In vitro viability of mouse 8-cell embryos vitrified in a simple solution of ethylene glycol

A study was made to determine if ethylene glycol (EG) can be used in a simple solution to vitrify mouse 8-cell embryos and to determine the critical factors that affect its success. Mouse 8-cell embryos were vitrified after exposure to 2M and 7M EG prepared in Dulbecco's phosphate buffered saline (PBS) with 10% heat-inactivated calf serum (CS). Mouse 8-cell embryos exposed to 2M EG for 2, 5 and 10 min, and to 7M EG for 2 and 5 min had survival rates similar to the untreated controls (93.3-100%). No significant difference in their survival rates in vitro was observed. Higher room temperatures (> 24 degrees C) at exposure before cooling resulted in poor development rates to the blastocyst stage. The survival rates of embryos vitrified after 2 min in 7M EG at 18-22 degrees C room temperature did not differ significantly from the control, but embryos vitrified after 5 min had significantly lower survival rates (p < 0.0001). In conclusion, effective vitrification of mouse 8-cell embryos can be achieved by initial exposure to 2M EG for 2-10 min followed by equilibration in 7M EG for 2 min at 18-22 degrees C room temperature. This study has shown that 7M EG in PBS with 10% CS is sufficient to provide cryoprotection of vitrified mouse 8-cell embryos and that exposure of the embryos to the vitrification solution at temperatures over 24 degrees C is critical to their subsequent development in vitro.