Effect of cholesterol on the motility and plasma membrane integrity of frozen equine spermatozoa after thawing.

The aim of the present study was to investigate the cryoprotectant properties of cholesterol after incorporation into the plasma membranes of equine spermatozoa. A cholesterol-methyl-beta-cyclodextrin complex was used to alter sperm plasma membrane cholesterol content. Ejaculates from six stallions were centrifuged in a non-fat skimmed milk glucose-sucrose extender (MK) or a modified Tyrode's medium (TALP). The sperm pellets were resuspended in the appropriate extender with or without added cholesterol (0.125 mmol cholesterol-methyl-beta-cyclodextrin complex l(-1)) and incubated at 24 degrees C for 15 min. After incubation, the aliquots were centrifuged and the sperm pellets were resuspended in lactose-EDTA-egg yolk freezing extender, frozen in static nitrogen vapour and stored at -196 degrees C. The straws were thawed and the motility and plasma membrane integrity of the spermatozoa were analysed. Addition of cholesterol to the incubation extenders improved the mean percentages of motile, progressively motile and rapidly motile spermatozoa in both the MK and TALP extenders containing cholesterol compared with extenders without cholesterol (P < 0.05). The percentage of spermatozoa with intact plasma membranes was higher in samples incubated in extenders containing cholesterol than in those without cholesterol (P < 0.05). The results of this study indicate that cyclodextrins can be used to incorporate cholesterol into equine sperm plasma membranes and that cholesterol incorporation imparts protection to the spermatozoa during cryopreservation.