*Article* **Effects of Ethanol Exposure during Lactation on Ultrasonic Vocalizations of Rat Pups upon Their Isolation: Increase in Pup Distress Calls †**

**Mohd. Ashik Shahrier 1,\* and Hiromi Wada <sup>2</sup>**


**Abstract:** Recording ultrasonic vocalizations (USVs) is a highly sensitive tool to study the dam–pup social relationships, and USV recordings have been used to study the effects of ethanol on pups. Gestational effects of ethanol on the emission of USVs in rat pups have been studied in our previous research. In the present study, the effects of ethanol given to dams during lactation on the acoustic parameters of USVs emitted by isolated pups were examined. Ethanol was administered to dams from postnatal days (PNDs) 5–21. From PNDs 11–21, the high- and low-ethanol-treated dams were exposed to ethanol-containing water (*v*/*v*) at concentrations of 30% and 15%, respectively. Tap water without ethanol (0%) was provided to the control dams. The pups in all three ethanol-treated groups were separated from the dam and littermates on PNDs 4, 8, 12, and 16, and USVs produced by the pups were recorded for 5 min. It was found that elevated distress USVs with longer duration and higher percentage of frequency modulations were displayed by the pups from the high-ethanol dams. Alterations in USVs were particularly evident in the pups with a reduced body weight at PND 12. This effect might be because high-ethanol dams showed significantly lower intake of higher ethanol-containing water, and consequently, produced lower amount of milk, as well as exhibited poor maternal care. Insufficient maternal care and malnutrition resulted in pup growth retardation and increased mortality rate in the high-ethanol group, which were not observed in the low-ethanol or control pups. Accordingly, the pups in the high-ethanol group experienced elevated negative emotionality during isolation from their dam and increased emission of USVs. Longer duration and increased frequency modulation of pup USVs are expected to be noticed by the dam and to initiate/increase proper maternal care. It is concluded that ethanol given to lactating mothers has more serious consequences on pup development than the gestational ethanol exposure, and has more harmful effects on pups.

**Keywords:** dam–pup interaction; ethanol; pup isolation; rat pup; ultrasonic vocalization
