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

The research suggests that employing a combination of 20 mM sucrose and 1% BSA based extender for chilling stallion sperm at 5 °C prior to vitrification is a feasible alternative to conventional sperm freezing method using glycerol.

Objectives and Methodology of the Study

• This study aimed to analyze the impact of non-permeable cryoprotective agents (CPAs) along with varying equilibration temperatures on the quality of stallion sperm post-vitrification.
• Experiment 1 of the study involved different concentrations of sucrose (20, 50, 100 mM or mmol/L) and bovine serum albumin (BSA 1%, 5%, 10%) combined with different temperatures for the equilibration (≈22 °C or 5 °C).
• The process of vitrification was executed by dropping a 30 μl sperm suspension directly into liquid nitrogen (LN).
• In Experiment 2, conventional sperm freezing using 2.2% of glycerol in 0.5 ml straws that were frozen in LN vapours was compared to the most desirable results produced by the sucrose and BSA extenders (and their combination).
• The sperm’s motility, plasma membrane integrity, and acrosome integrity were statistically compared across all treatments.

Findings of the Research

• The study found that equilibration of sperm at 5 °C using 20 mM sucrose (S20) or 1% BSA (BSA1) resulted in the most significant values for most of the assessed sperm variables, indicating greater viability.
• Using a combination of 20 mM sucrose and 1% BSA under 5 °C (S20 + BSA1/5 °C) led to notably higher values than freezing with glycerol when it came to total and progressive sperm motility, plasma membrane integrity, and intact-acrosomes, along with most of the kinetics assessed.
• This suggests that a combination of sucrose and BSA extenders at 5 °C is a promising alternative for the vitrification of stallion sperm compared to established sperm freezing methods.

Conclusion

• The research concludes that cooling stallion sperm at 5 °C using a mixture of 20 mM sucrose and 1% BSA based extender before vitrification represents a promising alternative to the conventional method of sperm freezing employing glycerol.