| This study focused on bovine zona-free somatic cell nuclear transfer.To obtain a simple and effective method for bovine somatic cloning,the different protocols and experiments were adopted to reach an optimized procedure.In this research,different culture methods of bovine oocytes maturation were compared,bovine ear fibroblast cells were separated and cultured,and the effects of different methods of enucleation and fusion on reconstructed embryos development were studied.1.Aspiration method was used to recover usable cumulus-oocyte complexes (COCs) from follicles of slaughtered bovine ovaries,and the research focused on impacts of training system of different variety and doses hormones which included PMSG,HCG,FSH,LH to bovine oocytes maturity rate.The result indicated that these hormones could promote maturation rate of the oocytes,and the highest maturation rate is 81.7%.2.Effects of oocytes maturation culture time on the expelling rates and attaching rates of first poly body were observed.When oocytes were cultured 18-19h,20-21h and 23-24h,the expelling rates of first poly body were 76.1%,81.7%and 82.2%and the attaching rates of the first poly body were 77.2%,64.1%and 41.3%respectively. The results indicated that the appropriate oocytes maturation time was 20-21h.3.To obtain a simple and effective method for enucleation,zona-free two-half cytoplasts manipulation and blind-cutting manipulation were compared.The rate of enucleation is 100%with zona-free two-half cytoplasts manipulation by fluorescent staining,except for the time cost is longer than blind-cutting manipulation,so the efficiency of enucleation is lower.4.Electric fusions were performed in 2.4Kv/cm,2.6Kv/cm and 2.8Kv/cm under the condition of 40μs,2 pulse per time,and 1 second interval,and the fusion rates were 77.55%,93.96%,68.98%respectively,the results indicated that the optimum parameter of electric fusion was 2.6Kv/cm.5.Study of chemic fusion:The results indicated that PEG with concentration of 40%or 45%or 50%can lead to fusion,and the highest rate of fusion is 53.33%when the concentration of PEG is 50%.6.Enucleated zone-free oocytes and somatic cells were fused to reconstructed embryos,and the embryos were activated by 7%ethanol combined with 6-DMAP and ionomycin combined with 6-DMAP and were cultured in WOWs(only pressed). When the embryos were activated by 7%ethanol,the cleavage rates and 8-cell development rates of two-half cytoplasts embryos were 61.2%and 3.27%,and the cleavage rates and 8-cell development rates of blind-cutting manipulation embryos were 70.8%and 7.56%.On the contrast,when the reconstructed embryos were activated by ionomycin,the cleavage rates and 8-cell development rates of two-half cytoplasts embryos were 46.5%and 0,and the cleavage rates and 8-cell development rates of the blind-cutting manipulation embryos were 56.1%and 3.12%.The results indicated that treatment with 7%ethanol combined with 6-DMAP was a better way to activate bovine reconstructed embryos,and the cleavage rates and 8-cell development rates of blind-cutting manipulation embryos were higher than the rates of two-half cytoplasts embryos.The results indicated the zone-flee cloning method might be easier to adopt than conventional cloning procedure for users and was an easy procedure,but the developmental system of zone-flee reconstruct embryos in vitro culturing needs to improve in the future.
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