Abstract: In small animals like mice, the monitoring of endocrine functions over time is constrained seriously by the adverse effects of blood sampling. Therefore, noninvasive techniques to monitor, for example, stress hormones in these animals are highly demanded in laboratory as well as in field research. The aim of our study was to evaluate the biological relevance of a recently developed technique to monitor stress hormone metabolites in fecal samples of laboratory mice. In total, six experiments were performed using six male and six female mice each. Two adrenocorticotropic hormone (ACTH) challenge tests, two dexamethasone (Dex) suppression tests and two control experiments [investigating effects of the injection procedure itself and the diurnal variation (DV) of glucocorticoids (GCs), respectively] were conducted. The experiments clearly demonstrated that pharmacological stimulation and suppression of adrenocortical activity was reflected accurately by means of corticosterone metabolite (CM) measurements in the feces of males and females. Furthermore, the technique proved sensitive enough to detect dosage-dependent effects of the ACTH/Dex treatment and facilitated to reveal profound effects of the injection procedure itself. Even the naturally occurring DV of GCs could be monitored reliably. Thus, our results confirm that measurement of fecal CM with the recently established 5alpha-pregnane-3beta,11beta,21-triol-20-one enzyme immunoassay is a very powerful tool to monitor adrenocortical activity in laboratory mice. Since mice represent the vast majority of all rodents used for research worldwide and the number of transgenic and knockout mice utilized as animal models is still increasing, this noninvasive technique can open new perspectives in biomedical and behavioral science.

Abstract: Reasons for performing study: In horses, serum cortisol concentration is considered to provide an indirect measurement of stress. However, it includes both free and bound fractions. The sampling method is also invasive and often stressful. This is not the case for salivary cortisol, which is collected using a more welfare-friendly method and represents a part of the free cortisol fraction, which is the biologically active form. Objectives: To compare salivary and serum cortisol assays in horses, in a wide range of concentrations, using an adrenocorticotropic hormone (ACTH) stimulation test, in order to validate salivary cortisol for stress assessment in horse. Methods: In 5 horses, blood samples were drawn using an i.v. catheter. Saliva samples were taken using swabs. Cortisol was assayed by radioimmunoassay. All data were treated with a regression method, which pools and analyses data from multiple subjects for linear analysis. Results: Mean ± s.d. cortisol concentrations measured at rest were 188.81 ± 51.46 nmol/l in serum and 1.19 ± 0.54 nmol/l in saliva. They started increasing immediately after ACTH injection and peaks were reached after 96 ± 16.7 min in serum (356.98 ± 55.29 nmol/l) and after 124 ± 8.9 min in saliva (21.79 ± 7.74 nmol/l, P<0.05). Discharge percentages were also different (225% in serum and 2150% in saliva, P<0.05). Correlation between serum and salivary cortisol concentrations showed an adjusted r2= 0.80 (P<0.001). The strong link between serum and salivary cortisol concentrations was also estimated by a regression analysis. Conclusions: The reliability of both RIAs and regression found between serum and salivary cortisol concentrations permits the validation of saliva-sampling as a noninvasive technique for cortisol level assessment in 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: Earlier studies have developed a clinical tool to evaluate objectively the function of the equine back. The ability to differentiate horses with back pain from asymptomatic, fully functioning horses using kinematic measures from this tool has not been evaluated. OBJECTIVES: To compare the kinematics of the back at walk and trot in riding horses with back dysfunction to the same parameters in asymptomatic sport horses. METHODS: The kinematics of the back in 12 horses with impaired performance and back pain were studied at walk and trot on a treadmill. Data were captured for 10 sees at 240 Hz. Range of movement (ROM) and intravertebral pattern symmetry of movement for flexion and extension (FE), lateral bending (LB) and axial rotation (AR) were derived from angular motion pattern data and the results compared to an earlier established database on asymptomatic riding horses. RESULTS: At walk, horses with back dysfunction had a ROM smaller for dorsoventral FE in the caudal thoracic region (T13 = 7.50 degrees, T17 = 7.71 degrees; P<0.05), greater for LB at T13 (8.13 degrees; P<0.001) and smaller for AR of the pelvis (10.97 degrees; P<0.05) compared to asymptomatic horses (FE-T13 = 8.28 degrees, FE-T17 = 8.49 degrees, LB-T13 = 6.34 degrees, AR-pelvis = 12.77 degrees). At trot, dysfunctional horses had a smaller (P<0.05) ROM for FE at the thoracic lumbar junction (T17 = 2.46 degrees, L1 = 2.60 degrees) compared to asymptomatic horses (FE-T17 = 3.07 degrees, FE-L1 = 3.12 degrees). CONCLUSIONS: The objective measurement technique can detect differences between back kinematics in riding horses with signs of back dysfunction and asymptomatic horses. The clinical manifestation of back pain results in diminished flexion/extension movement at or near the thoracic lumbar junction. However, before applying the method more extensively in practice it is necessary to evaluate it further, including measurements of patients whose diagnoses can be confirmed and long-term follow-ups of back patients after treatment. POTENTIAL RELEVANCE: Since the objective measurement technique can detect small movement differences in back kinematics, it should help to clinically describe and, importantly, objectively detect horses with back pain and dysfunction.

Abstract: Reasons for performing study: Little information exists on the immunological effects of transport or the use of supplements to minimise transport stress. Objectives: To establish baseline ranges and evaluate immunophenotypic and functional changes associated with transport and a nutritional ‘adaptogen’ supplement. Methods: Horses received either supplement (n = 10) or placebos (n = 9) during the 30 day study. After 28 days in stalls, 12 horses (6 supplement; 6 placebo) were transported for 24 h, then unloaded and recovered. Venous blood samples were collected on Days 1, 14 and 28 to establish baselines, and on Days 28, 29 and 30 to examine changes during transport and recovery. Results: Transport prompted elevations (P<0.05) in cortisol concentration, neutrophil count and white blood cell counts, while lymphocyte subpopulation counts (CD3+, CD4+, CD8+, CD21+) decreased (P<0.05). Normal phenotypic lymphocyte profiles returned within 24 h of recovery. Supplement effects on immunophenotype (CD21+ and CD8+) were observed in stabled horses (P<0.05), but not in transported horses. Conclusions: These results provide insights into the immunological mechanisms associated with long-term transport. Potential relevance: The existence of a small window of immunological uncertainty follows long-term transportation, enhancing the potential risk of infectious disease in susceptible individuals.