The Research Of Nuclear Transplantation Technology On Sheep

Somatic cell nuclear transfer technology is important for animal breeding,conservation and basic theory of protection of endangered species,scientific experiments and developmental biology. This study provides technical support on the application of somatic cell cloning in sheep breeding and variety improvement. Through the nuclear transfer studies ,we have found: compared with enzyme digestion, although it training a longer time, tissue block culture method can prevent cells polluted in process of primary culture and passage, besides,pure sheep skin fibroblast cell line can be obtained using a longer digestion time. Through contrast of two developmental medium we found, estrus sheep serum plays an important role on the growth of the oocyte, developmental medium of liquid B (15% estrus sheep serum, 10μg/ml FSH, 20μg/ml LH, 1.5μg/ml 17-?E2 of TCM199) was finally selected.Made physical activation on oocytes in the voltage range on 1000V/cm ~ 1800V/cm , found 1400V/cm voltage is the most appropriate, access to the cleavage rate, morula rate and blastocyst rate is the highest. In comparison of effect of physical activation and chemical activation on oocyte development, the cleavage rate and morulae / blastocysts ratio were 70.3% and 21.3% by physical activation (1400V/cm); in Ionomycin combined with 6-DMAP activation treatment,the cleavage rate and morulae \ blastocyst ratio were 80.1% and 38.3% ; in 7% ethanol combined with 6-DMAP activation treatment of sheep oocytes, the cleavage rate and morulae \ blastocyst ratio were 78.3% and 35.2%. Compared chemical activation with physical activation, the chemical activation method was found more effective than physical activation, Compared two chemical activation method , ionomycin combined 6-DMAP activation method has higher cleavage and morulae \ blastocyst rate than treatment of 7% ethanol combined 6-DMAP activation . In electrofusion experiments, compared integration effects in the range of the 1100 V / cm -1600 V / cm, obtained the highest fusion rate on 1200 V / cm, when the voltage is increased to 1400 V / cm, the cleavage rate up to 47.1% , but not high of morula. Finally we selected voltage 1200 V / cm -1300 V / cm as the best voltage. cloned embryo transfer was carried a total of 39 times, more than 290 pieces somatic cell cloned embryos were produced, 39 sheep recipients were transplanted , there were 10 sheep did not return to estrus after two sexual period , work on the care of pregnant sheep was paid attention, but fetal abortion still happened, so far produced only one lamb.