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.

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