It has been established that exercise causes oxidative stress in humans1,2 and rats.3 Therefore, reducing oxidative stress (ie, free radical production) and inflammation during exercise may improve recovery. This has led to several investigations evaluating the effect of antioxidant supplementation in humans during exercise.4–18 Several studies4–11 have revealed reductions in markers of oxidative stress with antioxidant supplementation, compared with placebo treatments. However, some studies15–18 have detected no effect of antioxidant supplementation on markers of running-induced oxidative stress. Most studies5,6,16,17,19,20 have detected no effect of antioxidant supplementation on markers of muscle damage after running, although some have.18,21 Several studies6,7,17,19,22 have detected no effect of antioxidant supplementation on markers of inflammation after prolonged running. Ingestion of a TCJB reduced markers of exercise-induced muscle damage in humans.23 The actions of cyclooxygenase-inhibiting flavonoids24,25 and anthocyanins associated with high antioxidant and anti-inflammatory activities of tart cherries24,26,27 were thought to be responsible for this effect, although this was not investigated.
Horses in various athletic competitions, such as endurance horses28–30 and racehorses,31,32 are exposed to oxidative stress and muscle damage. Shortening the recovery time after an exercise event would yield a substantial advantage to owners, trainers, and horses. Certain markers have been used in horses to estimate oxidative stress and inflammation. The TBARS test measures lipid peroxidation that results from oxidative stress, which results in formation of free radicals that react intracellularly to form other substances, such as malondialdehyde.29,32,33 Such substances can cause cellular damage that varies from mild to severe. Many aldehydes formed during lipid peroxidation, particularly malondialdehyde, are used for estimation of lipid peroxidation in biological membranes.34
Although plasma fibrinogen concentration has been used as a marker of exercise-induced inflammation in horses, the time between onset of inflammation and increases in plasma fibrinogen limits this marker's usefulness.35–37 Serum amyloid A has been identified38,39 as an inflammation marker that correlates well with clinical conditions in humans, and use of SAA concentration was recently validated for clinical application in horses with bacterial pneumonia.40 Acute-phase proteins such as SAA have been proposed as the most sensitive indicators of inflammation in horses.38,39
The purpose of the study reported here was to determine whether administering a TCJB to horses prior to exercise would reduce skeletal and cardiac muscle damage by influencing the inflammatory and oxidative stress response to exercise. A secondary objective was to determine whether administration of a TCJB would cause a positive test result in a routine equine drug-screening protocol.
Materials and Methods
Experimental design—A heparinized blood sample for drug screeninga,b was obtained from the left jugular vein of each horse upon entry into the study. Horses were then randomly allocated to receive either TCJB or placebo solution orally twice daily for 14 days. On day 14, a second blood sample for drug screening was obtained before submitting horses to a stepwise incremental exercise protocol. In addition, blood samples were obtained and separated into 4 aliquots to measure markers of skeletal muscle damage (via serum activities of CK and AST), oxidative stress (via the measurement of TBARS), and inflammation (via concentration of SAA). These blood samples were obtained prior to exercise, at each exercise intensity level, hourly after exercise for 4 hours, and then daily for 5 days. Finally, heparinized blood samples were obtained for assessment of cardiac muscle damage (via plasma concentration of cTnI) prior to exercise and 1, 3, 24, 48, 72, and 96 hours after exercise. After a 2-week washout period, each horse received the alternative treatment (TCJB or placebo) and the experiment was repeated.
Horses—Six unfit (ie, not in a training program) adult (mean age, 10 years; range, 5 to 17 years) sexually intact female horses (5 Thoroughbreds and 1 Warmblood [mean ± SD weight, 493 ± 33 kg]) were used in the study. The horses were determined to be in good condition via physical examination, video endoscopic examination of the upper portion of the respiratory tract, and evaluation of a CBC. Horses were kept in box stalls and were walked daily at 1.8 m/s for 1,600 m to become acclimatized to the treadmill and its surroundings. All procedures complied with federal and state regulations and approved local institutional animal care and use procedures.
Exercise protocol—A standardized exercise test reported to increase CK and AST in horses was used.41 Briefly, at time 0, the treadmill was started and accelerated to 4 m/s. At 2 minutes, the treadmill was inclined to a 6.3% slope. At 4 minutes, the treadmill was accelerated to 6 m/s and kept at that speed for 1 minute. At each subsequent minute, the treadmill was accelerated by 1 m/s until the horse was no longer capable of maintaining its position near the front of the treadmill.
Solutions—Solutions (1.42 L of a proprietary TCJBc or a placebo [1.42 L of a cherry-flavored solution]) were administered orally twice daily until the morning of the exercise test. Each solution had a 13% sugar content and was mixed with 2.7 kg of a concentrate (14% protein and 6% fat) for voluntary consumption by each horse.d In addition, horses were fed a timothy-clover grass hay mix.
cTnI, CK, and AST analyses—One sample per sampling time was analyzed for cTnI concentration in a validated point-of-care machinee by use of a 2-site ELISA that uses 2 monoclonal antibodies (caprine and murine) directed against human cTnI. Analytic sensitivity of the assay is 0.02 ng/mL. The reference range in horses is 0.00 to 0.06 ng/mL.f Sera for determination of CK and AST activities were submitted directly to the Clinical Pathology Laboratory at Cornell University for standard analyses.g
TBARS test—Concentration of malondialdehyde was estimated by the measurement of TBARS. Standards and plasma samples were prepared according to directions of the assay kit,h and absorbance was measured in triplicate at 532 nm.i
SAA—The SAA concentration was measured in duplicate by use of the latex agglutination method. Equine standard sera with SAA concentrations ranging from 0.8 to 400.0 μg/mL that were produced according to standard methods were used.42–44 The assay was performed on an automated analyzerj with polyclonal rabbit and monoclonal murine antibodies covalently bound to polystyrene latex particles.k The assay coefficients of variation at 35.5 μg/mL (n = 20 samples) and 257.3 μg/mL (20) were 0.9% and 0.8%, respectively.40 The assay detection limit was 0.1 μg/mL.
Statistical analysis—To control for variation among horses in the study and meet the assumptions of normality, response to administration of placebo solution or TCJB was measured as the difference from baseline values. To ensure comparability between the treatment and control groups prior to exercise, the outcomes (activities or concentrations of CK, AST, cTnI, TBARS, and SAA) were compared in samples obtained immediately prior to exercise by use of a 2-sample t test. An overall effect of treatment on each of these variables, while controlling for sampling time, was assessed by use of regression analysis with appropriate transformation (if deemed necessary). Thereafter, the experimental period was stratified into 2 phases (exercise phase and recovery phase) and stratified analysis was performed to determine whether the concentrations or enzyme activities measured during the 2 phases differed. Results are expressed as mean ± SEM values. For all comparisons, a value of P < 0.05 was considered significant.
Results
All horses consumed all of the feed mixture containing the TCJB or placebo solution. Some stall wall staining as well as red discoloration of the nose was evident in 5 of the 6 horses during administration of both solutions. Volume loss was estimated to be < 30 mL (approx 2% daily) for the placebo and TCJB solutions. No adverse effects were detected. There were no differences immediately prior to exercise between the 2 groups in any of the variables measured. During exercise, the maximum speeds reached after administration of the TCJB (mean, 11.3 ± 0.5 m/s) and the placebo solution (mean, 11.7 ± 0.7 m/s) were not significantly different. Maximum heart rates during each trial after TCJB (mean, 206 ± 4.7 beats/min) and placebo administration (mean, 212 ± 5.6 beats/min) were also not significantly different. By use of the equine drug-screen panel, no positive results were detected at entry into the study or after 2 weeks of administration of TCJB or placebo solution.
cTnI, CK, and AST—There was no significant effect of treatment or exercise on cTnI concentration in either group (Figure 1; Table 1). There was great variation in exercise-induced muscle damage among horses (Figures 2 and 3). The time to peak increase of AST activity was unaffected by treatment (placebo, 1,211 ± 686 minutes; TCJB, 490 ± 304 minutes). Activity of AST increased significantly (P = 0.011) with time, principally because of increase during the recovery phase. Compared with the control group, there was significantly less exercise-induced increase in AST activity, as measured from baseline, in TCJB-treated horses (P = 0.017). When the analysis was stratified, this difference was significant during the exercise (P = 0.009) and recovery (P = 0.014) phases.
Results of linear regression analysis of variables measured during strenuous treadmill exercise and a 5-day recovery period for 6 horses treated orally with TCJB or a placebo solution for 2 weeks prior to exercise.
Variable | Constant (intercept) | Effect of treatment (P value)a | Regression coefficient for sampling time (P value) |
---|---|---|---|
CK | −294.122 | 606.499 (0.063)a | 42.8607 (0.090)a |
AST | −102.228 | 122.558 (0.017)a | 12.9030 (0.001) |
cTnI | 0.00450 | 0.00167 (0.737) | 0.00001 (0.894) |
TBARS | 0.11909 | 0.42071 (0.104) | 0.14867 (< 0.001) |
SAA | −0.13236 | 0.06956 (0.565) | 0.02344 (0.014) |
P value for test of variable significance.
Similarly, the time to peak increase of CK activity was unaffected by treatment (placebo, 180 ± 40.0 minutes; TCJB, 159 ± 49.6 minutes [P = 0.2]). During the exercise phase, there was a mild increase in CK activity over time (P < 0.001) and no treatment effect was detected. As expected, most of the increase in CK activity occurred during the recovery phase in both groups, and horses treated with TCJB had an increase of lesser magnitude than control horses, although the difference did not reach significance (P = 0.054).
TBARS test—The exercise protocol resulted in oxidative stress, as measured via TBARS concentration, which increased overall with time (P < 0.001) because of increase during the exercise phase (P < 0.001) in both treatment groups. Although the TBARS value remained increased during the recovery phase, it did not change significantly during that phase in either group (Figure 4). There were no significant differences between TCJB and placebo groups at any time.
SAA—Exercise induced a significant (P = 0.014) increase in SAA concentration in both groups (Figure 5), which was not affected by treatment (Table 1). There were no effects of treatment (TCJB or placebo) on SAA values at any time.
Discussion
The strenuous exercise protocol used in the present study resulted in increased muscle enzyme activities in the unfit horses, as reported elsewhere.41 In addition, the exercise protocol was also associated with increased concentrations of SAA and TBARS, which are indicators of inflammation and oxidative stress, respectively. However, SAA concentrations increased only to a small degree, compared with other inflammatory processes such as pneumonia.40 Therefore, substantial inflammation might not have occurred during the study protocol.
A randomized crossover design was used because equine muscle enzyme activities,32,45 the key indicators of muscle damage used in this study, are known to have large individual variations. It is noteworthy that no horses had clinical signs of muscle damage, despite increased muscle enzyme activities, a finding that was also reported in another study.29
It has been hypothesized that oxidative stress, through free radical production, changes the permeability of muscle membranes.32,45,46 The TBARS test has been used as a measure of oxidative stress, and a wide range of values has been reported from measurements in humans18,47 and horses.28,32 In the present study, TBARS test values did not change significantly during the recovery period, which is similar to results reported for endurance horses.32
Increases in cTnI concentrations after exercise were mild, were not significant, and did not exceed the upper reference limit (0.12 ng/mL)48,f in any horse; increases were also not associated with sampling time. Therefore, it seems unlikely that substantial cardiac stress or damage was induced by the experimental protocol. Equine skeletal muscle has a weak cross-reactivity with the cTnI assay, and that cross-reactivity has been estimated to contribute 0.05% to 0.1% of the total cTnI concentration.49 Therefore, the mild increase in cTnI concentrations could have been caused by skeletal muscle damage.
Use of antioxidants such as vitamins E and C to modify oxidative stress, which is a proposed source of exercise-induced muscle damage, has not been associated with modifications of CK and AST activities in horses undergoing an 80-km endurance race.29 In the present study, oral administration of TCJB for 2 weeks prior to short-term strenuous exercise, closer in duration to Thoroughbred and Standardbred racing conditions than endurance competition, resulted in a lower magnitude of increase in the activity of AST, compared with administration of a placebo solution. Whether endurance horses would also have this effect if administered TCJB remains to be evaluated. Although the numerous antioxidants and anti-inflammatory agents in tart cherries have been proposed to be responsible for such effects,23 no direct treatment effect of TCJB on the markers of oxidative stress or inflammation was detected in the present study.
Because administration of TCJB was associated with increased AST activity of lower magnitude than in horses administered a placebo solution, it may have potential as a treatment for horses with exercise-induced myopathy. In addition, studies in fit horses are warranted.
ABBREVIATIONS
AST | Aspartate aminotransferase |
CK | Creatine kinase |
cTnI | Cardiac troponin I |
SAA | Serum amyloid A |
TBARS | Thiobarbituric acid reactive substances |
TCJB | Tart cherry juice blend |
Equine Drug Testing Laboratory, New York State Animal Health Diagnostic Center, Cornell University, Ithaca, NY.
Toxicology Laboratory, New York State Animal Health Diagnostic Center, Cornell University, Ithaca, NY.
CherryPharm Inc, Geneva, NY.
Legends Grow & Perform Textured Horse Feed, Southern States, Richmond, Va.
i-STAT 1, Heska Corp, Loveland, Colo.
Kraus MS, Jesty SA, Gelzer AR, et al. Characterization of cardiac troponin I as an indicator of cardiac damage in horses utilizing an i-STAT 1 analyzer (abstr). J Vet Intern Med 2007;21:604–605.
Clinical Pathology Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY.
OXItek, ZeptoMetrix Corp, Buffalo, NY.
Safire reader and Magellan software, Tecan, Zurich, Switzerland.
Hitachi 7020, Hitachi, Tokyo, Japan.
LZ test Eiken SAA, Eiken Chemical, Tokyo, Japan.
References
- 1.
Rietjens SJ, Beelen M, Koopman R, et al. A single session of resistance exercise induces oxidative damage in untrained men. Med Sci Sports Exerc 2007;39:2145–2151.
- 2.
Ascensão A, Rebelo A, Oliveira E, et al. Biochemical impact of a soccer match—analysis of oxidative stress and muscle damage markers throughout recovery. Clin Biochem 2008;41:841–851.
- 3.↑
Niu AJ, Wu JM, Yu DH, et al. Protective effect of Lycium barbarum polysaccharides on oxidative damage in skeletal muscle of exhaustive exercise rats. Int J Biol Macromol 2008;42:447–449.
- 4.
Bloomer RJ, Goldfarb AH, McKenzie MJ. Oxidative stress response to aerobic exercise: comparison of antioxidant supplements. Med Sci Sports Exerc 2006;38:1098–1105.
- 5.
Machefer G, Groussard C, Vincent S, et al. Multivitamin-mineral supplementation prevents lipid peroxidation during “The Marathon des Sables”. J Am Coll Nutr 2007;26:111–120.
- 6.
Mastaloudis A, Morrow JD, Hawkins DW, et al. Antioxidant supplementation prevents exercise-induced lipid peroxidation, but not inflammation, in ultramarathon runners. Free Radic Biol Med 2004;36:1329–1341.
- 7.
Kon M, Tanabe K, Akimoto T, et al. Reducing exercise-induced muscular injury in kendo athletes with supplementation of coenzyme Q10. Br J Nutr 2008;100:903–909.
- 8.
Bloomer RJ, Goldfarb AH, McKenzie MJ, et al. Effects of antioxidant therapy in women exposed to eccentric exercise. Int J Sport Nutr Exerc Metab 2004;14:377–388.
- 9.
Bryer SC, Goldfarb AH. Effect of high dose vitamin C supplementation on muscle soreness, damage, function, and oxidative stress to eccentric exercise. Int J Sport Nutr Exerc Metab 2006;16:270–280.
- 10.
Goldfarb AH, Bloomer RJ, McKenzie MJ. Combined antioxidant treatment effects on blood oxidative stress after eccentric exercise. Med Sci Sports Exerc 2005;37:234–239.
- 11.
Shafat A, Butler P, Jensen RL, et al. Effects of dietary supplementation with vitamins C and E on muscle function during and after eccentric contractions in humans. Eur J Appl Physiol 2004;93:196–202.
- 12.
Connolly DA, Lauzon C, Agnew J, et al. The effects of vitamin C supplementation on symptoms of delayed onset muscle soreness. J Sports Med Phys Fitness 2006;46:462–467.
- 13.
Warren JA, Jenkins RR, Packer L. Elevated muscle vitamin E does not attenuate eccentric exercise-induced muscle injury. J Appl Physiol 1992;72:2168–2175.
- 14.
Beaton LJ, Allan DA, Tarnopolsky MA, et al. Contraction-induced muscle damage is unaffected by vitamin E supplementation. Med Sci Sports Exerc 2002;34:798–805.
- 15.
Kingsley MI, Wadsworth DL, Kilduff JP, et al. Effects of phosphatidylserine on oxidative stress following intermittent running. Med Sci Sports Exerc 2005;37:1300–1306.
- 16.
Dawson B, Henry GJ, Goodman C, et al. Effect of vitamin C and E supplementation on biochemical and ultrastructural indices of muscle damage after a 21 km run. Int J Sports Med 2002;23:10–15.
- 17.
Kaikkonen J, Kosonen L, Nyyssönen K, et al. Effect of combined coenzyme Q10 and d-α-tocopheryl acetate supplementation on exercise-induced lipid peroxidation and muscular damage: a placebo-controlled double-blind study in marathon runners. Free Radic Res 1998;29:85–92.
- 18.
Rokitzki L, Logemann E, Sagredos AN, et al. Lipid peroxidation and antioxidative vitamins under extreme endurance stress. Acta Physiol Scand 1994;151:149–158.
- 19.
Peters EM, Anderson R, Nieman DC, et al. Vitamin C supplementation attenuates the increases in circulating cortisol, adrenaline and anti-inflammatory polypeptides following ultramarathon running. Int J Sports Med 2001;22:537–543.
- 20.
Mastaloudis A, Traber MG, Carstensen K, et al. Antioxidants did not prevent muscle damage in response to an ultramarathon run. Med Sci Sports Exerc 2006;38:72–80.
- 21.
Itoh H, Ohkuwa T, Yamazaki Y, et al. Vitamin E supplementation attenuates leakage of enzymes following 6 successive days of running training. Int J Sports Med 2000;21:369–374.
- 22.
Castell LM, Poortmans JR, Leclercq R, et al. Some aspects of the acute phase response after a marathon race and the effects of glutamine supplementation. Nutrition 1997;13:738–742.
- 23.↑
Connolly DA, McHugh MP, Padilla-Zakour OI, et al. Efficacy of a tart cherry juice blend in preventing the symptoms of muscle damage. Br J Sports Med 2006;40:679–683.
- 24.
Seeram NP, Bourquin LD, Nair MG. Degradation products of cyanidin glycosides from tart cherries and their bioactivities. J Agric Food Chem 2001;49:4924–4929.
- 25.
Wang H, Nair MG, Strasburg GM, et al. Cyclooxygenase active bioflavonoids from Balaton tart cherry and their structure activity relationships. Phytomedicine 2000;7:15–19.
- 26.
Blando F, Gerardi C, Nicoletti I. Sour cherry (Prunus cerasus L) anthocyanins as ingredients for functional foods. J Biomed Biotechnol 2004;2004:253–258.
- 27.
Tall JM, Seeram NP, Zhao C, et al. Tart cherry anthocyanins suppress inflammation-induced pain behavior in rat. Behav Brain Res 2004;153:181–188.
- 28.
Frankiewiez-Jozko A, Szarska E. Anti-oxidant level to exercise in the blood of endurance horses. Biol Sport 2000;17:217–227.
- 29.↑
Williams CA, Kronfeldt DS, Hess TM, et al. Antioxidant supplementation and subsequent oxidative stress of horses during an 80-km endurance race. J Anim Sci 2004;82:588–594.
- 30.
Marlin DJ, Fenn K, Smith N, et al. Changes in circulatory antioxidant status in horses during prolonged exercise. J Nutr 2002;132:1622S–1627S.
- 31.
Chiaradia E, Avellini L, Rueca F, et al. Physical exercise, oxidative stress and muscle damage in racehorses. Comp Biochem Physiol B Biochem Mol Biol 1998;119:833–836.
- 32.↑
White A, Estrada M, Walker K, et al. Role of exercise and ascorbate on plasma antioxidant capacity in thoroughbred race horses. Comp Biochem Physiol A Mol Integr Physiol 2001;128:99–104.
- 33.
Nielsen F, Mikkelsen BB, Nielsen JB, et al. Plasma malondialdehyde as biomarker for oxidative stress: reference interval and effects of life-style factors. Clin Chem 1997;43:1209–1214.
- 34.↑
Wong SH, Knight JA, Hopfer SM, et al. Lipoperoxides in plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric acid adduct. Clin Chem 1987;33:214–220.
- 35.
Giguère S, Hernandez J, Gaskin J, et al. Evaluation of white blood cell concentration, plasma fibrinogen concentration, and an agar gel immunodiffusion test for early identification of foals with Rhodococcus equi pneumonia (Erratum published in J Am Vet Med Assoc 2003;223:1300). J Am Vet Med Assoc 2003;222:775–781.
- 36.
Hulten C, Demmers S. Serum amyloid A (SAA) as an aid in the management of infectious disease in the foal: comparison with total leucocyte count, neutrophil count and fibrinogen. Equine Vet J 2002;34:693–698.
- 37.
Hulten C, Gronlund U, Hirvonen J, et al. Dynamics in serum of the inflammatory markers serum amyloid A (SAA), haptoglobin, fibrinogen and alpha2-globulins during induced noninfectious arthritis in the horse. Equine Vet J 2002;34:699–704.
- 38.
Malle E, De Beer FC. Human serum amyloid A (SAA) protein: a prominent acute-phase reactant for clinical practice. Eur J Clin Invest 1996;26:427–435.
- 39.
McAdam KP, Elin RJ, Sipe JD, et al. Changes in human serum amyloid A and C-reactive protein after etiocholanolone-induced inflammation. J Clin Invest 1978;61:390–394.
- 40.↑
Hobo S, Niwa H, Anzai T. Evaluation of serum amyloid A and surfactant protein D in sera for identification of the clinical condition of horses with bacterial pneumonia. J Vet Med Sci 2007;69:827–830.
- 41.↑
Rasanen LA. Exercise induced purine nucleotide degradation and change in myocellular protein release. Equine Vet J Suppl 1995;18:235–238.
- 42.
Nunokawa Y, Fujinaga T, Taira T, et al. Evaluation of serum amyloid A protein as an acute-phase reactive protein in horses. J Vet Med Sci 1993;55:1011–1016.
- 43.
Satoh M, Fujinaga T, Okumura M, et al. Sandwich enzyme-linked immunosorbent assay for quantitative measurement of serum amyloid A protein in horses. Am J Vet Res 1995;56:1286–1291.
- 44.
Stoneham SJ, Palmer L, Cash R, et al. Measurement of serum amyloid A in the neonatal foal using a latex agglutination immunoturbidimetric assay: determination of the normal range, variation with age and response to disease. Equine Vet J 2001;33:599–603.
- 45.
Hargreaves BJ, Kronfeld DS, Waldron JN, et al. Antioxidant status of horses during two 80-km endurance races. J Nutr 2002;132:1781S–1783S.
- 46.
McBride JM, Kraemer WJ. Free radicals, exercise and antioxidants. J Strength Cond Res 1999;13:175–183.
- 47.
Laaksonen DE, Atalay M, Niskanen L, et al. Blood glutathione homeostasis as a determinant of resting and exercise-induced oxidative stress in young men. Redox Rep 1999;4:53–59.
- 48.↑
Phillips W, Giguère S, Franklin RP, et al. Cardiac troponin I in pastured and race-training thoroughbred horses. J Vet Intern Med 2003;17:597–599.
- 49.↑
O'Brien PJ, Landt Y, Ladenson JH. Differential reactivity of cardiac and skeletal muscle from various species in a cardiac troponin I immunoassay. Clin Chem 1997;43:2333–2338.