Studies On Cryopreservation By Vitrification And Embryo Splitting Of Mouse Morulae

The studies were performed on the cryopreservation of mouse morula by vitrification, embryo splitting and embryo transfer (ET) .The in vitro and in vivo development of mouse morulae after cryopreservation in straw or open pulled straw (OPS) was examined. The fresh embryos without cryopreservation were used as controls. The blastocyst rate of vitrified mouse morula obtained from the best EFS40 groups in the 2-step straw method (96.40% ) was superior to that in the 1-step straw method (88.75% ) but didn't differ from that in controls (100%) . While the blastocyst rate of vitrified mouse morula in the 1-step OPS method (97.92% )were similar(P>0.05)to that in controls. In the 2-step OPS method, embryos were pretreated with 10%EG+10%DMSO for 30s before exposed to EDFS30, a vitrification solution, for 25s, then immersed in liquid nitrogen. The blastocyst rate of vitrified mouse morula after equilibration in 0.5mol/L sucrose for 5min was 100%.The vitrified mouse morulae (Experiment 1) obtained from the best OPS groups and fresh morulae (Experiment 2) were transferred to Day 2.5 to Day 3 pseudo-pregnant female mice. In Experiment 1, 34 fetuses were obtained from 5 of 10 recipients which had received 183 vitrified embryos, and the rate of live cubs in Experiment 1 (41.46%) was similar (p>0.05) to that in Experiment 2 (42.73%) .In embryo splitting, the mouse morulae were bisected in sucrose, the exported splitting solutions or mPBS. The rates of survival demi-morula after splitting in sucrose (69.53%) and exported splitting solutions (77.40%) were higher (p<0.05) than that (56.82%) in mPBS, but the rate of demi-morula developed to blastocysts and the cell number of blastocyst were similar (p>0.05) .The fresh mouse morulae were bisected following decompaction (Experiment 3 ) and other morulae without decompaction (Experiment 4) also splitted. The rate of survival demi-embryos after splitting (82.56%vs 57.50%) and the rate of demi-embryos developed to blastocysts (75.35%vs 50%) in Experiment 3 were higher (P<0.01) than those in Experiment 4.The mouse morulae were cryopreserved by vitrification in OPS (Experiment 5 ) or straw(Experiment 6) followed by splitting. The fresh embryos undergoing the same procedure served ascontrols The rate of survival demi-embryos after splitting and the rate of demi-embryos developed toblastocysts in Experiment 5 and 6 were similar (p>0.05) to those in controls, but the blastocyst rate incontrols (77.38%) was a little higher than that in Experiment 5 and 6 (70.22% and 68.52%) .The demi-embryos derived from vitrified morulae in OPS (Experiment 7) and fresh morulae (Experiment 8 ) after cultivation for 24h were transferred to Day 2.5 to Day 3 pseudo-pregnant female mice. In Experiment 7, 15 fetuses were obtained from 5 of 11 recipients which had received 191 demi-embryos. In Experiment 8, 4 of 10 recipients, which had received 182 demi-morulae, became pregnant, and 13 live cubs were obtained. And the rate of live fetuses between Experiment 7 (17.65 % ) and Experiment 8 (18.31%) showed no difference (p>0.05)...