Young, L., McCallum, R., Perreault, M. et al. 2024. Opportunistically using a Chronic Unpredictable Stress study to investigate ‘inactive-but-awake’ behaviour as a potential welfare indicator in laboratory rats. Applied Animal Behaviour Science 274, 106249.

Being awake but motionless, inactive-but-awake (IBA), has been suggested to reflect low arousal negative affect in several species. This preliminary study investigated IBA in rats exposed to Chronic Unpredictable Stress (CUS), opportunistically (to comply with the 3Rs) during biomedical research modelling depression. Twenty individually-housed Wistar females underwent a 6-week CUS protocol involving daily stressors (e.g. cold, food deprivation). Half, housed on high shelves and dosed with potential antidepressant, were assumed relatively less stressed by the CUS (sub-group ‘RLS’); half, on more anxiogenic low shelves and dosed with vehicle, were assumed relatively more stressed (‘RMS’). If IBA reflects negative affect, then IBA should be increased by CUS duration, and by being RMS rather than RLS: predictions tested by observing rats in their homecages during CUS Weeks 0, 2 and 4 (blind to treatment, sub-group and hypothesis). Assumptions that negative affect increased with sustained CUS, and being RMS compared to RLS, were checked via the rat-specific stress indicator, chromodacryorrhea (ocular porphyrin). Facial/postural changes were also assessed, in exploratory attempts to identify particularly welfare-sensitive sub-types of IBA. Additionally, we evaluated stereotypic behaviour: a hyper-active response to negative states that mouse data suggest may be an alternative to waking inactivity. We found that chromodacryorrhea was an easy-to-score cageside welfare indicator. Chromodacryorrhea was also highly sensitive to CUS (increasing with very large effect sizes). IBA increased with CUS duration, alongside chromodacryorrhea (the two significantly covarying in two of three observation weeks). Thus as predicted, IBA increased, paralleled by physiological stress, over weeks of aversive experience. RMS rats also showed more chromodacryorrhea than RLS, supporting the designation of these sub-groups as respectively relatively more versus less stressed. This group difference was not mirrored by IBA, however: a false negative result for this potential welfare indicator. Turning to sub-types of IBA, forms involving ocular squinting increased with CUS, while those involving hunched postures or backward-pointing ears did not. Furthermore, IBA involving squinting seemed higher in RMS than RLS rats. Stereotypic behaviour, in contrast, decreased with CUS, and negatively covaried with IBA, suggesting that hypo- and hyper-activity are alternative responses. Overall, these preliminary results thus suggest that IBA, especially sub-types involving ocular squinting, can indicate poor welfare in rats. This needs replicating with negative controls, male subjects, and other types of challenge, but cautiously adds to growing evidence that particular forms of inactivity can indicate negative affect. Welfare science/biomedical collaborations can also advance welfare understanding without involving additional animal-use.