Effect Of Different Growing Factors On Development Of Bovine Preimplantation Embryos And Derivation Of Embryonic Stem Cells

Recent years,a variety of animals have been used to a lot of experimental research,as well as livestock include cattle,sheep,pig,horse.Particularly,establishment of in vitro culture system create a remarkable achievement,it included in vitro maturation(IVM),in vitro fertilization(IVF),and in vitro development(IVD).But the mechanism of process still remained unclear.In this study,we used bovine oocytes as material,carry on IVM,IVF,IVD three process,to investigate the effect of the different signal pathway on the development of bovine embryos in vitro.First,based on the traditional IVM and IVF experiments,added termed 2i/Lif,it include Mek inhibitor PD0325901(PD03),glycogen synthase kinase 3 inhibition by CHIR99021(CH)and leukaemia inhibitory factor(lif);Y27632(Rock pathway)and hyaluronidase several factor compounds respectively in development medium in 48 hours after IVF,to observed the development of embryos.Second,count the cell number of inner cell mess(ICM)and trophectoderm(TE)of blastocysts,to study the effect of different signal pathway on the development of bovine embryos.Finally,we divided concentration of Y27632 into 10μM,50 μM and 100 μM to culture bovine embroys.we investigated the effect of different concentration of Y27632 on development of bovine embryos.The result showed 10 μM Y27632,Rock pathway,have significant function in whether the development of blastocysts or development of trophectoderm than 50um and 10um.Even embryonic stem cell(ESCs)studies have been carried out on animals such as rats,hamsters,minks,sheep,rabbits,pigs,cattle,however only mouse ESCs cells are truly pluripotent cells,and be recognized by naive ESCs.In study of bovine embryonic stem cells,still struggle to find the way to make ESCs like the mouse ESCs.In here,we used the AFR(Activin A,bFGF,KSR),AFY(Activin A,bFGF,Y27632),human and Pig ESCs culture medium to outgrowth of bovine blastocysts which derived from IVF,and observed development of bovine ESCs.We obtained three cell lines with AP positive,and cultured to passage four.The further detail analysis need to be explored in future research.