Recent advances in donkey sperm vitrification.

Artificial insemination (AI) with cryopreserved semen is an important tool to preserve endangered species, including European donkey breeds. Sperm vitrification is an alternative method to conventional freezing using high cooling rates and non-permeable cryoprotectant agents (CPAs). In donkeys, sperm vitrification was firstly developed in spheres by directly dropping the sperm (30 µl) into the liquid nitrogen. The vitrification media contained a combination of sucrose and bovine serum albumin as non-permeable CPAs and resulted in better sperm parameters after warming than extenders containing glycerol. Thereafter, sperm vitrification was optimized using an aseptic protocol, which consists of volumes up to 160 µl vitrified at 300 million sperm/ml using 0.25-ml straws with outer covers, obtaining similar sperm parameters as conventional freezing for total motility (52.7 ± 15.6% versus. 58.2 ± 16.1%), progressive motility (44.3 ± 15.0% versus. 44.7 ± 18.2%) and plasma membrane integrity (49.2 ± 11.2% versus. 55.4 ± 9.0%), respectively. In order to vitrify larger volumes of sperm, a procedure using 0.5-ml straws was evaluated; however, this methodology failed when compared to conventional freezing or other vitrification protocols, obtaining poor sperm quality after warming. Recently, a new methodology was developed for warming 0.25-ml straws in a water bath and after AI using the vitrified sperm, the uterine inflammatory response solved faster, and pregnancy rates were greater (22%) than frozen semen (10%) but not statistically different. In conclusion, all these findings confirm that sperm vitrification can be performed in donkeys as an alternative to conventional freezing for AI in jennies.
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