The Study Of Ovine Somatic Cell Nuclear Transfer And The Fragmantation Mechanisms Of Early Cloned Embryos

In this study, ovine oocytes maturated for different period in vitro were used as recipient cytoplasm; 4 to10 passages of skin fibroblast cells were used as donor cells. The effects of different maturated period of oocytes on enucleation, fusion, cleavage, marulae/blastocyst and pregnancy rates were investigated. In additions, the possible mechanisms of fragmentation in early NT embryos were also investigated. Transfer of 2- to 4-cell NT embryos to pseudo-pregnant recipients resulted in pregnancy, unfortunately, all of the pregnant recipients aborted. 1. Development of cloned ovine embryos in vitro/in vivo1.1 In this study, donor cells were prepared from the ear of an adult PollDorset. Karyotype analysis showed that 75.9% of fibroblast cells had normal chromosome number at passage 12, cells at 4 to 10 passages were used as donor cells.1.2 The enucleation rates of oocytes matured for different period did not differ significantly, the average rates were over 80%.1.3 Two different activation protocols were used in the present study. The results showed that the combination of ionomycin (Iono) and cycloheximide (CHX) resulted in significantly higher cleavage rate, marulae/ blastocyst development than the combination of A23187 and 6-dimethylaminipurine (6-DMAP). However, the blastocyst development was with no difference between these two activated protocols.1.4 When oocytes maturated for 20hr were used as recipient cytoplasm, the fusion rate was significantly lower than oocytes IVM for 22-24hr (51.2% vs 77.4%); the cleavage rate was also lower in 20 hr oocytes than 22-24 hr oocytes (70.8% vs 84.7%, respectively). The results showed that ovine oocyte maturated for 22-24hr in vitro could significantly increase the fusion and cleavage rates.2. Investigation of fragmentation in early NT embryos2.1 When using the same activated and fusion protocols, significant difference in fragmentation of NT embryos were observed between two cultural systems (SOFaa vs CR1aa). The CR1aa system seemed to cause higher frequency of fragmentation than SOFaa (50.6% vs 19.2%, respectively, p<0.05).2.2 When NT embryos were cultured in CR1aa medium, the stimulating voltages (140V/mm vs 180V/mm, respectively) did not affect embryo fragmentations.2.3 Under lower stimulating voltages (140V/mm), activated with 5.0μM Iono + CHX could cause a significantly higher rate of fragmentation than that of 2.5μM Iono + CHX (44.6% vs 29.7%, p<0.05).2.4 When cultured the embryos in SOFaa medium, there was no significantly different in rate of fragmentation between different activated protocols (Iono+CHX, A23187+6-DMAP).2.5 There was no significant difference in fragmentation when NT embryos wereactivated with different concentrations of Iono (2.5μM vs 5.0μM) in SOFaacultural system.In conclusion, 1) Fragmentation in early embryos was a common phenotype inovine somatic cell nuclear transfer, and had diverse frequency when differentexperiment protocols combined; 2) When using different Iono concentrations,CR1aa cultural system resulted in a higher rate of fragmentation than in SOFaa;3) SOFaa cultural system was more suitable for ovine NT embryo development.