Influence of Transportation on the Serum Concentrations of the Cardiac Biomarkers Troponin I and Creatine Kinase-myocardial Band (CK-MB) and on Cortisol and Lactate in Horses

The objective of this study was to determine the influence of transportation on the serum concentrations of the cardiac biomarkers troponin I (cTnI) and creatine kinase-myocardial band (CK-MB) and on cortisol and lactate in horses. For this purpose, 10 horses were transported for 300 km. Blood samples were collected 24 hours before transport (T0), just before transport (T1), during transport at 50 km (T2), 100 km (T3), 200 km (T4), and 300 km (T5). An additional blood sample (T6) was collected 24 hours after transport. The median resting basal cTnI values in the horses were at T0, 0.000 ± 0.007 ng/mL and at T1, 0.01 ± 0.007 ng/mL. The median resting basal CK-MB values in the horses were at T0, 0.19 ± 0.05 ng/mL and at T1, 0.16 ± 0.05 ng/mL. Statistical analyses showed no significant differences of cTnI and CK-MB among the measured values (T0–T6). On the other side, the cortisol and lactate concentrations increased significantly (P < .01) at T2, T3, T3, and T4 compared with the resting values at T0. At T6, cortisol and lactate concentrations had returned to pretransport values, with no statistically significant differences compared with pretransport concentrations. In conclusion, the 300-km transportation of the horses did not influence the levels of the cardiac biomarkers, cTnI and CK-MB. The serum concentrations of cortisol and lactate, on the other side, increased significantly. The possible influence of transportation for longer distances or under more stressful conditions (higher temperature or in horses not used for transportation) on cTnI and CK-MB concentrations needs to be further investigated.     

Circulating β-endorphin,adrenocorticotrophic hormone and cortisol levels of stallions before and after short road transport: stress effect of different distances

Background

Since transport evokes physiological adjustments that include endocrine responses, the objective of this study was to examine the responses of circulating β-endorphin, adrenocorticotrophic hormone (ACTH) and cortisol levels to transport stress in stallions.

Methods

Forty-two healthy Thoroughbred and crossbred stallions were studied before and after road transport over distances of 100, 200 and 300 km. Blood samples were collected from the jugular vein: first in a single box immediately before loading (pre-samples), then immediately after transport and unloading on arrival at the breeding stations (post-samples).

Results

An increase in circulating β-endorphin levels after transport of 100 km (P < 0.01), compared to basal values was observed. Circulating ACTH levels showed significant increases after transport of 100 km (P < 0.001) and 200 km (P < 0.001). Circulating cortisol levels showed significant increases after road transport over distances of 100, 200 and 300 km (P < 0.001). An effect of transport on β-endorphin, ACTH and cortisol variations was therefore evident for the different distances studied. No significant differences (P > 0.05) between horses of different ages and different breeds were observed for β-endorphin, ACTH and cortisol levels.

Conclusion

The results obtained for short term transportation of stallions showed a very strong reaction of the adrenocortical system. The lack of response of β-endorphin after transport of 200–300 km and of ACTH after transport of 300 km seems to suggest a soothing effect of negative feedback of ACTH and cortisol levels.     

The Potential Role of Training Sessions on the Temporal and Spatial Physiological Patterns in Young Friesian Horses

This study compares the circulating adrenocorticotrophin hormone (ACTH), cortisol, lactate, glucose, heart rate, respiratory rate, rectal temperature, and blood count values in initially 2-year-old horses subjected to dressage training schedule during three consecutive days per 2 weeks. Sixteen healthy Friesian horses were used and were considered dressage group. Six healthy young horses not involved in training programs were used as control group. Blood sampling were collected from the jugular vein in baseline condition (dressage group and control group) and after exercise, within 5 minutes of the end of the training session (dressage group). Compared to baseline values, results showed higher ACTH concentrations after the first day of the first training week (P < .005) and after the third day of the second week (P < .005); higher lactate concentrations after the second and the third day of the second week (P < .01); lower glucose concentrations after the third day of the first week (P < .01); higher HR, RR, and RT values and lower PLT count after different time points during both training weeks. One-way ANOVA showed significant training effect for ACTH (F = 7.605; P < .0001) and glucose (F = 3.505; P < .001) concentrations over time points. Two-way ANOVA showed a significant effect of dressage training sessions between the first and the second week for ACTH (F = 6.508; P < .001) and cortisol (F = 5.559; P < .0001) concentrations. From obtained data, it seems that the use of ACTH and cortisol changes for the assessment of effects of training in initially 2-year-old horses could be an ideal measure of quantitative and qualitative stress responses. The quantification at the same time of functional responses to stressful stimuli may offer a more objective measurement of dressage training effects.     

Circadian and circannual rhythms of cortisol,ACTH, and α-melanocyte-stimulating hormone in healthy horses

Cosinor analysis was used to evaluate whether pituitary and adrenal hormones exhibit circadian rhythmicity in horses. The effect of season and animal age on their respective rhythms was also determined. In addition, the usefulness of evaluating cortisol rhythmicity for the diagnosis of pituitary pars intermedia dysfunction (PPID) was assessed. Serum cortisol concentrations (P < 0.01), but not plasma ACTH or α-melanocyte-stimulating hormone (α-MSH), showed a significant circadian periodicity in horses. An effect of season on hormone concentration was observed with plasma ACTH and α-MSH concentration greater in the fall and cortisol concentration greater in the spring (P < 0.001). Age did not affect cortisol rhythm, but it did blunt the variation in cortisol concentration in horses, similar to what has been previously reported to occur in aged people and dogs. In addition, our results suggest that clinically and diagnostically normal, non–PPID-affected horses commonly have a loss of cortisol diurnal rhythm. Therefore, measurement of circadian rhythm is not an appropriate diagnostic test for PPID.     

Effects of road transportation on metabolic and immunological responses in Holstein heifers

This study examined the effects of road transportation on metabolic and immunological responses in dairy heifers. Twenty Holstein heifers in early pregnancy were divided into non‐transported (NT; n = 7) and transported (T; n = 13) groups. Blood was collected before transportation (BT), immediately after transportation for 100 km (T1) and 200 km (T2), and 24 h after transportation (AT). The T heifers had higher (P < 0.05) blood cortisol and non‐esterified fatty acid concentrations after T1 and T2 than did NT heifers. By contrast, the T heifers had lower (P < 0.05) serum triglyceride concentrations after T1 and T2 than had the NT heifers. The serum cortisol and triglyceride concentrations returned (P > 0.05) to the BT concentrations at 24 h AT in the T heifers. The granulocyte‐to‐lymphocyte ratio and the percentage of monocytes were higher (P < 0.05) after T2 in the T heifers than in the NT heifers, suggesting that transportation stress increased the numbers of innate immune cells. T heifers had higher (P < 0.01) plasma haptoglobin concentrations than NT heifers 24 h AT. In conclusion, transportation increased cortisol secretion and was correlated with increased metabolic responses and up‐regulation of peripheral innate immune cells in dairy heifers.     

Cortisol response to road transport stress in calm and nervous stallions

Cortisol may be useful as a marker in predicting how an animal will respond to stressful stimuli, thus providing information on animal's temperament. To quantify the level of transport stress and the effect of temperament on the adrenocortical response, the change in circulating cortisol levels was evaluated in 84 healthy experienced thoroughbred and crossbred stallions, mean age 11.4 ± 4.5 years old, after road transport in a commercial trailer (6 horses per load, stocking density: 2 m²/horse), over mean distances of 210 ± 11.8 km for about 3 hours. Several experienced caretakers were asked to complete 2 questionnaires, one that used a 5-point scale to subjectively evaluate temperament and another that used a 3-point scale to assess tendencies in response to ordinary care and daily management. The scores for the latter were defined as follows: a score of 1 indicated that the horse had never or rarely troubled the caretaker during management, 2 occasionally, and 3 usually. On this basis, the subjects were distinguished between calm and nervous stallions. Blood samples were taken same daytime (8.00 am), in single box, immediately before loading, then after transport and unloading. Serum cortisol concentrations were analyzed in duplicate by immunoenzymatic assay. Compared with basal levels, cortisol increases were observed in both calm (P < 0.001) and nervous (P < 0.05) stallions after transport. Repeated-measures analysis of variance showed significant effects of transport on cortisol changes (P < 0.001). Nervous subjects showed lower (P < 0.01) cortisol levels than did calm subjects after transport, and basal cortisol levels did not differ between calm and nervous subjects. No significant differences (P > 0.05) between different age, breed, and orientation were detected.The results showed that temperament could influence the adrenocortical responses of stallions after short-term transportation. The presence of the same staff for handling, loading, confinement, and unloading, the same veterinarian taking all blood samples, and the presence of cospecifics did not reduce the response to short transport stress both in calm and nervous stallions already accustomed to transport. Lower cortisol concentrations in nervous subjects might be because of failure of the adrenal cortex to respond normally to transport stress. Moreover, signs of transport stress were less pronounced in nervous stallions.     

Differential responses of Equus caballus and Equus asinus to infection with two pathogenic strains of equine infectious anemia virus

Most in vivo studies with equine infectious anemia virus (EIAV) have been performed in horses and ponies (Equus caballus) with little published information available detailing the clinical responses of donkeys (Equus asinus) to infection with this virus. Consequently, donkeys were inoculated with two strains of EIAV (EIAV(PV) and EIAV(WY)) which have been documented to produce disease in E. caballus. Four ponies, 561, 562, 564 and 567 and two donkeys, 3 and 5 were infected with EIAV(PV) and one horse (94-10) and one donkey (4) were infected with EIAV(WY). Although the horse and ponies all experienced clinical signs of disease, which in some cases were severe, the donkeys remained asymptomatic throughout a 365-day observation period, except for mild transient reductions in platelet counts. The results from serological assays, virus isolation from plasma and detection of plasma-associated viral RNA by RT-PCR, indicated that initial replication of EIAV(PV) and EIAV(WY) was lower in donkeys than in horses and ponies. This conclusion was confirmed using competitive RT-PCR, in which viral RNA levels in the plasma of EIAV(PV)-infected ponies was up to 100,000-fold higher than in infected donkeys during the first 20 days post-infection (dpi). Similar results were obtained in the EIAV(WY)-infected animals, in which viral RNA burdens in the donkey at 20 dpi were 1000-fold less than in the horse. However, infection of donkey and horse monocyte-derived macrophage cultures with EIAV(PV) demonstrated that these cells in vitro were equally susceptible to virus-induced cytopathic effects and yielded similar levels of progeny virus. This result suggests that factors other than host cell permissiveness mediate the clinical differences observed between horses and donkeys infected with EIAV(PV) or EIAV(WY).     

Cortisol release and heart rate variability in sport horses participating in equestrian competitions

Equestrian competitions require both physical activity and mental adaptation in horses. Cortisol, heart rate, and heart rate variability (HRV) are accepted stress parameters and, in this study, have been determined in horses (n = 13) participating in equestrian competitions for up to 3 consecutive days. Participation in competitions caused an increase in salivary cortisol concentrations (e.g., on day 1 from 1.0 ± 0.2 before to 2.2 ± 0.4 ng/mL after the competition, days 1 and 2: P < 0.001, day 3: P < 0.05) and an increase in heart rate (days 1 and 2: P < 0.001, day 3: P = 0.01). A consistent decrease in HRV occurred only in response to the final competition on day 3 (P < 0.01). When horses competing in dressage and show jumping were compared, cortisol release and HRV did not differ between groups, but after the competition, heart rate was lower in dressage than in show jumping horses (P < 0.05). Heart rate increased not only during the actual competition but already when horses were prepared in their stables (e.g., day 1: ?60 minutes, 38.6 ± 2; ?5 minutes, 77 ± 7; competition, 81 ± 10 beats per minute; P < 0.01). In conclusion, participation in equestrian competitions caused an increase in cortisol release and heart rate and a decrease in HRV variables. However, competitions were not a major stressor compared with other anthropogenic challenges such as transport, to which horses are exposed regularly.     

Changes in salivary and plasma cortisol levels in Purebred Arabian horses during race training session

Physical activity and stress both cause an increase in cortisol release ratio. The aim of this study was to evaluate the use of saliva samples for the determination of cortisol concentrations indicating the work‐load level in horses during race training. Twelve Purebred Arabian horses aged 3–5 years were studied during the routine training session. After the warm‐up, the horses galloped on the 800 m sand track at a speed of 12.8 m/s. Three saliva samples, and three blood samples were collected from each horse. Both types of samples were taken at rest, immediately after return from the track and after 30 min restitution. The concentrations of blood lactic acid (LA), and cortisol in saliva and plasma samples were measured and analyzed. Blood LA, plasma and salivary cortisol levels increased significantly after exercise (P < 0.05). Salivary cortisol concentration determined 30 min after the exercise correlated significantly with plasma cortisol level obtained immediately after exercise (P < 0.05) as well as measured 30 min after the end of exercise (P < 0.05). The determination of cortisol concentration in saliva samples taken from racehorses 30 min after the end of exercise can be recommended to use in field conditions to estimate the work‐load in racehorses.     

Effects of haul distance and stocking density on young suckling calves transported in Japan

We conducted applicability evaluation of recommendations of the European Union (EU) and the Royal Society for Prevention of Cruelty to Animals (RSPCA) for suckling calves. Nineteen calves (14 Holstein aged 11–26 days and five cross‐bred aged 13–45 days) were divided into six groups. Three groups were allocated into the stocking densities of 0.25, 0.35, 0.45 m²/head and were weekly transported 50, 100, 150 km in this order. Another three groups were allocated and transported in reverse order. Observation was conducted during and after transportation. Blood sampling was performed before and after transportation. The occurrences of lying and turning round on the truck were significantly associated with stocking density (P < 0.05), but not with haul distance. Scratching was observed only in one calf. Incidence of watery feces was not associated with both factors. There were significant effects of haul distance on the concentrations of plasma cortisol and noradrenaline, and serum aspartate aminotranserase (AST) and IgM (P < 0.05). These concentrations, except cortisol, were higher after transportation at 150 km compared to the pre‐transportation value (AST and IgM P < 0.05; noradrenaline P < 0.10). Results suggest that haul distances greater than 100 km should not be recommended even for suckling calves transported in Japan.     

Influence of riders’ skill on plasma cortisol levels of horses walking on forest and field trekking courses

The aim of this study was to evaluate the influence of rider's skill on the plasma cortisol levels of trekking horses on two courses, walking on field and forest courses (about 4.5 to 5.1 km each). Three riders of different skills did horse trekking (HT) in a tandem line under a fixed order: advanced‐leading, beginner‐second and intermediate‐last. A total of six horses were used and they experienced all positions in both courses; a total of 12 experiments were done. Blood samples were obtained before HT, immediately after and 2 h after HT. As a control, additional blood samples were obtained from the same horses on non‐riding days. Irrespective of the course and the rider's skill, the cortisol level before HT was higher than that of control (P < 0.05). In both courses, the cortisol levels immediately after HT ridden by the advanced rider were higher than that of control (P < 0.05). However, in every case, the cortisol level 2 h after HT was closely similar to the level of the control. Thus, we concluded the stress of trekking horse was not sufficient to disturb the circadian rhythm of the cortisol level, irrespective of the course and the rider's skill.     

Adrenocortical Response in Cows after Intramuscular Injection of Long‐Acting Adrenocorticotropic Hormone (Tetracosactide Acetate Zinc Suspension)

The objectives of this study were first to show adrenocortical response to a long‐acting adrenocorticotropic hormone preparation (tetracosactide acetate zinc suspension) (ACTH‐Z) and its effect on adrenocortical function in beef cows ( Experiment 1 ) and second to apply the ACTH‐Z challenge in dairy cows based on cortisol concentrations in milk collected at routine milking ( Experiment 2 ). In Experiment 1 , four beef cows in luteal phase were challenged with ACTH‐Z, and plasma cortisol concentrations were determined for 48 h after the injection at 30‐min to 2‐h intervals. A rapid ACTH test was conducted 3 days before and 2 h after the completion of ACTH‐Z injection for 48 h to investigate the effect on adrenocortical function. Plasma cortisol concentrations increased significantly 30 min after ACTH‐Z injection (p < 0.001), and the high cortisol levels were maintained for approximately 10 h after the injection. In Experiment 2 , eight dairy cows were subjected to ACTH‐Z challenge 1–2 weeks and 4–5 weeks post‐partum. Blood and milk samples were taken at morning and afternoon milking. All the cows showed a significant increase in cortisol concentrations in plasma as well as in skim milk 8 h after ACTH‐Z injection 1–2 weeks and 4–5 weeks post‐partum (p < 0.001). There was a significant correlation between plasma and skim milk cortisol concentrations 8 h after ACTH‐Z challenge (r = 0.74, p < 0.001). The results obtained in this study suggest that elevated levels of plasma cortisol are maintained for approximately 10 h after ACTH‐Z treatment without adverse effect on adrenocortical function and a long‐acting ACTH‐Z challenge based on cortisol concentrations in milk, which were collected at the morning and the afternoon milking, can be a useful tool to monitor adrenocortical function in cows.     

Relationship between serum cortisol levels and some physiological parameters following reining training session in horse

The changes of cortisol, red blood cells (RBC), hemoglobin (Hb), hematocrit (Hct), heart rate (HR), respiratory rate (RR) and rectal temperature (RT) were evaluated after a reining training session in eight Quarter Horses. All parameters were measured before exercise (T0), immediately after exercise (T1), 1 h after exercise (T2), 2 h after exercise (T3) and 24 h after exercise (T4). One‐way analysis of variance (ANOVA) for repeated measures, followed Bonferroni's post hoc test, showed a significant effect of the reining training session (P < 0.0001) on cortisol, RBC, Hb, Hct, HR, RR and RT. Simple linear regression analysis showed the positive correlation (P < 0.05) between cortisol changes and variations of studied parameters in T1, T3 and T4. Exercise‐induced cortisol concentrations reflect the physiological response of reining training, suggesting that the changes observed are useful to assess the performance in reining horses and their reining training adaptability.     

Effects of different positions during transport on physiological and behavioral changes of horses

The aim of this study was to evaluate the effect of different transport positions on some physiological parameters in racehorses and their behavior patterns during and after the journey. Twelve horses made 3-hour journeys of 200 km on the same route, with the same driver, and in 3 different positions: facing forward, backward, and sideways in relation to the direction of travel. Physiological and behavioral parameters were registered before, during, and after the journey. Horses were checked at 5 different times: at rest (T0), at loading (T1), at unloading (T2), and at 2 (T3) and 4 (T4) hours after return from the journey. At each check, heart rate, respiratory rate, and rectal temperature were measured and blood samples were collected by jugular vein puncture to assess cortisol, packed cell volume, total protein, albumin, glucose, creatinine, triglycerides, cholesterol, urea, creatine kinase, lactate dehydrogenase, alanine transaminase, aspartate transaminase, alkaline phosphatase, calcium, phosphorus, and chlorine. Loading and unloading were filmed. Behavioral patterns were recorded by direct observation, during the travel, 2 and 4 hours after arrival in a new stall. The same parameters were recorded at the same times (excluding loading and unloading) in a control group that did not travel. All data were analyzed using a repeated-measures analysis (analysis of variance). Loading produced an increase of heart rate and packed cell volume in comparison with rest values. Horses facing in the direction of travel during journey made fewer forward, backward, and sideways movements than others, whereas horses traveling sideways lost their balance and touched the stall rails less frequently. Highest serum cortisol concentration value was recorded soon after unloading horses that had faced in the direction of travel (P < 0.01). Two hours after return, horses that had traveled sideways revealed an increase of creatine kinase (P < 0.01). The traveling position in the vehicle did not appear to affect postjourney behavior. In comparison with the control group, the horses that had traveled consumed concentrate faster, spent more time eating hay, and drank more frequently in the first 2 hours after return from the journey. Front-facing position led to an increase in serum cortisol concentration, whereas the sideways position caused some muscular tension, which disappeared 4 hours after the journey. Although facing backward was the travel position that provoked the greatest number of horses’ movements, it did not have a negative effect on physiological and behavioral parameters during and after the journey. We concluded that for Standardbred trotters accustomed to travel, the latter may be the less stressful position during a 200-km transport.     

Changes in cortisol release and heart rate variability in sport horses during long-distance road transport

It is widely accepted that transport is stressful for horses, but only a few studies are available involving horses that are transported regularly and are accustomed to transport. We determined salivary cortisol immunoreactivity (IR), fecal cortisol metabolites, beat-to-beat (RR) interval, and heart rate variability (HRV) in transport-experienced horses (N = 7) in response to a 2-d outbound road transport over 1370 km and 2-d return transport 8 d later. Salivary cortisol IR was low until 60 min before transport but had increased (P < 0.05) 30 min before loading. Transport caused a further marked increase (P < 0.001), but the response tended to decrease with each day of transport. Concentrations of fecal cortisol metabolites increased on the second day of both outbound and return transports and reached a maximum the following day (P < 0.001). During the first 90 min on Day 1 of outbound transport, mean RR interval decreased (P < 0.001). Standard deviations of RR interval (SDRR) decreased transiently (P < 0.01). The root mean square of successive RR differences (RMSSD) decreased at the beginning of the outbound and return transports (P < 0.01), reflecting reduced parasympathetic tone. On the first day of both outbound and return transports, a transient rise in geometric HRV variable standard deviation 2 (SD2) occurred (P < 0.01), indicating increased sympathetic activity. In conclusion, transport of experienced horses leads to increased cortisol release and changes in heart rate and HRV, which is indicative of stress. The degree of these changes tended to be most pronounced on the first day of both outbound and return transport.     

Cortisol release,heart rate,and heart rate variability in transport-naive horses during repeated road transport

Domestic animals are often repeatedly exposed to the same anthropogenic stressors. Based on cortisol secretion and heart rate, it has been demonstrated that transport is stressful for horses, but so far, changes in this stress response with repeated road transport have not been reported. We determined salivary cortisol concentrations, fecal cortisol metabolites, cardiac beat-to-beat (RR) interval, and heart rate variability (HRV) in transport-naive horses (N = 8) transported 4 times over a standardized course of 200 km. Immunoreactive salivary cortisol concentrations always increased in response to transport (P < 0.001), but cortisol release decreased stepwise with each transport (P < 0.05). Concentrations of fecal cortisol metabolites increased from 55.1 ± 4.6 ng/g before the first transport to 161 ± 17 ng/g the morning after (P < 0.001). Subsequent transport did not cause further increases in fecal cortisol metabolites. In response to the first transport, mean RR interval decreased with loading of the horses and further with the onset of transport (1551 ± 23, 1304 ± 166, and 1101 ± 123 msec 1 d before, immediately preceeding, and after 60–90 min of transport, respectively; P < 0.05). Decreases in RR interval during subsequent transports became less pronounced (P < 0.001). Transport was associated with a short rise in the HRV variable standard deviation 2 (P < 0.001 except transport 1), indicating sympathetic activation. No consistent changes were found for other HRV variables. In conclusion, a transport-induced stress response in horses decreased with repeated transport, indicating that animals habituated to the situation, but an increased cortisol secretion remained detectable.     

Differentiating among horse (Equus caballus), donkey (Equus asinus) and their hybrids with combined analysis of nuclear and mitochondrial gene polymorphism

A novel and brief method of differentiating among horse (Equus caballus) and donkey (Equus asinus) and their hybrids (mule, E. asinus × E. caballus and hinny, E. caballus × E. asinus) with combined analysis of nuclear and mitochondrial gene polymorphism (CANMGP) was reported in the present report. A nuclear gene, protamine P1 gene of donkey was sequenced and compared with the known horse sequence from GenBank while a published equid mitochondrial gene, cytochrome b gene of donkey was compared with that of horse. In each of the two genes, a fixed nucleotide substitution within an exon that could be recognized by Dpn II restriction enzyme was found between the two species. Two pairs of primers were designed for amplifying the fragments within the two genes containing the informative nucleotide positions in 65 horses and 41 donkeys and 38 hybrids and conditions of polymerase chain reaction and restriction fragment length polymorphism (PCR‐RFLP) analysis were optimized. Horse, donkey and mule and hinny had their own specific cleavage patterns after the PCR‐RFLP analysis was performed, which made it very easy to identify them from each other. As multiplex PCR can be conducted with the two pairs of primers and only one restriction enzyme is involved in PCR‐RFLP analysis, the method described in the present study is a convenient way to identify horse and donkey and their hybrids. The idea involved in the method of CANMGP can be also used to differentiate other animal species or breeds and their hybrids.     

Reference intervals for biochemical and haematological parameters in mature domestic donkeys (Equus asinus) in the UK

Reference intervals (RIs) for haematology and serum biochemistry for donkeys in a temperate climate have been previously published using blood sample results from the resident population of a large donkey shelter in the UK. Periodic review of reference intervals is recommended to ensure their applicability to the patient population and changes in laboratory methods and technologies. The current study aimed to revise the previously published haematology and serum biochemistry values for the adult domestic donkey (Equus asinus) in the UK in the light of a change in analytical equipment at the Donkey Sanctuary laboratory, but also to refine the demography of the sample population with respect to age, physiology and clinical history. Clinical pathology results from 138 clinically healthy mature (4–24 years inclusive) female and castrated male donkeys selected from the resident population of the Donkey Sanctuary, were analysed retrospectively. The animals were blood sampled during the period February 2008 to June 2011 as part of a routine health screen prior to rehoming. Results for a total of 38 biochemical and haematological parameters were analysed including 3 previously unreferenced parameters in addition to those assessed in the previous study. The new reference intervals and median values show very poor transferability with recently derived reference intervals for non‐Thoroughbred horses and only limited transferability with reference intervals previously published for donkeys in the UK. Of particular note is a marked reduction in the upper reference limit for triglycerides of 2.8 mmol/l (from 4.3 mmol/l) since this parameter is used to decide when donkeys are at risk of developing hyperlipaemia. This study demonstrates the value of intermittent review of reference intervals and refinement of study populations. Notwithstanding the caution with which reference interval data from different laboratories should be compared, the lack of transferability of results between donkeys and horses highlights the importance of use of species‐appropriate reference intervals for clinical decision‐making.     

Physical, Hematological, and Biochemical Responses to Acute Intense Exercise in Polo Horses

The aim of this study was to evaluate the response of physical, hematological, and biochemical parameters after acute intense exercise in polo horses playing in an outdoor international competition. The game consisted of four periods (chukkas) and each period consisted a playing time of 7 minutes. Two matches were played everyday for a week. A total of 12 horses were examined. Each “high-goal” polo horse played one chukka a day for 4 days. Horses were clinically examined the day before the games started and then daily during the 4 days of their participation in the games. During these days, physical examination was performed and blood sample was collected at rest (T0), immediately (T1) after exercise, and after 30 minutes of exercise (T2). Blood samples were analyzed for total cell counts and for determination of creatine kinase, lactate dehydrogenase (LDH), aspartate aminotransferase, lactate, total proteins, calcium, magnesium, phosphorus , and cortisol. Data were evaluated using two-way analysis of variance. Exercise caused significant dehydration (P < .01), mucous membranes congestion, increased heart rate (P < .001), and capillary refill time (P < .001). It also caused increased value of the following parameters: hematocrit (P < .001), red blood cells (P < .001), hemoglobin (P < .001), white blood cells (P < .05), lymphocyte (P < .001), total proteins (P < .001), creatine kinase (P < .05), LDH (P < .01), lactate (P < .001), and cortisol (P < .01), and a decrease in the platelet count (P < .001), calcium (P < .01), phosphorus (P < .001), and magnesium (P < .001). All parameters returned within or near the reference range by 30 minutes postexercise. On the basis of these observations, data were considered indicative of a good response to an acute intense exercise. Moreover statistical results obtained were typical of a mixed aerobic/anaerobic metabolic pathway that is prevailing in this sport.     

Effects of dermal dexamethasone application on ACTH and both basal and ACTH-stimulated cortisol concentration in normal horses

There are no data available regarding the systemic (adverse) effects which might be induced by topical/dermal glucocorticoids (GCs) application in the horse. Besides their widespread use for the treatment of a variety of peripheral inflammatory disorders such as atopic dermatitis, eczemas or arthritis in the horse, their surreptitious application has become a concern in doping cases in competition/performance horses. Assessing both basal and ACTH‐stimulated plasma cortisol as well as basal ACTH concentrations following application of dexamethsone‐containing dermal ointment is necessary to determine influences on hypothalamus‐pituitary‐adrenal (HPA) axis. Ten clinically healthy adult standardbred horses (6 mares, 4 geldings) were rubbed twice daily each with 50 g dexamethasone‐containing ointment on a defined skin area (30 × 50 cm) for 10 days. RIA and chemiluminescent enzyme immuno‐metric assay were used to determine resting and ACTH‐stimulated plasma cortisol and basal ACTH concentrations, respectively. HPA feedback sensitivity and adrenal function were measured by a standard ACTH stimulation test. Dermal dexamethasone suppressed significantly the resting plasma cortisol level (to 75–98%) below baseline (P < 0.001) within the first 2 days and decreased further until day 10. ACTH stimulation test showed a markedly reduced rise in plasma cortisol concentrations (P < 0.001 vs. baseline). Plasma ACTH level decreased also during topical dexamethasone application. The number of total lymphocytes and eosinophil granulocytes was reduced, whereas the number of neutrophils increased. No significant change of serum biochemical parameters was noted. Dermal dexamethasone application has the potential to cause an almost complete and transient HPA axis suppression and altered leukocyte distribution in normal horses. The effects on HPA axis function should be considered in relation to the inability of animals to resist stress situations. The data further implicate that percutaneously absorbed dexamethasone (GCs) may cause systemic effects relevant to ‘doping’.