Activation and Penetration In Vitro of Pig Oocytes Treated with Calcium Ionophore

Abstract

The present study examined the effects of pre-treatment of pig oocytes with different concentrations (0-50 μM) of calcium ionophore A23187 (CaA) on their activation, development and penetration in vitro. Although untreated oocytes were not activated and did not cleave in culture, high proportions of treated oocytes did so and 20% of oocytes developed to the blastocyst stage when treated with 6.25 μM CaA for 2 min. However, these proportions were reduced in a concentration-dependent manner. When inseminated in vitro with 1 × 10⁶ spermatozoa/ml, the penetration rate of oocytes treated with 6.25 μM CaA was similar to that of untreated oocytes. However, fewer oocytes treated with 12.5 and 50 μM CaA were penetrated than untreated oocytes. On the other hand, the proportion of monospermy of oocytes treated with 6.25 μM CaA was higher than the values in oocytes not treated or treated with 50 μM CaA. The time required for zona dissolution of oocytes treated with 6.25 and 12.5 μM CaA was not different from that in untreated oocytes, but oocytes treated with 50μM CaA required a longer time than untreated oocytes, indicating that zona solubility by protease does not reflect penetrability of oocytes in vitro. When oocytes were inseminated with different concentrations (1-10 × 10⁶ cells/ml) of spermatozoa, the highest penetration rate was observed at 1 × 10⁶ cells/ml in untreated oocytes and a similar result was obtained in oocytes treated with 6.25 μM CaA. There was no difference in the rate of monospermy in untreated oocytes among different concentrations of spermatozoa, but in treated oocytes, higher proportions of monospermy were observed at 0.5-5 × 10⁶ than 10 × 10⁶ cells/ml. At 1 × 10⁶ cells/ml, the proportion of monospermy was higher in treated than untreated oocytes. These results suggest that pre-treatment of pig oocytes with 6.25 μM CaA, an appropriate concentration, inhibits polyspermic penetration in vitro when insemination occurs with spermatozoa at a concentration of 1 × 10⁶ cells/ml.