Study On Bovine Somatic Cell Nuclear Transfer

The method still requires sophisticated and con- siderable skills from the operator, putting cloning beyond the reach of many laboratories.In this research we want to set up simple and high-performance nuclear transfer methodsform important link of boving cloning.1. Proliferation-promoting rule of bovine ear fibroblast cells were studied by protracting the growth curve. The result indicates that, bovine ear fibroblast cells growth proliferative detention period is 1-3 days, the exponential phase of growth is 3-7 days, the platform phase of growth is 7-9 days.2. The detection of Bax and Bcl-2 protein in five different passages of bovine ear fibroblast cells, the result indicates that the five passages of bovine ear fibroblast cells all have the expression of Bax and Bcl-2 protein, and in Bcl-2 protein, the 12 passages of bovine ear fibroblast cells significantly (P<0.05) higher than that other passages. In Bax protein, the 10 passages of bovine ear fibroblast cells significantly (P<0.05) higher than 8 passages and 12 passages,to follow the passages grouth the Bax and Bcl-2 protein expression have invariably development change.3.To optimize the time and method of enucleation, the oocytes were stained by Hoechst33342 to localize the chromosomes and the extent of migration at different stages. The purpose of staining was to optimize the procedure of bovine nuclear transfer and increased the enucleation rate of bovine somatic cell nuclear transfer. The results showed that: The rate of oocytes maturation in vitro cultured 17h showed no significant difference compared with that of oocytes cultured 19h in vitro (65.48% and 67.49%, respectively, P>0.05). The rate of oocytes which polar body migrated and MII chromotin over 30 degrees in the group of in vitro maturation culture for 19h was 17.21%. Meanwhile, the rate of oocytes which polar body migrated at the same angle was only 7.3% in the group of in vitro maturation culture for 17h. There was significant difference between the two groups (P<0.05). There was no significant difference on the rate of fusion of reconstructed embryos, cleavage, and development of blastocyst between the two groups. Therefore, it is feasible to construct bovine somatic cell nuclear transfer embryos using blind enucleation method for oocytes cultured 17h in vitro.4. Bovine reconstructed embryos were obtained from ear fibroblast cells of two different origins (donor), donor A or donor B. The result indicates that, within these two granulose cells, donor cell origin can not significantly(P>0.05) affect the result of bovine nuclear transfer, however, fusion rate in donor B is higher than that in donor A.5. Bovine reconstructed embryos were obtained from serum-starved or not starved donor cells. The development rates were not significantly (P>0.05)different between two methods. The result indicates that, serum-starved or not can not significantly affect bovine nuclear transfer.6. Bovine reconstructed embryos were cultured in atmosphere consists of 5%CO2 -AIR or 5%CO2-5%O2-90%N2. The development rates were not significantly(P>0.05) different between two methods.However, all development rates in atmosphere containing 5%CO2-5%O2-90%N2 are a bit higher than another one. The result indicates that, atmosphere of 5%CO2-5%O2-90%N2 may have active effect on later development of bovine reconstructed embryos.