Equilibrium vitrification of oocytes using low concentrations of cryoprotectants

In order to make dry ice transportation of vitrified embryos practical, a near-equilibrium vitrification was developed using a cryoprotectant solution (EDFS10/10a), by which mouse embryos at various stages were vitrified in a near-equilibrium environment. EDFS10/10a consisted of 10% (v/v) ethylene glycol, 10% (v/v) Me₂SO, 0.4 M sucrose and 24% (w/v) Ficoll PM70. This method exhibited the benefits of slow freezing and vitrification, with a low risk of osmotic injury. In this study, we investigated whether mouse oocytes are vitrifiable with EDFS10/10a in a highly dehydrated/concentrated state, and whether they can remain fertilizable and developing into embryos after vitrification. When mature mouse oocytes were vitrified in liquid nitrogen and after 4-28 days of storage at -80 °C, high survival rates were observed (88-99%). Vitrified and warmed oocytes were subjected to partial zona dissection and in vitro fertilized. The rate of 2-cell stage was 80-82%. Blastocyst formation rate was 55-70% which was similar to that of embryos derived from fresh oocytes. After the 2-cell embryos were transferred to recipient mice, the implantation and offspring rates did not differ significantly from those of embryos derived from fresh oocytes, indicating that vitrified oocytes retained the developmental ability. Therefore, it is possible to vitrify mouse oocytes in a near-equilibrium state using EDFS10/10a and conveniently transported using dry ice.