A comparison of different cryoprotectant solutions and thawing methods for the cryopreservation of embryos of mice and rats

Abstract

The proper choice of the cryoprotectant and thawing method affects the efficiency of cryopreservation. A freezing-thawing method aimed at the preservation of blastomere cells was evaluated in experiments with ICR mice. The cleavage-stage embryos of ICR mice, GC rats, and OXYS rats were collected on Day 3 of pregnancy and frozen in plastic straws according to the standard procedure. We compared the effect of permeating (ethylene glycol and glycerol) and nonpermeating (sucrose) cryoprotectants and their combinations on the survival rate of embryos after thawing. We also compared the effect of rapid (water bath, 10 s, 37°С) and slow (40 s, room temperature; then 40 s, 30°С) thawing methods. The viability of the embryos of mice and rats after cryopreservation was evaluated by their in vitro culturing after thawing. Our data prove that slow thawing is more suitable for mice embryos and provides a higher survival rate; the addition of sucrose to the basic cryoprotectant (ethylene glycol or glycerol) improves the parameters of the in vitro cultures of embryos after thawing, especially if glycerol is used as the basic cryoprotectant. This freezing-thawing method (glycerole and sucrose as the cryoprotectant solution and slow thawing) was used for cryopreservation of GC and OXYS rats. As a result, the survival rate of embryos after freezing was 68–83.3% and the rate of in vitro development after thawing was 64.7–66.6%.