In vitro penetration assay of fresh and frozen/thawed porcine sperm by co-incubation with homologous immature oocytes

To develop a method for assessment of porcine sperm fertility using co-incubation with immature homologous oocytes, we examined oocyte penetration by fresh and frozen/thawed sperm after co-incubation. Cumulus-oocyte complexes (COCs) were dissected from follicles with a diameter of 2-6 mm and washed in m-M 199. COCs were introduced into a droplet of m-M199. Also, COCs denuded by 0.50% hyaluronidase and pipetting (denuded oocytes, DOs) were introduced into a droplet of m-M199. Sperm was collected by gloved-hand method from four boars (one Large White, one Duroc and 2 hybrid). Collected semen was washed and diluted to 3 * 10**8 cells/ml in m-M199. Semen suspension was pre-incubated for 3 hrs at 38.5 deg C under 5% CO2 in humidified air and used as fresh sperm. The collected semen was frozen, thawed, and diluted to 3 * 10**8cells/ml in m-M199. This sperm suspension was used as frozen/thawed (F/T) sperm. The sperm concentration in a droplet for co-incubation with immature porcine oocytes was 1 * 10**6cells/ml for fresh sperm, 1 * 10**7cells/ml for F/T sperm. Co-incubation was performed for 18-20 hrs at 38.5 deg C under 5% CO2 in humidified air. After co-incubation, oocytes were fixed and stained for assessment of sperm penetration. Rates of COCs or DOs penetration of fresh and F/T sperm were 50.9% or 54.6% and 16.7% or 28.0%, respectively. The penetration rates of F/T sperm were significantly lower (roh < 0.001) than those of fresh sperm. The mean number of fresh sperm per a penetrated COC and a DO was 9.3 and 14.5, respectively. And the number per a DO was significantly higher (roh < 0.01) than that per a COC. The mean number of F/T sperm per a penetrated COC and DO was 1.9 and 1.7, respectively. Although there was no significant difference between them, the number of F/T sperm was significantly lower (roh < 0.001) than that of fresh sperm. The results of the present study indicated that rate of oocyte penetration and number of sperm per a penetrated oocyte after co-incubation with boar sperm and immature oocytes reflected fertility of boar semen.