Effect of cryoprotectants and their concentration on post-thaw survival and development of expanded mouse blastocysts frozen by a simple rapid-freezing procedure

摘要

Blastocysts were frozen using ethylene glycol (EG) or propylene glycol (PG) at concentrations of 4.5, 6.0 or 7.0 mol/litre in the presence of 0.5 mol/litre sucrose. For both cryoprotectants, re-expansion and hatching rate were highest (P<0.05) for 7.0 mol. When the 2 cryoprotectants were compared, a higher (P<0.05) rate of hatching was obtained for EG at 7.0 mol than for PG at the same concentration. The survival and development of embryos frozen with the best concentration of each cryoprotectant pre-exposed to a low concentration (1.5 mol/litre + 0.25 mol/litre sucrose) of the respective cryoprotectant or ascending concentrations of sucrose were also compared. The in vivo development of embryos frozen with the best protocol (pre-exposure to 1.5 mol followed by 7.0 mol EG) was compared with that of non-frozen embryos. The highest rates of re-expansion (91%) and hatching (86%) of expanded blastocysts were achieved by pre-exposing embryos to a low concentration of EG and then freezing in 7.0 mol EG. Embryos frozen using this procedure were transferred to recipient females. The percentage of live fetuses did not differ between frozen-thawed and non-frozen (49 vs. 57%) embryos. It was concluded that simple rapid freezing with dehydration in ascending sucrose concentrations or pre-equilibration in a low concentration of EG or PG followed by exposure to the respective cryoprotectant at 7.0 mol resulted in high rates of survival and development of expanded blastocysts. Freezing using 7.0 mol EG afterpre-equilibration was the most effective protocol.