Equilibrium Vitrification: Conclusion

Therefore, we composed new vitrification solutions (EFSc solutions) in which the concentration of sucrose was increased to 3.1 mol/kg water. The increase in the sucrose concentration increases not only the molality (moles/kilogram water) of sucrose but also that of ethylene glycol, because the increase in the volume of sucrose decreases the volume of water. Eventually,…

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Equilibrium Vitrification: DISCUSSION

When they were placed in ethanol at —80°C, they remained transparent after 3 min but became opaque after 24 h. This strongly suggests that small, invisible ice crystals had not formed in the transparent solutions in LN2. If the crystals had formed, the solution would have turned opaque quickly at —80°C. Therefore, we consider that…

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Equilibrium Vitrification: RESULTS 2

The swelling rate of embryos was significantly higher with EFS30c (26%-71%) than with EFS35c (4%-7%) and EFS40c (3%-4%). Accordingly, survival rates were high with EFS35c and EFS40c (82%-85%) but not with EFS30c (0-49%). In the next experiment, we examined whether embryos vitrified with EFS35c or EFS40c and then kept at —80°C for 4 days remain…

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Equilibrium Vitrification: RESULTS

Assessment of In Vivo Survival To examine the developmental potential of vitrified embryos in vivo, a total of 62 two-cell embryos were collected from C57BL/6J mice, vitrified with EFS35c, kept at — 80°C for 4 days, and recooled in LN2. A total of 8-10 embryos were loaded into each straw, and two to three straw…

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Equilibrium Vitrification: Warming and Recovery of Vitrified Embryos

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,…

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Equilibrium Vitrification: Vitrification Solutions

We tried to develop new vitrification solutions based on the original EFS solution, PB1 medium containing ethylene glycol, Ficoll, and sucrose. The solution was first developed for the vitrification of mouse morulae and has proven effective for mouse embryos at various developmental stages, as well as for embryos of many mammalian species. In the present…

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Equilibrium Vitrification: MATERIALS AND METHODS

Recently, we have shown that the rate of survival of vitrified mouse morulae decreased when they were kept at —80°C for 3 min (25%-75%), 60 min (24%-66%), or 24 h (0-14%) before recovery. This shows that embryos were vitrified with a considerable extent of supercooling (in nonequilibrium), and the osmolality of the vitrification solution is…

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