Parthenogenetic Embryo Culture And Somatic Cell Nuclear Transfer In Bovine

The effect of EGF(epithelial growth factor) on the maturation of oocyte nuclear and cytoplasm is studied by adding EGF in culture media in vitro. In addition, it is also examined by parthenogenetic activation. It is necessary to add various serums or BSA(bovine serum album)in culture media, but these when to be added hasn't reported in our country. One of the main factors, which impact embryo development of nuclear transfer, is the synchronization of cell cycle. Usually, the way of serum starvation is common in embryo nuclear transferring. In this way, cell cycle is synchronized in GO/G1 period. Ros, the inhibitor of cdk2, can make cells be in Gl period by inhibiting the activation of cdk2 kinase. Ceils are back into S period again if they are taken from Ros media. In this paper, the development of nuclear transfer embryo is studied which is treated by serum starvation, Ros media, non-starvation respectively. Meanwhile, the difference of reconstructed embryo development between fibroblasts and granular cells as donor cell is also studied. The result is as follows.l.The maturation rates of oocytes are examined 24 hours after adding EGF 0,10,30,50mg/ml respectively in maturation media. Oocytes are placed in the media (including 5μmol/L lonomycin).activated 5min,and then cultured 4 hours in the media(including 6-DMAP and CB),fmally in CR1 media, after 7-8 days, rates of blastocyst development are examined .Results show that EGF(30ng/ml) can improve the maturation of bovine oocytes and enhance the development rate of bovine parthenogentic blastocyst. So EGF can improve nuclear maturation and cytoplasmic maturation of bovine oocytes.2.Adding 10%(V/V) FBS in the media in 0,second, fourth, sixth day after matured bovine oocytes are parthenogentized in Vitro, blastocyst rate are examined in seventh or eighth day. Results indicate that the best time to add FBS is in the fourth day, but in the second day, the development of parthenogenetic embryos is blocked.3.By cytoplasmic injection, the development of reconstructed embryos, using fibroblasts and granular cells as donor cells that are treated by serum starvation, non-starvation and Ros media, is studied. Results are as follows. (1) The development rate of bovine fibroblast reconstructed embryos in non-starvation group and Ros group is higher significantly than that which is treated by serum starvation(p<0.05), the difference between Ros group and non-starvation group is not significant(P>0.05). (2) In a similar way, bovine granular cells are treated, the difference of development rate between Ros group and serum starvation group is not significant (P>0.05). Additionally, the development rate in non-starvation group is higher significantly than other two (P<0.05).4.The difference between two type of cells, which are treated by serum starvation and non-starvation, is not significant (P>0.05). But after they are induced by Ros, the blastocyst development rate, using bovine granular cells as donor cells is higher significantly than using bovine fibroblast cells (PO.05). In conclusion, EGF can improve the maturation of bovine oocyte; the oocyte cytoplasmic maturation can be examined by parthenogenesis; the development rate of bovine fibroblast reconstructed embryos, which are treated by non-starvation or Ros media is higher than other two.