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EQUINE VETERINARY JOURNAL Equine vet. J . (1991) 23 (3) 219-223
Exercise induced hormonal and metabolic changes in Thoroughbred horses: effects of conditioning and acepromazine J. F. FREESTONE, K. J. WOLFSHEIMER, S. G. KAMERLING', G. CHURCHt, J. HAMRA' and C. BAGWELL' Department of Veterinary Clinical Sciences, *Department of Veterinary Physiology, Pharmacology and Toxicology and tDepartment of Experimental Statistics, Louisiana State University, Baton Rouge, Louisiana, 70803-8422,USA.
Summary Nine Thoroughbred horses were assessed to determine the normal response of insulin, glucose, cortisol, plasma potassium (K) and erythrocyte K through conditioning and to exercise over 400 and 1O , OO m. In addition, adrenaline, noradrenaline, cortisol, plasma K, erythrocyte K and L-lactate concentrations were evaluated in response to maximal exercise with and without the administration of acepromazine. Conditioning caused no obvious trends in plasma K, erythrocyte K, insulin or glucose concentration. Serum cortisol increased (P c 0.05) from the initial sample a t Week 1 to Weeks 4 and 5 (attributed to a response to training), and then decreased. During conditioning, three horses had low erythrocyte K concentrations (c 89.3 mmol/litre). Further work is needed to define the significance of low erythrocyte K concentrations in the performance horse. In all tests maximal exercise increased plasma K, glucose and cortisol concentrations, whereas insulin and erythrocyte K concentrations decreased. Thirty minutes following exercise, plasma K and erythrocyte K concentrations returned to resting values, whereas glucose and cortisol concentrations continued to increase and the insulin concentration also was increased. The magnitude of the changes varied for pre-conditioned vs postconditioned exercise tests and the duration of exercise. The administration of acepromazine prior to exercise over 1O , OO m failed to alter the circulating noradrenaline and adrenaline concentrations in anticipation of exercise or 2 mins following exercise. Acepromazine administration, however, did cause lower L-lactate concentration 2 mins (P c 0.03) and 30 mins (P 2 0.005) following exercise. Also, erythrocyte K showed a delayed return to baseline levels at 30 mins post exercise. Further evaluation of these trends may help explain the beneficial role acepromazine plays in limiting signs of exertional rhabdomyolysis when administered prior to exercise.
Rose 1987; Snow, Mason, Ricketts and Douglas 1983). However, in man marked differences occur when evaluating the endocrine response of submaximal and maximal exercise (Howlett 1987). The endocrine and biochemical parameters in many of the equine studies also have been measured independently and often in an uncontrolled environment such as a racecourse. The investigators in this study controlled the management, conditioning and testing conditions of the subjects. All horses were exercised maximally, and the conditioning programme was designed to reproduce the exercise performed by Thoroughbred racehorses in training. The investigators sought answers to the following specific questions: 1) what is the normal response of insulin, glucose, cortisol, plasma potassium and erythrocyte potassium following maximal exercise; 2) do the baseline concentrations of the variables measured change during conditioning and 3) what is the response of noradrenaline and adrenaline, as well as other measured variables, prior to and following maximal exercise with and without acepromazine. Acepromazine was studied for its effect on decreasing the incidence of exertional rhabdomyolysis. Current treatment trends to limit exertional rhabdomyolysis using acepromazine are unsubstantiated, because no mechanism of action has been identified for its success. Two hypotheses for acepromazine's apparent success have been proposed: 1) acepromazine is a vasodilator, inhibiting a receptors and increasing arterial diameter and volumetric flow rate, thereby increasing blood flow (Walker and Geiser 1986) and 2) acepromazine tranquilises nervous horses. Some researchers suggest that these nervous horses are predisposed to exercise-associated muscle damage (Hodgson 1987). In this study, we hypothesise that acepromazine may modify the release of catecholamines in anticipation of exercise and in response to exercise. The catecholamines are potent vasoconstrictors and may decrease blood supply to muscle, which then may lead to hypoxic muscle damage. Materials and methods
Introduction Horses THIS study was performed to evaluate the response of insulin, cortisol, glucose, plasma potassium (K) and erythrocyte K to conditioning and maximal exercise tests in the Thoroughbred horse. Although other studies have evaluated the response of endocrine and other biochemical parameters to exercise, the exercise regimen in these studies has varied, changing in type (eg endurance, swimming, treadmill), intensity (eg submaximal, maximal) and duration (Judson, Frauenfelder and Mooney 1983; Dybdal, Gribble and Madigan 1980 Garcia and Beech 1986; Church, Evans, Lewis and
Nine healthy Thoroughbred horses, consisting of five males and four females, with a mean age of 4.2 k 0.4 years were used in this study. All horses had received previous race training but had not been conditioned or raced for nine months prior to the study. The horses were clinically normal as assessed by a complete blood count, serum biochemistry profile and physical examination. All horses had been vaccinated against Eastern encephalomyelitis virus, Western encephalomyelitis virus, tetanus and influenza virus, treated
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with an anthelmentic (Ivermectin per os), and tested negative for equine infectious anaemia virus on agar gel immunodihsion. Testing and conditioning timetable Exercise tests were performed prior to and 11 weeks following a conventional race conditioning programme. Exercise tests were begun at approximately 07:30 and completed at 1000. The experimental timetable is shown in Table 1. Exercise testing Preparation of the horses for exercise tests was identical. Horses were fed their evening ration and then food was withheld prior to testing. The following morning, blood samples were collected from horses at rest in their stalls, then the animals were saddled and taken to the track. Once mounted and on the track, the horses trotted for 1,200 m and cantered for 800 m in a clockwise direction (warm-up). At this point the animals were stopped, turned in the opposite direction and asked for a maximal effort over either 400 or 1,OOO m (depending on the test). The horses were exercised maximally over either 400 or 1,OOO m in an attempt to standardise each exercise test. Prior to the last exercise test (Week 16), acepromazine (Promace; Fort Dodge Laboratories, Fort Dodge, Iowa) was administered intravenously (iv) (7 mg per horse) after the resting blood sample collection. The acepromazine dosage of 7 mg was selected after consultation with veterinarians who reported using a prophylactic dose ranging from 5 to 10 mg prior to exercise. They cited the temperament of the horse and the incidence and severity of exertional rhabdomyolysis as factors determining dosage. Acepromazine administered at a dose of 0.066 m a g bodyweight (bwt) is effective within 5 mins, and lasts at least 30 mins (Walker and Geiser 1986). Therefore, after a 20-min post drug interval, the horses in this study were subjected to the 1,OOO m exercise test. Between each test, the horses continued to exercise to a mild degree on the track and in the swimming pool. Blood sample collection At Week 0 during the conditioning period and prior to any experimental intervention, baseline samples were collected at 10:00 (2 h after feeding). During the training period, samples were collected once weekly at 07:30 to 08:00, prior to feeding. On exercise test days, samples were collected prior to (baseline), and then 2 mins and 30 mins following the test. An additional sample was collected for noradrenaline and adrenaline prior to the exercise tests over 1,OOO m, when the horses were saddled and in clear view of the racetrack. Sample analysis Serum insulin, cortisol and plasma glucose, potassium and erythrocyte potassium were determined during conditioning, as well TABLE 1: Exercise testing and 6onditioning regimen for nine horses Week 1 Alternate daily 400 m trot or 80 m swim 2 Pre-conditioning excercise - 400 rn 2-13 Horses conditioned six days/week 1,600 rn -4 to 5 mins/day for 4 days/week, adding 800 rn each week until 4,000 m/day Speed increased over 11 weeks until 1,600 m/3 mins reached Two days/week horses swam, increasing to 24 laps in 20 mins of an 11 m pool by Week 13 14 Post conditioning exercise test -400 m 15 Post conditioning exercise test - 1,000 rn 16 Post conditioning exercise test - 1,000 m following acepromazine
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as during the 400 and 1,OOO m exercise tests. In addition, L-lactate concentration was measured in the 1,OOO m exercise tests. Serum insulin and plasma glucose concentrations were not determined following acepromazine administration. Plasma adrenaline and noradrenaline concentrations with and without acepromazine were determined in only seven horses. Approximately 10 ml of blood was drawn into plain vacutainer tubes to determine insulin and cortisol concentration. Heparinised tubes were used to measure erythrocyte K, plasma K and L-lactate concentration and EDTA tubes for glucose. EDTA tubes were spun immediately for plasma glucose measurement (System MA, Beckman, Fullerton, California). Plasma K and erythrocyte K were determined with flame photometry (Kalina Flame; Beckman) using the method of Muylle et a1 (1983). L-lactate concentration was determined using an ultraviolet, enzymatic method (Sigma Chemical Co, St Louis, Missouri). Adrenaline and noradrenaline concentrations were made in plasma from heparinised evacuated vacutainer tubes with 60 mg/ml of glutathione added at the rate of 20 @/ml of blood. adrenaline and noradrenaline concentrations were determined by high performance liquid chromatography (Hardee, Lai, Semrad and Trim 1982). Hormonal validation Insulin values for serum samples were determined by radioimmunoassay with a commercially available kit used in man (Insulin Coat-a-Count; Diagnositc Products, Los Angeles, California). Specificity for equine insulin was evaluated by demonstration of dilutional parallelism between standard solutions and serial dilutions of endogenous insulin in equine serum (r = 0.9974). Biological specificity was demonstrated by revealing that iv glucose stimulated an increase in serum insulin concentrations in healthy horses and ponies. Accuracy was demonstrated by the addition of porcine insulin to equine serum in concentrations from 4 to 235 piu/ml. Linear regression analysis of the recovery curve demonstrated a correlation coefficient (r) of 0.9999. Low end sensitivity for the assay, defined as the apparent concentration 3 sd below the counts at maximum binding, was 1.9 piu/ml. The intra-assay precision for 20 replicates of equine serum with a mean concentration of 14.1 piu/ml and 16.3 piu/ml revealed coefficients of variation of 15.6 per cent and 13.8 per cent respectively. The inter-assay coefficient of variation for the same samples for six runs was 17.8 per cent and 6.6 per cent respectively. The reference range of insulin concentration in equine serum determined from 12 healthy horses was 2.6 to 12.1 piu/ml. The mean was 5.6 piu/ml. Cortisol values for serum samples were determined by radioimmunoassay with a commercially available kit used in man (Cortisol Coat-a-Count; Diagnostic Products, Los Angeles, California). Specificity for equine cortisol was evaluated by demonstrating dilutional parallelism between standard solutions and serial dilutions of endogenous cortisol. Biological specificity was demonstrated in a horse by showing a two-fold increase in endogenous cortisol after the injection of 100 iu intramuscularly (im) of ACTH (60 to 143 ng/ml). Also, the concentration of endogenous cortisol decreased to less than 4.5 ng/ml and 5.2 ng/ml at 15 and 24 h respectively, following 40 @ k g im of dexamethasone (normal resting cortisol values ranged from 19.3 to 83.8 ng/ml with a mean of 57 ng/ml f 2.1). The intra-assay precision for 20 replicates of equine plasma at three concentrations (18.7, 113.7 and 435.5 ng/dl) revealed coefficients of variation of 14.7, 5.0 and 3.7 per cent, respectively. The inter-assay coefficients of variation for eight assays of three equine plasma samples were 16.5 per cent, 4.2 per cent and 7.1 per cent (18.3, 127.3 and 406.4 ng/dl, respectively). Statistical analyses Data were analysed using repeated measures, two-way analysis of variance, using the Huynh-Feldt test statistic adjusted for sphericity.
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Weekly means were compared in the conditioning regimen using Duncan's multiple range test. Means in the exercise tests were compared univariately with least square means for times within groups. The noradrenaline and adrenaline data were not distributed normally. The noradrenaline data were transformed by square root prior to performing the repeated measures analysis of variance. Noradrenaline data were reported using means and confidence intervals by Sokal and Rohlf (1981). Adrenaline concentrations were analysed non-parametrically using the Kruscal-Wallis test. Significance was reported at P 50.05 level.
Results Conditioning Mean (f sd) plasma K concentrations were within normal limits over the 11 weeks of conditioning, ranging from 3.3 f 0.5 mmol/litre to 3.8 f 0.2 mmol/litre. Plasma K, erythrocyte K, plasma glucose and serum insulin concentrations showed no significant differences over time for the 11 weeks of conditioning (Table 2). Three horses had consistently low erythrocyte K concentrations during conditioning (Horses 1 [x = 81.6 mmol/litre], 7 [x = 78.6 mmol/litre] and 8 [x = 81.0 mmol/litre]). The three horses with low erythrocyte K concentrations had normal plasma K concentrations at all sample times. Serum cortisol concentration ranged from 52.2 f 12.8 ng/ml at Week 1 to a peak of 67.6 f 10.2 ng/ml by Week 5. The increase in serum cortisol concentration occurred gradually and was significantly higher than baseline (Week 1) by Weeks 4 and 5 of conditioning (P 20.05) and then the concentration began to decrease (Table 2). Pre-conditioning vs post conditioning exercise tests over 400 m Maximal exercise induced significant changes over time for plasma K, plasma glucose, serum insulin and cortisol (P I 0,0001) and erythrocyte K (P I 0.0014). Also, there were significant changes between the pre-conditioned and post conditioned groups for serum cortisol concentration (P I 0.0012). In both the conditioned and unconditioned group, serum cortisol increased from baseline concentrations to 2 mins following exercise and then increased further to the 30 min sample. After conditioning, serum cortisol concentrations were greater at baseline (P I 0.001), 2 mins post exercise (P I 0.01) and 30 mins post exercise (P 20.01) than the concentrations in the unconditioned horses. The percentage change from baseline to 2 mins following exercise was similar for both groups (20 per cent unconditioned and 24 per cent conditioned). However, by 30 mins after exercise, the unconditioned horses showed a 70 per cent increase in serum cortisol concentration from baseline values compared to a 52 per cent increase in the conditioned horses. No differences were noted between the pre-
conditioned and post-conditioned horses exercising over 400 m for glucose, K, erythrocyte K or serum insulin concentration. The exercise was performed in 23.6 f 1.4 secs and 23.1 f 1.3 secs in the pre- and post conditioned groups, respectively. Exercise tests for conditioned horses: 400 vs I ,ooOm Exercise induced significant alterations in plasma K (P I O.OOOl), erythrocyte K (P 2 0.01), cortisol (P I 0.0001), insulin (P IO.0001) and glucose (P 2O.OOOl) over time. Differences were noted between the 400 m and 1,000 m groups for plasma K (P I 0.009) and glucose (P I 0.02). After the 1,000 m test, plasma K was significantly higher at 2 mins (P 10.0003) and 30 mins (P 50.04) following exercise than after the 400 m test. After both tests, erythrocyte K decreased following maximal exercise and then increased. Also, after the 1,OOO m test, glucose concentration was significantly higher 2 mins following exercise than after the 400 m test (P I 0.05) (Table 3). there were no differences between the groups for insulin or cortisol concentration. The mean speeds for the exercise periods were 17.3 m/sec for the 400 m test and 15.2 m/sec for the 1,OOO m test. Maximal exercise over I ,OOO m with and without acepromazine for conditioned horses Following the administration of acepromazine, alterations in behaviour were observed in all horses. %o horses with anxious and nervous temperaments were more relaxed and easier to manage both before and during exercise after acepromazine was administered. Also, these horses showed no adverse effects during exercise, and in some cases even improved their performance time. In contrast, horses relaxed prior to exercise became even more relaxed, and at times were difficult to motivate during exercise. One of these horses ran a very slow exercise test time. Horses receiving acepromazine performed the test in a mean time of 66.4 f 3.2 secs compared to 65.8 f 1.4 secs for the 1,OOO m test. In both groups, mean maximal heart rates (Hippocard, PEH 2,000 Bioengineering, Zurich, Switzerland) exceeded 200 beatdmin (bpm) during exercise (205 f 4.9 bpm with acepromazine and 208.1 f 5.4 bpm without acepromazine). This study demonstrated that exercise performed over 1,OOO m with and without acepromazine showed significant time effects for plasma K (P I 0.0001), erythrocyte K (P I0.02), cortisol (P I 0.0001), adrenaline (P IO.OOOl), noradrenaline (P I 0.03) and Llactate concentration (P I0.0001). Only L-lactate concentration differed between the groups (P I 0.02). Plasma L-lactate concentration increased immediately following exercise in both groups and then decreased to the 30 min sample. With acepromazine, plasma L-lactate concentration was significantly lower 2 mins (P I0.03) and 30 mins after exercise (P I 0.0005) TABLE 3: Response to maximal exercise over 400 m vs 1,000 m post conditioning in nine horses
TABLE 2: Weekly alterations in plasma potassium (PK), erythrocyte potassium (RBCK), insulin, glucose and cortisol concentrationsin nine horses during Conditioning
Distance
Week
400 rn
1 2 3 4 5 6 7 8
9 10
PK RBCK (mmol/litre) (mmolllitre)
3.6f0.6 3.8f0.2 3.7f0.3 3.6f0.4 3.7f0.5 3.5f0.3 3.6f0.3 3.7f0.5 3.3f0.5 3.5f 0.4
87.5 f 8.0 88.8 f 5.1 90.1 f 6 . 8 89.1 f 9 . 7 85.8 f 8.0 88.8 f 9.1 86.2 f 6.7 87.7 f 9.3 88.8 f 7.8 86.0 f 6.1
Insulin (piu/ml)
Glucose (mmol/litre)
Cortisol (ng/ml)
6.0 f 3.2 5.1 f 1.2 5.6 f 1.4 4.4 f 0.7 5.0 f 2.0 4.7 f 1.o 4.4 f 0.8 4.6 f 0.8 4.6 f 1.9 5.6 f 1.9
4.6 f 0.4 4.5f0.3 4.4f0.4 4.3f0.2 4.5 f 0.4 4.5 f 0.4 4.5 f 0.3 4.4 f 0.4 4.5 f 0.5 4.5f0.3
52.2 f 12.8 59.2f 8.6 64.1 f l l . 1 66.0f 9.4' 67.6 f 10.2* 61.6 f 15.5 63.3 f 12.5 57.7 f 16.7 59.1 f 13.4 60.0f 9.4
Data expressed as means f sd. 'Significantly different to Week 1 (P I 0.05)
Baseline
l m e (mins) post exercise: 2 30
PK (mmol/litre) 3.4 f0.3 5.0 f 0.6a 3.7 f 0.2 RBCK (rnmol/litre) 88.4 f 7.6 84.3 f 6.8a 90.5 f 6.2 Insulin (Fiulrnl) 7.1 f 3.V 5.4 f 1.9 8.8 f3.4a Glucose (mmol/litre) 4.5 f 0.5 5.4 f 0.5a 6.0 f 1.3a Cortisol (ng/ml) 69.4 f 21 .O 87.1 f 22.7a 104.8 f 18.2b
1,000m PK (mrnol/litre) 3.7 f0.4 5.6 f 0.3at 4.0 f 0.4' RBCK (mmoWlitre) 84.7 f 5.7 82.5 f 5.4a 83.7 f 4.4 Insulin (piu/ml) 5.2 f 1.3 4.4 k 1.4 7.9 f 3.4a Glucose (mmol/litre) 4.5 f 0.2 6.1 f0.9a' 6.4 f 0.7a Cortisol (nglrnl) 65.2 f 12.0 85.3f 18.6a 116.6 f 21.2b Data expressed as mean f sd. Means significantly higher between the 400 m and 1,000 m groups P I 0.05; t P I 0.001. Values in the same line with letter superscripts (a,b) are significantly different, P50.05. PK: plasma postassium; RBCK: erythrocyte potassium
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been reported in response to exercise in the horse (Harris and Snow 1988). Following the completion of exercise, K quickly declines, controlled by extrarenal factors including insulin, the sympathetic nervous system and acid-base balance (Bia and DeFronzo 1981). These extrarenal factors can influence the translocation of K intracellularly or extracellularly. In addition, another contributing factor to the plasma K concentration is the shift of erythrocyte K to the extracellular fluid. In this study, erythrocyte K was consistently decreased following all maximal exercise tests. The increase in hydrogen ion concentration following maximal exercise may stimulate K movement from erythrocytes, and a suppressed insulin Discussion concentration may in turn, allow this to happen (Hespel et a1 1986). In this study, maximal exercise resulted in increased glucose Effects of conditioning concentrations 2 mins after exercise and then a further increase at Throughout the conditioning period there was no significant change the 30-min sample. In addition, serum insulin was decreased 2 min from baseline measurements in plasma K, erythrocyte K, serum following exercise; this effect appears erroneous because of the insulin or plasma glucose concentration. Three horses had increase in plasma glucose. However, similar trends have been noted erythrocyte K concentrations consistently below 83.9 mmoMitre, a in insulin concentration following maximal exercise in man, and in value determined by Muylle et a1 (1984) to be low. These authors other horses (Howlett 1987; Church et a1 1987; Mikines et a1 suggested that low erythrocyte K concentrations correlate with low 1988a). Serum insulin rebounded to increased levels by 30 mins muscle K concentrations and thus may impair performance (Muylle after exercise. The rebound insulinaemia following exercise is et a1 1983; Muylle et a1 1984). The three horses with low thought to be caused by 1) withdrawal of sympathetic neural erythrocyte K concentrations showed a normal response to exercise inhibition of pancreatic B cells and 2) elevations in endogenous in that their plasma K concentrations increased. Although no genetic opioid peptides stimulating insulin release (Farrell, Sonne, Mikines pattern has been observed in horses with low erythrocyte K and Galbo 1988). If glucose increases following maximal exercise concentrations, a definite hereditary disorder occurs in sheep and insulin is initially suppressed, then other factors must be (Tucker and Ellory 1970). Further studies on the hereditability of involved in the glucoregulatory response. The glucoregulatory high or low erythrocyte K concentrations may prove interesting, response to exercise in this study was characterised by a fall in particularly if an association is observed between low erythrocyte K insulin, and an increase in adrenaline, noradrenaline and cortisol concentrations and poor performance. Conditioning did lead to an concentrations. Glucagon was not measured, but increases in man, initial increase in serum cortisol concentration at Weeks 4 and 5 and probably horses, following exercise (Vranic et a1 1984). compared with its lowest concentration at Week 1, and this was Adrenaline and neural noradrenaline release appear to be most significant in increasing glycogenolysis in the liver, as well as in attributed to possible adaptation to conditioning. exercising and non-exercising muscle (Richter et a1 1982; Hoelzer et a1 1986a). Similarly, the fall in insulin and rise in glucagon Effect of exercise support the increase in glucose concentration (Hoelzer et a1 1986b). During exercise, K is released from working skeletal muscles Serum cortisol concentration increased immediately following (Kjellmer 1965; Van Beaumont et a1 1973). Following all exercise maximal exercise and increased further at the 30-min sample. This tests in this study, as plasma K increased, erythrocyte K declined. trend was common in all exercise trials. Similar findings in response Research has demonstrated that the intensity of the exercise and the to maximal exercise have been reported in man and horses time taken to collect samples following exercise will influence both (Thornton 1985; Church et a1 1987; Howlett 1987). In man, a work the plasma K and erythrocyte K concentration measured (Hespel et intensity of 60 per cent of maximal oxygen uptake is the critical a1 1986; Harris and Snow 1988). Therefore, the directional changes point for exercise-induced elevations in cortisol (Howlett 1987). in plasma K and erythrocyte K in this study might have been more Several studies have shown that physical conditioning potentiates pronounced if the samples had been collected sooner than 2 mins the action of cortisol, resulting in a smaller increase in post-exercise following exercise. ACT'H and cortisol (Buono, Yeager and Sucec 1987; Church et a1 Very high levels of plasma K (in excess of 10 mmol/litre) have 1987; Luger et al 1987). In the present study, a similar trend was
(Table 4). Individual noradrenaline concentrations were highly variable at all collection times. Plasma noradrenaline (P 5 0.005) and adrenaline (P 5 0.02) were increased 2 mins following exercise over baseline concentrations and the groups were not significantly different. Plasma noradrenaline concentrations were decreased (P 5 0.04) at the 30 min post exercise sample in the acepromazine treated group compared to the non-treated group, in which noradrenaline concentrations remained increased. Administration of acepromazine failed to alter responses in anticipation of exercise.
TABLE 4: Response to maximal exercise over 1,000 m with and without acepromazine administration (7 ma ivl
N Control PK (mmolllitre) RBCK (mmolllitre) Cortisol (nglml) L-lactate (mmolllitre) 7 Adrenaline (pglml) Noradrenaline (pglml) Acepromazine 9 PK (mmolllitre) RBCK (mmolllitre) Cortisol (nglml) L-lactate (mmolllitre) 7 Adrenaline (pglml) Noradrenaline (pglml) 9
Baseline
Immediately pre-exercise
Time (mins) post exercise: 2
30
3.7 f 0.4 84.7 f 5.7 65.2 f 12 1.4 f 0.4 333 f 815.7 876(392,1552)
ND ND ND ND 143.7 f 227.2 412(110,906)
5.6 f 0.3a 82.5 f 5.4 85.3 f 18.6a 21.8 f 1.7b 988.7 f 669.S 131O(697,2116)a
4.0 f 0.4 83.7 f 4.4 116.6f21.2b 19.2 f 3.7at 125.9 f 175.2 1303(692,2107)a
3.6 f 0.3 85.8 It 7.4 77.0 f 14.8 1.6 f 0.4 61.7 f 101 222(26,610)
ND ND ND ND 105.3 f 213.4 443(125,949)
5.4 f 0.4a 84.8 f 8.4 96.1 f 11.4a 19.1 f 2.6b 1004.6 f 614.3a 1140(576,190 1)a
3.9 f 0.3 83.7 k 7.7 112.3 f 19.6b 14.6 f 5.3a 61.4 f 99.1 329(69,778)'
Data expressed as mean It sd, except noradrenaline for which confidence limits are reported. Means significantly higher between the control and acepromazine treated groups, * P 50.05,t P IO.001. Values in the same line with letter superscripts (a,b) are significantly different, P20.05. PK: plasma potassium; RBCK: erythrocyte potassium; ND: not determined
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noted in horses exercised over 400 m prior to and after conditioning. In this study, noradrenaline and adrenaline increased 2 mins following exercise over 1,OOO m, as previously reported (Evans, Smith and Weil-Malherbe 1956). Plasma noradrenaline concentrationswere highly variable and difficult to interpret because of the excitable temperament of Thoroughbred racehorses. Plasma adrenaline, on the other hand, was less variable. Horses showed no obvious trend in plasma adrenaline and noradrenaline concentrations in anticipation of exercise. Effect of acepromazine
Acepromazine administration prior to exercise resulted in behaviour changes in all horses. This was most marked in horses with excitable temperaments. Even at a low dosage (7 mg iv) the horses became more manageable, easier to ride and in some cases showed improved performance. Horses that were normally relaxed and easy to handle became more relaxed and were in some cases difficult to motivate, adversely affecting their performance. The administration of acepromazine prior to exercise did iiot alter the circulating catecholamines in anticipation of exercise or in the immediate postexercise period. Therefore, our study does not support the hypothesis that acepromazine limits the incidence of exertional rhabdomyolysis by suppressing circulating catecholamines. The decreased noradrenaline concentration in the acepromazine-treated horses 30 mins following exercise may have been a drug effect or individual variation. Perhaps the most interesting result of this study concerns the responses of L-lactate concentration and erythrocyte K following acepromazine administration. This may warrant further investigation. In the acepromazine-treated horses, L-lactate concentrations were decreased compared to the control group at 2 mins and 30 mins post exercise. This suggests that acepromazine may decrease the production and/or increase the clearance of Llactate. One of the oldest suggested pathogenic mechanisms for the development of exercise-associated rhabdomyolysis is the accumulation of lactic acid, although other authors have suggested that lactate accumulation is an improbable cause of muscle damage (Carlstrom 1932; Lindholm, Johansson and Kjaersgaard 1974; Hanis and Snow 1986). Another change following acepromazine administration was the delayed return of erythrocyte K to normal concentrations 30 mins after exercise, a trend noted in all other exercise tests. Because the promazine tranquilisers influence the extracellular to intracellular movement of electrolytes, these tranquilisers have been beneficial in the treatment of secretory diarrhoea (Rabbani, Greenough, Holmgren and Kirkwood 1982). By influencing electrolyte movements, acepromazine may alter neuromuscular irritability and impair the development of exertional rhabdomyolysis(Hanis and Snow 1986; C o h a n et a1 1978).
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Evans, C.L., Smith, D. F. G . and Weil-Malherbe. H. (1956)The relation between sweating and the catechol content of blood. J. Physiol. 132,542-552. Farrell, P. A,, Sonne, B., Mikines, K. and Galbo. H. (1988)Stirnulatory role for endogenous opioid peptides on postexercise insulin secretion in rats. J. appl. Physiol. 65,744-749.
Garcia, M.C. and Beech, J. (1986)Endocrinologic, hematologic, and heart rate changes in swimming horses. Am. J. vet. Res. 47,2004-2006. Hardee, G. E.,Lai, J. W., Semrad. S. D. and Trim, C. M. (1982)Catecholamines in equine and bovine plasmas. J. vef.Pharmacol. Therap. 5,279-284. Harris, P. E. and Snow, D. H. (1986)5 i n g up the loose ends of equine rhabdomyolysis. Equine vet. J. 5,346-348. Harris, I? and Snow, D. H.(1988)The effects of high intensity exercise on the plasma concentration of lactate, potassium and other electrolytes. Equine ref. J. 20. 109113.
Hespel, P.. Lijnen, I?, Fiocchi, R.. Denys, B., Lissens, W.. M'Buyamba-Kabangu, J. R. and Amery. A. (1986) Cationic concentrations and transmembrane fluxes in erythrocytes of humans during exercise. J. appl. Physiol. 61,3743. Hodgson. D. R. (1987)Exertional rhabdomyolysis. In: Current Therapy in Equine Medicine. Ed: N. E. Robinson. W. B. Saunders, Philadelphia. pp 487490. Hoelzer, D. R., Dalsky, G. P., Schwartz, N. S.. Clutter, W. E., Shah, S. D., Holloszy, J. 0. and Cryer, P. E. (1986a) Epinephrine is not critical to prevention of hypoglycemia during exercise in humans. Am. J.Physiol. 251,E104-EI 10. Hoelzer, D. R., Dalsky. G. P.. Clutter, W. E., Shah, S. D.. Holloszy, J. 0. and Cryer, P. E. (1986b) Glucoregulation during exercise: hypoglycemia is prevented by redundant glucoregulatory systems, sympathochromaffin activation, and changes in islet hormone secretion. J. clin. Invesi. 77,212-221. Howlen, T. A. (1987)Hormonal responses to exercise and training: a short review. Clin. Endocrin. 26,723-742.
Judson, G . J., Frauenfelder, H. C. and Mooney, G . J. (1983)Biochemical changes in Thoroughbred racehorses following submaximal and maximal exercise. In: Equine Exercise Physiology. Eds: D. E. Snow, S. B. G . Persson and R. J. Rose, Granta Editions, Cambridge. pp 408415. Kjellmer. 1. (1965)The potassium ion as a vasodilator during muscular exercise. Acfa. Physiol. Scand. 63,460-468.
Lindholm, A.. Johansson, H.-E. and Kjaersgaard, I? (1974)Acute rhabdomyolysis ("typing-up") in Standardbred horses: a morphological and biochemical study. Acta vef.scand. 15,325-339. Luger, A., Deuster, P. A., Kyle, S. B., Gallucci, W. T.. Montgomery, L. C., Gold, I? W.. G., I? (1987)Acute hypothalamic-pituitary-adrenal Loriaux, D. L. and C ~ U S O S responses to the stress of treadmill exercise. New Engl. J. Med. 316,1309- 13 15. Mikines. K. J., Farrell, I? A., Sonne, B.. Tronier, B. and Galbo. H. (1988a) Postexercise dose-response relationship between plasma glucose and insulin secretion. J. appl. Physiol. 64.988-999.
Muylle, E., Van den Hende. C.. Nuynen. J.. Oyaert. W. & Vlaminck, K. (1983) F'reliminary studies on the relationship of red blood cell potassium concentration and performance. In: Equine Exercise Physiology. Eds: D. E. Snow, S. B. G. Persson and R. J. Rose. Granta Editions, Cambridge. pp 360-370. Muylle, E., Van den Hende, C., Nuynen. J., Deprez, P., Vlaminck, K. and Oyaert, W. (1984)Potassium concentration in equine red blood cells: normal values and correlation with potassium levels in plasma. Equine vet. J. 16,447-449. Rabbani, G. H., Greenough. W. B., 111, Holmgren, J. and Kirkwood. B. (1982) Controlled hial of chlorpromazine as antisecretory agent in patients with cholera hydrated intravenously. Br. Med. J. 284, 1361-1364. Richter, E. A.. Ruderman, N. B.. Gavras, H., Belur, E. R. and Galbo. H. (1982)Muscle glycogenolysis during exercise: dual control by epinephrine and contractions. Am. J. Physiol. 242,E25-E32. Snow, D. H., Mason, D. K.. Ricketts. S. W. and Douglas, T. A. (1983)Post race blood biochemistry in Thoroughbreds. In: Equine Exercise Physiology. Eds: D. E. Snow, S . G. B. Persson and R. J. Rose. Granta, Cambridge. pp 389-399. Sokal, R. R. and Rohlf, F. J. (1981)Assumptions of analysis of variance: Logarithmic transformation. In: Biometry. Eds: R. R. Sokal and F. J. Rohlf. W. H. Freeman and Co. San Francisco. pp 419423. Thornton. J. R. (1985)Hormonal responses to exercise and training. Ver. Clin. N. Am. 1,
477496. Tucker, E. M. and Elloly, J. C. (1970)The M-L blood group system and its influence on red cell potassium levels in sheep. Anim. BloodGrp. & Bif~chem.Genet. 1, 101-112. Van Beaumont, W., Strand. J. C., Petrofsky, J. S.. Hipskind. S. G . and Greenleaf. J. E. (1973)Changes in total plasma content of electrolytes and proteins with maximal exercise. J.appl. Physiol. 34, 102-106. Vranic, M., Gauthier, C.. Bilinski, D.. Wasserman, D., El Tayeb, K., Hetenyi, G.. Jr. and Lickley. L. A. (1984)Catecholamine responses and their interactions with other glucoregulatory hormones. Am. J. Physiol. 247,E145-EI56. Walker, M. and Geiser. D. (1986) Effects of acetylpmmazine on the hemodynamics of the equine metatarsal anery, as determined by two-dimensional real-time and pulsed Doppler ultrasonography.Am. J. vet. Res. 47,1075-1078.
Received for publication: 10.10.89 Accepted: 6.6.90
EQUINE VETERINARY JOURNAL Equine vet. J . (1991) 23 (3) 219-223
Exercise induced hormonal and metabolic changes in Thoroughbred horses: effects of conditioning and acepromazine J. F. FREESTONE, K. J. WOLFSHEIMER, S. G. KAMERLING', G. CHURCHt, J. HAMRA' and C. BAGWELL' Department of Veterinary Clinical Sciences, *Department of Veterinary Physiology, Pharmacology and Toxicology and tDepartment of Experimental Statistics, Louisiana State University, Baton Rouge, Louisiana, 70803-8422,USA.
Summary Nine Thoroughbred horses were assessed to determine the normal response of insulin, glucose, cortisol, plasma potassium (K) and erythrocyte K through conditioning and to exercise over 400 and 1O , OO m. In addition, adrenaline, noradrenaline, cortisol, plasma K, erythrocyte K and L-lactate concentrations were evaluated in response to maximal exercise with and without the administration of acepromazine. Conditioning caused no obvious trends in plasma K, erythrocyte K, insulin or glucose concentration. Serum cortisol increased (P c 0.05) from the initial sample a t Week 1 to Weeks 4 and 5 (attributed to a response to training), and then decreased. During conditioning, three horses had low erythrocyte K concentrations (c 89.3 mmol/litre). Further work is needed to define the significance of low erythrocyte K concentrations in the performance horse. In all tests maximal exercise increased plasma K, glucose and cortisol concentrations, whereas insulin and erythrocyte K concentrations decreased. Thirty minutes following exercise, plasma K and erythrocyte K concentrations returned to resting values, whereas glucose and cortisol concentrations continued to increase and the insulin concentration also was increased. The magnitude of the changes varied for pre-conditioned vs postconditioned exercise tests and the duration of exercise. The administration of acepromazine prior to exercise over 1O , OO m failed to alter the circulating noradrenaline and adrenaline concentrations in anticipation of exercise or 2 mins following exercise. Acepromazine administration, however, did cause lower L-lactate concentration 2 mins (P c 0.03) and 30 mins (P 2 0.005) following exercise. Also, erythrocyte K showed a delayed return to baseline levels at 30 mins post exercise. Further evaluation of these trends may help explain the beneficial role acepromazine plays in limiting signs of exertional rhabdomyolysis when administered prior to exercise.
Rose 1987; Snow, Mason, Ricketts and Douglas 1983). However, in man marked differences occur when evaluating the endocrine response of submaximal and maximal exercise (Howlett 1987). The endocrine and biochemical parameters in many of the equine studies also have been measured independently and often in an uncontrolled environment such as a racecourse. The investigators in this study controlled the management, conditioning and testing conditions of the subjects. All horses were exercised maximally, and the conditioning programme was designed to reproduce the exercise performed by Thoroughbred racehorses in training. The investigators sought answers to the following specific questions: 1) what is the normal response of insulin, glucose, cortisol, plasma potassium and erythrocyte potassium following maximal exercise; 2) do the baseline concentrations of the variables measured change during conditioning and 3) what is the response of noradrenaline and adrenaline, as well as other measured variables, prior to and following maximal exercise with and without acepromazine. Acepromazine was studied for its effect on decreasing the incidence of exertional rhabdomyolysis. Current treatment trends to limit exertional rhabdomyolysis using acepromazine are unsubstantiated, because no mechanism of action has been identified for its success. Two hypotheses for acepromazine's apparent success have been proposed: 1) acepromazine is a vasodilator, inhibiting a receptors and increasing arterial diameter and volumetric flow rate, thereby increasing blood flow (Walker and Geiser 1986) and 2) acepromazine tranquilises nervous horses. Some researchers suggest that these nervous horses are predisposed to exercise-associated muscle damage (Hodgson 1987). In this study, we hypothesise that acepromazine may modify the release of catecholamines in anticipation of exercise and in response to exercise. The catecholamines are potent vasoconstrictors and may decrease blood supply to muscle, which then may lead to hypoxic muscle damage. Materials and methods
Introduction Horses THIS study was performed to evaluate the response of insulin, cortisol, glucose, plasma potassium (K) and erythrocyte K to conditioning and maximal exercise tests in the Thoroughbred horse. Although other studies have evaluated the response of endocrine and other biochemical parameters to exercise, the exercise regimen in these studies has varied, changing in type (eg endurance, swimming, treadmill), intensity (eg submaximal, maximal) and duration (Judson, Frauenfelder and Mooney 1983; Dybdal, Gribble and Madigan 1980 Garcia and Beech 1986; Church, Evans, Lewis and
Nine healthy Thoroughbred horses, consisting of five males and four females, with a mean age of 4.2 k 0.4 years were used in this study. All horses had received previous race training but had not been conditioned or raced for nine months prior to the study. The horses were clinically normal as assessed by a complete blood count, serum biochemistry profile and physical examination. All horses had been vaccinated against Eastern encephalomyelitis virus, Western encephalomyelitis virus, tetanus and influenza virus, treated
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with an anthelmentic (Ivermectin per os), and tested negative for equine infectious anaemia virus on agar gel immunodihsion. Testing and conditioning timetable Exercise tests were performed prior to and 11 weeks following a conventional race conditioning programme. Exercise tests were begun at approximately 07:30 and completed at 1000. The experimental timetable is shown in Table 1. Exercise testing Preparation of the horses for exercise tests was identical. Horses were fed their evening ration and then food was withheld prior to testing. The following morning, blood samples were collected from horses at rest in their stalls, then the animals were saddled and taken to the track. Once mounted and on the track, the horses trotted for 1,200 m and cantered for 800 m in a clockwise direction (warm-up). At this point the animals were stopped, turned in the opposite direction and asked for a maximal effort over either 400 or 1,OOO m (depending on the test). The horses were exercised maximally over either 400 or 1,OOO m in an attempt to standardise each exercise test. Prior to the last exercise test (Week 16), acepromazine (Promace; Fort Dodge Laboratories, Fort Dodge, Iowa) was administered intravenously (iv) (7 mg per horse) after the resting blood sample collection. The acepromazine dosage of 7 mg was selected after consultation with veterinarians who reported using a prophylactic dose ranging from 5 to 10 mg prior to exercise. They cited the temperament of the horse and the incidence and severity of exertional rhabdomyolysis as factors determining dosage. Acepromazine administered at a dose of 0.066 m a g bodyweight (bwt) is effective within 5 mins, and lasts at least 30 mins (Walker and Geiser 1986). Therefore, after a 20-min post drug interval, the horses in this study were subjected to the 1,OOO m exercise test. Between each test, the horses continued to exercise to a mild degree on the track and in the swimming pool. Blood sample collection At Week 0 during the conditioning period and prior to any experimental intervention, baseline samples were collected at 10:00 (2 h after feeding). During the training period, samples were collected once weekly at 07:30 to 08:00, prior to feeding. On exercise test days, samples were collected prior to (baseline), and then 2 mins and 30 mins following the test. An additional sample was collected for noradrenaline and adrenaline prior to the exercise tests over 1,OOO m, when the horses were saddled and in clear view of the racetrack. Sample analysis Serum insulin, cortisol and plasma glucose, potassium and erythrocyte potassium were determined during conditioning, as well TABLE 1: Exercise testing and 6onditioning regimen for nine horses Week 1 Alternate daily 400 m trot or 80 m swim 2 Pre-conditioning excercise - 400 rn 2-13 Horses conditioned six days/week 1,600 rn -4 to 5 mins/day for 4 days/week, adding 800 rn each week until 4,000 m/day Speed increased over 11 weeks until 1,600 m/3 mins reached Two days/week horses swam, increasing to 24 laps in 20 mins of an 11 m pool by Week 13 14 Post conditioning exercise test -400 m 15 Post conditioning exercise test - 1,000 rn 16 Post conditioning exercise test - 1,000 m following acepromazine
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as during the 400 and 1,OOO m exercise tests. In addition, L-lactate concentration was measured in the 1,OOO m exercise tests. Serum insulin and plasma glucose concentrations were not determined following acepromazine administration. Plasma adrenaline and noradrenaline concentrations with and without acepromazine were determined in only seven horses. Approximately 10 ml of blood was drawn into plain vacutainer tubes to determine insulin and cortisol concentration. Heparinised tubes were used to measure erythrocyte K, plasma K and L-lactate concentration and EDTA tubes for glucose. EDTA tubes were spun immediately for plasma glucose measurement (System MA, Beckman, Fullerton, California). Plasma K and erythrocyte K were determined with flame photometry (Kalina Flame; Beckman) using the method of Muylle et a1 (1983). L-lactate concentration was determined using an ultraviolet, enzymatic method (Sigma Chemical Co, St Louis, Missouri). Adrenaline and noradrenaline concentrations were made in plasma from heparinised evacuated vacutainer tubes with 60 mg/ml of glutathione added at the rate of 20 @/ml of blood. adrenaline and noradrenaline concentrations were determined by high performance liquid chromatography (Hardee, Lai, Semrad and Trim 1982). Hormonal validation Insulin values for serum samples were determined by radioimmunoassay with a commercially available kit used in man (Insulin Coat-a-Count; Diagnositc Products, Los Angeles, California). Specificity for equine insulin was evaluated by demonstration of dilutional parallelism between standard solutions and serial dilutions of endogenous insulin in equine serum (r = 0.9974). Biological specificity was demonstrated by revealing that iv glucose stimulated an increase in serum insulin concentrations in healthy horses and ponies. Accuracy was demonstrated by the addition of porcine insulin to equine serum in concentrations from 4 to 235 piu/ml. Linear regression analysis of the recovery curve demonstrated a correlation coefficient (r) of 0.9999. Low end sensitivity for the assay, defined as the apparent concentration 3 sd below the counts at maximum binding, was 1.9 piu/ml. The intra-assay precision for 20 replicates of equine serum with a mean concentration of 14.1 piu/ml and 16.3 piu/ml revealed coefficients of variation of 15.6 per cent and 13.8 per cent respectively. The inter-assay coefficient of variation for the same samples for six runs was 17.8 per cent and 6.6 per cent respectively. The reference range of insulin concentration in equine serum determined from 12 healthy horses was 2.6 to 12.1 piu/ml. The mean was 5.6 piu/ml. Cortisol values for serum samples were determined by radioimmunoassay with a commercially available kit used in man (Cortisol Coat-a-Count; Diagnostic Products, Los Angeles, California). Specificity for equine cortisol was evaluated by demonstrating dilutional parallelism between standard solutions and serial dilutions of endogenous cortisol. Biological specificity was demonstrated in a horse by showing a two-fold increase in endogenous cortisol after the injection of 100 iu intramuscularly (im) of ACTH (60 to 143 ng/ml). Also, the concentration of endogenous cortisol decreased to less than 4.5 ng/ml and 5.2 ng/ml at 15 and 24 h respectively, following 40 @ k g im of dexamethasone (normal resting cortisol values ranged from 19.3 to 83.8 ng/ml with a mean of 57 ng/ml f 2.1). The intra-assay precision for 20 replicates of equine plasma at three concentrations (18.7, 113.7 and 435.5 ng/dl) revealed coefficients of variation of 14.7, 5.0 and 3.7 per cent, respectively. The inter-assay coefficients of variation for eight assays of three equine plasma samples were 16.5 per cent, 4.2 per cent and 7.1 per cent (18.3, 127.3 and 406.4 ng/dl, respectively). Statistical analyses Data were analysed using repeated measures, two-way analysis of variance, using the Huynh-Feldt test statistic adjusted for sphericity.
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Weekly means were compared in the conditioning regimen using Duncan's multiple range test. Means in the exercise tests were compared univariately with least square means for times within groups. The noradrenaline and adrenaline data were not distributed normally. The noradrenaline data were transformed by square root prior to performing the repeated measures analysis of variance. Noradrenaline data were reported using means and confidence intervals by Sokal and Rohlf (1981). Adrenaline concentrations were analysed non-parametrically using the Kruscal-Wallis test. Significance was reported at P 50.05 level.
Results Conditioning Mean (f sd) plasma K concentrations were within normal limits over the 11 weeks of conditioning, ranging from 3.3 f 0.5 mmol/litre to 3.8 f 0.2 mmol/litre. Plasma K, erythrocyte K, plasma glucose and serum insulin concentrations showed no significant differences over time for the 11 weeks of conditioning (Table 2). Three horses had consistently low erythrocyte K concentrations during conditioning (Horses 1 [x = 81.6 mmol/litre], 7 [x = 78.6 mmol/litre] and 8 [x = 81.0 mmol/litre]). The three horses with low erythrocyte K concentrations had normal plasma K concentrations at all sample times. Serum cortisol concentration ranged from 52.2 f 12.8 ng/ml at Week 1 to a peak of 67.6 f 10.2 ng/ml by Week 5. The increase in serum cortisol concentration occurred gradually and was significantly higher than baseline (Week 1) by Weeks 4 and 5 of conditioning (P 20.05) and then the concentration began to decrease (Table 2). Pre-conditioning vs post conditioning exercise tests over 400 m Maximal exercise induced significant changes over time for plasma K, plasma glucose, serum insulin and cortisol (P I 0,0001) and erythrocyte K (P I 0.0014). Also, there were significant changes between the pre-conditioned and post conditioned groups for serum cortisol concentration (P I 0.0012). In both the conditioned and unconditioned group, serum cortisol increased from baseline concentrations to 2 mins following exercise and then increased further to the 30 min sample. After conditioning, serum cortisol concentrations were greater at baseline (P I 0.001), 2 mins post exercise (P I 0.01) and 30 mins post exercise (P 20.01) than the concentrations in the unconditioned horses. The percentage change from baseline to 2 mins following exercise was similar for both groups (20 per cent unconditioned and 24 per cent conditioned). However, by 30 mins after exercise, the unconditioned horses showed a 70 per cent increase in serum cortisol concentration from baseline values compared to a 52 per cent increase in the conditioned horses. No differences were noted between the pre-
conditioned and post-conditioned horses exercising over 400 m for glucose, K, erythrocyte K or serum insulin concentration. The exercise was performed in 23.6 f 1.4 secs and 23.1 f 1.3 secs in the pre- and post conditioned groups, respectively. Exercise tests for conditioned horses: 400 vs I ,ooOm Exercise induced significant alterations in plasma K (P I O.OOOl), erythrocyte K (P 2 0.01), cortisol (P I 0.0001), insulin (P IO.0001) and glucose (P 2O.OOOl) over time. Differences were noted between the 400 m and 1,000 m groups for plasma K (P I 0.009) and glucose (P I 0.02). After the 1,000 m test, plasma K was significantly higher at 2 mins (P 10.0003) and 30 mins (P 50.04) following exercise than after the 400 m test. After both tests, erythrocyte K decreased following maximal exercise and then increased. Also, after the 1,OOO m test, glucose concentration was significantly higher 2 mins following exercise than after the 400 m test (P I 0.05) (Table 3). there were no differences between the groups for insulin or cortisol concentration. The mean speeds for the exercise periods were 17.3 m/sec for the 400 m test and 15.2 m/sec for the 1,OOO m test. Maximal exercise over I ,OOO m with and without acepromazine for conditioned horses Following the administration of acepromazine, alterations in behaviour were observed in all horses. %o horses with anxious and nervous temperaments were more relaxed and easier to manage both before and during exercise after acepromazine was administered. Also, these horses showed no adverse effects during exercise, and in some cases even improved their performance time. In contrast, horses relaxed prior to exercise became even more relaxed, and at times were difficult to motivate during exercise. One of these horses ran a very slow exercise test time. Horses receiving acepromazine performed the test in a mean time of 66.4 f 3.2 secs compared to 65.8 f 1.4 secs for the 1,OOO m test. In both groups, mean maximal heart rates (Hippocard, PEH 2,000 Bioengineering, Zurich, Switzerland) exceeded 200 beatdmin (bpm) during exercise (205 f 4.9 bpm with acepromazine and 208.1 f 5.4 bpm without acepromazine). This study demonstrated that exercise performed over 1,OOO m with and without acepromazine showed significant time effects for plasma K (P I 0.0001), erythrocyte K (P I0.02), cortisol (P I 0.0001), adrenaline (P IO.OOOl), noradrenaline (P I 0.03) and Llactate concentration (P I0.0001). Only L-lactate concentration differed between the groups (P I 0.02). Plasma L-lactate concentration increased immediately following exercise in both groups and then decreased to the 30 min sample. With acepromazine, plasma L-lactate concentration was significantly lower 2 mins (P I0.03) and 30 mins after exercise (P I 0.0005) TABLE 3: Response to maximal exercise over 400 m vs 1,000 m post conditioning in nine horses
TABLE 2: Weekly alterations in plasma potassium (PK), erythrocyte potassium (RBCK), insulin, glucose and cortisol concentrationsin nine horses during Conditioning
Distance
Week
400 rn
1 2 3 4 5 6 7 8
9 10
PK RBCK (mmol/litre) (mmolllitre)
3.6f0.6 3.8f0.2 3.7f0.3 3.6f0.4 3.7f0.5 3.5f0.3 3.6f0.3 3.7f0.5 3.3f0.5 3.5f 0.4
87.5 f 8.0 88.8 f 5.1 90.1 f 6 . 8 89.1 f 9 . 7 85.8 f 8.0 88.8 f 9.1 86.2 f 6.7 87.7 f 9.3 88.8 f 7.8 86.0 f 6.1
Insulin (piu/ml)
Glucose (mmol/litre)
Cortisol (ng/ml)
6.0 f 3.2 5.1 f 1.2 5.6 f 1.4 4.4 f 0.7 5.0 f 2.0 4.7 f 1.o 4.4 f 0.8 4.6 f 0.8 4.6 f 1.9 5.6 f 1.9
4.6 f 0.4 4.5f0.3 4.4f0.4 4.3f0.2 4.5 f 0.4 4.5 f 0.4 4.5 f 0.3 4.4 f 0.4 4.5 f 0.5 4.5f0.3
52.2 f 12.8 59.2f 8.6 64.1 f l l . 1 66.0f 9.4' 67.6 f 10.2* 61.6 f 15.5 63.3 f 12.5 57.7 f 16.7 59.1 f 13.4 60.0f 9.4
Data expressed as means f sd. 'Significantly different to Week 1 (P I 0.05)
Baseline
l m e (mins) post exercise: 2 30
PK (mmol/litre) 3.4 f0.3 5.0 f 0.6a 3.7 f 0.2 RBCK (rnmol/litre) 88.4 f 7.6 84.3 f 6.8a 90.5 f 6.2 Insulin (Fiulrnl) 7.1 f 3.V 5.4 f 1.9 8.8 f3.4a Glucose (mmol/litre) 4.5 f 0.5 5.4 f 0.5a 6.0 f 1.3a Cortisol (ng/ml) 69.4 f 21 .O 87.1 f 22.7a 104.8 f 18.2b
1,000m PK (mrnol/litre) 3.7 f0.4 5.6 f 0.3at 4.0 f 0.4' RBCK (mmoWlitre) 84.7 f 5.7 82.5 f 5.4a 83.7 f 4.4 Insulin (piu/ml) 5.2 f 1.3 4.4 k 1.4 7.9 f 3.4a Glucose (mmol/litre) 4.5 f 0.2 6.1 f0.9a' 6.4 f 0.7a Cortisol (nglrnl) 65.2 f 12.0 85.3f 18.6a 116.6 f 21.2b Data expressed as mean f sd. Means significantly higher between the 400 m and 1,000 m groups P I 0.05; t P I 0.001. Values in the same line with letter superscripts (a,b) are significantly different, P50.05. PK: plasma postassium; RBCK: erythrocyte potassium
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been reported in response to exercise in the horse (Harris and Snow 1988). Following the completion of exercise, K quickly declines, controlled by extrarenal factors including insulin, the sympathetic nervous system and acid-base balance (Bia and DeFronzo 1981). These extrarenal factors can influence the translocation of K intracellularly or extracellularly. In addition, another contributing factor to the plasma K concentration is the shift of erythrocyte K to the extracellular fluid. In this study, erythrocyte K was consistently decreased following all maximal exercise tests. The increase in hydrogen ion concentration following maximal exercise may stimulate K movement from erythrocytes, and a suppressed insulin Discussion concentration may in turn, allow this to happen (Hespel et a1 1986). In this study, maximal exercise resulted in increased glucose Effects of conditioning concentrations 2 mins after exercise and then a further increase at Throughout the conditioning period there was no significant change the 30-min sample. In addition, serum insulin was decreased 2 min from baseline measurements in plasma K, erythrocyte K, serum following exercise; this effect appears erroneous because of the insulin or plasma glucose concentration. Three horses had increase in plasma glucose. However, similar trends have been noted erythrocyte K concentrations consistently below 83.9 mmoMitre, a in insulin concentration following maximal exercise in man, and in value determined by Muylle et a1 (1984) to be low. These authors other horses (Howlett 1987; Church et a1 1987; Mikines et a1 suggested that low erythrocyte K concentrations correlate with low 1988a). Serum insulin rebounded to increased levels by 30 mins muscle K concentrations and thus may impair performance (Muylle after exercise. The rebound insulinaemia following exercise is et a1 1983; Muylle et a1 1984). The three horses with low thought to be caused by 1) withdrawal of sympathetic neural erythrocyte K concentrations showed a normal response to exercise inhibition of pancreatic B cells and 2) elevations in endogenous in that their plasma K concentrations increased. Although no genetic opioid peptides stimulating insulin release (Farrell, Sonne, Mikines pattern has been observed in horses with low erythrocyte K and Galbo 1988). If glucose increases following maximal exercise concentrations, a definite hereditary disorder occurs in sheep and insulin is initially suppressed, then other factors must be (Tucker and Ellory 1970). Further studies on the hereditability of involved in the glucoregulatory response. The glucoregulatory high or low erythrocyte K concentrations may prove interesting, response to exercise in this study was characterised by a fall in particularly if an association is observed between low erythrocyte K insulin, and an increase in adrenaline, noradrenaline and cortisol concentrations and poor performance. Conditioning did lead to an concentrations. Glucagon was not measured, but increases in man, initial increase in serum cortisol concentration at Weeks 4 and 5 and probably horses, following exercise (Vranic et a1 1984). compared with its lowest concentration at Week 1, and this was Adrenaline and neural noradrenaline release appear to be most significant in increasing glycogenolysis in the liver, as well as in attributed to possible adaptation to conditioning. exercising and non-exercising muscle (Richter et a1 1982; Hoelzer et a1 1986a). Similarly, the fall in insulin and rise in glucagon Effect of exercise support the increase in glucose concentration (Hoelzer et a1 1986b). During exercise, K is released from working skeletal muscles Serum cortisol concentration increased immediately following (Kjellmer 1965; Van Beaumont et a1 1973). Following all exercise maximal exercise and increased further at the 30-min sample. This tests in this study, as plasma K increased, erythrocyte K declined. trend was common in all exercise trials. Similar findings in response Research has demonstrated that the intensity of the exercise and the to maximal exercise have been reported in man and horses time taken to collect samples following exercise will influence both (Thornton 1985; Church et a1 1987; Howlett 1987). In man, a work the plasma K and erythrocyte K concentration measured (Hespel et intensity of 60 per cent of maximal oxygen uptake is the critical a1 1986; Harris and Snow 1988). Therefore, the directional changes point for exercise-induced elevations in cortisol (Howlett 1987). in plasma K and erythrocyte K in this study might have been more Several studies have shown that physical conditioning potentiates pronounced if the samples had been collected sooner than 2 mins the action of cortisol, resulting in a smaller increase in post-exercise following exercise. ACT'H and cortisol (Buono, Yeager and Sucec 1987; Church et a1 Very high levels of plasma K (in excess of 10 mmol/litre) have 1987; Luger et al 1987). In the present study, a similar trend was
(Table 4). Individual noradrenaline concentrations were highly variable at all collection times. Plasma noradrenaline (P 5 0.005) and adrenaline (P 5 0.02) were increased 2 mins following exercise over baseline concentrations and the groups were not significantly different. Plasma noradrenaline concentrations were decreased (P 5 0.04) at the 30 min post exercise sample in the acepromazine treated group compared to the non-treated group, in which noradrenaline concentrations remained increased. Administration of acepromazine failed to alter responses in anticipation of exercise.
TABLE 4: Response to maximal exercise over 1,000 m with and without acepromazine administration (7 ma ivl
N Control PK (mmolllitre) RBCK (mmolllitre) Cortisol (nglml) L-lactate (mmolllitre) 7 Adrenaline (pglml) Noradrenaline (pglml) Acepromazine 9 PK (mmolllitre) RBCK (mmolllitre) Cortisol (nglml) L-lactate (mmolllitre) 7 Adrenaline (pglml) Noradrenaline (pglml) 9
Baseline
Immediately pre-exercise
Time (mins) post exercise: 2
30
3.7 f 0.4 84.7 f 5.7 65.2 f 12 1.4 f 0.4 333 f 815.7 876(392,1552)
ND ND ND ND 143.7 f 227.2 412(110,906)
5.6 f 0.3a 82.5 f 5.4 85.3 f 18.6a 21.8 f 1.7b 988.7 f 669.S 131O(697,2116)a
4.0 f 0.4 83.7 f 4.4 116.6f21.2b 19.2 f 3.7at 125.9 f 175.2 1303(692,2107)a
3.6 f 0.3 85.8 It 7.4 77.0 f 14.8 1.6 f 0.4 61.7 f 101 222(26,610)
ND ND ND ND 105.3 f 213.4 443(125,949)
5.4 f 0.4a 84.8 f 8.4 96.1 f 11.4a 19.1 f 2.6b 1004.6 f 614.3a 1140(576,190 1)a
3.9 f 0.3 83.7 k 7.7 112.3 f 19.6b 14.6 f 5.3a 61.4 f 99.1 329(69,778)'
Data expressed as mean It sd, except noradrenaline for which confidence limits are reported. Means significantly higher between the control and acepromazine treated groups, * P 50.05,t P IO.001. Values in the same line with letter superscripts (a,b) are significantly different, P20.05. PK: plasma potassium; RBCK: erythrocyte potassium; ND: not determined
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noted in horses exercised over 400 m prior to and after conditioning. In this study, noradrenaline and adrenaline increased 2 mins following exercise over 1,OOO m, as previously reported (Evans, Smith and Weil-Malherbe 1956). Plasma noradrenaline concentrationswere highly variable and difficult to interpret because of the excitable temperament of Thoroughbred racehorses. Plasma adrenaline, on the other hand, was less variable. Horses showed no obvious trend in plasma adrenaline and noradrenaline concentrations in anticipation of exercise. Effect of acepromazine
Acepromazine administration prior to exercise resulted in behaviour changes in all horses. This was most marked in horses with excitable temperaments. Even at a low dosage (7 mg iv) the horses became more manageable, easier to ride and in some cases showed improved performance. Horses that were normally relaxed and easy to handle became more relaxed and were in some cases difficult to motivate, adversely affecting their performance. The administration of acepromazine prior to exercise did iiot alter the circulating catecholamines in anticipation of exercise or in the immediate postexercise period. Therefore, our study does not support the hypothesis that acepromazine limits the incidence of exertional rhabdomyolysis by suppressing circulating catecholamines. The decreased noradrenaline concentration in the acepromazine-treated horses 30 mins following exercise may have been a drug effect or individual variation. Perhaps the most interesting result of this study concerns the responses of L-lactate concentration and erythrocyte K following acepromazine administration. This may warrant further investigation. In the acepromazine-treated horses, L-lactate concentrations were decreased compared to the control group at 2 mins and 30 mins post exercise. This suggests that acepromazine may decrease the production and/or increase the clearance of Llactate. One of the oldest suggested pathogenic mechanisms for the development of exercise-associated rhabdomyolysis is the accumulation of lactic acid, although other authors have suggested that lactate accumulation is an improbable cause of muscle damage (Carlstrom 1932; Lindholm, Johansson and Kjaersgaard 1974; Hanis and Snow 1986). Another change following acepromazine administration was the delayed return of erythrocyte K to normal concentrations 30 mins after exercise, a trend noted in all other exercise tests. Because the promazine tranquilisers influence the extracellular to intracellular movement of electrolytes, these tranquilisers have been beneficial in the treatment of secretory diarrhoea (Rabbani, Greenough, Holmgren and Kirkwood 1982). By influencing electrolyte movements, acepromazine may alter neuromuscular irritability and impair the development of exertional rhabdomyolysis(Hanis and Snow 1986; C o h a n et a1 1978).
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Received for publication: 10.10.89 Accepted: 6.6.90
