Effect Of5-Azacytidine On Production In Vitro Of Porcine Cloned Embryos

Although cloned animals can be produced successfully, but there are still many technical issues that need to research during SCNT procedure in the future. DNA methylation is one of the important patterns in mammals’ epigenetic, and involved in the regulation of gene expression, so it is essential for normal mammalian development. If the donor cell was not completely demethylated during early embryo stages, and the re-methylation of embryo was occurred earlier, this may affect the normal development of cloned embryos.5-azacytidine (5-azaC); a DNA methylation inhibitor, is a pyrimidine analogue, which can enhance the DNA methyltransferases (DNMT) bind with cytosine, which leads to reduce DNMT activity in cells, In this study,5-azaC was added to medium, using many different ways, to observe the effect of5-azaC on donor cells, cloned embryos, IVF embryos and parthenogenetic embryos. The results are as follows:1、Effect of5-azaC on the donor cellsIn this experiment, Porcine cumulus cells are nuclear donor cells,5-azaC was added to medium of cumulus cells, in order to raise the nuclear of donor cells re-programming capabilities by changing DNA methylation states, which is a new way, can be adopted to achieve high efficiency production of cloned embryos. However,5-azaC is toxic, and therefore the cultivation of cells in the laboratory is particularly sensitive. So it is important to determine the suitable concentration and appropriate treatment time. Cumulus cells were treated with0,0.01,0.05,0.1and1μiM5-azaC for72h. Cell morphology was observed by optical microscope, moreover cell proliferation was observed by growth curve, and cell apoptosis was detected by comet assay. These observations have shown that the morphology, growth and apoptotic of cells did not show any significant changes at low concentrations, but by using higher concentration (1μM of5-azaC), There have been noticeable changes in the morphology of cells and that have prevented its growth to become apoptotic as observed by comet assay. This result indicates that when using concentrations of less than1μM of5- azaC less harmful effects on the cumulus cells will occur.2、Effect of5-azaC on porduction in vitro of porcine embryosIn this study, porcine SCNT embryos, IVF embryos and parthenogenetic embryos as research object, we detected the effect of5-azaC on the development of porcine cloned embryos, IVF embryos, and parthenogenetic embryos. We evaluated rate of oocytes matured in vitro, enucleated oocytes, survival reconstructed embryos and4-cell or8-cell cloned embryos, the results showed that pbl exclusion rate of oocytes in vitro was76.9%, enucleation rate was82.9%, survival rate of reconstructed embryos was84.8%and survival rate of4cell and8-cell cloned embryos all were100%.We observed rate of cleavage and morula of cloned embryos from donor cells treated with0～1μM5-azaC for72h, the result showed that cleavage rate appeared to be slightly higher at0.01μM5-azaC group, but contrast to the control group, the difference was not significant(73.8%VS67.1%, P>0.05). However, development of cloned embryos supplemented with1μM5-azaC was decreased. We also observed rate of cleavage、8-cell stage and morula of cloned embryos treated with0,0.1,110μM5-azaC for48h, the result showed that rate of cleavage(71.7%VS66.7%) and8-cell stage embryos (47.8%VS44.4%) were higher at1μM5-azaC group, but contrast to the control, the difference was not significant(P>0.05), However, development of embryos of10μM5-azaC concentration decreased significantly(43.2%VS66.7%),(4.5%VS17.8%),(P<0.05). Then to treat IVF embryos with0,0.1,1,10μM5-azaC for48h or60h, it showed that cleavage rate of IVF embryos treated with1μM5-azaC for48h were higher significantly than the control group(80.0%VS60.3%, P<0.05), but morula were not higher significantly(25.0%VS23.1%, P>0.05). However, whether dealing with48h or60h, post-development of IVFembryos at10μM5-azaC concentrations was reduced. Then we observed development of parthenogenetic embryos treated with0～10μM5-azaC for48h or60h, it was observed that cleavage rate of embryos treated with1μM5-azaC for48h or60h was higher significantly than the control group(81.8%VS62.9%and83.3%VS63.3%; P<0.05), but morular was not significantly higher (30.3%VS24.3and29.2%VS24.5%; P>0.05). Development of embryos at10μM5-azaC concentrations was reduced.(6)To compare the effect5-azaC on the development of embryos derived from IVF and parthenogenetic at the same concentration but different treatment time, only good pairs of0.1μM and1μM were selected fou the comparision, it was noticed that there have been no significant changes in embryos when dealing with the same concentration for48h or60h.(P>0.05). the results from the above indicates it was futil to improve development of cloned embryos,when doner cell-cumulus cell was treated with0～1μM5-azaC, or the culture medium of embryo treated with0～10μM5-azaC for48h. but it was helpful to improve the rate of cleavage of IVF and parthenogenetic embryos through the medium adding5-azaC, but morula rate was not higher significantly, However, the development capabilities of porcine SCNT, IVF and parthenogenetic embryos were reduced at high concentrations of5-azaC.