Influence of combined activation treatments on the success of bovine nuclear transfer using young or aged oocytes.

This study was determined if the combined activation of young or aged oocytes influence their development. The 16-cell stage in vitro maturation/fertilization embryos were exposed to 10 microL nocodazole for 18-20 h, blastomeres that divided within 3 h after the release from nocodazole were used as synchronized donor blastomeres. Metaphase II oocytes were enucleated at 20-22 h post onset of maturation. Enucleated oocytes were divided into 2 groups: oocytes activated at 24 h (young) and oocytes activated at 38 h (aged). In both groups (young and aged), one group of oocytes was activated in 7% ethanol alone for 5 min (alone) and the other group (combination) was activated in ethanol and subsequently incubated in 5 micrograms/ml cycloheximide in TCM199 for 6 h (combination). Electrofusion was carried out at 30 h (young) and 44 h (aged). The nuclear morphology of the blastomere-oocyte complexes at 1 h post-fusion and their development to the blastocyst stage after 6 days of culture in modified synthetic oviduct fluid were examined. Interphase and swollen nuclei were observed at 1 h post-fusion following nuclear transfer to the cytoplasm from young oocytes of combined activation and aged oocytes of combined and ethanol alone activation. When young oocytes were treated with the combined activation method, the reconstituted embryos had a significantly higher developmental rate to the blastocyst stage than the aged oocyte groups (P < 0.05). We conclude that the combined activation of young oocytes leads to a more efficient development of bovine nuclear transfer embryos.