139 INTRACELLULAR GLUTATHIONE CONCENTRATION AND IN VITRO DEVELOPMENT OF BOVINE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE

Abstract

During in vitro development of bovine embryos, a large proportion of embryos fail to develop to the blastocyst stage. Brilliant cresyl blue (BCB) stain determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes. It was previously reported that BCB positive oocytes are better developed to the blastocyst stage than BCB-negative oocytes for IVF (Alm et al. 2005 Theriogenology 63, 2194–2205). The objective of this study was to compare the intracellular glutathione (GSH) level and the developmental competence of bovine oocytes selected by BCB. Bovine ovaries were obtained at a local slaughterhouse and cumulus–oocytes complexes (COCs) were collected from follicles. In this study, we only used COCs with a compact cumulus investment. The concentration of BCB was reported in a previous study (Rodriguez-Gonzalez et al. 2002 Theriogenology 57, 1397–1409). The COCs were exposed to 26 µm of BCB for 90 min at 38.5°C in humidified air. Treated oocytes were divided into BCB-positive (colored cytoplasm) and BCB-negative (colorless cytoplasm). The selected COCs (BCB-positive and negative COCs) were matured in TCM-199 supplemented with 10% FBS, and then inseminated using frozen semen (1 × 10⁶ cells mL^–1). Subsequently, presumptive zygotes were cultured in vitro in CR1-aa medium supplemented with 0.3% BSA. After 3 days of IVC, cleaved embryos were transferred to CR1-aa medium supplemented with 10% FBS and cultured for an additional 4 days at 38.5°C, 5% CO₂ in air. Before (geminal vesicle (GV) stage) and after IVM (metaphase II (MII) stage), the COCs were denuded mechanically to prepare the samples for GSH assay, and then stored at –70°C until used. In the GV- and MII-stage oocytes, intracellular concentration of GSH was measured according to Mertens et al. (2005 Reprod. Domest. Anim. 40, 126–130). This experiment was replicated at least three times. GSH concentrations were analyzed by Student's t-test and the blastocyst formation rates were analyzed by chi-square analysis using the SAS 8.01 program (SAS Institute, Inc., Cary, NC, USA). Differences among treatment effects were considered significant at P < 0.05. The GSH concentration of BCB-negative oocytes (4.5 ± 0.7 pmol/oocyte; n = 105) was significantly (P < 0.05) lower than that of BCB-positive oocytes (7.3 ± 0.7 pmol/oocyte; n = 78) before IVM. After 24 h IVM, no differences in the GSH concentration were observed between the BCB-positive group (13.5 ± 0.5 pmol/oocyte; n = 85) and the BCB-negative group (12.6 ± 2.6 pmol/oocyte; n = 110). However, the percentage of embryos developed to the blastocyst stage in the BCB-positive group was significantly higher compared with that of the BCB-negative group (23.5 ± 3.6 v. 9.8 ± 2.1%, respectively; P < 0.05). Here, we concluded that intracellular GSH plays an important role in embryonic development to the blastocyst stage in vitro.