The ultrasonic vocalizations (USVs) of house mice (Mus musculus) have a surprisingly complex structure and have been actively studied as a model for applied questions in biomedical science. Despite this, the functional significance of different USV types and their use during natural social behaviour in diverse groups remains unclear. In this pilot long-term study, we examined the ultrasound activity of female mice housed in stable groups within an enriched environment, focusing on how behavioural context influences USV time-frequency characteristics, call type occurrence, and nonlinear phenomena. Our findings show that USVs are mainly produced in bouts, and bouts containing ten or more variable calls always accompanying direct social interactions. USV variables and types varied markedly depending on the interaction context. During aggressive encounters, USVs and entire bouts became longer, vocalization time and frequency modulation increased, while peak and minimum fundamental frequencies decreased. Additionally, aggressive context was characterized by a higher prevalence of multi-component and complex-low USVs (with peak frequency < 50 kHz), and increased nonlinear phenomena. These results suggest that USV features closely reflect emotional arousal (intensity) and probably also valence (positive/negative) in female mice during social hierarchy formation and maintenance. Notably, the relationship between arousal and nonlinear vocal phenomena in mice USVs follows patterns observed in audible vocalizations of other mammals (increase with arousal). In the future, complex-low USVs could potentially serve as non-invasive indicators of negative emotional expression in groups of female mice. This opens new possibilities for acoustic home-cage monitoring aimed at welfare assessment and other applied uses.
