Research On The In Vitro Culture And Vitrification Of Mammaliam Embryos

In vitro culture and vitrification of mouse and sheep IVP embryos were studied in this paper.Based on improvement of culture system ,FVP embryos from mouse 1-cell embryos and IVF oocytes of sheep were cryopreserved successfulry.The embryos of Kunming mouse show 2-cell block in vitro.The effects of several culture media on overcoming the 2-block in embryo development were studied.The modified M2 and M16,in which glucose and phosphate were excluded and 5.55mM (lg/1) fructose. 0.127mM EDTA. 2.5mM taurine.2mM glutamine and 2% essential amino acid(EAA.50x) and 1% nonessential amino acid(NEA,100*)were added,were shown to overcome the 2-cell block and support the development to blastocyst stage.mM16 shew highest blastocyst rate in 80% of the 2-cell embryos,significantly higher than that of other groups (p<0.01). mM2 (glutamine,EAA and NEA were added) improved blastocyst rate compared to A mM2(without glutamine,EAA and NEA),50% and 46%respectively,but no difference found.M16+TE(2.5mM taurine and 0.127mM EDTA were added in M16) and M16+T (2.5mM taurine were added in M16) were also shown to be effective to overcome the 2-cell blockand 63% and 19% of 2-cell developed to blastocyst stage after 96 hours culture.We concluded that glutamine ,EAA and NEA have beneficial effect and taurine play an important role in overcoming the 2-cell block during Kunming mouse embryo development.Factors affecting the development of micro-injected mouse embryos were studied.The results shew that blastocyst rate of microinjected 1-cell embryos was 35%,lower than non-treated group(46%).but no significant difference(p>0.05);blastocyst rate of micromanipulated embryos cultured in vitro(72h)was lower than that of embryos cultured in vivo(72h),25% and 38% respectively,no difference found(p>0.05);mM2,as a microinjection and transfer operation mediunymproved pregnant rate(62.5%) than M2(35.9%)(p<0.05).Mouse morula and early blastocysts were vitrified with EFS30 and EFS40.The results shew that two-step vitrification of early blastocysts with EFS30 got highest development rate afterthawing(88.9%);The development rate of early blastocysts cultured in vitro had no difference compared to embryos cultured in vivo with two-step vitrification.IVF embryos of sheep also show development block,happening in 8-16-cell stage. Stripption degree of cumulus cells of matured oocytes with normal cumulus cells before insemination had no significant effect on FVF of oocytes and development of embryos in vitro.The optimal concention of heparin and caffeine in fertilization medium was lOIU/ml heparin+lOmmol/ml caffeine when oocytes were transfered to culture medium at 6h after insemination.In whickblastocyst rate was 32.1%, higher than other group.Taurine could improve development of sheep embryos ,5mM suitable for the requirement of sheep embryos.The blastocyst rate in simple medium mSOF had no difference with that in TCM-199.Different freezing and thawing methods of sheep embryos were studied.Survical rate of one-step vitrified blastocysts was 50%,significant lower than two-step and slow freezing(p<0.05):two step and slow freezing shew same effect and survival rates were 73% and 81% respective!}'.,without difference ; development rates of vitrified embryos with one-step and two-step thawing had no difference(76.5% and 80.0% respectively).