Incubator Unit; B.T.C. Engineering, Cambridge, England; 36 rpm) at 37°C. After the pre-culture period (2–4 hr; maximum 4 hr), alcohol exposure was started by transferring the embryos into a medium containing 6 µl/ml of 95% ethanol (approximately 400 mg/dL, or 88 mM) throughout 44 hrs. Before the experiment, the medium alcohol levels in the setting were tested using an Analox alcohol analyzer (Analox Instruments USA, Lunenburg, MA). The control group was cultured in the medium with no ethanol. On Day 2, the culture medium was changed 22 hr after the start of the exposure with the same treatments described above. All cultures were terminated 44 hr from the beginning of treatment. A total of 27 embryos were used in the current; of which 9 were used for MeDIP analysis (control, ALC-NTC and ALC-NT, n = 3 each) in the same run (control, ALC-NTC and ALC-NT, n = 3 each) (see below), and 18 for embryo morphology (control, ALC-NTC and ALC-NT, n = 6 each). A comparable number of control embryos were always included in each batch of culture for each experiment; all arrays were performed in one batch. The concentration of ethanol in the medium over 24 hr was tested
