Development of porcine blastocysts from in vitro-matured and activated haploid and diploid oocytes

The purpose of this study was to determine the developmental capacity of electro-activated porcine oocytes. Follicular oocytes collected from gilts at local slaughterhouses were matured for 48 h and were then subjected to a single square pulse of direct current for 100 mu sec at 1,500 V/cm for activation. To obtain activated diploid oocytes, some were treated with 5.0 mu g/ml cytochalasin B for 4 h immediately after electro-activation. The frequency of activation ranged from 96 to 100%. While 91% of activated oocytes that had not been treated with cytochalasin B had 2 polar bodies and a nucleus (haploids), 92% of the oocytes treated with cytochalasin B had only the first polar body and 2 nuclei (diploids). Haploids and diploids were further cultured in TCM-199 medium that contained 10% (v/v) heat-treated fetal calf serum (FCS) and 0.1 mg/ml sodium pyruvate (mTCM) or in Whitten's medium plus 0.4% (w/v) bovine serum albumin (BSA). The frequency of abnormal oocytes was significantly higher in mTCM (83%) than in Whitten's medium (65%) 96 h after the electroactivation (p<0.01), suggesting that Whitten's medium supported the development of activated oocytes beyond the morula stage. In all cases, several oocytes developed to the blastocyst stage 144 h after electro-activation (1 to 12%). The frequency was significantly higher in the case of diploids cultured in Whitten's medium (12%) (P<0.01) than in the case of haploids cultured in Whitten's medium (4%), or in the case of haploids cultured in mTCM (1%). The mean number of nuclei per blastocyst was significantly lower in mTCM (haploids, 15; diploids, 16.1) than in Whitten's medium (haploids, 35.7; diploids, 40.1; P<0.01), suggesting that the number of nuclei in blastocysts was affected by the culture medium.