In the planning phase of the study, we assumed that the modification of horse environment from wild setting to paddock restraining could have had a direct influence on both horse behavior and stress parameters. Unexpectedly, the mares did not change any behavioral aspect when observed in collective paddocks during the event compared to wild environment.
A different result was obtained recently by Ruet and collaborators 20 that studied the reverse of the present study. Individual box housing is supposed to potentially compromise animal welfare: for this reason, a common practice is to offer horses a short-term period on pasture with conspecifics to alleviate the impact of long-term deprivation triggered by box shelter. When on pasture, horses increased the expression of natural behaviors such as locomotion, exploration and social behaviors which may have not been easily expressed when stabled in boxes; in addition, they did not display any stereotypies and aggressive behaviors towards humans. Interestingly, during the initial days on pasture horses were unresponsive to the environment in a significant manner than what was observed when stabled in individual boxes. After a proper period of adaptation, such behavior reverted to the level previously observed when horses were in boxes. Authors concluded that pasture can positively influence the welfare state of horses usually stabled individually, but also that several days of adaptation are needed, probably due to the novelty of the environmental and the needing to establish social conditions.20
Conversely from Ruet and collaborators conclusions, in the present study the behavioral evaluation of horses did not change when passing form wild to paddock. A possible explanation to our finding could be that the mares were moved into collective paddocks and not in single boxes, maintaining their herd.
Differently from behavioral observations, objective salivary parameters of horse stress namely BChol and Cortisol significantly changed during the study, while sAA remained unchanged. Regarding sAA, it is considered a potential salivary stress marker of acute and chronic stress in people especially in those sensitive to a negative social assessment.28,29 In horses experimenting an acute stress, for example immediately after a sudden scare, sAA increase, to return at lower level after few minutes.30 Since in our study horses were not exposed to an acute stress, the lack of change we observed could be due to the lack of reliability of sAA in evaluating non-acute stress in horses. Butyrylcholinesterase, a serine hydrolase biochemically related to the cholinergic enzyme acetylcholinesterase, is widely distributed in the nervous system, and it seems to be involved in neural function serving as a co-regulator of cholinergic transmission. The function of BChol in saliva is not fully understood but it has been associated with acute stress in humans, rats, and pigs.31 In our study, Bchol salivary concentrations were significantly higher in saliva samples collected in the wild compared to samples collected on the same mares restrained in paddocks. A similar trend was observed in salivary cortisol showing the highest mean values when the mares stayed in the wild. Salivary cortisol represents the unbound biologically active fraction of total plasma cortisol, that is mainly bound to carrier proteins, thus reflecting the hypothalamic–pituitary–adrenal (HPA) axis activation.32,33
The unexpected findings of the present study can be explained by an adaptive approach called “coping strategy” that defines the behavioral and physiological struggles of animal to face a new, potentially stressful situation. The coping strategy attracts the interest of different authors dealing with applied horse welfare and behavior. 34 When a change in environment occurs, as in the present study, animals need to cope with their environment using both behavioral and physiological stress responses. Different parameters are related to such coping strategy and may be monitored over time such as changes in form and frequency of behavioral response, changes in animals-based index (such as heart rate, body temperature, hormones, neutrophils and lymphocytes ratio, and other blood indicators). In this context, the implementation and study of novel biomarkers as sAA, salivary cortisol, and Bchol that can be easily measured and may serve as indicators over time, supporting in the understanding of individual adaptive capacity to stressful events. 35
Environment has a major impact on horse welfare and behavior (influencing for example the chance of feeding, protection from weather condition, and sleep-awake cycle)36 and horse may reveal specific behavior patterns to adapt to challenging situations especially in man-made environment 37.
The results of the present study provide new knowledge in the multidimensional approach toward horse behavior understanding, hopefully providing valuable information for specific, possibly challenging situations. Animals living in man-made environments (as paddocks in the present study), are believed to be influenced by different kinds of stressor agents that causes adaptive responses.38 On the other hand, it could also be hypothesized that although life in the wild allows horses to shows their natural behavior in freedom, at the same time this could represents a stress factor, linked to the need for managing hierarchical relationships within the herd, and the exposure to feral animals, predators, and weather changes, might explain these findings.
We aimed to appraise the relationship between behavioral and physiological factors involved in the horse stress response and, surprisingly, salivary biomarkers revealed that stress level was higher in the wild.
It should also be underlined that a large heterogeneity between the studies exists, because of the lack of standardized methods for measuring salivary cortisol, heterogeneity of studied populations and the bias of risk factors. In human being, for example, adolescents with severe symptoms of depression have not different concentration of salivary cortisol when compared to adolescents with mild symptoms of depression but had a significantly higher ratio of morning to evening levels of salivary cortisol.39 This should be taken in account in future studies also in veterinary research. The findings of the present study are related to a typical Italian horse breed and the same evaluation considering different breeds/attitudes is warmly welcome, to provide more details to interpret horse behavior. Exploring coping strategies in horses and in any captive animal can be successful when a multidimensional approach including behavioral, neural, hormonal, and hematological measures is considered. Knowledge on stress and related coping styles can provide valuable information to prevent potentially challenging situations, in order to maximize animal welfare.