Penetration in vitro of zona-free pig oocytes by homologous and heterologous spermatozoa

Abstract

We examined the penetrability of pig, rat and bull spermatozoa into zona-free pig oocytes. Frozen-thawed boar spermatozoa penetrated into both zona-intact and zona-free oocytes with similar efficacy in a modified Tris-buffered medium (mTBM) supplemented with BSA and caffeine, but not in medium without caffeine. Rat epididymal spermatozoa did not readily penetrate into zona-free pig oocytes in mTBM with BSA. However, when a modified Krebs–Ringer bicarbonate solution was used, penetration rate varied with sperm concentrations at insemination: 79% of the oocytes were penetrated at 1.0×10 6 cells/ml, but very few at 0.1×10 6 and 10.0×10 6 cells/ml. In all oocytes penetrated, no activation was observed and the sperm nucleus was fully decondensed but did not transform into a male pronucleus. Frozen-thawed bull spermatozoa were also found to penetrate into zona-free pig oocytes in mTBM with BSA, caffeine and heparin: higher penetration rates were obtained with 1.0×10 6 and 10.0×10 6 spermatozoa/ml compared with 0.1×10 6 spermatozoa/ml. The penetration rate with 1.0×10 6 spermatozoa/ml was stable in five different bulls. All oocytes penetrated were activated and male pronuclear formation was observed in 57–79% of the penetrated oocytes. These results suggest that capacitation or the acrosome reaction is required for boar, rat, and possibly, bull spermatozoa to penetrate into zona-free pig oocytes. Bull spermatozoa can easily induce activation of pig oocytes and form male pronuclei, but rat spermatozoa cannot do so, indicating species differences in the ability of spermatozoa to activate pig oocytes and to transform to male pronuclei in the ooplasm.