Warming and Recovery of Vitrified Embryos
Ethanol was cooled at —80°C by placing a Dewar flask containing ethanol in a freezer at —80°C for 24 h or more. The vitrified straw sample was taken out of LN2 (—196°C) and then immersed in ethanol precooled at —80°C. The sample was kept at that temperature for 4, 7, or 10 days and then warmed in water at 25°C. As the vitrified control, the vitrified sample was warmed without being kept at —80°C. In some experiments, some samples kept at —80°C for 4 days were recooled in LN2 directly or after being kept in LN2 gas before recovery.
When warmed from —80°C, the sample was directly immersed in water at 25°C. When warmed from the LN2 temperature, the sample was kept in air at room temperature (25°C) for 10 sec to allow it to pass through the glass transition temperature of the cytoplasm (approximately — 130°C) slowly, and then it was immersed in water at 25°C. When the crystallized S-PB1 medium in the straw began to melt in water (after ~7 sec), the straw was taken out and wiped dry, and both ends were cut off. The contents containing embryos were recovered quickly in an empty watch glass by flushing the straw with 0.8 ml of S-PB1 medium using a 1-ml syringe at 25°C and agitated gently to promote dilution of the vitrification solution. Embryos were collected and transferred into fresh S-PB1 medium under paraffin oil. At about 5 min after perfusion, the embryos were transferred into fresh PB1 medium under paraffin oil. Upon transfer to PB1 medium, embryos were observed as to whether they had swelled or not, because swelling is a feature of the formation of intracellular ice. fully
Assessment of In Vitro Survival
As the basic medium for culturing embryos, M16medium supplemented with 10 iM ethylenediaminetetraacetic acid-Na, 1 mM glutamine, and 10 iM p-mercaptoethanol was used. Three drops (0.2 ml each) of modified M16 medium were placed in a culture dish under paraffin oil and equilibrated in a humidified CO2 incubator (5% CO2, 95% air) at 37°C overnight.
Vitrified embryos were recovered and cultured in modified M16 medium. As the solution control, embryos were pretreated with EFS20a for 2 min at 25°C, then treated with vitrification solution (EFS30a-EFS50a, EFS30c-EFS50c) for 60 sec at 25°C as for vitrification, and then recovered without cooling. As the fresh control, fresh embryos were cultured without any treatment.
After 1 h of culture, the morphology of each blastomere of the embryos was observed under a dissecting microscope. Morphological survival was expressed as the total number of morphologically normal blastomeres per total number of blastomeres of recovered embryos. The survival of embryos was assessed by their ability to develop into expanded blastocysts during 96 h of culture.