54 EFFECT OF THAWING TEMPERATURE ON POST-THAW SURVIVAL OF BOAR SPERM

Abstract

The survival of cryopreserved sperm is strongly influenced by the warming rate. Aims of the study were to examine the time taken by the extender inside the straw to reach 39°C and to evaluate the effect of thawing temperature on post-thaw survival of boar sperm. The sperm samples were diluted in egg yolk-based freezing extender containing trehalose (100 mM) and 0.25% Equex STM™. The samples were cryopreserved using the straw-freezing procedure (Buranaamnuay et al. 2009 Reprod. Domest. Anim. 44, 69–73). To examine the change of extender temperature inside the straw, sealed 0.5-mL straws with the extender were immersed in water (40, 60, or 80°C) after freezing and the temperature inside the straw was measured using a 2-channel digital record thermometer (TNA-140, Tasco Japan Co. Ltd., Osaka, Japan). After frozen sperms were thawed at different temperatures for the time taken by the extender inside the straw to reach 39°C, the sperms were analysed for motility, viability, and acrosome integrity. Statistical analysis from 5 replicated trials was performed by ANOVA. Average time taken to reach 39°C in 40, 60, and 80°C water was 36, 13, and 9 s, respectively (n = 8). There were 2 phases of the rates of change of temperature (RCT) inside the straw: rapid at the beginning of the course of thawing (–196 to approximately 0°C) and relatively slower at the liquid phase. The RCT was very rapid during the first 2 s, with a mean rate of 73.3, 81.3, and 86.9°C s^–1 in 40, 60, and 80°C water, respectively. The RCT was not significantly different among the 3 groups between 2 and 6 s after the start of warming, whereas the RCT was significantly higher in 80°C water compared with 60 and 40°C from the next second (P < 0.01). Starting from 12th second, RCT was significantly higher in 60°C water compared with 40°C water (P < 0.01). When frozen sperms were thawed at 40, 60, or 80°C for 36, 13, or 9 s, respectively, however, there were no significant differences in post- thaw parameters (motility and viability) of sperms thawed in different temperatures. The motility of sperms thawed at 40, 60, and 80°C water was 41.2, 42.5, and 30.0%, whereas the viability was 42.3, 47.6, and 42.6%, respectively. The percentage of sperms with intact acrosomes was 54.8, 64.2, and 68.0%, respectively. There was a trend of increased acrosome integrity when the sperms were thawed at higher temperature (P = 0.06). In conclusion, this preliminary result suggests that rapid thawing may affect acrosome integrity rather than the motility and viability of cryopreserved sperm. However, additional observations are necessary to reach further conclusions.