Study On The Buffalo Somatic Cell Nuclear Transfer And Methylation Detection

Effects of 5-Aza-dc(5-Aza-2-deoxycytidine, a DNA methyl-transferaseinhibitor) on DNA methylation in buffalo fibroblasts were investigated. Cellswere grown to confluence at passage 6 and subsequently treated with 5-Aza-dc(0μM, 0.005μM, 0.01μM, 0.03μM, 0.09μM) for 72h. The levels of total cellularDNA methylation were quantified by flow cytometry through measuring thefluorescent intensity of cells after incubation in anti-5-methycytosine primaryantibodies and fluorescent-conjugated secondary antibodies. The resultsindicated that the levels of DNA methylation in cultured buffalo fibroblastsdecreased by treatment with 5-Aza-dc.Effects of 5-Aza-dc on buffalo somatic cell nuclear transfer was examined.buffalo donor fibroblasts were treated with 5-Aza-dc(0μM, 0.005μM, 0.01μM,0.03μM, 0.09μM) for 72h. The 0.09μM group was significant lower than theother four groups in the fusion rate(55.3% vs 72.1%, 68.2%, 75.3%, 73.4%,P<0.05), There were not siginificant difference in cleavage rate of reconstructedembryos among the five groups (P>0.05). The blastocyte development rate ofreconstructed embryos was the highest in group treated with 0.03μM 5-Aza-dcand was significant higher than that of the other four groups(26.5% vs 15.6%,14.4%, 17.5%, 6.02%, P<0.05); 0.03μM 5-Aza-dc treated with buffalo donorfibroblasts for 0h, 24h, 48h, 72h, 96h, respectively. There were not siginificantdifference in the fusion rate among the five groups (P>0.05). The 96h groupwas significant lower than the 72h group in the cleavage rate(74.3% vs 85.1%,P<0.05), However, the blastocyte development rate of reconstructed embryoswas the highest in group treated with 0.03μM 5-Aza-dc for 72h and wassignificant higher than that of the control group(25.6% vs 14.6%, P<0.05).These results showed that treatment of buffalo donor fibroblasts with 0.03μM5-Aza-dc for 72h can improve their subsequent somatic cell nuclear transfer efficiency.The objective of this study was to determine the individual nuclei DNAmethylation pattern of the SCNT and IVF buffalo embryos during the earlyembryogenesis. The levels of DNA methylation were semi-quantitative byconfocal microscopy through measuring the fluorescent intensity of thedevelopmental stages embryos after using double immunostaining. The resultsshowed that the average levels of DNA methylation of the individual nuclei inthe SCNT embryos were higher than that of the IVF embryos at the samedevelopmental stage and hypermethylation was also observed in SCNT embryoscompared with IVF embryos at the 4-cell, 8-cell and morula stages(P<0.05). Inboth types of embryos, the relative levels of DNA methylation individual nucleidecreased from the 2-cell stage until the 8-cell stage. Then levels of DNAmethylation at the morula stage increased and reached the greatest level at theblastocyst stage. However, it was shown that insufficient DNA demethylationfrom the 2-cell stage in buffalo SCNT embryos. These results indicated that anaberrant DNA methylation pattern in SCNT buffalo early embryos.