Studies on Factors Affecting the Development of Embryos from Somatic Cell Nuclear Transplantation in the Fish

For comparison of hatching rate,fertilized eggs of Misgurnus anguillicaudatus were cultured in different mediums.The results showed that normal and total fries in 1×Holtfreter were the highest,reached 54.8% and 59.2%,respectively.There was no difference in hatching rate of normal fries between 1/2×Holtfreter,1/10×Holtfreter,cold boiled water and changed mediums.They reached 48.0%,49.6%,50.0% and 48.8%,respectively.Rate of abnormal fries was the highest in cold boiled water,reached 8.8%.PBS was not suitable for cultured medium.Mechanic injury had no considerable effects on hatching rate of embryos (73.39% vs.75.70%),but abnormal fries increased considerably (8.8% vs.0).Cultured cells from caudal fin of gynogenetic bighead carp (Aristichthys nobilis) were used as donor cells,and unfertilized eggs of Misgurnus anguillicaudatus were used as recipient eggs.Microsatellite marker analysis showed that the nuclei of nuclear transfer embryos came from the donor nuclei,the nucleus contained in the recipient eggs was rejected or degraded.The passage number of cultured cells critically affected developmental rate of nuclear transfer embryos.With the increase of the passage number,developmental rate of nuclear transfer embryos was decreased.Serial nuclear transfer facilitated reprogramming of nuclear transfer embryos.Developmental rate could be heightened by serial nuclear transfer,especially for the first serial nuclear transfer of somatic cells,rate of late blastula of nuclear transfer embryos increased abruptly from 7.69% to 43.84%.There was no considerable difference in developmental rate of nuclear transfer embryos between Misgurnus anguillicaudatus and Paramisgurnus dabryanus unfertilized recipient eggs (7.69% vs.7.02%).