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: Reliable physiological markers for performance evaluation in sport horses are missing. To determine the diagnostic value of plasma ACTH and cortisol measurements in the warmblood horse, 10 initially 3-yr-old geldings of the Hannovarian breed were either exposed to a training schedule or served as controls. During experimental Phase 1, horses were group-housed, and half of the horses were trained for 20 wk on a high-speed treadmill. During Phase 2, groups were switched and one group was trained for 10 wk as during Phase 1, whereas the control group was confined to boxes. During Phase 3 horses were initially schooled for riding. Thereafter, all horses were regularly schooled for dressage and jumping, and half of the horses received an additional endurance training for 24 wk. During all phases horses were exposed at regular intervals to various standardized treadmill exercise tests. During and after the tests frequent blood samples were taken from an indwelling jugular catheter for determination of ACTH and cortisol. Treadmill exercise increased both hormones. Maximum ACTH concentrations were recorded at the end of exercise, and maximum cortisol levels were recorded 20 to 30 min later. Except for one test there were no differences in ACTH levels between trained horses and controls. There was no significant effect of training on the cortisol response (net increase) to treadmill exercise in any of the tests during Phase 1. During Phase 2 higher cortisol responses were recorded in controls than in trained horses (P < .05) after 10 wk of training (controls confined to boxes). During Phase 3 plasma cortisol responses were also higher in controls than in trained horses (P < .05 after 6, 18, and 24, P < or = .07 after 12 wk of training) when the inclination of the treadmill was 5%, but not at 3%. There was no overlap in net cortisol responses at 30 min between trained and untrained horses. An ACTH application after 24 wk of training resulted in higher cortisol responses in controls than in trained horses (P < or = .05), without any overlap between the groups at 30 min after ACTH. Plasma cortisol responses to either treadmill exercise or ACTH injection may be a reliable physiological marker for performance evaluation. Prerequisites are sufficient differences in training status and sufficient intensity of exercise test conditions.

Abstract: The global positioning system (GPS) has been used to record activity and monitor habitat use in many animal species. In the horse (Equus caballus) the monitoring of activity and feeding patterns has been used to assess the impact of environmental factors on behaviour and welfare. In free-ranging animals GPS can provide such information but the accuracy and reliability of these devices has yet to be confirmed. The aim of this study was: 1) to compare the results of visual observation with GPS recordings of the horse’s head and neck position (head up (HU) and down (HD)) used to quantify time spent grazing; 2) to test the use of GPS collars to monitor activity patterns where distance, speed and location paths were recorded. In both studies two animals were fitted with Lotek GPS 3300S collars (with integrated GPS data logger and removable battery pack) round the top of the neck. In study 1 two horses were fitted with collars and turned loose into a 20x40m sand arena for 45 minutes. Feed balls and hay were provided (in nets and on the ground) to encourage movement and feeding behaviour for comparison using the two methods (observation from digital video recordings and GPS). HD was recorded by the GPS collars for a significantly longer time (interpreted as feeding/grazing time) than that recorded by observation (p=0.004). However when the visual observation was split into HU, HD and also head in mid-way position (HMW), where the nose of the horse was level or just above the chest, then no difference between the collar (HU and HD) and visual observation for (HU and HD+HMW) was found. It is likely that when in HMW the GPS collar may not be sufficiently angled to trigger the sensor to record HU or the collar may move on the neck. Conclusions relating to time spent feeding should be treated with caution. In study 2, the collars were fitted to two ponies with access to 2.02 hectares of lowland grazing. Activity (distance travelled and speed) and location was recorded for 2 days. The total distance travelled by the ponies in 24 hours (2.84km) and their average speed (4.04m/minute) was calculated and showed no significant difference between day and night. The total area was split into four equal segments and there was no significant difference in the time the ponies spent in each area although they were found to move at slower speeds and stand for longer in some areas. Movement paths could be identified by inputting the GPS collar data into ArcGIS and viewed on Google Maps. There was a high level of comparability observed between the two ponies confirming behavioural synchronicity. As in other species, the use of GPS collars to monitor the movement and location of horses/ponies was found to be effective, but data relating to head position did not provide a reliable means of recording the time spent feeding.

Abstract: Wrinkles above the eye ball are common in domestic horses but may differ in number and shape both between and within individuals. They are caused by contraction of the inner eye brow raiser, and some people working with horses call them “worry wrinkles”, considering them to reflect emotional states. However, as yet no study has formally investigated the relationship between eye wrinkles and emotional state in horses.
The aim of the present study was to induce states of different emotional valence and to assess whether positive emotional states would reduce the expression of eye wrinkles while negative emotional states would increase it. Sixteen horses were confronted in a balanced order with two presumably positively and two negatively valenced situations each. Positive situations included anticipation of a food reward (FA) and petting (P), negative situations included food competition (FC) and waving a plastic bag (PB). Each situation lasted for 60s (TRT) and was preceded by a 60s control phase (CON). Throughout CON and TRT pictures of the eyes were taken, and for each horse four pictures per situation (FA, P, FC, PB) and phase (CON and TRT) were randomly selected (n = 512) and scored in random order and blind to treatment for six outcome variables: overall impression (qualitative), number, angle and markedness of eye wrinkles, presence of eye white, and shape of eye lid.
Data were analysed separately for the right and left eye using linear mixed effects models (angle, number), generalised linear mixed models (eye white, markedness), and ordered logistic regression (qualitative, shape of eye lid), with “situation” (FA, P, FC, PB), “phase” (CON, TRT) and their two-way interaction as fixed effects.
Expression of eye wrinkles did not vary consistently across “situation” and “phase”. Independent of phase, eye white appeared less frequently during P than during FA (z=-3.15, p=0.009), FC (z=-2.94, p=0.02), and PB (z=4.17, p<0.001) in the left eye and during PB (z=4.10, p 0.001) in the right eye. Similarly, wrinkles were less marked during P compared to the other situations in the left eye (FA: z=3.15, p=0.009; FC: z=-2.94, p=0.017; PB: z=4.17, p<0.001) and compared to PB in the right eye (z=4.10, p=0.001), while no differences between situations occurred in number of wrinkles, overall impression and shape of eye lid for both eyes. Consistent with our hypothesis, P induced relaxation of the underlying muscle in the right eye resulting in a wider angle compared to its control phase (interaction situation*phase: F3,10=3.71, p=0.055; post-hoc comparison: z=-3.57, p=0.009), while FC induced muscle contraction, resulting in a sharper angle in the left eye (interaction situation*phase: F3,11=6.57, p=0.011; z=3.73, p=0.005).
We conclude that emotional valence may affect characteristics of eye winkle expression in horses which might therefore be a promising indicator of horses’ emotional states, but further research is needed to validate the relevant outcome variables.