The Study Of The Developmental Potential Of In Vitro Fetrilization, Somatic Cell Nuclear Transfer And Recloning Embryos

Recloning has been realized in different mammals. However, the recloningefficiency of various species was different due to the species and experimentalmethods. Recently studies have shown that epigenetic reprogramming takes animportant role in the development of cloned embryos. Inadequete reprogranmmingwould lead to the abnormal expression of some critical genes, and the development ofcloned embryos was arrested. In order to determine the affect of recloning technologyin pig, the efficiency of recloning pig and developmental protential of pig recloningembryos were detected, and IVF and SCNT embryos were took as the control groups.In order to detect embryonic gene expression more accurately, a single-embryo qPCRtechnology was established in our study. Using this technology we detected thepluripotency marker gene expression in pig recloning embryos at early developmentalperiods, and the data would be referened for pig cloning research. Experimentalresults are as follows:The pig SCNT and recloning embryos were transplanted into61and11of surrogateLandrace sows. Finally,46piglets were birthed and9survived healthy in SCNTgroup, however, in recloning group19piglets were birthed and only2survived. Thepregnancy and survival rates of recloning piglets declined significantly from36.4%to10.5%and19.6%to10.5%, respectively (p<0.05).Further, in order to analysis the reasons of the low recloning efficiency in pig, wedetected the early developmental potential in recloning embryos, IVF and SCNTembryos were took as the control groups. The finds were that during the period of2-cell embryo develop to4-cell embryo, the developmental potencial in IVF, SCNT,and recloning embryos were86.1%,80.7%, and57.9%. It indicatied thedevelopmental potential of recloning embryos at2-cell stage declined significantlycompared to the control groups (p<0.05). However, the potential in recloning embryos at other stages have not significantly difference. And then the total numbers of cells inblastocysts were compared during the three groups through Hoeches33342staining.The number of cells in IVF, SCNT, and recloning blastocysts were58±8.9,39±9.8,and25±7.0, respectively (p<0.05). Those finds revealed that the lower developmentalpotential in pig recloning embryos is one of factors that lead to the lower recloningefficiency in pig.In order to analysis pig recloning embryos at gene expression level, we build asingle-embryo amplified cDNA library. And the amplified library was detected usingqPCR, it showed that relative abundance of genes between the unamplified andamplified library have not significantly difference. The results indicated that multiplegenes in amplified cDNA library could be quantitative analyzed using qPCR.Finally, pluripotency genes (Oct4, Nanog, Sox2, and Klf4) in the three groups at2-cell,4-cell, morula, and blastocyst stages were determined. In comparison to IVFand SCNT embryos, the levels of Oct4and Klf4in recloning embryos were lower atthe give stages. At2-cell stage, Nanog and Sox2expressed significantly higher inrecloning embryos than that in IVF and SCNT embryos. In contrast, at morula andblastocyst stages the levels of Nanog and Sox2declined significantly in recloningembryos compared to IVF and SCNT embryos. It indicated that the abnormalexpression of pluripotency genes (Oct4, Nanog, Sox2, and Klf4) in recloning embryoswould be responsible for the lower developmental potential.