Influence of nocodazole on the development of donor blastomeres from 16-cell stage bovine embryos in nuclear transfer.

The aim of the present study was to establish a reliable procedure with nocodazole treatment for the synchronous cleavage of blastomeres of bovine embryos used as nuclear donors for nuclear transfer. Sixteen-cell stage embryos derived from in vitro-maturation, fertilization and culture were used. In three initial experiments, embryos were incubated in mTCM-199+FCS with various concentrations (0-20 microM) of nocodazole under 5% CO2 in air. The concentrations required to arrest the blastomeres in the mitotic phase were examined. The effects of 10 microM nocodazole were also examined by observation of the division rate of blastomeres after the removal of nocodazole. Ninety percent (90%) of the blastomeres were arrested in the mitotic phase when embryos were exposed to 10 and 20 microM nocodazole. Exposure to 10 microM nocodazole had the highest blastomere-cleavage rate (47%). When the exposure period to 10 microM nocodazole was prolonged to 36 hr, the division rate of the blastomeres decreased. Furthermore, the effects of 2 culture conditions (mTCM-199 under 5% CO2 in air vs modified synthetic oviduct fluid medium under 5% CO2, 5% CO2 and 90% N2) were compared on the division rate of blastomeres of embryos exposed to 10 microM nocodazole for 12 hr. When the embryos were exposed to nocodazole in mSOF, the division rate of blastomeres was improved to about 60%. The blastomeres produced by this treatment condition were used as nuclear donors and the developmental potential of the reconstituted embryos was investigated. The developmental rate to the blastocyst stage was 30.1% (58/193). Five embryos were transferred to 5 recipient cows and 2 of the 5 recipients (40%) became pregnant. Subsequently, one normal calf was born.