Caffeine is a drug of the group of methylxanthines, substances that stimulate the CNS.1,2 The Association of Racing Commissioners International does not approve of the therapeutic use of caffeine in horses, and when it is found in the urine or other biological fluids, it is classified as a class 2 illegal substance, characterized as doping.1,3 However, because of the natural presence of caffeine in teas, coffees, chocolates, and rations, professionals who are involved in the day-to-day practice of equestrian sports argue that the detection of low blood and urinary concentrations of caffeine in athletic horses can be associated with environmental contaminations and have no effect on the results of equestrian competitions.3 Currently, investigations of caffeine and its interference with athletic performance in aerobic and anaerobic exercises are performed in a systematic manner in humans2,4 as well as horses3,5; most of the studies on horses use the Thoroughbred breed in their experiments.3,5
In humans, the ergogenic effect of caffeine has been described, and there is consensus on its beneficial effects in aerobic and resistance exercises.2 During intense exercise of short duration, however, there is controversy over the improvement of athletic performance after the administration of caffeine.2 In horses, caffeine was reported to improve the response of cardiovascular function as a result of the stimulation of the CNS.5 In contrast, authors of another study3 did not observe effects on cardiorespiratory variables of horses after the administration of caffeine at a dose of 2.5 mg/kg; horses in that study3 underwent an IET following caffeine administration. The objective of the study reported here was to determine the effects of acute administration of caffeine on physiologic variables of Arabian horses during exercise with increasing intensity on a treadmill, by examining metabolic, endocrine, hematologic, and cardiovascular variables.
Materials and Methods
Animals—In this study, 12 trained horses of the Arabian breed were used, which included geldings and females from the Horse Breeding Sector of the College of Agricultural and Veterinary Sciences, São Paulo State University, Jaboticabal, Brazil. Horses had a mean ± SE body weight of 390 ± 25.4 kg and age of 8.6 ± 3.3 years. Horses selected for study were evaluated by clinical examination and laboratory tests including a CBC and serum biochemical analysis. Horses had no clinical signs compatible with skeletal muscle lesions and were deloused and dewormed on a quarterly basis. The study followed the Ethical Principles in Animal Experimentation adopted by the Brazilian College of Animal Experimentation and was approved by the institutional animal care and use committee of the university.
Conditioning protocol—The duration of the physical conditioning period was 8 weeks. Each horse performed in a climate-controlled room, which contained a high-performance treadmill.a Before initiation of the training program, horses underwent a 30-day period of adaptation to handling. The training program was conducted exclusively on the treadmill; it has been verified that training on a treadmill is effective for the physical conditioning of horses.6 The velocity (intensity) was set at 80% of the velocity at which the blood lactate concentration reached 4 mmol/L. Horses were exercised 3 times/wk with 48 hours of rest between training sessions. The distance for each velocity step of the IET was 10 km.
Groups—The 12 horses were randomly assigned to 2 experimental groups of 6 horses each. A crossover design was used, thereby compounding data for the control (n = 12) and caffeine-treated (12) groups. Horses underwent 2 physical tests separated by a 10-day interval. Anhydrous caffeine was dissolved in sterile saline (0.9% NaCl) solution. Thirty minutes before the first IET, 6 control horses received saline solution IV and 6 caffeine-treated horses received caffeineb IV at a dose of 5.0 mg/kg. In the second IET, the procedure was the same but reversed for the 2 groups.
Incremental exercise test—Horses were adapted to exercise on a high-performance treadmillb and then underwent the exercise test for a duration of 30 minutes. The warm-up exercise was first performed for 4 minutes at a speed of 4 m/s, which was then increased at 1-minute intervals to 5, 6, 7, 8, 9, and 10 m/s. At the stage of maximum velocity, the exercise continued with deceleration, returning to a velocity of 3 m/s for 20 minutes, which corresponded to the active cooling-down period. From the velocity of 5 m/s onward, the IET was performed with the treadmill at an incline of 10%. The cooling-down phase was performed without an incline.
Blood sample collection—A standard operating procedure for blood sample collection was created to establish proper procedures for collection, processing, and storage. Blood was collected 15 seconds before the end of each velocity step of the IET. Prior to the exercise, the area close to the left jugular vein was shaved and aseptically prepared for venous catheterization, which was always performed 12 hours before the exercise test. An extension tube was connected to a 12-gauge catheter to facilitate blood collection with the horse in motion. After each collection, the entire assembly was flushed with 2.5% heparin solution.
Heart rate—Heart rate was determined with a digital heart rate monitorc; 3 measurements were obtained at each velocity step (at onset, middle, and final). Means obtained at each velocity step of the IET were plotted and analyzed by linear regression to determine velocities at which heart rates were 180 and 200 beats/min.
Lactate—Blood lactate concentration was determined in 0.5 mL of blood that was separated and processed in Eppendorf tubes containing 1.0 mL of 1% sodium fluoride. The hypotonic solution caused hemolysis and inhibition of glycolysis, thereby preventing coagulation and lactate production by erythrocytes. Blood lactate concentration was determined by use of an electrochemical lactate analyzerd in which the samples were assayed in duplicate. Blood was collected 15 seconds before the end of each exercise step. Mean values were plotted against velocity, and exponential regression analysis was used to determine the velocities at which blood lactate concentration was 2 and 4 mmol/L.
Hematologic variables—The Hct, hemoglobin concentration, RBC count, and WBC count were measured at velocity steps of 0, 4, 6, 8, and 10 m/s. Blood cell counts were determined by use of a dilutere and counter.f The Hct was performed by use of the microhematocrit method, and hemoglobin concentration was determined by use of the colorimetric cyanmethemoglobin method.g All blood samples were collected in vacuum tubes,h and assays were performed in duplicate.
Glucose—Three-milliliter blood samples were reserved for determination of blood glucose concentration. Glucose assays were processed with anticoagulanti containing the glycolysis inhibitor potassium fluoride and later analyzed by use of spectrophotometry.j
Cortisol and insulin—Ten-milliliter blood samples were obtained in vacuum tubes containing sodium heparin and immediately centrifuged under refrigerationk; plasma samples were stored at −20°C. Plasma cortisol and insulin concentrations were determined by solid-phase radioimmunoassay by use of a commercial kit.l The inter- and intra-assay coefficients of variation for insulin were 10.3% and 8.6%, respectively. The inter- and intra-assay coefficients of variation for cortisol were 11.6% and 9.7%, respectively.
Statistical analysis—The effect of exercise on physiologic variables was evaluated by use of an ANOVA for repeated measures, with the aim of determining significant differences for each velocity step of the IET, followed by the Tukey test when necessary. A Student t test for paired samples was also performed to compare differences among groups. A value of P ≤ 0.05 was considered significant.
Results
Evaluation of performance—Velocities at which heart rates were 180 and 200 beats/min increased significantly by 10% (P = 0.046) and 11.7% (P = 0.038), respectively, in caffeine-treated horses, compared with control horses (Figure 1). Heart rate increased significantly (P < 0.001) during the exercise phase in all horses and returned toward baseline values in the cooling-down period.
Graphic representation of the changes in heart rate (A) and blood lactate concentration (B) in 6 horses during incremental exercise on a treadmill after caffeine treatment (5 mg/kg, IV; CAF; 12 samples) or saline (0.9% NaCl) solution treatment (IV; C; 12 samples). #Significantly (P < 0.05) higher values in control horses, compared with caffeine-treated horses. *Significantly (P < 0.05) higher values in caffeine-treated horses, compared with control horses.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1670
Graphic representation of the changes in heart rate (A) and blood lactate concentration (B) in 6 horses during incremental exercise on a treadmill after caffeine treatment (5 mg/kg, IV; CAF; 12 samples) or saline (0.9% NaCl) solution treatment (IV; C; 12 samples). #Significantly (P < 0.05) higher values in control horses, compared with caffeine-treated horses. *Significantly (P < 0.05) higher values in caffeine-treated horses, compared with control horses.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1670
Graphic representation of the changes in heart rate (A) and blood lactate concentration (B) in 6 horses during incremental exercise on a treadmill after caffeine treatment (5 mg/kg, IV; CAF; 12 samples) or saline (0.9% NaCl) solution treatment (IV; C; 12 samples). #Significantly (P < 0.05) higher values in control horses, compared with caffeine-treated horses. *Significantly (P < 0.05) higher values in caffeine-treated horses, compared with control horses.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1670
Velocities at which blood lactate concentrations were 2 or 4 mmol/L decreased significantly by 25.4% (P = 0.001) and 24.2% (P = 0.009), respectively, in caffeine-treated horses, compared with control horses (Figure 1). Beginning at a velocity of 5 m/s, blood lactate concentrations were significantly (P < 0.001) higher in caffeine-treated horses, compared with control horses, for all velocity steps of the IET. Caffeine-treated horses had significantly (P < 0.001) higher maximum blood lactate concentrations than control horses (Table 1). No significant differences in blood lactate concentrations were found between mares and geldings.
Mean ± SEM indices of athletic performance following IV administration of caffeine (5 mg/kg) in 12 horses exercising with increasing intensity on a treadmill.
| Horses | No. of samples | V180 (m/s) | V200 (m/s) | V2 (m/s) | V 4(m/s) | Peak blood lactate (mmol/L) | Maximum Hct (%) |
|---|---|---|---|---|---|---|---|
| Control | 12 | 6.20 + 0.20 | 7.26 + 0.25 | 5.69 + 0.33 | 8.36 + 0.41 | 8.10 + 0.76 | 52 + 0.6 |
| Caffeine treated | 12 | 6.82 ± 0.32* | 8.11 ± 0.32* | 4.31 ± 0.27* | 6.23 ± 0.28* | 17.73 ± 1.73* | 55 ± 0.7 |
Significant (P < 0.05) difference between control and caffeine-treated horses.
V180 and V200 = Velocities at which heart rate was 180 and 200 beats/min, respectively. V2 and V4 = Velocities at which blood lactate concentration was 2 and 4 mmol/L, respectively.
Hematologic values—In all horses, the Hct significantly (P < 0.001) increased during all exercise phases, with a significant (P < 0.001) decrease during the active cooling-down period (Table 2). At exercise test times of 4, 6, 8, 15, 20, and 30 minutes (corresponding to velocities of 4, 6, 8, 3, 3, and 3 m/s, respectively), caffeine-treated horses had significantly (P = 0.009, 0.05, 0.039, 0.016, 0.006, and 0.001, respectively) higher Hct than control horses. No significant difference was found between groups in RBC count, hemoglobin concentration, and WBC count.
Mean ± SEM biochemical variables following IV administration of caffeine (5 mg/kg) in 12 horses exercising with increasing intensity on a treadmill.
| Variable | Horses | Time (min) | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Exercise | Cooling-down time | ||||||||
| Rest | 4 | 6 | 8 | 10 | 15 | 20 | 30 | ||
| Hct (%) | C | 38 ± 1a | 45 ± 0.6b | 49 ± 0.4c | 50 ± 0.6c | 52 ± 0.6c | 49 ± 1c | 46 ± 1b | 43 ± 1b |
| CAF | 42 ± 1a | 51 ± 1b,* | 52 ± 0.6b,* | 54 ± 0.7c,d,* | 55 ± 0.7d | 52 ± 1b,* | 51 ± 1b,* | 48 ± 1b,* | |
| Glucose (mmol/L) | C | 4.54 ± 0.37a | 4.03 ± 0.16a | 3.94 ± 0.17a | 4.21 ± 0.20a | 4.71 ± 0.23a | 6.79 ± 0.46b | 6.77 ± 0.34b | 6.94 ± 0.35b |
| CAF | 4.83 ± 0.10a | 4.59 ± 0.14a,* | 4.60 ± 0.12a,* | 4.97 ± 0.16a,* | 5.79 ± 0.33a,* | 9.25 ± 0.40b,* | 8.40 ± 0.51b,* | 8.01 ± 0.31b,* | |
| Insulin (pmol/L) | C | 56 ± 7a | 40 ± 6a,b | 43 ± 6b,c | 37 ± 3b,c | 22 ± 4d | 48 ± 13b,c | 51 ± 11a,b | 85 ± 11a |
| CAF | 28 ± 5a,* | 18 ± 2b,* | 17 ± 3b,* | 13 ± 3b,c,* | 10 ± 2b,c,* | 3 ± 1d,* | 15 ± 4b,c,* | 14 ± 7b,c,* | |
| Cortisol (nmol/L) | C | 404 ± 50a | 442 ± 54a | 412 ± 35a | 492 ± 49b | 471 ± 55b | 495 ± 58b | 501 ± 65b | 439 ± 35a |
| CAF | 274 ± 18a,* | 287 ± 25a,* | 300 ± 15a,* | 314 ± 21a,* | 323 ± 29a,* | 354 ± 24b,* | 343 ± 27a,* | 272 ± 16a,* | |
Significant (P < 0.05) difference between control and caffeine-treated horses.
C = Control horses (12 samples). CAF = Caffeine-treated horse (12 samples).
Different letters in the same row indicate significantly (P < 0.05) differentvalues.
Insulin—During exercise, plasma insulin concentrations significantly (P < 0.001) decreased in both groups of horses (Table 2). At exercise test times of 0, 4, 6, 8, 10, 15, 20, and 30 minutes (corresponding to velocities of 0, 4, 6, 8, 10, 3, 3, and 3 m/s, respectively), control horses had significantly (P = 0.011, 0.019, 0.006, 0.003, 0.033, 0.036, < 0.001, and 0.006, respectively) higher plasma insulin concentrations than caffeine-treated horses.
Glucose—In all horses, blood glucose concentrations were significantly (P < 0.001) higher during the cooling down period, compared with during increasing velocities of exercise (Table 2). At exercise test times of 4, 6, 8, 10, 15, 20, and 30 minutes (corresponding to velocities of 4, 6, 8, 10, 3, 3, and 3 m/s, respectively), caffeine-treated horses had significantly (P = < 0.001, 0.002, 0.029, 0.013, 0.001, 0.034, and 0.021, respectively) higher blood glucose concentrations than control horses.
Cortisol—Exercise significantly (P < 0.001) altered plasma cortisol concentrations in all horses (Table 2). At exercise test times of 0, 4, 6, 8, 10, 15, 20, and 30 minutes (corresponding to velocities of 0, 4, 6, 8, 10, 3, 3, and 3 m/s, respectively), control horses had significantly (P = 0.016, 0.037, 0.026, 0.004, 0.035, 0.029, 0.037, and 0.008, respectively) higher plasma cortisol concentrations than caffeine-treated horses.
Discussion
In the present study, caffeine at a dose of 5 mg/kg, IV, was administered 30 minutes before exercise. This dose was chosen on the basis of findings in a previous study7 that evaluated spontaneous locomotor activity of horses in an automated behavior chamber. At a dose of 5 mg/kg, authors of that study7 detected an increase in excitability evidenced by the occurrence of increased locomotor activity 25 minutes after IV administration of caffeine.
In humans, ergogenic effects from the ingestion of caffeine have been described.2 However, findings in horses are contradictory.3,5 In the present study, it was observed that exercise alone resulted in an increase in the Hct. This finding is most likely related to splenic contraction caused by the increased amounts of circulating catecholamines that are inherent to exercise.8 The release of adrenaline and noradrenaline in response to caffeine may explain the increase in the Hct in caffeine-treated horses in our study. Alternatively, caffeine may act as a phosphodiesterase inhibitor, thereby increasing intracellular cAMP concentrations and activating β-adrenergic receptors.9,10
With respect to lactate, blood concentrations at maximum effort of the IET were highest in caffeine-treated horses, compared with control horses. This finding indicates that lactate production increased with administration of caffeine before exercise, which also can be explained by an increase in catecholamines, causing glycogenolysis and anaerobic glycolysis in muscle fibers.11 This finding is in accordance with that in a study5 that revealed caffeine can increase anaerobic metabolism during intense exercise.
The relationship between increased blood lactate concentrations and treadmill velocity is frequently used for evaluating the conditioning of athletic horses.12 The higher the velocity at which blood lactate concentration reaches 4 mmol/L, the better the aerobic capacity of a horse.13 In our study, velocities at which blood lactate concentrations were 2 or 4 mmol/L were lower in caffeine-treated horses, compared with control horses. Therefore, the administration of caffeine apparently diminishes the aerobic metabolism of horses and provides further evidence that caffeine can enhance the anaerobic potential during intense exercise of short duration.
In this study, Arabians were chosen because of the lack of knowledge involving caffeine and exercise in this breed, because most of the studies on this subject had been performed with Thoroughbreds.3,5,7,8 Comparatively, Arabians possess a higher percentage of type I fibers than Thoroughbreds; these fibers provide better capacity for aerobic activities of long duration.14 These oxidative fibers produce less lactate, compared with fiber types IIa and IIx. Results obtained from caffeine-treated horses in the present study revealed that these horses had higher blood glucose and lactate concentrations, compared with control horses, and lower velocities at which blood lactate concentrations were 2 or 4 mmol/L.
Additional evidence of caffeine-induced recruitment of fast-twitch fibers is the fact that, in the present study, in which horses underwent aerobic training, higher blood lactate concentrations were found in caffeine-treated horses, compared with those in control horses. This is contrary to what has been found in Thoroughbreds3 under similar study conditions.
The relation between velocity and heart rate is usually used in the evaluation of athletic potential.15 Although there have been study3,5 results that indicate that caffeine causes positive chronotropism in the heart, results of the present study indicate that the velocities at which heart rate was 180 and 200 beats/min were higher in caffeine-treated horses, compared with those in control horses, indicating a reduction in heart rate in caffeine-treated horses. These results may reflect a vagal reflex response,16 resulting in a positive inotropism effect of caffeine.17,18 Another possible explanation is that caffeine, as a methylxanthine, caused bronchodilation and improved airflow to lower airways, resulting in lower heart rates at any given velocity, thereby increasing the velocity at which heart rate was 180 and 200 beats/min.
Carbohydrates, in the form of muscle glycogen and blood glucose, are important substrates for skeletal muscle contraction. In this study, the dynamics of glucose were evaluated during intense exercise after the administration of caffeine. The plasma glucose concentration increased in the later velocity steps of the IET (recovery), probably reflecting the increase in glycogenolysis and neoglycogenesis as a result of adrenergic activity.11,19 Additionally, insulinemia was significantly lower for caffeine-treated horses, compared with control horses. This finding can be explained by the adrenergic action, exacerbated by caffeine, on the α2-receptors present on B cells of the pancreas that, when stimulated, inhibit the secretion of insulin.20 The importance of lower plasma insulin concentrations is related to the maintenance of blood glucose concentrations during exercise by means of increasing glycogenolysis, which in turn contributes to the prevention of the induction of fatigue mechanisms.21,22 Moreover, because the development of increased plasma insulin concentrations after feeding may induce hypoglycemia before or during exercise,23 the results obtained in this study indicate that caffeine may help in the reduction of postprandial hyperinsulinemia.
Another point to be considered with respect to the increase in sympathetic neuronal activity induced by the administration of caffeine during intense physical activity is the relation between the increased blood glucose and lactate concentrations. Evidenced by studies in humans24 and horses,11 the action of catecholamines enhances the availability of glucose as well as the production of lactate.
The adrenocortical response related to exercise has been exhaustively studied because of its interference with metabolism with respect to the mobilization of the energy substrate for muscle contraction.25 During intense exercise, increases in plasma cortisol concentrations may result in hepatic and muscular neoglycogenesis, thereby increasing the bioavailability of glucose, mainly for the CNS. This may contribute to athletic performance by postponing fatigue as a result of hypoglycemia in the CNS.26 In a study27 conducted in humans, results were contradictory to those of our study. In that study,27 caffeine induced increases in plasma cortisol concentrations in humans. Potentially, a lower response of the hypothalamic-pituitary-adrenal axis of caffeine-treated horses, compared with humans, could provide an explanation for the differing study results. Caffeine, as a psychostimulant that improves motor and cognitive functions,28 could contribute to the decreased response induced by exercise in horses in terms of cortisol production. This hypothesis is supported by findings in another study29 that revealed that, in humans, the administration of levodopa, a drug that enhances dopaminergic activity, also decreases cortisol concentrations.
Results obtained in the present study regarding blood lactate, blood glucose, and plasma cortisol concentrations indicate that caffeine promotes improved adaptation of Arabian horses during short intense exercise. Despite the effect of caffeine in diminishing the aerobic energy metabolism, it increased anaerobic glycolysis.
ABBREVIATION
| IET | Incremental exercise test |
Galloper treadmill, Sahinco LTDA, Palmital, Brazil.
Sigma Aldrich, St Louis, Mo.
S610, Polar, Port Washington, New York.
Yellow Springs Instrument Co, Yellow Springs, Ohio.
CELM Companhia Equipadora de Laboratorios Modernos, Barueri, Brazil.
DC 510, CELM Companhia Equipadora de Laboratorios Modernos, Barueri, Brazil.
Kit, Labtest Diagnóstica, Lagoa Santa, Brazil.
EDTA, 5.0 mL, Vacutainer BD, Chacara Santo Antonio, Brazil.
Glistab Cat 29, Labtest Diagnóstica, Lagoa Santa, Brazil
Bio 2000, Bioplus, Barueri, Brazil.
Multispeed refrigerated centrifuge PK121R, ALC, Princeton, NJ.
Coat-a-count, Diagnostic Products Corp, Los Angeles, Calif.
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