Vitrification of Donkey Sperm: Is It Better Using Permeable Cryoprotectants?

SIMPLE SUMMARY: Conventional donkey sperm-freezing using permeable cryoprotectants has been successfully performed, and good sperm parameters have been obtained after thawing. Unfortunately, artificial insemination of jennies with cryopreserved semen has given unsatisfactory results. Vitrification by directly dropping the sperm into the liquid nitrogen following the spheres methodology has been developed in human beings as an alternative to conventional freezing. This technique has shown to be a species-specific methodology and the concentration of cryoprotectants should be optimized in donkeys. Additionally, in this study, a permeable cryoprotectant (glycerol) has been tested for the first time for donkey sperm vitrification. According to our findings, vitrification of donkey sperm was effectively carried out using an extender supplemented with sucrose or bovine serum albumin (BSA) as non-permeable agent. When glycerol, a permeable agent, was compared to sucrose 0.1 M and BSA 5%, sperm quality significantly decreased. Therefore, donkey sperm vitrification in the absence of permeable agents obtained better results and gives a new approach to create a pattern for future studies of fertility trials. ABSTRACT: Vitrification by direct exposure of sperm to liquid nitrogen is increasing in popularity as an alternative to conventional freezing. In this study, the effect of permeable cryoprotectant agents for donkey sperm vitrification was compared to an extender containing non-permeable cryoprotectants. First, three different concentrations of sucrose (0.1, 0.2, and 0.3 molar, M) and bovine serum albumin, BSA (1, 5, and 10%) were compared. Secondly, the concentration of non-permeable agents producing the most desirable results was compared to an extender containing glycerol as permeable agent. Vitrification was performed by dropping 30 μL of sperm suspension directly into LN2 and warming at 42 °C. Sperm motility (total, TM; and progressive, PM) and plasma membrane integrity, PMI (mean ± SEM) were statistically compared between treatments. Sucrose 0.1 M showed a significantly higher percentage of total sperm motility (21.67 ± 9.22%) than sucrose 0.2 M (14.16 ± 4.50%) and 0.3 M (8.58 ± 6.22%); and no differences were found in comparison to the control (19.71 ± 10.16%). Vitrification with sucrose 0.1 M or BSA 5% obtained similar results for TM (21.67 ± 9.22% vs. 19.93 ± 9.93%), PM (13.42 ± 6.85% vs. 12.54 ± 6.37%) and PMI (40.90 ± 13.51% vs. 37.09 ± 14.28); but both showed higher percentages than glycerol (TM = 9.71 ± 4.19%; PM = 5.47 ± 3.17%; PMI = 28.48 ± 15.55%). In conclusion, donkey sperm vitrification in spheres using non-permeable cryoprotectants exhibited better sperm motility and viability parameters after warming than sperm vitrification using extenders containing permeable cryoprotectants.