Abstract
We substantially improved the cryosurvival rate of unfertilized mouse oocytes by em-ploying a pre-vitrification 2-step equilibration with sucrose (0.25 M and 0.5 M for 5 min each) at room temperature. As a result, 96.4% of the oocytes vitrified in a DPS-1 solution (2.75 M dimethylsulphoxide, 2.75 M propylene glycol, and 1.0 M sucrose) were recovered normally after warming, and 85.2% of the recovered oocytes were successfully fertilized by fresh spermatozoa. The in vitro developmental rate of these fertilized eggs into the blastocysts was 81.4% which was comparable to that (83.0%) of its solution control. Using this technique, we next examined the developmental rate of vitrified mouse oocytes that were fertilized by frozen mouse epididymal spermatozoa. Though the in vitro fertilization rate was relatively low (55.8%), the in vitro develop-ment of these fertilized eggs into the 2-cell stage was 93.5%, comparable to the solution control (95.6%). The in vivo developmental rate after transfer into recipient females was 54.2%, which was also comparable to the rate (57.8%) of eggs derived from fertilization between fresh oocytes and spermatozoa. We successfully demonstrated that fetal development is possible from cryopreserved oocytes and spermatozoa in mice.
