63 STEPWISE THAWING PRESERVES MITOCHONDRIA MEMBRANE POTENTIAL OF BOAR SPERMATOZOA EXTENDED IN GLYCEROL-FREE MEDIUM CONTAINING TREHALOSE

Abstract

Motility and penetrability of spermatozoa are significantly affected by the energy produced in mitochondria. The survival of cryopreserved spermatozoa is influenced by the warming rate as well. The objective in the present study was to evaluate the effect of stepwise thawing on post-thaw motility and mitochondria activity of boar spermatozoa. The sperm samples collected from a Berkshire boar with high fertility at an AI centre were diluted in egg yolk-based and glycerol-free freezing extender containing 100 mM trehalose and 0.25% Equex STM™. The samples were cryopreserved using the straw freezing procedure. Best thawing durations in high temperatures were examined in a previous study. Straws were immersed in 80, 70, and 60°C water for 6, 8, and 10 s and continued to thaw in 39°C for 54, 52, and 50 s, respectively. Sperms thawed at 39°C for 60 s were considered as the control. Frozen-thawed spermatozoa were analysed for motility with computer-assisted semen analysis and mitochondrial membrane potential (MMP) with JC-1/PI staining (Table 1). Data were analysed with ANOVA and Pearson correlation. Motility and MMP of frozen-thawed sperm were significantly lower than fresh samples. Motility was fairly high compared with frozen-thawed controls, when thawed rapidly in 2-steps, though the difference was not significant. The MMP was also significantly higher when thawed in 2-steps from 80 or 70°C, then to 39°C, as compared with frozen-thawed controls, and it was positively correlated with the thawing temperature (r = 0.64; P < 0.01). There was a positive trend between thawing temperature and motility, but it was not significant (r = 0.37; P = 0.11). In conclusion, our results suggest that stepwise thawing with a rapid thawing at the beginning preserves post-thaw motility and MMP of boar spermatozoa. Table 1.Effect of thawing temperature on post-thaw motility and mitochondrial membrane potential (MMP)1