Comparison of Whole and Centrifuged Egg Yolk Added to Kenney's and Lactose-EDTA Extenders for Donkey Semen Cryopreservation

Egg yolk (EY) has been the most widely used external cryoprotectant for donkey spermatozoa; however, it interferes with semen evaluation. Using clarified EY in equine freezing extenders has been successful, but it is difficult to apply in mountainous areas where mules are bred. Thus, the objective of this study was to evaluate the use of centrifuged EY for deep-freezing donkey spermatozoa. Twelve ejaculates were collected from four jacks of proven fertility. Seminal plasma was removed and, before freezing, samples were rediluted in four extenders, each with 7% dimethylformamide (DMF): (1) Kenney extender with 20% EY (Kenney-Y); (2) Kenney extender with 20% centrifuged EY (Kenney-CY); (3) EDTA-glucose extender with 20% EY (EDTA-Y); and (4) EDTA-glucose extender with 20% centrifuged EY (EDTA-CY). After thawing, motility, membrane function, and acrosome status were evaluated. Total and progressive motility observed in Kenney-Y and EDTA-Y was higher (P < .05) than that in Kenney-CY and EDTA-CY. The percentage of frozen-thawed sperm with functional membranes and intact acrosomes was higher (P < .05) in the samples extended with EY when compared with those extended with centrifuged EY. In addition, samples extended with centrifuged EY showed a higher (P < .05) proportion of sperm with nonfunctional membranes and percent of detached acrosomes. This study has shown that it is possible to freeze donkey semen with 7% DMF using a rapid curve of temperature decrease and a manual freezing procedure. However, addition of centrifuged EY to semen freezing extenders had an unexpected detrimental effect on sperm motility, membrane function, and acrosome status.