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Clenbuterol (β

Clenbuterol, (RS)-1-(4-amino-3,5-dichlorophenyl)-2-(tert-butylamino)ethan-1-ol, as Ventipulmin is an FDA approved β2 agonist medication for the management of airway obstruction in horses. Administration above the FDA approved doses for clenbuterol produces repartitioning effects, which have led to restrictions on its use in human athletics and Quarter Horse and Thoroughbred racing. Clenbuterol, however has long been used therapeutically at FDA approved doses in Harness racing. The goal of this study was to identify a withdrawal time guideline for its use at FDA approvsed dose levels in Harness racing, where horses may start at seven-day intervals. Eight healthy, moderately fit Standardbred horses (4 mares, 4 geldings, weight 491±40 kg, age 13±2 years) were administered 0.8 μg/kg of clenbuterol as Ventipulmin syrup twice daily (BID) for three days. Blood samples were collected prior to dosing and at 1, 24, 48 and 96 h post administration. Clenbuterol was quantified in all samples using the New York Drug Testing and Research Laboratory ISO-17025 Racing and Medication Testing Consortium (RMTC) accredited quantitative procedure. The lower limit of quantitation of the method was 1.0 pg/ml, and three data points at 96 h post administration were censored. One horse developed diarrhoea and data from this horse was excluded from the overall analysis. Plasma regulatory thresholds were calculated using the 95/95 tolerance method and Gauss Camp Meidell at P=0.05 and P=0.001. Horses were also evaluated for effects of clenbuterol on body composition using body mass and ultrasound measurements of rump fat thickness. There were no effects (P>0.05) of clenbuterol on any of the measures including fat mass and fat free mass and thus no repartitioning effect was observed. The pharmacokinetic data and the 96 h data set support the therapeutic use of clenbuterol in Harness horses at the FDA approved 0.8 μg/kg BID dose for three days and suggest a 41 pg/ml regulatory threshold for a 96 h withdrawal time with a P=0.001 probability of randomly exceeding this regulatory threshold.

The objective of this study was to investigate the changes in Glut-4 expression in gluteus muscle, and in the plasma concentrations of hormones and metabolites during the suckling period, after weaning, and after exercise training in foals. Our hypotheses were Glut-4 abundance will decrease following metabolite and hormonal concentration between neonatal period and late development but will increase with exercise training. Eight clinically normal Standardbred foals were used in this study, and they stayed with their mothers from birth until weaning at 6 months. After weaning, the foals were randomly divided in two groups: an exercise group (EX) (n=4; ~282 kg) which trained 3 d/wk for 12 wks and a control group (n=4; ~271 kg) without structured exercise. Venous blood samples were obtained immediately after birth (0.05). Plasma lactate, insulin and cortisol concentrations decreased during the first three months of lactation while glucose and leptin concentrations increased (P0.05). In conclusion, Glut-4 abundance in gluteus muscle was highest at the birth and decreased during development and exercise training for 12 weeks did not change its’ abundance.

A commonly held belief is that lactate production during exercise limits performance via fatigue. The current study aimed to assess the effects of a supplement marketed as performance enhancing (Lactanase®) on equine exercise performance and plasma lactate concentrations. Lactanase was expected to alter exercise performance and plasma lactate. Eight healthy, unfit Standardbred horses (four geldings and four mares, 9±3 years, 489±33 kg, mean ± standard error) were administered Lactanase (5 doses of 25 g) top-dressed on a commercial pelleted ration or unsupplemented pellets (Control) in a cross-over design experiment. Doses were administered over a four-day period and horses performed a graded exercise test on the fourth day following the final dose of either Lactanase or control. Blood samples (20 ml) were obtained via jugular catheter 30 min prior to exercise, immediately prior to exercise, in the last 10 s of each incremental exercise step, and 5 and 60 min post-exercise. Samples were frozen at -80 °C until further analysis. Data were analysed using a two-way repeated measures ANOVA. Statistically different means were separated using Student-Newman-Keuls and Dunnetts. No differences (P>0.05) in total run time were observed following Lactanase administration. Plasma lactate increased (P0.05) of Lactanase on the plasma lactate response during exercise. Similarly, supplementation did not affect (P>0.05) plasma glucose or total protein at any time point. Exercise did increase plasma total protein (P

Cobalt is a required trace element in animals, but administration in excess is considered dangerous and potentially performance enhancing in equine athletes. This study seeks to determine if cobalt may actually act as a performance enhancing drug (PED) by altering biochemical parameters related to red blood cell production as well as markers of aerobic and anaerobic exercise performance. In addition, for adequate regulation of naturally occurring substances, such as cobalt, its distribution among the population must be defined. In order to identify this distribution, plasma Cobalt was determined from 245 Standardbred horses with no cobalt supplementation from farms in New York and New Jersey, including horses at the Rutgers University Equine Science Center. Samples were analysed by Inductively Coupled Plasma Mass Spectrometry. Seven healthy, race fit Standardbreds (4 geldings, 3 mares, age: 5±3 years, ~500 kg) were used for the PED experiment. An incremental graded exercise test (GXT) to measure maximal aerobic capacity (V̇O2max) and markers of performance, measurement of plasma volume and blood volume as well as the measurement of lactate, erythropoietin (EPO), and various blood haematological factors were determined 7 days prior to cobalt administration. Each horse was administered a sterile solution of cobalt salts (50 mg of elemental Co as CoCl2 in 10 ml of saline, IV) at 9 AM on three consecutive days via the jugular vein. Blood samples were obtained from the contralateral jugular vein before and at 1, 2, 4 and 24 h after administration. Plasma and blood volume were measured one day after the last dose of cobalt, and a post administration GXT was performed the next day. Horses were observed for signs of adverse effects of the cobalt administration (agitation, sweating, increased respiration, etc.). Plasma cobalt concentration increased from a pre-administration mean of 1.6±0.6 to 369±28 μg/l following 3 doses of the cobalt solution (P0.05). There were no observed adverse effects.

metabolomics is the high-throughput, multiparametric identification and classification of hundreds of low molecular weight metabolites in a biological sample. Ultimately, metabolites are the downstream readouts of cellular signalling, transcriptomic and proteomic changes that can provide a comprehensive view of tissue and organismal phenotype. The popularity of metabolomics in human sport and exercise has been gaining over the past decade and has provided important insights into the energetic demands and mechanistic underpinnings of exercise and training. To the contrary, metabolomics in the field of equine exercise physiology is lagging despite the horse's superior aerobic and muscular capabilities, as well as its prominence in competitive sport. As such, this narrative review aims to describe metabolomics, its routine implementation, the various analytical methods applied and the state of its use in the equine athlete. Sufficient attention will be paid to methodological considerations, as well as gaps in the equine literature, particularly with regard to the skeletal muscle metabolome. Finally, there will be a brief discussion of the future directions and barriers to metabolomics use in the athletic horse. A thorough understanding of the metabolomics changes that occur in the equine athlete with exercise will undoubtedly help to improve horse management and health across the lifespan.

This study tested the hypothesis that 12 weeks of training would significantly alter maximal aerobic capacity (VO2max), running capacity, and body composition in mature Standardbred horses, and that these alterations would be maintained over an additional 60 weeks of training. It was also hypothesised that there would be a reversal of the training-induced adaptations with 20 weeks of detraining. Eight rested Standardbred horses (n=4 mares, n=4 geldings, 3-8 years) were trained for 72 weeks and then undertook 20 weeks of detraining in groups of four per two-acre paddocks. Body composition, VO2max, run time to fatigue, and distance completed were assessed before training, at 12 and 72 weeks of training, and after 20 weeks of detraining (92 weeks). VO2max (P

The purpose of this study was to examine the effects of an external nasal strip on thermoregulation during submaximal exercise in Standardbred horses. While several studies have been conducted to determine the effects of the external nasal strip on airway resistance, exercise induced pulmonary hemorrhage, gas exchange, and time to fatigue in maximally exercising horses, the effects of the nasal strip on equine thermoregulation have not yet been examined. It was hypothesized that the application of an external nasal strip would alter central venous temperature (T

Objective, automated early lameness detection plays an important role for animal well-being. The work in this paper uses horse locomotion data collected by wearable inertial measurement units, extracts gait cycle routines and constructs a multi-layer classifier for horse lameness detection, identification, and evaluation. Multi-layer classifier (MLC) is based on support vector machine and K-Nearest-Neighbors methods. Each layer is independently designed and works as a binary classifier. Horse gait classification and limb lameness detection and evaluation are then handled by each layer successively. Experiment results show that the MLC achieves 94 % detection accuracy and also generates superior performance than a deep convolutional neural network-based multiclass classifier in terms of various assessment criteria.

Sturkie's Avian Physiology is a highly regarded textbook for the study of comparative poultry physiology. Less well known, however, is the contribution of Paul D. Sturkie (1909-2002) as a pioneer in the experimental physiology of avian species. His seminal research on the cardiovascular and hemodynamic controls of chickens and egg-laying hens had a notable impact on the poultry industry and breeding practices of farmers. The purpose of this article is to highlight the contributions and practical insights of Paul D. Sturkie to the field of poultry science.

Two groups of unfit Standardbred mares (adult: 9-14 years, 540 kg, n=7) and old (20-25 years, 530 kg, n=5) were used to test two hypotheses, first, that aging and training would alter plasma and muscle glutamine [Gln] and glutamate [Glu] and second, that aging and training would alter Glut-4 expression in skeletal muscle. All animals were housed on pasture with free access to grass and all received hay and supplementation with a commercially prepared supplement (15% crude protein and 3.00 Mcal/kg dry matter) in individual stalls. Mares were fed to meet or exceed NRC (2007) nutrient recommendations for moderate to heavy exercise. The mares were exercise trained in a free-stall motorised circular exercise machine for 30 min/d, 5 d/week, for 8 weeks. Work intensity during training was set at a relative intensity of ~60% of the maximum heart rate, previously determined during an incremental exercise test (GXT). Blood samples and muscle biopsies (gluteus) obtained before and after 8 weeks of training were used for measurement of [Gln], [Glu] and Glut-4 abundance. Samples were collected before the initiation of training and at 24 h after cessation of last bout of exercise in the training period. All samples were immediately frozen in liquid nitrogen and stored at -80 °C until enzymatic analysis for [Gln], [Glu] and Western Blot analysis for Glut-4 protein abundance. Data were analysed by one-way or two-way ANOVA for repeated measures and the Pearson correlation method. Post-hoc differences were identified with the Tukey test. Significance was set at P0.05) in muscle [Glu] due to aging. Training decreased (P0.05) due to training. Glut-4 abundance analysis did not differ (P>0.05) between the young adult and old horses; however, there was a trend (P=0.063) towards an effect of training when samples from both groups were pooled. It was concluded that training and aging produce changes in plasma and muscle [Gln], which may affect immune function in athletic horses, but not in Glut-4.

While exercise has been found to change the faecal microbiome (FM) in laboratory animals exposed over weeks, no studies have identified immediate changes in the FM associated with short spans of intense exercise, ~5 min. The purpose of this study was to test the hypothesis that acute intense exercise would alter the FM in horses. Each horse performed two rounds of testing undergoing both a graded exercise test (GXT) and a parallel standing control (SC) trial before (GXT1 and SC1) and after (GXT2 and SC2) 12 weeks of exercise training. Rectal faecal samples were taken 24 h before and after testing. Bacterial community analysis was done by sequencing the 16s rRNA (V3-V4) region via Illumina Miseq. The relative abundance of the genus Clostridium significantly decreased in SC1 (P0.05). Our results indicate that very intense acute exercise does not alter the faecal microbiome of the Standardbred race horse and that 12 weeks of exercise training does not alter that response.

Effects of social isolation, sham clipping, and novel object exposure on muscular and behavioral responses were investigated in horses. Humans increase muscular tension under psychologically or physically demanding situations and thus, similar conditions were expected to alter stress responses in horses. Eight mature Standardbreds (4 mares, 4 geldings) were exposed to 3 min of social isolation (ISO), sham clipping (CLIP), novel object (NOV), and control (CON) conditions in a replicated 4×4 Latin Square experimental design with 10 min washout periods. Surface electromyography (EMG) and stress-related behaviors were recorded continuously. Median frequency (MF) and average rectified value (ARV) of the EMG signal were calculated for the first, middle, and final 10 sec of each period. EMG data were log transformed prior to analysis with a mixed model, repeated measures ANOVA. Behavior data were analyzed using a negative binomial distribution mixed model ANOVA. Significantly different means were separated using Tukey’s method. More stress-related behaviors (P < 0.05) were observed during ISO (3.25 ± 0.26, LSM ± SE) than CON (1.46 ± 0.29) or CLIP (1.50 ± 0.36). ISO tended (P = 0.054) to produce more stress-related behaviors than NOV (2.31 ± 0.28). CLIP and ISO produced higher (P < 0.05) ARV than CON or NOV in the left and right masseter. CLIP elicited the highest (P < 0.05) MF in the left and right masseter with ISO resulting in lower (P < 0.05) MF than CON. ARV was higher (P < 0.05) in the left cervical trapezius during ISO as compared to all other conditions and in the right as compared to CON and CLIP. In the right cervical trapezius, ISO elicited higher (P < 0.05) MF than CON. Increased stress-related behaviors indicate a stress response was induced. Elevated muscular activity suggests muscular tension can be used in assessing stress.

Anecdotal evidence suggests high horse turnover is a concern in the field of equine assisted services (EAS) with potential negative effects on sustainability. Members of key EAS organizations (American Hippotherapy Association; Professional Association of Therapeutic Horsemanship, International; eagala; Certified Horsemanship Association for a total of 26,000 recipients) were surveyed to characterize horses in EAS programs, their use, and causes of retirement. We expected respondents to indicate horses remained in their programs for relatively short periods of time and list behavior as a leading cause of retirement. The survey (QualtricsⓇ) was distributed via email using a repeat contact strategy of 4 contacts spaced 2–4 wk apart. Descriptive statistics were computed in SAS (v 9.4). Kendall's correlation with Bonferroni correction was used to analyze relationships of retirement causes and longevity with selection procedures and workload. Respondents (n = 176 completed responses; 0.14% response rate) reported 12 ± 8 (mean ± SD) horses and ponies were active in their programs. Animals ranged in age from less than 5 yr (2 ± 5% of animals in program) to 36–40 yr (0.1 ± 1.3%) with the greatest number falling between 16 and 20 yr (34% ± 22%). Donation (40 ± 34% of animals in program), purchase from private owner (23 ± 31%), and free lease (18 ± 26%) were reported as the most common means of acquiring horses and ponies. Animals had been active in the program for less than one yr (4.5 ± 12% of animals in program) to over 20 yr (4.4 ± 18%) with the greatest percentage of animals active for 1–5 yr (37 ± 35%) followed by 6–10 yr (31 ± 31%). Respondents indicated horses and ponies typically worked anywhere from 0 to 1 h/wk (0.6% of respondents) to 42–48 h/wk (0.6%) with the largest number working 4–8 h/wk (20%). When asked reasons for retiring horses and ponies, respondents ranked (1 = most frequent; 7 = least frequent) age (2, 1–3; median, IQR), behavior (2, 1–3), and unsoundness (2, 1–3) as the most frequent reasons followed by burnout (2.5, 1–4), other health issues (3, 2–3), death (4, 2–4), lease expiration (5, 3.5–5), and other reasons. Selection procedures and workload were not significantly corelated with reason for retirement or longevity. Further research is needed to understand how burnout, a behaviorally assessed state, differs from behavioral reasons for retirement and its implications. Collection and analysis of other variables, potentially at an individual animal level, is needed to further our understanding of factors contributing to horse longevity and retirement.

There are many choices for methods of extracting bacterial DNA for Next Generation Sequencing (NGS) from fecal samples. Here, we compare our modifications of a phenol/chloroform extraction method plus an inhibitor removal solution (C3) (ph/Chl+C3) to the PowerFecal® DNA Isolation Kit (MoBio-K). DNA quality and quantity coupled to NGS results were used to assess differences in relative abundance, Shannon diversity index, unique species, and principle coordinate analysis (PCoA) between biological replicates. Six replicate samples, taken from a single ball of horse feces manually collected from the rectum, were subjected to each extraction method. The Ph/Chl+C3 method produced 100× higher DNA yields with less shearing than the MoBio-K method. To assess the methods, the two method samples were sent for sequencing of the bacterial V3-V4 region of 16S rRNA gene using the Illumina MiSeq platform. The relative abundance of Bacteroidetes was greater and there were more unique species assigned to this group in MoBio-K than in Ph/Chl+C3 (P

Exercise has a significant effect on different physiological systems in the body of human and animals. Only limited numbers of published studies in laboratory animals or humans have shown the effect of exercise on the gut microbiota, and no studies have shown this effect in horses. In this study, 8 horses (4 mares, 4 geldings) were exercise trained for 12 weeks, and 4 additional mares were used as a parallel seasonal control. To identify bacterial community changes over time for both groups, rectal faecal samples were collected, DNA was extracted, and the 16S rRNA gene (V3-V4) was sequenced using the Illumina Miseq platform. One-way ANOVA, Shannon diversity index, and Principal Coordinate Analysis (PCoA) were used to identify differences between and among samples. The exercise training group showed significant changes in the levels of Bacteroidetes, Proteobacteria, and Spirochaetes phyla (P0.05). Moreover, with training two genera significantly changed in their relative abundance over time, namely Clostridium and Dysgonomonas (P

An antioxidant derived from a variety of melon purported to be high in superoxide dismutase was fed to horses and the effect on performance and recovery was measured following repeated daily bouts of intense exercise. In a cross-over design study, six unfit Standardbred mares were fed this antioxidant at a rate of 2.0 IU/kg body weight (BW)/d as part of the daily ration for 4 weeks before undergoing a simulated race test (SRT) repeated over a 3-day period. The SRT, designed to simulate the training of Standardbreds, consisted of a 2 min warm-up at 6 m/s followed by a high speed run to fatigue at 125% of the speed calculated to correspond to the horse’s maximal aerobic capacity (VO2max) as determined in a baseline incremental exercise test. Measurements included VO2 and the VO2 recovery time, VO2 at the point of recovery, run time, as well as plasma lactate, and plasma cortisol. Oxidative stress was measured by assessing malondialdehyde (MDA). The results from the study demonstrated that there were no differences (P>0.05) in mean values for the plasma concentrations of lactate, cortisol, or MDA. However, when supplemented with this antioxidant, there was an observable pattern with a small-to-medium effect size of an increase average run time of 14 s. Another important observation was that despite the longer run time, there was a moderate effect for a faster VO2 recovery time. The data suggest that supplementation with 2.0 IU/kg BW of this antioxidant had a modest but non-significant effect on performance and recovery time. Additionally, it did not negatively impact physiology in untrained Standardbred mares.

Six Standardbred mares (age 12±2 years, body weight 502±63 kg; mean ± standard deviation) were given 1.6 µg/kg acylated human ghrelin or vehicle treatment as an intravenous bolus in a randomised, cross-over design to test the hypothesis that exogenous ghrelin infusion would increase feed intake and alter metabolic parameters of energy homeostasis, leptin, glucose, insulin and cortisol. After the horses had initial access to hay cubes for 1.5 h, doses were given and hay cubes were available once again. Leftover feed was weighed 6 times over each of the 24 h testing periods. Blood samples for measurement of active ghrelin, growth hormone, leptin, glucose, insulin and cortisol were taken at time 0 (immediately before infusion) and 20, 40, 60, 80, 100, 120, 240, 480, and 720 min post-infusion. Every 10 min, the horses’ behaviour was recorded for eating, drinking, resting, and other behaviours. Ghrelin infusion did not increase (P≯0.05) feed intake in the mares as a group, but did increase feed intake (P

This study tested the hypotheses that age-induced alteration in cortisol, ACTH, and glucose concentrations are due to differences in the response of the hypothalamic-pituitary-adrenal axis and that exercise training would attenuate these differences. Six old (22.0±0.7 yr; mean±SE) and 6 young (7.3±0.6 yr) unfit Standardbred mares ran 3 graded exercise tests (GXT): before (GXT1), after 8 wk of training (GXT2), and at study end at 15 wk (GXT3). Mares trained 3 d/wk at 60% maximum heart rate. Each mare underwent 5 endocrine stimulation tests pre- and posttraining: 1) control (CON), 2) adrenocorticotropin hormone (ACTHtest), 3) combined dexamethasone suppression/ACTH (DEX/ACTH), 4) dexamethasone suppression (DEX), and 5) combined DEX/corticotropin releasing factor (DEX/CRF). For CON, there was no difference in plasma cortisol between age groups pretraining (P=0.19), but young mares had a 102% higher mean (P=0.02) plasma cortisol concentration than old mares posttraining. The pretraining ACTHtest showed young mares had a 72% higher (P=0.05) overall plasma cortisol concentration compared to old. There was no overall age difference in cortisol in the posttraining ACTHtest, but old mares still had lower cortisol concentrations at 30 min during the test, suggesting decreased adrenal response to ACTH stimulation. There was no difference in cortisol response between old and young mares in DEX, DEX/ACTH, or DEX/CRF tests. Young mares had higher (P=0.02) overall plasma cortisol concentration posttraining in response to DEX/ACTH, but old mares showed no change. In CON and DEX/CRF, there were no age differences in plasma ACTH concentration, pre- or posttraining. Pretraining, there was no age difference in glucose response to DEX, but posttraining old mares had a 4% (P=0.04) lower overall plasma glucose concentration compared to young. Posttraining, old mares had lower mean plasma glucose concentrations during DEX compared to pretraining (P=0.02), but there was no change pre- vs. posttraining in young mares (P=0.19). Old and young mares had lower plasma glucose concentrations posttraining during DEX/ACTH (P

The chronic bradycardia seen in several species after intense exercise training may be due to autonomic mechanisms, non-autonomic mechanisms, such as increased pre-load, or a combination of the two. Thirteen, healthy, unfit Standardbred mares were split into two groups: young (age 12±1 yr; mean ± standard error, n=8) and old (age 22±1 yr, n=5) to test the hypothesis that there would be age and training related differences in resting heart rate (RHR), intrinsic heart rate (IHR), maximal heart rate (HRmax) and plasma volume (PV). Mares were trained 3 d/wk at 60% HRmax for 20 min and gradually increased to exercising 5 d/wk at 70% HRmax for 30 min and RHR, IHR, HRmax, and PV were measured prior to and after the 8 wk training period. There were no age related differences (P≯0.05) between young and old mares before (41±2 vs. 42±2 beats per minute (bpm); 86±5 vs. 80±4 bpm) or after training (35±1 vs. 34±1 bpm; 81±6 vs. 78±2 bpm) for RHR and IHR respectively. RHR was decreased (Pmax than the old horses (P

Clenbuterol was intended as a treatment for respiratory diseases in horses, but has been used in multiple species, including humans, for its repartitioning of fat to lean effects (free fatty acids are released from adipose tissue to be used by tissues of higher priority). In the horse industry clenbuterol application is restricted to the treatment of chronic obstructive pulmonary disease and reactive airway disease (heaves). Negative effects of clenbuterol exposure include a decrease in maximum oxygen intake and increased muscle fatigue upon exercise. As a result of these and other negative effects, clenbuterol remains strictly controlled by the US Food and Drug Administration.

The purpose of this study was to determine the metabolic effect(s) of four graded exercise tests (GXT) performed on the underwater treadmill (UWT), and compare them to the results from performing the standard Bruce protocol on a traditional land treadmill (LT). Twelve male Division I college athletes performed the stand Bruce protocol on a LT and 4 different GXT protocols on the UWT. Each test was performed to volitional max. oxygen consumption (VO2), heart rate (HR), respiratory exchange ratio (RER) and blood lactate (BL), which were measured at regular intervals throughout each of the GXTs. A two-way ANOVA with repeated measures was used to determine any main effect among the variables within the protocols and within the stages. Established effects were identified further using Tukey's post-hoc analysis. VO2and HR were positively correlated in all the GXTs, (r=0.992−0.999). When comparing the UWT GXTs to the Bruce, significant (P≤0.001) differences were found within certain stages for each of the dependent variables. While the Bruce resulted in attainment of VO2max, (mean = 55.72±5.92 ml/kg/min), only one of the UWT GXTs resulted in the same level of metabolic stress. At maximum speed and water velocity, the UWT produced a mean VO2of 53.9±3.68 ml/kg/min. HR and RER both increased by stage in all the GXTs as did BL. The onset of blood lactate accumulation (OBLA) was identified in all of the GXTs with the exception of one of the UWTs. Mean BL at OBLA ranged from, 3.12+1.31 to 5.98+4.24 mmol/dl. Our results suggest that an UWT GXT protocol can be developed that would produce metabolic results similar to the Bruce through stage 5. Buoyancy and variation in biomechanics need to be studied further for their effects on energy metabolism while performing work on the UWT. However, this study provides a better understanding of the metabolic demands at different treadmill speeds and water jet settings with the UWT.

This study tested the hypothesis that old and young mares exhibit different endocrine responses to a frequently sampled intravenous glucose tolerance test (FSIGT) and different cytokine profiles in blood, adipose and muscle tissues. It was also hypothesised that exercise training alters endocrine and tissue cytokine profiles. Pilot data from 15 mixed background horses indicated tissue differences in cytokine profiles. For the main study, six old (22.0±0.7 years) and six young (7.3±0.6 years; mean±SE) unfit Standardbred mares were tested pre- and post-training. Exercise training occurred three days/week for 15 weeks at ~60% maximum heart rate. Plasma insulin and glucose concentrations were measured via radioimmunoassay and enzyme-electrode interface, respectively. Samples of blood, middle gluteal muscle (RM), and subcutaneous adipose tissue from the neck (NF) were collected pre- and post-training for mRNA quantification. Minimal model analysis of FSIGT, repeated measures ANOVA and Pearson Product Moment were used to analyse data. The null hypothesis was rejected when P≤l0.10. Post-training, old and young mares improved insulin sensitivity (SI) (P=0.08, P=0.01, respectively) and disposition index (P=0.04, P

In order to understand how gaited horses use their energy during exercise, a standardised field gaited test (SFGT) was developed to assess energy expenditure of four beat gaited horses independently of size, sex or breed. This work aimed at developing such an SFGT, using as main measurement parameter the heart rate (HR) of horses during the SFGT performance. Thirty-one four beat gaited horses were evaluated and divided into two groups: FIT (conditioned) and UNFIT (not conditioned). Horses were submitted to the SFGT and their heart rates were measured with a heart rate monitor as follows: right after being mounted, at the beginning of pre-test (HRSADDLE); at 5, 10, 15, 20, 25 and 30 minutes of four beat gait dislocation; and at 15 minutes after the recovery period (T+15). Maximum HR (HRMAX); HR percentage over 150 beats per minute (HR%≯150), HR percentage over 170 beats per minute (HR%≯170), and average HR during the four beat gait stage (HRM@M) of SFGT were calculated. Results were analysed by ANOVA for repeated measures. Where significant differences were observed, ‘T’ test was performed and significance was set at 5%. The FIT group presented HRMAX, HR+15, HRM@M, HR%≯150 and HR%≯170 rates lower (PMAX (R=−0.67; PMAX and HR+15 (R=0.60; P

Summary Reasons for performing study: Mangalarga-Marchador is a popular 4-gaited Brazilian horse breed; however, there is little information about their metabolic and physiological response to exercise. Objectives: To measure physiological and metabolic responses of the Mangalarga-Marchador to a simulated marcha field test and to compare these responses between 2 types of marcha gaits (picada and batida). Methods: Thirteen horses were used in the study and randomly assigned to either the picada or batida gait for the simulated marcha field test (speed ∼3.2 m/s; 30 min; load ∼80 kg). Measurements: Included body composition, heart rate (HR), respiratory rate (RR), glucose (GLUC), lactate (LACT), packed cell volume (PCV), total plasma protein (TPP), albumin, urea, creatinine, total and HDL cholesterol, triglycerides, creatine kinase, alanine, glutamate and glutamine (GLN). Measurements were obtained pretest (control/fasting), immediately after simulation (T0), and 15 (T15), 30 (T30) and 240 (T240) min after the simulation. Lactate (LACT) was measured at T0, T15 and T30. Data were analysed using ANOVA, Tukey's test and t tests with significance set at P

To help resolve the mechanistic bases for haematological adaptations (~28% increase in red blood cell volume) of equids to high altitude (3800 m, barometric pressure Pb, 487 mm Hg) and exercise, plasma erythropoietin concentration ([EPO]) was measured at rest and following exercise in six, moderately fit equids (four Arabians, one Quarter Horse and one Shetland Pony; four females and two males; age 9.0 ± 4.5 years (mean ± SD)). [EPO] was measured on 2 days at 225 m (i.e. ~sea level; Pb, 743 mm Hg), over the course of a 10-day altitude exposure, and then again for 2 days after return to sea level. A standard track exercise test (submaximal, speed set-to-heart rate of 110 (trot), 150 (canter), 180 (gallop) bpm) was performed 2 days pre-high-altitude exposure and on three separate days at high altitude. In addition, a maximal incremental exercise test was performed on a high-speed motor-driven treadmill at sea level and 2 days following return to sea level from high altitude. Resting [EPO] increased from 28 ± 29 at sea level to 144 ± 46 mU ml− 1 (P 0.05 vs. pre-high altitude) and did not change over the remaining 8 days at high altitude nor over the 2 days after return to sea level. [EPO] was not significantly altered by acute exercise at sea level or at 3800 m. These results indicate that [EPO] increases rapidly (though transiently) in response to hypobaric hypoxia but not to acute exercise, and that exercise does not appear to potentiate the altitude response. Thus, if any [EPO]-derived haematological adaptations to high altitude are present, these appear to result from a transient ~4-fold elevation of [EPO] rather than any sustained increase in this signalling mechanism, at least in the equid.

Summary Reasons for performing study: Studies have demonstrated increases in mRNA expression for inflammatory cytokines following exercise in horses and have suggested those markers of inflammation may play a role in delayed onset muscle soreness. However, measurement of mRNA expression in white blood cells is an indirect method. No studies to date have documented the cytokine response to exercise directly in muscle in horses. Hypothesis: This study tested the hypothesis that exercise increases cytokine markers of inflammation in blood and muscle. Methods: Blood and muscle biopsies were obtained from 4 healthy, unfit Standardbred mares (∼500 kg). The randomised crossover experiment was performed with the investigators performing the analysis blind to the treatment. Each horse underwent either incremental exercise test (GXT) or standing parallel control with the trials performed one month apart. During the GXT horses ran on a treadmill (1 m/s increases each min until fatigue, 6% grade). Blood and muscle biopsies were obtained 30 min before exercise, immediately after exercise and at 0.5, 1, 2, 6 and 24 h post GXT or at matched time points during the parallel control trials. Samples were analysed using real time-PCR for measurement of mRNA expression of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1 (IL-1). Data were analysed using t tests with the null hypothesis rejected when P0.10) in IL-1, IL-6, IFN-gamma or TNF-alpha during control. Exercise induced significant increases in IFN-gamma, IL1 and TNF-alpha in blood and significant increases in IFN-gamma, IL-6 and TNF-alpha in muscle. There were no significant changes in mRNA expression of IL-1 in muscle or IL-6 in blood following the GXT. These cytokine markers of inflammation all returned to preGXT levels by 24 h post GXT. Conclusion: High intensity exercise results in a transient increase in the expression of inflammatory cytokines in muscle and blood.

Summary Reasons for performing study: Many nutraceuticals are used as equine supplements without their efficacy having been scientifically tested. Black tea, cranberries, orange peel and ginger are a few of those nutraceuticals that warrant further study. Objective: To test the effects of single doses of black tea, cranberry, orange peel and ginger extract on markers of oxidative stress and antioxidant status following exercise in horses. Methods: In Study 1, 9 mature, healthy but unfit Standardbred mares were administered 2 l of a control (water), orange peel extract (30 g extract) or decaffeinated black tea extract (28 g extract). In Study 2 the same mares were administered 2 l of a control (water), cranberry extract (30 g extract) or ginger extract (30 g extract). In each study, mares were given the extracts via nasogastric tube 1 h before performing a graded exercise test (GXT), in a randomised crossover design with at least 7 days between GXTs. Blood samples were collected at rest, at fatigue, and 1 and 24 h post exercise and analysed for lipid hydroperoxides (LPO), total glutathione (GSH-T), glutathione peroxidase (GPx), α-tocopherol (TOC), β-carotene (BC) and retinol. Data were statistically analysed using a repeated measures ANOVA. Results: In Study 1 there was no effect of treatment for LPO, GSH-T, GPx, TOC or BC. Retinol was higher for both tea (P = 0.0006) and water (P = 0.004) than for orange peel. In Study 2 there was no treatment effect for LPO, GPx, GSH-T, RET, BC or TOC. Conclusions: The results show that a single dose of various nutraceuticals in exercising horses do not produce an effect on either oxidative stress or antioxidant status and further investigation is needed as to whether long-term supplementation would enhance these effects.

Summary Reason for performing study: Older horses have an increased risk of hyperthermia due to impaired cardiovascular function. While many studies have investigated thermoregulation in horses during exercise, none have investigated the effects of ageing. Objective: To test the hypothesis that there is a difference in thermoregulation during exercise and plasma volume (PV) in young and old horses. Methods:Study 1: 6 young (Y, 7.7 ± 0.5 years) and 5 old (O, 26.0 ± 0.8 years) unfit Standardbred mares (507 ± 11 kg, mean ± s.e.) ran on a treadmill (6% grade, velocity calculated to generate a work rate of 1625 watts) until core temperature reached 40°C. Core (CT), skin (ST), rectal temperature (RT) and heart rate (HR) were measured every min until 10 min post exertion. Packed cell volume (HCT), lactate (LA) and plasma protein (TP) were measured in blood samples collected before, at 40°C and every 5 min until 10 min post exercise. Sweat loss was estimated using bodyweight. Study 2: Plasma volume was measured in 26 young (8.2 ± 0.7 years) and 8 old (26.6 ± 0.7 years) Standardbred mares (515 ± 12 kg) using Evans Blue dye. Pre-exercise blood (rBV) and red cell (rRCV) volumes were calculated using PV and HCT. Data analysis utilised repeated measures ANOVA and t tests and data are expressed as mean ± s.e. Results: Old horses reached 40°C faster (998 ± 113 vs. 1925 ± 259 s; P 0.05) post exercise. Age did not alter (P>0.05) CT, ST, RT, LA, HCT or TP. Plasma volume was greater in Y vs. O horses (P

The objective of this experiment was to test the hypothesis that ageing and training alter plasma concentrations of the peptide hormone leptin. The rationale for the study is based on prior investigations performed at Rutgers University, where published reports documented that ageing disrupts the immune and endocrine responses to acute exercise. The training period for mature young (7.3 ± 0.6 years; n = 6) and old (22 ± 0.7 years; n = 6) (mean ± SE) Standardbred mares was the duration of the summer of 2009 at the Rutgers University Equine Exercise Physiology Laboratory. Mares exercise trained in groups of six in a free-stall motorized circular equine exercise machine for 30 min, three times per week. Each mare performed a graded exercise test before beginning the training, after 8 weeks and after 16 weeks of training. There was no difference in body mass due to age (P>0.10). Training caused an increased body mass in both old and young mares (P

This study tested the hypothesis that phenylbutazone would block the exercise-induced increase in cytokine markers of inflammation in blood. Blood samples were obtained from unfit Standardbred mares (age 10 ± 4 years, ~500 kg) before and after three different trials (standing control (CON), n = 9; exercise with phenylbutazone (EX-bute), n = 9; and exercise with water, n = 9). Comparisons were made for data collected in three trials, one where each horse underwent an incremental exercise test (graded exercise test (GXT)) where they were administered water as a placebo, a GXT following phenylbutazone administration (2 g given orally 2 h before the GXT) or standing parallel control where they stood quietly in stalls. During the GXT, horses ran on a treadmill (1 m s − 1 increases each min until fatigue, 6% grade). Blood samples were obtained 30 min before exercise, immediately after exercise and at 0.5, 1, 2, 4 and 24 h post-GXT or at matched time points during the parallel control trials. Samples were analysed using real-time PCR for measurement of mRNA expression of interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and interleukin (IL)-6 in samples collected during all three trials, and for IL-1 and IL-10 in samples collected for the CON and EX-bute trials. Data were analysed using ANOVA for repeated measures, and where appropriate, post hoc separation of means utilized the Student–Newman–Keuls test. The null hypothesis was rejected when P P >0.05) in IL-1, IL-6, IFN-γ or TNF-α during CON or following phenylbutazone administration. During the water trial, exercise resulted in significant increases in IFN-γ, IL-1 and TNF-α. It was concluded that high-intensity exercise results in a transient increase in the expression of inflammatory cytokines in blood that is blocked when phenylbutazone is administered to horses.

Summary The purpose of the present study was to determine whether the training-induced hypervolemic response seen in the horse is associated with aldosterone-mediated renal mechanisms affecting sodium conservation during the first days of training. Five healthy, Standardbred mares (weight 450–500 kg, age 4–8 years) that were unfit, but accustomed to running on the treadmill, were used to test the hypothesis that repeated submaximal exercise would alter plasma aldosterone (ALDO) concentration and renal excretion of electrolytes in horses within the first 3 days of training. The experiment consisted of a 2 week housing equilibration period followed by a 1 week control period and a 3 day exercise training period (30 min/day at 60% VO2max). During control, ALDO and renal fluid and electrolyte losses were measured for 24 h on 3 separate days. Renal function (urine volume [UV], 24 h excretion of Na+, K+ and CI- [UNA+V, UK+V, UClV], clearance of Na+ [CNa+], K+ [CK+] and Cl− [CCl-], creatinine [CCr], osmotic substances [Cosm], and solute-free water [FWC], and the fractional excretion of Na+, K+ and Cl−) and ALDO were measured for an additional 3 consecutive days during the training period. There were no differences (P>0.05) in any variable during the control period. Plasma volume increased (+18.7%, P

Summary It has long been known that body mass and, more specifically, lean body mass are strongly correlated with maximal oxygen uptake (VO2max) in man and animals. However, there are no data to date describing this phenomenon in the horse. The purpose of this paper is to examine the relationship between body composition and VO2max in the horse. Twenty-three healthy and unfit Standardbred mares performed an incremental exercise test (GXT) to measure VO2max. Rump fat thickness (RTH), a measure of fat covering, was measured using B-mode ultrasound. Plasma volume, total blood volume and red cell volume were determined, using the Evan's Blue dye dilution technique and packed cell volume. VO2max was correlated with body mass (r = 0.541; P

The purpose of the study was to determine whether 12 weeks of exercise training would affect plasma glucose and plasma insulin concentrations in young and older Standardbred mares. Eighteen healthy, unfit mares representing 3 age groups were used: young (Y = 6.8 +/- 0.4 years; n = 6), middle-aged (MA = 15.2 +/- 0.4; n = 6), and old (O = 27.0 +/- 0.2; n = 6). Pre- and post-training incremental exercise tests (GXT) were performed to measure plasma glucose and insulin concentration from immediately after, until 120 min postexercise. Training consisted of exercise 3 days/week (weeks 1-8) and 4 days/week (weeks 9-12) at a submaximal intensity (approximately 60% of pretraining HRmax) for approximately 30 min/day. Old mares had lower levels of glucose after the GXT when compared to Y and MA mares (P0.05). There was also a time-by-age interaction (P = 0.003) in that, at 120 min post-GXT, glucose levels had not returned to pre-exercise values. Plasma glucose concentrations, in response to acute exercise, were not altered by training for any age group (P0.05). Prior to exercise training, O mares had higher plasma insulin compared to Y and MA mares at 120 min postexercise (P0.05), and insulin was also elevated in O mares compared to all other time points at 120 min post-GXT (P0.05). Training resulted in increased plasma insulin concentrations at 120 min post-GXT in all age groups (P0.05). In conclusion, age affected glucose and insulin responses to acute exercise prior to and after 12 weeks of exercise training. With regards to insulin, 12 weeks of exercise training resulted in a postexercise rebound hyperinsulinaemia, which may be related to an increased need for glycogen repletion in the muscle. These factors are important in considering the ability of an older horse to tolerate exercise.

Summary Several body systems make critical contributions to the defence of blood volume, mean arterial pressure and plasma tonicity. These mechanisms ensure adequate blood flow to the working muscles and vital tissues along with the provision of adequate fluid volume for sweating and thermoregulation. The present paper integrates data from several recent studies that examined the neuroendocrine control of cardiovascular function in exercising horses. These studies focused on the effects of exercise on plasma concentrations of atrial natriuretic peptide (ANP), arginine vasopressin (AVP), plasma renin activity (PRA) and aldosterone (ALDO), hormones associated with the regulation of blood pressure, the distribution of blood flow and the long term control of vascular fluid volume. Data were collected during incremental and endurance exercise, following frusemide administration and after splenectomy. Increases in ANP during exercise were associated with increased atrial pressure and stretching of the atrium. Increases in PRA and ALDO are correlated with increases in sympathetic activity during incremental exercise and with decreases in plasma Na+ and CI− concentrations. Plasma AVP increases later in exercise in response to fluid losses, increases in plasma osmolality, and decreases in right atrial pressure. AVP has a minimal effect on renal function during exercise and may be more important during recovery, when it stimulates thirst and drinking. Frusemide and splenectomy decrease atrial pressure, resulting in lower plasma ANP and greater plasma AVP concentrations during steady-state submaximal exercise. The ANP and AVP responses are part of the neuroendocrine action of the cardiopulmonary baroreflex which appears to mediate the accommodation of the mobilised splenic reserve in the horse. In summary, exercise induced increases in ANP, AVP, PRA and ALDO in the horse appear to play an important role in cardiovascular homeostasis.

The present study tested the hypothesis that lactate concentration ([La− ]) would differ between sample sites and between assay techniques that used different analytical substrates. Six clinically normal adult (two Thoroughbreds, three Standardbreds and one Quarter Horse) mares weighing between 435 and 560 kg were used in the study. Each mare performed an incremental exercise test (graded exercise test, GXT) where it ran on a treadmill at a fixed 6% grade. The GXT started at 3 m s− 1 for 1 min with increased in speed by 1 m s− 1 every 60 s until the horses completed the final 10 m s− 1 step. Jugular vein, pulmonary arterial and carotid arterial blood samples (14 ml) were collected before exercise and during the last 10 s of each step of the GXT. [La− ] was measured in whole blood (WB, no manipulations), total blood (TB, where the red blood cells were lysed) and plasma. Data were used to calculate the velocity to produce [La− ] of 4 mmol l− 1 (VLA4) and 10 mmol l− 1 (VLA10). Statistical analysis utilized a three-way ANOVA and, where appropriate, the Holm–Sidak or the Student Neuman–Keuls method for post hoc comparisons. The null hypothesis was rejected when P 0.05) between jugular vein and pulmonary artery. Within method, there was a difference (P 0.05) between WB (6.54 ± 0.36) and plasma (6.04 ± 0.64). Further analysis of the data demonstrated that the method and sample site influenced (P

Ten mature Standardbred mares (9–13 years, ,522 kg) were used to test the hypothesis that pelleted dietary supplementation would alter total plasma bicarbonate (tCO2) concentrations. All the mares used in this study were unconditioned, but were familiarized to the laboratory setting and running on a treadmill. Each of the ten mares was semi-randomly assigned one of four dietary treatments. The four treatments (oats as control and three pelleted feed supplements: Drive, Omolene and Strategy) were administered in a crossover fashion throughout a 4-week testing period. These products were chosen based on the frequency of their use by Standardbred/Thoroughbred owners and trainers in New Jersey. The horses underwent a simulated race test (SRT) on a treadmill (6% grade) at the end of each administration period. During the SRT, horses ran for 2 min at 4 m s 21 , 2 min at the speed previously shown to correspond to VO2max and 2 min at 4 m s 21 . Blood was collected before supplement treatment (24 h), 10 min prior to exercise and at 0, 60 and 90 min post-exercise. Plasma concentrations of bicarbonate, sodium, potassium and chloride were measured using a Beckman ELISE analyser. The major finding of this study indicates that the plasma [tCO2], chloride and sodium concentrations were not altered by the dietary supplements studied (P . 0.05). There were differences (P , 0.05) in plasma [tCO2] across sampling intervals (2 4h , 2 10 min, þ0 min, þ60 min and þ90 min) that were attributable to acute exercise (mean ^ SE: 34.4 ^ 0.9, 33.2 ^ 1.1, 20.2 ^ 0.8, 31.5 ^ 0.8, 30.3 ^ 1.6 mmol l 21 ). There was a slight effect of treatment (P ,2 0.05) on potassium levels. However, exercise was the main factor that caused substantial changes (P , 0.05) in the plasma tCO2, potassium, haematocrit and total protein concentrations. It was concluded that the pelleted diet supplements examined do not alter plasma [tCO2] in horses.

Clenbuterol is a beta(2)-agonist and potent selective bronchodilator that is used to treat bronchospasm in the horse. The drug is normally administered to horses orally as a syrup formulation. Once absorbed into the systemic circulation, clenbuterol has the potential to cause many side effects, including a repartitioning effect and major alterations in cardiac and skeletal muscle function. Recent studies have also reported that clenbuterol can affect bone and the immune, endocrine and reproductive systems. A great deal of information has been published on the beneficial effects of short term therapeutic doses of clenbuterol on the equine respiratory system, although there is limited information about chronic administration, particularly since this has been associated with adverse physiological effects on other systems. This review summarizes the relevant understanding of clenbuterol for clinicians and horse owners who may administer this drug to pleasure and performance horses.

Previous studies of the effect of age on maximal aerobic capacity (maximal rate of oxygen consumption, VO2max )i n horses have only grouped horses (young, middle-aged and old) for statistical analysis. Those experiments were not designed to determine a break point due to age. The purpose of this study was to utilize data collected over the last 15 years to test the nature of the association between age and VO2max, and secondarily, to determine whether there is a ‘breakpoint’ in the age-related decline in aerobic capacity. Data were acquired from 50 unfit Standardbred mares ranging in age from 4 to 29 years, and were used in a retrospective regression analysis in order to characterize the age-related decline in VO2max. All VO2max values were measured using the same incremental graded exercise test protocol. The data were analyzed using a curvilinear regression analysis predicting VO2max from age, and the break point was determined using univariate simple contrasts. There was a significant decline in VO2max with age (R 2 ¼ 0.554; P , 0.001). The data analysis demonstrated a notable downturn in cardiopulmonary function between the ages of 18 and 20 years (P , 0.001). Such results indicate similar responses to age between humans and horses. These results have implications for improving training methods in active older horses.

Glutamine is concentrated within skeletal muscle, where it has been proposed to play a regulatory role in maintaining protein homeostasis. The work presented here addressed the hypothesis that glutamine would be the most abundant free alpha-AA in plasma and skeletal muscle in the foal during the first year of life. Glycine, however, was the most abundant free alpha-AA in plasma at birth and between 3 and 12 mo of age. The concentration of glutamine, the second most abundant AA at birth, increased through the first 7 d (P < 0.05) and then returned to values similar to those at birth. This resulted in glutamine being the most abundant free alpha-AA in plasma from 1 d through 1 mo of age. The most abundant free alpha-AA in skeletal muscle at birth was glutamine, but the concentration fell by more than 50% by d 15 and continued to decrease, reaching about one-third of the original values by 1 yr of age (P < 0.05). Glutamine synthetase was barely detectable in skeletal muscle at birth, but the abundance increased rapidly within 15 d of birth. The concentration of glycine, the second most abundant alpha AA in muscle at birth, decreased by about 40% by d 15 (P < 0.05) and then stabilized at this value throughout the year. In contrast, glutamate, alanine, and serine concentrations, the third, fourth, and fifth most abundant free alpha-AA in muscle at birth, respectively, increased to new stable concentrations between 3 and 6 mo of age (P < 0.05). This resulted in alanine being the most abundant free alpha-AA in skeletal muscle at 12 mo of age, followed by glutamate, glutamine, and glycine. The decrease in intramuscular glutamine content, particularly during the first 2 wk after birth, is not compatible with a regulatory role for glutamine in muscle protein synthesis because it occurred at the time of maximum growth in these animals. The findings that, at certain times of development, glutamine was not the most abundant free alpha-AA in the foal is novel and signifies that intramuscular glutamine may have functions specific to muscle type and mammalian species.

Methoxy polyethylene glycol–epoetin β (pegylated epoetin β, peg-epoetin β, Mircera®) is an erythropoiesis-stimulating agent that is synthesized by pegylating epoetin β. It is prohibited in racehorses and humans because it enhances their performance. It is detectable in human urine and plasma using the isoelectric focusing–double-blotting (IEF–DB) method that has been widely applied in human doping control laboratories to detect doping with recombinant erythropoietins (EPOs). Fifty micrograms of peg-epoetin β were administered intravenously to a 450 kg female Thoroughbred, and blood was collected for 48 h. The plasma was processed by immunopurification followed by IEF–DB. The analytical procedures required about 2 days. No peg-epoetin β was detected in the baseline plasma collected prior to drug administration. It was readily detected in samples collected at 2, 4, 6, 8, 24 and 48 h. The isoforms showed a characteristic pattern that differed from epoetin β and was unlike any other known erythrocyte-stimulating agent or recombinant EPO. No peg-epoetin β was detected in plasma from five control horses. The plasma samples were also tested by a rapid, automated and chemiluminescent immunometric assay for EPO (Immulite®). The samples collected from the treated horse at 2–48 h contained the equivalent of approximately 200–800 pg ml− 1 of peg-epoetin β. The samples from the ten control horses were negative. These data show that a small dose of peg-epoetin β is detectable for 48 h by two different methods that are based on two different principles (chemiluminescence immunoassay and IEF–DB). The Immulite® assay is suitable as a screen and the IEF–DB assay as a confirmation method. These studies need to be expanded to other horses. The screen and confirmation tests, if applied, will probably eliminate the use of peg-epoetin β as a horse doping agent.