Effects of cryopreservation on the acrosomal status of stallion spermatozoa

The effects of cryopreservation on the acrosomal status of equine spermatozoa were investigated. Ejaculates (n=10) from six stallions were processed fresh, after cooled storage at 4-6 degrees C for 24 h in either a milk-based or lactose-EDTA freezing extender and after freeze-thawing in lactose-EDTA extender in liquid nitrogen at either 5 x 10(7) or 2 x 10(8) spermatozoa ml(-1). All samples were incubated in TALP-TEST for 2 h at 39 degrees C in 5% CO2. Subsamples were challenged with calcium ionophore A23187 for 10 min. The acrosomal status of the spermatozoa was evaluated by staining the spermatozoa with FITC-conjugated Pisum sativum agglutinin, with ethidium homodimer as a nuclear counterstain. Sperm cell viability was assessed with Hoechst 33258. The data were analysed by a general linear model ANOVA (P < 0.05). Treatment with calcium ionophore did not affect the percentage of acrosome reactions. The samples containing 5 x 10(7) and 2 x 10(8) spermatozoa ml(-1) that were frozen in lactose-EDTA extender in liquid nitrogen had higher percentages of spermatozoa in intermediate categories of acrosomal staining than did any other treatment. The percentage of acrosome-reacted cells was also higher overall for these samples. The percentage of viable cells was lowest for the sperm samples frozen in lactose-EDTA extender, and lower in semen stored in either a milk-based or lactose-EDTA freezing extender than in fresh semen. In conclusion, freeze-thawing resulted in a high percentage of acrosomal changes and a significant decrease in sperm viability.