Abstract: Horses have always been animals used for companionship, work, transportation, and performance purposes over the history of humanity; there are different ways of managing horses, but studies on how horse welfare is influenced by different activities and managements are scanty. Understanding how the management, the environment, and the different uses of horses can affect the level of stress and well-being is important not only for people associated with horses. Three groups of horses with different management, environments, and activities were selected: (1) stabled horses ridden frequently, (2) horses that perform public order service under the Italian state police, and (3) free-ranging horses. Cortisol analysis was carried out on horsehair samples using liquid chromatography coupled to hybrid orbitrap high-resolution mass spectrometry (LC-HRMS/MS), a laboratory technique used for the first time to quantify horsehair cortisol. The selection of horses to be included in the three groups was carried out by including only subjects with positive welfare assessment in accordance with the horse welfare assessment protocol (AWIN). These analyses demonstrated that the cortisol levels detected in the horsehair of free-ranging animals were significantly higher compared to those detected in stabled and working horses. These results may have been a consequence of complex environmental, managerial, and behavioral factors, which should be worth further investigation

Abstract: Glucocorticoids (GCs; i.e. cortisol/corticosterone) are a central component of the stress response and thus their measurement is frequently used to evaluate the impact of stressful situations. Their metabolites from faeces of various animal species are more and more taken as a non-invasive aid to assess GC release and thus adrenocortical activity. The current literature review includes an extensive collection (1327 papers) and evaluation (see also Supplementary Tables) of the literature on faecal cortisol/corticosterone metabolite (FCM) analysis published to date. It aims at giving reference for researchers interested in implementing FCM analysis into their study or seeking to improve such methods by providing background knowledge on GC metabolism and excretion, conveying insights into methodological issues and stating caveats of FCM analysis and by highlighting prerequisites for and some examples of a successful application of such methods. Collecting faecal samples and analysing FCMs may appear simple and straightforward, but researchers have to select and apply methods correctly. They also need to be aware of the many pitfalls and potentially confounding factors and, last but not least, have to carefully interpret results. Applied properly, measurement of FCMs is a powerful non-invasive tool in a variety of research areas, such as (stress) biology, ethology, ecology, animal conservation and welfare, but also biomedicine.

Abstract: Abstract Stress is a generic concept describing the body's reaction to external stimuli, including both physiological and psychological factors. Therefore, by definition, the assessment of psychological stress in the exercising horse encompasses the problem of teasing apart the psychological and physiological factors both of which result in stress responses. The present study reviews the existing literature on various measures of stress taken specifically in the context of equitation science. Particular attention has been paid to short-term effects, and commonly used measurements of short-term stress include heart rate, a number of heart rate variability parameters, blood or saliva cortisol levels, eye temperature, and various behaviour parameters including in particular behaviour patterns presumably indicative of conflict with the rider's/trainer's aids. Inspection of the individual studies' results revealed that disagreement between these different measures of stress is commonplace. For physiological parameters, the largest proportion of agreement (i.e. both parameters simultaneously indicated either higher, insignificant or lower stress compared to a control treatment) was found for heart rate and heart rate variability parameters, while generally limited agreement was found for cortisol. It appears that cortisol levels may not be particularly useful for assessing/assessment of the valence of a situation in the exercising horse as cortisol levels are predominantly linked to activation and exercise levels. Although heart rate variability parameters reflect in theory more closely sympathovagal balance compared to cortisol levels, great care has to be taken regarding the use of appropriate time-frames, appropriate raw data correction methods as well as the use of appropriate equipment. In spite of its wide-spread and apparently successful use, popular equipment may in fact not be accurate enough under field conditions. Eye temperature is another promising parameter for assessment of psychological stress, but the technique is likewise susceptible to application errors. Given the high susceptibility of physiological parameters to errors at various experimental stages, behavioural rather than physiological parameters may in fact provide more accurate measures of valence when conducting experiments in the exercising horse. Behavioural parameters that appear to be particularly practical in assessing stress in ridden horses' behaviour are associated with frequencies of behaviour indicative of conflict. However, while increased frequencies of are a good indicator of stress, the absence of conflict behaviour does not provide proof of the absence of stress due to the possible occurrence of conditions such as Learned Helplessness. In future studies, the above issues should be taken into consideration when designing experiments to assess psychological stress in ridden horses.

Abstract: Stress in humans has been reported to be associated with a decrease in the salivary immunoglobulin A (s-IgA) levels enabling the possible use of s-IgA to assess stress. Prolonged stress, if reliably assessed in a non-invasive manner, may be used to assess animal welfare. This study analysed groups of dogs undergoing physical and temperamental training and s-IgA levels were measured by rocket immunoelectrophoresis in prospective samples. Behavioural assessment was carried out and cortisol levels in saliva were measured by ELISA. A significant negative correlation (P < 0.007) between the logarithmic cortisol concentrations and s-IgA levels in saliva was recorded. The behavioural assessment of the dogs agreed well with the biochemical markers. It is concluded that IgA levels in saliva may be a useful marker of dog well-being and that stress results in decreased s-IgA levels.

Abstract: In order to improve the housing conditions of stallions in individual boxes by offering a possibility to have more social contact, the Swiss national stud farm tested a new box system for horses, allowing increased physical contact with the neighbouring stallion. The aim of this part of the study was to investigate whether this type of housing system (named “social box”) potentially induces a change in stress reactions in stallions compared to conventional boxes. Therefore faecal cortisol metabolite (FCM) concentration was measured as a non-invasive parameter to assess endocrine responses related to this new environment.
Four groups each consisting of eight adult Freiberger breeding stallions were included in the test design. Every stallion spent three weeks in a conventional box and in a social box respectively (cross-over design). The conventional box consisted of a separation wall with a lower opaque part and an upper part with vertical barriers (5 cm between barriers), allowing visual and olfactory contact but strongly limiting tactile contact. The separating wall of the social box consisted of two lateral sections, one part being opaque to the ceiling and the second part consisting of vertical barriers (30 cm between barriers), allowing the horse to have physical contact with its neighbour or to avoid it.
In horses, FCM concentration reflects an average level of circulating cortisol over a period of approximatively 24h. Faecal samples were collected the day following integration in social / conventional boxes, reflecting the potential stress induced by increased social interactions during the integration. In order to asses potential chronical stress, faeces samples were also collected in week one, two and three after the integration into the social / conventional box (in total: 4 samples per horse and housing system). The samples were immediately stored at -20°C until they were analysed. The samples were not analysed in the laboratory until the end of the experiment, therefore the duration of conservation in the freezer varied from 40 to 429 days.
A considerable percentage of data from groups 1 and 2 was below the detection limit (<0.8 ng/g) (Tab. 1). Thus the statistical analysis was conducted with the FCM concentration from groups 3 and 4 (n horses = 16) which contained no values below the detection limit.
Tab. 1: Details about FCM values and storage time for the 4 groups of stallions
Group Storage duration [d] Proportion of data below the detection limit (<0.8 ng/g) Mean [ng/g] Median [ng/g]
Group 1 384-429 55.6 % 2.2 0
Group 2 315-360 25.5 % 5.8 6.3
Group 3 41-79 0.0 % 8.7 8.0
Group 4 40-85 0.0 % 5.8 5.4
Despite the impressive social interactions observed between the stallions directly after being introduced into the social boxes, we did not find any differences in FCM concentration between the stallions being introduced into the conventional box and the social box on the day of integration (social box: n samples = 16, mean±SD: 6.9±4.7 ng/g; conventional box: n samples = 16, mean±SD: 9.0±11.2 ng/g; Wilcoxon signed rank test V = 70, p = 0.94).
Overall the samples taken during integration and in week one, two and three did not show evidence of changes in FCM concentration in either housing system over a longer period of time (social box: n samples = 64, mean±SD: 7.9±6.2 ng/g; conventional box: n samples = 64, mean±SD: 6.6±3.4 ng/g; Linear mixed model (LMM), p = 0.56).
Our results suggest that the possibility of having physical contact with a conspecific does not induce changes in FCM concentration in breeding stallions. The considerable percentage of values below the detection limit in groups 1 and 2 seemed to correlate with the increasing duration of storage before analysis. During the IESM Network Meeting 2015, we would like to discuss possible methodological issues and the possibilities to correctly integrate these low values in the statistical analysis.