Abstract: Non-invasive techniques to monitor stress hormones in small animals like mice offer several advantages and are highly demanded in laboratory as well as in field research. Since knowledge about the species-specific metabolism and excretion of glucocorticoids is essential to develop such a technique, we conducted radiometabolism studies in mice (Mus musculus f. domesticus, strain C57BL/6J). Each mouse was injected intraperitoneally with 740 kBq of 3H-labelled corticosterone and all voided urine and fecal samples were collected for five days. In a first experiment 16 animals (eight of each sex) received the injection at 9 a.m., while eight mice (four of each sex) were injected at 9 p.m. in a second experiment. In both experiments radioactive metabolites were recovered predominantly in the feces, although males excreted significantly higher proportions via the feces (about 73%) than females (about 53%). Peak radioactivity in the urine was detected within about 2h after injection, while in the feces peak concentrations were observed later (depending on the time of injection: about 10h postinjection in experiment 1 and about 4h postinjection in experiment 2, thus proving an effect of the time of day). The number and relative abundance of fecal [3H]corticosterone metabolites was determined by high performance liquid chromatography (HPLC). The HPLC separations revealed that corticosterone was extensively metabolized mainly to more polar substances. Regarding the types of metabolites formed, significant differences were found between males and females, but not between the experiments. Additionally, the immunoreactivity of these metabolites was assessed by screening the HPLC fractions with four enzyme immunoassays (EIA). However, only a newly established EIA for 5alpha-pregnane-3beta,11beta,21-triol-20-one (measuring corticosterone metabolites with a 5alpha-3beta,11beta-diol structure) detected several peaks of radioactive metabolites with high intensity in both sexes, while the other EIAs showed only minor immunoreactivity. Thus, our study for the first time provides substantial information about metabolism and excretion of corticosterone in urine and feces of mice and is the first demonstrating a significant impact of the animals' sex and the time of day. Based on these data it should be possible to monitor adrenocortical activity non-invasively in this species by measuring fecal corticosterone metabolites with the newly developed EIA. Since mice are extensively used in research world-wide, this could open new perspectives in various fields from ecology to behavioral endocrinology.

Abstract: REASONS FOR PERFORMING STUDY: It is recognised that the amount of psychological stress that an animal encounters determines the degree of response of the hypothalamic-pituitary-adrenal (HPA) axis. In human athletes, the added emotive stress of competition is an important element in the adrenal response. The aim of this study was to examine the effect of show-jumping as well as dressage on stress levels by comparing horses' stress response at a horse show compared to their familiar home. METHODS: Fifty-one horses involved in competition were used. EDTA blood samples were collected before exercise, upon arrived to the schooling area (control), and k over a jump or dressage course. After sampling, plasma was separated and stored at -80 degrees C until determinations of cortisol and ACTH were performed. Fourteen healthy horses not involved in competition were used as control group. RESULTS: Competition induced a significant increase in cortisol and ACTH responses in both, jumping and dressage horses and this effect was more apparent in dressage horses. When horses were most experienced, cortisol and ACTH responses were much lower. CONCLUSION: This study shows that competition elicits a classic physiological stress response in horses and that different training programmes induce different responses. It suggests that horses involved in competition can provide a good model to study the exercise-induced stress response.

Abstract: Ground reaction force (GRF) measurements are often normalised to body mass to facilitate inter-individual comparisons. The objective of this study was to explore the effect of a rider on the GRFs and fetlock joint kinematics of trotting horses. The subjects were 5 dressage-trained horses and 3 experienced dressage riders. Ground reaction force measurements and sagittal view videotapes were recorded as the horses trotted at the same velocity in hand (3.49 +/- 0.52 m/s) and with a rider (3.49 +/- 0.46 m/s). Data were time-normalised to stance duration. Ground reaction force measurements were expressed in absolute terms and normalised to the system mass (horse or horse plus rider). All the horses showed changes in the same direction when comparing the ridden condition with the in-hand condition. There was an increase in the absolute peak vertical GRFs of the fore- and hindlimbs with a rider. However, the mass-normalised peak vertical GRFs were lower for the ridden condition, with the peak occurring later in the forelimbs and earlier in the hindlimbs compared with the inhand condition. Maximal fetlock angle and its time of occurrence were similar for the 2 conditions, but the fore fetlock joint was more extended during the later part of the stance phase in ridden horses. The presence of a rider appeared to affect the GRFs and fetlock joint kinematics differently in the fore- and hindlimbs, and the ridden horse did not seem to be equivalent to a proportionately larger horse. This should be considered when normalising for body mass in studies comparing horses in hand and ridden horses.

Abstract: REASONS FOR PERFORMING STUDY: Dehydration and heat stress are serious welfare issues for equids working in developing countries. There is a lack of any standardised method or validated interpretation of the skin tent test in horses and donkeys. Owners of dehydrated and heat-stressed animals often depend on veterinary examination for identification of these conditions, leading to delays in treatment and unnecessary reliance on external sources to effect welfare improvement. OBJECTIVES: To validate a standardised skin tent test for dehydration and a behavioural measure of heat stress in working equids; and to examine the effect of heat stress and dehydration on tripping and staggering behaviour. METHODS: The study was carried out on 130 working horses and donkeys in Pakistan. Associations between skin tent and blood parameters (packed cell volume [PCV], serum total protein [TP], serum osmolality), clinical parameters, resting and drinking behaviour were examined. Heat stress behaviour (increased respiratory rate and depth, head nodding, flared nostrils, apathy) was observed in conjunction with rectal temperature. Tripping and staggering were assessed using a simple obstacle course. RESULTS: In both species, heat stress behaviour was significantly associated with increased rectal temperature (P<0.001). A positive skin tent test was not significantly associated with PCV or TP, although in donkeys it was significantly associated with lower serum osmolality (P<0.001). More animals age >15 years had a positive skin tent than those in younger age groups (P = 0.037). Very thin horses were more likely to have a positive skin tent than those in thin or moderate condition (P = 0.028). There was no significant correlation between skin tent and tripping or staggering in either species. CONCLUSIONS AND POTENTIAL RELEVANCE: Heat stress behaviour is related to increased body temperature in working horses and donkeys. Owners may use this to make judgements regarding rest and cooling, precluding the need to seek veterinary attention. The skin tent test for dehydration used in this study did not show a significant relationship with PCV or TP. However, the use of blood parameters to validate the skin tent test may be confounded by anaemia, hypoproteinaemia or electrolyte depletion. Alternative methods are needed to confirm or refute the validity of the skin tent test in working equids.

Abstract: REASONS FOR PERFORMING STUDY: Equine lameness is commonly evaluated when the horse is being ridden, but the influence of the rider on the lameness has not been documented. OBJECTIVE: To document the effect of 2 riders of different training levels on the vertical movement of the head and croup. METHODS: Twenty mature horses were ridden at trot by an experienced dressage rider and a novice rider, as well as trotted in hand. Kinematic measurements of markers placed on the horse's head and sacral bone were carried out. The asymmetries of the vertical head and sacral bone motion were calculated as lameness parameters and compared with paired t tests. RESULTS: Trotting in hand, 17 horses showed forelimb lameness (1-4/10) and 13 hindlimb lameness (1-2/10). Intra-individually, 11 horses showed significant differences in forelimb lameness and 4 horses showed significant differences in hindlimb lameness when ridden. Over all horses, hindlimb lameness increased significantly under the dressage rider compared to unridden horses. CONCLUSIONS: The presence of a rider can alter the degree of lameness; however, its influence cannot be predicted for an individual horse. POTENTIAL RELEVANCE: In order to evaluate mild lameness, horses should be evaluated at trot both under saddle and in hand. If lameness is exacerbated, a second rider may be helpful; the level of training of the rider should be taken into consideration.