In a previous study1 of highly trained Alaskan sled dogs that ran a distance of 160 km on each of 5 consecutive days, there was no cumulative depletion of skeletal MG during the repeated periods of prolonged submaximal exercise. This occurred despite substantial MG metabolism during the initial exercise period and consumption of a diet that provided an estimated 15% of daily digestible energy intake from carbohydrates.1 It was suggested that compensatory adjustments prompted by the initial period of prolonged exercise resulted in preservation of MG during subsequent periods of exercise. However, it was not determined whether preservation of MG resulted from a more rapid rate of glycogen replenishment during the brief recovery periods or from a progressive decline in glycogen use during continued exercise.
Results of other studies2,3 of rodents undergoing repeated episodes of high-intensity exercise indicated that rapid replenishment of MG during transient recovery periods prevented cumulative depletion of MG. In contrast, in human athletes who consumed a mixed or high-carbohydrate diet, consecutive days of prolonged running exercise at 80% 
O2max resulted in cumulative MG depletion.4,5 This finding might reflect dependence on MG as the prime energy substrate during exercise because of the diet composition and the high-intensity nature of the exercise challenge. In runners who consumed a diet that did not meet their predicted carbohydrate expenditure, glycogen depletion decreased on successive days of running; after several days of exercise, glycogen replenishment was 26% greater in these athletes, compared with runners who consumed a diet designed to meet their carbohydrate expenditure.5 Repeated episodes of exercise reportedly impede MG supercompensation in humans who consume a highcarbohydrate diet and have substantial impacts on energy substrate metabolism and cytokine responses during exercise.6–10 The high-fat, low-carbohydrate diet that is typically consumed by sled dogs might therefore have an important impact on muscle substrate metabolism and regulatory responses to repeated exercise. Additionally, sled dogs perform multiday exercise at lower intensities (conditions that induce approx 40% O2max). However, to our knowledge, reports of studies of glycogen depletion and repletion in any species undergoing repeated episodes of prolonged nonresistance exercise at intensities that approximate 40% Vo2max do not exist.
In dogs, free fatty acids in plasma reportedly supply 70% to 90% of energy demand during prolonged submaximal exercise, with plasma glucose contributing a further 10% to 15% to the energy demand.11 Increases in BUN concentration suggest that protein substrates may also be involved in meeting energy requirements in exercising sled dogs.12 Additionally, MT has been reported to represent a critical energy substrate in exercising dogs.13 Therefore, it is possible that dogs that are fed a high-fat diet and undergo prolonged submaximal exercise might preserve MG stores through attenuation of glycogen use during repeated episodes of exercise. However, an increased rate of MG replenishment during periods of rest and food intake might also contribute to maintenance of MG concentrations.
The objective of the study of this report was to determine the pattern of depletion and replenishment of MG and MT in aerobically conditioned sled dogs during repeated periods of running (over a cumulative distance of 560 km) and during the first 96 hours of the recovery period. We hypothesized that highly trained Alaskan sled dogs would maintain MG concentrations during repeated episodes of prolonged submaximal exercise, and that this MG preservation would be attributable to decreased use of MG during exercise and compensatory use of MT among several potential alternative substrates.
Materials and Methods
Dogs—Fifty-four healthy Alaskan sled dogs from 1 kennel that were in training for multiday endurance racing were used in the study. Dogs ranged in age from 2 to 8.5 years; there were 33 sexually intact males and 21 sexually intact females. Mean body weight was 23.4 ± 2.7 kg. Dogs were selected by 2 mushers on the basis of their degree of fitness and the mushers' assessment of the dog's ability to successfully complete the distances required. All procedures were approved by the Oklahoma State University Institutional Animal Care and Use Committee according to the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals.
Prior to study commencement, 6 of the 54 dogs were randomly selected to participate as nonexercising control animals (4 males and 2 females; mean weight, 23.9 ± 3.5 kg). Until the time of study commencement, control dogs and the dogs that were selected to participate in the exercise program were trained and fed in an identical manner. During a 5-month prestudy training period, all dogs consumed 3 meals/d; each week, training typically occurred on 4 days. The distance of the training runs progressively increased to a maximum of 80 km in a single run by the end of the training period. All dogs were rested from training for 9 days prior to the start of the study because of extremely cold temperatures that precluded safe training.
Experimental design—The experimental design was planned to include 48 dogs that repeatedly ran distances of 70 km to a remote location on 4 consecutive days, returning to the kennel each day after completion of 140 km. Dogs were randomly divided into 3 teams of 16 animals; teams commenced running at 30- to 60-minute intervals. The dogs pulled a lightly laden sled and a musher for a distance of 70 km across snow in approximately 5.5 hours, which was estimated to correspond to an exercise intensity of 30% to 40% of O2max.14 Dogs rested for 6 to 8 hours between each 70-km run, during which time they consumed 2 meals (at the start and end of the rest period). Thus, the cumulative distances completed by the dogs within 24, 48, 72, and 96 hours were 140, 280, 420, and 560 km, respectively. After the final episode of exercise, dogs were allowed to rest for as long as 96 hours.
The 6 control dogs did not participate in any of the periods of exercise bouts; prior to commencement of exercise (0 hours) by the other dogs, muscle biopsy specimens were collected from the control dogs. Muscle biopsy specimens were obtained from 1 hind limb of 6 randomly selected dogs as soon as possible after exercise ceased at cumulative distances of 140, 280, 420, and 560 km and from the other hind limb of the same dogs 6 to 8 hours later. All dogs undergoing biopsy procedures were removed from the exercise program, and teams were reorganized to maintain a constant number (≥ 12) of dogs in a team. The timing of the biopsy procedures was intended to correspond closely to the start and finish of the rest period provided after each 140-km run. Meal ingestion by dogs undergoing biopsy and those continuing in the exercise program was matched as closely as possible. Thus, dogs undergoing biopsy were allowed to consume a full meal as soon as they recovered from anesthesia following the first procedure; however, dogs were not offered a meal prior to the second procedure to minimize the development of complications during or after anesthesia. In dogs that completed the entire 560-km distance, a biopsy specimen from a single limb was obtained at 28, 50, and 96 hours of recovery (samples were collected from 6 dogs at each time point, allowing for possible premature attrition of 6 dogs).
The experimental design was altered during the study to accommodate the premature withdrawal of 10 dogs as a result of musculoskeletal problems and the refusal of a single team of dogs to continue exercise beyond a distance of 510 km. To accommodate these events, no dogs underwent muscle biopsy and removal from the study at 280 km; the 13 dogs in the team that ceased exercise prematurely were randomly allocated to 1 of 2 groups that underwent muscle biopsy from a single limb at 50 or 96 hours of recovery.
Muscle biopsy procedure—For muscle biopsy, each dog was anesthetized with propofol (4 mg/kg) administered via an IV catheter that was placed in a cephalic vein. The dog was positioned in lateral recumbency; the hair over a small area in the central region of the biceps femoris muscle (approx midfemur location) was clipped, and the skin was aseptically prepared. A stab incision was made through the skin and fascia of the biceps femoris muscle by use of a No. 10 scalpel blade. Two to 3 muscle samples were then obtained from the center of the biceps femoris muscle (approx 1.0 to 1.5 cm from the skin surface) by use of a 12-gauge, 1-handed biopsy needle.a Muscle tissue samples were gently blotted and then immediately weighed on a microbalance to ensure that a minimum of 60 mg of muscle tissue was obtained from each dog. Fresh samples were divided approximately in half before being frozen in liquid nitrogen and stored at −80°C for later analysis of MG and MT concentrations. None of the dogs undergoing a biopsy procedure participated in any further exercise during the study.
Diet formulation—During the 5-month prestudy training period, all dogs consumed 3 meals/d. During the study, exercising dogs consumed 4 meals/d, which included a meal given at the start and finish of each 70-km run. Dogs that were recovering from a muscle biopsy procedure and dogs that were continuing to exercise were fed in an identical manner. A so-called phantom meal (an uneaten meal retained for analysis) was collected at each feeding time within the 3-day period prior to the study, throughout the study, and for 72 hours after the final run was completed. Proximate analysis of phantom meal samples was performed by a commercial laboratoryb; carbohydrate content of the food was assumed to approximate the nitrogen-free extract and was calculated from the analysis results as 100% minus the percentages of moisture, crude protein, crude fat, crude fiber, and ash.15 Meals consisted of variable combinations of beef, liver, tripe, supplemental fat, water, and commercial dried dog food.c
Muscle sample analysis—Muscle samples were freeze-dried and dissected free of blood, fat, and connective tissue by use of a stereomicroscope. Muscle glycogen and MT concentrations (mmol/kgDW) were determined via previously described methods.16,17
Statistical analysis—The data were analyzed by use of ANOVA techniques with commercial statistical software.d To assess muscle substrate use, the differences in MG and MT concentrations before and after completion of the first 140 km were compared with the corresponding differences in those variables before and after the completion of the final 140 km (ie, cumulative distance of 420 to 560 km) via an ANOVA with a priori testing of the hypothesis that the differences would be equal. Replenishments of MG and MT (calculated as mmol/kgDW/h) were compared among the 3 groups of dogs from which paired biopsy samples were obtained after completion of 140, 480, or 560 km. Significance was set at a value of P < 0.05. Results are reported as mean ± SD.
Results
Thirty-eight of the 48 dogs that were initially included in the study ran a distance that allowed them to be used in the investigative procedures. The remaining 10 dogs were removed prematurely from teams as a result of various musculoskeletal problems that developed during running. Data were not collected from those dogs. The mean ± SD pace of the teams during runs was 12.6 ± 1.4 km/h (range, 11.1 to 15.4 km/h); the mean rest period between 70-km runs was 6.7 ± 0.097 hours.
Diet analysis—During the 3 days prior to commencement of the study, the estimated consumption of all dogs was 3,300 cal/24 h (energy content as follows: protein, 27%; fat, 39%; and carbohydrate, 34%) divided among 3 meals each day. During the 4-day exercise period, the calorie consumption of running dogs increased to approximately 5,000 cal/24 h (energy content as follows: protein, 27.7 ± 2.8%; fat, 38.8 ± 2.6%; and carbohydrate, 33.5 ± 1.7%) divided among 4 meals each day.
After recovery from anesthesia, dogs that underwent muscle biopsy immediately after exercise consumed an estimated 1,500 cal (energy content as follows: protein, 30.2 ± 3.3%; fat, 43.6 ± 6.5%; and carbohydrate, 26.2 ± 5.4%) at 0.7 ± 0.03 hours after ceasing exercise. The exercising dogs that did not undergo muscle biopsy consumed an identical meal within 20 minutes of exercise cessation.
During the first 24 hours of recovery after completion of the exercise phase of the study, dogs consumed approximately 4,700 calories (energy content as follows: protein, 31%; fat, 34%; and carbohydrate, 35%) divided among 4 meals. During the 2 successive 24-hour periods of recovery, dogs consumed an estimated 3,300 cal/24 h (energy content as follows: protein, 28%; fat, 39%; and carbohydrate, 33%) divided among 3 meals/d.
Muscle biopsy procedure—In dogs selected for paired muscle biopsy procedures after running 140, 420, or 560 km, muscle tissue samples were first obtained 0.4 ± 0.17 hours after exercise cessation. The biopsy of the other hind limb of the same dogs was performed 7.2 ± 0.13 hours after exercise cessation.
Thirteen dogs successfully completed the cumulative distance of 560 km and were randomly assigned to 1 of 2 groups; 6 dogs underwent the described paired biopsy procedures immediately after exercise, and 7 dogs underwent a single muscle biopsy procedure at 28.2 ± 0.35 hours after exercise ceased. A single team comprised of 13 dogs refused to continue exercise beyond 510 km. Dogs in that team were randomly allocated to 1 of 2 groups; a single muscle biopsy procedure was performed in 7 dogs at 50.2 ± 0.4 hours after exercise cessation and in 6 dogs at 98.08 ± 0.28 hours after exercise cessation. As a result of the loss of 3 samples during transport, MG quantification was performed on biopsy samples collected from 6 dogs at 28 and 50 hours of the recovery period and from 5 dogs at 98 hours of the recovery period. Including procedures in the control dogs, 62 biopsy procedures were performed; the mean muscle tissue sample weight obtained from each dog was 74 ± 10 mg (MG concentration was measured in 59 samples, and MT concentration was measured in 62 samples). No adverse consequences associated with anesthesia or the biopsy procedures were evident in any dog.
MG concentrations—The mean MG concentration in control dogs at 0 hours exceeded 300 mmol/kgDW (Figure 1). Compared with the control value, the concentration of MG in tissue samples collected immediately after exercise was significantly decreased in dogs that ran 140 km; this value was not different from the concentration in dogs that ran 420 km. The concentration of MG in tissue samples collected immediately after exercise in dogs that ran 560 km was significantly greater than the values in dogs that ran the lesser distances and was not significantly different from the MG concentration in control dogs. After rest and consumption of a meal in dogs undergoing paired biopsy procedures after completing distances of 140, 420, and 560 km, the MG concentrations were 233 ± 26 mmol/kgDW, 314 ± 36 mmol/kgDW, and 378 ± 66 mmol/kgDW, respectively. Assuming that MG concentrations in dogs that underwent biopsy procedures were representative of the remaining study dogs, a significantly (P < 0.001) greater amount of MG was depleted during the initial 140-km run (approx 238 mmol/kgDW) of the exercise challenge, compared with that depleted during final 140-km run (16 mmol/kgDW). The MG concentration increased to 122% of the resting control value at 28 hours of the recovery period and remained similarly increased at 50 and 98 hours of the recovery period (although the increase was only significantly greater than control value at 50 hours of the recovery period). The calculated rate of glycogen replenishment during the 3 approximately 7-hour rest periods (between 140-km runs) did not differ significantly among dogs regardless of distance run (140 km completed, 13.5 ± 6.7 mmol/kgDW/h; 420 km completed, 15.5 ± 5.0 mmol/kgDW/h; and 560 km completed, 11.0 ± 4.6 mmol/kgDW/h).
Mean ± SD MG concentrations in aerobically conditioned Alaskan sled dogs assessed in muscle biopsy specimens collected before (at 0 hours from 6 unexercised control dogs) and immediately after running cumulative distances of 140, 420, and 560 km (within 24, 72, and 96 hours; 6 dogs at each time point) and at 28, 50, and 98 hours of recovery (at 118 [n = 6 dogs], 140 [6], and 188 [5] hours after study commencement). Solid double-headed horizontal arrows indicate periods of exercise (70-km runs); final double-headed horizontal arrow indicates the recovery period after the exercise portion of the study. a–dValues with different letters are significantly (P < 0.05) different.
Citation: American Journal of Veterinary Research 69, 8; 10.2460/ajvr.69.8.1097
MT concentrations—The mean MT concentration in control dogs at 0 hours exceeded 25 mmol/kgDW (Figure 2). Compared with the control value, the concentration of MT in tissue samples collected immediately after exercise was significantly decreased in dogs that ran 140 km; concentrations remained similarly lower than control value in dogs that ran 420 or 560 km. After rest and consumption of a meal in dogs undergoing paired biopsy procedures after completing distances of 140, 420, and 560 km, the MG concentrations were 10.3 ± 4.9 mmol/kgDW, 10.7 ± 5.9 mmol/kgDW, and 12.3 ± 3.4 mmol/kgDW, respectively. Assuming that MT concentrations in dogs that underwent biopsy procedures were representative of the remaining study dogs, a significantly (P = 0.024) greater amount of MT was depleted during the initial 140-km run (approx 18.5 mmol/kgDW) of the exercise challenge, compared with that depleted during the final 140-km run (4.6 mmol/kgDW). At 28, 50, and 98 hours of the recovery period, MT concentration not significantly different from the control value. The calculated rate of replenishment of MT during the 3 approximately 7-hour rest periods between 140-km runs did not differ significantly among dogs regardless of distance run (140 km completed, 0.47 ± 0.69 mmol/kgDW/h; 420 km completed, 0.15 ± 0.68 mmol/kgDW/h; and 560 km completed, 0.8 ± 0.53 mmol/kgDW/h). The coefficient of variation for repeated measurement of MTG concentration in tissue samples was 19%.
Mean ± SD MT concentrations in aerobically conditioned Alaskan sled dogs assessed in muscle biopsy specimens collected before (at 0 hours from 6 unexercised control dogs) and immediately after running cumulative distances of 140, 420, and 560 km (within 24, 72, and 96 hours; 6 dogs at each time point) and at 28, 50, and 98 hours of recovery (at 118 [n = 7 dogs], 140 [7], and 188 [6] hours after study commencement). Solid double-headed horizontal arrows indicate periods of exercise (70-km runs); final double-headed horizontal arrow indicates the recovery period after the exercise portion of the study. See Figure 1 for key.
Citation: American Journal of Veterinary Research 69, 8; 10.2460/ajvr.69.8.1097
Discussion
Results of a previous study1 of highly trained Alaskan sled dogs indicated that a gradual replenishment of MG occurred during a 5-day period of repeated prolonged exercise, despite a limited carbohydrate intake. Findings of the present study suggest that this phenomenon results from dramatic attenuation of glycogen usage during repeated episodes of prolonged lowintensity exercise in fit sled dogs that are consuming a high-fat diet. Assuming that MG concentrations in dogs that underwent biopsy procedures were representative of the other dogs in our study, then during the first 140-km run, dogs used approximately 64% of stored MG. However, despite running at a similar pace during each run, dogs used only 5% of their estimated glycogen stores during the final 140-km run; furthermore, there was minimal difference in MG concentration before and after the final episode of exercise, which was in distinct contrast to the changes detected during the initial episode of exercise. The calculated rates of glycogen replenishment during the rest periods between consecutive episodes of exercise bouts did not differ. Together with the evident depletion of MT, these data support development of a progressive increase in MG concentration because of enhanced use of other substrates during exercise and recovery. Prior findings of increases in plasma BUN, free fatty acids, and postexercise ketone concentrations in sled dogs after a similar exercise challenge further support the hypothesis that lipid and protein substrates are critical alternative energy sources for exercising sled dogs.12,e In the present study, significantly more MT was used during the first 140-km run, compared with the amount used during the final 140-km, which suggests that extramuscular substrates likely play an important role in supporting muscular work in sled dogs that are undergoing repeated episodes of prolonged exercise. Results of another study11 of dogs undergoing prolonged submaximal exercise indicated that plasma free fatty acids are the predominant energy substrate used at lower exercise intensity in this species.
A study18 of human cyclists who consumed a high-fat diet revealed an increased capacity for fat metabolism and a 4-fold decrease in MG use during exercise, compared with cyclists who consumed a standard diet; these changes had no negative impact on performance.18 A fat-rich diet is associated with increased limb muscle uptake of very–low-density lipoprotein-triacylglycerol and fatty acids during submaximal exercise in trained humans, in conjunction with a significant depression of muscle glycogenolysis.19 Furthermore, consumption of a high-fat diet has a synergistic effect with conditioning in rats, resulting in increased submaximal running endurance.20 The combination of a high-fat intake and extensive endurance training of the dogs in the present study likely enhanced the capacity to use fat-based substrates during exercise and may have promoted extensive sparing of MG. A high-fat diet may therefore have important benefits for athletes competing in endurance events of ≥ 2 days' duration and may possibly ameliorate the reported negative impact of repeated exercise on MG supercompensation.7,18 In the sled dogs in the present study, MG replenishment occurred steadily and MG concentrations at 50 hours of recovery after 4 consecutive days of prolonged exercise were significantly higher than the value recorded in unexercised control dogs. Although dogs that underwent muscle biopsy procedures at 50 and 98 hours of the recovery period completed only 20 km of the final 70-km run before refusing to continue running, MG concentrations at 50 and 98 hours of the recovery period were not different from the MG concentration at 24 hours of the recovery period in dogs that completed the final 70-km run. Furthermore, although dogs were rested for 9 days prior to the study because of restrictive cold-weather conditions, MG concentrations measured in the control dogs and the absolute decrease in MG concentration during the initial episode of exercise were comparable to previously reported1 values in similarly trained sled dogs that were rested from training for only 2 days; this suggests that the unscheduled rest period had minimal impact on the outcomes of the present study.
The similar rates of MG replenishment detected during the rest periods between the episodes of exercise was surprising, given that degree of glycogen depletion incurred during exercise purportedly dictates glycogen replenishment rate, at least during the early hours of the recovery period.21 However, it is possible that prolonged high-fat intake may alter glucose and glycogen metabolism, thereby reducing the impact of glycogen depletion on regulatory responses. A high-fat diet alters oxidative carbohydrate disposal and insulin-stimulated glycogen synthesis in skeletal muscle, but these effects may be dependant on the provision of a period of adaptation to the altered diet of several weeks' duration.22–24
Proximate analysis of the diet fed to dogs in the present study revealed a higher contribution of carbohydrate to daily caloric intake (33%) than that reported for the diet used in an earlier study by our group, wherein carbohydrate content of the diet was estimated from known feed components (15%).1 This difference may not only partly reflect discrepancies between the methods of diet analysis (proximate analysis vs component estimation), but may also reflect an increase in the proportion of dried dog food included in the diet implemented by the mushers, compared with the proportion included in the diet during the previous study. Nevertheless, the absolute fat and protein contents of the diets used in the 2 studies were similar. Despite the difference in dietary carbohydrate intake, absolute MG values in rested or exercised dogs were similar in both studies. This finding suggests that the increase in dietary carbohydrate intake in the present study was not large enough to alter muscle substrate metabolism significantly, compared with the effect of the diet in the previous study, or perhaps that repeated exercise has a more dramatic impact on regulatory processes than dietary composition. Among the dogs of the present study, the increased dietary carbohydrate intake may have been disposed of via oxidation of blood glucose as an energy source, replenishment of hepatic glycogen and MG stores during recovery, and the synthesis of glycogen in nonactive muscle fibers during exercise (which occurs in athletically trained humans25). Carbohydrate substrates can contribute to energy demand even in dogs performing aerobic exercise.26 However, in a study27 of dogs that were exercising at 40% of O2max, 77% of energy demand was supplied via oxidation of fat.
Apart from the potential impact of dietary composition, it is likely that the ability to limit muscle glycogenolysis during exercise in dogs is related to the capacity of the species for aerobic metabolism as well as the extent of preexisting endurance training.27 In a previous investigation1 of dogs from the kennel involved in the present study, the proportion of type 1 highly oxidative fibers in skeletal muscle tissue was greater than proportions reported for other dog breeds, which could represent breed differences, training influences, or both.28 Although dogs are no more capable of performing work at high intensities without metabolism of carbohydrate fuel sources than are species that have less capacity for aerobic metabolism, their greater aerobic metabolic capacity allows them to run at relatively higher speeds with preferential use of fatty substrates.26 Nonetheless, unconditioned dogs do have significant differences in carbohydrate and fat metabolism during exercise, compared with findings in aerobically conditioned dogs; therefore, endurance training still plays a critical role in optimizing innate aerobic ability.26
On the basis of results of the present study, skeletal muscle glycogenolysis appears to be greatly reduced in Alaskan sled dogs undergoing consecutive days of prolonged submaximal exercise in favor of the use of alternative energy sources, including MT and likely other extramuscular substrates that were not measured in our study. This phenomenon occurred rapidly, and MG use had almost ceased by the fourth day of exercise; thus, the use of alternative energy sources is likely related to a combination of innate ability, diet composition, and intensive endurance training.
ABBREVIATIONS
| kgDW | Kilogram of dry weight |
| MG | Muscle glycogen |
| MT | Muscle triglyceride |
| O2max | Maximum oxygen consumption per unit time |
PGI EZ Core, Products Group International Inc, Lyons, Colo.
Servi-Tech Laboratories, Dodge City, Kan.
Eukanuba Adult Large Breed Premium Performance, The Iams Co, Dayton, Ohio.
PC SAS, version 8.2, SAS Institute Inc, Cary, NC.
Hinchcliff KW, Jose-Cunilleras E, Davis MS, et al. Muscle triglyceride concentration and fat metabolism during endurance exercise by sled dogs (abstr). Physiologist 2004;47:302.
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