85 THE EFFECT OF 17a-ETHINYL ESTRADIOL EXPOSURE OF IN VITRO-CULTURED BOVINE MORULAE ON SUBSEQUENT EMBRYONIC DEVELOPMENT AND QUALITY

Research has shown that 17-a-ethinyl oestradiol (EE2), an important component of most oral birth-control pills, acts as a xeno-oestrogen after being released into the environment through urine and feces. Although this emphasizes the need for the evaluation of its toxicity, several oocyte and embryo-toxic effects have been reported (Beker-Van Woudenberg et al. 2012). Therefore, the aim of the present study was to evaluate the effect of EE2 exposure during bovine early embryonic development, specifically at the morula stage (18 h), on subsequent embryonic development and quality. Bovine cumulus–oocyte complexes (COC) from 2- to 6-mm-diameter follicles were matured in groups of 50 in 500 µL of TCM with 20 ng mL–1 epidermal growth factor (EGF) for 24 h and subsequently fertilized in groups of 100 in 500 µL of fertilization medium for 22h (5% CO2, 38.5°C). Presumptive zygotes were denuded and cultured in groups of ±25 in 50 µL of SOF with ITS (5 µg mL–1 insulin, 5 µg mL–1 transferrin, 5 ng mL–1 selenium) and 2% BSA, covered with mineral oil (5% O2, 5% CO2, 38.5°C). Subsequently, embryonic developmental stage was determined at 135 h post-insemination (p.i.). Sole embryos at morula stage were selected and randomly allocated to treatment groups (n) divided over 5 replicates: (1) Control (67), (2) solvent control: 0.1% ethanol (49), (3) 10 ng mL–1 EE2 (49), or (4) 10 µg mL–1 EE2 (63). The morulas were cultured individually in 30 µL of standard SOF medium supplemented with the desired concentrations ethanol or EE2 (5% O2, 5% CO2, 38.5°C) in 96-well half-area culture plates, without oil coverage for 18 h. Following exposure, embryos were cultured singly in standard culture medium for 2 more days. Subsequently, developmental competence was evaluated and blastocyst rates calculated (blastocyst rate = total blastocyst/number of grade 1 selected morula). Expanded (EB) and hatched blastocysts (HB) were fixed with 4% paraformaldehyde, and total cell number and apoptotic cell ratio were determined by DAPI and TUNEL staining (13 EB and 7 HB per treatment). Comparable blastocyst rates were obtained in all treatment groups: solvent control (77.8%), 10 ng mL–1 EE2 (69.0%), and 10 µg mL–1 EE2 (84.0%) compared with the controls (88.2%; P > 0.05; binary logistic regression). In addition, no significant effect of treatment could be found on total cell number or apoptotic cell ratio: solvent control (149.70 ± 23.47 and 3.46 ± 1.73), 10 ng mL–1 EE2 (154.75 ± 23.26 and 3.22 ± 1.35), and 10 µg mL–1 EE2 (150.50 ± 26.69 and 4.23 ± 1.85) compared with the controls (145.02 ± 24.71 and 3.07 ± 2.03; P > 0.05; two-way ANOVA). Although our results show no immediate statistical significant effect of short-term EE2 exposure during the morula stage in in vitro culture on subsequent blastocyst development and quality, additional research is necessary to find out if EE2 may affect gene-expression patterns, eventually resulting in still unknown embryotoxic effects that might turn up during later embryonic development.