pH7.4) supplemented with 20 units/ml penicillin and 20 units/ml streptomycin (Sigma, St. Louis, MO), and gassed with 5% O2, 5% CO2, and 90% N2 in a rotating culture system (B.T.C. Precision Incubator Unit, B.T.C. engineering, Cambridge, England, 36 rpm) for 2 h. After 2 h, treatment was initiated by transferring embryos into the same medium with or without 88 mM ethanol in isotonic buffer. The bottles were gassed for an additional 20 h with 5% O2, 5% CO2, and 90% N2, and then between 22 h and 46 h with 20% O2, 5% CO2, and 75% N2. The culture medium in alcohol and control cultures was replaced with fresh medium (with or without ethanol, respectively) 22 h after the start of the treatment. In this culture system, it was previously determined that the media alcohol concentration declined from 88 mM to 44 mM over the course of the experiment. Alcohol concentrations in this range (44-88 mM) have been commonly used in whole embryo cultures to generate FAS-related structural malformations [41,42,67] in multiple strains of mice [29], and are comparable to blood alcohol concentrations produced by in vivo doses of acute ethanol injections that produce teratogenic effects in mice during this
