The effect of cryopreservation on the capacitation status and epithelial cell attachment capability of dog spermatozoa

Freezing and cooling of spermatozoa during cryopreservation for artificial insemination causes ultrastructural changes in the acrosome and plasma membrane which reduces longevity and fertility. Cryopreservation-induced capacitation-like changes and reduced ability of spermatozoa to bind to the cells of the reproductive tract of the bitch may contribute to the reduced fertility of cryopreserved spermatozoa. Previous studies in the dog have investigated the effects of extending and cooling spermatozoa on the plasma membrane but often only after freeze-thawing and not in conjunction with an assessment of their ability to bind to uterine tube epithelial explants. This study investigated the effect of each stage of the cryopreservation process on capacitation and attachment to the reproductive tract of the bitch. The capacitation status of spermatozoa was studied over time after cryopreservation using a chlortetracycline and Hoechst 33258 stain. The ability of spermatozoa to bind to uterine tube epithelial explants was studied using Hoechst 33342 stain. Extending, cooling and freeze-thawing promoted capacitation and decreased the spermatozoal binding ability. The effect of each stage appeared to be cumulative with the freeze-thawing stage being the most dramatic. The results suggested that the cumulative effect of each stage of the cryopreservation process results in the promotion of capacitation before spermatozoa have reached the site of fertilisation, and therefore spermatozoa have reduced ability to attach to epithelial cells. These effects are contributory factors to the reduced fertility of cryopreserved spermatozoa.