The Effects Of Porcine Oocyte Source And Supplement In Vivo-matured Follicular Fluid Or SDF1 During In Vitro Maturation On The Cloned Pig Embryo Development

Pig somatic cell nuclear transfer(SCNT)technology has important application value in the breeding and conservation of agricultural animals,the rescue of endangered animals,biomedicine and medicine.However,the current birth efficiency of SCNT pigs is only about 1%.The low cloning efficiency seriously limits the popularization and application of pig SCNT technology.The abnormal development of SCNT embryos was found to be due to incomplete reprogramming of their genomes.During the developmental period of early cloned embryos,the reprogramming and differentiation of their genomes mainly depend on the large amount of m RNA and protein regulation stored by oocytes during the maturation.Therefore,the reprogramming ability of oocytes is a significant factor for the successful development of pig SCNT embryo.At present,pig SCNT relies heavily on in vitro matured(IVTM)oocytes produced by in vitro maturation(IVM),and whether in vivo matured(IVVM)oocytes can improve the efficiency of pig cloning is still unknown.In addition,10% of slaughter house-derived immature follicle-derived fluid(IFF)is currently supplemented in culture of porcine oocyte IVM.However,the ovaries from the source of IFF are often transported in vitro for a long time,and frequently suffer from cold and heat stress that reduces the abundance of beneficial substances or accumulates negative regulatory factors on oocyte quality,which reduces the effect of oocyte IVM.And these follicles from the IFF are still in a physiological state that is not yet ovulating,which result in a significant difference between IFF and In vivo-matured follicle-derived fluid(MFF).It is also unknown whether adding MFF instead of IFF into the IVM medium can improve the developmental efficiency of cloned pig embryos.In order to improve the developmental efficiency of cloned embryos,it is necessary to identify the factors in MFF are responsible.Therefore,the purpose of this study is to(1)compare the in vitro developmental efficiency of cloned embryos from IVTM and IVVM pig oocytes;(2)compare the effects of 10% IFF and 10% MFF supplementation on the quality of pig oocytes and in vitro developmental efficiency of cloned embryos in vitro;(3)screen and identify the potential factors in MFF that play an important role in improving the quality of porcine oocytes,and explore whether supplementation of potential factors in vitro maturation medium can improve the quality of porcine oocytes and the in vitro development of cloned embryos.The results show that(1)the in vitro developmental efficiency of cloned embryos from pig IVVM oocytes was significantly higher than that of the IVTM group;(2)supplementation of 10% MFF in IVM process could effectively reduce the reactive oxygen species(ROS)level in oocytes,up-regulate the expression of oocyte quality-related genes,and improve the developmental ability of SCNT embryos produced by IVTM oocytes compared with10% IFF;FST in MFF is significantly lower than IFF,while SDF1 is significantly higher than IFF according to parallel reaction monitoring(PRM)results;(3)supplementation of 20ng/m L SDF1 in IVM culture medium could significantly improve the development potential of oocytes,reduce the ROS level of oocytes,improve the potential of mitochondria in oocytes,promote the expression of oocyte quality-related and anti-apoptotic genes,and inhibit the expression of pro-apoptotic genes.The study demonstrated that pig IVTM oocytes have less potential to support the development of cloned embryos than IVVM oocytes and provided two procedures to optimize the culture medium of oocyte IVM,and provided technical support for improving pig oocyte quality and promoting developmental capacity of SCNT embryo.