The degree of necrosis in dead pups was highly variable. Dead pups were not examined further. On PCD 35, areola/nipple number and location were noted for each (male and female) pup in a blinded fashion. Pups were weaned (females on PCD 43 and males on PCD 44) and housed in unisexual groups of two to three littermates. Pups were placed in cages numbered and arranged in treatment blocks according to the assignment of the mother. Pups were given ear punch identification marks according to the mother’s treatment, and picric acid marks to identify them within a litter.
After pups were weaned, dams were killed using a CO2 anesthetic overdose, the uterus was removed, and uterine scars were counted to assess pup rearing success relative to the number of fetal implantations. Postimplantation loss was calculated as
Beginning on PCD 46, female pups were examined daily for the vaginal opening. Pups were weighed on the day of vaginal opening. Beginning on PCD 61, male pups were examined for preputial separation (detachment of the prepuce from the glans penis). The prepuce was gently retracted far enough to note either the presence of a constriction at the base of the glans penis, or connective tissue that prevented the prepuce from being retracted to the base of the glans. Note that complete preputial separation (PPS) was distinguished from incomplete PPS in which portions of the prepuce remained attached to the glans, typically via a thread along the ventral midline of the phallus.