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

Experiments were conducted to develop a simple rapid-freezing protocol for expanded mouse blastocyst-stage embryos. The effect of type of cryoprotectant (ethylene glycol and propylene glycol) and its concentrations (4.5, 6.0 and 7.0 mol/L each with 0.5 mol/L sucrose) on morphological survival and development in vitro were studied. The survival and development of embryos frozen with best concentration of each cryoprotectant pre-exposed to either a low concentration (1.5 mol/L with 0.25 mol/L sucrose) of the respective cryoprotectant or ascending concentrations of sucrose were also compared. The in vivo development of embryos frozen with best protocol (pre-exposure to 1.5 mol followed by 7.0 mol ethylene glycol) was compared with nonfrozen embryos. The rate of re-expansion and hatching was influenced by the type and concentration of the cryoprotectant. A significantly higher re-expansion and hatching rate was achieved at 7.0 mol of both cryoprotectants compared with 4.5 and 6.0 mol of the respective cryoprotectants. When comparing 2 cryoprotectants, a higher (P < 0.05) rate of hatching was obtained with ethylene glycol at 7.0 mol compared with a similar concentration of propylene glycol. The highest re-expansion (91%) and hatching (86%) of expanded blastocysts was achieved with pre-exposure of embryos to a low concentration of ethylene glycol followed by freezing in the same cryoprotectant at 7.0 mol. The transfer of embryos frozen using this protocol resulted in the development of live fetuses. The proportion of live fetuses in the pregnant recipients with frozen-thawed embryos were not different from those transferred nonfrozen embryos (49 vs 57%). It may be concluded that simple rapid-freezing with dehydration in ascending sucrose concentrations or pre-equilibration in a low concentration of ethylene glycol or propylene glycol followed by exposure to the respective cryoprotectant at 7.0 mol resulted in high survival and development of expanded blastocysts. Ethylene glycol at 7.0 mol with pre-equilibration is, however, most effective for cryopreservation of this stage in the mouse.