. phytofirmans
(27)
Brachiaria A. brasilense 31.49 % in the relative content
ruziziensis of water in leaves
(32)
Lolium Pseudomonas fluorescens 63 and 51 % in the production of
multiflorum and Bacillus subtilis dry mass of plants and biomass,
respectively
(33)
Brachiaria Burkholderia pyrrocinia 770 %, 300 %, 17 % in root
brizantha and Pseudomonas biomass, dry matter and
fluorescens chlorophyll, respectively
(34)
Panicum Azospirillum brasilense 23 % in biomass
virgatum L.
(37)
Avena saliva Sinorhizobium meliloti, 10.34 and 28.92 % in height and
L. Bacillus megaterium, root length (28.92 %)
Enterobacter sp., A.
chroococcum,
Pseudomonas sp.
(41)
Pennisetum Klebsiella sp., 52 %, 170 %, 134 % in shoot
clandestinum Beijerinckia sp., length, shoot dry weight and
Achromobacter sp. root length, respectively
(42)
Megathyrsus Bacillus sp. 30.8 % and 12.7 % in biomass
maximus production and height,
respectively
(43)
Avena saliva Providencia rettgeri, 81.19 %, 26.89 %, 10.94 % in
L. Advenella incenata, height, root length and
Acinetobacter chlorophyll, respectively.
calcoaceticus, Serratia
plymuthica,
453
Acinetobacter
calcoaceticus
(44)
Avena saliva Bacillus thuringiensis and 92 % in germinated seeds
L. B. thuringiensis
(45)
Phleum Bacillus subtilis 26.6 % and 63.8 % in shoots and
pratense L. roots, respectively
(46)
Pennisetum Sphingomonas, Pantoea, 116.01 % increase in shoot dry
purpureum Bacillus and Enterobacter weight
Schumach
(47)
Sorghum Azotobacter sp. and 21.5 % and 16.8 % in crude
bicolor L. Burkholderia sp. protein and dry matter
digestibility, respectively
Source: prepared based on the indicated quotations.
The positive response of the plant with the inoculation of diazotrophs is mainly due to two
main conditions; first, because it favors the availability of nitrogen in the soil, which is an
element that is part of proteins, amino acids, DNA, RNA, cytochromes, nucleic acids, and
chlorophyll(2,21); and second, because of the production of secondary metabolites of bacterial
origin such as: a) auxins that are involved in cell growth, differentiation, and division(16), b)
gibberellins, which are hormones involved in the regulation of cell division and elongation,
seed germination, bud appearance and stem growth(48), c) cytokines, which are related to the
regulation of cell growth(48), d) siderophores, which are compounds that can bind to iron,
making it available for use in metabolic processes(26) and e) biosurfactants, which are
chemical agents that form micelles and allow better interaction between the membrane of
microorganisms and nutrients dissolved in the soil and in rhizo-depositions(49).
Of these biomolecules, auxins are the most studied; indole-acetic acid stands out, which is
synthesized from tryptophan, which can be derived from the following pathways: indole-3-
acetonitrile, indole-3-acetamide, indole-3-pyruvic acid or tryptamine(48,50). This hormone is
produced by some diazotrophs, for example: Stenotrophomonas spp., Pseudomonas spp.(49),
Azospirillum spp.(51), Azotobacter spp., and Pseudomonas spp.(26). Its main effect is related
to the modification of the structure, elongation and increase of forage root biomass(37), which
favors the absorption of nutrients.
The hormonal stimulus that can be indirectly caused by the application of diazotrophs to the
plant can favor its phenotypic plasticity in shady environments(23), in drought conditions(15)
or saline soils(46). Physiologically, tolerance to stress conditions is related to an increase in
the activity of the superoxide dismutase and catalase enzymes that eliminate H from free
radicals generated under stressful conditions(32). An increase in the contents of proline,
glutathione reductase(42), and ACC-deaminase(46) has also been reported.
454
On the other hand, greater availability of N in the soil due to bacterial effect allows the plant
to increase the production of chlorophyll as it is part of its chemical structure, which leads to
an increase in the photosynthetic rate of the plant and consequently in the production of
biomass(32). Compositionally, it can promote the crude protein content of forage(1) and the
production of unsaturated fatty acids(14).
Despite the aforementioned synergisms, antagonistic responses are reported with the
inoculation of diazotrophs(2), due to the effect of nitrogenase inactivation due to exposure to
high doses of N. Nevertheless, the lack of response may also be due to a low dose of inoculant
applied(23), which can be inhibited by allelopathic control of the plant, which generates low
survival, adaptation, and persistence of the inoculated microorganisms. In fact, the variability
among the ecosystem can limit the response of bacteria because the BNF process occurs only
in favorable environments that allow the persistence of the alpha-proteobacterial taxonomic
group(9,51).
Conclusions
BNF is the main source of N in perennial meadows where synthetic N is not applied and in
areas of severe drought where the plant manages to maintain its growth thanks to structural
adaptations such as the reduction of aerial material to increase root length. The specific
signaling mechanisms that allow the expression of proteins for the production of hormones
and enzymes that make these modifications possible and potentiate microbial communities
specialized in BNF to favor plant survival under extreme conditions are unknown. However,
it has been identified that the species of Brachiaria spp. and Pennisetum spp. have high
potential to contribute to the BNF process due to the persistence of alpha proteobacteria in
the rhizosphere and in the tissue of roots, stems, and leaves.
Azospirillum spp. and Azotobacter spp. are highlighted, but of these, Azospirillum brasilense
has the greatest potential to fix N due to the ability to infect forage tissue, which eventually
facilitates its survival. Nonetheless, it is unknown whether the colonization of this isolate
along with other endophytic microorganisms resists the plant’s defense system during
prolonged exposure times, and perhaps this is related to the lack of productive response with
the application of some inoculants. This is why the biotechnological development of these
products aims at the study of native microorganisms to avoid a negative allelopathic response
by the plant.
Increased dry matter with the application of biofertilizers is the main response observed
according to the review analysis, this effect may eventually allow shorter grazing intervals
and the intensification of rotations in livestock systems. It has also been observed that the
application of diazotrophs can stimulate the phenotypic plasticity of the plant in shaded
455
conditions, which is why the use of biofertilizers can be a cost-effective option in

silvopastoral systems.
There are still challenges such as ensuring positive interactions between applied
microorganisms and native strains, developing biofertilizers combined with chemical
fertilizers and biostimulants, reducing the technical costs of isolation, massification and
obtaining the final product, formulating products by crop and according to the stage of
growth, using monitoring methods for the detection and quantification of persistent bacterial
populations that allow adjusting the dosage and frequency of use of biofertilizers according
to management, crop, environmental conditions and soil type, and encouraging their
application in farm systems as an ecosystem service.
Acknowledgements and conflict of interest
To Minciencias call 779 of 2017, royalties from the Government of the department of Boyacá
– Colombia for financial support and the University of Cauca project ID. 5150 for technical
support, also to the Pedagogical and Technological University of Colombia UPTC and the
Research Group in Biochemistry and Animal Nutrition GIBNA.
The authors have no conflict of interest.
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461
Technical note
Frequency of seropositivity against porcine circovirus type 2 (PCV2) in

the metropolitan area of Monterrey, Nuevo León, and its peripheral area
José Pablo Villarreal-Villarreal a
César Dávila-Martínez a
Heidi Giselle Rodríguez-Ramírez a*
a
Universidad Autónoma de Nuevo León. Facultad de Medicina Veterinaria y Zootecnia.
Campus de Ciencias Agropecuarias, Colonia Ex-Hacienda el Canadá, General Escobedo,
Nuevo León, México.
*Corresponding author: rdzmvz@gmail.com
Abstract:
Porcine circovirus type 2 (PCV2) is a DNA-type virus that has an affinity for cells of the
immune system and generates lymphocyte depletion so it favors the development of diseases
caused by other opportunistic agents, it is also related to the generation of different
syndromes. Therefore, this virus causes major illnesses on the pig industry; nevertheless,
PCV2-associated syndromes can easily be prevented through the proper application of
biosecurity and vaccination measures. On the other hand, small-scale production units
(SPUs) often lack this type of preventive management, as well as routine surveillance by a
veterinarian. Although PCV2 is considered a widely distributed virus, there are no reports of
its presence in SPUs in Nuevo León. The presence of antibodies against PCV2 was
determined using a commercial kit and complete blood count was performed on the animals.
A total of 48 SPUs were found, with 91.67 % positivity and 89.7 % seropositivity in the
animals. In the complete blood count, it was found that HGB and HCT were decreased in
individuals who were positive for antibodies compared to negative ones (P=0.03 and P=0.01,
respectively); on the contrary, the value of total white blood cells was found to be decreased
in individuals who were negative for the presence of antibodies against PCV2 (P=0.01).
462
Keywords: Backyard, PCV2, Porcine circovirus type 2, ELISA, Seroprevalence, Pigs.
Porcine circovirus type 2 (PCV2) is a single-stranded DNA virus that only infects pigs, so it
has no zoonotic significance. PCV2 is a pathogen that is implicated in the development of
different syndromes, such as post-weaning wasting syndrome, dermatitis and nephropathy
syndrome, as well as reproductive failure(1). It is recognized that infection with this virus is
a predisposing factor for syndromes, however, it requires co-infection with another pathogen
to trigger the disease process. Among the risk factors that increase the possibility of
introducing the agent into the pig population and its dissemination are: low birth weight, low
weaning weight, as well as those related to facilities and management practices, such as
housing a large number of animals in a small space, contact between pigs and hygiene(2).
PCV2 has an affinity for the cells of the immune system, especially macrophages and
lymphocytes(3); in fact, one of the expected clinical findings during infection by this virus is
the reduction of total leukocyte counts, as well as lymphopenia(1,4,5). Among the diagnostic
methods for this virus are PCR and immunohistochemistry (IHC)(6); for the latter, lymphatic
tissue is used to detect antigens of the virus, which indicates the presence of the virus in the
target cells. It has been confirmed that lymphocyte depletion in lymphatic tissue is related to
the activation of apoptosis through the pathways of caspases 3 and 8 within lymphocytes(3),
although it is not ruled out that there are other mechanisms involved. Lymphocyte depletion
induces a state of immunosuppression that is also aggravated by co-infection with other
pathogens, such as porcine parvovirus, PRRS virus, and others(7), which allows the
appearance of the aforementioned syndromes.
In Mexico, small-scale production units (SPUs) are still present in some areas. The
characteristics of these systems present in the metropolitan area of Monterrey, Nuevo León,
have previously been researched, and among them, the lack of biosecurity, medication and
routine surveillance by a veterinarian has been confirmed. The aforementioned conditions
favor the entry of pathogens into production units, as well as their subsequent perpetuation
in the environment; nevertheless, PCV2 is especially important since being
immunosuppressive, it allows the appearance of clinical manifestations of secondary
infections in some cases(8-10). Therefore, this study aimed to determine the presence of
antibodies against PCV2 in backyard pigs that did not have previous vaccination against this
pathogen, so a cross-sectional study was carried out, which included pigs in SPUs from 9
municipalities corresponding to the metropolitan area of Monterrey, Nuevo León.
463
The small-scale production system (also known as backyard or artisanal) was defined as one
in which pig rearing activities will be carried out in the home of the owners of the animals,
or as a complementary economic activity, that is, one that will not represent the main family
income. The minimum number of samples was calculated with the WinEpi software under
the following considerations: an expected prevalence of 92 % was considered, which was
taken from a previous report in Mexico(11), a margin of error of 5 %, and a confidence level
of 95 % for an unknown population, yielding a minimum number of 114 samples. Once the
SPUs were identified, permission was requested from the owners to take samples from the
animals and a questionnaire was applied at the end. Samples were collected from May 2019
to March 2020. Pigs of different ages were included; however, sampling in pregnant females
was avoided to prevent the risk of abortion, as well as in suckling piglets to avoid detection
of maternal antibodies.
For sampling, the pigs were physically restrained. During immobilization, the body condition
of each individual was rated on a scale of 1 to 5. Two blood samples were taken from the
jugular vein, one in a collection tube with EDTA and the other in a collection tube with serum
separator. The samples with EDTA were processed in the clinical laboratory of the
Veterinary Hospital of Small Species (HVPE, for its acronym in Spanish) of the Autonomous
University of Nuevo León under the standard procedure on the KONTROLab 5R+Vet
equipment.
The serum was separated from the clot at 1,000 rpm for 10 min at 4 °C and then fractionated
into 500 μL aliquots and stored at -80 °C until later use. For the detection of antibodies against
PCV2, a commercial kit (Bio Check®) was used according to the manufacturer’s
instructions. This kit has a sensitivity of 92.1 % and a specificity of 95.6 %. The absorbance
of the samples was read at 405 nm on the Awareness technology Chromate® equipment
(Awareness technology Inc.).
Data were captured in a spreadsheet to determine the percentage of seropositivity. A

Student’s T-test was performed to determine the difference between the means of the
hematological parameters between the antibody-positive and antibody-negative groups, as
well as of the frequency of the animals’ body condition. Statistical analysis was performed
using the GraphPad Prism 6 software (San Diego, CA). A P-value ≤ 0.05 was considered
significant.
There were localized 48 SPUs, in which access to sample was allowed, which were in the
municipalities of Apodaca, Cadereyta Jiménez, García, General Escobedo, Hidalgo, Juárez,
Santiago and Salinas Victoria. It was found that, in 44 of the sites sampled, at least one animal
tested positive for antibodies, so the percentage of positivity was 91.67 %. The rest of the
production units corresponded to four sites where no animal was detected as positive for
464
antibodies against PCV2. In all municipalities, it was possible to detect positive production
units.
A total of 204 animals were sampled, of which the presence of antibodies against PCV2 was
confirmed in 183, resulting in 89.7 % seropositivity. Some studies have previously been
carried out in Mexico, where 92.29 % positivity for antibodies against PCV2 was determined
among animals and 98.14 % positivity among production units with at least one positive
animal(11), so, in congruence with findings of other authors, seropositivity against this virus
was found ubiquitously in the SPUs in the metropolitan area of Monterrey, Nuevo León, and
its peripheral area.
Figure 1: A) Map of Mexico. B) Map of Nuevo León. Each red dot represents a sampled
municipality. C) Identification of sampled municipalities
On the other hand, different research groups have explored the presence of PCV2 through
the use of real-time PCR; an example is in Brazil where they have found the presence of the
virus genome in 15.6 %(8) of the lung samples studied. On the other hand, by using
quantitative PCR, a 90 % prevalence has been detected in Colombia by using white blood
cell samples(9). In Spain, another group of researchers have taken on the task of identifying
the presence of the virus in technified production units in different areas using quantitative
PCR in environmental samples, which included swabs from the surfaces of pens, workers’
boots and even inside the offices of five production units(12), finding a 42.9 % positivity rate,
thus reiterating the easy spread of this virus and its wide dissemination in the environment.
The seroprevalence results obtained were entered into the WinEpi platform, and the
sensitivity and specificity specifications provided by the kit manufacturer were also entered
465
in order to estimate the positive and negative predictive values. The platform yielded a
positive predictive value of 99.9 % and a negative predictive value of 25.4 %, as well as an
actual prevalence of 97.3 %.
In this study, pregnant females were not sampled due to the risk of inducing abortions or,
where applicable, preterm births. Nonetheless, there are studies in the context of other
infectious agents, such as influenza A, in which it has been shown that females with a higher
number of births have a greater immune experience due to their age, as well as a higher
concentration of specific antibodies(13). Therefore, it is to be assumed that, if positivity was
found in animals of other ages, there is also seropositivity among females. On the other hand,
pigs are animals that are born agammaglobulinemic unless they are exposed to an agent in
utero, therefore, the intake of colostrum is important for their survival, since in this way they
acquire IgG and IgA from the mother(14); in this study, piglets that had not been weaned were
not included because the presence of maternal antibodies can be detected through the ELISA
method without representing seroconversion due to exposure to the agent.
Regarding the body condition of the animals, only one animal with condition of 1 (0.41 %),
49 animals with condition of 2 (20.16 %), 86 with condition of 3 (35.39 %) and 107 animals
with condition of 4 (44.03 %) were observed. No animals with a condition of 5 were
observed. Mean body condition in the antibody-positive group was compared with the
negative group (n= 139 animals), but no difference was found (P>0.05). Since vaccination
against PCV2 was not reported for any of the animals, it is presumed that the presence of
antibodies is due to seroconversion due to previous immune experience against the field
virus, however, these antibodies could be fulfilling a protective role against the development
of PCV2-associated syndromes. It has been shown that vaccination does not always prevent
viremia, but it does reduce systemic viral load in vaccinated individuals(15). In addition to the
above, a group of researchers demonstrated that vaccination has a positive effect on the
cellular and humoral immune response even in animals that previously had viremia(16), that
is, that had been infected before being vaccinated.
Although a high frequency of antibody positivity was found in this study, no animals with
apparent clinical symptoms were found. Most of the animals had medium to good body
condition; it was not possible to differentiate antibody-negative animals from positive ones
by body condition either. Other researchers have found that the presence of the virus on farm
is not necessarily compatible with the presence of PCV2-associated syndromes(12), and
although this virus is recognized as necessary to trigger associated syndromes, its presence
alone is not sufficient to produce the disease(17).
On the other hand, the means of the hematological parameters in the antibody-positive group
were compared with the negative group, and in terms of the red line, a significant difference
was found for hemoglobin (HGB) (P=0.03) and hematocrit (HCT) (P=0.01), which were
466
found to be decreased in the positive group compared to the antibody-negative group (Figure
2).
Figure 2: Comparison of hematological parameters in the red line
The number of individuals included in each analysis were: A= 124, B= 124, C= 141, D= 141, E= 141, F=
141.
For the white line (Figure 3), a decrease in total white blood cells was found in the group
negative for antibodies against PCV2 compared to those who were seropositive (P=0.01).
Although there were found differences in the decreased parameters of HGB and HCT in the
positive individuals and total white blood cells in greater amounts in the antibody-positive
compared to the negative ones, all three parameters were within the expected normal ranges
in both groups. Interestingly, it was found a decrease in the total leukocyte count in the
antibody-negative group; nevertheless, the finding suggests that these pigs could be in a state
of infection and even viremia in which they have yet to develop antibodies; likewise, it must
be taken into consideration that most of the individuals who remained negative for the
presence of antibodies were in places where at least one positive animal was found, so it is
very likely that they will have contact with the virus at some point in their lives.
467
Figure 3: Comparison of hematological parameters in the white line
The number of individuals included in each analysis were: A= 147, B=81, C=134, D= 81, E= 134.
In conclusion, there is presence of antibodies against PCV2 in a large proportion of the SPUs
and in the pigs within them, which are located in the metropolitan area of Monterrey, Nuevo
León, and its peripheral area; however, this does not imply the presence of clinical symptoms.
Acknowledgements
We are grateful for the financial support for the research provided by the Autonomous
University of Nuevo León in the PAICyT 2021 call, to carry out this project.
Conflicts of interest
The authors declare that there is no conflict of interest.
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Technical note
Prevalence and infection intensity of honey bee (Apis mellifera) viral

diseases in six regions of the state of Jalisco, Mexico
Ana Karen Ramos-Cuellar a
Álvaro De la Mora b
Francisca Contreras-Escareño c
Nuria Morfin d
José María Tapia-González e
José Octavio Macías-Macías e
Tatiana Petukhova f*
Adriana Correa-Benítez a
Ernesto Guzman-Novoa b
a
Universidad Nacional Autónoma de México. FMVZ, Departamento de Medicina y
Zootecnia de Abejas. Ciudad de México, México.
b
University of Guelph. School of Environmental Sciences, 50 Stone Road East, Guelph, ON,
N1G 2W1, Canadá.
c
Universidad de Guadalajara. CUCSur, Depto. Prod. Agríc., Autlán, Jal., México.
d
University of British Columbia. Dept. Biochem. Mol. Biol., Vancouver, BC, Canadá.
e
Universidad de Guadalajara. CUSur, Depto. Cienc. Natur., Cd. Guzmán, Jal., México.
f
University of Guelph. Department of Population Medicine, Guelph, ON, Canadá.
*Corresponding author: tpetukho@uoguelph.ca
471
Abstract:
Jalisco is one of the foremost honey-producing states in Mexico. However, there is no

information on viral diseases that affect honey bees (Apis mellifera) in the different
beekeeping regions of the state. The objective of this study was to determine the prevalence
and intensity of four viral diseases of Apis mellifera during the spring, in six regions of
Jalisco. Bee samples from 79 colonies were analyzed, of which, 66 % and 38 % were positive
for black queen cell virus (BQCV) and deformed wing virus (DWV), respectively. Two viral
diseases were not detected, those caused by the Israeli acute paralysis virus (IAPV) and the
chronic bee paralysis virus (CBPV). The infection levels of BQCV were relatively low but
elevated for DWV, with infection intensities 8,000 higher than those of BQCV. The
prevalence of DWV was significantly higher in the regions of the Highlands, Center, and
South, while for BQCV there were no differences between regions. For infection intensity,
there were no differences between regions for DWV, but there were for BQCV. The regions
with the highest infection levels were the South and Center. Surveys during other seasons of
the year are recommended to identify possible seasonal viral effects on the bees and to design
control strategies.
Keywords: Apis mellifera, Deformed wing virus, Black queen cell virus.
Viral diseases of honey bees (Apis mellifera) are increasingly being associated with colony
losses(1), so it is important to know their prevalence and distribution to be able to control
them. More than 20 viruses are known to infect honey bees, but few of them appear to have
a serious impact on their health. Among them, we can mention deformed wing virus (DWV),
black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), and chronic bee
paralysis virus (CBPV)(2,3). In Mexico, the presence of DWV, IAPV and BQCV(4,5) has been
reported in the high plateau region. Moreover, DWV and IAPV were identified in Varroa
destructor mites, with DWV being the most prevalent in both, bee and mite samples(4). Not
much is known about honey bee viral diseases in Mexico, and the information about them, is
limited to a few regions of a few states. For Jalisco’s case, there are still no official reports
about the distribution and levels of viruses in honey bee colonies of the state’s different
beekeeping regions. It would be important to know this information because Jalisco is one of
the foremost honey producing states in Mexico, ranking third in 2021 with 6,073 t(6).
For beekeeping purposes, Jalisco has been divided into six different regions that vary in
topography and climate, including the regions of the Highlands, Center, North, Sierra Amula,
472
South and Southeast. More than half of the state’s producers and hives are located in the
South and Southeast regions(7). Because there is no information on the prevalence and
infection intensity of honey bee viral diseases for the different regions of Jalisco, it seems
relevant to conduct surveys to identify and quantify viruses in honey bee colonies from those
regions, as well as to find out if there is a relationship between viruses and regions. Therefore,
the objective of this study was to determine the prevalence and infection levels of the main
viral diseases that affect adult honey bees, including DWV, BQCV, IAPV and CBPV, in
samples of bees from colonies of six regions from Jalisco, Mexico.
Adult bee samples were collected during early spring, in the months of March, April and
May 2018, from 12 to 16 colonies of each of the six beekeeping regions of Jalisco. In each
apiary, three colonies were randomly selected and sampled. A total of 81 colonies from 27
apiaries were sampled, although data were obtained from only 79 colonies. Two samples of
three bees each were collected from the entrance of each hive. The bees were introduced into
2 mL microfuge tubes containing RNAlater® (Thermo Scientific; Mississauga, ON, Canada)
to preserve viral RNA. The samples were transported in coolers with freezing packs and were
stored at -70° C until processed.
The molecular analyses to diagnose and quantify viral infections were conducted at the
Honey Bee Research Centre, School of Environmental Sciences, University of Guelph, in
Guelph, Ontario, Canada. First, the presence of DWV, BQCV, IAPV and CBPV, was
determined by RT-PCR. RNA was extracted from three bees per sample with TRIzol (Fisher
Scientific; Mississauga, ON, Canada), as per the manufacturer’s instructions. cDNA was
synthesized with the RevertAidTM H Minus First Strand kit (Fermentas; Burlington, ON,
Canada), following the manufacturer’s instructions.
The PCR reactions were carried out using a Master thermocycler (Eppendorf; Mississauga,
ON, Canada). Each reaction contained 1.5 µL of 10x pH buffer for PCR (New England
BioLabs; Pickering, ON, Canada), 1 µL of both primers (10 mM), 0.2 µL 5U/µL of Taq
polymerase (New England BioLabs; Pickering, ON, Canada), 2 µL of cDNA and 8.8 µL of
dH2O. The primer sequences and amplification cycles used were those described in previous
studies for DWV(4,8), BQCV(9), IAPV(10) and CBPV(11). The PCR products were separated by
electrophoresis on agarose gels and the amplified bands were photographed with a digital
camera under UV light. Additionally, viral copies of DWV and BQCV were quantified with
real time PCR (qRT-PCR). The other two viruses were not detected and therefore, not
quantified. The calibration standard curve for DWV and BQCV was created using a 300 bp
synthetic gene fragment or gBlock® (Integrated DNA technologies; Coralville, IO, USA) for
each virus. The lyophilized of the synthetic genes (500 ng) were diluted with 50 µL of
nuclease free dH2O to obtain an initial concentration of 10 ng/µL that was used for serial
dilutions from 109 to 102 viral copy numbers.
473
The qRT-PCR reactions were done using a BioRad CFX96TM thermocycler (Bio-Rad
Laboratories; Mississauga, ON, Canada) with PowerUp™ SYBRgreen™ (Supermix 2X)
(Applied Biosystems; Foster City, CA, USA) on 96-well PCR plates (Hard-Shell®). The
reactions had a final volume of 20 µL that contained the following. For DWV, 10 µL of
Supermix 2X (Applied Biosystems; Foster City, CA, USA), 0.4 µL of both primers (200nM),
7.2 µL of nuclease free dH2O (Invitrogen; Burlington, ON, Canada) and 2 µL of cDNA or
the synthetic gene dilutions. For BQCV, 10 µL of Supermix 2X, 0.8 µL of primers (400nM),
6.4 µL of nuclease free dH2O and 2 µL of cDNA or the synthetic gene dilutions. The primer
sequences and amplification cycles were those described in previous studies for DWV(12) and
BQCV(13).
The thermocycler software calculated the efficiency, determination coefficient (R2), and the
slope of the viral RNA standard curve. To calculate the amount of viral RNA in the serial
dilutions the following equation was used:
Number of viral RNA copies = (ng of synthetic gene) (6.022x1023) /(length of synthetic gene)
(1x109) (650 D). Where: 650 D is the average weight of a base pair and 6.022x1023 is the
Avogadro number(14). A graph using Ct values with the initial number of RNA viral copies
and the number of DWV and BQCV copies of the samples was calculated using a regression
equation.
To determine if there were differences between regions for viral prevalence, the data were
analyzed with comparative tests for equality of proportions, using the Benjamini-Hochberg
correction. Also, the data on infection intensity were subjected to Shapiro-Wilk and Bartlett
tests to analyze the assumptions of normality and homoscedasticity, respectively. The data
did not comply with the assumptions and thus, were log transformed and subjected to
analyses of variance. When significance was detected, the regional means were compared
with t tests using the Benjamini-Hochberg correction. All the statistical analyses were
performed with the R 3.3.1 program (Foundation for Statistical Computing, Vienna, Austria).
Two viruses were detected in the honey bee samples from all regions of Jalisco, BQCV
(Figure 1) and DWV (Figure 2). The other two viruses, IAPV and CBPV, were not detected.
Of the detected viruses, the prevalence of BQCV at the state level was 66 % and that of DWV
was 38 %. The prevalence and intensity of the viral infections identified are shown in Table
1.
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Figure 1: Photograph of an agarose gel that shows bands of 698 bp of black queen cell
virus (BQCV) in columns 1, 2, 5, 6, 7 and 8. A honey bee gene (RpS5) is used as a control
in the RT-PCR reaction
Figure 2: Photograph of an agarose gel that shows bands of 642 bp of deformed wing virus
(DWV) in columns 2, 4 and 6. A honey bee gene (RpS5) is used as a control in the RT-
PCR reaction
Table 1: Prevalence and mean intensity of viral infections that affect honey bee colonies in
the state of Jalisco, Mexico
Pathogen N Prevalence (%) Intensity ± E.E.1
Deformed wing virus 79 38.0 4,083.40 ± 2,676.051
Back queen cell virus 79 65.8 0.49 ± 0.233
1
Number of viral copies per µg of RNA x 106
At the regional level, DWV prevalence in colonies from the Southeast and North regions was
only 8 %, which was significantly lower than those of colonies from the South, Sierra Amula,
and Center regions (P<0.05, Table 2). For DWV infection intensity, there were no significant
differences between colonies of different regions (F5,73= 0.64, P=0.67).
475
Table 2: Prevalence and mean infection intensity of deformed wing virus (DWV) in adult
workers of honey bee colonies in different regions of the state of Jalisco, Mexico
Region N Prevalence (%) Intensity ± E.E.1
Highlands 12 33.3 a,b 368.81 ± 239.91
Center 12 66.7 a 955.33 ± 611.20
Sierra Amula 15 60.0 a 14,652.31 ± 13,740.36
North 12 8.3 b 176.67 ± 85.12
South 16 50.0 a 955.19 ± 476.21
Southeast 12 8.3 b 5,778.83 ± 3,860.24
1
Number of viral copies per µg of RNA x 106.
ab
Different literals indicate significant differences based on tests for equality of proportions using the
Benjamini-Hochberg correction (P<0.05).
BQCV was detected in 42 to 81 % of the colonies from the different studied regions, but
there was no significant difference between regions for BQCV prevalence (P>0.05, Table 3).
However, for BQCV infection intensity, regions varied significantly (F5,73= 7.14, p< 0.01).
For example, the South region had colonies with infection intensities significantly higher
than those of colonies from the rest of the regions, except the Center region that was second
for BQCV levels. The North region had the colonies with the lowest titers of BQCV
infections.
Table 3: Prevalence and mean infection intensity of black queen cell virus (BQCV) in adult
workers of honey bee colonies in different regions of the state of Jalisco, Mexico
Region N Prevalence (%) Intensity ± E.E.1
Highlands 12 41.7 0.12 ± 0.07 b,c
Center 12 66.7 0.37 ± 0.21 a,b
Sierra Amula 15 66.7 0.06 ± 0.05 c,d
North 12 58.3 0.02 ± 0.01 d
South 16 81.2 1.94 ± 1.08 a
Southeast 12 75.0 0.03 ± 0.01 d
1
Number of viral copies per µg of RNA x 106.
abcd
Different literals indicate significant differences based on ANOVA and t tests using the Benjamini-
Hochberg correction (P<0.05) of log transformed data.
In Mexico, there is little information about the presence of honey bee viral diseases, since
viruses like DWV, IAPV, and BQCV were molecularly diagnosed for the first time just a
decade ago in the Mexican high plateau(4,5), but nothing is known about the prevalence or
infection intensity of these viruses in almost all states of the country. In northern Mexico,
IAPV, DWV, sac brood bee virus (SBV), Kashmir bee virus (KBV), and filamentous virus
(FV) were reported in colonies from the state of Chihuahua, but its prevalence and infection
intensity were not determined(15,16). Therefore, the results of this study are a reference point
for future research in Mexico’s regions of beekeeping importance.
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In other countries of the Americas, several prevalence rates of honey bee viruses have been
reported. However, most colonies of other countries have in common the prevalence of DWV
and BQCV. For example, in Uruguay, 100% of the analyzed colonies were infected with
DWV and BQCV(17,18). In Argentina and Chile, the most prevalent honey bee virus was
DWV, which was detected in 35 y 37 % of the colonies sampled, respectively(19,20). In Cuba,
DWV was the most prevalent virus, which was detected in 91 % of the colonies surveyed,
but BQCV was not detected(21). In Colombia, both viruses were detected at a prevalence of
19.9 and 10.6 %, respectively(22). In North America, the prevalence of eight honey bee viruses
was determined during six years in the USA, and in every single year, DWV was the most
common of all viruses at a prevalence that ranged between 65 and 92 %, closely followed by
BQCV with a prevalence range of 60 to 92 %(23).
Regarding viral infection intensities, with the exemption of reports from the USA and
Canada, no study so far conducted in Central America, the Caribbean, or Mexico, has
reported infection levels of honey bee viruses in different regions of a state, like this study
does. Therefore, to the best of our knowledge, this is the first study to report infection levels
of honey bee viruses at a regional level.
The prevalence of viruses varied between regions. DWV prevalence was significantly lower
(8 %) in colonies of the Southeast and North regions than in other regions. Conversely, the
prevalence of DWV in the regions South, Sierra Amula, and Center, was over 50 %.
However, there were no differences for the intensity of infections of DWV between regions
because viral infection levels were high in all regions. No differences in the prevalence of
BQCV were found between colonies of different regions, however, their infection levels
varied between colonies from one region to another. The colonies of the South region had
higher BQCV infection levels than the colonies of the rest of the regions, except for the
Center region. Regarding infection intensity, mean DWV infection levels were very high,
with 4083.4 X 106 viral copies per µg of RNA, whereas for BQCV infection levels were
relatively low, with 0.49 X 106 viral copies per µg of RNA. Thus, the intensity of DWV
infection in honey bees from Jalisco was approximately 8,000 times higher than that of
BQCV infection.
Some of the factors that could have influenced the differences in viral prevalence and
infection intensity in honey bee colonies between Jalisco’s regions include environmental
effects, bee genotype, and possibly different viral strains. Regarding climatic effects, it is
known that DWV infections are more prevalent and intense in colonies located in temperate
climates than in colonies established in tropical climates(24). The authors of the cited study
proposed that this occurs because colder climates favor the transmission and replication of
DWV and could reduce the immune responses of bees, making them more susceptible to the
virus. The authors also found an effect of the interaction between climate and parasitism by
V. destructor, a mite that is strongly related to the prevalence and infection intensity of DWV,
477
since it not only serves as a vector of the virus, but the virus multiplies in the mite’s
tissues(25,26). Therefore, colonies with greater V. destructor infestation rates tend to have
higher DWV prevalence and infection intensity than colonies with low mite infestation
levels(27). Furthermore, the genotype of bees varies with their degree of Africanization. It has
been shown that the intensity of infection caused by DWV and BQCV is higher in colonies
with bees of European mitotype or morphotype than in colonies with bees of African
mitotype or morphotype(28). It is possible that the colonies that were less infected with viruses
in this study, had a greater degree of Africanization than the more infected ones. However,
this hypothesis would have to be investigated in future studies.
The high levels of DWV infection found in this study are concerning because if beekeepers
neglect their V. destructor control measures, the prevalence and intensity of DWV infections
could increase. It is known that together with Varroa parasitism, this virus can weaken
colonies until they collapse(1). Therefore, it is essential to emphasize the importance of
implementing an adequate control strategy for V. destructor infestations to keep DWV
infections as low as possible in honey bee colonies. Regarding BQCV, although it had a high
prevalence in colonies of most regions, its infection levels were low. However, this study
was seasonal, and thus, studies would have to be carried out throughout an entire year and
for several years, to confirm if it is a virus that could represent potential damage to the
beekeeping industry in Jalisco.
In conclusion, the most prevalent honey bee virus in the state of Jalisco was BQCV, which
was detected in 66 % of the colonies, while DWV was detected in 38 % of them. Infection
levels of DWV were high (8,000 times higher than those of BQCV). The regions with the
highest DWV prevalence were Center, South, Highlands, and Sierra Amula. Regarding the
intensity of DWV infections, there were no significant differences between regions. There
were also no significant differences between regions for BQCV prevalence, but there were
for infection intensity of this virus. The regions with the highest infection levels were the
South and Center regions. Additional studies are recommended with surveys conducted
during different seasons of the year and for several years, to find out under what conditions
and seasons, viruses could be harmful to the beekeeping industry, and to design control
strategies.
Acknowledgments and conflict of interest
The authors thank the 42 beekeepers that kindly allowed the collection of honey bee samples
from their colonies. Sara Dino, Ulises Nuño, Shaira Alvarado and Miriam Rangel, helped
with sample collection. This study was partially financed by CUSur research funds granted
478
to J.T. and by the University of Guelph Pinchin fund granted to E.G. The authors declare not
to have conflict of interest.
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Revista Mexicana de Ciencias Pecuarias
Edición Bilingüe
Rev. Mex. Cienc. Pecu. Vol. 15 Núm. 2, pp. 249-482, ABRIL-JUNIO-2024 Bilingual Edition
ISSN: 2448-6698
CONTENIDO
CONTENTS
ARTÍCULOS / ARTICLES Pags.

Estudio de la Estructura y Diversidad genética de ganado Holstein del sistema familiar en México
Study of the Genetic Structure and Diversity of Holstein cattle in the small holder system in Mexico
Felipe de Jesús Ruiz-López, José G. Cortés-Hernández, José Luis Romano-Muñoz, Fernando Villaseñor-González, Adriana García-Ruiz........…..........….........................................................................…......... 249
Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 15 Núm. 2, pp. 249-482, ABRIL-JUNIO-2024
Efecto de diferentes protocolos de castración en indicadores productivos de cerdos: meta-análisis
Effect of various castration protocols on production indicators in pigs: meta-analysis
Humberto Rafael Silva-Santos, Francisco Ernesto Mar�nez-Castañeda, Gregorio Álvarez-Fuentes,María de la Salud Rubio-Lozano, María Elena Trujillo-Ortega.....................................................................267
Acumulación de materia seca, rendimiento y calidad nutricional del forraje de híbridos de maíz cosechados a diferentes días después de la siembra
Dry matter accumulation, yield, and nutritional quality of forage of corn hybrids harvested at different days after sowing
Diego Eduardo Ramírez Gu�érrez, José de Jesús Olmos Colmenero, Alfonso Peña Ramos, Juan Isidro Sánchez Duarte, Ernesto Medina Núñez, Silviano Gallardo Ramírez, Omar Iván Santana............…….287
Análisis por microscopía electrónica y difracción de rayos X de enterolitos de equinos en el valle de Aburrá, Antioquia, Colombia
Electron microscopy and X-ray diffraction analysis of equine enteroliths from the Aburrá Valley in Antioquia, Colombia
Sergio Andrés Vélez Gil, Juan José Pa�ño Marulanda, José Ramón Mar�nez Aranzales.................….....……...................…….....…….....…….....…….....…...............................................…….....................................302
Prevalencia y factores de riesgo asociados a Cryptosporidium spp. en bovinos de leche de Chiquinquirá (Colombia)
Prevalence and risk factors associated with Cryptosporidium spp. in dairy cattle in Chiquinquirá (Colombia)
Diana M. Bulla-Castañeda, Deisy J. Lancheros Buitrago, Leneth B. Castañeda Sedano, Rosa I. Higuera Piedrahita, Martin O. Pulido-Medellin...................................................................….…..310
Influence of the type of container and traditional methods on the long-term storage of honey produced by
stingless Scaptotrigona mexicana: bioactive compounds and antioxidant properties
Influencia del tipo de recipiente y de los métodos tradicionales en el almacenamiento a largo plazo de la miel producida por
Scaptotrigona mexicana sin aguijón: compuestos bioactivos y propiedades antioxidantes
Naida Juárez-Trujillo, Simón Carrouché, María Remedios Mendoza-López, Juan L. Monribot- Villanueva, José A. Guerrero-Analco, Maribel Jiménez-Fernández………….…....................................................323
Un efecto novedoso del extracto acuoso de semillas de Pimpinella anisum sobre garrapatas de perros domésticos (Canis lupus familiaris)
A novel effect of aqueous extract of Pimpinella anisum seeds on ticks of domestic dogs (Canis lupus familiaris)
William Fernando Várguez-Tec, Sara Luz Nahuat-Dzib, Julia Cano-Sosa, Lorena Reyes-Vaquero, Edgar E. Lara-Ramirez, Benjamín Abraham Ayil-Gu�érrez, Angel Virgilio Domínguez-May...........................344
Conocimiento socio-ecológico de la actividad apícola en la Costa Chica de Guerrero, México

Socio-ecological knowledge of the beekeeping activity in the Costa Chica region of Guerrero, Mexico
José Cámara-Romero, William Cetzal-Ix, Luis Alaniz-Gu�érrez, Agus�n Rojas-Herrera, José Aparicio- López, Columba Rodríguez-Alviso...….......…...........................................................................................360
Prevalencia de Fasciola hepatica y Calicophoron spp. en vacunos de crianza extensiva del distrito Florida (Amazonas), Perú
Prevalence of Fasciola hepatica and Calicophoron spp. in extensively reared cattle in the Florida district (Amazonas), Peru
Medali Cueva-Rodríguez, Teófilo Torrel, Cris�an Hobán, Wuesley Alvarez-García, Flor Mejía, Luis Vargas-Rocha................................................……..…..……......................................................................…..... 376
Influence of feedlot living space on production variables, carcass and meat quality traits in Holstein steers
Influencia del espacio vital del corral de engorda en las variables de producción, rasgos de calidad de la canal y la carne en novillos Holstein
Ana Mireya Romo-Valdez, Cris�na Pérez-Linares, Francisco Gerardo Ríos-Rincón, Fernando Figueroa-Saavedra, Alberto Barreras-Serrano,
Beatriz Isabel Castro-Pérez, Eduardo Sánchez-López, Georgina Valen�na Cervantes Cazarez........….......…..........….....................................................…............................................................................……….. 393
REVISIONES DE LITERATURA / REVIEWS

Lenteja de agua (Lemna minor): potencial alimentario y ambiental. Revisión
Common duckweed (Lemna minor): food and environmental potential. Review
Olga Jaimes Prada, Olga Lora Díaz, Katherine Tache Rocha......................................................…........……....……....……....……....……....……....……....……....……....……....……....……....……....……....…….......…….........… 404
Implicación de las Fusariotoxinas en la producción avícola. Revisión

Gabriela Guadalupe Gómez Verduzco, Ernesto Ávila González, Guillermo Téllez Isaías, Juan Carlos Del Río García, Jacqueline Uribe Rivera............................................................................................…...... 425
Contribución de gramíneas forrajeras a la fijación biológica de nitrógeno y su respuesta a la inoculación de diazótrofas. Revisión
Contribution of forage grasses to biological nitrogen fixation and their response to diazotroph inoculation. Review
Dania Fonseca López, Nelson Vivas Quila, Raúl Cuervo Mulet, Carlos Eduardo Rodríguez Molano………....…………....…………....….……....……......................................................................................................… 446
NOTAS DE INVESTIGACIÓN / TECHNICAL NOTES

Frecuencia de seropositividad contra el circovirus porcino tipo 2 (PCV2) en el área metropolitana de Monterrey, Nuevo León y su área periférica
Frequency of seropositivity against porcine circovirus type 2 (PCV2) in the metropolitan area of Monterrey, Nuevo León, and its peripheral area
José Pablo Villarreal-Villarreal, César Dávila-Mar�nez, Heidi Giselle Rodríguez-Ramírez.…………....…………....…………....…………....….….......….......….......….......….......….......….......…..........…….............…….........… 462
Prevalencia e intensidad de virosis de abejas melíferas (Apis mellifera) en seis regiones del estado de Jalisco, México
Prevalence and infection intensity of honey bee (Apis mellifera) viral diseases in six regions of the state of Jalisco, Mexico
Ana Karen Ramos-Cuellar, Álvaro De la Mora, Francisca Contreras-Escareño, Nuria Morfin, José María Tapia-González,
José Octavio Macías-Macías, Ta�ana Petukhova, Adriana Correa-Benítez, Ernesto Guzman-Novoa.….......….........…...…….................................................................................................................................. 471
Rev. Mex. Cienc. Pecu. Vol. 15 Núm. 2, pp. 249-482, ABRIL-JUNIO-2024

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Rev. Mex. Cienc. Pecu. Vol. 15 Núm. 2, pp. 249-482, ABRIL-JUNIO-2024

TIPOGRAFÍA Y FORMATO: Oscar L. Rodríguez Rivera

VISITE NUESTRA PÁGINA EN INTERNET

REV. MEX. CIENC. PECU. VOL. 15 No. 2 ABRIL-JUNIO-2024

Estudio de la Estructura y Diversidad genética de ganado Holstein del sistema familiar

Efecto de diferentes protocolos de castración en indicadores productivos de cerdos:

Acumulación de materia seca, rendimiento y calidad nutricional del forraje de híbridos

Análisis por microscopía electrónica y difracción de rayos X de enterolitos de equinos en

Prevalencia y factores de riesgo asociados a Cryptosporidium spp. en bovinos de leche

Un efecto novedoso del extracto acuoso de semillas de Pimpinella anisum sobre

Conocimiento socio-ecológico de la actividad apícola en la Costa Chica de Guerrero,

Prevalencia de Fasciola hepatica y Calicophoron spp. en vacunos de crianza extensiva

Lenteja de agua (Lemna minor): potencial alimentario y ambiental. Revisión

Contribución de gramíneas forrajeras a la fijación biológica de nitrógeno y su respuesta

Frecuencia de seropositividad contra el circovirus porcino tipo 2 (PCV2) en el área

Prevalencia e intensidad de virosis de abejas melíferas (Apis mellifera) en seis regiones

INSTRUCTIONS FOR AUTHORS

Revista Mexicana de Ciencias Pecuarias is a scientific Title page

VIII) Steel RGD, Torrie JH. Principles and procedures of

Study of the Genetic Structure and Diversity of Holstein cattle in the

Felipe de Jesús Ruiz-López a

José Luis Romano-Muñoz a

*Corresponding author: garcia.adriana@inifap.gob.mx

Keywords: Genetic diversity, Traces of selection, Inbreeding, Family dairy system.

Material and methods

Results and discussion

Type of ROH Length Number of ROHs Percentage

Unique - 5,628 35.86

Table 2: Frequency of runs of homozygosity (ROH) at different lengths

Length (Mb) Number Percentage

Figure 3: Genomic inbreeding (FROH) percentages by lactation number at the time of

Figure 4: Percentage of chromosome covered by runs of homozygosity (ROH)

Figure 5: Percentage of runs of homozygosity (ROH) on each chromosome

Conclusions and implications

Acknowledgments and funding source

Project funded by INIFAP-CENIDFyMA with the name “Development of a sustainable

The authors declare that there are no conflicts of interest.

2. Sainz-Sánchez PA, López-González F, Estrada-Flores JG, Martínez-García CG, Arriaga-

3. Villamar AL, Oliveira CE. Situación actual y perspectiva de la producción de leche de

4. Ceballos FC, Hazelhurst S, Ramsay M. Assessing runs of Homozygosity: a comparison of

9. Moravčíková N, Kasarda R, Kadlečík O, Trakovická A, Halo M, Candrák, J. Runs of

11. Ferenčaković M, Hamzić E, Gredler B, Solberg TR, Klemetsdal G, Curik I, et al.

12. Gurgul A, Szmatoła T, Topolski P, Jasielczuk I, Żukowski K, Bugno-Poniewierska M.

13. Qanbari S, Simianer H. Mapping signatures of positive selection in the genome of

18. Alexander DH, Novembre J, Lange K. Fast model-based estimation of ancestry in

19. Li N, Stephens M. Modeling linkage disequilibrium and identifying recombination

21. Ferenčaković M, Sölkner J, Curik I. Estimating autozygosity from high-throughput

24. Cortes-Hernández JG, Ruiz-López FJ, Vásquez-Peláez CG, García-Ruiz A. Runs of

27. Mcquillan R, Leutenegger A, Abdel-rahman R, Franklin CS, Pericic M, Barac-lauc, et

28. Cortes-Hernández J, García-Ruiz A, Vásquez-Peláez CG, Ruiz-Lopez FdJ. Correlation

32. Marras G, Gaspa, G, Sorbolini S. Dimauro C, Ajmone-marsan P, Valentini A, et al.

33. Mastrangelo S, Tolone M, Gerlando RD, Fontanesi L, Sardina MT, Portolano B.

34. Szmatoła T, Gurgul A, Ropka-Molik K, Jasielczuk I, Zabek T, Bugno-Poniewierska M.

Effect of various castration protocols on production indicators in pigs:

Humberto Rafael Silva-Santos a

Francisco Ernesto Martínez-Castañeda b

María de la Salud Rubio-Lozano d

María Elena Trujillo-Ortega e*

* Corresponding author: elenam@unam.mx

Keywords: Immunocastration, GnRH, Meta-analysis, Carcass, Stockbreeding.

The standard immunization protocol consists of two subcutaneous doses at 12 and 16 wk of

Material and methods

Figure 1: Keyword combinations for article searches