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

Vocalisations are known indicators of emotional arousal in animals, but their assessment 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 (11 females, 7 males and 2 of unknown sex) underwent a short-term isolation test with two phases aimed at eliciting progressive and increasing levels of arousal in response to separation from conspecifics: partial isolation, where lambs maintained visual, acoustic and tactile contact with conspecifics through a fence, followed by 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 than in partial isolation (321 vs 27 in total), validating higher arousal. Eye temperature also increased from partial to full isolation (however only in lambs classified as short and not in tall 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 vocalisations, 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 shorter but not taller lambs. Overall, lamb vocalisations indicated arousal and were correlated with bodily activation, while co-variations with physiological measures depended on behaviour and individual traits.