The beta-agonist clenbuterol in mane and tail hair of horses

REASONS FOR PERFORMING STUDY: The beta2-agonist clenbuterol is commonly administered for therapeutic purposes in the horse, but its use an an anabolic agent is illegal. Clenbuterol can be detected in blood and urine for a relatively short period after administration and detection in hair could enhance the analytical range and be used to determine the history of clenbuterol application. HYPOTHESIS: That detection in mane or tail hair is possible over an extended period. METHODS: Four horses received 0.8 microg clenbuterol hydrochloride/kg bwt b.i.d. for 10 days. Four other horses were used as untreated controls. Blood, urine, mane and tail hair samples were taken on Day 0 (before) and 5, 10, 30, 35, 40, 60, 90, 120, 150 and 360 days after start of treatment. Gas chromotography/high resolution mass spectrometry (GC/HRMS) was developed for clenbuterol analysis: limit of detection was 0.2 pg/mg; intra-assay repeatability limit r = 0.06 (confidence level 95%); interassay repeatability limit r = 0.03 (confidence level 95%). Prior to treatment, clenbuterol was absent from all samples analysed. RESULTS: Clenbuterol was detectable as early as Day 5 in tail and mane hair of Segment 1 (0-20 mm from the roots) and was maximal on Day 90. However, as time progressed, shift into lower 20 mm segments was observed. On Day 360, the maximum concentration (up to 21 pg/mg) was located in Segment 13, i.e. 26-28 cm from roots of hair. Clenbuterol was not detectable in blood or urine after Day 30. Mane and tail hair results were very similar. CONCLUSIONS: The study showed that the beta-agonist clenbuterol can be found in mane and tail hair of horses after extended periods. POTENTIAL RELEVANCE: It will be possible to detect clenbuterol in breeding and show horses where anabolic drugs have been used illegally to improve conformation. This method may also be helpful to monitor therapeutic clenbuterol treatment.     

Concentrations of toxic metals and essential minerals in the mane hair of healthy racing horses and their relation to age

Concentrations of trace elements (As, Al, Pb, Cd, Hg, Se, Si, P, Na, K, Ca, Mg, Fe, Cu, Zn, Mn, Cr, Ni and Mn) in the mane hair obtained from 9 female and 15 male healthy racing Thoroughbred horses aged 2-5 years were analyzed by the inductively coupled plasma atomic emission spectrometry (ICP-AES) method. No significant differences between the female and male horses were observed in the mean concentrations of those minerals. Significantly positive correlations with age were observed in Cd (r=0.546, p<0.01) and Mo (r=0.733, p<0.001). Significantly negative correlations with age were observed in Hg (r= -0.726, p<0.001), Mn (r= -0.450, p<0.05) and Fe (r=-0.642, p<0.01). This reference range of trace elements in the mane hair of racing horses should be used to assess disease and the nutritional status in equine practice.     

Relationship between trace elements status in mane hair and atrial fibrillation in horse

To investigate the relationships between trace elements concentrations in hair and atrial fibrillation (AF) in horses, concentrations of nineteen trace elements were detected in hair using the particle induced X-ray emission (PIXE) method. The horses were assigned to either control (n=22, no abnormalities) or AF groups (n=5) based on electrocardiograph findings. The mean concentrations of Ca and Zn in the hair of the AF group were significantly higher than those in the control group. The Zn/Cu ratio of the hair in the AF group (29.8 +/- 5.5) was significantly higher than that in the control group (23.4 +/- 2.2, P<0.05). The results of the present study suggest that there is a relationship between elevated concentrations of Ca and Zn in hair and AF.     

Interactions of morphine and isoflurane in horses

OBJECTIVE: To quantitate dose- and time-related magnitudes of interactive effects of morphine (MOR) and isoflurane (ISO) in horses and to characterize pharmacokinetics of MOR in plasma and the ventilatory response to MOR during administration of ISO. ANIMALS: 6 adult horses. PROCEDURE: Horses were anesthetized 3 times to determine the minimum alveolar concentration (MAC) of ISO in O2 and then to characterize the change in anesthetic requirement as defined by the alteration in ISO MAC following IV administration of saline (0.9% NaCl) solution and 2 doses of MOR (low dose, 0.25 mg/kg; high dose, 2.0 mg/kg). Arterial blood samples were obtained before and after MOR and analyzed. RESULTS: Mean +/- SD baseline ISO MAC was 1.43 +/- 0.06%. The ISO MAC did not change with time after administration of saline solution. Effects of MOR on ISO MAC varied. Maximal change in MAC ranged from -20.2 to +28.3% and -18.9 to +56.2% after low and high doses of MOR, respectively. Typical half-life of MOR in plasma was 40 to 60 minutes and related to dose. Mean PaCO2 increased from 70 mm Hg before MOR to 88 to 102 mm Hg for 30 to 240 minutes after the high dose of MOR. Recovery from anesthesia after administration of the high dose of MOR was considered undesirable and dangerous. CONCLUSIONS AND CLINICAL RELEVANCE: Our results do not support routine clinical use of MOR administered IV at dosages of 0.25 or 2.0 mg/kg as an adjuvant to anesthesia in horses administered ISO.     

Pharmacokinetics and protein binding of morphine in horses

Morphine could be detected in horses dosed with 0.1 mg of drug/kg of body weight for up to 48 hours in blood and 144 hours in urine. This dose of morphine elicited no observable effects and is a suggested analgesic dose. Computer analysis revealed that a 3-compartment open system was the best fitting model with a serum half life (t1/2(beta)) of 87.9 minutes and a urine t1/2(beta) of 101.1 minutes. Binding to equine serum proteins was linear over a drug concentration range of 3.88 X 10(-5)M to 3.50 X 10(-8)M and averaged 31.6%. In RBC-partitioning experiments, 78.1% of the drug was found in the plasma fraction. The data indicated that a horse should not be given morphine closer than 1 week before a race.     

Mineral intake and hair analysis of horses in arizona

To evaluate the effect of diet on hair mineral content, 61 feeds, 31 pasture grasses, 29 mineral supplements, 31 water samples and hair samples from 391 horses from 31 ranches in Arizona were analyzed for fifteen minerals. Based on dietary histories and estimated intakes when free choice access was involved, mineral intake of the horses was calculated and compared to mineral content of the hair. Data were evaluated by regression analysis for correlations between hair and dietary minerals and variations in hair mineral content attributable to coat color, age, sex and breed.¹¹,¹²,¹³,¹⁴

There were differences (P<.05) in hair mineral content of horses of different breeds, coat colors, ages and sex, regardless of dietary intake. However, there were some significant correlations between feed mineral intake and hair mineral content. A few weak simple correlations (Ca:Ca, P:P, Na:Na, K:K) were found, but complex mineral correlations (Ca:P, Ca:Na, Zn:K, Fe:Zn etc.) were more common and had higher correlation coefficients (Table 7). Variations in mineral intake explained only 30% of the variation for a given hair mineral, with the exception of manganese. The multiple relationships between minerals and their effect on metabolism appear to have a greater effect on hair mineral content than a single mineral deficiency or excess¹⁷,¹⁸,²⁰,²¹ and may offer better insight to the interpretation of mineral nutrition than simple one to one correlations.     

Reverse tolerance to the stimulant effects of morphine in horses

Horses were given 4 intravenous injections of morphine sulfate, 0.5 mg per kg, spaced two days apart. The motor response to morphine was measured by counting the number of steps taken with the left foreleg during a 4 minute interval at various times after the injection. By the third injection of morphine there was a significant increase in motor response. This sensitization or reverse tolerance persisted for several weeks after the last injection. Cross-sensitization also developed to the stimulant effects of apomorphine. This finding suggests that sensitization to the stimulant effects of morphine may involve dopamine receptors in the central nervous system.     

Effects of peri-operative morphine administration during halothane anaesthesia in horses

OBJECTIVE: To study the effects of morphine on haemodynamic variables, blood gas values and the requirement for additional anaesthetic drugs in horses undergoing surgery. STUDY DESIGN: Prospective randomized study. METHODS: Thirty-eight client-owned horses, ASA(American Society of Anesthesiologists) category I or II, undergoing elective surgical procedures, were studied. Horses were divided between two groups, and were paired according to operation, anaesthetist, body position during surgery, mass and breed. Group M+ received morphine by intravenous (IV) injection (0.15 mg kg(-1)) before induction of anaesthesia and then by infusion (0.1 mg kg(-1) hour(-1)) throughout anaesthesia. Group M- received the same anaesthetic technique (pre-anaesthetic medication with romifidine (100 microg kg(-1)) IV; induction with ketamine (2.2 mg kg(-1)) and diazepam (50 microg kg(-1)) IV; maintenance with halothane), except that morphine was excluded. Both groups received flunixin IV (1.1 mg kg(-1)) before surgery. Both groups also received 50% nitrous oxide for the first 10 minutes of anaesthesia. During anaesthesia, end-tidal halothane was maintained at 0.9% (+/-0.1%) in both groups. Heart rate (HR) and respiratory rate (fr), systolic, mean and diastolic arterial pressures were recorded every 5 minutes. Arterial blood samples were analysed every 20 minutes. Additional anaesthetics (ketamine and midazolam) were administered whenever the horse moved. Dobutamine was infused to maintain mean arterial pressure (MAP) > 58 mm Hg, but was discontinued when MAP reached 68 mm Hg. Mechanical ventilation was imposed when PaCO(2) exceeded 9.3 kPa (70 mm Hg). RESULTS: Haemodynamic data (HR and MAP) and blood gas measurements were analysed using repeated measure analysis using a mixed covariance pattern model (SAS version 8.2). A Student's t-test was used to investigate differences between groups in the doses of additional anaesthetics required. There were no significant differences between M+ or M- groups in MAP (p = 0.65), HR (p = 0.74), PaO2 (p = 0.40) or PaCO2 (p = 0.20). Fewer horses in the M+ group received additional anaesthetics (15.8% compared to 21.1% in M- group), and the mean dose of ketamine required was higher in the M- group (mean +/- SD: M-, 0.93 +/- 0.70; M+, 0.45 +/- 0.17). These differences were not statistically significant (p = 0.28). CONCLUSIONS: Pre-anaesthetic and peri-operative morphine administration is not associated with significant haemodynamic or ventilatory changes. Horses receiving morphine tended to receive fewer and lower doses of additional anaesthetic drugs, although this was not statistically significant.     

Effects of epidural morphine on gastrointestinal transit in unmedicated horses

ObjectiveTo evaluate the effect of epidural morphine on gastrointestinal (GI) motility in horses.Study designRandomly ordered crossover design.AnimalsSix healthy adult horses weighing 585 ± 48 kg (mean ± SD).MethodsHorses were randomly assigned to receive either 0.2 mg kg^?1 morphine or an equal volume (0.04 mL kg^?1) of saline epidurally (the first inter coccygeal space) with 2 weeks between treatments. The horses were stabled, fed a standardized diet and allowed water ad libitum throughout the duration of the study. Radiopaque spheres were administered by stomach tube. Xylazine 0.2 mg kg^?1 intravenously was administered prior to epidural injection. Heart rate, respiratory rate, GI sounds score and behavior score were recorded before drug administration and after epidural injection at 4, 8, 12, 18, 24 hours and every 12 hours thereafter for 6 days. Feces were weighed, radiographed and the number of spheres counted. Data were analyzed using a mixed effect model.ResultsAt no time did horses exhibit signs of colic or show significant differences between treatments regarding heart rate, respiratory rate, GI sounds score, behavior score, or cumulative number of spheres. The concentration of spheres per kg of feces was significantly lower (p < 0.05) for the morphine group at 18 and 24 hours. Using the centroid of the curves (spheres kg^?1 plotted versus time) the average transit time after saline epidural was 38 hours and after morphine it was 43 hours. The weight of feces hour^?1 was significantly lower (p < 0.05) at only 4 and 8 hours after morphine.Conclusions and clinical relevanceEpidural morphine, at a dose of 0.2 mg kg^?1, temporarily reduced GI motility but did not cause ileus or colic in this small group of healthy unfasted horses. Care should be taken when extrapolating these data to situations in which other factors may also affect GI motility.     

Comparison of hair follicle histology between horses with pituitary pars intermedia dysfunction and excessive hair growth and normal aged horses

Background –  Pituitary pars intermedia dysfunction (PPID) in older equids is commonly recognized by a long hair coat that fails to shed. Objective –  The aim of this study was to compare hair follicle stages in PPID‐affected horses with excessively long hair coats with the stages of normal aged horses (controls) and to compare hair follicle stages in PPID‐affected horses after 6 months of treatment with pergolide mesylate with those of control horses. Animals –  Eight PPID‐affected horses and four normal, age‐matched, control horses. Methods –  Skin biopsies were collected from the neck and rump of PPID‐affected and control horses. A diagnosis of PPID was established based on hair coat changes and supportive overnight dexamethasone suppression test results. Skin biopsies were repeated after 6 months of treatment with pergolide. The number of hair follicles in anagen (A) or telogen (T) was counted for each skin biopsy using transverse sections. Results –  Pretreatment biopsies had a greater percentage of A follicles (neck 96%, rump 95%) and a lower percentage of T follicles (neck 4%, rump 5%) in PPID‐affected horses than in control horses (A, neck 15%, rump 25%; and T, neck 85%, rump 75%). After treatment with pergolide, all PPID‐affected horses had improved shedding, and the percentages of A follicles (neck 69%, rump 70%) and T follicles (neck 31%, rump 30%) were not different from untreated control horses (A, neck 68%, rump 82%; and T, neck 32%, rump 18%). Conclusions –  These findings document that excessive hair growth (hypertrichosis) in PPID‐affected horses is due to persistence of hair follicles in A. Furthermore, treatment with pergolide improved shedding and reduced the percentage of A follicles in PPID‐affected horses.     

Heritability of hair whorl position on the forehead in Konik horses

There are studies on the relationship between the position and shape of hair whorls on bovine forehead and phenotypic traits. According to anecdotal beliefs by horse users and handlers, temperamental traits may be related to the position of hair whorls in horses. No previous research on the mechanisms of inheritance of hair whorls has been performed, so the aim of the present study was to determine the heritability of the position of the hair whorl on the forehead of Konik horses. The horses (n = 362) were classified into five groups based on the whorl position on forehead with respect to the top and bottom eye lines. The estimated heritability of hair whorl position was 0.753 (SE = 0.056). Heritability adjusted for the discontinuity of the trait was 0.836. The results show that hair whorl position in Konik Polski horses is highly heritable. The possible relationship between position of hair whorls on the forehead and other morphological traits needs further research and should be interpreted with caution.     

Detection and pharmacokinetics of tetrahydrogestrinone in horses

The anti-doping rules of national and international sport federations ban any use of tetrahydrogestrinone (THG) in human as well as in horse sports. Initiated by the THG doping scandals in human sports a method for the detection of 3-keto-4,9,11-triene steroids in horse blood and urine was developed. The method comprises the isolation of the analytes by a combination of solid phase and liquid–liquid extraction after hydrolysis and solvolysis of the steroid conjugates. The concentrations of THG in blood and urine samples were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
A THG excretion study on horses was conducted to verify the method capability for the analysis of postadministration urine samples. In addition, blood samples were collected to allow for determination of the pharmacokinetics of THG in horses. Following the administration of a single oral dose of 25 μg THG per kg bodyweight to 10 horses, samples were collected at appropriate intervals. The plasma levels of THG reached maximal concentrations of 1.5–4.8 ng/mL. Twenty-four hours after the administration plasma levels returned to baseline. In urine, THG was detectable for 36 h. Urinary peak concentrations of total THG ranged from 16 to 206 ng/mL. For the 10 horses tested, the mean plasma clearance of THG was 2250 mL/h/kg and the plasma elimination half-life was 1.9 h.     

Comparison of cervical epidural morphine with intravenous morphine administration on antinociception in adult horses using thermal threshold testing

ObjectiveTo compare the antinociceptive effects of morphine administered via cervical epidural catheter to intravenously administered morphine using a thermal threshold (TT) testing model in healthy adult horses.Study designProspective, randomized, blinded experimental study.AnimalsA total of six university-owned adult horses.MethodsHorses were instrumented with a cervical (C1–C2) epidural catheter and TT testing device with probes at withers and thoracic limb coronary bands. All horses underwent three TT testing cycles including cervical epidural morphine administration (treatment EpiM; 0.1 mg kg^–1), systemic morphine administration (treatment SystM; 0.1 mg kg^–1) and no morphine administration (treatment Control). Baseline TT was established prior to treatments, and TT was tested at 15, 30, 60, 90, 120, 150, 180, 240, 300, 360, 420, 480, 600 and 720 minutes following treatment. Horses underwent a 5 day washout period between treatments and the order of treatment was randomized. Differences between treatments were analyzed with repeated measures anova.ResultsSystemic and epidural morphine administration resulted in significantly higher TT values compared with baseline and control treatment. The duration of effect was significantly longer in treatment EpiM (10–12 hours) than in treatment SystM (1.5–2.0 hours). Horses in treatment EpiM had significantly higher TT values at time points 180–600 minutes (withers) and 300–600 minutes (coronary band) than horses in treatment SystM.Conclusions and clinical relevanceCervical epidural administration of morphine provided antinociceptive effects as measured by increased TT for 10–12 hours compared with 1.5–2.0 hours for intravenously administered morphine. No complications or adverse effects were noticed following epidural placement of a C1–C2 catheter and administration of morphine. The use of a cervical epidural catheter can be considered for analgesia administration in treatment of thoracic limb and cervical pain in the horse.     

Problems associated with perioperative morphine in horses: a retrospective case analysis

Objective To identify the incidence of adverse effects caused by morphine 100–170 µg kg^?1 administration during surgery in horses. Design Retrospective case record analysis (1996–2000). Animals Eighty‐four healthy (ASA 1 or 2) horses, mean age 5.5 ± 3.1 (SD) years (2 months to 16 years), mean weight 524 ± 14 kg (100–950). Methods Physiological data and evidence of complications were collected from the anaesthetic records of all animals anaesthetized with romifidine, ketamine, diazepam and halothane and undergoing laryngeal surgery or orchiectomy at the Royal (Dick) School of Veterinary Studies. Cases were divided into those receiving (group M+; n = 18) and those not receiving morphine (M?; n = 29), and the data compared. Values for heart and respiratory rate and mean arterial pressure were compared at 15‐minute intervals between 30 and 120 minutes after induction using anova for repeated measures. The incidence of intraoperative problems was compared using Fisher's exact test. Recovery scores were compared using Student's unpaired t‐test. The records of a further 37 horses undergoing umbilical herniorrhaphy (n = 5), arthroscopy (n = 29) or tarsal arthrodesis (n = 3) were also studied but not analysed statistically due to disparate treatment distribution. Results There were no significant differences between the M+ and M? groups. The incidence of post‐operative complications such as box‐walking and colic were similar in each group. Conclusions Morphine doses of 100–170 µg kg^?1 do not increase the risk of problems when used to provide perioperative analgesia in horses anaesthetized with romifidine, ketamine, diazepam and halothane. Clinical relevance Morphine provides an acceptable and relatively inexpensive way to provide perioperative analgesia in horses.     

The cardiorespiratory effects of morphine and butorphanol in horses anaesthetised under clinical conditions

Twenty-five horses admitted for minor orthopaedic or soft tissue surgery were anaesthetised with detomidine, ketamine and halothane. Heart rate, arterial blood pressure, respiratory rate, tidal volume, minute volume, blood gases and occlusion pressures were measured before and for 30 mins after intravenous (iv) injection of saline, butorphanol 0.05 mg/kg bodyweight (bwt) or morphine 0.02 or 0.05 mg/kg bwt. Drug or saline treatment induced no significant changes from pre-treatment values within a group for arterial blood pressure, heart rate, respiratory rate, arterial carbon dioxide tension, arterial oxygen tension and occlusion pressure. In conclusion, both morphine and butorphanol at the stated doses cause no adverse effects on the cardiovascular and respiratory systems of anaesthetised horses.     

The effects of morphine on the recovery of horses from halothane anaesthesia

OBJECTIVE: To investigate the effects of peri-operative morphine on the quality and duration of recovery from halothane anaesthesia in horses. STUDY DESIGN: Prospective randomized study. ANIMALS: Twenty-two client owned horses, ASA category I or II. METHODS: Horses undergoing elective surgical procedures were divided into two groups and paired according to procedure, body position during surgery, body mass and breed. Group M+ received morphine by intravenous injection (0.15 mg kg(-1)) before induction of anaesthesia and then by infusion (0.1 mg kg(-1) hour(-1)) during anaesthesia. Group M- received the same anaesthetic agents except that morphine was excluded. At the end of surgery, the horses were placed in a recovery box and allowed to recover without assistance. Recoveries were recorded on videotape, beginning when the anaesthetist left the recovery box, and ending when the horse stood up. Recoveries were assessed from digital video recordings by three observers, unaware of treatment. The time to first movement, attempting and attaining sternal recumbency and standing were recorded. The quality of various aspects of the recovery was assessed to produce a total recovery score; high numerical values indicate poor recoveries. The duration of anaesthesia and the total dose of morphine administered were recorded. RESULTS: The mean morphine dose (95% CI) was 147 (135-160) mg [equivalent to 0.27 (0.25-0.29) mg kg(-1)]. The recovery scores (median, 95% CI) for the M- and M+ groups were 25, 19-41 and 20, 14-26, respectively. Total score increased as duration of anaesthesia increased, independent of treatment. Untreated (M-) horses made more attempts to achieve sternal recumbency: mean number of attempts (95% CI) for M- was 4.5 (2.7-6.2) compared with 2.0 (1.4-2.6) (M+). Untreated horses made more attempts to stand (2.1, 1.6-2.6) compared with the morphine recipients (1.3, 1.1-1.5). Time to standing (in minutes) was significantly (p = 0.0146) longer for the untreated (31.3, 24.3-38.3) compared with treated animals (26.6, 20.9-32.3). The interval between the first movement in recovery to the time at standing was significantly (p < 0.001) longer for M- (14.5, 12.1-16.9 minutes) compared with M+ animals (7.4, 5.0-9.8 minutes). CONCLUSIONS AND CLINICAL RELEVANCE: Recoveries from anaesthesia in the morphine recipients were characterized by fewer attempts to attain sternal recumbency and standing, and a shorter time from the first recovery movement to the time of standing.