All experimental protocols were approved by the Marmara University Animal Care and Use Committee (Project no: SAGC-DRP-031210-0275). Eighteen female and six male adult Wistar albino rats (250–300 g) were obtained from the Marmara University (MU) Animal Centre (DEHAMER). They were randomised into three groups of eight animals (six females, two males) each and treated as follows: Group 1 (control group) was given tap water with a fluoride concentration <0.05 mg/L, group 2 (moderate-fluoride group) was given fluoride as NaF 30 mg/L in drinking water, and group 3 (high-fluoride group) was given fluoride as NaF 100 mg/L in drinking water. The animals were maintained in the laboratory at 22 ± 2 °C with a 12-h dark/light cycle and relatively humidity of 60– 70 % and were fed a standard diet throughout the experiment. Drinking water was available ad libitum.

The animals were acclimatised to the laboratory conditions for 1 week before the experiments began and were housed in plastic cages with stainless steel grill tops. When the experiment began, female and male rats were caged together for mating. The animals in the experimental groups received fluoridated water during the mating and breeding periods. The presence of a vaginal plug was taken as an indicator of pregnancy. When a plug was observed, that female rat was placed in a cage marked with the date of breeding and was considered pregnant. The pregnant rats in the experimental groups continued to receive fluoridated water during gestation and until the 21st day after delivery (the pups were exposed to fluoride via the placenta and lactation). After weaning, 63 male pups born in the control group and two experimental groups were selected randomly and divided into nine subgroups (n = 7), which received the same water regimens as the groups into which they were born. The pups were followed for 1, 3, or 5 months after birth. The average water consumption and body weight of all pups were recorded daily. At the end of each experiment, the rats were anesthetised with an intraperitoneal injection of ketamine and chlorpromazine 100 mg/kg and sacrificed by cardiac puncture.