D. J. Marlin

Muscle buffering capacity and dipeptide content in the Thoroughbred horse, Greyhound dog and man

1. Muscle buffering capacity (beta m) and dipeptide content were measured in locomotory muscles of the Thoroughbred horse, Greyhound dog and Man. 2. Beta m and carnosine contents were highest in the horse. Anserine was only found in dog muscle. 3. The higher beta m in horse and dog muscle, compared with man, appears to be predominantly due to higher muscle contents of histidine containing dipeptides in these species.

Changes in Circulatory Antioxidant Status in Horses during Prolonged Exercise

Prolonged low-medium intensity exercise is associated with increased oxidative stress in humans. We hypothesized that competitive equine endurance racing would induce changes in circulatory antioxidants and produce systemic oxidative stress. Forty horses competing in a 140-km endurance race in warm conditions [shade temperature 15-19 degrees C; 62-88% relative humidity (%RH)] were sampled before (Pre), immediately after exercise (End) and at approximately 16 h into recovery (+16 h). Plasma ascorbic acid concentration was not different between Pre [11.1 (median); 4.6-20.3 micromol/L (range)] and End [9.7; 3.0-38.9 (range) micromol/L] but was significantly decreased at +16 h (5.5; 2.8-15.5 micromol/L; P < 0.05). Total red cell hemolysate glutathione (TGSH) concentration was significantly reduced by exercise (Pre 1261; 883-1532 micromol/L; End 1065; 757-1334 micromol/L; P < 0.05) and at +16 h recovery (1032; 752-1362 micromol/L; P < 0.05). Glutathione redox ratio was unchanged by exercise but was significantly decreased at +16 h compared with that at both Pre and End (P < 0.05). The concentration of total barbituric acid reactive substances (TBARS) in plasma was increased compared with that at Pre (309; 66-1048 nmol/L), both at End (408; 170-1196 nmol/L; P < 0.05) and +16 h (380; 99-1161 nmol/L; P < 0.05). alpha-Tocopherol was unchanged by exercise or recovery. Mean race speed was 16.5 +/- 1.6 km/h and ranged from 13.9 to 19.7 km/h. Mean speed during competition in horses that completed the full 140 km (n = 28) was significantly correlated with end of exercise ascorbic acid (r = 0.622; P = 0.0004). Although there were increases in creatine phosphokinase (CK), aspartate aminotransferase (AST) and TBARS and a loss of TGSH, this study failed to demonstrate evidence of classical oxidative stress.

Effects of exercise intensity and environmental stress on indices of oxidative stress and iron homeostasis during exercise in the horse.

The effects of prolonged variable-intensity and short-term high-intensity exercise on indices of oxidative stress and iron homeostasis were compared in six fit horses under cool [20°C, 40% relative humidity (RH)] or hot/humid (30°C, 80% RH) environmental conditions. The exercise protocols were designed to simulate equine competition, including racing (intense exercise) or the speed and endurance phase of a 3-day event (prolonged exercise). Increased plasma concentrations of lipid hydroperoxides and haemolysate concentrations of oxidised glutathione (GSSG) were measured within 30 min of the completion of exercise, indicating production of reactive oxygen species (ROS) and lipid membrane peroxidation. The horses were unable to complete the prolonged exercise protocol at high temperature and humidity. This coincided with higher maximal values of lipid hydroperoxides [138.2 (17.7) μM and GSSG [110.6 (18.2) μM], compared to high-intensity [105.2 (14.9) μM and 63.6 (8.6) μM, respectively] or prolonged [100.7 (18.7) μM and 86.2 (9.1) μM, respectively] exercise performed under cooler environmental conditions. Significant correlations were found between the duration of the final stage of exercise during hot/humid environmental conditions and increased levels of lipid hydroperoxides (r = 0.85), GSSG (r = 0.94), xanthine (r = 0.92) and uric acid (r = 0.96). Excerise also decreased the iron (Fe)-binding antioxidant activity of the plasma and increased the total plasma Fe levels, although this was only significant for prolonged exercise in ambient conditions. There was no detectable free Fe in the plasma at any stage of exercise. Other changes in biochemical parameters had returned to pre-exercise levels within 24 h after exercise. The results show that exercise can induce changes in biochemical parameters that are indicative of oxidative stress in the fit horse and that this was, exacerbated during exercise at high temperature and humidity.

Exercise-associated oxidative stress

Abstract During exercise a number of potential sources exist for the production of reactive oxygen species such as superoxide anions, hydrogen peroxide and hydroxyl radicals. Oxidative stress has been defined as a disturbance in the pro-oxidant-antioxidant balance in favour of the former, leading to potential damage (Sies 1991). Oxidative stress does not always result in oxidative damage. However, oxidative stress may result in oxidative damage to lipids, protein and DNA and consequently decrease athletic performance. Here we review the evidence for oxidative stress following exercise and the effects of exercise on the enzymatic and non-enzymatic antioxidant systems in a number of species including the horse. The effects of antioxidant supplementation on oxidative stress and performance during exercise are also evaluated.

Muscle ATP loss and lactate accumulation at different work intensities in the exercising Thoroughbred horse

SummaryThe effect of 2 min treadmill exercise, at speeds of 6–12 in ·st-1 on an incline of 5°, upon muscle adenine nucleotide loss and lactate accumulation was studied in six Thoroughbred horses. Minimal change occurred in the adenosine triphosphate (ATP) content of the middle gluteal muscle at speeds of 10 m·s− or less, but significant loss (up to 47%) had occurred in all horses by 12 m·s−1. The decline in ATP significantly correlated with the accumulation of muscle lactate, beginning shortly after the accumulation of 40 mmol·kg− dry muscle lactate. Decline in muscle ATP was mirrored closely by the appearance of ammonia, and to a lesser extent, hypoxanthine and uric acid in plasma. The results suggest that peak accumulation of any of these, or simply the concentration at a specified recovery time, may be used as a measure of ATP loss in the musculature as a whole. This was not so in the case of xanthine, which may also be formed from the degradation of guanidine nucleotides. An ln-ln plot of plasma ammonia against treadmill speed indicated a break point in accumulation between 8 and 9 m·−. The kinetics of ammonia accumulation with speed differed from those of lactate.

Estimation of the carnosine content of different fibre types in the middle gluteal muscle of the thoroughbred horse.

1. Skeletal muscle samples were obtained by needle biopsy from one of two depths of the m. gluteus medius in a group of twenty race‐trained thoroughbred horses. 2. The content of carnosine was determined in each muscle sample, part of which was used for histochemical analysis. Fibres were classified as type I, type IIA or type IIB on the basis of the pH dependent lability of the myosin ATPase reaction. 3. Muscle samples with a higher type II fibre section area (FSA) have a higher carnosine content than those with a higher type I FSA. 4. Multiple linear regression analysis was used to estimate the mean carnosine content of individual fibre types. The results estimated a mean carnosine content in type I fibres of 54 mmol (kg dry muscle (DM))‐1, in type IIA fibres 85 mmol (kg DM)‐1 and in type IIB fibres 180 mmol (kg DM)‐1. 5. Based on the estimated values of single fibre carnosine content, there was close concordance between the estimated and the measured carnosine content of mixed fibre samples. 6. It would appear from this and other studies that carnosine has an important role as a physico‐chemical buffer in equine middle gluteal muscle and that this is greatest in type IIB fibres, where it may account for up to 50% of physico‐chemical buffering of H+ produced by muscle in the pH range 7.1‐6.5.

Metabolic adaptation to a fat-supplemented diet by the thoroughbred horse

Following 10 weeks of fat supplementation a group of aerobically trained thoroughbred horses exhibited a significant decrease in postprandial plasma triacylglycerol concentration. This decrease was associated with a mean 50% increase in plasma total lipase activity following pentosan polysulfate administration and an increase in postprandial plasma cholesterol concentration. A significant increase in the activity of muscle citrate synthase (EC 4.1.3.7), expressed as a ratio to the total fractional area occupied by type I and type IIa muscle fibres, was also observed. No significant change in the concentration of resting muscle glycogen or triacylglycerol occurred as a result of fat supplementation. These results suggest that there was improved management of the fat load and that the triacylglycerol-clearing capacity of the horses was increased as a result of fat supplementation. It is suggested that the increase in plasma total lipase activity following pentosan polysulfate administration may have reflected an increase in muscle lipoprotein lipase (EC 3.1.1.34) activity, which would increase the capacity of muscle for free fatty acid uptake from circulating triacylglycerol-rich plasma lipoproteins. Fat supplementation may also enhance the oxidative capacity of muscle, as suggested by the significant increase in muscle citrate synthase and the trend towards an increase in beta-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35) following 10 weeks of fat supplementation.

Frequency domain analysis of heart rate variability in horses at rest and during exercise

The pattern of variation in heart rate on a beat-to-beat basis contains information concerning sympathetic (SNS) and parasympathetic (PNS) contributions to autonomic nervous system (ANS) modulation of heart rate (HR). In the present study, heart period (RR interval) time series data were collected at rest and during 3 different treadmill exercise protocols from 6 Thoroughbred horses. Frequency and spectral power were determined in 3 frequency bands: very low (VLF) 0-< or = 0.01, low (LO) >0.01-< or = 0.07 and high (HI) >0.07-< or = 0.5 cycles/beat. Indicators of sympathetic (SNSI = LO/HI) and parasympathetic (PNSI = HI/TOTAL) activity were calculated. Power in all bands fell progressively with increasing exercise intensity from rest to trot. At the gallop VLF and LO power continued to fall but HI power rose. SNSI rose from rest to walk, then fell with increasing effort and was lowest at the gallop. PNSI fell from rest to walk, then rose and was highest at the gallop. Normalised HI power exceeded combined VLF and LO power at all gaits, with the ratio HI to LO power being lowest at the walk and highest at the gallop. ANS indicators showed considerable inter-horse variation, and varied less consistently than raw power with increasing physical effort. In the horses studied, the relationship between power and HR changed at exercise intensities associated with heart rates above approximately 120-130 beats/min. At this level, humoral and other non-neural mechanisms may become more important than autonomic modulation in influencing heart rate and heart rate variability (HRV). HRV at intense effort may be influenced by respiratory-gait entrainment, energetics of locomotion and work of breathing. HRV analysis in the frequency domain would appear to be of potential value as a noninvasive means of assessing autonomic modulation of heart rate at low exercise intensities, only. The technique may be a sensitive method for assessing exercise response to experimental manipulations and disease states.

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.

Effect of chronic airway inflammation and exercise on pulmonary and systemic antioxidant status of healthy and heaves-affected horses.

In heaves-affected horses the relation between oxidant status, airway inflammation (AI) and pulmonary function (PF) is unknown. The oxidant status of blood and pulmonary epithelial lining fluid (PELF) of healthy (H, n = 6) and heaves-affected horses in clinical remission (REM, n = 6) and in crisis (CR, n = 7) was assessed at rest, during and after standardised exercise test by measurement of reduced and oxidised glutathione, glutathione redox ratio [GRR%]; uric acid and 8-epi-PGF2alpha. Oxidant status was related to PF parameters (mechanics of breathing and arterial blood gas tension) and Al parameters (bronchoalveolar lavage [BAL] neutrophil % and AI score). Haemolysate glutathione was significantly different between groups and was correlated with PF and AI parameters; GRR in PELF was increased during CR and was correlated with PF and AI parameters. Exercise induced an increase of plasma uric acid that was significantly higher both in REM and CR. PELF 8-epi-PGF2alpha was significantly increased in CR and correlated with PF and AI parameters. These results suggest that oxidative stress occurring in heaves is correlated with PF and AI and may be locally assessed by PELF glutathione status, uric acid and 8-epi-PGF2alpha. Systemic repercussions are reflected by assay of GSH in resting horses and by uric acid in exercising horses.