Concentrations of non-permeable cryoprotectants and equilibration temperatures are key factors for stallion sperm vitrification success

Vitrification is based on rapid freezing by direct exposure of sperm to liquid nitrogen (LN2). This study evaluated the effect of non-permeable CPAs and equilibration temperature on stallion sperm quality after vitrification. In Experiment 1, different concentrations of sucrose (20, 50, 100 mM; mmol/L) and bovine serum albumin (BSA 1%, 5%, 10%) were compared including different temperatures for the equilibration (≈22 °C or 5 °C). Vitrification was performed dropping 30 μl sperm suspension directly into LN2. In Experiment 2, conventional sperm freezing using 2.2% of glycerol in 0.5 ml straws, frozen in LN2 vapours, was compared to the sucrose and BSA extenders (and its combination) producing the most desirable results. Sperm motility, plasma membrane and acrosome integrity were statistically compared between treatments. Vitrification after sperm cooling at 5 °C with sucrose 20 mM (S20) or BSA 1% (BSA1) resulted in the greatest values (mean ± SEM) for most of the sperm variables assessed. With use of the combination (S20 + BSA1/5 °C), there were greater values (P<0.001) than freezing with glycerol for total (55.67 ± 2.99 vs 35.41 ± 2.96) and progressive sperm motility (38.32 ± 3.05 vs 14.42 ± 1.80), plasma membrane integrity (66.61 ± 2.69 vs 49.16 ± 2.60), intact-acrosomes (49.19 ± 2.60 vs 14.91 ± 1.57) and most of the kinetics assessed, respectively. In conclusion, stallion sperm can be vitrified after cooling at 5 °C using a combination of 20 mM sucrose and 1% BSA based extender and this is a promising alternative compared with conventional sperm freezing using glycerol.