117 EFFECT OF STEPWISE DILUTION ON THE VIABILITY OF FROZEN–THAWED BOVINE OOCYTES MATURED IN VITRO

Abstract

The purpose of this study was to evaluate the effect of stepwise dilution on the viability of frozen–thawed bovine oocytes matured in vitro. Oocytes matured in vitro were denuded and equilibrated in modified TCM-199 (m199: 11 mmol L^-1 HEPES, 9 mmol L^-1 Na-HEPES, 5 mmol L^-1 NaHCO₃, 20% (v/v) calf serum) supplemented with 10% (v/v) glycerol for 15 min at room temperature (RT). Then they were exposed to m199 with 10% glycerol and 0.25 mol L^-1 sucrose and loaded into 0.25-mL plastic straws. The straws were sealed and seeded at -6°C, cooled at the rate of 0.33°C min^-1 to -25°C, and plunged into LN₂. For thawing, the straws were first held in air at RT for 10 s, followed by immersion in 30°C water for 10 s. In the first experiment, frozen-thawed oocytes were subjected to cryoprotectants in 5 different manners of dilution. In the non-step dilution, the oocytes (n = 60) were put into m199 for 5 min. In the single-step dilution, the oocytes (n = 37) were transferred to 0.25 mol L^-1 sucrose in m199 for 5 min. In the two-step dilution, the oocytes (n = 56) were transferred to 0.5 and then 0.25 mol L^-1 sucrose in m199 for 5 and 5 min, respectively. In the three-step dilution, the oocytes (n = 57) were transferred to 0.75, 0.5, and 0.25 mol L^-1 sucrose in m199 for 1, 5, and 5 min, respectively. In the four-step dilution, the oocytes (n = 52) were transferred to 1.0, 0.75, 0.5, and 0.25 mol L^-1 sucrose in m199 for 1, 1, 5, and 5 min, respectively. After dilution, all of the oocytes were washed twice in TCM-199 supplemented with 5% fetal bovine serum for 5 min and cultured for 1 h to assess the morphology. The rate of morphological normal oocytes in the four-step dilution (94.2%) was significantly (P < 0.05) higher than that in other groups (non-, single-, two-, and three-step dilution: 61.7%, 73.0%, 78.6%, and 77.2%). In the second experiment, non-frozen (control, n = 170) and frozen–thawed oocytes (n = 145) with four-step dilution were fertilized and cultured in vitro (Kuwayama 1992 J. Reprod. Fert. 96, 187–193). To assess fertilization, some of the oocytes were fixed at 10 h after insemination. Cleavage and blastocyst rates were determined on Day 2 and Day 8 after fertilization (Day 0), respectively. There was no difference (P > 0.05) between control and frozen–thawed oocytes in the fertilization rate (88.0% vs. 93.1%). Some of the frozen–thawed oocytes cleaved and developed to blastocysts (44.0% and 11.2%), although the rates were significantly (P < 0.01) lower than those in control (71.7% and 35.0%). These results indicate that stepwise dilution of frozen–thawed oocytes improves the recovery of oocytes with normal morphology, and that the oocytes maintain the abilities to be fertilized and develop to blastocysts.