| In present study, interspecies cloned embryos were constructed using goat fetal fibroblasts as nuclear donor and in vitro matured sheep oocytes as nuclear recipient. The effect of FSH and ECS on maturation of sheep oocytes and the parameter of electrofusion were studied. Moreover, at various developmental stage (1-, 2-, 4-, 8-cell and blastocyst) of interspecies cloned embryos, the origin and percentage of two types of mtDNA was examined by using PCR-RFLP.Results obtained as follow:1. When adding 0.2 IU/mL FSH, the maturation and parthenogenetic activation rate were 72.5 % and 77.3 % respectively, higher than control (without FSH) and other test groups(P<0.05), and the maturation and parthenogenetic activation rate of test group markedly higher than that of control group(P<0.05). These results indicated that the effect of FSH on promoting maturation and cleavage rate of sheep oocyte was conspicuous, and there was dose-dependent relationship between the effect and concentration of FSH.2. Adding different period ECS into the basal medium with FSH at it's optimal concentra -tion, when adding 10 % D1 or D3 ECS, the maturation and parthenogenetic activation rate were higher than control and D5,D7 group(P< 0.05) (D1 ECS: 79.1 % and 76.5 %, D3 ECS: 75.8 % and 74.4 %), the blastocysts rate of 5 % D7 ECS was 44.4 %, higher than control and D1 group(P<0.05), but there was no difference compared with D3 and D5 group(P> 0.05). Results showed that the effect of ECS on in vitro maturation and cleavage of sheep oocyte was decreased, and the effect of it on development of blastocysts was strengthened, with the development of pubescence. Further studys showed that when adding 10 % or 15 % ECS (P<0.05), there was no difference in maturation and parthenogenetic activation rate of sheep oocyte.3. Under the condition of one time electric pulse, pulse 20μs, there was no difference in fusion, cleavage and blastocyst rate of reconstructed embryos. when electric field intensity was 1.2 kv/cm or 1.4 kv/cm, the fusion rate (58.82 % and 61.36 % respectively)was markedly lower (P<0.05), compared with the electric field intensity was 1.6 kv/cm(73.68 %) or 1.8 kv/cm (80.61 %). However, the cleavage rates of the former two electric field intensity(68.33 % and 72.22 respectively) were markedly higher than that of the last two electric field intensity(47.14 % and 31.65 % respectively)(P<0.05). Therefore, the optimal electric field intensity was between 1.2 kv/cm and 1.4 kv/cm。4. When using two times electric pulse(1.4 kv/cm, 20μs), the fusion and cleavage rates of reconstructed embryos were higher than that of one and three times electric pulse. Results showed that in this experiment, the effect of two times electric pulse was better than others. After fusion, the reconstructed embryos were cultured in maturation medium. It was benefit for cleavage, when cultured between 60 and 120 minutes in maturation medium, and the blastocysts rate was 17.72 %, when cultured 90 minutes.5. In present study, goat(capra hircus)-sheep(Ovisaries L.) cloned embryos were constructed by fusing goat fetal fibroblasts (GFFs) into sheep oocytes, and then cultured in vitro to investigate the mtDNA origin and proportion in reconstructed embryos. Moreover, at each stage of 1- (immediately after fused), 2-, 4-, 8-cell and blastocyst, the copy number of mtDNA from GFF and sheep oocyte was examined by using PCR-restriction fragment length polymorphism(PCR-RFLP). Results showed that the proportions of nuclear donor to recipient mtDNA in 1-cell,2-cell,4-cell and 8-cell reconstruction embryos were (2.5±0.8) %, (2.8±0.6) %, (1.9±0.4) % and (1.7±0.6) % respectively and there was no differrence (P> 0.05), however, in heteroplasmic blastocysts, the proportion of nuclear donor mtDNA declined to(0.3±0.1)%, and the difference is significant compared with that before 8-cell stage(P< 0.05). According the results above, we strongly argued a mechanism that donor-cell-derived mitochondria were degraded for the depression of bioenergetic functions, and then selectively eliminated during the embryogenesis of interspecies cloned embryos.
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