
TY  - JOUR
A1  - Hamawaki, A. 
A1  - Hamano, S. 
A1  - Yoshikawa, M. 
A1  - Matsukawa, K. 
T1  - 117 EFFECT OF STEPWISE DILUTION ON THE VIABILITY OF FROZEN&#8211;THAWED BOVINE OOCYTES MATURED <i>IN VITRO</i>
JF  - Reproduction, Fertility and Development
JO  - Reprod. Fertil. Dev.
Y1  - 2006
VL  - 19
IS  - 1
SP  - 176
EP  - 176
KW  - 
UR  - https://doi.org/10.1071/RDV19N1AB117 
N2  - The purpose of this study was to evaluate the effect of stepwise dilution on the viability of frozen&#8211;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&#8211;thawed oocytes (n = 145) with four-step dilution were fertilized and cultured in vitro (Kuwayama 1992 J. Reprod. Fert. 96, 187&#8211;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&#8211;thawed oocytes in the fertilization rate (88.0% vs. 93.1%). Some of the frozen&#8211;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&#8211;thawed oocytes improves the recovery of oocytes with normal morphology, and that the oocytes maintain the abilities to be fertilized and develop to blastocysts. 
ER  -

