Evidence that frozen/thawed ram spermatozoa show accelerated capacitation in vitro as assessed by chlortetracycline assay

We have compared the time-course of in vitro capacitation, as assessed by chlortetracycline (CTC) binding pattern, in membrane-intact fresh and frozen/thawed ram spermatozoa. Samples were filtered through Sephadex G-10 for the enrichment in motile/viable spermatozoa, and combined staining was carried out using Hoechst 33258 for membrane integrity and CTC for capacitation stage. Freezing and thawing did not alter the CTC distribution pattern of membrane-intact spermatozoa. On the other hand, after incubation under capacitating conditions (i.e., 3 washings in PBS/BSA and 2-h incubation in modified synthetic oviductal fluid), the CTC pattern from frozen-thawed intact spermatozoa significantly differed from the corresponding fresh controls, as evidenced by an increase in the percentage of form I (noncapacitated) and Form III (capacitated) spermatozoa. In addition, after incubation, frozen/thawed samples showed an enhanced ability to undergo acrosomal exocytosis in response to the calcium ionophore A23187 in comparison with that of incubated fresh spermatozoa. These results suggest that freezing and thawing of ram spermatozoa provoke structural and/or functional alterations that result in an increased proneness to capacitation and acrosome reaction, a fact to be kept in mind for the timing of both in vivo and in vitro fertilizing procedures.