Avy mice were obtained from a colony that has been maintained with sibling mating and forced heterozygosity for the Avy allele for over 220 generations, resulting in a genetically invariant background [Waterland and Jirtle, 2003 (link)]. Virgin a/a dams, 6 weeks of age, were randomly assigned to one of four phytoestrogen-free AIN-93G diets (diet 95092 with 7% corn oil substituted for 7% soybean oil; Harlan Teklad, Madison, WI): (1) standard diet (n = 11 litters, 86 total offspring, 39 Avy/a offspring); (2) standard diet supplemented with 50 ng BPA/kg diet (n = 14 litters, 107 total offspring, 48 Avy/a offspring); (3) standard diet supplemented with 50 μg BPA/kg diet (n = 9 litters, 67 total offspring, 32 Avy/a offspring); (4) standard diet supplemented with 50 mg BPA/kg diet (n = 13 litters, 91 total offspring, 45 Avy/a offspring). All diet ingredients were supplied by Harlan Teklad except BPA, which was supplied by NTP (National Toxicology Program, Durham NC). The mg dosage was formulated to be an order of magnitude lower than the dietary administered maximum nontoxic threshold in rodents (200 mg/kg BW/day) [Takahashi et al., 2003 (link)], whereas the ng and μg BPA dosages were used to potentially capture the physiologically relevant range of human exposure.
Following 2 weeks on their respective diets, at 8 weeks of age a/a virgin dams were mated with Avy/a males, 7–8 weeks of age. All animals were housed in polycarbonate-free cages and provided ad libitum access to diet and BPA-free water. The dams remained on the assigned diets throughout pregnancy and lactation. At postnatal day 22 (d22), a/a and Avy/a offspring were weighed and tail-tipped. In addition, at d22, a single observer visually classified Avy/a offspring coat color phenotype into one of five categories based on proportion of brown fur: yellow (<5% brown), slightly mottled (between 5 and 40% brown), mottled (~50% brown), heavily mottled (between 60 and 95% brown), and pseudoagouti (>95% brown). Tail tissue was collected for analysis from all offspring.
Animals used in this study were maintained in accordance with the Guidelines for the Care and Use of Laboratory Animals [Institute of Laboratory Animal Resources, 1996] and were treated humanely and with regard for alleviation of suffering. The study protocol was approved by the University of Michigan Committee on Use and Care of Animals.
Following 2 weeks on their respective diets, at 8 weeks of age a/a virgin dams were mated with Avy/a males, 7–8 weeks of age. All animals were housed in polycarbonate-free cages and provided ad libitum access to diet and BPA-free water. The dams remained on the assigned diets throughout pregnancy and lactation. At postnatal day 22 (d22), a/a and Avy/a offspring were weighed and tail-tipped. In addition, at d22, a single observer visually classified Avy/a offspring coat color phenotype into one of five categories based on proportion of brown fur: yellow (<5% brown), slightly mottled (between 5 and 40% brown), mottled (~50% brown), heavily mottled (between 60 and 95% brown), and pseudoagouti (>95% brown). Tail tissue was collected for analysis from all offspring.
Animals used in this study were maintained in accordance with the Guidelines for the Care and Use of Laboratory Animals [Institute of Laboratory Animal Resources, 1996] and were treated humanely and with regard for alleviation of suffering. The study protocol was approved by the University of Michigan Committee on Use and Care of Animals.
