Effects of hyperthermia and acidosis on mitochondrial production of reactive oxygen species.

Exercise is associated with the development of oxidative stress, but the specific source and mechanism of production of pro-oxidant chemicals during exercise has not been confirmed. We used equine skeletal muscle mitochondria to test the hypothesis that hyperthermia and acidosis affect mitochondrial oxygen consumption and production of reactive oxygen species (ROS). Skeletal muscle biopsies were obtained at rest, after an acute episode of fatiguing exercise, and after a 9-wk conditioning program to increase aerobic fitness. Mitochondrial oxygen consumption and ROS production were measured simultaneously using high-resolution respirometry. Both hyperthermia and acidosis increased nonphosphorylating (LEAK) respiration (5.8× and 3.0×, respectively, P < 0.001) and decreased efficiency of oxidative phosphorylation. The combined effects of hyperthermia and acidosis resulted in large decreases in phosphorylating respiration, further decreasing oxidative phosphorylation efficiency from 97% to 86% (P < 0.01). Increased aerobic fitness reduced the effects of acidosis on LEAK respiration. Hyperthermia increased and acidosis decreased ROS production (2× and 0.23×, respectively, P < 0.001). There was no effect of acute exercise, but an aerobic conditioning program was associated with increased ROS production during both nonphosphorylating and phosphorylating respiration. Hyperthermia increased the ratio of ROS production to O2 consumption during phosphorylating respiration, suggesting that high-temperature impaired transfer of energy through the electron transfer system despite relatively low mitochondrial membrane potential. These data support the role of skeletal muscle mitochondria in the development of exercise-induced oxidative stress, particularly during forms of exercise that result in prolonged hyperthermia without acidosis.NEW & NOTEWORTHY The results of this study provide evidence for the role of mitochondria-derived ROS in the development of systemic oxidative stress during exercise as well as skeletal muscle diseases such as exertional rhabdomyolysis.

Effects of Feeding Coenzyme Q10-Ubiquinol on Plasma Coenzyme Q10 Concentrations and Semen Quality in Stallions.

Although coenzyme Q10 (CoQ10) serves as an antioxidant and energy source for spermatozoa when added to stallion semen before cooling or freezing, the effects of feeding CoQ10 on semen quality have not been studied. We assessed the effects of daily oral ingestion of CoQ10-ubiquinol by stallions on their plasma CoQ10 concentrations and semen quality. Seven mature Andalusian stallions ate 1g ubiquinol/day for 4 weeks followed by a 4-week washout period. Four horses initially completed an additional 4-week control period without ubiquinol. Blood was sampled weekly for determination of plasma CoQ10 concentrations. Ejaculates were collected every two weeks and assessed for total motility (TM), progressive motility (PM), and viability (V) after cooling for 24hours (T1), immediate cryopreservation (T2), and cryopreservation after 24hours cooling (T3). Ingesting ubiquinol resulted in an increase in plasma CoQ10 concentration (P < .001). Two weeks of CoQ10-ubiquinol resulted in improved V with all treatments (T1: P = .007; T2: P = .05; T3: P = .01) and PM with T3 (P = .04). In five stallions, TM and PM were also improved for T1 (P = .01 and P = .02, respectively) and TM increased with T2 (P = .03). Overall, semen quality parameters increased within the first 2 weeks of supplementation, plateaued at the end of the 4-week supplementation period and persisted after discontinuing ubiquinol until the end of the sampling period (8 weeks). Feeding 1 g CoQ10-ubiquinol for 4 weeks to breeding stallions improved semen quality after cooling and freezing in 5 of 7 stallions. This could be important for improving reproductive efficiency in stallions.

Validation of a Rapid Bronchoalveolar Lavage Fluid Colorimetric Evaluation for Assessing the Severity of Exercise-Induced Pulmonary Hemorrhage in Horses in Field Conditions.

Diagnosis and assessment of severity of exercise-induced pulmonary hemorrhage (EIPH) relies on postexercise visualization of fresh blood in the airways via tracheobronchoscopic examination (TBE) and/or counting erythrocytes in bronchoalveolar lavage fluid (BALFRBC). Determining the BALFRBC is more sensitive than TBE but its usefulness is hampered by the need to have BALFRBC counted at a laboratory. We explored the feasibility of evaluating the severity of EIPH by using a color chart comprised of five shades of red and matching those colors with the color of BALF immediately following collection. To validate the technique, sets of ten BALF samples with known BALFRBC numbers were created and randomly shown to two groups of 18 observers who independently matched the color of the BALF with one of the shades of red displayed on the screen of a smartphone. Interobserver and intra-observer agreements regarding colors were > 0.9. The utility of the color chart was further validated under field conditions at two barrel racing events where 63 BALF samples were collected from 21 horses and BALF color was graded independently by three observers. The number of BALFRBC in the 63 samples ranged from 25-1,100,000/µL. EIPH was diagnosed in 39 samples based on the detection of color, and all 5 colors were matched multiple times with BALF samples. Overall, the color of the BALF was related to the number of BALFRBC. Colorimetric evaluation of BALF represents a practical and reliable option for rapid postexercise assessment of the presence and severity of EIPH.

Effects of exercise, furosemide, blood depletion, and reinfusion on body fluid compartment volumes in horses.

High pulmonary blood pressure contributes to exercise-induced pulmonary hemorrhage. The objective of this study was to use bioimpedance spectroscopy to assess body fluid compartment volumes under 3 conditions in 6 racehorses: i) Pre- and post-supramaximal treadmill exercise (control); ii) Exercise 4 hours after furosemide (0.5 mg/kg body weight, IV); iii) Exercise, removal of ~14 L of blood and subsequent reinfusion of the blood. Statistical analysis used linear mixed effects models. Body compartment volumes did not change during the control runs. Total body water (TBW) (P = 0.007, P = 0.007), extracellular fluid (ECF) (P = 0.003, P = 0.003), and intracellular fluid (ICF) volumes (P = 0.04, P = 0.04) decreased pre- and post-exercise following furosemide administration. The ICF trended to decrease (P = 0.07) after slow removal of blood. Blood reinfusion increased TBW (P = 0.02, P = 0.02) and ICF (P = 0.005, P = 0.005) pre- and post-exercise.

Effets de l'exercice, du furosémide, de la diminution sanguine et de la ré-infusion sur les volumes des liquides corporels compartimentés chez les chevaux. Une pression sanguine pulmonaire élevée contribue à des hémorragies pulmonaires induites par l'exercice. L'objectif de la présente étude était d'utiliser la spectroscopie à bio-impédance pour évaluer les volumes des liquides corporels compartimentés sous trois conditions chez six chevaux de course : i) Pré- et post-supramaximal exercice au tapis roulant (témoin); ii) Exercice 4 h après administration de furosémide (0,5 mg/kg de poids corporel, IV); iii) Exercice, retrait d'environ 14 L de sang et ré-infusion subséquente du sang. Les analyses statistiques utilisaient des modèles linéaires à effets mixtes. Les volumes des compartiments corporels n'ont pas changé durant les essais témoins. Les volumes de la quantité totale d'eau corporelle (TBW) (P = 0,007, P = 0,007), de liquide extracellulaire (ECF) (P = 0,003, P = 0,003) et liquide intracellulaire (ICF) (P = 0,04, P = 0,04) ont diminué pré- et post-exercice à la suite de l'administration de furosémide. L'ICF avait tendance à diminuer (P = 0,07) à la suite du lent retrait de sang. La ré-infusion de sang augmenta la TBW (P = 0,02, P = 0,02) et l'ICF (P = 0,005, P = 0,005) pré- et post-exercice.(Traduit par Dr Serge Messier).

Relationship between tracheobronchoscopic score and bronchoalveolar lavage red blood cell numbers in the diagnosis of exercise-induced pulmonary hemorrhage in horses.

BACKGROUND: Exercise-induced pulmonary hemorrhage (EIPH) is diagnosed and its severity assessed by post-exercise tracheobronchoscopy, and enumeration of bronchoalveolar lavage fluid red blood cells (BALFRBC). Minimal information is available regarding the relationship of tracheobronchoscopy score to BALFRBC number. OBJECTIVE: Evaluate the relationship between BALFRBC number and tracheobronchoscopy scores and determine their diagnostic sensitivities. ANIMALS: Nine sedentary horses, 21 fit Thoroughbreds, 129 Barrel Racers. METHODS: Normal BALFRBC number and the effect of bronchoalveolar lavage (BAL) on it were evaluated by performing 2 BALs 24 hours apart in sedentary horses. Tracheobronchoscopy followed by BAL was performed 247 times on 150 horses after treadmill, racetrack, or barrel racing exercise. Lastly, a BALFRBC diagnostic threshold number that optimized the geometric mean of the sensitivity and precision (F1-score) was determined using Bayesian analysis. RESULTS: No increase in BALFRBC occurred after the second BAL (mean ± SD, 304 ± 173/µL). Tracheobronchoscopy scores ranged from 0 (n = 112) to 4 (n = 4) and BALFRBC ranged from 102 to 4605268/µL. Spearman correlation between tracheobronchoscopy score and BALFRBC was weak (P < .001; rs = 0.42) with large ranges of BALFRBC associated with each tracheobronchoscopy score. The highest F1-score occurred for a BALFRBC threshold number = 992/µL. Seventy-five tracheobronchoscopy scores equaled 0 although BALFRBC number was ≥992/µL. Sensitivity of tracheobronchoscopy for diagnosing EIPH was poor (0.59; 95% confidence intervals [CI], 0.49-0.68), compared to BALFRBC number ≥992/µL (0.93; 95% CI, 0.88-0.96). CONCLUSIONS AND CLINICAL IMPORTANCE: False negatives are common with tracheobronchoscopy. Follow-up determination of BALFRBC may be indicated for tracheobronchoscopy scores = 0 before EIPH can be ruled out.

Assessment of two methods to determine the relative contributions of the aerobic and anaerobic energy systems in racehorses.

A prospective, randomized, controlled study was designed to determine relative aerobic and anaerobic (lactic and alactic) contributions at supramaximal exercise intensities using two different methods. Thoroughbred racehorses (n = 5) performed a maximal rate of oxygen consumption (V̇o2max) test and three supramaximal treadmill runs (105, 115, and 125% V̇o2max). Blood lactate concentration (BL) was measured at rest, every 15 s during runs, and 2, 5, 10, 20, 30, 40, 50, and 60 min postexercise. In method 1, oxygen demand was calculated for each supramaximal intensity based on the V̇o2max test, and relative aerobic and anaerobic contributions were calculated from measured V̇o2 and the accumulated oxygen deficit. In method 2, aerobic contribution was calculated using the trapezoidal method to determine V̇o2 during exercise. A monoexponential model was fitted to the postexercise V̇o2 curve. Alactic contribution was calculated using the coefficients of this model. Lactate anaerobic contribution was calculated by multiplying the peak to resting change in BL by 3. Linear mixed-effects models were used to examine the effects of exercise intensity and method (as fixed effects) on measured outcomes (P ≤ 0.05). Relative aerobic and anaerobic contributions were not different between methods (P = 0.20). Horses' mean contributions were 81.4, 77.6, and 72.5% (aerobic), and 18.5, 22.3, and 27.4% (anaerobic) at 105, 115, and 125% V̇o2max, respectively. Individual alactic anaerobic energy was not different between supramaximal exercise intensities (P = 0.43) and was negligible, contributing a mean of 0.11% of the total energy. Relative energy contributions can be calculated using measured V̇o2 and BL in situations where the exercise intensity is unknown. Understanding relative metabolic demands could help develop tailored training programs. NEW & NOTEWORTHY Relative energy contributions of horses can be calculated using measured V̇o2 and BL in situations where the exercise intensity is unknown. Horses' mean contributions were 81.4, 77.6, and 72.5% (aerobic), and 18.5, 22.3, and 27.4% (anaerobic) at 105, 115, and 125% of V̇o2max, respectively. Individual alactic capacity was unaltered between supramaximal exercise intensities and accounted for a mean contribution of 0.11% of energy use.

Exercise-induced pulmonary hemorrhage in barrel racing horses in the Pacific Northwest region of the United States.

BACKGROUND: Exercise-induced pulmonary hemorrhage (EIPH) refers to bleeding from the lungs in association with strenuous exercise. It has been documented in race horses but little information exists on EIPH in barrel racing horses. HYPOTHESIS/OBJECTIVES: Our goals were to evaluate the presence of EIPH in barrel racing horses and estimate its prevalence in the Pacific Northwest. ANIMALS: 149 barrel racing horses enrolled at events in WA (11), ID (3), and MT (33). METHODS: Observational cross-sectional study. Data collected included signalment, history of illness, respiratory disease, race division, and pre-race medications. Endoscopy was performed and tracheobronchoscopic (TBE) EIPH score was assigned based on quantity of blood in the trachea (0 = no blood to 4 = abundance of blood within the trachea). After TBE, bronchoalveolar lavage (BAL) was performed. Erythrocyte (red blood cell, RBC) counts were obtained from bronchoalveolar lavage fluid (BALF). Statistical analysis included linear and logistic regression, Fisher's exact t test, and calculation of correlation coefficient. Significance was set at P < .05. RESULTS: The prevalence of EIPH based on TBE EIPH score was 54%. When based on BALF RBC count >1,000 cells, EIPH prevalence was 66%. Race time did not significantly affect the presence of EIPH. A significant (P < .0001) positive linear relationship between the TBE and BAL erythrocyte count was identified, but its strength was poor (r2 = .15). CONCLUSIONS AND CLINICAL IMPORTANCE: EIPH occurs in over 50% of barrel racing horses in the Pacific Northwest. Precise determination of the impact of EIPH on health of barrel racers requires further study.

Comparison of Fick and thermodilution cardiac output determinations in standing horses.

The Fick and thermodilution (TD) methods are two currently popular techniques for determination of cardiac output (CO) in adult horses. To our knowledge, a comparison of these two techniques has not been reported. Six healthy, resting, fit, adult horses of either sex and weighing 516.5+/-33.2 kg (mean+/-SD) were instrumented to enable measurement of cardiac output. Resting CO was determined by the Fick method and by thermodilution while the horses stood quietly in the stocks. Fick and thermodilution CO measurements were repeated under conditions of increased cardiac output achieved with the use of a dobutamine infusion (5 microg kg(-1) min(-1), IV), and again under conditions of decreased CO induced by administration of xylazine (0.5 mg/kg, IV). Fick and thermodilution cardiac outputs were compared using Bland-Altman analysis for repeated measures. The mean of the differences+/-1.96SD (bias and precision) between the two techniques was 1.88+/-24.17 L/min. Variability between measurements with the two techniques was decreased to 3.41+/-46.78 mL kg(-1) min(-1) when CO was normalized for body size by calculation of cardiac index.

Effects of a polymerized ultrapurified bovine hemoglobin blood substitute administered to ponies with normovolemic anemia.

The development of ultrapurified hemoglobin-based oxygen carriers has eliminated many problems associated with whole-blood transfusions in other species. We hypothesized that the administration of polymerized ultrapurified bovine hemoglobin (PUBH) would result in improved hemodynamic parameters in ponies with normovolemic anemia without adverse effects on renal function or coagulation times. Normovolemic anemia was induced in 6 healthy adult ponies. Over a 3-day period, at least 45 mL/kg of whole blood was withdrawn from each pony until a target PCV of <12% was attained. Plasma was separated from the red blood cells via centrifugation and readministered to the ponies on each day. After the final plasma transfusion, 15 mL/kg of hetastarch (control, n = 6) or 15 mL/kg of PUBH (treatment, n = 6) was administered at 10 mL/kg/h IV. Administration of PUBH at a rate of 10 mL/kg/h was not associated with any adverse effects in 5 of the 6 ponies. One pony experienced an anaphylactoid reaction during infusion of PUBH. The reaction, characterized by intense pruritus, tachycardia, and tachypnea resolved shortly after stopping the infusion. Ponies receiving PUBH had significantly lower cardiac indices (P = .03) and heart rates (P = .002) than control animals. A significantly greater increase in central venous pressure was observed in the PUBH group compared to the hetastarch group (P = .02). No adverse renal or coagulation effects were observed with PUBH infusion. These results suggest that PUBH improves hemodynamics and oxygen transport parameters in horses experiencing normovolemic anemia. Patients should be monitored closely during infusion for any adverse reactions.

Effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation.

OBJECTIVE: To investigate the effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation. SAMPLE POPULATION: Blood samples from 6 Thoroughbreds. PROCEDURE: The degree of fluorescence associated with binding of fluorescein isothiocyanate (FITC)-conjugated anti-human fibrinogen antibody and FITC-annexin V in unactivated and adenosine diphosphate (ADP)-, platelet activating factor (PAF)-, and A23187-activated platelet samples in unfixed and 0.5, 1.0, and 2.0% formaldehyde-fixed samples was assessed by use of flow cytometry. RESULTS: In samples incubated with FITC-anti-human fibrinogen antibody prior to fixation, addition of 2.0% formaldehyde resulted in a 30% increase in total fluorescence in ADP- and PAF-activated samples and a 60% increase in A23187-activated samples. Fixation for 24 hours prior to addition of antibody resulted in reduced fluorescence of samples containing antihuman fibrinogen antibody for all 3 concentrations of formaldehyde in PAF-activated samples. The addition of all 3 concentrations of formaldehyde after incubation with FITC-annexin V resulted in significant increases in fluorescence in unactivated and activated platelet samples. As length of fixation time increased, there was a gradual increase in fluorescence that was significant at 24 hours. CONCLUSIONS: Because fixation with 2.0% formaldehyde results in significant changes in fluorescence in activated platelet samples containing anti-fibrinogen antibody, lower concentrations of formaldehyde should be used to fix equine platelet samples. Formaldehyde-fixed platelet samples should be analyzed within 12 hours of fixation to avoid artifactual increases in fluorescence. Fixation of samples containing FITC-annexin V should be avoided because of significant increases in fluorescence that may interfere with interpretation of results.

Measurement of the activation of equine platelets by use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody.

OBJECTIVE: To investigate the potential use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody for detection of the activation of equine platelets by use of flow cytometry. SAMPLE POPULATION: Platelets obtained from 6 Thoroughbreds. PROCEDURE: Flow cytometry was used to assess platelet activation as indicated by detection of binding of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-thrombospondin antibody to unactivated and ADP-, collagen-, platelet activating factor (PAF)-, and A23187-activated equine platelets. Human platelets were used as control samples. Determination of 14C-serotonin uptake and release was used to assess the extent of platelet secretion. RESULTS: Anti-human thrombospondin antibody failed to bind to equine platelets. Annexin V bound to platelets activated with PAF or A23187 when platelets had undergone secretion. Anti-human fibrinogen antibody bound to ADP-, PAF-, and A23817-activated platelets, but binding was not dependent on platelet secretion. The extent of binding of anti-fibrinogen antibody was less in equine platelets, compared with that for human platelets, despite maximal stimulation. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of equine platelets can be detected by use of fluorescent-labeled annexin V and anti-human fibrinogen antibody but not by use of anti-human thrombospondin antibody. These flow cytometric techniques have the potential for detection of in vivo platelet activation in horses at risk of developing thrombotic disorders.