Oocyte penetration by fresh or stored diluted boar spermatozoa before and after in vitro capacitation treatments.

This study examined whether washing and preincubation of boar spermatozoa was necessary to achieve high in vitro penetration rates of pig oocytes. In experiment 1, diluted, sperm-rich fractions, stored for 24 h at 16 degrees C (stored diluted sperm) were used. After centrifugation once at 50 g for 3 min, the supernatant was concentrated at 1200 g for 3 min. The pellets were washed (1200 g for 3 min) 0, 1, or 2 times in saline-BSA solution and preincubated for 0, 20, or 40 min in modified Medium 199 before insemination of immature oocytes. In experiment 2, immature and ovulated oocytes were inseminated with untreated (unwashed and nonpreincubated sperm from concentrated pellet resulting from centrifugation of supernatant fraction obtained by initial low-speed centrifugation) or treated (washed 2 times in saline-BSA solution and preincubated for 40 min), diluted spermatozoa that had been stored and processed as described above. In experiment 3, freshly undiluted or stored diluted spermatozoa were untreated or treated and used to penetrate immature oocytes. In experiment 4, immature oocytes were exposed to freshly undiluted or stored diluted spermatozoa from untreated or treated samples, and, at various times after insemination, oocytes were examined for evidence of penetration. High penetrability rates were obtained when untreated, stored diluted spermatozoa were used. Type of oocyte (immature vs. ovulated) did not affect penetrability regardless of whether untreated or treated, stored diluted spermatozoa were used. Penetration rates and number of spermatozoa per oocyte were lower (p < 0.05) using stored diluted spermatozoa that were washed twice and preincubated 40 min than when freshly undiluted or untreated, stored diluted spermatozoa were used. First evidence of penetration of oocytes by untreated or treated spermatozoa (freshly undiluted or stored diluted) was observed 3 h after insemination. Results indicate that, under the in vitro conditions studied, boar spermatozoa undergoes capacitation and a true acrosome reaction during coincubation with oocytes even when not washed or preincubated.