The Study On WAGYU Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer(SCNT) is a promising technology with potential applications in rapid multiplication of elite animal breed, transgenic animal production, conservation of valuable breeding stock, generating functionally pharmaceutical proteins and studies of basic research involved in development biology, cellular biology, embryology and many other fields.Therefore, the potential benefits of this technique in research, industry and agriculture are vast. However, the cloning efficiency is still low, generally ranging of 5-10%.In this study, we aimed to improve the in vitro developmental potency of WAGYU SCNT embryos. We established the WAGYU fibroblast cell lines originated from WAGYU adult ear tissue and fetal tissue, and performed SCNT using these fibroblasts as donor cells.Experiment One was aimed to establish the fibroblast cell lines from WAGYU adult ear tissue and fetal tissue. We established 5 adult ear tissue fibroblasts lines(from two females and one bull) and 3 fetal tissue fibroblasts lines(from two fetus), and compared their morphology, growth characteristics and ploidy. These experiment results showed that two newly established WAGYU fetal and ear skin fibroblast cell lines were stable and had normal biological characteristics.Experiment Two was aimed to improve the in vitro development of WAGYU SCNT embryos by changing several procedures. First, we compared developmental rates of cloned embryos originated from 2 different donor cell lines(fetal and ear skin fibroblasts). The cleavage rates and blastocyst rates were not significantly different between both 2 cell lines. When donor cells were prepared by direct use of freezing and thawing after cells reached 90%confluence, the cleavage rate(90.7%) was higher than cells being prepared inconventional method(84.4%), although no significant difference was observed in terms of blastocyst rates(27.2% and 31.0%). Demecolcine treatment resulted in better enucleation rate(treatment vs. control: 96.0% vs.79.5%), but no difference was shown in cleavage rate and blastocyst rate.Demecolcine treatment effect was better when COCs were cultured for 2h in demecolcine-supplemented medium after 16 h of IVM(protrusion rate 57.5%)than in control(4.7%) and other method(IVM 16 h and remove cumulus cells and culture 2 h in DEM)(54.5%). More cleaved embryos(90.6%) and blastocyst(27.5%) were produced by zona free method than by using perivitelline injection method(63.8% and 12.0%, respectively). Also, the cleavage rate and blastocyst rate in triplet fusion(2 ooplasm and 1 donor cell)were higher than in couplet fusion(1 ooplasm and 1 donor cell). WAGYU cloned embryo were transferred into the recipient animals and totally 4 calves were born(2 calves originated from WAGYU ear fibroblasts, and the other 2calves from WAGYU fetal fibroblast), but from which, one calf died at birth and one calf died a week after birth.Experiment Three was aimed to examine the effect of CUDC-101, a histone deacetylase inhibitor(HDACi), on in vitro development of WAGYU cloned embryos. When donor cells were treated with more than 5μM CUDC-101, the cleavage rate, blastocyst rate and total cell number were all higher than those in control or 2μM concentration. However, in vitro development of WAGYU cloned embryos became lower when treated with high concentration(more than 100μM) of CUDC-101, and moreover, there was not appeared any blastocyst in 250 and 500μM treatment. CUDC-101 treatment for 24 h was better than control or for 48 h. The expression level of Oct4 and Cdx2 gene in CUDC-101 treatment was significantly higher than that in control. On the contrary, the expression of DNMT1 and HDAC1, which are related withepigenetics in early embryos, was significantly downregulated in CUDC-101 treatment.In conclusion, the newly established WAGYU fibroblasts were successfully used in SCNT. The direct use of WAGYU fibroblasts after thawing, which were stored after reaching 90% confluence, could make cell preparation to be easy in SCNT. Also, demecolcin treatment could be effectively used in SCNT enucealtion without any detrimental effect. Zonafree NT method in WAGYU SCNT was favor in not only manipulation but in vitro development of SCNT embryos. CUDC-101 treatment of WAGYU fibroblasts could improve the in vitro development of SCNT embryos, which were related to gene expression including Oct4, Cdx2, DNMT1 and HDAC1.