ABSTRACT: Horses have the ability to generate a remarkable repertoire of facial expressions, some of which have been linked to the affective component of pain. This study describes the facial expressions in healthy horses free of pain before and during transportation and social isolation, which are putatively stressful but ordinary management procedures. Transportation was performed in 28 horses by subjecting them to short-term road transport in a horse trailer. A subgroup (n = 10) of these horses was also subjected to short-term social isolation. During all procedures, a body-mounted, remote-controlled heart rate monitor provided continuous heart rate measurements. The horses' heads were video-recorded during the interventions. An exhaustive dataset was generated from the selected video clips of all possible facial action units and action descriptors, time of emergency, duration, and frequency according to the Equine Facial Action Coding System (EquiFACS). Heart rate increased during both interventions (p<0.01), confirming that they caused disruption in sympato-vagal balance. Using the current method for ascribing certain action units (AUs) to specific emotional states in humans and a novel data-driven co-occurrence method, the following facial traits were observed during both interventions: eye white increase (p<0.001), nostril dilator (p<0.001), upper eyelid raiser (p<0.001), inner brow raiser (p = 0.042), tongue show (p<0.001). Increases in 'ear flicker' (p<0.001) and blink frequency (p<0.001) were also seen. These facial actions were used to train a machine-learning classifier to discriminate between the high-arousal interventions and calm horses, which achieved at most 79% accuracy. Most facial features identified correspond well with previous findings on behaviors of stressed horses, for example flared nostrils, repetitive mouth behaviors, increased eye white, tongue show, and ear movements. Several features identified in this study of pain-free horses, such as dilated nostrils, eye white increase, and inner brow raiser, are used as indicators of pain in some face-based pain assessment tools. In order to increase performance parameters in pain assessment tools, the relations between facial expressions of stress and pain should be studied further.
