PARTHENOGENETIC DEVELOPMENT OF BOVINE OOCYTES MATURED IN-VITRO FOR 24-HR AND ACTIVATED BY ETHANOL AND CYCLOHEXIMIDE

This research was undertaken to improve development of parthenogenetic embryos following various combined treatments of ethanol and cycloheximide. In Experiment 1 in vitro matured oocytes (IVM, 24 hr) were treated with 7% ethanol for 5 min followed by incubation in 10 mu g/ml cycloheximide in Medium 199 for 0 (control), 5, 10, and 20 hr. Development to 2-8 cells following culture for 3 days was similar among treated groups (32-41%; P > 0.05), which was higher than that of controls (6%; P < 0.05). Experiment 2 compared pre-ethanol exposures for 0, 1, 2.5, and 5 min, followed by 5 hr cycloheximide treatment on activation development. One- to 5-min groups resulted in 42-44% cleavage contrasted to 1-12% for controls (P < 0.05). Experiment 3 examined the effect on oocyte development of ethanol and different concentrations of cycloheximide (0, 1, 5, and 10 mu g/ml). Cleavage to 2-8 cells was similar among the 5 and 10 mu g/ml cycloheximide groups (36% and 42%, P > 0.05) but lower (P < 0.05) for the 1 mu g/ml group (24%) and the controls (2-13%). When 5 mu g/ml cycloheximide was used (Experiment 4), pre-exposure to ethanol (1, 2.5, and 5 min) resulted in more oocytes cleaved (38-41%) than in the cycloheximide alone group (0%) or the control (0%, P < 0.05). Experiment 5 tested blastocyst development of the activated oocytes with or without cytochalasin B treatment. Oocytes developed to blastocysts were 0%, 14%, 3%, and 3% (P < 0.05), respectively, for control, treatment with ethanol and cycloheximide in the presence, or absence of cytochalasin B, or electrical pulse plus cycloheximide. In conclusion, the combined ethanol and cycloheximide treatment supported high rates of parthenogenetic development using 24 hr IVM bovine oocytes. Blastocyst rate was significantly higher when cytochalasin B was added to the combined activation regimen. (C) 1994 Wiley-Liss, Inc.