Stress in horses: The story told by saliva

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Cortisol levels in saliva are often used in studies to determine if horses are stressed, but researchers in France and Spain have identified other options.

María Contreras-Aguilar and her colleagues found that levels of the enzyme butyrylcholinesterase in saliva showed particular promise.

Not only did levels of butyrylcholinesterase rise with acute stress, but the biggest rises seen in the horses used in the study were in those that showed the most behavourial signs of stress.

“Butyrylcholinesterase appeared to be the most reliable predictor of behavioural responses,” the study team reported in the open-access journal Animals.

The researchers noted that while emotionality is usually assessed by measuring behavioural patterns, biomarkers could provide additional information about stress response.

In their pilot study, the researchers set about measuring the behavioural responses of riding horses to a fright, and how that affected a series of salivary biomarkers related to stress, including salivary alpha-amylase, lipase, total esterase, butyrylcholinesterase, adenosine deaminase, and cortisol.

Nine riding horses, aged 5 to 22, were used, with saliva collected via a gauze-filled plastic tube that had a hole through it to allow saliva in. To collect a sample, it was worn like a bit for one minute, being clipped to the halter.

Saliva was obtained for analysis when the horse was standing in its stall, after it had been led to the indoor arena for the test, at the time of the stress event (when a person suddenly opened an umbrella in the arena), and 30 minutes and 40 minutes later.

Each horse’s behaviour was recorded for a minute from the time they witnessed the umbrella opening. Common behavioural signs of stress were assessed and an index of emotionality was applied to determine the level of arousal for each horse.

The researchers found significant changes in most of the biomarkers evaluated after the induced stress.

Butyrylcholinesterase was assessed as the most reliable predictor of behavioural responses, as it correlated with the index of emotionality and the occurrence of alarm signals.

They also found that low salivary alpha-amylase values were more closely related to quietness behaviours.

“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 alphaamylase, for the evaluation of acute stress in horses,” they concluded.

They added that although there is evidence that butyrylcholinesterase is directly released after acute stress in other species, further studies are necessary to assess the possible physiological mechanisms relating to its increased levels in saliva and stress-related behaviour in horses.

The study team comprised Contreras-Aguilar, Séverine Henry, Caroline Coste, Fernando Tecles, Damián Escribano, Jose Cerón and Martine Hausberger. They are variously affiliated with the University of Rennes in France, the Campus of Excellence Mare Nostrum in Spain, and the University of Murcia, also in Spain.

Changes in Saliva Analytes Correlate with Horses’ Behavioural Reactions to An Acute Stressor: A Pilot Study
María D. Contreras-Aguilar, Séverine Henry, Caroline Coste, Fernando Tecles, Damián Escribano, Jose J. Cerón and Martine Hausberger.
Animals 2019, 9(11), 993;

The study, published under a Creative Commons License, can be read here.


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