Stallion spermatozoa surviving freezing and thawing experience membrane depolarization and increased intracellular Na

In order to gain insight of the modifications that freezing and thawing cause to the surviving population of spermatozoa, changes in the potential of the plasma membrane (Em) and intracellular Na⁺ content of stallion spermatozoa were investigated using flow cytometry. Moreover, caspase 3 activity was also investigated and the functionality of the Na⁺ -K⁺ ATPase pump was investigated before and after freezing and thawing. Cryopreservation caused a significant (p < 0.001) increase in the subpopulation of spermatozoa with depolarized sperm membranes, concomitantly with an increase (p < 0.05) in intracellular Na⁺ . These changes occurred in relation to activation of caspase 3 (p < 0.001). Cryopreservation reduced the activity of the Na-K⁺ pump and inhibition of the Na⁺ -K⁺ ATPase pump with ouabain-induced caspase 3 activation. It is concluded that inactivation of Na⁺ -K⁺ ATPase occurs during cryopreservation, an inhibition that could play a role explaining the accelerated senescence of the surviving population of spermatozoa.