av Abanico veterinario Abanico vet 2007-428X 2448-6132 Sergio Martínez González 10.21929/abavet2021.8 00106 Artículos originales Determinación de la calidad del semen criopreservado con lecitina de soya o yema de huevo, en machos cabríos 0000-0002-0110-4536 Moreno-Avalos Silvestre 1 0000-0002-5105-1508 Veliz-Deras Francisco 2 0000-0002-8566-7474 Calderon-Leyva Guadalupe 1 0000-0002-9198-5372 Contreras-Villarreal Viridiana 2 0000-0001-9273-8931 Guillen-Muñoz Juan 2 0000-0003-2239-7398 Angel-García Oscar * 2 Departamento de Producción Animal, Universidad Autónoma Agraria Antonio Narro. Torreón, Coahuila, México. Universidad Autónoma Agraria Antonio Narro Departamento de Producción Animal Universidad Autónoma Agraria Antonio Narro Torreón Coahuila Mexico Departamento de Ciencias Médico Veterinarias, Universidad Autónoma Agraria Antonio Narro. Torreón, Coahuila, México. Universidad Autónoma Agraria Antonio Narro Departamento de Ciencias Médico Veterinarias Universidad Autónoma Agraria Antonio Narro Torreón Coahuila Mexico Autor responsable y de correspondencia: Ángel-García Oscar. Periférico y Carretera a Santa Fé SN, Colonia Valle Verde, Torreón, Coahuila, México, CP 27052. Correo. angelgarciao@hotmail.com, velizderas@gmail.com, gcalderon06@hotmil.com, dra.viridianac@gmail.com, mvz_guillen@hotmail.com

Clave:2020-75

 30 04 2021 Jan-Dec 2021 11 e106 31 08 2020 20 01 2021 Este es un artículo publicado en acceso abierto bajo una licencia Creative Commons RESUMEN:

El objetivo fue comparar la calidad del semen caprino criopreservado con diferentes tratamientos a base de lecitina de soya o yema de huevo. El semen fue colectado de machos cabríos Alpinos (n=4), se utilizaron dos diluyentes comerciales: AndroMed® (1% de lecitina de soya, LS); Optidyl® con 20% (v/v) de Tris-yema de huevo; TY), y un tercer diluyente a base de citrato-yema de huevo (CY), en semen fresco (SF) y después fue enfriado de 37 a 4 °C durante 2 h; semen refrigerado (SR), posteriormente se llenaron pajillas con semen y se congelaron en nitrógeno líquido a -196 °C (SC). No existieron diferencias (p>0.05) entre diluyentes en el SF respecto a motilidad masal (MM; 4.7±0.26), viabilidad espermática (VE; 74.1±1.66) y motilidad individual (MI; 62.3±4.0). En el mismo sentido, para el SR no existió diferencia (p>0.05) entre diluyentes respecto a MM=3.83±0.4, y MI= 52.1±6.0, sin embargo, la VE varió (p<0.05) de acuerdo al diluyente, observando la menor viabilidad en LS vs. CY y TY (51.0±13.0 vs 71.3±3.0 y 69.0±3.1). Respecto al SC, la MM, MI y VE favorecieron (p<0.05) al diluyente TY vs. LS y CY (2.4±0.5, 32.5±8.3, 41.3±13.0). Los resultados mostraron una mejor crio-preservación del semen caprino con el diluente Tris-yema respecto al de lecitina de soya.

 Palabras clave: Lecitina de soya diluyente semen caprino INTRODUCCIÓN

Los diluyentes tradicionales que son agregados al semen para la preservación de la viabilidad y fertilidad de los espermatozoides durante la crioconservación incluyen yema de huevo (Lima-Verde et al., 2017); lo anterior, debido a que la yema de huevo protege al espermatozoide de los daños inducidos por la crioconservación durante el enfriamiento, congelación y descongelación al interactuar directamente con la membrana plasmática (Andrabi et al., 2008; Akçay et al., 2012; Sieme et al., 2016). Sin embargo, en los últimos años, frecuentemente se ha opinado en contra del uso de la yema de huevo debido a la gran variabilidad de sus componentes, lo que hace que la evaluación de sus beneficios sea compleja (Kulaksız et al., 2010); además se ha tratado de evitar el uso de diluyentes de origen animal, ya que podrían ser una posible ruta de transmisión de enfermedades (Lima-Verde et al., 2017; Ansari et al., 2017).

En lo que se refiere a la yema de huevo, algunos componentes indeseables (hormonas esteroides y sus moléculas precursoras) se consideran perjudiciales para la integridad del espermatozoide (Akhter et al., 2012; Lima-Verde et al., 2017). El componente principal de la yema de huevo que protege a la membrana espermática es la lipoproteína de baja densidad (LDL). Se ha demostrado que los fosfolípidos o lecitina de la yema de huevo hace a los espermatozoides menos sensibles al enfriamiento (Zeron et al., 2002). De manera particular, en el macho cabrío, existen interacciones negativas entre los fosfolípidos de la yema de huevo y la glándula bulbouretral, esta glándula secreta con el plasma seminal una enzima coagulante de la yema de huevo, la cual cataliza la hidrólisis de la lecitina de la yema de huevo en ácidos grasos y lisolecitina, que son citotóxicos (Ngoma et al., 2016). Por lo anterior, se han utilizado sustitutos de la yema de huevo químicamente definidos sin ser de origen animal (El-Sisy et al., 2018; Gamal et al., 2016), elaborados a base de lecitina de soya y que pueden ser alternativas potenciales para la criopreservación del semen (Akhter et al., 2012).

Se cree que los liposomas actúan de manera similar a las lecitinas de la yema de huevo o la leche (Belala et al., 2016). En el búfalo de agua, no se encontraron diferencias en términos de integridad acrosomal cuando se utilizaron diluyentes a base de yema de huevo, lecitina o liposomas de soya (Kumar et al., 2015; Singh et al., 2013). En este contexto, merecen atención especial los diluyentes elaborados en base a yema de huevo en cuanto a sus componentes y su efecto en comparación con los diluyentes basados en liposomas. Debido a que el efecto de los componentes mencionados anteriormente es poco conocido sobre la calidad del semen crioconservado en el macho cabrío, nos planteamos el objetivo de comparar los efectos de diluyentes a base de lecitina de soya o yema de huevo sobre la calidad del semen, conservado por refrigeración y congelación.

 MATERIAL Y MÉTODOS <italic>General</italic>

Todos los métodos y manejo de las unidades experimentales utilizadas en este estudio fueron en estricto acuerdo con los lineamientos para el uso ético, cuidado y bienestar de animales en investigación a nivel internacional (FASS, 2010) y nivel nacional (NAM, 2002) con número de referencia de aprobación institucional UAAAN-UL con clave 38111- 425501002-2431.

Localización y animales

El experimento se realizó en el norte de México, en el Centro Caprino de la Universidad Autónoma Agraria Antonio Narro (26° de Latitud Norte y 104° de Longitud Oeste), durante la época reproductiva (enero). El área de estudio se encuentra a una altitud 1120 msnm, con una precipitación media anual de 230 mm y con temperatura promedio de 24 °C, máxima de 41 °C en mayo y junio, y mínima de -1 °C en diciembre y enero (CONAGUA, 2015). Se utilizaron machos cabríos adultos de la raza Alpino-francés (n = 4, de 1.5 a 2 años de edad), homogéneos en cuanto a peso vivo (PV; 75.0 ± 0.32 kg) y condición corporal (CC; 3.5 ± 0.10 unidades); con fertilidad probada previo al estudio experimental a través de evaluaciones frecuentes de calidad seminal. Durante el periodo experimental, los machos se alimentaron dos veces al día (800 h y 1800 h), a libre acceso con una dieta basada en heno de alfalfa (18% PC, 1.95 Mcal de EM) y 100 g de concentrado comercial (21% PC,1.7 Mcal EM) en base a sus requerimientos nutricionales (NRC, 2007). Los machos tuvieron libre acceso al agua limpia y sales minerales y un periodo de adaptación de 2 semanas previas a la investigación.

 Colección y procesamiento del semen

El semen fue colectado por la mañana (800 a 1000 h) cada 3 d, durante tres semanas, se usó como estímulo para la extracción de semen una hembra en actividad estral. El semen fue recolectado con una vagina artificial estándar para ovinos y caprinos, mantenida a una temperatura de 38 °C, por lo que se precalentó a 42 °C previo a la recolección. Se colectaron un total de 24 eyaculados, después de cada extracción el semen fue sumergido inmediatamente en baño maría a 37 °C para su posterior análisis macroscópico y microscópico durante los siguientes 10 minutos.

 <italic>Preparación de diluyentes y proceso de congelado</italic>

De un total de 24 eyaculados (6 eyaculados por macho) y cada eyaculado se dividió en tres alícuotas en partes iguales para ser procesado para la criopreservación, utilizando dos diluyentes comerciales: AndroMed® (Minitübe, Tiefenbach, Alemania; con 1% de lecitina de soya; LS); Optidyl® (CRYO-VET, Francia; con 20% (v/v) de Tris-yema de huevo; TY), y un tercer diluyente a base de citrato-yema de huevo fresco (CY) obtenido de acuerdo a la técnica utilizada por Salamon y Maxwell, (2000).

En el experimento fueron consideradas únicamente muestras con un volumen > 0.5 mL, una concentración de 2.5x109 mL, motilidad masal ≥ 3.0 y viabilidad ≥ 70%. Posteriormente las muestras ya diluidas fueron sometidas a 3 procesos para su evaluación: semen fresco (SF); semen refrigerado (SR, equilibrado a 4 °C por 2 horas); y semen congelado (SC). Después de refrigerar el semen se llenaron las pajillas de 0.25 mL y para el proceso de congelación se colocaron a 7 cm de nitrógeno líquido (NL, - 140°C) por 10 min; después se sumergieron directamente en el NL (-196°C) y se almacenaron hasta su análisis (Jerez et al., 2016). En cada uno de los estados de conservación (SF, SR y SC), el semen se analizó, inmediatamente y cada 15 minutos durante el proceso de conservación, para evaluar la motilidad masal e individual y la viabilidad. En el caso del SC, éste se analizó 24 horas después, para lo cual, la pajilla se descongeló, sumergiéndola en agua atemperada (37º C) durante 30 s.

Variables evaluadas

La motilidad masal (MM; %), se evaluó con el uso de una plataforma precalentada (37°C), colocando una gota de semen puro (20 μl) sobre una lámina portaobjetos en el microscopio óptico con objetivo de 10x, y de acuerdo con el movimiento observado se asignó un puntaje de escala arbitraria de 1 a 5, donde 1 = 25% y 5 = 100% de espermatozoides móviles (Mahsud et al., 2013). La motilidad individual (MI; %), se determinó en base a la proporción de espermatozoides progresivamente móviles, para ello, se colocó una gota (10µL) de semen sobre una lámina portaobjetos y fue cubierta con una laminilla cubreobjetos; posteriormente se observó al microscopio con objetivo de 40x. La viabilidad espermática (VE; %), se evaluó mediante el uso de la técnica de tinción con eosina-nigrosina (Kafi et al., 2004), se observaron al menos 200 espermatozoides por muestra mediante microscopio óptico, utilizando el objetivo de 100X, y se calculó el porcentaje de células vivas (sin teñir) y de células muertas (teñidas de color rosa). Todas las evaluaciones fueron realizadas siempre por el mismo evaluador calificado.

 <italic>Análisis estadístico</italic>

Los datos fueron analizados mediante un ANOVA usando el procedimiento Modelo Lineal General (GLM). Las medias obtenidas de los parámetros seminales fueron comparadas usando una prueba de t. Se contempló el efecto del uso de los diferentes diluyentes, los estados del proceso de crioconservación y la interacción de estos. Todos los datos fueron analizados utilizando el paquete estadístico SAS V9.1 (SAS, 2005). Las diferencias fueron consideradas significativas a un valor de P≤0.05.

 RESULTADOS Y DISCUSIÓN

Los resultados de los diferentes parámetros evaluados para determinar la calidad del semen diluido con LS, TY o CY, en semen fresco (SF), refrigerado durante 2 h (SR) y semen congelado (SC) se resumen en el cuadro 1.

Medias (±EEM) para la calidad del semen criopreservado de machos cabríos Alpinos- frances diluido con lecitina de soya o yema de huevo
Parámetros MM(escala, 1-5) MI (%) VE (%)
Semen Fresco
LS 4.6±0.2a 63.0±1.2a 76.0±2.0a
TY 4.8±0.3a 62.5±1.4a 75.0±0.0a
CY 4.8±0.3a 61.3±4.3a 71.3±4.0a
Semen Refrigerado
LS 3.1±1.0a 44.0±12.0ab 51.0±13.0bc
TY 4.4±0.1a 57.5±2.5a 71.3±2.4a
CY 4.0±0.2a 55.0±3.5a 69.0±3.1ab
Semen Congelado
LS 1.0±0.4c 11.0±6.4c 11.0±7.1d
TY 2.4±0.5b 32.5±8.3b 41.3±13.0c
CY 0.5±0.3c 6.3±3.8c 2.5±2.5d

Tratamientos: Andromed® (LS; 1% de lecitina de soya), Optidyl® (TY: Tris- 20% de yema de huevo) o citrato- yema de huevo (CY). Motilidad masal (MM; escala,1-5), Motilidad individual (MI; %), Viabilidad espermatica (VE; %). abcd Superíndices desiguales entre filas indican diferencia estadística significativa (P≤0.05).

En el SF, se obtuvieron valores similares entre los grupos (P>0.05) en cada una de las variables evaluadas [MM (4.7 ± 0.26 %), VE (74.1 ± 1.66 %) y MI (62.3 ±4.0 %)]. Los resultados sugieren que la composición de los diluyentes utilizados en este estudio afecta la calidad de los espermatozoides posterior al proceso de descongelamiento. Sin embargo, la calidad seminal post descongelación se vio más afectada cuando se utilizó el diluyente a base LS, en comparación con el TY. Estos resultados son similares a los reportados en equinos y cérvidos; en los cuales se muestra una mayor calidad seminal cuando se utilizan diluyentes a base de yema de huevo (Pillet et al., 2012; Stewart et al., 2018). Es probable que los resultados encontrados se deban a que el componente Tris- yema de huevo ayuda a disminuir la generación de especies reactivas de oxígeno (ROS) manteniendo así el potencial de integridad de la membrana al reducir el choque térmico causado por los cambios de temperatura en el proceso de crioconservación (Alcay et al., 2016; Seifi-Jamadi et al., 2017). Lo anterior puede estar relacionado con el componente efectivo de la yema de huevo que es la lipoproteína de baja densidad (20%), que contiene el TY y funciona como fracción crioprotectora que ayuda a mantener el semen de mejor calidad (Amirat et al., 2004; Forouzanfar et al., 2010; Alcay et al., 2016).

Al comparar los efectos de los diluyentes en las condiciones de SR, no existieron diferencias (P>0.05) en la MM y MI (3.83±0.4 % y 52.1±6.0 %, respectivamente); sin embargo, el porcentaje de VE fue más bajo con el diluyente a base de LS comparado con el TY y CY (51.0±13.0 vs 71.3±3.0 y 69.0±3.1 respectivamente; P<0.05).

Los resultados reportados en el presente experimento referentes al diluyente TY concuerdan con lo reportado por Celeghini et al. (2008), que indican una mayor integridad del acrosoma de espermatozoides de toro, y Konyak et al. (2018) quienes encontraron que después del equilibrio y la congelación del semen de machos cabríos, la motilidad espermática es significativamente mayor al utilizar el diluyente Tris con 20% de yema de huevo, respecto al conformado por 1% de lecitina de soya. Al respecto, se ha comprobado que la lecitina de soya no es capaz de evitar la peroxidación lipídica que ocurre durante el proceso de enfriamiento del espermatozoide (Salmani et al., 2013).

Previas investigaciones en ovinos han demostrado que el semen diluido con lecitina de soya presenta daño en la membrana espermática, y en consecuencia daño a nivel mitocondrial, lo que resulta en una menor movilidad y fertilidad de los espermatozoides (Del Valle et al., 2012 ; Lima-Verde et al., 2017). Konyak et al. (2018) atribuye la baja en la calidad espermática del semen expuesto a la LS a diferencias en la concentración de la lecitina de soya utilizada, en relación a esto, Forouzanfar et al. (2010) observaron en semen de carneros que los diluyentes que contenían concentraciones de 1% de lecitina presentaban mayor viabilidad espermática, comparado con los de 2% de lecitina, y además que el rango 1 a 1.5% de lecitina de soya en el diluyente mostraron mejores características del semen después de la preservación.

En el SC, los valores de MM, MI y VE fueron superiores en el semen diluido con TY (2.4±0.5, 32.5±8.3, 41.3±13.0, respectivamente; P≤0.05) en comparación con el semen diluido con LS y el CY que disminuyeron su MM, MI y VE drásticamente (1.0.0±0.4, 11.0±6.4, 11.0±7.1 y 0.5±0.3±, 6.3± 3.8 y 2.5± 2.5 respectivamente; P≤0.05). De igual forma los resultados en nuestro estudio en el CY post-descongelación fue inferiores en comparación con el TY. Es probable que estos resultados estén asociados a los componentes del diluyente, específicamente al porcentaje de yema de huevo, en el diluyente CY fue a una concentración del 15%, y en el TY al 20%. Estos resultados concuerdan con Fourouzanfar et al. (2010), quienes reportan que la motilidad y viabilidad espermática post-descongelación son superiores cuando se utiliza una concentración de yema de huevo al 20% que cuando se utiliza al 15%. Del mismo modo, otros trabajos confirman que el aumento en la concentración de la yema de huevo mejora la preservación de la calidad espermática (Amirat et al., 2004; Forouzanfar et al., 2010; Alcay et al., 2016). La razón de este mejoramiento puede ser debido a que los fosfolípidos que contiene la yema de huevo como la fosfatidilcolina, son importantes para el mantenimiento de la integridad de la membrana espermática durante el proceso de congelación y post-descongelación (Mousa et al., 2002; Amirat et al., 2004; Forouzanfar et al., 2010; Alcay et al., 2016). Por lo que es probable que un alto porcentaje de yema de huevo mejore la viabilidad espermática observada en el TY, y ésta contribuyera a mantener los niveles de ácidos grasos polinsatrurados necesarios para la membrana de los espermatozoides, siendo menos susceptibles a la peroxidación lipídica destructiva (Cerolini et al., 2001; Kaeoket et al., 2010).

Por otra parte, la mala calidad seminal post-descongelación del CY, pudo deberse a que la yema de huevo tiene alto riesgo de sufrir una contaminación microbiana, lo cual pudo disminuir la calidad seminal, post-descongelación (Aboagla et al., 2004; Kulakzis et al., 2010); y al ser la yema de huevo comercial (CY), pudo no tener una buena calidad sanitaria, lo que perjudicó la calidad seminal post-congelación. Otro factor que pudo afectar la calidad del semen del CY, es la dieta y el manejo de las aves productoras (Lima- Verde et al., 2017). En efecto, varios estudios han mostrado que las yemas de huevo de diferentes especies de aves muestran una variación en sus componentes, lo que resulta en diferentes efectos en el proceso de criopreservación sobre los espermatozoides (Trimeche et al., 1997; Bathgate et al., 2006; Singh et al., 2013). Además, la yema de huevo puede contener metabolitos dañinos y endotoxinas que afectan la viabilidad de los espermatozoides (Vidal et al., 2013), así como hormonas esteroides que reducen la motilidad espermática (El-Sisy et al., 2018). Previos estudios de laboratorio revelan que, mediante eliminación de algunos componentes en la yema de huevo por centrifugación; además de ciertas sustancias en la yema que inhiben la respiración y la motilidad espermática, sugiriendo el reemplazo la yema de huevo entera por la fracción crioprotectora (Amirat et al., 2004).

 CONCLUSIONES

Los resultados del presente estudio no mostraron diferencias estadísticas significativas entre el uso de los diferentes diluyentes para la conservación del SF y SR; sin embargo, el Tris-yema de huevo obtuvo una mayor motilidad masal e individual y viabilidad post- descongelación en comparación con el diluyente a base de lecitina de soya durante el proceso de crioconservación del semen de machos cabríos Alpinos

 AGRADECIMIENTOS

Los autores agradecen el apoyo financiero brindado a la Secretaria de Agricultura y Desarrollo Rural y al Consejo Nacional de Ciencia y Tecnología (SADER-CONACYT, México) por el apoyo financiero otorgado a través del Fondo Sectorial de Investigación en Materia Agrícola, Pecuaria, Acuacultura, Agrobiotecnología y Recursos Fitogeneticos, 2017-04-291691.

 LITERATURA CITADA Aboagla EME, Terada T. 2004. Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa. Theriogenology. 62(6):1160-1172. ISSN: 0093-691X. https://doi.org/10.1016/j.theriogenology.2004.01.013 Aboagla EME Terada T. 2004 Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa Theriogenology 62 6 1160 1172 0093-691X 10.1016/j.theriogenology.2004.01.013 Akçay E Kulaksız R, Daşkin A, Çebi Ç, Tekin K. 2012. The effect of different dilution rates on post-thaw quality of ram semen frozen in two different egg-yolk free extenders. Slovenian Veterinary Research. 49(2):97-102. ISSN: 1580-4003. https://www.academia.edu/24162140/The_Effect_of_Different_Dilution_Rates_on_Post_Thaw_Quality_of_Ram_Semen_Frozen_in_Two_Different_Eggyolk_Free_Extenders Akçay E Kulaksız R Daşkin A Çebi Ç Tekin K. 2012 The effect of different dilution rates on post-thaw quality of ram semen frozen in two different egg-yolk free extenders Slovenian Veterinary Research 49 2 97 102 1580-4003 https://www.academia.edu/24162140/The_Effect_of_Different_Dilution_Rates_on_Post_Thaw_Quality_of_Ram_Semen_Frozen_in_Two_Different_Eggyolk_Free_Extenders Akhter S, Ansari MS, Andrabi SMH, Rakha BA, Ullah N, Khalid M. 2012. Soya‐lecithin in extender improves the freezability and fertility of buffalo (Bubalus bubalis) bull spermatozoa. Reproduction in Domestic Animals. 47(5):815-819.ISSN: 1439-0531.xhttps://doi.org/10.1111/j.1439-0531.2011.01973.x Akhter S Ansari MS Andrabi SMH Rakha BA Ullah N Khalid M. 2012 Soya‐lecithin in extender improves the freezability and fertility of buffalo (Bubalus bubalis) bull spermatozoa Reproduction in Domestic Animals 47 5 815 819 1439-0531.x 10.1111/j.1439-0531.2011.01973.x Alcay S, Gokce E, Toker MB, Onder NT, Ustuner B, Uzabacı E, Cavus S. 2016. Freeze- dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa. Cryobiology. 72(3): 269-273. ISSN: 0011-2240. https://doi.org/10.1016/j.cryobiol.2016.03.007 Alcay S Gokce E Toker MB Onder NT Ustuner B Uzabacı E Cavus S. 2016 Freeze- dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa Cryobiology 72 3 269 273 0011-2240 10.1016/j.cryobiol.2016.03.007 Amirat L, Tainturier D, Jeanneau L, Thorin C, Gérard O, Courtens JL, Anton M. 2004. Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl®, a commercial egg yolk extender. Theriogenology. 61(5): 895-907. ISSN: 0093- 691X. https://doi.org/10.1016/S0093-691X(03)00259-0 Amirat L Tainturier D Jeanneau L Thorin C Gérard O Courtens JL Anton M. 2004 Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl®, a commercial egg yolk extender Theriogenology 61 5 895 907 0093-691X 10.1016/S0093-691X(03)00259-0 Andrabi SMH, Ansari MS, Ullah N, Anwar M, Mehmood A, Akhter S. 2008. Duck egg yolk in extender improves the freezability of buffalo bull spermatozoa. Animal Reproduction Science. 104(2-4): 427-433. ISSN: 0378-4320. https://doi.org/10.1016/j.anireprosci.2007.07.003 Andrabi SMH Ansari MS Ullah N Anwar M Mehmood A Akhter S. 2008 Duck egg yolk in extender improves the freezability of buffalo bull spermatozoa Animal Reproduction Science 104 2-4 427 433 0378-4320 10.1016/j.anireprosci.2007.07.003 Ansari MS, Rakha BA, Akhter S. 2017. Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation. Reproduction in Domestic Animals. 52(6):992-997. ISSN: 1439-0531. https://doi.org/10.1111/rda.13008 Ansari MS Rakha BA Akhter S. 2017 Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation Reproduction in Domestic Animals 52 6 992 997 1439-0531 10.1111/rda.13008 Bathgate R, Maxwell WMC, Evans G. 2006. Studies on the effect of supplementing boar semen cryopreservation media with different avian egg yolk types on in vitro post‐ thaw sperm quality. Reproduction in Domestic Animals. 41(1):68-73. ISSN: 1439-0531. https://doi.org/10.1111/j.1439-0531.2006.00623.x Bathgate R Maxwell WMC Evans G. 2006 Studies on the effect of supplementing boar semen cryopreservation media with different avian egg yolk types on in vitro post‐ thaw sperm quality Reproduction in Domestic Animals 41 1 68 73 1439-0531 10.1111/j.1439-0531.2006.00623.x Belala R, Briand-Amirat L, Vinciguerra L, Tainturier D, Kaidi R, Thorin C, Bencharif D. 2016. Effect of equilibration time on the motility and functional integrity of canine spermatozoa frozen in three different extenders. Research in Veterinary Science. 106: 66-73. ISSN:0034-5288. https://doi.org/10.1016/j.rvsc.2016.03.010 Belala R Briand-Amirat L Vinciguerra L Tainturier D Kaidi R Thorin C Bencharif D. 2016 Effect of equilibration time on the motility and functional integrity of canine spermatozoa frozen in three different extenders Research in Veterinary Science 106 66 73 0034-5288 10.1016/j.rvsc.2016.03.010 Celeghini ECC, de Arruda RP, de Andrade AFC, Nascimento J, Raphael CF, Rodrigues PHM. 2008. Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin. Animal Reproduction Science. 104(2-4):119-131. ISSN:0378-4320. https://doi.org/10.1016/j.anireprosci.2007.02.001 Celeghini ECC de Arruda RP de Andrade AFC Nascimento J Raphael CF Rodrigues PHM. 2008 Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin Animal Reproduction Science 104 2-4 119 131 0378-4320 10.1016/j.anireprosci.2007.02.001 Cerolini S, Maldjian, A, Pizzi F, Gliozzi T. M. 2001. Changes in sperm quality and lipid composition during cryopreservation of boar semen. Reproduction. 121(3):395-401. ISSN: 1741-7899. https://doi.org/10.1530/rep.0.1210395 Cerolini S Maldjian A Pizzi F Gliozzi T. M. 2001 Changes in sperm quality and lipid composition during cryopreservation of boar semen Reproduction 121 3 395 401 1741-7899 10.1530/rep.0.1210395 CONAGUA. 2015. Normales climatológicas por estación. Ciudad de México: Servicio Meteorológico Nacional, Comisión Nacional del Agua. https://smn.conagua.gob.mx/es/ CONAGUA 2015 Normales climatológicas por estación Ciudad de México Servicio Meteorológico Nacional, Comisión Nacional del Agua https://smn.conagua.gob.mx/es/ Del Valle I, Gómez‐Durán A, Holt WV, Muiño‐Blanco T, Cebrián‐Pérez JA. 2012. Soy lecithin interferes with mitochondrial function in frozen‐thawed ram spermatozoa. Journal of Andrology. 33(4):717-725. ISSN: 1939-4640. https://doi.org/10.2164/jandrol.111.014944 Del Valle I Gómez‐Durán A Holt WV Muiño‐Blanco T Cebrián‐Pérez JA. 2012 Soy lecithin interferes with mitochondrial function in frozen‐thawed ram spermatozoa Journal of Andrology 33 4 717 725 1939-4640 10.2164/jandrol.111.014944 El-Sisy GA, El-Badry DA, El-Sheshtawy RI, El-Nattat WS. 2018. Effects of Phoenix dactylifera pollen grains extract supplementation on post-thaw quality of Arabian stallion semen. Bulgarian Journal of Veterinary Medicine. 21(1):40-49. ISSN: 1.311-1477. https://doi.org/10.15547/bjvm.1044 El-Sisy GA El-Badry DA El-Sheshtawy RI El-Nattat WS. 2018 Effects of Phoenix dactylifera pollen grains extract supplementation on post-thaw quality of Arabian stallion semen Bulgarian Journal of Veterinary Medicine 21 1 40 49 1.311-1477 10.15547/bjvm.1044 FASS. 2010. Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching, 3rd ed. Federation Animal Science Society, Savoy, IL, USA. ISBN: 978-956-14-2161-5. https://books.google.com.mx/books?id=EA1QDwAAQBAJ&printsec=frontcover&dq=Guide+for+the+Care+and+Use+of+Agricultural+Animals+in+Agricultural+Research+and+Teaching&hl=es&sa=X&ved=2ahUKEwi8svisttjrAhUIW60KHUttBHMQ6AEwAnoECAUQAg#v=onepage&q=Guide%20for%20the%20Care%20and%20Use%20of%20Agricultural%20Animals%20in%20Agricultural%20Research%20and%20Teaching&f=false FASS 2010 Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching 3 Federation Animal Science Society Savoy, IL, USA 978-956-14-2161-5https://books.google.com.mx/books?id=EA1QDwAAQBAJ&printsec=frontcover&dq=Guide+for+the+Care+and+Use+of+Agricultural+Animals+in+Agricultural+Research+and+Teaching&hl=es&sa=X&ved=2ahUKEwi8svisttjrAhUIW60KHUttBHMQ6AEwAnoECAUQAg#v=onepage&q=Guide%20for%20the%20Care%20and%20Use%20of%20Agricultural%20Animals%20in%20Agricultural%20Research%20and%20Teaching&f=false Forouzanfar M, Sharafi M, Hosseini S M, Ostadhosseini S, Hajian M, Hosseini L, Nasr-Esfahani MH. 2010. In vitro comparison of egg yolk-based and soybean lecithin- based extenders for cryopreservation of ram semen. Theriogenology. 73(4):480-487. ISSN: 0093-691X. https://doi.org/10.1016/j.theriogenology.2009.10.005 Forouzanfar M Sharafi M Hosseini S M Ostadhosseini S Hajian M Hosseini L Nasr-Esfahani MH. 2010 In vitro comparison of egg yolk-based and soybean lecithin- based extenders for cryopreservation of ram semen Theriogenology 73 4 480 487 0093-691X 10.1016/j.theriogenology.2009.10.005 Gamal A, El-Maaty AMA, Rawash ZM. 2016. Comparative blood and seminal plasma oxidant/antioxidant status of Arab stallions with different ages and their relation to semen quality. Asian Pacific Journal of Reproduction. 5(5):428-433. ISSN: 2305-0500. https://doi.org/10.1016/j.apjr.2016.07.006 Gamal A El-Maaty AMA Rawash ZM. 2016 Comparative blood and seminal plasma oxidant/antioxidant status of Arab stallions with different ages and their relation to semen quality Asian Pacific Journal of Reproduction 5 5 428 433 2305-0500 10.1016/j.apjr.2016.07.006 Jerez R, González N, Olaciregui M, Luño V, de Blas I, Gil L. 2016. Use of soy milk combined with different cryoprotectants for the ram semen cryopreservation. Small Ruminant Research. 134:34-38. ISNN: 0921-4488. https://doi.org/10.1016/j.smallrumres.2015.12.003 Jerez R González N Olaciregui M Luño V de Blas I Gil L. 2016 Use of soy milk combined with different cryoprotectants for the ram semen cryopreservation Small Ruminant Research 134 34 38 0921-4488 10.1016/j.smallrumres.2015.12.003 Kaeoket K, Chanapiwat P, Tummaruk P, Techakumphu M. 2010. Supplemental effect of varying L-cysteine concentrations on the quality of cryopreserved boar semen. Asian Journal of Andrology. 12(5):760. https://dx.doi.org/10.1038/aja.2010.48 Kaeoket K Chanapiwat P Tummaruk P Techakumphu M. 2010 Supplemental effect of varying L-cysteine concentrations on the quality of cryopreserved boar semen Asian Journal of Andrology 12 5 760 760 10.1038/aja.2010.48 Kafi M, Safdarian M, Hashemi M. 2004. Seasonal variation in semen characteristics, scrotal circumference, and libido of Persian Karakul rams. Small Ruminant Research. 53(1-2):133-139. ISSN: 0921-4488. https://doi.org/10.1016/j.smallrumres.2003.07.007 Kafi M Safdarian M Hashemi M. 2004 Seasonal variation in semen characteristics, scrotal circumference, and libido of Persian Karakul rams Small Ruminant Research 53 1-2 133 139 0921-4488 10.1016/j.smallrumres.2003.07.007 Konyak P, Mandal A, Mondal M, Bhakat C, Das SK, Rai S, Karunakaran M. 2018. Preservation of black Bengal buck semen in soybean lecithin based chemically defined extender. Indian Journal of Animal Research. 52(8):1151-1154. ISSN: 0976-0555. https://doi.org/10.18805/ijar.B-3335 Konyak P Mandal A Mondal M Bhakat C Das SK Rai S Karunakaran M. 2018 Preservation of black Bengal buck semen in soybean lecithin based chemically defined extender Indian Journal of Animal Research 52 8 1151 1154 0976-0555 10.18805/ijar.B-3335 Kulaksiz R, Çebi Ç, Akçay E, Daşkın A. 2010. The protective effect of egg yolk from different avian species during the cryopreservation of Karayaka ram semen. Small Ruminant Research. 88(1):12-15. ISSN: 0921-4488.https://doi.org/10.1016/j.smallrumres.2009.11.014 Kulaksiz R Çebi Ç Akçay E Daşkın A. 2010 The protective effect of egg yolk from different avian species during the cryopreservation of Karayaka ram semen Small Ruminant Research 88 1 12 15 0921-4488 10.1016/j.smallrumres.2009.11.014 Kumar P, Saini M, Kumar D, Balhara AK, Yadav SP, Singh P, Yadav PS. 2015. Liposome-based semen extender is suitable alternative to egg yolk-based extender for cryopreservation of buffalo (Bubalus bubalis) semen. Animal Reproduction Science. 159:38-45. ISSN: 0378-4320. https://doi.org/10.1016/j.anireprosci.2015.05.010 Kumar P Saini M Kumar D Balhara AK Yadav SP Singh P Yadav PS. 2015 Liposome-based semen extender is suitable alternative to egg yolk-based extender for cryopreservation of buffalo (Bubalus bubalis) semen Animal Reproduction Science 159 38 45 0378-4320 10.1016/j.anireprosci.2015.05.010 Lima-Verde IB, Johannisson A, Ntallaris T, Al‐Essawe E, Al‐Kass Z, Nongbua T, Morrell, JM. 2017. Effect of freezing bull semen in two non‐egg yolk extenders on post‐thaw sperm quality. Reproduction in Domestic Animals. 53(1): 127-136. ISSN: 1439-0531 https://doi.org/10.1111/rda.13080 Lima-Verde IB Johannisson A Ntallaris T Al‐Essawe E Al‐Kass Z Nongbua T Morrell JM. 2017 Effect of freezing bull semen in two non‐egg yolk extenders on post‐thaw sperm quality Reproduction in Domestic Animals 53 1 127 136 1439-0531 10.1111/rda.13080 Mahsud T, Jamil H, Qureshi ZI, Asi MN, Lodhi LA, Waqas MS, Ahmad A. 2013. Semen quality parameters and selected bio-chemical constituents level in plasma of Lohi rams. Small Ruminant Research. 113(1): 175-178. ISSN: 0921-4488. https://doi.org/10.1016/j.smallrumres.2013.04.004 Mahsud T Jamil H Qureshi ZI Asi MN Lodhi LA Waqas MS Ahmad A. 2013 Semen quality parameters and selected bio-chemical constituents level in plasma of Lohi rams Small Ruminant Research 113 1 175 178 0921-4488 10.1016/j.smallrumres.2013.04.004 Moussa M, Martinet V, Trimeche A, Tainturier D, Anton M. 2002. Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen. Theriogenology. 57(6):1695-1706. ISSN: 0093-691X. https://doi.org/10.1016/S0093-691X(02)00682-9 Moussa M Martinet V Trimeche A Tainturier D Anton M. 2002 Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen Theriogenology 57 6 1695 1706 0093-691X 10.1016/S0093-691X(02)00682-9 NAM. 2002. Guide for the Care and Use of Laboratory Animals. Co-produced by the National Academy of Medicine-Mexico and the Association for Assessment and Accreditation of Laboratory Animal Care International, 1st ed. Harlan Mexico, DF, Mexico. ISBN: 978-0-309-15400-0. https://books.google.com.mx/books?id=GyxkAgAAQBAJ&printsec=frontcover&dq=Guide+for+the+Care+and+Use+of+Laboratory+Animals.+ISBN&hl=es&sa=X&ved=2ahUKEwjr1pGOtNjrAhUEPq0KHRA3AU0Q6AEwAHoECAIQAg#v=onepage&q=Guide%20for%20the%20Care%20and%20Use%20of%20Laboratory%20Animals.%20ISBN&f=false NAM 2002 Guide for the Care and Use of Laboratory Animals. Co-produced by the National Academy of Medicine-Mexico and the Association for Assessment and Accreditation of Laboratory Animal Care International 1 Harlan Mexico DF, Mexico 978-0-309-15400-0 https://books.google.com.mx/books?id=GyxkAgAAQBAJ&printsec=frontcover&dq=Guide+for+the+Care+and+Use+of+Laboratory+Animals.+ISBN&hl=es&sa=X&ved=2ahUKEwjr1pGOtNjrAhUEPq0KHRA3AU0Q6AEwAHoECAIQAg#v=onepage&q=Guide%20for%20the%20Care%20and%20Use%20of%20Laboratory%20Animals.%20ISBN&f=false Ngoma L, Kambulu L, Mwanza M. 2016. Factors Influencing goat’s semen fertility and storage: A Literature Review. Journal of Human Ecology. 56(1-2):114-125. ISSN: 2456-6608. https://doi.org/10.1080/09709274.2016.11907045 Ngoma L Kambulu L Mwanza M. 2016 Factors Influencing goat’s semen fertility and storage: A Literature Review Journal of Human Ecology 56 1-2 114 125 2456-6608 10.1080/09709274.2016.11907045 NRC. 2007. Nutrient Requirements of Small Ruminants: Sheep, Goats, Cervids and New World Camelids. National Research Council, National Academies Press, Washington, USA. ISBN: 978-0-309-47323-1. https://www.nap.edu/catalog/11654/nutrient-requirements-of-small-ruminants-sheep-goats-cervids-and-new NRC 2007 Nutrient Requirements of Small Ruminants: Sheep, Goats, Cervids and New World Camelids National Research Council, National Academies Press Washington, USA 978-0-309-47323-1 https://www.nap.edu/catalog/11654/nutrient-requirements-of-small-ruminants-sheep-goats-cervids-and-new Pillet E, Labbe C, Batellier F, Duchamp G, Beaumal V, Anton M, Magistrini M. 2012. Liposomes as an alternative to egg yolk in stallion freezing extender. Theriogenology. 77(2):268-279. ISSN: 0093-691X. https://doi.org/10.1016/j.theriogenology.2011.08.001 Pillet E Labbe C Batellier F Duchamp G Beaumal V Anton M Magistrini M. 2012 Liposomes as an alternative to egg yolk in stallion freezing extender Theriogenology 77 2 268 279 0093-691X 10.1016/j.theriogenology.2011.08.001 Salamon S, Maxwell W MC. 2000. Storage of ram semen. Animal Reproduction Science. 62(1-3):77-111. ISSN: 0378-4320. https://doi.org/10.1016/S0378-4320(00)00155-X Salamon S Maxwell W MC. 2000 Storage of ram semen Animal Reproduction Science 62 1-3 77 111 0378-4320 10.1016/S0378-4320(00)00155-X Salmani H, Nabi MM, Vaseghi-Dodaran H, Rahman MB, Mohammadi-Sangcheshmeh A, Shakeri M, Zhandi M. 2013. Effect of glutathione in soybean lecithin-based semen extender on goat semen quality after freeze-thawing. Small Ruminant Research.112(1-3):123-127. ISSN:0921-4488. https://doi.org/10.1016/j.smallrumres.2012.12.015 Salmani H Nabi MM Vaseghi-Dodaran H Rahman MB Mohammadi-Sangcheshmeh A Shakeri M Zhandi M. 2013 Effect of glutathione in soybean lecithin-based semen extender on goat semen quality after freeze-thawing Small Ruminant Research 112 1-3 123 127 0921-4488 10.1016/j.smallrumres.2012.12.015 SAS (Statistical Analysis System) 2005. Statistical Analysis Software SAS/STAT®. version 9.1, Cary, N.C., USA: SAS Institute Inc. SAS (Statistical Analysis System) 2005 Statistical Analysis Software SAS/STAT®. version 9.1 Cary, N.C., USA SAS Institute Inc Seifi-Jamadi A, Ahmad E, Ansari M, Kohram, H. 2017. Antioxidant effect of quercetin in an extender containing DMA or glycerol on freezing capacity of goat semen. Cryobiology. 75:15-20. ISSN: 0011-2240. https://doi.org/10.1016/j.cryobiol.2017.03.002 Seifi-Jamadi A Ahmad E Ansari M Kohram H 2017 Antioxidant effect of quercetin in an extender containing DMA or glycerol on freezing capacity of goat semen Cryobiology 75 15 20 0011-2240 10.1016/j.cryobiol.2017.03.002 Sieme H, Oldenhof H, Wolkers WF. 2016. Mode of action of cryoprotectants for sperm preservation. Animal Reproduction Science. 169:2-5. ISSN: 0378-4320. https://doi.org/10.1016/j.anireprosci.2016.02.004 Sieme H Oldenhof H Wolkers WF. 2016 Mode of action of cryoprotectants for sperm preservation Animal Reproduction Science 169 2 5 0378-4320 10.1016/j.anireprosci.2016.02.004 Singh MAHAK, Ramteke SS, Ghosh SK, Prasad JK, Rajoriya JS. 2013. Efficacy of egg yolk from three avian species on semen freezability of Tharparkar bull. Indian Journal Animals Reproduction. 34(2):25-28. ISSN: 0970-2997. https://scholar.google.com/citations?user=ot596g4AAAAJ&hl=en#d=gs_md_cita-d&u=%2Fcitations%3Fview_op%3Dview_citation%26hl%3Den%26user%3Dot596g4AAAAJ%26citation_for_view%3Dot596g4AAAAJ%3AWF5omc3nYNoC%26tzom%3D300 Singh MAHAK Ramteke SS Ghosh SK Prasad JK Rajoriya JS. 2013 Efficacy of egg yolk from three avian species on semen freezability of Tharparkar bull Indian Journal Animals Reproduction 34 2 25 28 0970-2997 https://scholar.google.com/citations?user=ot596g4AAAAJ&hl=en#d=gs_md_cita-d&u=%2Fcitations%3Fview_op%3Dview_citation%26hl%3Den%26user%3Dot596g4AAAAJ%26citation_for_view%3Dot596g4AAAAJ%3AWF5omc3nYNoC%26tzom%3D300 Stewart JL, Shipley CF, Ellerbrock RE, Schmidt L, Lima FS, Canisso IF. 2018. Physiological variations in reproductive and metabolic features of white-tailed deer (Odocoileus virginianus) bucks throughout the rutting season. Theriogenology. 114(1):308-316. ISSN: 0093-691X. https://doi.org/10.1016/j.theriogenology.2018.04.015 Stewart JL Shipley CF Ellerbrock RE Schmidt L Lima FS Canisso IF. 2018 Physiological variations in reproductive and metabolic features of white-tailed deer (Odocoileus virginianus) bucks throughout the rutting season Theriogenology 114 1 308 316 0093-691X 10.1016/j.theriogenology.2018.04.015 Trimeche A, Anton M, Renard P, Gandemer G, Tainturier D. 1997. Quail egg yolk: a novel cryoprotectant for the freeze preservation of Poitou jackass sperm. Cryobiology. 34(4):385-393. ISSN: 0011-2240. https://doi.org/10.1006/cryo.1997.2009 Trimeche A Anton M Renard P Gandemer G Tainturier D. 1997 Quail egg yolk: a novel cryoprotectant for the freeze preservation of Poitou jackass sperm Cryobiology 34 4 385 393 0011-2240 10.1006/cryo.1997.2009 Vidal AH, Batista AM, da Silva ECB, Gomes WA, Pelinca MA, Silva SV, Guerra MMP. 2013. Soybean lecithin-based extender as an alternative for goat sperm cryopreservation. Small Ruminant Research. 109(1):47-51. ISSN: 0921-4488. https://doi.org/10.1016/j.smallrumres.2012.07.022 Vidal AH Batista AM da Silva ECB Gomes WA Pelinca MA Silva SV Guerra MMP. 2013 Soybean lecithin-based extender as an alternative for goat sperm cryopreservation Small Ruminant Research 109 1 47 51 0921-4488 10.1016/j.smallrumres.2012.07.022 Zeron Y, Tomczak M, Crowe J, Arav A. 2002. The effect of liposomes on thermotropic membrane phase transitions of bovine spermatozoa and oocytes: implications for reducing chilling sensitivity. Cryobiology. 45(2):143-152. ISSN: 0011-2240. https://doi.org/10.1016/S0011-2240(02)00123-2 Zeron Y Tomczak M Crowe J Arav A. 2002 The effect of liposomes on thermotropic membrane phase transitions of bovine spermatozoa and oocytes: implications for reducing chilling sensitivity Cryobiology 45 2 143 152 0011-2240 10.1016/S0011-2240(02)00123-2 Original Article Determination of the quality of semen cryopreserved with soy lecithin or egg yolk, in male goats

Code:2020-75

 ABSTRACT:

The objective was to compare the quality of cryopreserved goat semen with soy lecithin or egg yolk. The semen was collected from male goats (n=4), two commercial diluents AndroMed® (1% soy lecithin, SL); Optidyl (20% (v/v) Tris-egg yolk; TY), and a citrate-egg yolk-based diluent (CY) were used in fresh semen (FS) and then cooled from 37 to 4 °C for 2 h (refrigerated semen, RS), afterwards straws were filled with semen and frozen in liquid nitrogen at -196 °C (FS). There were no differences (p>0.05) between diluents in the FS in the mass motility (MM; 4.7±0.26), sperm viability (SV; 74.1±1.66) and individual motility (MI; 62.3±4.0). In the same sense, for the RS there was no difference (p>0.05) between diluents with respect to MM (3.83±0.4) and MI (52.1±6.0), however, the SV varied (p<0.05) according to the diluent, observing the lowest viability in SL vs CY and TY (51.0±13.0 vs 71.3±3.0 and 69.0±3.1). Regarding FS the MM, MI and SV obtained better values (p<0.05) with the diluent TY vs SL and CY (2.4±0.5, 32.5±8.3, 41.3±13.0). The results showed a better cryopreservation of goat semen with the diluent Tris-yolk compared to that of soy lecithin.

 Keywords: Soy lecithin diluent goat semen INTRODUCTION

Traditional diluents that are added to semen for the preservation of sperm viability and fertility during cryopreservation include egg yolk (Lima-Verde et al., 2017). This is due to the fact that the egg yolk protects the sperm from the damage induced by cryopreservation during cooling, freezing and thawing by interacting directly with the plasma membrane (Andrabi et al., 2008; Akçay et al., 2012; Sieme et al., 2016). However, in recent years, there has been frequent opinion against the use of egg yolk due to the great variability of its components, which makes the evaluation of its benefits complex (Kulaksız et al., 2010); Furthermore, an attempt has been made to avoid the use of diluents of animal origin, since they could be a possible route of disease transmission (Lima-Verde et al., 2017; Ansari et al., 2017).

Regarding the egg yolk, some undesirable components (steroid hormones and their precursor molecules) are considered detrimental to the integrity of the sperm (Akhter et al., 2012; Lima-Verde et al., 2017). The main component of egg yolk that protects the sperm membrane is low-density lipoprotein (LDL). Phospholipids or lecithin in egg yolk have been shown to make sperm less sensitive to cooling (Zeron et al., 2002). In particular, in the male goat, there are negative interactions between the phospholipids of the egg yolk and the bulbourethral gland, this gland secretes with the seminal plasma a coagulating enzyme of the egg yolk, which catalyzes the hydrolysis of the lecithin of the yolk in fatty acids and lysolecithin, which are cytotoxic (Ngoma et al., 2016). Therefore, chemically defined substitutes for egg yolk have been used without being of animal origin (El-Sisy et al., 2018; Gamal et al., 2016), made from soy lecithin and which can be alternatives potentials for the cryopreservation of semen (Akhter et al., 2012).

Liposomes are believed to act similarly to lecithins in egg yolk or milk (Belala et al., 2016). In water buffalo, no differences were found in terms of acrosomal integrity when diluents based on egg yolk, lecithin or soy liposomes were used (Kumar et al., 2015; Singh et al., 2013). In this context, diluents based on egg yolk deserve special attention in terms of their components and their effect compared to diluents based on liposomes. Due to the fact that the effect of the aforementioned components is little known on the quality of cryopreserved semen in the male goat, we set ourselves the objective of comparing the effects of diluents based on soy lecithin or egg yolk on the quality of semen, preserved by refrigeration and freezing.

 MATERIAL AND METHODS General

All the methods and management of the experimental units used in this study were in strict accordance with the guidelines for the ethical use, care and welfare of animals in research at the international level (FASS, 2010) and national level (NAM, 2002) with number UAAAN-UL institutional approval reference with code 38111-425501002-2431.

 Location and animals

The experiment was carried out in northern Mexico, at the Goat Center of the Antonio Narro Autonomous Agrarian University (26° North Latitude and 104° West Longitude), during the reproductive season (January). The study area is at an altitude of 1120 meters above sea level, with an average annual rainfall of 230 mm and an average temperature of 24 °C, a maximum of 41 °C in May and June, and a minimum of -1° in December and January. (CONAGUA, 2015). Adult male goats of the Alpine-French breed (n = 4, 1.5 to 2 years old), homogeneous in terms of live weight (LW; 75.0 ± 0.32 kg) and body condition (CC; 3.5 ± 0.10 units) were used; with proven fertility prior to the experimental study through frequent evaluations of seminal quality. During the experimental period, males were fed twice a day (800 h and 1800 h), with free access, with a diet based on alfalfa hay (18% PC, 1.95 Mcal of ME) and 100 g of commercial concentrate (21% PC, 1.7 Mcal ME) based on their nutritional requirements (NRC, 2007). The males had free access to clean water and mineral salts and an adaptation period of 2 weeks prior to the investigation.

 Collection and processing of semen

The semen was collected in the morning (800 to 1000 h) every 3 days, for three weeks, a female in oestrous activity was used as a stimulus for the extraction of semen. The semen was collected with a standard artificial vagina for sheep and goats, kept at a temperature of 38 °C, so it was preheated to 42 °C prior to collection. A total of 24 ejaculates were collected, after each extraction the semen was immediately immersed in a water bath at 37 °C for subsequent macroscopic and microscopic analysis during the next 10 minutes.

 Preparation of diluents and freezing process

Out of a total of 24 ejaculates (6 ejaculates per male) and each ejaculate was divided into three equal parts aliquots to be processed for cryopreservation, using two commercial diluents: AndroMed® (Minitübe, Tiefenbach, Germany; with 1% lecithin from soy; SL); Optidyl® (CRYO-VET, France; with 20% (v/v) of Tris-egg yolk; TY), and a third diluent based on citrate-fresh egg yolk (CY) obtained according to the technique used by Salamon and Maxwell, (2000).

Only samples with a volume> 0.5 mL, a concentration of 2.5x109 mL, mass motility ≥ 3.0, and viability ≥ 70% were considered in the experiment. Subsequently, the already diluted samples were submitted to 3 processes for evaluation: fresh semen (FS); chilled semen (RS, equilibrated at 4 °C for 2 hours); and frozen semen (FS). After refrigerating the semen, the 0.25 mL straws were filled and for the freezing process they were placed in 7 cm of liquid nitrogen (NL, -140 °C) for 10 min; then they were immersed directly in the NL (-196 °C) and stored until analysis (Jerez et al., 2016). In each of the conservation states (FS, RS and FS), the semen was analyzed, immediately and every 15 minutes during the conservation process, to evaluate the mass and individual motility and viability. In the case of FS, it was analyzed 24 hours later, for which the straw was thawed, immersing it in tempered water (37º C) for 30 s.

 Variables evaluated

Mass motility (MM;%) was evaluated with the use of a preheated platform (37 °C), placing a drop of pure semen (20 μl) on a slide in the optical microscope with a 10x objective, and according to With the observed movement, an arbitrary scale score of 1 to 5 was assigned, where 1=25% and 5=100% motile sperm (Mahsud et al., 2013). Individual motility (MI ;%) was determined based on the proportion of progressively mobile spermatozoa. For this, a drop (10µL) of semen was placed on a slide and covered with a coverslip slide; later it was observed under a microscope with a 40x objective. Sperm viability (SV;%), was evaluated by using the eosin-nigrosin staining technique (Kafi et al., 2004), at least 200 sperm per sample were observed by light microscope, using the 100X objective, and the percentage of live cells (unstained) and dead cells (stained pink) was calculated. All evaluations were always carried out by the same qualified evaluator.

<italic>Statistical analysis</italic>

Data were analyzed by ANOVA using the General Linear Model (GLM) procedure. The means obtained from the seminal parameters were compared using a t test. The effect of the use of different diluents, the states of the cryopreservation process and their interaction were considered. All data were analyzed using the statistical package SAS V9.1 (SAS, 2005). Differences were considered significant at a value of P≤0.05.

 RESULTS AND DISCUSSION

The results of the different parameters evaluated to determine the quality of semen diluted with SL, TY or CY, in fresh semen (FS), refrigerated for 2 h (RS) and frozen semen (FS) are summarized in Table 1.

Means (± SEM) for the quality of cryopreserved semen from French Alpine goats diluted with soy lecithin or egg yolk
Parameters MM (escala,1-5) MI (%) SV (%)
Fresh Semen
SL 4.6±0.2a 63.0±1.2a 76.0±2.0a
TY 4.8±0.3a 62.5±1.4a 75.0±0.0a
CY 4.8±0.3a 61.3±4.3a 71.3±4.0a
Refrigerated Semen
SL 3.1±1.0a 44.0±12.0ab 51.0±13.0bc
TY 4.4±0.1a 57.5±2.5a 71.3±2.4a
CY 4.0±0.2a 55.0±3.5a 69.0±3.1ab
Frozen Semen
SL 1.0±0.4c 11.0±6.4c 11.0±7.1d
TY 2.4±0.5b 32.5±8.3b 41.3±13.0c
CY 0.5±0.3c 6.3±3.8c 2.5±2.5d

Treatments: Andromed® (SL; 1% soy lecithin), Optidyl® (TY: Tris- 20% egg yolk) or citrate- egg yolk (CY). Mass motility (MM; scale, 1-5), Individual motility (MI;%), Sperm viability (SV;%). abcd Unequal superscripts between rows indicate significant statistical difference (P≤0.05).

In the FS, similar values were obtained between the groups (P> 0.05) in each of the evaluated variables [MM (4.7 ± 0.26%), SV (74.1 ± 1.66%) and MI (62.3 ± 4.0%)]. The results suggest that the composition of the diluents used in this study affects the quality of the sperm after the thawing process. However, post-thaw semen quality was more affected when SL-based diluent was used compared to TY. These results are similar to those reported in horses and cervids; in which a higher seminal quality is shown when using egg yolk-based diluents (Pillet et al., 2012; Stewart et al., 2018). It is likely that the results found are due to the fact that the Tris-egg yolk component helps to reduce the generation of reactive oxygen species (ROS), thus maintaining the integrity potential of the membrane by reducing the thermal shock caused by temperature changes in the cryopreservation process (Alcay et al., 2016; Seifi-Jamadi et al., 2017). This may be related to the effective component of the egg yolk which is the low-density lipoprotein (20%), which contains the TY and functions as a cryoprotective fraction that helps to maintain the better quality semen (Amirat et al., 2004; Forouzanfar et al., 2010; Alcay et al., 2016).

When comparing the effects of the diluents in the RS conditions, there were no differences (P> 0.05) in the MM and MI (3.83 ± 0.4% and 52.1 ± 6.0%, respectively); however, the SV percentage was lower with the SL-based diluent compared to TY and CY (51.0 ± 13.0 vs 71.3 ± 3.0 and 69.0 ± 3.1 respectively; P <0.05).

The results reported in the present experiment regarding the TY diluent agree with that reported by Celeghini et al. (2008), which indicate greater integrity of the bull sperm acrosome, and Konyak et al. (2018) who found that after balancing and freezing the semen of goats, sperm motility is significantly higher when using the Tris diluent with 20% egg yolk, compared to that made up of 1% soy lecithin. In this regard, it has been proven that soy lecithin is not capable of preventing lipid peroxidation that occurs during the sperm cooling process (Salmani et al., 2013).

Previous research in sheep has shown that semen diluted with soy lecithin presents damage to the sperm membrane, and consequently damage at the mitochondrial level, which results in less mobility and fertility of sperm (Del Valle et al., 2012; Lima-Verde et al., 2017). Konyak et al. (2018). (2018) attributes the low sperm quality of semen exposed to SL to differences in the concentration of soy lecithin used, in relation to this, Forouzanfar et al. (2010) observed in rams semen that diluents containing concentrations of 1% lecithin had higher sperm viability, compared to 2% lecithin, and also that the range 1 to 1.5% soy lecithin in the diluent showed better semen characteristics after preservation.

In FS, MM, MI and SV values were higher in semen diluted with TY (2.4 ± 0.5, 32.5 ± 8.3, 41.3 ± 13.0, respectively; P≤0.05) compared to semen diluted with SL and CY that their MM, MI and SV drastically decreased (1.0.0 ± 0.4, 11.0 ± 6.4, 11.0 ± 7.1 and 0.5 ± 0.3 ±,6.3 ± 3.8 and 2.5 ± 2.5 respectively; P≤0.05). Similarly, the results in our study in post- thaw CY were lower compared to TY. It is likely that these results are associated with the components of the diluent, specifically the percentage of egg yolk, in the CY diluent it was at a concentration of 15%, and in the TY at 20%. These results agree with Fourouzanfar et al. (2010), who report that post-thaw sperm motility and viability are higher when using a 20% egg yolk concentration than when using 15%. Similarly, other studies confirm that the increase in the concentration of egg yolk improves the preservation of sperm quality (Amirat et al., 2004; Forouzanfar et al., 2010; Alcay et al., 2016). The reason for this improvement may be due to the fact that the phospholipids contained in the egg yolk, such as phosphatidylcholine, are important for the maintenance of the integrity of the sperm membrane during the freezing and post-thawing process (Mousa et al., 2002; Amirat et al., 2004; Forouzanfar et al., 2010; Alcay et al., 2016). Therefore, it is likely that a high percentage of egg yolk improves the sperm viability observed in TY, and this contributes to maintaining the levels of polinsaturated fatty acids necessary for the sperm membrane, being less susceptible to destructive lipid peroxidation (Cerolini et al., 2001; Kaeoket et al., 2010).

On the other hand, the poor post-thaw semen quality of CY could be due to the fact that the egg yolk has a high risk of suffering microbial contamination, which could decrease post-thaw semen quality (Aboagla et al., 2004; Kulakzis et al., 2010) and being the commercial egg yolk (CY), it could not have a good sanitary quality, which impaired post- freezing semen quality. Another factor that could affect the quality of the CY semen is the diet and management of the producing birds (Lima-Verde et al., 2017). Indeed, several studies have shown that the egg yolks of different species of birds show a variation in their components, resulting in different effects in the cryopreservation process on sperm (Trimeche et al., 1997; Bathgate et al., 2006; Singh et al., 2013). Furthermore, egg yolk can contain harmful metabolites and endotoxins that affect sperm viability (Vidal et al., 2013), as well as steroid hormones that reduce sperm motility (El-Sisy et al., 2018). Previous laboratory studies reveal that, by eliminating some components in the egg yolk by centrifugation; in addition to certain substances in the yolk that inhibit respiration and sperm motility, suggesting the replacement of the whole egg yolk by the cryoprotective fraction (Amirat et al., 2004).

 CONCLUSION

The results of the present study did not show statistically significant differences between the use of the different diluents for the conservation of FS and RS. However, Tris-yolk obtained higher individual and mass motility and post-thaw viability compared to soy lecithin-based diluent during the cryopreservation process of Alpine goat semen.

 ACKNOWLEDGEMENTS

The authors are grateful for the financial support provided to the Ministry of Agriculture and Rural Development and the National Council of Science and Technology (SADER- CONACYT, Mexico) for the financial support granted through the Sectorial Fund for Research in Agricultural, Livestock, Aquaculture, Agrobiotechnology and Plant Genetic Resources, 2017-04-291691.