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Quick freezing of unfertilized mouse oocytes using ethylene glycol with sucrose or trehalose

Unfertilized mouse oocytes were frozen by directly plunging them into liquid nitrogen vapour after equilibration in a freezing medium containing 3 mol ethylene glycol l^–1 with 0.25 mol sucrose or trehalose l^–1 for 5–40 min. After thawing and dilution of the cryoprotectant, oocytes of normal morphology were inseminated in vitro and the effect of equilibration period on the rates of fertilization and development in vitro was examined. Regardless of the equilibration in the freezing medium, no significant difference was observed on the fertilization rate of frozen–thawed oocytes. However, higher fertilization and higher normal fertilization rates were obtained with equilibration in 3 mol ethylene glycol l^–1 with either 0.25 mol sucrose l^–1 or trehalose for 20 and 40 min than with 5 and 10 min equilibration. Development rates to two-cell embryos and expanded blastocysts of in vitro fertilized frozen–thawed oocytes that were equilibrated in the freezing medium for 20 and 40 min were significantly higher (P < 0.05 or P < 0.01) than with 5 min equilibration. Development in vivo was assessed by transferring blastocysts derived from unfertilized oocytes frozen by the optimum treatment (20 min equilibration in the freezing medium before freezing) into the uterine horns of day 3 pseudopregnant female recipients. The development rate of frozen–thawed oocytes to the blastocyst stage after insemination in vitro was significantly lower than that of the non-frozen control (P < 0.001). However, transfer of the blastocysts derived from frozen-thawed oocytes to the uterine horns of the recipients resulted in fetal development and implantation rates similar to those of the control. The overall development rates to fetuses of blastocysts derived from in vitro fertilization of mouse oocytes frozen after 20 min equilibration in 3 mol ethylene glycol l^–1 with 0.25 mol sucrose l^–1 or trehalose were 20.3 and 22.5%, respectively.

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