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

While vocalizations are widely recognized as indicators of emotional arousal across animal species, concurrent validation of changes in vocalisation structure using physiological and behavioural measures recorded simultaneously is still lacking in numerous species including sheep. Here, we examined the vocal expression of negative emotional arousal in sheep using both stress-related behaviours to quantify bodily activation and eye temperature measured via infrared thermography, as non-invasive indicators of arousal. 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, with complete separation. During full isolation, lambs exhibited increased 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 compared to large ones). Calls emitted in full isolation were characterised by higher frequencies, noisiness (higher chaos) and lower durations. When combining behavioural and physiological assessment of arousal and test they impact on spectro-temporal structure of vocalizations, we found that bodily activation, but not eye peak temperature, impacted the frequency distribution and noisiness of the calls. Call duration increased with eye temperature, but only in lambs exhibiting a bodily activation high enough and the mean of the second formant increased with eye temperature in smaller, but not in larger, lambs. Hence, lamb vocalizations indicate arousal and are correlated with bodily activation, while co-variations with physiological measures depended on behaviour and individual traits.