Intratracheal clenbuterol in the horse: its pharmacological efficacy and analytical detection

Clenbuterol, a beta2 agonist/antagonist, is the only bronchodilator approved by the US Food and Drug Administration for use in horses. The Association of Racing Commissioners International classifies clenbuterol as a class 3 agent, and, as such, its identification in post-race samples may lead to sanctions. Anecdotal reports suggest that clenbuterol may have been administered by intratracheal (IT) injection to obtain beneficial effects and avoid post-race detection. The objectives of this study were (1) to measure the pharmacological efficacy of IT dose of clenbuterol and (2) to determine the analytical findings in urine in the presence and absence of furosemide. When administered intratracheally (90 microg/horse) to horses suffering from chronic obstructive pulmonary disease (COPD), clenbuterol had effects that were not significantly different from those of saline. In parallel experiments using a behavior chamber, no significant effects of IT clenbuterol on heart rate or spontaneous locomotor activity were observed. Clenbuterol concentrations in the urine were also measured after IT dose in the presence and absence of furosemide. Four horses were administered i.v. furosemide (5 mg/kg), and four horses were administered saline (5 mL). Two hours later, all horses were administrated clenbuterol (IT, 90 microg), and the furosemide-treated horses received a second dose of furosemide (2.5 mg/kg, i.v.). Three hours after clenbuterol dose (1 h after hypothetical 'post-time'), the mean specific gravity of urine samples from furosemide-treated horses was 1.024, well above the 1.010 concentration at which furosemide is considered to interfere with drug detection. There was no interference by furosemide with 'enhanced' ELISA screening of clenbuterol equivalents in extracted and concentrated samples. Similarly, furosemide had no effect on mass spectral identification or quantification of clenbuterol in these samples. These results suggest that the IT dose of clenbuterol (90 microg) is, in pharmacological terms, indistinguishable from the dose of saline, and that, using extracted samples, clenbuterol dose is readily detectable at 3 h after dosing. Furthermore, concomitant dose of furosemide does not interfere with detection or confirmation of clenbuterol.     

Pharmacokinetics and pharmacodynamics of furosemide after oral administration to horses

Furosemide is the most common diuretic drug used in horses. Furosemide is routinely administered as IV or IM bolus doses 3-4 times a day. Administration PO is often suggested as an alternative, even though documentation of absorption and efficacy in horses is lacking. This study was carried out in a randomized, crossover design and compared 8-hour urine volume among control horses that received placebo, horses that received furosemide at 1 mg/kg PO, and horses that received furosemide at 1 mg/kg IV. Blood samples for analysis of plasma furosemide concentrations, PCV, and total solids were obtained at specific time points from treated horses. Furosemide concentrations were determined by reversed-phase high-performance liquid chromatography with fluorescent detection. Systemic availability of furosemide PO was poor, erratic, and variable among horses. Median systemic bioavailability was 5.4% (25th percentile, 75th percentile: 3.5, 9.6). Horses that received furosemide IV produced 7.4 L (7.1, 7.7) of urine over the 8-hour period. The maximum plasma concentration of 0.03 microg/mL after administration PO was not sufficient to increase urine volume compared with control horses (1.2 L [1.0, 1.4] PO versus 1.2 L [1.0, 1.4] control). There was a mild decrease in urine specific gravity within 1-2 hours after administration of furosemide PO, and urine specific gravity was significantly lower in horses treated with furosemide PO compared with control horses at the 2-hour time point. Systemic availability of furosemide PO was poor and variable. Furosemide at 1 mg/kg PO did not induce diuresis in horses.     

Effect of furosemide on urine specific gravity and osmolality in thoroughbred racehorses

Postrace urine samples from thoroughbred horses were examined to compare osmolality and specific gravity between horses treated with furosemide and those not treated. Samples were assigned to groups in relation to reported medication (furosemide) status, race finish position, and distance of race. Urine osmolality was significantly (P <.05) lower in samples from horses treated with furosemide when compared with untreated horses. Specific gravity determinations are less precise at measuring urine osmolality at lower levels (1.01 g/ml or less). The measurement of osmolality is a superior method for determining the urine solute concentration and facilitating the regulation of furosemide.     

Estimation of the probability for exceeding thresholds of urine specific gravity and plasma concentration of furosemide at various intervals after intravenous administration of furosemide in horses

OBJECTIVE: To estimate the probability of concurrently exceeding thresholds for plasma concentration of furosemide and urine specific gravity after IV administration of furosemide in horses. ANIMALS: 12 mature healthy Thoroughbred (n = 6) or Quarter Horse (6) mares. PROCEDURE: Venous blood was collected from each horse prior to and 0.25, 0.5, 0.75, 1, 2, 3, 4, 4.5, 5, and 6 hours after IV administration of 250 mg (first experiment) or 500 mg (second experiment) of furosemide. Urine was collected hourly between 1 and 6 hours after administration of furosemide at both doses. Concentrations of furosemide were determined by use of an ELISA. Concentration of furosemide and urine specific gravity was modeled as a function of time, accounting for inter- and intrahorse variabilities. On the basis of pharmacokinetic and specific gravity data, the probability of exceeding a concentration of 100 ng of furosemide/ml as a function of time was determined, using a semiparametric smooth functional averaging method. A bootstrap approach was used to assess the inherent variation in this estimated probability. RESULTS: The estimated probability of exceeding the threshold of 100 ng of furosemide/ml and urine specific gravity < 1.012 was approximately 0% between 4.0 and 5.5 hours after IV administration of 250 mg of furosemide/horse, and ranged from 0 to 1% between 4 and 5.5 hours after IV administration of 500 mg of furosemide/horse. The probability of a horse being falsely identified as in violation of regulatory concentrations was inversely associated with time. CONCLUSIONS AND CLINICAL RELEVANCE: Coupling plasma furosemide concentration with urine specific gravity testing will greatly reduce the chance that some horses are misclassified as being in violation of regulatory concentrations.     

Efficacy of furosemide in the treatment of exercise-induced pulmonary hemorrhage in Thoroughbred racehorses

The repeatability of endoscopic observations of exercise-induced pulmonary hemorrhage (EIPH) and the efficacy of furosemide as a prophylactic treatment of horses with EIPH were studied in Thoroughbred race horses after consecutive breezes (at or near maximum speed, approx 16 m/s). Of 56 horses examined greater than or equal to 2 times, 21 (38%) had identical EIPH scores, whereas 26 (46%) and 9 (16%) had scores that differed by greater than or equal to 1 grade. In 56 nontreated horses, there was good agreement between 2 consecutive observations (K = 0.59, Z = 4.54, P less than 0.001). Similar comparisons after placebo (saline solution) treatment of 21 horses yielded fair to good agreement, whereas poorer agreement was seen after furosemide treatment of 23 horses. Comparison of average and maximum EIPH scores of 44 horses with a minimum of 4 observations (2 nontreated, 1 saline-treated, and 1 furosemide-treated) indicated that although furosemide did not stop EIPH, it did reduce the EIPH score in 28 (64%) horses.     

Inheritance of racing performance of Thoroughbred horses

Horse racing is a contest between horses, usually held for the purpose of betting. Thoroughbred horse racing is the most diffused form of horse racing throughout the world. Thoroughbred is one of the most versatile of horse breeds and has influenced the development of many other breeds. Thoroughbred horses served as a foundation stock for the development of the light horse breeds. The two types of horse racing are flat racing and jumping races/steeplechases. The measures of racing performance are broadly classified into three categories. They are time and its several variations, handicap or similar performance ratings and earnings. One common measure of the performance of racehorses evaluated genetically is racing time or final time. The heritability estimates differed according to method of estimation, age, sex, track and distance. Time measure generally had a heritability in the range of 0.1 to 0.2 with the higher values for shorter races. For handicap and earning measures the heritabilities reported were generally higher in the range of 0.3 to 0.4; hence these may be considered in genetic evaluation of racing performance of Thoroughbred horses. The average generation interval of Thoroughbred horses was 11.2 ± 4.5 and 9.7 ± 3.8 years for males and females respectively, which limits the genetic progress in racing horses. However, the major advantage is that the racing performance may be evaluated in both males and females and repeated observations can be obtained on the same animal in relatively short periods. These factors coupled with the reasonable heritability of some measures of racing performance, suggest that mass selection based on performance tests would be the selection procedure of choice to improve the racing performance of Thoroughbred horses. In general, the inbreeding at the rate that is usually practised in Thoroughbred population does not enable much gene fixing. However, practice of close inbreeding may be avoided, even though it still fascinates breeders at subconscious level.     

Age at first start and racing career of a cohort of Australian Standardbred horses

OBJECTIVE Compare the career profiles of a cohort of Standardbred horses that first raced as 2-year-olds with those that started their racing careers at a later age. METHOD Retrospective analysis of the racing records of all foals born in New South Wales in the 2000 foaling season. RESULTS The career records of 999 horses were analysed. Almost half (43.9%) first raced as 2-year-olds and one-third (33.9%) as 3-year-olds. The median career duration for horses that first raced as 2-year-olds was 2.93 years (interquartile range (IQR) 2.70-3.16), which was significantly greater than the median for horses that first raced at 3, 4 or ≥5 years old (P < 0.001). Males, and horses that first raced as 2-year-olds, earned significantly more prize money than females or horses that started racing aged ≥3 years (P < 0.001). The population median number of career starts was 28.0 (IQR 8-64). Males, and horses that first raced as 2-year-olds, had significantly more career starts than females or horses that started racing aged ≥3 years (P < 0.001). CONCLUSION This study found no evidence suggesting that racing as a 2-year-old had a deleterious effect on a horse's racing career.     

Association between exercise-induced pulmonary hemorrhage and performance in Thoroughbred racehorses

OBJECTIVE: To determine whether exercise-induced pulmonary hemorrhage (EIPH) was associated with racing performance inThoroughbred horses not medicated with furosemide and not using nasal dilator strips. DESIGN: Observational cross-sectional study. ANIMALS: 744 two- to 10-year-old Thoroughbred horses racing in Melbourne, Australia. PROCEDURE: Horses were enrolled prior to racing, and a tracheobronchoscopic examination was performed after 1 race. Examinations were recorded on videotape, and presence and severity (grade 0 to 4) of EIPH were subsequently determined by 3 observers blinded to the horses' identity. Race records were abstracted for each horse examined. RESULTS: Overall, 52.1% of horses eligible for participation in the study were examined, and horses that were examined did not differ from horses that were not examined in regard to age, sex distribution, or proportion of horses that won or finished in the first 3 positions. Horses with EIPH grades < 1 were 4.0 times as likely to win, 1.8 times as likely to finish in the first 3 positions, and 3.03 times as likely to be in the 90th percentile or higher for race earnings as were horses with grades > 2. Horses with EIPH grades > 1 finished significantly farther behind the winner than did horses without EIPH. However, odds that horses with grade 1 EIPH would win or finish in the first 3 positions were not significantly different from odds for horses without EIPH. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that EIPH is associated with impaired performance in Thoroughbred racehorses not medicated with furosemide and not using nasal dilator strips.     

Resting concentrations of cardiac troponin I in fit horses and effect of racing

ObjectivesTo determine normal resting values for cardiac troponin I (cTnI) in healthy Standardbred, Thoroughbred and Warmblood horses and investigate if racing has an influence on cTnI concentrations.BackgroundMeasuring cTnI concentrations in plasma is the gold standard for detecting myocardial injury in humans. Cardiac troponin I is highly conserved between species and has gained interest as a marker for cardiac injury in horses. Increased levels of cTnI have been reported in association with endurance and short-term strenuous exercise on a treadmill in horses. However, the effect of true racing conditions has not yet been reported.Animals, materials and methodsBlood samples for analysis of cTnI concentrations in plasma were collected from 67 Standardbred racehorses, 34 Thoroughbred racehorses and 35 Warmblood dressage horses at rest. Blood samples were also collected prior to and after racing in 22 Standardbred racehorses and 6 Thoroughbred racehorses.ResultsAll horses except one had resting plasma cTnI concentrations <0.022 μg/L. Mild increases in cTnI concentrations were seen in some horses 1–2 h after the race (1/17 Standardbreds and 2/6 Thoroughbreds) as well as 10–14 h after the race (4/21 Standardbreds and 1/6 Thoroughbreds).ConclusionsResting cTnI concentrations in horses are low but mildly elevated cTnI concentrations may be detected in some horses 1–14 h after racing. These findings could be of importance when evaluating horses with suspected cardiac disease that recently have performed hard exercise.     

Effects of furosemide on the racing times of Thoroughbreds

The effects of furosemide on the racing times of 79 horses without exercise-induced pulmonary hemorrhage (EIPH) and 52 horses with EIPH were investigated. Racing times were adjusted to 1-mile equivalent racing times by 2 speed handicapping methods, and analysis of covariance was used to adjust actual racing times by winning time and distance for each race. All 3 methods of determining racing time indicated that geldings without EIPH had significantly faster racing times (P less than 0.05) when given furosemide before racing than when furosemide was not given before racing. Females and colts without EIPH were determined to have faster racing times when furosemide was given before racing, but the difference was not significant. Geldings with EIPH had significantly faster racing times (P = 0.0231) when given furosemide before racing, as determined by one of the speed handicapping methods. There was a strong correlation (range 0.9314 to 0.9751) between the 1-mile equivalent racing times, as determined by the 2 speed handicapping methods for horses with and without EIPH. Furosemide failed to prevent the development of EIPH in many horses that were previously considered to be EIPH-negative. When given furosemide, 62 (25.3%) of 235 EIPH-negative horses were EIPH-positive after racing. Furosemide had questionable efficacy for prevention of EIPH in known EIPH-positive horses. Thirty-two (61.5%) of 52 EIPH-positive horses given furosemide before a race remained EIPH-positive after that race.     

Detection, quantification, and pharmacokinetics of furosemide and its effects on urinary specific gravity following IV administration to horses

Furosemide is a potent loop diuretic used for the prevention of exercise-induced pulmonary hemorrhage in horses. This drug may interfere with the detection of other substances by reducing urinary concentrations, so its use is strictly regulated. The regulation of furosemide in many racing jurisdictions is based on paired limits of urinary SG (<1.010) and serum furosemide concentrations (>100 ng/ml). To validate this regulatory mechanism, a liquid chromatography/mass spectrometry/mass spectrometry method employing a solid-phase extraction procedure and furosemide-d5 as an internal standard was developed. The method was used to determine the pharmacokinetic parameters of furosemide in equine serum samples and its effects on urinary SG after IV administration (250 mg) to 10 horses. Pharmacokinetic analysis showed that serum concentrations of furosemide were well described by a two-compartmental open model. Based on results in this study, it is very unlikely for horses to have serum furosemide concentrations greater than 100 ng/ml or urine SG less than 1.010 at 4 hours after administration (250 mg IV). However, it should be remembered that urine SG is a highly variable measurement in horses, and even without furosemide administration, some horses might naturally have urine SG values less than 1.010.     

Quantitative evaluation of subchondral bone injury of the plantaro‐lateral condyles of the third metatarsal bone in Thoroughbred horses identified using nuclear scintigraphy: 48 cases

Reasons for performing study: Increased radio‐isotope uptake (IRU) in the subchondral bone of the plantaro‐lateral condyle of the third metatarsus (MTIII) is a commonly reported scintigraphic finding and potential cause of lameness in UK Thoroughbred racehorses in training and has not been fully documented. Objectives: To characterise lameness attributable to IRU of the subchondral bone of MTIII, compare the scintigraphic findings of these horses with a normal population and evaluate the use of scintigraphy as an indicator of prognosis. Hypothesis: IRU will be in significantly higher in horses with subchondral bone injury and will be related to prognosis and future racing performance. Methods: Data were analysed from 48 horses in which subchondral bone injury of the plantaro‐lateral condyle of MTIII had been diagnosed using nuclear scintigraphy and that met the inclusion criteria. Data recorded included age, sex, trainer, racing discipline, lameness assessment, treatment regimes, radiographic and scintigraphic findings, response to diagnostic analgesia where performed and racing performance pre‐ and post diagnosis. Region of interest (ROI) counts were obtained for the plantar condyle and the mid diaphysis from the latero‐medial view, the ratio calculated and then compared with a control group of clinically unaffected horses. Results: The mean condyle mid‐diaphysis ROI ratio was significantly (P<0.001) higher in the affected population and with positively correlation (P = 0.024) with the level of lameness. The presence of radiographic findings had no significant effect on the ROI ratio. Conclusion: Subchondral bone injury of the plantar lateral condyles of MTIII is a significant cause of lameness in UK Thoroughbred racehorses. Nuclear scintigraphy is a useful diagnostic imaging modality in the detection of affected horses but is a poor indicator of prognosis for the condition. Potential relevance: Better understanding of the clinical manifestations, diagnosis of and prognosis for subchondral bone injury will benefit the Thoroughbred industry in the UK.     

Hydrocortisone levels in the urine and blood of horses treated with ACTH.

An investigation was undertaken to demonstrate whether therapeutic treatment with ACTH raises hydrocortisone (cortisol) levels in horse urine above the limit (1000 ng/ml) established by the International Conference of Racing Authorities with the aim of controlling the abuse of cortisol and ACTH in equine sports. ACTH (200 iu) was administered i.m. to 3 Thoroughbred horses; urine and blood samples were collected at intervals afterwards and analysed by an immunoenzymatic system (ELISA) and HPLC-MS. To ascertain post exercise cortisol levels in untreated horses, 101 urine and 103 serum samples were taken from horses immediately after racing and analysed by ELISA. The peak urine level of cortisol, detected 8 h after ACTH administration, was around 600 ng/ml using either ELISA or HPLC-MS. The peak serum cortisol concentration was found to be around 250 ng/ml by ELISA, but consistently less by HPLC-MS. Mean cortisol levels in post race horses were 135.1+/-72.1 ng/ml in urine and 90.1+/-41.7 ng/ml in serum. High levels of the metabolite 20beta-dihydrocortisol in urine and the cortisol precursor 11beta-desoxycortisol in serum were found. The latter showed high cross-reactivity with cortisol on ELISA. In our experiment, treatment with ACTH 200 iu i.m. did not raise urinary cortisol levels above the 1000 ng/ml threshold proposed by the ICRA.     

Assessment of the efficacy of an abductor muscle prosthesis for treatment of laryngeal hemiplegia in horses

Four variations of abductor muscle prosthesis for treating laryngeal hemiplegia were evaluated in 153 horses by questionnaire, and in the 100 Thoroughbred racehorses in this group survival analysis was used to compare their racing performances and earnings with those of 400 control horses. The questionnaire indicated that the technique which included a ventriculectomy and 2 prostheses was regarded as being the most successful (P less than 0.01) and resulted in the least residual stertor (P less than 0.001). Survival analysis showed that there was no significant difference between the treated group of horses and the control horses (P greater than 0.05).     

Comparison of serum and urinary concentrations of clenbuterol with and without concomitant administration of furosemide in horses

Furosemide is frequently used to control or prevent exercise-induced pulmonary hemorrhage in performance horses. The bronchodilating agent clenbuterol is also commonly used as a treatment for inflammatory airway disease in performance horses. Use of both medications is regulated by many racing authorities. The effects of concomitant administration of furosemide and clenbuterol on the pharmacokinetics of clenbuterol have not been well characterized. A study was designed to evaluate the influence of furosemide on serum and urine concentrations of clenbuterol after oral administration of clenbuterol and intravenous administration of furosemide in horses. Results indicated that urinary concentrations of clenbuterol in horses treated concomitantly with furosemide and clenbuterol were increased, whereas serum concentrations of the drug were decreased. These effects persisted during the study period and varied among horses.     

Assessment of prognosis for racing after carpal surgery in 210 Thoroughbreds

The efficacy of treating carpal lesions by arthrotomy was evaluated in 210 Thoroughbred racehorses, using survival analysis to compare their racing performances and earnings with those of 840 control horses. The treated horses were significantly inferior with respect to races contested, and wins plus places (P less than 0.001) and races won (P less than 0.01). There was no difference with respect to earnings (P less than 0.1); after adjusting for other factors, arthritis, site of fracture and presence of a displaced chip had no effect on racing performance in horses with a single-site lesion involving a chip.     

Effect of dose of furosemide on plasma tCO2 changes in standardbred horses

Nine Standardbred horses of similar athletic fitness (six mares, three geldings), ranging from 4 to 11 years of age, were used to determine the effects of 0, 250, or 500 mg intravenously administered furosemide on plasma tCO₂ changes over time. All horses were either currently racing or in advanced stages of race training before entering a qualifying race. Horses were randomly allotted to one of the three treatment levels of furosemide during 3 consecutive weeks. Jugular venous samples were obtained from horses at rest in box stalls before and hourly for 6 hours after administration of furosemide. Body weights of horses ranged from 356 to 456 kg, and the mean was 417 kg. Thus, the dose of furosemide received by each horse ranged from 0.55 to 0.70 mg/kg body weight for the 250-mg injections and from 1.1 to 1.4 mg/kg body weight for the 500-mg injections. Furosemide caused metabolic alkalosis in the horses. Least square means (±SEM) were determined and horses had adjusted plasma tCO₂ of 32.2, 33.9, and 34.7 ± 0.41 for the 0-, 5-, and 10-mL doses of furosemide, respectively. The type 3 tests of hypotheses found that there was a difference (P < .0001) across time, a difference (P = .0016) according to furosemide dose, and a difference (P < .0001) according to treatment × hour. There was no difference (P > .05) according to week or treatment × week. These data suggest that either 250 or 500 mg furosemide given to Standardbred race horses induces statistically similar metabolic alkalosis.     

Scrutiny of antimicrobial use in racing horses with allergic small airway inflammatory disease

Antimicrobials had been administered to 38/55 (69%) racing standardbred and Thoroughbred horses with poor performance, subsequently diagnosed with nonseptic inflammatory airway disease. Horses with cough were more commonly treated (P = 0.02). In almost all cases, no clinical signs suggested that bacterial infection was present. Inappropriate use of antimicrobials was common.     

The association of 2‐year‐old training milestones with career length and racing success in a sample of Thoroughbred horses in New Zealand

Reasons for performing study: There is increasing evidence that exercise early in life has a positive effect on musculoskeletal health. At present, there is little whole population research investigating the effect of racing as 2‐year‐olds on future racing career. Objectives: To investigate the association between attaining training milestones as 2‐year‐olds with length of career and racing success in Thoroughbred horses in New Zealand. Methods: Retrospective data were obtained of the 2001/02‐born Thoroughbred foal crop. The 3 training milestones were: registered with a trainer, trialled and raced. The association of the training milestones with career length was measured using the outcomes: number of race starts and number of years raced, in a Cox regression model. Logistic regression models analysed the association of the training milestones with the outcomes: won or placed in a race. Linear regression was performed to assess the association of training milestones with total career earnings. Results: Of 4683 horses in the population; 3152 horses were registered with a trainer, 2661 horses trialled and 2109 horses raced. Horses that raced as 2‐year‐olds had significantly (P<0.001) more race starts than those first raced as 3‐year‐olds or older, this was also true when the 2‐year‐old year data were omitted. Horses that raced as 2‐year‐olds had significantly (P<0.001) more years racing. Horses registered with a trainer, trialled or raced as 2‐year‐olds were more likely to have won or been placed in a race than those that achieved the milestones as 3‐year‐olds or older. Horses that first trialled and raced as 2‐year‐olds had greater total earnings than those that first trialled or raced at a later age. Conclusions and potential relevance: Two‐year‐old training milestones had a strong association with positive racing career outcomes. Horses in training or racing as 2‐year‐olds may have better musculoskeletal health throughout life than horses that are first in training or racing at a later age.     

Subclinical leptospirosis may impair athletic performance in racing horses

The infection by Leptospira in horses, in both its acute disease and subclinical forms, is very common, particularly in endemic regions. Therefore, the objective of this study was to evaluate the effects of subclinical leptospirosis in the athletic performance of racing thoroughbred horses. Athletic performance of 119 racing Thoroughbred horses from Rio de Janeiro, Brazil, was calculated by assigning a point value for the results in racing (performance index (PI)), and serology for leptospirosis was conducted. A total of 85 (71.4?%) horses showed reactive titers (??100), and of which 52 had high titers (34 with 400 and 18 with ??800). Although those animals had high titers against Leptospira, no clinical signs associated with leptospirosis were observed. Seventeen (89.5?%) out of the 19 horses with substandard performance were seroreactive with high titers, in contrast with 35?% of seroreactivity in horses with good athletic performance (P?<?0.0001). Additionally, seroreactivity to leptospirosis was more often observed in horses with substandard athletic performance in contrast to those with good performance (P?<?0.0001, odds ratio 15.8). The Average PI of this group increased to 133?% after treatment (P?<?0.0001). Leptospirosis may impair performance in racing horses, and antibiotic therapy may improve the performance of affected animals.