Changes in Saliva Analytes Correlate with Horses’ Behavioural Reactions to An Acute Stressor: A Pilot Study

SIMPLE SUMMARY: Emotionality is an individual characteristic defined as the propensity to respond to stress-inducing stimuli such as fear-inducing objects or social separation. Evaluation of emotionality in horses is important as it may impact their learning performance. Although emotionality is usually assessed by measuring behavioural patterns, biomarkers could provide additional information about stress response, especially with respect to its temporal dynamics. In this study, behavioural responses were measured as well as a panel of salivary biomarkers related to stress, including salivary alpha-amylase, lipase, total esterase, butyrylcholinesterase, adenosine deaminase, and cortisol, in riding horses after acute experimental stress (the sudden opening of an umbrella). We found significant changes in most of the salivary biomarkers evaluated after the induced stress, where increases in butyrylcholinesterase were more closely related to behavioural responses to acute stress and low salivary alpha-amylase values were more closely related to quietness behaviours. Therefore, this preliminary research provides information about the relationship between behaviour patterns and biomarkers of stress in saliva in horses, and opens the possibility of wider use of selected biomarkers in saliva, such as butyrylcholinesterase or alpha-amylase, for the evaluation of acute stress in horses. ABSTRACT: Acute stress induces an array of behavioural reactions in horses that vary between individuals. Attempts to relate behavioural patterns and physiological responses have not always given clear-cut results. Here, we measured the changes in a panel of salivary components: salivary alpha-amylase (sAA), lipase, total esterase (TEA), butyrylcholinesterase (BChE), adenosine deaminase (ADA), and cortisol, and their potential link with horses’ behaviours after acute stress. Saliva samples were collected in nine riding horses subjected to a test consisting of opening an umbrella. Saliva sampling was obtained at a basal time point in the stall (T1), in the test indoor arena (T2), at a time of stress (T3), and 30 min (T4) and 60 min (T5) later. The horses’ behaviour was recorded at T3 for 1 min. sAA, lipase, TEA, and BChE showed significant changes along time, increasing at T3 for BChE, and decreasing at T4 for sAA and BChE. Butyrylcholinesterase appeared to be the most reliable predictor of behavioural responses, as it correlated with the index of emotionality, of laterality, and the occurrence of alarm signals, while sAA decreased when horses expressed quieter behaviours. These first results bring promising lines for novel, more precise physiological markers of acute stress in horses that can bridge the gap between behaviour and physiology.