232 PARTHENOGENETIC DEVELOPMENT AND PLOIDY OF BOVINE OOCYTES FOLLOWING VARIOUS CHEMICAL ACTIVATION REGIMENS

Abstract

Bovine oocytes treated using various combinations of ionomycin (Ion), cycloheximide (CHX), and cytochalasin B (CCB) were evaluated for developmental rates and ploidy status. Metaphase II oocytes were allocated 5 treatment groups, and the groups were treated as follows: Group 1: 5 µm Ion for 5 min; Group 2: Ion + 10 µg mL^–1 CHX for 5 h; Group 3: Ion + 10 µg mL^–1 CHX + 5 µg mL^–1 CCB for 1 h + 10 µg mL^–1 CHX for 4 h; Group 4: Ion + 10 µg mL^–1 CHX + 5 µg mL^–1 CCB for 3 h + 10 µg mL^–1 CHX for 2 h; and Group 5: Ion + 10 µg mL^–1 CHX + 5 µg mL^–1 CCB for 5. Difference among groups was analyzed using one-way ANOVA by SPSS 10.0 (SPSS, Inc., Chicago, IL, USA). In Experiment 1, 430 oocytes in 4 replicates were compared for the extrusion rate of the second polar body (PB) at 8 h after Ion treatment among groups. Group 5 exhibited significantly (P < 0.05) lower rates of second PB extrusion than did Groups 1–4 (22% v. 53–67%). Experiment 2 compared the rates of cleavage at 48 h and development to the blastocyst stage at 216 h after Ion treatment among groups. A total of 536 oocytes were used in 5 replicates. Parthenotes in Group 1 showed lower rates of cleavage and blastocyst development than those in other groups (20% and 1% v. 53–67% and 6–31%). Among the groups, parthenotes in Group 5 showed significantly (P < 0.05) higher blastocyst development. In Experiment 3, at 8 h after Ion treatment, oocytes from Groups 2, 3, and 5 were divided into two subgroups based on the presence or absence of the second PB, and assessed for cleavage rates and ploidy in 239 2-cell-stage parthenotes in 4 replicates, as described earlier by King et al. (1979 Vet. Sci. Commun. 3, 51–56). The cleavage rates did not differ among activation treatments, or by the presence or absence of the second PB in any activation group. The haploid rate was significantly (P < 0.05) higher in Group 2 than in Groups 3 and 5 (38% v. 19% and 0%, respectively). The diploid rate was significantly (P < 0.05) higher in Group 5 than in Groups 2 and 3 (88% v. 69% and 45%, respectively). In Experiment 4, the diploid rate of Group 2 blastocyst-stage parthenotes was 100% (4/4), whereas the diploid rates of Groups 3 and 5 blastocyst-stage parthenotes were 50% (6/12) and 71% (17/24), respectively, but the rates did not differ among groups. These results indicate that oocyte activation by CHX/CCB for 5 h after Ion treatment could enhance parthenogenetic development in bovines with higher rates of diploidy by preventing the extrusion of the second PB.