Cryopreservation Of Mouse Embryos By Vitrification

The techniques for cryopreservation of mouse embryos by vitrification were systematically studied The vitrification solution EDT was made up of ethylene glycol, dex.tran and trehalose. Mouse embryos at various developmental stages were vitrified with various EDT solutions at room temperature ( 250C ). The work would provide an alternative method for cryopreservation of HBsAg-transgenic murine embryos and of rare and endangered animal embryos.Vitrification and devitrification tests of cryoprotectants demonstrated the feasibility of vitrification solution EDT. Toxicity tests showed dextran and trehalose solutions had no effects on in vitro development of morulae after exposed for 5-20 mm at 250C. The toxicity of solution EDT mainly resulted from ethylene glycol, and related to the concentration of ethylene glycol and equilibration time.Zygote vitrification tests demonstrated the developmental rate of the groups of EDT3O-one-step method with 2 mm equilibration(63%), EDT3O-two-step method with I mm equilibration(66%), EDT4O-one-step method with I mm equilibration(53%) and EDT4O-two-step method with I mm equilibration(55%) were best in each treated group, but significantly different from those ofcontrols(p0.05).Blastocyte vitrification tests demonstrated the groups of EDT3O-one-step method with 1 mm equilibration, EDT3O-two-step method with I mm equilibration, EDT4O-one-step method with I mm equilibration and EDT4O-two-step method with 0.5 mm equilibration had the best developmental percentages in each treated group after cultured in vitro for 24 hours, 90%, 91%, 91% and 89% respectively, and had no significant difference from those of controls(p>O.05). But their hatched percentages were 48%, 50%, 49% and 51% respectively after in vitro culture for 48 hours, and there was a significant difference in hatched percentages of these groups and controls(p<0.0 1).The transplantation of best-development-rate frozen-embryos into recipients uteri demonstrated neither the proportions of recipients becoming pregnant nor the proportions of live young based on embryos tranferred to all recipients or pregnant recipients were significantly different from the proportions in the controls(p>0.05).This showed EDT vitrification solution had no effects on in vivo development of mouse embryos.In a word, our study demostrated the vitrification method with EDT solution was simple,2economical and efficient. The best survuval rate of each stage thawed-embryos existed in groups of Zygote EDT3O-two-step method with I mm equilibration(66%), of Morulae EDT3O-two-step method with I mm equilibration(94%) and EDT4O-two--step method with 0.5 mm equilibration(94%), of Blastocyte EDT3O-two-step method with I mm equilibration(91%) and EDT4O-one-step method with I mm equilibration(91%).