166 THE EFFECT OF TEMPERATURE DURING STORAGE OF IN VITRO-MATURED BOVINE OOCYTES IN A HEPES-BUFFERED MEDIUM ON DEVELOPMENTAL COMPETENCE

Abstract

The objective of this study was to clarify the effect of the temperature during liquid storage in in vitro matured (IVM) bovine oocytes. IVM bovine oocytes were stored in Eppendorf tube containing 1 mL HEPES TCM-199 supplemented with 10% (v/v) new born calf serum at different temperatures (4°C, 15°C, 25°C, and 38.5°C) for 20 h. The developmental rates of stored and not stored (control) oocytes to the blastocyst stage, cell numbers in resultant blastocysts, and fertilization normality were evaluated after in vitro fertilization and in vitro culture. The ATP content, reduced glutathione (GSH) content, and apoptosis rates in oocytes were also determined in stored and control groups. At least 3 replicates were conducted for each experiment. The data were analysed by 1-way ANOVA followed by post hoc Fisher’s protected least significantly difference test. Percentage data were transformed to arc-sine before analysis. All of the storage groups (4, 15, 25, and 38.5°C groups, respectively) showed significantly lower blastocyst developmental rates (8.5, 14.9, 19.3, and 24.5%, respectively) compared with the control group (39.8%; P < 0.05). Within the storage groups, the 25°C and the 38.5°C groups exhibited the greatest rate of blastocyst formation. In contrast, the total cell number of the 38.5°C group was significantly lower than that of control group (P < 0.05), whereas that of the 25°C group was similar with the control group. The frequency of normal emission of the second polar body (2PB) was significantly greater in the control group compared with the storage groups (P < 0.05). The 2PB emission rate was significantly lower in the 38.5°C group compared with the 4°C group (P < 0.05) but not different from those of the 15°C and 25°C storage groups. The percentage of male pronuclear formation in the control group was significantly higher than those in the stored groups (P < 0.05) except for the 25°C group. During storage at 4°C, the ATP content was significantly decreased compared with the control group (1.3 v. 1.7 pmol; P < 0.05); however, in the 25°C and 38.5°C groups, the ATP content (2.0 and 1.9 pmol, respectively) was significantly higher than that in the control group (1.7 pmol; P < 0.05); whereas the 15°C group showed the same ATP level compared with the control group. Storage of oocytes for 20 h reduced the GSH content compared with the control group without storage (P < 0.05); however, there were no significant differences among storage groups. Annexin-V staining revealed increased incidences of early apoptotic oocytes in the 4°C and 15°C groups (P < 0.05) compared with other groups. In conclusion, based on the embryo developmental competence of stored oocytes and quality of resultant blastocysts, 25°C was determined as the most suitable temperature for temporal storage of matured bovine oocytes.

The study was supported by the NARO Institute of Livestock and Grassland Science, Japan (N32G4126), and the Royal Golden Jubilee-PhD scholarship (2.B.TS/53/F.2).