C. A. Roberts

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.

Mechanics of breathing during strenuous exercise in Thoroughbred horses

The changes induced by exercise on the mechanics of breathing, as well as the simultaneous changes occurring in arterial blood gas tensions and in respiratory gas exchange were investigated in 6 healthy thoroughbred horses, performing a treadmill exercise of increasing intensity. Respiratory airflow and tidal volume (VT) were measured with ultrasonic flowmeters. Pleural pressure changes were measured by an oesophageal balloon catheter. Gas concentration of the expired air was analysed with a mass spectrometer; the oxygen consumption (VO2) and the carbon dioxide output (VCO2) were computed breath-by-breath. Arterial blood gas values were obtained by sampling from the carotid artery. Between rest and fast gallop VT, respiratory frequency, expired minute ventilation (VE), VO2, VCO2, total pulmonary resistance (RL), mechanical work of breathing (Wrm) and PaCO2 increased significantly while PaO2 decreased significantly. The Wrm.VO2(-1) ratio in galloping horses increased exponentially with VE. This, together with the relationship between the changes in PaO2 and in PaCO2 and the increase in the ventilatory mechanics parameters, suggests that the mechanics of breathing may be one of the factors constraining further increase in ventilation in exercising healthy horses.

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.

Breath Condensate Hydrogen Peroxide Correlates with Both Airway Cytology and Epithelial Lining Fluid Ascorbic Acid Concentration in the Horse

The relationship between hydrogen peroxide (H2O2) concentration in expired breath condensate (EBC) and cytology of the respiratory tract obtained from tracheal wash (TW) or bronchoalveolar lavage (BAL), and epithelial lining fluid (ELF) antioxidant status is unknown. To examine this we analysed the concentration of H2O2 in breath condensate from healthy horses and horses affected by recurrent airway obstruction (RAO), a condition considered to be an animal model of human asthma. The degree of airway inflammation was determined by assessing TW inflammation as mucus, cell density and neutrophil scores, and by BAL cytology. ELF antioxidant status was determined by measurement of ascorbic acid, dehydroascorbate, reduced and oxidised glutathione, uric acid and α-tocopherol concentrations. RAO-affected horses with marked airway inflammation had significantly higher concentrations of breath condensate H2O2 than control horses and RAO-affected horses in the absence of inflammation (2.0±0.5 μmol/l, 0.4±0.2 μmol/l and 0.9±0.2 μmol/l H2O2, respectively; p<0.0001). The concentration of breath condensate H2O2 was related inversely to the concentration of ascorbic acid in ELF (r=-0.80; p<0.0001) and correlated positively with TW inflammation score (r=0.76, p<0.0001) and BAL neutrophil count (r=0.80, p<0.0001). We conclude that the concentration of H2O2 in breath condensate influences the ELF ascorbic acid concentration and provides a non-invasive diagnostic indicator of the severity of neutrophilic airway inflammation.

Antioxidant and inflammatory responses of healthy horses and horses affected by recurrent airway obstruction to inhaled ozone.

REASONS FOR PERFORMING STUDY Inhaled ozone can induce oxidative injury and airway inflammation. Horses affected by recurrent airway obstruction (RAO) have a decreased pulmonary antioxidant capacity, which may render them more susceptible to oxidative challenge. It is currently unknown whether RAO-affected horses are more susceptible to oxidative stress than those unaffected by RAO. OBJECTIVES To determine whether ozone exposure induces greater oxidative stress and airway inflammation in RAO-affected horses in remission than in healthy horses. METHODS Seven healthy control horses and 7 RAO-affected horses were exposed to 0.8 ppm ozone for 2 h at rest. RESULTS At baseline, bronchoalveolar lavage fluid (BALF) ascorbic acid concentrations were lower in RAO-affected horses than healthy controls. Ozone appeared to preferentially oxidise glutathione rather than ascorbic acid 6 h after exposure. Individual healthy and RAO-affected horses demonstrated oxidation of BALF glutathione after ozone exposure. Overall, RAO-affected horses did not demonstrate increased oxidative stress following ozone exposure, compared with healthy horses. Ozone did not induce significant airway inflammation in either group. CONCLUSIONS RAO-affected horses in remission are not more sensitive to ozone despite a decreased pulmonary antioxidant capacity. Sensitivity to ozone appears to be independent of initial pulmonary antioxidant status. POTENTIAL RELEVANCE Horses with high susceptibility to oxidative stress may benefit from antioxidant supplementation.

Stride length and respiratory tidal volume in exercising thoroughbred horses

The purpose of this study was to test the hypothesis that locomotor-respiratory coupling in horses may provide a basis for the appropriate matching of lung ventilation to running speed and metabolic power. This was achieved by determining the effect on respiratory frequency and tidal volume in thoroughbred horses of changing running speed while maintaining a constant metabolic demand by also varying the incline of the treadmill. This procedure was performed at three different speeds at two different levels of metabolic rate. It appears that, in response to metabolic demands which are not directly related to speed, horses can control tidal volume independently of stride length but they do not vary respiratory frequency independently of stride frequency. When running at 0 degrees incline, however, they probably do not operate an elevated tidal volume, because of the increased energetic costs such an elevated tidal volume would incur.

Respiratory responses of mature horses to intravenous lobeline bolus.

The respiratory stimulant lobeline has been used in equine clinical practice to increase inspiratory and expiratory airflow rates at rest in order to facilitate investigation of both lower and upper airway function. Some of the responses to lobeline in the pony have been reported, but the detailed time course, effect of dose, possible side effects and reproducibility associated with lobeline administration have not been described in the horse. Respiratory airflow rates and oesophageal pressure were measured with a Fleisch No. 5 pneumotachometer and lightweight facemask and a microtip pressure transducer catheter, respectively. The output of the Fleisch pneumotachometer was calibrated for flow rates up to +/- 70 l/s. Seven mature horses with no clinical signs of respiratory disease were studied. Investigations were conducted to determine: (1) the responses to different doses of lobeline (0.15, 0.20, 0.25 and 0.30 mg/kg bwt) as a rapid i.v. bolus (6 horses); (2) arterial blood gases during and after lobeline administration (0.20 mg/kg bwt; 3 horses); and (3) the reproducibility of lobeline-stimulated hyperpnoea (5 horses; 2 doses of 0.20 mg/kg bwt lobeline, 15 min apart). All horses tolerated the lobeline-stimulated hyperpnoea well, although one always coughed or snorted at the onset. Mild tremor was noted following the highest dose in several horses. Apnoea of approximately 40 s was common after the hyperpnoea. Both tidal volume (VT) and frequency (fR) increased with lobeline dose. During peak hyperpnoea at a dose of 0.30 mg/kg bwt, peak inspired flow rate (PIF), peak expired flow rate (PEF) and minute ventilation (VE) were mean +/- s.e. 41+/-5 l/s, 61+/-10 l/s and 920+/-99 l/min, respectively. The hyperpnoea also caused marked changes in arterial PaO2, PaCO2 and pHa at 90 s after lobeline (0.20 mg/kg bwt) administration (mean +/- s.e. 146.0+/-6.9 mmHg, 20.6+/-0.8 mmHg and 7.707+/-0.020, respectively) compared to at rest (mean +/- s.e. 104.0+/-4.0 mmHg, 50.6+/-2.8 mmHg and 7.432+/-0.012). Dynamic lung compliance (Cdyn) was unaltered by lobeline administration. The lobeline-induced hyperpnoea was highly reproducible, with no significant difference in any of the parameters during 2 stimulations 15 min apart. Lobeline induced highly reproducible responses without any apparent adverse effects and may be useful in the investigation of pulmonary function in healthy horses and those with airway disease.

Investigation of the incidence and type of injuries associated with high-speed treadmill exercise testing

REASONS FOR PERFORMING STUDY During the past 20 years, treadmill exercise testing has played an important role in both the study of equine exercise physiology and the investigation of poor athletic performance. However, it has been suggested that some trainers and veterinarians may be reluctant to refer horses for treadmill exercise testing because of fears that horses may be at increased risk of musculoskeletal injury during treadmill exercise. OBJECTIVE To investigate the incidence and types of injuries sustained by horses undergoing treadmill exercise. METHODS Data were collated from 9 centres in the UK, France and Belgium, and the prevalence and types of injury were established. RESULTS A total of 2305 records were reviewed, with 2258 horses performing treadmill exercise. There was an overall injury rate of 5.4%. However, the majority of injuries sustained were minor in nature (4.7%). Only 13 horses (0.6%) sustained major injuries in association with treadmill exercise. These included 5 cases of severe exercise-induced myopathy, 4 fractures (of which 1 was catastrophic), 2 tendon injuries, 1 case with undiagnosed severe lameness and 1 with marked exacerbation of a previously diagnosed lameness. Two other major incidents were reported but were not directly associated with treadmill exercise (one had iliac thrombosis and one collapsed and died as a result of a pulmonary embolism). CONCLUSIONS This study confirms that the majority of horses undergo treadmill exercise without incident. The majority of injuries that did occur were minor in nature and the incidence of major injuries was similar to that reported during competition elsewhere. POTENTIAL RELEVANCE Treadmill exercise is a safe procedure and does not appear to pose an increased risk of injury in comparison with overground exercise.

Pulmonary Bioavailability of Ascorbic Acid in an Ascorbate-synthesising Species, the Horse

Vitamin C (ascorbic acid) is a non-enzymatic antioxidant important in protecting the lung against oxidative damage and is decreased in lung lining fluid of horses with airway inflammation. To examine possible therapeutic regimens in a species with ascorbate-synthesising capacity, we studied the effects of oral supplementation of two forms of ascorbic acid, (each equivalent to 20 mg ascorbic acid per kg body weight) on the pulmonary and systemic antioxidant status of six healthy ponies in a 3 2 3 Latin square design. Two weeks supplementation with ascorbyl palmitate significantly increased mean plasma ascorbic acid concentrations compared to control (29 - 5 and 18 - 7 w mol/l, respectively; p <0.05 ). Calcium ascorbyl-2-monophosphate, a more stable form of ascorbic acid, also increased mean plasma ascorbic acid concentrations, but not significantly (23 - 1 w mol/l; p =0.07 ). The concentration of ascorbic acid in bronchoalveolar lavage fluid increased in five out of six ponies following supplementation with either ascorbyl palmitate or calcium ascorbyl-2-monophosphate compared with control (30 - 10, 25 - 4 and 18 - 8 w mol/l, respectively; p <0.01 ). Neither supplement altered the concentration of glutathione, uric acid or f -tocopherol in plasma or bronchoalveolar lavage fluid. In conclusion, the concentration of lung lining fluid ascorbic acid is increased following ascorbic acid supplementation (20 mg/kg body weight) in an ascorbate-synthesising species.