Int. J. Environ. Res. Public Health 2010, 7 2036 from blood cells by centrifugation at 1,500 × g for 10 minutes and stored frozen at −20C until assayed for estradiol-17β (E2). Ovulation Induction, Embryo Recovery and in vitro Culture Ovulation was induced at approximately 48 hours post PMSG in each of the remaining treated and control mice with an IP injection of 5 IU of human chorionic gonadotropin (hCG). Induced mice were subsequently placed with proven breeder males in a 1:1 ratio. The following morning, female mice were inspected for the presence of vaginal plugs as evidence of mating. Five mated females in the HU treatment or control group were sacrificed by CO2 asphyxiation immediately post detection of vaginal plugs (approximately 14–16 h post hCG), oviducts excised and flushed with Whitten’s medium (WM; [13]) containing 0.1% hyaluronidase and each flushing examined with a Nikon TMD inverted microscope at 200× magnification for the presence of cumulus masses. Each oviductal flushing was incubated at room temperature for approximately 10 minutes to permit the dispersion of cumulus cells from ovulated ova (one-cell embryos or non-fertilized oocytes). An ovum was considered fertilized if it contained 2 pronuclei in their cytoplasm and 2 polar bodies in the perivitelline space. Fertilized and non-fertilized ova were separated from the cumulus cells using pulled pipettes with the aid of an Olympus dissecting microscope. The total number of fertilized and non-fertilized oocytes per mouse was counted and considered as ovulation rate. The remaining mated mice were sacrificed at 26 to 28 hours after detection of vaginal plugs, ovaries excised and dissected free of fat tissue and weighed. Oviducts were removed and flushed with WM [13] lacking bovine serum albumin (BSA). All procedures involving animal care, anesthesia, euthanasia, and tissue collection were approved by the Meharry Medical College Institutional Animal Use and Care Committee. The flushings were examined with a Nikon TMD inverted microscope at 200× magnification for the presence of 2-cell embryos. Figure 1 depicts a photomicrograph of a normal 2-cell mouse embryo. Normal 2-cell mouse embryos, each defined as an embryo with two blastomeres of equal size with two polar bodies in the perivitelline space and encompassed by an intact zona pellucida were pooled among mice within treatment and washed in several droplets of WM containing 1% BSA (Sigma Chemical Co., St. Louis, MO). Figure 1. Normal 2-cell embryo with two equal blastomeres (B), one of two polar bodies (PB) encompassed by an intact zona pellucida (ZP; magnification = 400X). PB