During subsequent observations, the numbers of delivered pups were noted. Animals showing vaginal bleeding or having pups scattered throughout the cage and uncleaned (blood on the pups and dam) or placentas in the bedding were considered to be in the process of delivery. In addition, signs of dystocia were also noted. To reduce observer effects on parturition the litter and nests were not disturbed, and the dams or pups were not handled.
Anogenital distance and body weight were measured in male and female pups on PCD 25 (PND 3). Measurements were conducted in a manner in which the observer was blind to the identity of the pups. Pups were manually restrained under a Leica MZ6 dissecting scope. Skin between the phallus and tail-base was extended maximally (using care not to stretch the skin) and anogenital distance measurements were made to the nearest 0.1 mm using a 10-mm ocular graticule. Scope magnification at 15X (1.5 X 10) was calibrated using a 1-mm stage micrometer with 0.01-mm divisions. After PCD 25, dead pups were removed from the mother’s cage daily until the time of weaning. Partially cannibalized pups were counted as dead.