Kazutaka Mukai

Effect of training and detraining on monocarboxylate transporter (MCT) 1 and MCT4 in Thoroughbred horses

The aim of this study was to investigate the effects of training and detraining on the monocarboxylate transporter (MCT) 1 and MCT4 levels in the gluteus medius muscle of Thoroughbred horses. Twelve Thoroughbred horses were used for the analysis. For 18 weeks, all the horses underwent high‐intensity training (HIT), with running at 90–110% maximal oxygen consumption ( ) for 3 min, 5 days week−1. Thereafter, the horses either underwent detraining for 6 weeks by either 3 min of moderate‐intensity training (MIT) at 70% , 5 days week−1 (HIT‐MIT group) or stall rest (HIT‐SR group). The horses underwent an incremental exercise test, was measured and resting muscle samples were obtained from the middle gluteus muscle at 0, 18 and 24 weeks. The content of MCT1 and MCT4 proteins increased after 18 weeks of HIT. At the end of this period, an increase was noted in the citrate synthase activity, while phosphofructokinase activity remained unchanged. After 6 weeks of detraining, all these indexes returned to the pretraining levels in the HIT‐SR group. However, in the HIT‐MIT group, the increase in the MCT1 protein content and citrate synthase activity was maintained after 6 weeks of MIT, while the MCT4 protein content decreased to the pretraining value. These results suggest that the content of MCT1 and MCT4 proteins increases after HIT in Thoroughbred horses. In addition, the increase in the MCT1 protein content and oxidative capacity induced by HIT can be maintained by MIT of 70% , but the increase in the MCT4 protein content cannot be maintained by MIT.

Effect of acute exercise on monocarboxylate transporters 1 and 4 in untrained and trained Thoroughbreds

OBJECTIVE To evaluate the effects of a single incremental exercise test (IET) on mRNA expression and protein content of monocarboxylate transporter (MCT) 1 and MCT4 in the gluteus medius muscle of Thoroughbreds. ANIMALS 12 Thoroughbreds (6 males and 6 females; age, 3 to 4 years). PROCEDURES Horses underwent an IET before and after 18 weeks of high-intensity exercise training (HIT). Horses were exercised at 90% of maximal oxygen consumption for 3 minutes during the initial 10 weeks of HIT and 110% of maximal oxygen consumption for 3 minutes during the last 8 weeks of HIT. Gluteus medius muscle biopsy specimens were obtained from horses before (baseline), immediately after, and at 3, 6, and 24 hours after the IET. RESULTS Expression of MCT1 and MCT4 mRNA was upregulated at 3 and 6 hours after the IET in muscle specimens obtained from horses prior to HIT (untrained horses) and at 6 hours after the IET in muscle specimens obtained from horses after HIT (trained horses). For both untrained and trained horses, MCT1 and MCT4 protein contents were increased at 6 hours after the IET and did not differ at 24 hours after the IET, compared with those at baseline. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that a single IET resulted in transient increases in MCT1 and MCT4 mRNA expression and protein content in untrained and trained horses. These results may be important for the elucidation of exercise-induced alterations in lactate metabolism.

Effects of three warm‐up regimens of equal distance on V̇o2 kinetics during supramaximal exercise in Thoroughbred horses

REASONS FOR PERFORMING STUDY Several studies have indicated that even low-intensity warm-up increases O(2) transport kinetics and that high-intensity warm-up may not be needed in horses. However, conventional warm-up exercise for Thoroughbred races is more intense than those utilised in previous studies of equine warm-up responses. OBJECTIVES To test the hypothesis that warm-up exercise at different intensities alters the kinetics and total contribution of aerobic power to total metabolic power in subsequent supramaximal (sprint) exercise in Thoroughbred horses. METHODS Nine well-trained Thoroughbreds ran until fatigue at 115% of maximal oxygen consumption (VO2max) 10 min after warming-up under each of 3 protocols of equal running distance: 400 s at 30% VO2max (LoWU), 200 s at 60% VO2max (MoWU) and 120 s at 100% VO2max (HiWU). Variables measured during exercise were rates of O(2) and CO(2) consumption/production (VO2,VO2), respiratory exchange ratio (RER), heart rate, blood lactate concentration and accumulation rate and blood gas variables. RESULTS VO2 was significantly higher in HiWU than in LoWU at the onset of the sprint exercise and HR was significantly higher in HiWU than in LoWU throughout the sprint. Accumulation of blood lactate, RER, P(a)CO(2) and PvCO2 in the first 60 s were significantly lower in HiWU than in LoWU and MoWU. There were no significant differences in stroke volume, run time or arterial-mixed venous O(2) concentration. CONCLUSIONS These results suggest HiWU accelerates kinetics and reduces reliance on net anaerobic power compared with LoWU at the onset of the subsequent sprint.

Comparison of net anaerobic energy utilisation estimated by plasma lactate accumulation rate and accumulated oxygen deficit in Thoroughbred horses

REASONS FOR PERFORMING STUDY Accumulated O(2) deficit (AOD) and plasma lactate accumulation rate (PLAR) are alternative methods for estimating net anaerobic energy utilisation (NAEU) in exercising horses. How they compare or their accuracy is unknown. OBJECTIVES We hypothesised net anaerobic energy utilisation calculated by PLAR (NAUE(PLAR)) is equivalent to NAUE estimated by AOD (NAUE(AOD)). METHODS Six Thoroughbred horses ran at identical supramaximal speeds (118% aerobic capacity) until exhaustion for 2 runs while breathing normoxic (NO, 21% O(2)) or hyperoxic (HO, 26% O(2)) gas. Jugular blood was sampled at 15 s intervals to measure plasma lactate concentration. Horses also ran at incremental submaximal speeds from 1.7-11.0 m/s to determine the linear relationship between speed and O(2) consumption to estimate O(2) demand for AOD calculations. RESULTS Maximum O(2) consumption of horses increased 11.6 ± 2.3% in HO and NAEU(PLAR) and NAUE(AOD) decreased 38.5 ± 8.0% and 46.2 ± 17.7%, respectively. The NAEU(PLAR) in NO was 114.5 ± 27.4 mlO(2) (STPD) equivalent/kg bwt contributing 23.5 ± 3.7% to total energy turnover and in HO was 70.9 ± 19.8 mlO(2) (STPD) equivalent/kg bwt contributing 14.6 ± 3.8% to total energy turnover. The NAUE(AOD) in NO was 88.6 ± 24.3 mlO(2) (STPD) equivalent/kg bwt contributing 19.9 ± 2.1% to total energy turnover and in HO was 56.2 ± 19.1 mlO(2) (STPD) equivalent/kg bwt contributing 10.9 ± 4.3% to total energy turnover. Overall, NAEU(AOD) was systematically biased -23.5 ± 16.8 mlO(2) (STPD) equivalent/kg bwt below NAEU(PLAR). Total energy demand estimated by PLAR was 11.1 ± 5.4% greater than that estimated by AOD and was higher in every horse. CONCLUSIONS The NAUE(PLAR) estimates average 40.0 ± 29.6% higher than NAUE(AOD) and are highly correlated (r(2) = 0.734), indicating both indices are sensitive to similar changes in NAEU. Accuracy of the estimates remains to be determined. Multiple considerations suggest NAUE(AOD) may underestimate total energy cost during high-speed galloping, thus biasing low the AOD estimate of NAEU.

Effect of growth on monocarboxylate transporters and indicators of energy metabolism in the gluteus medius muscle of Thoroughbreds

OBJECTIVE To examine the changes in monocarboxylate transporter (MCT) 1 and MCT4 content and in indicators of energy metabolism in the gluteus medius muscle (GMM) of Thoroughbreds during growth. ANIMALS 6 Thoroughbreds (3 males and 3 females). PROCEDURES Samples of GMM were obtained when horses were 2, 6, 12, and 24 months old. Muscle proteins were separated via SDS-PAGE; amounts of MCT1 and MCT4 and peroxisome proliferator-activated receptor-γ coactivator-1α content were determined by use of western blotting. Muscle activities of phosphofructokinase and citrate synthase were measured biochemically; lactate dehydrogenase isoenzymes were separated by agarose gel electrophoresis and quantified. RESULTS Compared with findings when horses were 2 months old, MCT1 protein content in GMM samples obtained when the horses were 24 months old was significantly higher; however, MCT4 protein content remained unchanged throughout the study period. Peroxisome proliferator-activated receptor-γ coactivator-1α content was significantly increased at 24 months of age and citrate synthase activity was increased at 6 and 24 months of age, compared with findings at 2 months. Phosphofructokinase activity remained unaltered during growth. The percentage contributions of lactate dehydrogenase 1 and 2 isoenzymes to the total amount of all 5 isoenzymes at 12 and 24 months of age were significantly higher than those at 2 months of age. CONCLUSIONS AND CLINICAL RELEVANCE Changes in protein contents of MCTs and the lactate dehydrogenase isoenzyme profile in GMM samples suggested that lactate usage capacity increases with growth and is accompanied by an increase in the oxidative capacity in Thoroughbreds.

Effects of high-intensity training on lipid metabolism in Thoroughbreds

OBJECTIVE To investigate the effects of high-intensity training (HIT) on carbohydrate and fat metabolism in Thoroughbreds. ANIMALS 12 Thoroughbreds (3 to 4 years old; 6 males and 6 females). PROCEDURES Horses performed HIT for 18 weeks. They ran at 90% or 110% of maximal oxygen consumption ((V)O(2max)) for 3 minutes (5 d/wk) and were subjected to incremental exercise testing (IET) before and after training. Blood samples were collected during IET, and muscle samples were obtained from the gluteus medius muscle immediately after IET. Phosphofructokinase, citrate synthase, and β-3-hydroxyacyl CoA dehydrogenase (β-HAD) activities were measured to determine glycolytic and oxidative capacities. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and fatty acid translocase (FAT/CD36) protein contents were detected via western blotting. Metabolome analysis was performed via capillary electrophoresis-electrospray ionization mass spectrometry to measure substrate concentrations related to carbohydrate metabolism. RESULTS Peak speed during IET and (V)O(2max) increased after HIT. Activities of citrate synthase and β-HAD increased after HIT, whereas phosphofructokinase activity remained unchanged. The PGC-1α and FAT/CD36 protein contents increased after HIT, but plasma lactate concentration and the respiratory exchange ratio decreased after HIT. The plasma free fatty acid concentration increased after HIT, whereas the glucose concentration was not altered. Fructose 1,6-diphosphate, phosphoenolpyruvate, and pyruvate concentrations decreased after HIT. CONCLUSIONS AND CLINICAL RELEVANCE HIT caused an increase in oxidative capacity in equine muscle, which suggested that there was a decreased reliance on carbohydrate utilization and a concomitant shift toward fatty acid utilization during intensive exercise.

Effects of warm-up intensity on oxygen transport during supramaximal exercise in horses

OBJECTIVE To determine whether warm-up exercise at different intensities alters kinetics and total contribution of aerobic power to total metabolic power in subsequent supramaximal exercise in horses. ANIMALS 11 horses. PROCEDURES Horses ran at a sprint until fatigued at 115% of maximal oxygen consumption rate (VO(2max)), beginning at 10 minutes following each of 3 warm-up protocols: no warmup (NoWU), 1 minute at 70% VO(2max) (moderate-intensity warm-up [MoWU]), or 1 minute at 115% VO(2max) (high-intensity warm-up [HiWU]). Cardiopulmonary and blood gas variables were measured during exercise. RESULTS The VO(2) was significantly higher in HiWU and MoWU than in NoWU throughout the sprint exercise period. Blood lactate accumulation rate in the first 60 seconds was significantly lower in MoWU and HiWU than in NoWU. Specific cardiac output after 60 seconds of sprint exercise was not significantly different among the 3 protocols; however, the arterial mixed-venous oxygen concentration difference was significantly higher in HiWU than in NoWU primarily because of decreased mixed-venous saturation and tension. Run time to fatigue following MoWU was significantly greater than that with NoWU, and there was no difference in time to fatigue between MoWU and HiWU. CONCLUSIONS AND CLINICAL RELEVANCE HiWU and MoWU increased peak values for VO(2) and decreased blood lactate accumulation rate during the first minute of intense exercise, suggesting a greater use of aerobic than net anaerobic power during this period.

Use of an implantable transducer to measure force in the superficial digital flexor tendon in horses at walk, trot and canter on a treadmill

REASONS FOR PERFORMING STUDY Although the main cause of injury to the superficial digital flexor tendon (SDFT) is assumed to be high intensity loading of the tendon, to date the forces exerted on the SDFT during cantering have never been measured. OBJECTIVE To measure the force exerted on the SDFT at walk, trot and canter on a treadmill. METHODS Arthroscopically implantable force probes (AIFP) were implanted in the SDFT of the left and right forelimbs of 8 Thoroughbred horses (480-565 kg). The output of the AIFP was calibrated using the SDFT force calculated by inverse dynamics and an in vitro model of the lower forelimb at trot. The AIFP output was recorded at 1000 Hz at the walk, trot and canter (9 m/s) on a flat treadmill. RESULTS AIFP data were analysed successfully in 13 measurement sessions at the walk and trot, in the leading forelimb in 8 sessions at canter and in the trailing forelimb in 5 sessions at canter. The mean ± s.d. maximal force in the SDFT was 3110 ± 1787 N at the walk, 5652 ± 2472 N at the trot, 7030 ± 2948 N in the leading forelimb and 6453 ± 2940 N in the trailing forelimb at canter. CONCLUSIONS The force in the SDFT increases with running speed from the walk to the canter. The force in the SDFT could not be measured at the gallop. Further study is needed to determine the force in the SDFT at high speed, because it is important for preventing injuries to the SDFT to limit overloading of this tendon.

The Effects of Inclination (Up and Down) of the Treadmill on the Electromyogram Activities of the Forelimb and Hind limb Muscles at a Walk and a Trot in Thoroughbred Horses

ABSTRACT It is important to know the effects of the inclination of a slope on the activity of each muscle, because training by running on a sloped track is commonly used for Thoroughbred racehorses. The effects of incline (from −6 to +6%) on the forelimbs and hind limbs during walking and trotting on a treadmill were evaluated by an integrated electromyogram (iEMG). The muscle activities in the forelimbs (5 horses) and hind limbs (4 horses) were measured separately. Two stainless steel wires were inserted into each of the brachiocephalicus (Bc), biceps brachii (BB), splenius (Sp), and pectoralis descendens (PD) in the forelimb experiment and into the longissimus dorsi (LD), vastus lateralis (VL), gluteus medius (GM), and biceps femoris (BF) in the hind limb experiment. The EMG recordings were taken at a sampling rate of 1,000 Hz. At a walk, the iEMG values for the forelimb were not significantly different under any of the inclinations. In the hind limb, the iEMG values for the GM and BF significantly decreased as the inclination decreased. At a trot, the iEMG values for the Bc in the forelimb significantly decreased as the inclination of the treadmill decreased. In the hind limb, the iEMG values for the LD, GM, and BF significantly decreased as the inclination decreased. Uphill exercise increased the iEMG values for the Bc, LD, GM, and BF, while downhill exercise resulted in little increase in the iEMG values. It was concluded that the effects of inclination on the muscle activities were larger for the uphill exercises, and for the hind limb muscles compared with the forelimb muscles.

Effect of High-Intensity Training in Normobaric Hypoxia on Thoroughbred Skeletal Muscle

Hypoxic training is believed to increase endurance capacity in association with hypoxia inducible factor-1α (HIF-1α), a modulator of vascular endothelial growth factor-A (VEGF-A), and to influence activation of satellite cells (SCs). However, the effect of hypoxic training on SC activation and its relation to angiogenesis has not been thoroughly investigated. Eight Thoroughbred horses were subjected to normoxic (FIO2 = 21%) or hypoxic (FIO2 = 15%) training for 3 days/week (100%  V˙O2max) for 4 weeks. Incremental exercise tests (IET) were conducted on a treadmill under normoxia and the maximal oxygen consumption (V˙O2max) and running distance were measured before and after each training session. Muscle biopsy samples were obtained from the gluteus medius muscle at 6 scheduled times before, during, and one week after IET for immunohistochemical analysis and real-time RT-PCR analysis. Running distance and V˙O2max, measured during IET, increased significantly after hypoxic training compared with normoxic training. Capillary density and mRNA expression related to SC activation (e.g., myogenin and hepatocyte growth factor) and angiogenesis (VEGF-A) increased only after hypoxic training. These results suggest that increases in mRNA expression after training enhance and prolong SC activation and angiogenesis and that nitric oxide plays an important role in these hypoxia-induced training effects.