Effect of Cryoprotectant Gradient Equilibration Method on In Vitro Survival Rate and Pregnancy Rate of Bovine IVF Blastocysts.

Abstract

The objective of this study was to examine the cryoprotectant gradient equilibration method for the survival rate and pregnancy rate. Day 7 to 9 embryos (fertilization=0) were randomly allocated and equilibrated in (A) a one step equilibration group directly plunged into 10% glycerol solution, (B) a three step equilibration group with 3.3%, 6.7% and 10% glycerol solution or (C) a gradient equilibration group as four different concentrations of glycerol solution were loaded in a small test tube: 10%, 6.7%, 3.3% and 0% from the bottom to the top. In Exp. 2, day 7 and 8 embryos were randomly allocated to (A) and (C) groups. After 20 min, the embryos from each group were transferred into 10% glycerol with 0.2 M sucrose solution, and then loaded into 0.25 ml plastic straws. The straws were frozen in an alcohol bath type freezer. The straws were thawed in a 38°C water bath after 10 second air thawing. In Exp. 1, embryos were incubated with TCM199 and 5% calf serum for 48 h in a 38.8°C incubator. In Exp. 2, straws containing embryos were non-surgically and directly transferred into cow uterus. The in vitro survival rate of the gradient equilibration group was higher (P<0.05) than that of the three step equilibration group. The survival rate of day 9 embryos was lower (P<0.01) than day of 7 embryos. The pregnancy rates of the gradient equilibration group tended to be higher than those of the one step equilibration group.