205 Glucose-6-Phosphate Dehydrogenaze Activity in Ovine Oocytes Is Associated With Pre-Implantation Embryonic Development in Vitro

Abstract

A large proportion of ovine oocytes fail to develop into viable embryos following maturation, fertilization, and culture in vitro. Accurate, fast, and noninvasive predictors of ovine oocyte quality are therefore in urgent need for oocyte selection before in vitro maturation (IVM). Recent studies have shown that oocyte competence can be predicted through the presence of the glucose-6-phosphate dehydrogenase (G6PD) enzyme, as indicated by brilliant cresyl blue (BCB), a dye that can be degraded by G6PD. Thus, oocytes that have completed their growth phase show decreased G6PD activity and exhibit cytoplasm with a blue colouration (BCB+), whereas growing oocytes are expected to have a high level of G6PD, which results in colourless cytoplasm (BCB–). The brilliant cresyl blue staining test, as a noninvasive intrinsic criterion, has been successfully used to identify the more competent oocytes in various species. Therefore, this study aimed to investigate whether BCB staining, as an indicator of G6PD activity, can be used to select developmentally competent ovine oocytes before IVM and thereby increase the efficiency of in vitro embryo production. Ovine ovaries were obtained from a local slaughterhouse and transported to the laboratory, where cumulus–oocyte complexes (COC) were recovered by slicing the ovaries. Only oocytes with one or more complete layers of unexpanded cumulus cells and a homogeneous cytoplasm were used. The COC were exposed to 26 mM BCB diluted in modified Dulbecco’s PBS for 90 min at 39°C in humidified air. After BCB exposure, the COC were examined under a stereomicroscope and divided into 2 groups: BCB+ (blue cytoplasm, low G6PD activity) and BCB– (colourless cytoplasm, high G6PD activity). Cumulus–oocyte complexes in the control group were incubated for IVM directly after selection, without exposure to BCB dye. After IVM, oocytes were subjected to IVF followed by embryo culture for 7 days (5% CO2, 39°C, humidified air). Results were analysed by a chi-square test, and P < 0.05 was considered statistically significant. The proportion of oocytes that cleaved by Day 2 after insemination was significantly (P < 0.05) higher for the control and BCB+ groups [67.3% (68/101) and 71.7% (81/113), respectively] than for the BCB– group [50.5% (46/91)]. Significant differences among groups were also observed on Day 7 after fertilization, when the embryos reached the blastocyst stage of development. The BCB+ group yielded a significantly (P < 0.05) higher proportion of blastocysts [34.5% (39/113)] than both the control [20.8% (21/101)] and BCB– [4.3% (4/93)] groups. In addition, the blastocyst rate of development in the control group was significantly (P < 0.05) higher than that for the BCB– group. In conclusion, results of this study show that selection of ovine oocytes based on G6PD activity through the BCB test can be used as an efficient predictor of in vitro embryonic developmental competence. This positive predictive parameter of oocyte quality may also be useful in increasing the efficiency of blastocyst production during in vitro embryo production procedures in the ovine.