Plasma Coenzyme Q10 and Tocopherols in Thoroughbred Race Horses: Effect of Coenzyme Q10 Supplementation and Exercise

The fundamental role of coenzyme Q10 (CoQ10) in cellular bioenergetics is well established in humans and several other species, yet there is no information about its role in horses. In continuation of our study of serum CoQ10 in Thoroughbred race horses, we examined the effect of CoQ10 supplementation and exercise on changes in plasma CoQ10 and α- and γ-tocopherol concentrations. Two groups of horses, five per group, were used. One group received 1.9 g of CoQ10 daily and the second group 3.4 g as a dietary supplement. Both groups were subjected to graduated training sessions that involved low-intensity exercise (gallop) followed by high-intensity exercise (breeze). CoQ10 supplementation resulted in a significant increase in plasma CoQ10 during low-intensity exercise, whereas the increase following high-intensity exercise was markedly attenuated in both groups (P < .05). There were no changes in plasma α- and γ-tocopherol concentrations following the two exercise sessions. γ-Tocopherol constituted approximately 5% of α-tocopherol; this is the first report of plasma γ-tocopherol in horses. The attenuated response of plasma CoQ10 in horses receiving CoQ10 supplementation following high-intensity exercise may be attributed to its consumption during severe oxidative stress. This finding indicates that in horses not receiving any CoQ10 supplementation, high-intensity exercise would result in a marked depletion of endogenous CoQ10 in plasma, which could compromise their health and physical performance. This documents the importance of CoQ10 in horses. The study also establishes the safety and tolerability of high-dose CoQ10 in Thoroughbred race horses.     

The Effect of Exercise and Nutritional Supplementation on Proinflammatory Cytokine Expression in Young Racehorses During Training

The inflammatory response to vigorous exercise ranges from the mild symptoms of delayed-onset muscle soreness to debilitating injuries affecting soft tissue, joint, and bone. Although there is a great deal of information available on the inflammatory response to exercise in human athletes, less information is available regarding the inflammatory response to exercise in young horses undergoing training for racing careers. Here, we assessed the cytokine response to exercise in a group of young Thoroughbred racehorses during their initial training. Because there is interest in nonpharmacologic approaches to control or ameliorate exercise-induced inflammation, we also examined the anti-inflammatory effect of a nutritional supplement fed to half of the horses undergoing training. Twenty-five Thoroughbred horses aged 2 years were followed through their initial race training. Peripheral blood samples were collected at various times during the exercise for the quantitation of lactic acid, oxidative stress, and inflammatory cytokine gene expression. There was an intensity-dependent effect of exercise on lactate, malondialdehyde, and proinflammatory cytokine gene expression. Although training itself was associated with an overall reduction in inflammatory markers, horses receiving the supplement exhibited further reductions in their indicators of inflammation. As such, this study provides novel evidence of nutritional supplementation reducing postexercise inflammation.     

Effect of nutritional antioxidant supplementation on systemic and pulmonary antioxidant status,airway inflammation and lung function in heaves-affected horses

An oxidant/antioxidant imbalance in favour of oxidants has been identified as playing a decisive role in the pathogenesis of chronic inflammatory airway diseases. Nutritional antioxidant supplementation might reduce oxidative damage by enhancement of the antioxidant defence, thereby modulating inflammatory processes. In a placebo-controlled, blind study, it was tested whether a dietary antioxidant supplement administered for 4 weeks would improve lung function and reduce airway inflammation in heaves-affected horses. Eight horses in clinical remission of heaves were investigated at rest and after a standardised exercise test before and after treatment with an antioxidant supplement (consisting of a mixture of natural antioxidants including vitamins E and C and selenium from a variety of sources) or placebo (oatfeed pellets without additive). Pulmonary function and exercise tolerance were monitored; systemic and pulmonary lining fluid uric acid, glutathione and 8-epi-PGF(2alpha) were analysed, and bronchoalveolar lavage (BAL) cytology and inflammatory scoring of the airways were performed. The antioxidant treatment significantly improved exercise tolerance and significantly reduced endoscopic inflammatory score. Plasma uric acid concentrations were significantly reduced, suggesting downregulation of the xanthine-dehydrogenase and xanthine-oxydase pathway. Haemolysate glutathione showed a nonsignificant trend to increase, while plasma 8-epi-PGF(2alpha) remained unchanged. Pulmonary markers and BAL cytology were not significantly affected by antioxidant supplementation. The present study suggests that the antioxidant supplement tested modulated oxidant/antioxidant balance and airway inflammation of heaves-affected horses.     

Low dietary silicon supplementation may not affect bone and cartilage in mature,sedentary horses

As osteoarthritis is a major cause of lameness in horses in the United States, improving collagen health prior to onset and increasing collagen turnover within affected joints could improve health- and welfare-related outcomes. Through its positive effects on bone mineral content and density and its role in increasing collagen synthesis, silicon (Si) may slow the development and progression of osteoarthritis, thereby reducing lameness. This study evaluated the hypothesis that Si supplementation would increase cartilage turnover through increased collagen degradation and formation markers, as well as bone formation markers, resulting in reduced lameness severity when compared with controls. Ten mature Standardbred geldings were assigned to either a Si-treated (SIL) or control (CON) group and group-housed on pasture for 84 d. Horses were individually fed to ensure no cross-contamination of Si other than what was present in the environment. For the duration of the study, SIL horses received a Si–collagen supplement at the rate of 0.3 g supplement/(100 kg body weight day). Serum samples were taken weekly for osteocalcin, and plasma samples were taken on days 0, 42, and 84 for plasma minerals. On days 0, 42, and 84, subjective and objective lameness exams were performed, and radiographs and synovial fluid samples were taken from reference and osteoarthritic joints. Plasma minerals were similar in both groups and were lower on day 84 than on day 0 (P < 0.05). Si supplementation, fed at the manufacturer’s recommended rate, did not improve lameness or radiographs when compared with controls, and supplemented horses did not show greater collagen degradation and/or synthesis markers in synovial fluid than controls, indicating that cartilage turnover remained unaffected. However, a minimum beneficial threshold and range for Si supplementation standardized to body weight need to be established.     

Copper,Zinc-Superoxide Dismutase Activity in Exercising Horses Fed Two Forms of Trace Mineral Supplements

Nine mature horses were used to evaluate superoxide dismutase (SOD) response to a low-intensity standard exercise test and the effect of copper (Cu) and zinc (Zn) supplement form on enzyme activity. The modified switchback experiment was conducted over seven 28-day periods. Horses were conditioned to perform an average daily workload of 3.93 kg∗km∗10⁻³ with a digestible energy (DE) demand of approximately 29 Mcal. Following a controlled Cu and Zn repletion-depletion diet sequence, horses were fed a diet intended to be marginally deficient in Cu and Zn with the minerals supplemented in either sulfate or organic chelate forms. Horses were then subjected to a standardized exercise test to enable evaluation of SOD activity during exercise and immediate recovery. In the final analyses, mineral contents of the experimental diets were significantly different (P < .05). The mean mineral concentration of the sulfate diet was 6.28 ppm Cu and 35.99 ppm Zn, while the chelate diet contained 23.62 ppm Cu and 90.67 ppm Zn. Even so, resting SOD activity was not altered by diet (1793.47 ± 323.00 U/g Hb for the sulfate diet, 1355.70 ± 148.32 U/g Hb for the organic-chelate diet), nor did SOD activity change in response to the low-intensity workout. The SOD activity was unchanged by feeding a Cu and Zn supplemented diet to horses fed to be in a mineral-deficient state. These exercise bouts did not appear to have a significant impact on SOD activity. Other types of oxidative stress should be considered when trying to evaluate the antioxidant responsiveness of this enzyme.     

Oxidative Stress Biomarkers and Erythrocytes Hemolysis in Well-Trained Equine Athletes Before and After Exercise

The chronic exposure to regular exercise training seems to increase resistance to oxidative stress and improves the antioxidant defense system. The purpose of the present study was to investigate the effect of an exercise test of moderate intensity on oxidative stress biomarkers, antioxidant enzymes activity, and osmotic resistance of erythrocytes in well-trained equine athletes. Eighteen middle-aged horses of Ukrainian warmblood (8.3 ± 1.6 years) and Holsteiner (7.4 ± 1.9 years) breeds were used in this study. All horses have been in regular training for several years. The exercise test induced a significant increase of erythrocyte values, hemoglobin concentration, and hematocrit in horses of both breeds. Regular training induces activation the antioxidant enzymes and thereby can reduce oxidative stress in athletic horses. Our results suggest that the exercise test in horses of both breeds attenuates oxidative stress and accompanied with a significant decrease of lipid peroxidation and oxidatively modified proteins in erythrocytes after exercise. The findings of the present study demonstrated the elevated level of erythrocytes' catalase and glutathione reductase in Ukrainian warmblood horses, as well as decreased level of superoxide dismutase and glutathione reductase in Holsteiner horses reporting changes in levels of exercise-induced oxidative stress biomarkers in horses of both breeds. Statistically significant differences in the percentage of hemolyzed erythrocytes between pre-exercise and postexercise tests were observed and thereby signifying an oxidative stress-dependent impairment of erythrocyte stability. Our data suggest that oxidative stress and enzymatic antioxidant defense biomarkers can be used for the monitoring of fitness level, health benefits, and performance of equine athletes.     

Prognostic Significance of Oxidative Stress Markers in Colitis Associated with Phenylbutazone Administration in Draft Horses

The present study was carried out to assess the prognostic significance of oxidative stress markers in draft horses with colitis associated with phenylbutazone administration under field condition. For this purpose, a total of 40 native breed draft horses were studied. Based on case history, physical examination, and postmortem findings, diagnosis was made. According to the clinical outcome, horses were categorized into survivors and nonsurvivors. Clinically, there was significant association between nonsurvivors and anorexia (P < .01), stasis of intestinal motility (P < .01), melena (P < .01), and diarrhea (P < .001). Biochemically, malondialdehyde and nitric oxide showed a significant increase (P < .05) in nonsurvivors compared with survivors, whereas activities of superoxide dismutase showed a significant decrease (P < .05). Analysis of receiver operating characteristic curve indicated a high sensitivity and specificity of malondialdehyde, nitric oxide, and superoxide dismutase levels (P < .001) to predict the clinical outcome of colitis. Additionally, total protein and albumin showed a significant decrease in nonsurvivors compared with survivors; however, aspartate aminotransferase, sorbitol dehydrogenase, urea, and creatinine showed an increase (P < .05). The present results suggest that estimation of oxidative stress markers and antioxidant parameters may be useful predictors of the clinical outcomes of colitis associated with phenylbutazone administration in draft horses.     

Biochemical and Antioxidant Changes in Plasma, Serum, and Erythrocytes of Horses before and after a Jumping Competition

We designed an experiment to determine the concentrations of a marker of lipid peroxidation in erythrocytes of horses submitted to jumping competitions. Erythrocytes of exercised horses showed a significant increase in the concentration of thiobarbituric acid-reactive species content immediately after exercise (P < .001), which returned to normal levels 24 hours after exercise. Nonprotein sulfhydryl groups and superoxide dismutase activity (EC 1.15.1.1) in erythrocytes were significantly higher 24 hours after exercise, as compared with the resting period and control group (P < .001). Immediately after exercise, horses had increased serum concentrations of uric acid (P < .002) and plasma lactate, as well as increased creatine kinase (EC 2.7.3.2) and lactate dehydrogenase (EC 1.1.1.28) activities (P < .001), as compared with resting period and control group. All parameters returned to normal values 24 hours after exercise, except for uric acid serum levels that remained increased (P < .001). We conclude that the oxidative stress in erythrocytes of exercised horses may contribute to tissue damage. In addition, our results showed that horses submitted to a jumping competition showed higher production of free radicals and as a consequence, lipid peroxidation.     

Gender Differences of Oxidative Stress Biomarkers and Erythrocyte Damage in Well-Trained Horses During Exercise

The aim of this study was to determine the effects of gender differences on the blood oxidative stress biomarkers, antioxidant defenses, and resistance of erythrocytes to hemolytic agents of trained horses before and after exercise. The study was carried out on nine mares and 14 stallions of Ukrainian Warmblood well-trained horses, involved in jumping, eventing, and dressage. Oxidative stress biomarkers, antioxidant defenses, and osmotic resistance of erythrocytes were assessed. Trained stallions showed a decrease in lipid peroxidation and higher glutathione reductase activity, whereas mares presented a higher superoxide dismutase activity after exercise. The resistance of erythrocytes was similar in female and male. No statistically significant differences were observed in the percentage of hemolyzed erythrocytes between after and before exercise. A correlation between the oxidative stress biomarkers and antioxidant defenses in the stallions after exercises were observed, which may indicate a protective response of superoxide dismutase and catalase against exercise-induced oxidative stress.     

Antioxidant and haematological biomarkers in different groups of horses supplemented with polyunsaturated oil and vitamin E

Oxidative stress has been correlated with pathologies that impair the performance of athlete horses. The aim of this study was to assess the effects of supplementation with a mixture of polyunsaturated oil and vitamin E on the antioxidant and haematological biomarkers of horses. Horses under maintenance care (n = 6) and horses in training (n = 10) received 100 and 300 ml of the oil mixture respectively. Supplementation was provided for a period of 8 weeks, together with isocaloric inclusion. Blood samples were collected at three time periods (pretest, after 4 weeks and after 8 weeks) to analyse the following: the red blood cell count (RBCc); haemoglobin (Hb); haematocrit (HT); leucocytes; lymphocytes; platelets; the mean corpuscular volume (MCV); the mean corpuscular haemoglobin concentration (MCHC); the standard deviation of the red blood cell distribution width (RDW‐SD); the coefficient of variation of the red blood cell distribution width (RDW‐CV); glutathione peroxidase (GPx); superoxide dismutase (SOD); uric acid (UrAc); total plasma proteins (TPP); and creatine kinase (CK). After the 8 weeks of supplementation, animals under maintenance care exhibited significant increases in SOD, UrAc, the white blood cell count (WBCc), the RDW‐SD and the RDW‐CV (p < 0.05). The animals in training exhibited increases in GPx, SOD and UrAc (p < 0.05). In conclusion, supplementation with polyunsaturated oil and vitamin E increases blood antioxidants among animals under maintenance and in training, with different trends, while contributing to the fight against oxidative stress in each group analysed.     

Systemic and pituitary pars intermedia antioxidant capacity associated with pars intermedia oxidative stress and dysfunction in horses

OBJECTIVE: To determine whether a deficiency in systemic or local (pars intermedia) antioxidant capacity is associated with pituitary pars intermedia oxidative stress and pituitary pars intermedia dysfunction (PPID) in horses. SAMPLE POPULATION: Blood samples from 20 horses with PPID and 20 healthy client-owned horses, archived paraffin-embedded adrenal gland and substantia nigra tissues from 20 horses, and pituitary gland tissue from 16 horses. PROCEDURES: Total glutathione, superoxide dismutase, and glutathione peroxidase activities were determined in RBCs. Accumulation of a systemic marker of oxidative stress (3-nitrotyrosine) was assessed in plasma and formalin-fixed, paraffin-embedded adrenal gland and substantia nigra tissues. Local antioxidants (total and manganese superoxide dismutase, glutathione peroxidase, and total glutathione) were measured in pars intermedia tissues. RESULTS: No significant differences existed in systemic antioxidant enzyme activity or accumulation of 3-nitrotyrosine between horses with PPID and control horses. In pituitary gland tissues, glutathione peroxidase activity was increased in horses with oxidative stress, whereas total glutathione concentration and superoxide dismutase activity remained unchanged. There was an age-associated decrease in manganese superoxide dismutase activity in the pars intermedia. CONCLUSIONS AND CLINICAL RELEVANCE: There was no evidence of systemic accumulation of oxidative stress markers or deficiencies in antioxidant capacity in horses with PPID, suggesting that these are unlikely to be major predisposing factors in the development of PPID. Manganese superoxide dismutase activity in the pars intermedia decreased significantly with increasing age. Role of an age-associated decrease in antioxidant capacity for the pars intermedia in the development of PPID in horses warrants further investigation.     

Effect of prolonged branched-chain amino acid supplementation on metabolic response to anaerobic exercise in standardbreds

This study investigated the effect of prolonged BCAA supplementation on metabolic response to a 1600m run on treadmill in Standardbred trotters. Four trained Standardbreds were divided into two groups and assigned in a 2×2 Latin square design. Both groups were fed and exercised similarly: one group received an oral amino acids supplement (12 g leucine, 9 g isoleucine and 9 g valine) 30 minutes prior to exercise and immediately after, the other group received a placebo. The horses received the supplement 3 days per week for 5 weeks. In the last week horses performed an anaerobic exercise test on an inclined (3.5%) high-speed treadmill. The exercise consisted of a 15 minute warm-up phase immediately followed by a 1600 m run at maximal speed (heart rate > 200 beats/min). Blood samples were collected pre-exercise, after exercise and during recovery (10 min, 30 min, and 24 h), and analyzed for lactate, ammonia, total protein, urea, uric acid, creatinine, free fatty acids (FFA), creatine kinase (CK), lactate dehydrogenase (LDH), aspartate amino transferase (AST). Heart rate was continuously recorded during exercise and recovery. No statistical differences between the groups were observed for all the considered parameters. Nevertheless, BCAA supplementation resulted in a higher plasma ammonia and urea concentrations as reported in previous studies in humans and rats. These data suggest that a BCAA supplementation are not effective in enhancing performance in healthy and well-fed horses.     

A preliminary study on the changes in some potential markers of muscle-cell degradation in sub-maximally exercised horses supplemented with a protein and amino acid mixture

In this preliminary study, time-dependent changes in plasma CK and AST activity, tyrosine (Tyr), 3-methyl-histidine (3mHis), glucose and lactate concentrations were analysed in nine horses under two different conditions. Furthermore, intramuscular concentrations of Tyr, 3mHis and activities of cathepsin B, acid phosphatase (ACP), glucose-6-phosphate dehydrogenase (G6PDH) and mRNA expression of ubiquitin were determined at the same time. After studying the effects of exercise alone, the effects of exercise and feeding of an experimental protein/amino acid (AA) supplement were analysed. Horses were submitted to a total of four standardised exercise tests (SETs) of high intensity. Potential markers of muscle break down were determined prior to, immediately after, 4 and 18 h after exercise. The experiment was subdivided into two consecutive periods of 3 weeks. In each period, two SETs were performed. In the second period, horses were fed with the protein/AA supplement within 1 h after exercise. Significant changes in plasma, intramuscular Tyr levels and mRNA expression of ubiquitin were caused both by time in relation to exercise and by treatment with the protein/AA supplement. The experimental supplement significantly decreased the 4-h post-exercise expression of ubiquitin mRNA in muscle. Only a borderline increase of markers of lysosomal involvement was seen and CK and AST activity generally showed their normal post-exercise patterns. A clear post-exercise reduction of this CK activity, however, was not observed after supplementation with the protein/AA mixture. The current findings indicate that horses might benefit from protein and AA supplementation directly after training by decreasing post-exercise proteolysis. The results support that further studies should be performed to characterize changes in equine protein metabolism caused by exercise including underlying molecular mechanisms.     

Influence of an n‐3 long‐chain polyunsaturated fatty acid‐enriched diet on experimentally induced synovitis in horses

Dietary n‐3 long‐chain polyunsaturated fatty acid (LCPUFA) supplementation has previously been shown to modify joint‐related inflammation in several species, although information in the horse is lacking. We investigated whether dietary supplementation with n‐3 LCPUFA would modify experimentally induced synovitis in horses. Twelve, skeletally mature, non‐pregnant mares were randomly assigned to either a control diet (CONT) or an n‐3 long‐chain fatty acid‐enriched treatment diet (N3FA) containing 40 g/day of n‐3 LCPUFA for 91 days. Blood samples taken on days 0, 30, 60 and 90, and synovial fluid collected on days 0 and 90 were processed for lipid composition. On day 91, joint inflammation was stimulated using an intra‐articular (IA) injection of 100 ng of recombinant equine IL‐1beta (reIL‐1β). Synovial fluid samples taken at post‐injection hours (PIH) 0, 4, 8 and 24 were analysed for prostaglandin E₂ (PGE₂), matrix metalloproteinase (MMP) activity and routine cytology. Synovium and articular cartilage samples collected at PIH 8 were analysed for gene expression of MMP 1 and MMP 13, interleukin‐1beta (IL‐1β), cyclooxygenase 2 (COX‐2), tumour necrosis factor‐alpha and the aggrecanases, a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)‐4 and ADAMTS‐5. A 90‐day feeding period of n‐3 LCPUFA increased serum phospholipid and synovial fluid lipid compositions of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) compared to CONT horses. The reIL‐1β injection caused an inflammatory response; however, there was no effect of dietary treatment on synovial fluid PGE2 content and MMP activity. Synovial tissue collected from N3FA horses exhibited lower expression of ADAMTS‐4 compared to CONT horses. Despite the presence of EPA and DHA in the synovial fluid of N3FA horses, dietary n‐3 LCPUFA supplementation did not modify synovial fluid biomarkers compared to CONT horses; however, the lower ADAMTS‐4 mRNA expression in N3FA synovium warrants further investigation of n‐3 LCPUFA as a joint therapy.     

Form of Vitamin E Supplementation Affects Oxidative and Inflammatory Response in Exercising Horses

Vitamin E is an essential antioxidant that may benefit athletes by reducing oxidative stress and influencing cytokine expression. Supplements can be derived from natural or manufactured synthetic sources. This study aimed to determine (1) if supplemental vitamin E is beneficial to exercising horses and (2) if there is a benefit of natural versus synthetic vitamin E. After 2 weeks on the control diet (vitamin E–deficient grain and hay), 18 horses were divided into three groups and fed the control diet plus (1) 1000 IU/d synthetic α-tocopherol (SYN-L), (2) 4000 IU/d synthetic α-tocopherol (SYN-H), or (3) 4000 IU/d RRR-α-tocopherol (natural source [NAT]). On day 7, horses began a 6-week training protocol, with standard exercise tests (SETs) performed before and after the 6-week protocol. Venous blood samples were collected on days 0, 7, 29, and 49. Horses fed NAT had higher α-tocopherol (P < .05) at post-SET1 through post-SET2. Plasma thiobarbituric acid–reactive substance levels were lower in NAT versus SYN-L horses after SET2 (P = .02). Serum aspartate aminotransferase was lower after exercise in NAT horses versus SYN-L and SYN-H (P = .02), and less reduction in stride duration was seen after exercise in NAT as compared with SYN-L and SYN-H (P = .02). Gene expression of tumor necrosis factor α was lower in NAT compared with SYN-H (P = .01) but not SYN-L. In conclusion, feeding higher levels of natural vitamin E source resulted in higher serum α-tocopherol levels as well as some improvement in oxidative and inflammatory response and improved functional outcomes in response to an exercise test.     

Effect of Repeated Arthrocentesis on Cytology of Synovial Fluid

Repeated arthrocentesis is necessary to diagnose and monitor the evolution of joint diseases, but the procedure may worsen any inflammation and lead to an alteration in synovial fluid. The aim of this study was to determine the effect of repeated arthrocentesis on synovial fluid cytology in healthy horses with normal joints. The experimental study was approved by Ethics Committee (University of Pisa, Italy). Four horses were enrolled in this study on the basis of inclusion criteria and underwent repeated arthrocentesis of the intercarpal joint of both left and right forelimbs. The synovial fluid samples were processed for total protein concentration, total nucleated cell count, and differential leukocyte count. Data distribution was performed with the Komolgorov–Smirnov test, and a Friedman test for repeated measures and Dunn's test as post hoc were performed in order to verify differences related to sampling times comparing each time point. Significance was set at P < .05. All horses remained free of lameness throughout the study period. Statistical differences were found for macrophage and lymphocyte related to sampling time. Our results support the finding that repeated arthrocentesis does not induce detectable synovial fluid alterations. Although mild statistically significant changes in macrophage and lymphocyte populations were found, the values were always within normal ranges, suggesting that these changes were not clinically significant. Moreover, the cytologic alterations rapidly solved. In conclusion, repeated arthrocentesis does not cause long term and clinically relevant alterations in synovial fluid cytology in healthy horses with normal joints.     

Effect of Treadmill Exercise and Hydrogen-rich Water Intake on Serum Oxidative and Anti-oxidative Metabolites in Serum of Thoroughbred Horses

The present study aimed to clarify changes of oxidative stress and antioxidative functions in treadmill-exercised Thoroughbred horses (n=5, 3 to 7 years old), using recently developed techniques for measurement of serum d-ROMs for oxidative stress, and BAP for antioxidative markers. Also, the effect of nasogastric administration of hydrogen-rich water (HW) or placebo water preceding the treadmill exercise on these parameters was examined. Each horse was subjected to a maximum level of treadmill exercise in which the horses were exhausted at an average speed of 13.2 ± 0.84 m/sec. Blood samples were taken 4 times, immediately before the intake of HW or placebo water at 30 min preceding the treadmill exercise, immediately before the exercise (pre-exercise), immediately after the exercise (post-exercise) and at 30 min following the exercise. In all horses, both d-ROMs and BAP values significantly increased at post-exercise. The increase in d-ROMs tended to be lower in the HW trial, as compared to the placebo trial at pre-exercise. The increase in BAP was considerable at approximately 150% of the pre-exercise values in both the HW and placebo treatment trials. The BAP/d-ROMs ratio was significantly elevated at post-exercise in both treatment trials, while a significant elevation was also observed at pre-exercise in the HW trial. BAP, d-ROM, and the BAP/d-ROM ratio tended to decline at 30 min after the exercise, except BAP and BAP/d-ROMs in the placebo trial. These results demonstrate that the marked elevation of oxidative stress and anitioxidative functions occurred simultaneously in the intensively exercised horses, and suggest a possibility that HW has some antioxidative efficacy.     

Effects of exercise and oral antioxidant supplementation enriched in (n-3) fatty acids on blood oxidant markers and erythrocyte membrane fluidity in horses

The aim of this study was to investigate in a placebo-controlled field study the effect of a (n-3)-vitamin supplementation on erythrocyte membrane fluidity (EMF), oxidant/antioxidant markers and plasmatic omega3/omega6 fatty acid ratio (FAR) in 12 eventing horses. Venous blood was sampled at rest before (PRE) and after (POST) a three week treatment period with either the supplement (group S, n=6) or a placebo (group P, n=6) as well as after 15min (POST E15') and 24h (POST E24h) after a standardised exercise test. The following markers were analysed: EMF, plasma antioxidant capacity of water and lipid soluble components, ascorbic acid, uric acid (UA), glutathione (reduced: GSH, oxidised: GSSG), vitamin E (Vit E), beta-carotene, glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity, selenium, copper (Cu), zinc (Zn), oxidised proteins (Protox), lipid peroxides (Pool) and FAR. EMF did not differ between group S and P after treatment, but GPx remained unchanged in group S whereas it decreased in group P and plasma Cu/Zn ratio remained unchanged whereas it increased in group P. FAR were significantly increased in group S. Exercise induced a significant decrease of EMF (POST vs. E24h) in both groups, but which was significantly lower at E15' in group S than in group P. Exercise induced a significant increase of UA and ACW (POST vs. E15') and Protox (POST vs. E24h) in both groups. An exercise-related decrease in GSH and Pool (POST vs. E15') was found in group P, whereas Vit E and FAR (POST vs. E24h) significantly decreased in both groups. The study showed that exercise induced a decrease in EMF in horses associated with changes of blood oxidative balance. The (omega-3)-vitamin supplementation tested improved the oxidative balance poorly but delayed the exercise-induced decrease of EMF and increased the FAR.     

Relationship of oxidative stress in skeletal muscle with obesity and obesity-associated hyperinsulinemia in horses

In horses, hyperinsulinemia and insulin resistance (insulin dysregulation) are associated with the development of laminitis. Although obesity is associated with insulin dysregulation, the mechanism of obesity-associated insulin dysregulation remains to be established. We hypothesized that oxidative stress in skeletal muscle is associated with obesity-associated hyperinsulinemia in horses. Thirty-five light breed horses with body condition scores (BCS) of 3/9 to 9/9 were studied, including 7 obese, normoinsulinemic (BCS ≥ 7, resting serum insulin < 30 μIU/mL) and 6 obese, hyperinsulinemic (resting serum insulin ≥ 30 μIU/mL) horses. Markers of oxidative stress (oxidative damage, mitochondrial function, and antioxidant capacity) were evaluated in skeletal muscle biopsies. A Spearman’s rank correlation coefficient was used to determine relationships between markers of oxidative stress and BCS. Furthermore, to assess the role of oxidative stress in obesity-related hyperinsulinemia, markers of antioxidant capacity and oxidative damage were compared among lean, normoinsulinemic (L-NI); obese, normoinsulinemic (O-NI); and obese, hyperinsulinemic (O-HI) horses. Increasing BCS was associated with an increase in gene expression of a mitochondrial protein responsible for mitochondrial biogenesis (estrogen-related receptor alpha, ERRα) and with increased antioxidant enzyme total superoxide dismutase (TotSOD) activity. When groups (L-NI, O-NI, and O-HI) were compared, TotSOD activity was increased and protein carbonyls, a marker of oxidative damage, decreased in the O-HI compared to the L-NI horses. These findings suggest that a protective antioxidant response occurred in the muscle of obese animals and that obesity-associated oxidative damage in skeletal muscle is not central to the pathogenesis of equine hyperinsulinemia.