Abstract: The ability of horses to learn and remember new tasks is fundamentally important for their use by humans. Fearfulness may, however, interfere with learning, because stimuli in the environment can overshadow signals from the rider or handler. In addition, prolonged high levels of stress hormones can affect neurons within the hippocampus; a brain region central to learning and memory. In a series of experiments, we aimed to investigate the link between performance in two learning tests, the baseline level of stress hormones, measured as faecal cortisol metabolites (FCM), fearfulness, and social rank. Twenty-five geldings (2 or 3 years old) pastured in one group were included in the study. The learning tests were performed by professional trainers and included a number of predefined stages during which the horses were gradually trained to perform exercises, using either negative (NR) or positive reinforcement (PR). Each of the learning tests lasted 3 days; 7min/horse/day. The NR test was repeated in a novel environment. Performance, measured as final stage in the training programme, and heart rate (HR) were recorded. Faeces were collected on four separate days where the horses had been undisturbed at pasture for 48h. Social rank was determined through observations of social interactions during feeding. The fear test was a novel object test during which behaviour and HR were recorded. Performance in the NR and PR learning tests did not correlate. In the NR test, there was a significant, negative correlation between performance and HR in the novel environment (rS=-0.66, P<0.001, i.e. nervous horses had reduced performance), whereas there was no such correlation in the home environment (both NR and PR). Behavioural reactions in the fear test correlated significantly with performance in the NR test in the novel environment (e.g. object alertness and final stage: rS=-0.43, P=0.04), suggesting that performance under unfamiliar, stressful conditions may be predicted by behavioural responses in a fear test. There was a negative correlation between social rank and baseline stress hormones (rS=-0.43, P=0.04), i.e. high rank corresponded to low FCM concentrations, whereas neither rank nor FCM correlated with fearfulness or learning performance. We conclude that performance under stressful conditions is affected by activation of the sympathetic nervous system during training and related to behavioural responses in a standardised fear test. Learning performance in the home environment, however, appears unrelated to fearfulness, social rank and baseline FCM levels.

Abstract: Stress responses play a key role in allowing animals to cope with change and challenge in the face of both environmental certainty and uncertainty. Measurement of glucocorticoid levels, key elements in the neuroendocrine stress axis, can give insight into an animal’s well-being and can aid understanding ecological and evolutionary processes as well as conservation and management issues. We give an overview of the four main biological samples that have been utilized [blood, saliva, excreta (feces and urine), and integumentary structures (hair and feathers)], their advantages and disadvantages for use with wildlife, and some of the background and pitfalls that users must consider in interpreting their results. The matrix of choice will depend on the nature of the study and of the species, on whether one is examining the impact of acute versus chronic stressors, and on the degree of invasiveness that is possible or desirable. In some cases, more than one matrix can be measured to achieve the same ends. All require a significant degree of expertise, sometimes in obtaining the sample and always in extracting and analyzing the glucocorticoid or its metabolites. Glucocorticoid measurement is proving to be a powerful integrator of environmental stressors and of an animal’s condition.

Abstract: Many breeding organisations include a subjective scoring of rideability by a professional rider into their evaluation of sports horses, but the consistency and reliability of the scoring system is debateable. The aim of this study was to investigate (i) whether professional riders agree in their scoring of rideability, and (ii) whether rideability scores are affected by rein tension, horse conflict behaviour, heart rate, and salivary cortisol, and (iii) whether riders induce different levels of conflict behaviour and physiological responses in the horses. Ten professional, female riders each rode 10 dressage horses (level M German scale; n = 100 combinations) through a standardised dressage test (10 min warm-up followed by a 4-min test) and subsequently scored the horses for rideability on the official 1-10 scale (1 = poor to 10 = excellent) from the Danish Riding Federation. Rein tension, horse heart rate, saliva cortisol and conflict behaviour were measured for each rider-horse pair. The riders were inconsistent in their scoring of rideability to the individual horses, e.g. scores for one of the horses ranged from 1 to 8. There was a significant effect of rider (P = 0.003) and the frequency of conflict behaviour (undesired head movements: P < 0.001, breaking the gait: P = 0.013, and other evasive behaviour: P = 0.032) on rideability scores, i.e. the more conflict behaviour the lower the score. There was no significant effect of rein tension and the physiological measures on rideability scores. However, there was a significant effect of rider on rein tension, horses' heart rate and increases in saliva cortisol concentrations and a tendency for some types of conflict behaviour, suggesting that some riders induced more discomfort in the horses. Future studies could help shed light on which elements of riding style are particularly important for sports horse welfare. In conclusion, this study found a large variation in rideability scores assigned to ten sports horses by ten professional riders. Rideability scores were dependent on the level of horse conflict behaviour, but not rein tension and physiological measures. Further studies are needed to improve the objectivity, consistency and reliability of rideability assessment of sports horses.

Abstract: A multitude of endocrine mechanisms are involved in coping with challenges. Front-line hormones to overcome stressful situations are glucocorticoids (GCs) and catecholamines (CAs). These hormones are usually determined in plasma samples as parameters of adrenal activity and thus of disturbance. GCs (and CAs) are extensively metabolized and excreted afterwards. Therefore, the concentration of GCs (or their metabolites) can be measured in various body fluids or excreta. Above all, fecal samples offer the advantages of easy collection and a feedback-free sampling procedure. However, large differences exist among species regarding the route and time course of excretion, as well as the types of metabolites formed. Based on information gained from radiometabolism studies (reviewed in this paper), we recently developed and successfully validated different enzyme immunoassays that enable the noninvasive measurement of groups of cortisol or corticosterone metabolites in animal feces. The determination of these metabolites in fecal samples can be used as a powerful tool to monitor GC production in various species of domestic, wildlife, and laboratory animals.

Abstract: The capercaillie (Tetrao urogallus), the largest grouse species in the world, is decreasing in numbers in major parts of its distribution range. Disturbances by human outdoor activities are discussed as a possible reason for this population decline. An indicator for disturbances is the increase of the glucocorticoid corticosterone, a stress hormone, which helps to cope with life-threatening situations. However, repeated disturbances might result in a long-term increase of the basal corticosterone concentration, which can result in detrimental effects like reduced fitness and survival of an animal. To measure corticosterone metabolites (CMs) noninvasively in the droppings of free-living capercaillies, first an enzyme immunoassay (EIA) in captive birds had to be selected and validated. Therefore, the excretion pattern of intravenously injected radiolabeled corticosterone was determined and 3H metabolites were characterized. High-performance liquid chromatography (HPLC) separations of the samples containing peak concentrations revealed that corticosterone was extensively metabolized. The HPLC fractions were tested in several EIAs for glucocorticoid metabolites. The physiological relevance of this method was proved after pharmacological stimulation of the adrenocortical activity. Only the recently established cortisone assay, measuring CMs with a 3,11-dione structure, detected an expressed increase of concentrations following ACTH stimulation. To set up a sampling protocol suited for the field, we examined the influence of various storage conditions and time of day on concentrations of CMs.