FUNCTIONAL-ANALYSIS USING CHLORTETRACYCLINE FLUORESCENCE AND IN-VITROFERTILIZATION OF FROZEN-THAWED EJACULATED BOAR SPERMATOZOA INCUBATED IN A PROTEIN-FREE CHEMICALLY-DEFINED MEDIUM

Cumulus-enclosed pig oocytes were matured in vitro, freed from cumulus cells, and inseminated with frozen-thawed ejaculated spermatozoa in a chemically defined protein-free medium containing 37.0 mmol NaHCO3 l( -1) and 5 mmol caffeine l(-1). When the medium was supplemented with I mg polyvinylalcohol (PVA) ml(-1), more penetrated oocytes were observ ed 14 h after insemination with 7-12 x 10(6) cells ml(-1) than with 4- 5 x 10(6) cells ml(-1) and the incidence of polyspermy reflected the s perm concentration used. Varying the NaHCO3 concentration but maintain ing the sperm concentration at 8x10(6) cells ml(-1) resulted in signif icantly more oocytes being penetrated in media containing 45.83-50.25 than 37.0-41.42 mmol NaHCO3 l(-1); there were no significant differenc es in the incidence of either male pronuclear formation or polyspermy. In medium containing 45.83 mmol NaHCO3 l(-1), the inclusion of PVA at 0-5 mg ml(-1) had no effect on proportions of penetrated oocytes, mal e pronuclear formation or polyspermy. However, when spermatozoa from t hree different boars were evaluated, the penetration and male pronucle ar formation rates were highly variable, unlike the incidence of polys permy. Penetration of cumulus-free oocytes was first detected at 6 h. When spermatozoa were incubated for 6 h in the absence of oocytes, mot ility, but not vitality, decreased whether or not PVA was included in the medium. Chlortetracycline (CTC) fluorescence analysis of the capac itation state indicated a rapid decline in the proportion of live unca pacitated, acrosome-intact cells and a rapid rise in the proportion of live capacitated, acrosome-reacted cells during the first hour. Small er changes in the distribution of CTC patterns occurred during the lat er stages, suggesting that the rapidly responding cells were non-ferti lizing, owing to damage by freeze-thawing, and that the fertilizing sp ermatozoa were drawn from the remaining pool of cells which underwent capacitation more slowly. This is the first report indicating that cap acitation of frozen-thawed ejaculated boar spermatozoa and penetration of oocytes matured in culture are possible in a chemically defined, p rotein-free medium.