Effect of activation treatments of recipient oocytes on subsequent development of bovine nuclear transfer embryos

Effects of recipient oocyte activation methods on the development of nuclear transfer (NT) embryos were investigated. In Exp. 1, cell-cycle phase of serum-starved bovine cumulus cells was examined by flow cytometry. Majority (95.5%) of medium-sized (16-20 microm) cells that made up 56% of total cells was at the G0/G1 phase. NT embryos were constructed by electric fusion with the medium-sized serum-starved cumulus cells and bovine oocytes of 3 different preparations: enucleated oocytes treated with calcium ionophore A 23187 for 5 min and cycloheximide for 5 hr (A 23187/CHX), those treated with ethanol for 7 min and cycloheximide for 2 hr(ethanol/CHX) and those without treatment. In Exp. 2 and 3, developmental competence of NT embryos constructed with A 23187/CHX- and ethanol/CHX-treated oocytes was compared to that of NT embryos constructed with non-treated oocytes, respectively. Further, nuclear behavior in 3 different NT embryos was examined in Exp. 4. Within 1 hr after fusion, majority of the NT embryos constructed with non-treated oocytes showed condensed chromosome. Three hours after fusion, about 50% of NT embryos constructed with non-treated or ethanol/CHX-treated oocytes showed a single pronucleus-like structure. NT embryos constructed with ethanol/CHX-treated oocytes showed similar rates of fusion, cleavage and blastocyst formation to those of the non-treated oocytes. In contrast, NT embryos constructed with A 23187/CHX-treated oocytes did not show any pronucleus-like structure and showed lower cleavage rate and no development to blastocysts. The results indicate that ethanol/CHX-treated oocytes could support development of somatic cell NT embryos to the blastocyst stage at a similar rate to that of non-treated oocytes.