Simple SummaryDuring the last decades, the detrimental effects of exposure to tobacco smoke on children and mothers have been emphasized. Conversely, not as much attention has been paid to the risks for pets, even though second-hand smoke can affect them too. Cotinine, the primary metabolite of nicotine, has recently been found in the serum and hair of dogs living with smoking owners. The purpose of this study was to investigate the possible transplacental crossing of cotinine in bitches exposed to passive smoke during pregnancy by measuring cotinine in amniotic fluid, newborn hair, and maternal hair and serum at birth. Cotinine was measurable in all matrices and samples collected during the Caesarean section, with higher concentrations in exposed dogs than those not exposed. These findings highlight the transfer of cotinine from mother to fetus and warn that owners should be more aware of possible adverse health consequences for puppies.AbstractActive and passive smoking in pregnant women is associated with perinatal morbidity and mortality risk, including abortion, preterm birth, low birthweight, and malformations. No data are available on intrauterine exposure to smoking during pregnancy in dogs. This study aimed to fill this gap by exploring the detectability and quantity of cotinine, the major metabolite of nicotine, in maternal (serum and hair) and newborn (amniotic fluid and hair) biospecimens collected at birth in dogs. For this purpose, twelve pregnant bitches, six exposed to the owner’s smoke and six unexposed, were enrolled. A further six non-pregnant bitches exposed to passive smoke were included to investigate the role of pregnancy status on cotinine uptake. Exposed dogs, dams, and puppies had greater cotinine concentrations than unexposed ones. Although without statistical significance, serum and hair cotinine concentrations were higher in pregnant compared to non-pregnant bitches, suggesting a different sensitivity to tobacco smoke exposure during gestation. The present results provide evidence for cotinine transplacental passage in the dog. It is conceivable that fragile patients such as pregnant, lactating, and neonate dogs may be more susceptible to the harmful effects of second-hand smoke exposure. Owners should be sensitized to the risk of smoke exposure for their pets.