Cryopreservation of dog semen: The effects of Equex STM paste on plasma membrane fluidity and the control of intracellular free calcium

Understanding cryoinjury of dog spermatozoa is crucial to preserving fertilizing ability. This study examined flow cytometric indicators of sperm function to explore the reported benefits of Equex STM paste. The motility of cryopreserved spermatozoa immediately and 1 h after thawing was higher in the extender containing 0.5% Equex; no significant differences between the two extenders were observed regarding viability, acrosomal integrity and intracellular Ca2+ concentration. The proportion of spermatozoa having high membrane fluidity increased significantly post-thawing. The interaction between time after thawing and treatment was significant for plasma membrane fluidity. Dilution in a commercial diluent for transport before processing caused a significant increase in intracellular Ca2+, which may affect functional survival. No significant difference with or without Equex was detected in plasma membrane fluidity. However, a significant interaction between Equex and dogs was detected. A significant decrease in intracellular Ca2+ was detected in the live cell population both after dilution in Andersenʹs buffer and again after cooling and equilibration. One hour post-thaw, the proportion of live spermatozoa with high calcium concentration increased to a similar proportion as that seen in diluted semen; the interaction between diluent and dog was significant. The results suggest that Equex in the diluent benefited motility after cryopreservation. Live spermatozoa with high intracellular Ca2+ after cryopreservation seem to have a favoured survival in the first hour after thawing. Nevertheless, survival after cryopreservation was severely compromised, explaining the relatively poor fertility of cryopreserved dog semen.