Studies On The Nuclear Transfer Of Bovine Somatic Cells

Factors related to nuclear transfer bovine adult somatic cells were examined in this study. When electronic field intensity and pulse number were fixed at 100 v/mm and 2, 10 H-s of pulse duration resulted in significantly more cytoplasts fused with granulose cells (46.4%) and fibroblasts (50.3%) in comparison with 30 (as of pulse duration (26.0% and 35.0% resp, P<0.05). The cleavage rate of embryos reconstructed by fusing granulose cells to cytoplasts was significantly higher than that of embryos reconstructed by intracytoplasm injection of granulose cells (73.6% vs 55.3%, P<0.05), but direct injection of fibroblasts into cytoplasts resulted in significantly more reconstructed embryos cleaved than electronic fusion (70.4% vs 55.5%, P<0.05). In calculating blastocysts from total recipient oocytes, the blastocyst yield of direct injection was significantly higher than that of electronic fusion for either granulose cells (10.2% vs 4.4%, P<0.05) or fibroblasts (11.2% vs 3.0%, P<0.05). Serum starvation treatment of donor cells increased the survival rate of cytoplasts injected with granulose cells (90.2% vs 75.9%, P<0.05), but decreased the cleavage rate of embryos reconstructed by direct injection of fibroblasts (68.5% vs 81.3%, P<0.05). Addition of 7% ethanol to the injection medium containing 8% PVP and fibroblasts resulted in a significant increase in the cleavage rate of reconstructed embryos derived from fibroblasts (80.9% vs 65.5%, P<0.05). Significantly less recipient oocytes enucleated at Telophase II (Til) fused with granulose cells in comparison with the recipient oocytes enucleated at Metaphase II (29.2% vs 42.9%, P<0.05), the average blastocyst yield calculating from total recipient oocytes enucleated at Til after nuclear transfer of granulose cells and fibroblasts was also significantly lower than that of recipient oocytes enucleated at Mil (2.8% vs 9.1%, P<0.05). High percentage of embryos reconstructed by injection of fibroblasts developing to blastocysts was obtained when they were activated using ionomycin (5 |umol/L, 5min) at 3h after injection (14.0%) in comparison with the reconstructed embryos activated at Oh and 6h (4.8%, P<0.05).In conclusions, (1) The optimum electronic parameters for nuclear transfer of granulose cells and fibroblasts is proved to be 100 v/mm of field intensity, lO^s of pulse duration and 2 of pulses. (2) Intracytoplasmic injection is more efficient than electronic fusion in nuclear transfer of bovine granulose cells and fibroblasts. (3) Serum starvation treatment of donor cells may be unnecessary for nuclear transfer of bovine granulose cells and fibroblasts. (4) Activation of donor cells prior to injection can improve the development of nuclear transfer embryos. (5) Oocytes enucleated at Mil are superior to oocytes enucleated at Til in using as recipients for the nuclear transfer of somatic cells. (6) Interaction of somatic donor cells with recipient cytoplasmic factors for a certain period may be important for the development of reconstructed embryos.