CO2 gas. The gravid uterus was removed. Decidual tissues and the Reichert membrane were removed carefully and immediately immersed in the PBS containing 4% fetal bovine serum (Sigma, St. Louis, MO), leaving the visceral yolk sac and a small piece of the ectoplacental cone intact. Three embryos bearing 3–5 somites (E8.25 embryo) were placed in a sterile bottle (20 ml) containing a culture medium that consisted of 70% immediately centrifuged heat-inactivated rat serum (Harlan Sprague-Dawley, Inc.,) and 30% PB1 buffer (137 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 8 mM Na2HPO4, 1.47 mM KH2PO4, 0.9 mM CaCl2, 5.6 mM glucose and 0.33 mM sodium pyruvate; pH 7.4). Embryos were then supplemented with penicillin and streptomycin (20 units/ml and 20 microgram/ml, respectively; Sigma). Bottles were gassed at 0 to 22 hr with 5% O2, 5% CO2 and 90% N2 and at 22 to 44 hr with 20% O2, 5% CO2 and 75% N2 in a rotating culture system (B.T.C. Precision 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
