Study On Cryopreservation Of Mouse 2-Cell Embryos By Vitrification

The method of vitrification cryopreservation of mouse 2-cell embryos was studied. Several vitrification solutions named EFS30, EFS40, EDFS30 and EDFS40 were prepared. The solution contains ethylene glycol, Ficoll, sucrose and DMSO. Straw method was used to vitrify and cryopreservate in vivo mouse 2-cell embryos at room temperature (20℃ or 25℃). OPS method was used to vitrify the embryo at 25 ℃ or 37℃. The development abilities of thawed embryos were also assessed both in vivo and in vitro.The study of toxicities of the vitrification solutions showed that when embryos were equilibrated in PBS for 20min, the blastocyst rate was 70.73%. When embryos were equilibrated in 0.5mol/L sucrose for 5~20min, the blastocyst rates ranged from 65.71%~58.33%. When embryos were equilibrated in FS, 10%EG and 10%E+10%D for 5~20min, the blastocyst rates were between 61.54%~57.14%. The results indicate there were no effect on 2-cell embryos developed to blastocysts. However when embryos were equilibrated in EFS or EDFS vitrification solution for 0.5min, the blastocysts rates were between 61.53%~62.50%. When equilibration time was lmin, the blastocysts rates were 60.00%~60.42%. When the time was 2min, the blastocysts rates were 56.25%~61.70%. When the time was 5min, the blastocysts rates were 47.50%~61.70%. When the time prolonged for 10~20min, the blastocysts rates declined to 47.62%~0%.The results of cryopreservation of mouse 2-cell embryos by straw and OPS vitrification method in this study showed that when the 2-cell embryos were equilibrated in EFS40 at 20℃ for lmin (one-step straw method) before cryopreservation, the blastocysts rate was only 35.00%. There was a significant difference (P<0.01) from the control (65.00%). When 2-cell embryos were pretreated in 10%E+10%D for 5min then treated in EFS for 30s (two-step), the blastocysts rates were from 47.72%~48.78%. At a temperature of 25℃, the blastocysts rate for two-step method was 52.17%. There was no significant difference (P>0.05) compared with the control. When the temperature was at 25℃, the EFS30 OPS two-step method had a blastocyst rate of 62.22%, the highest rate in all groups. After the morulae cultured from thawed embryos from the best straw group and OPS group were transferred, all groups have obtained offspring.The cell number for blastocyst cultured from thawed embryos vitrified between straw and OPS two-step method (47.25+6.81; 55.42+8.96), but was no significant difference(P>0.05) compared with the control (67.43 + 9.05).