Acoustic markers of negative arousal in lambs: evidence from behavioural and eye thermal profiles

Abstract Vocalizations are known indicators of emotional arousal in animals, but validation using simultaneously collected physiological and behavioural measures remains limited to a few species. This study investigated sheep vocal expression of negative emotional arousal using stress-related behaviours and eye temperature as non-invasive arousal indicators. To this aim, twenty lambs underwent a short-term isolation test with two phases aimed at eliciting different levels of arousal in response to separation from conspecifics: partial isolation, where lambs maintained visual, acoustic and tactile contact with conspecifics through a fence, and full isolation (complete separation). During full isolation, lambs expressed higher bodily activation—spending more time running, jumping, and changing state behaviours—and produced more open-mouthed bleats (321 vs 27) than in partial isolation, validating higher arousal. Eye temperature also increased from partial to full isolation (however only in small lambs and not large ones). Calls emitted in full isolation were characterised by higher frequencies, were less tonal (more chaos) and had shorter durations. When combining behavioural and physiological assessment of arousal and testing their impact on the spectro-temporal structure of vocalizations, we found that bodily activation, but not eye peak temperature, impacted on the frequency distribution and tonality of the calls. Call duration increased with eye temperature, but only in lambs expressing high bodily activation, while the mean of the second formant increased with eye temperature in smaller but not larger lambs. Overall, lamb vocalizations indicate arousal and are correlated with bodily activation, while co-variations with physiological measures depended on behaviour and individual traits. Highlights Animal calls indicate emotional arousal, but few species have validated measures Lamb calls became higher in frequency, less tonal and shorter with negative arousal Calls indicated emotional arousal and were correlated with bodily activation Changes in calls with eye temperature depended on behaviour and individual traits Competing Interest Statement The authors have declared no competing interest. Footnotes Emails: nc.zoemiot{at}gmail.com, prenaud{at}irit.fr, silvana.mattiello{at}unimi.it, avelyne.villain{at}inrae.fr ↵* co-senior authors We have revised the manuscript to improve clarity, methodological transparency, and theoretical framing. In the Materials and methods section, we clarified the experimental design, including sample composition, selection criteria, testing procedures, and technical aspects of data collection (e.g., infrared thermography), thereby enhancing reproducibility. The Introduction has been revised to better situate the study within the existing literature. Finally, we have more explicitly addressed the study's limitations while highlighting its contribution as a meaningful and exploratory step that can inform future research.