Optimización de la metodología de preservación del esperma del asno andaluz (Equus Asinus)

La raza asnal Andaluza está catalogada actualmente como raza autóctona española en peligro de extinción (RD 2129/2008, Catálogo Oficial de Razas de Ganado de España). Hasta la fecha, sólo 94 garañones están considerados como individuos de alto valor genético (DAD-IS-FAO 2013). En este contexto, es necesaria la aplicación de técnicas de reproducción asistida como la inseminación artificial y la criopreservación de espermatozoides con el objetivo de mejorar la variabilidad genética de la raza y evitar así su extinción. Para ello, consideramos imprescindible realizar un estudio previo de las características reproductivas y los parámetros seminales propios de la raza. Hoy en día, en el análisis de las características seminales de numerosas razas de mamíferos ha sido objeto de gran interés el estudio de las subpoblaciones espermáticas presentes en los eyaculados, observándose una correlación directa entre la presencia de determinadas subpoblaciones espermáticas, la resistencia a la criopreservación y las tasas de fertilidad obtenidas. Por lo tanto, es importante estudiar la distribución de las subpoblaciones espermáticas que conforman el eyaculado del asno Andaluz para establecer la calidad espermática de las muestras seminales recolectadas, así como para predecir su congelabilidad y fertilidad. Actualmente, son escasos los estudios publicados sobre refrigeración y congelación del semen de burro, siendo inexistentes en el caso de la raza asnal Andaluza. Por ello, es necesario establecer un protocolo adecuado, así como seleccionar aquellos diluyentes y crioprotectores con los que obtener muestras seminales con buena calidad tras los procesos de refrigeración y crioconservación. En la primera publicación se estudiaron las características seminales (volumen, pH, concentración, movimiento y morfología espermática) del eyaculado del asno andaluz con el objetivo de poder establecer los parámetros de normalidad propios de la raza. Asimismo, se comprobó la existencia de diferentes subpoblaciones de espermatozoides dentro del eyaculado del asno andaluz así como la influencia del individuo y del eyaculado dentro de un mismo individuo en su frecuencia de distribución. Además, se investigó la correlación existente entre la edad y el peso corporal del garañón, las características de calidad espermática y la distribución de las subpoblaciones espermáticas. Para ello, se recolectaron un total de 60 eyaculados de 12...The Andalusian donkey (Equus asinus) is currently considered in Spain as an endangered breed (Real Decreto 2129/2008, regulation of the National Catalogue of Endangered Species). Currently, only 94 individuals are considered as breeding males (DAD-IS-FAO 2013). In such situation, the application of assisted reproductive technologies such as artificial insemination and semen cryopreservation becomes necessary for improving gene distribution and for reducing the effects of excessive inbreeding. However, for this purpose, a previous study of the reproductive features and/or semen characteristics of the Andalusian donkey is required. Nowadays, the identification of sperm subpopulations within mammalian ejaculates has become an issue of the utmost interest for the sound evaluation of the ejaculates. In this sense, a direct relationship between the presence of discrete motile sperm subpopulations, the resistance to cryopreservation and the fertility rates obtained has been demonstrated. Therefore, it is interesting to characterize the distribution of subpopulations in the ejaculate of the Andalusian donkey in order to study the relationship between the quality and the reproductive performance of semen samples, and to predict its freezability and fertility. To date, published studies concerning on cooling or freezing donkey semen are scarce, and, to our knowledge, no previous studies reporting of Andalusian donkey semen cryopreservation have been published. Consequently, it is important to develop a suitable technique and select those extenders and cryoprotectants which provide the highest sperm quality after cooling or freezing Andalusian donkey spermatozoa. In the first study the physiological sperm characteristics of Andalusian donkey (gel-free volume, pH, sperm concentration, motility and morphology) raw ejaculates were assessed to establish the average values normal for this donkey breed. Moreover, we studied the existence of sperm subpopulations with specific motion characteristics in raw Andalusian donkey ejaculates, evaluate the effects of individual donkey and ejaculates within the same male on the distribution of the subpopulations found, and explore the relationship between the age and the body weight of donkey donors, the sperm quality parameters, and the sperm subpopulations structure. To this end, 60 ejaculates (5 ejaculates per donkey) were collected from 12 Andalusian donkeys whose..

Effect of single layer centrifugation using Androcoll-E-Large on the sperm quality parameters of cooled-stored donkey semen doses

The aim of this study was to determine the effect of single layer centrifugation (SLC) using Androcoll-E-Large on donkey sperm quality parameters after 24 h of cool-storage. Ejaculates were collected from Andalusian donkeys and then cooled at 5°C. SLC was carried out after 24 h of cool-storage using Androcoll-E-Large. In the first experiment, all sperm parameters assessed (total and progressive sperm motility, viability, sperm morphology and sperm kinematics VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were statistically compared between semen samples processed or not with Androcoll-E-Large. Significant differences ( P<0.05) were found between SLC-selected and unselected semen samples for all parameters assessed, obtaining better results after SLC. In the second experiment, semen samples were classified in two groups according to their sperm progressive motility (PM) before SLC. Then, the increments obtained in semen quality parameters after SLC were compared between groups. No significant differences were found between groups, indicating that SLC improved the sperm quality parameters of entire set of semen samples processed with independence to their original PM. In conclusion, SLC with Androcoll-E-Large can be used in donkeys, increasing the sperm quality of cooled-stored donkey semen doses after 24 h of cool storag

Localization of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa

The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorial-distal regions of the sperm. The amount of hybridized signal in the ALS in the sperm of donkey (Equus asinus) was 1.3 times greater than in stallion (Equus caballus), and the length of the comet tail obtained in donkey sperm was 1.6 times longer than that observed in stallion (P < 0.05); however, these differences were not appreciated in somatic cells. In conclusion, ALS localization in sperm is not a randomized event and a different pattern of ALS distribution occurs for each species. These results suggest that ALS represents a species-specific issue related to chromatin organization in sperm and somatic cells in mammalian species, and they might diverge even with very short phylogenetic distances.Depto. de Medicina y Cirugía AnimalFac. de VeterinariaTRUEpu