Nuclear transfer in cattle: effect of nuclear donor cells, cytoplast age, co-culture, and embryo transfer.

There are many factors affecting the efficiency of nuclear transfer technology. Some are evaluated here using our novel approach by enucleating oocytes at 20-22 hr after in vitro maturation (IVM), culturing the enucleated oocytes (cytoplasts) for 8-10 hr or 18-20 hr to gain activation competence and then conducting nuclear transfer. In the first experiment, we demonstrated that cumulus cell (CC) monolayer can support some cloned embryos to develop into morulae or blastocysts. Co-culture with CC and bovine oviduct epithelial cell (BOEC) monolayers resulted in no differences (P > 0.05) in supporting the development of cloned embryos (Experiment 2). When in vitro matured oocytes were enucleated at 22 hr after IVM followed by nuclear transfer 18-20 hr later, cleavage and morula or blastocyst development of the cloned embryos were similar to those resulting from the enucleated oocytes which had been matured in vivo (Experiment 3). Frozen embryos as nuclear donor cells worked equally well as fresh embryos for cloning in embryo development which was superior to IVF embryos (Experiment 4). However, fresh embryos resulted in a higher proportion (P < 0.05) of blastomere recovery than did frozen of IVF embryos. Finally, embryo transfer of cloned embryos from our procedure produced a viable calf, demonstrating the commercial value of this novel approach of the technology.