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Survival depends on the cell type and its ability to endure stresses like of cooling and thawing. The aim of this study was to examine the survival rate of embryonic cell death and DNA fragmentation by controlling the time of equilibration exposure to equilibrium during freezing of in vitro matured porcine embryos. In vitro fertilization (IVF) embryos were exposed to Equilibration solution (HEPES within TCM199, 10% dimethylsulfoxide (DMSO), 10% ethylene glycol (EG)) and vitrification solution (20% DMSO, 20% EG, 0.5 mol/L sucrose) processing the three-step method. Embryos were loaded on open cryotop sheet and minimal volume of vitrification solution surrounding the embryo. Plunge the cryotop into liquid nitrogen quickly. For vitrification and warming steps were performed on a heated plate at 37°C. The experiment, different of vitrification solution exposure time used in the different protocols. The reagent composition of the three-step method used in the experiment was the same. A total of 82 in vitro fertilization embryo were randomly requested for fresh embryo group (control, n=27), conventional method group (Group 1, Equilibration time 15 min, n=27) or method of reducing time group (Group 2, Equilibration time 5 min and 5 min, n=28). Group 2 reduced the survival rate (10%), apoptosis (15%) and mitochondrial DNA fragmentation (17%) (p<0.05) compared with group 1. But there was no significant differences with cell number and pattern among three groups (control, group 1, group 2). These result suggest that time adjustment at the equilibration solution in vitrification may increase the quality of in vitro fertilization embryo.