IDENTIFICATION OF CAPACITATION IN BOAR SPERMATOZOA BY CHLORTETRACYCLINE STAINING

The functional status of boar spermatozoa undergoing capacitation in v itro was investigated. Two fluorescent stains were used: chlortetracyc line (CTC) and a FITC-conjugated lectin (FITC-PSA). The first has been used for the direct identification of the capacitated boar spermatozo a, while the second, based on the identification of capacitated sperma tozoa by their ability to undergo zona-induced acrosome reaction (AR), was used to confirm and validate the CTC assay in this species. Sperm atozoa obtained from 5 different boars was washed and incubated under capacitating conditions. Aliquots of spermatozoa were collected at 0, 90 and 180 min of incubation and then stained with CTC or FITC-PSA. Af ter CTC staining, 3 different fluorescent patterns were observed: Patt ern A with the fluorescence uniformly distributed on the sperm head, P attern B with the fluorescence concentrated in the post-acrosomial reg ion, and Pattern C with the fluorescence concentrated in the acrosomia l region. The percentage of spermatozoa displaying fluorescent Pattern A decreased throughout the incubation while that of spermatozoa with Pattern C showed a concomitant progressive increase. Pattern B fluores cence remained unchanged throughout the maturation period. Exposure to zonae pellucidae (ZP) brought back the levels of Pattern C fluorescen ce to basal values. Since only the capacitated spermatozoa are believe d to react to ZP, this observation together with the rising incidence of Pattern C throughout maturation suggests that fluorescence in the a crosomial region identifies capacitated spermatozoa. The analysis of a crosome integrity carried out with FITC-PSA showed that the proportion of zona-induced AR was nearly the same as that of spermatozoa display ing Pattern C, thus confirming that CTC staining is suitable for the d etection of boar sperm capacitation. In the second part of this study, CTC was used to investigate the effects of sperm origin and storage o n the capacitation process. Our finding demonstrates that capacitation kinetics show wide variations In sperm samples derived from different boars; moreover, capacitation is also affected by sperm storage. Whil e fresh semen showed a progressive increase in capacitated spermatozoa , ranging from low levels at the beginning of the culture to 46 % at t he end of incubation, the refrigerated semen had a relatively high per centage of capacitated spermatozoa at the beginning of culture, but th is proportion increased only slightly during the following 90 to 180 m in of treatment. These data indicate that CTC can be used to identify capacitated boar spermatozoa, and, because of its rapid and easy execu tion, it can be used routinely to identify the optimal capacitation ti me for different sperm samples.