Vitrification of mouse 8-cell embryos with glycerol as a cryoporotectant

Glycerol at a concentration of 6.85 M has been used for cryopreservation of 8-cell mouse embryos with the aim to induce formation of ice-free glass after plunging into liquid nitrogen. Before treatment with this concentration at approximately 0 degrees C, embryos were pre-equilibrated in 1.37 M glycerol at ambient temperature. It was found that the main source of damage to embryos is due to treatment with a high concentration of glycerol and osmotic events during its dilution. Cooling and warming of embryos per se induce little or no harm to their capacity to form expanded blastocysts after 48 h in vitro. Best results (together 169/198, 85.4%) were obtained, when both time (not more than 15 min) and temperature (approximately 0 degrees C) of exposure of embryos to vitrification media were controlled properly.