Studies On The Nuclear Transfer Of Buffalo

Methods of buffalo somatic cell nuclear transfer (SCNT) and factors affecting the efficiency of buffalo SCNT were investigated in this study.1. Effects of enucleation methods on somatic cell nuclear transfer of buffalo were studied. Buffalo oocytes maturated 22h in vitro were enucleated by blind-sucking, point-hiting and Spindle-View System respectively. The enucleation rate of oocytes using Spindle-View System (95.0%) was significantly higher than that of blind-sucking (65.1%) and point-hiting(81.8%) (P<0.01). However, there were not significant difference in the fusion ration, cleavage rate and the blastocyst yield of reconstructed embryos among the three enucleation methods (P>0.05). These results indicate that the point-hit can be replacement of Spindle-View system for enucleation of oocytes in con sideration of equipment costs.2. Methods of reverse nuclear transfer (RNT) were studied. The embryos reconstructed by RNT could develop to blastocyst. But the cleavage rate of and blastocyst rate of reconstructed embryos were significant lower than that of electric fusion nuclear transfer (54.0% vs 75.1%, P<0.01\ 6.9% vs 15.9%, P<0.05).3. Effects of series nuclear transfer on buffalo SCNT were examined. The fusion rate of series nuclear transfer was higher than that of original nuclear transfer (87.9% vs 76.2%, P<0.05), but the cleavage rate and blastocyst yield were not different each other. Thus, it is necessary to do more research on series nuclear transfer to improve the efficiency of buffalo SCNT.4. Nuclear transfer method of intact-cell intracytoplasmic microinjectionwas investigated. The embryo reconstructing efficiency by intact-cell intracytoplasmic microinjection was higher than that by electric fusion (87.1% vs 81.1% , P<0.05), but the cleavage rate of reconstructed embryos was lower in former than the latter (49.5% vs 71.8%, P<0.01). The blastocyst development rate of reconstructed embryos and the total efficiency of nuclear transfer were not significantly different each other (21.6% vs 19.3, P>0.05;18.8% vs 15.7%, P>0.05). The results indicate the intact-cell intracytoplasmic microinjection method can be employed in buffalo nuclear transfer.5. Effects of activation time after donor cell injection on buffao SCNT was examined. The appropriate activation time after donor cell injection was proved to be 3h.6. Effects of the dithiothreitol (DTT) on the buffalo SCNT were investigated. Treatment of donor cell or reconstructed embryos for 30 min with 8 mmol/L DTT did not affect the cleavage rate and blastocyst yield of embryos reconstructed.7. Effects of donor cells preserved in low temperature on the buffalo SCNT were studied. Storage of donor cells at 4°C for 12h did not affect the blastocyst yield of reconstructed embryos The result indicates that donor cells can be preserved in low temperature for certain peirod before nuclear transfer.