The APR has been associated with many infectious and heritable disease processes in dogs and humans. The acute-phase components most often evaluated in dogs are haptoglobin, CRP, and serum amyloid A, with CRP being the most widely evaluated for a variety of acute and chronic inflammatory processes. Changes in CRP concentrations can be subtle, as is the case in inflammatory bowel disease (40 to 100 μg/mL), or extremely robust, as is the case for infectious agents (> 300 μg/mL).1-8 The increase in serum concentrations of CRP can be highly variable, which may reflect a systemic response to the degree of inflammatory stimuli.
Rigorous exercise in humans results in an APR, particularly in individuals who participate in events associated with endurance-related exercise. Ultramarathon and triathlon participants have the most consistent APR, including severe hypoferritinemia, hypoalbuminemia, and increases in concentrations of CRP and other acute-phase proteins.9–11 In many instances, a strong increase in CRP concentrations can be measured well into recovery and can provide a marker for the APR of exercise.9–15 There has been much debate regarding the kind of exercise that induces this response. It has been observed that concentric exercise (eg, weight lifting or typical training for a sporting activity) does not induce an APR, whereas prolonged eccentric exercise (triathlons and marathons) induces a robust APR with increases in CRP concentrations preceding an increase in IL-6 concentrations, which is thought to help drive this APR.9–17 This increase in CRP concentrations often precedes a progressive decrease in serum concentrations of iron and albumin, which suggests that dramatic increases in concentrations of CRP may be a potential marker for chronic fatigue or overtraining syndrome.11 However, depending on the study evaluated, the increase in IL-6 or CRP concentrations can be 4 to 50 times as high as the baseline concentration and may be brief or extended14; therefore, the role of IL-6 in the induction of exercise-induced APR and the potential use of IL-6 or CRP concentrations as markers of exercise stress have been contested.14,18,19
The most pronounced increases in CRP concentrations and decreases in serum concentrations of iron and albumin are evident in ultramarathon participants because of the rigorous nature of the exercise.11 However, an athlete that often surpasses the exertion of a human ultramarathon participant is an endurance sled dog. Endurance sled dogs participate in races that require running 128 to 160 km each day. To our knowledge, there has been no evaluation of the APR in these elite canine athletes. Because of the rigorous nature of the exercise these dogs perform, evaluation of these dogs may provide a better understanding of the APR during exercise through investigation of the relationship among IL-6, CRP, albumin, and iron concentrations. We hypothesized that sled dogs participating in a 557-km endurance race would have increases in serum concentrations of CRP and IL-6 and marked decreases in serum concentrations of serum iron and albumin as well as serum biochemical changes similar to those detected in other studies20–23 of endurance sled dogs that raced in Alaska.
Materials and Methods
Animals—Three teams (12 mixed-breed sled dogs/team) were enrolled in the study. All dogs passed a physical examination performed before the race. Exclusion criteria included any dog with an elevated total or segmented WBC count or a postexercise (1 hour after racing) rectal temperature of > 39.4°C. All procedures complied with the standards set forth by the Cornell University Institutional Animal Care and Use Committee.
Two of the dog teams (teams 1 and 2) had exercised (ran approx 32 km) within 24 hours before collection of a prerace blood sample (collected from each dog 48 hours before the start of the endurance race). The remaining team (team 3) had not run within 3 days of collection of the prerace blood sample. Each team of dogs had trained by running between 2,320 and 2,880 km during the 3 to 4 months preceding the race. The race consisted of 557 km over mountainous terrain. Day 1 of the race consisted of 80 km, after which the dogs were allowed to continue the race the following day for the remaining 477 km. At the halfway point, each team was required to take a mandatory rest period of 8 hours. The finishing times for teams 1, 2, and 3, respectively, were 4 days, 10 hours, and 23 minutes; 4 days, 10 hours, and 26 minutes; and 5 days, 1 hour, and 16 minutes.
Blood sample collection, CBC, and serum biochemical analysis—A blood sample (10 mL) was collected from a jugular vein of each dog 48 hours before the start of the endurance race. Samples were collected by use of a syringe and 20-gauge needle; 3 mL was placed in a 3-mL tube containing potassium EDTA, and the remaining 7 mL was placed in a coagulant tube. The EDTA-anticoagulated blood samples collected before the race were stored at 4°C and transported to a commercial laboratorya for analysis within 24 hours after collection. The CBC results were generated with an automated analyzer.b Within 20 minutes after completion of the race, 5 mL of blood was collected from a cephalic vein of each dog, and rectal temperatures were measured after 30 minutes of rest for the 25 dogs that completed the race. Of the 25 dogs that completed the race, 12 were from team 1, 6 were from team 2, and 7 were from team 3. Serum was obtained within 2 hours after collection of blood samples via centrifugation at 2,400 × g for 5 minutes; serum was harvested and transferred to 3 separate cryovials to prevent the need for thawing and refreezing of samples when performing assays. All serum samples were immediately frozen (-20°C) and stored until 24 hours after the race, at which time they were shipped on dry ice via overnight delivery to the primary investigators' laboratory at Cornell University, where they were stored at −80°C for 2 weeks until analysis. Serum biochemical analysis was performed at the Cornell University Animal Health Diagnostic Center Clinical Pathology Laboratory by use of a biochemical analyzerc on samples obtained from each dog before and immediately after racing.
CRP ELISA—A canine CRP kitd that has been validated24,25 for use on canine serum was used to measure CRP concentrations. The kit was used in accordance with the manufacturer's suggestions, with all samples from the same dog being analyzed in duplicate on the same plate. All samples obtained after exercise were diluted 1:10, as suggested by the manufacturer, to ensure that concentrations were within the linear portion of the standard curve (5-60 μg/mL).
Cytokine analysis—A canine cytokine kite that consisted of beads coated with antibodies against IL-6 and TNF-α was shipped at 4°C to the primary investigators' laboratory. The canine cytokine kit has been used by other groups and was evaluated in our laboratory by use of normal canine serum samples to establish an intra-assay coefficient of variation of 5.8%. Values established by the manufacturer26 included an interassay coefficient of variation of 15.6% and intra-assay coefficient of variation of 3.9%, with a sensitivity of 4.1 pg/mL. Data generated were assessed against the linear portion of the standard curve because all values were < 400 pg/mL, and all standards less than this concentration yielded linear results. Initial evaluation of the assay (used in accordance with the manufacturer's directions) revealed that normal canine serum had IL-6 and TNF-α concentrations less than the detectable limit of the assay unless more serum was used than was indicated in the directions. Therefore, we doubled the serum required for the assay and decreased the assay buffer to generate data within the lower limit of detection for IL-6 (4.1 pg/mL) and adjusted the results accordingly. Each serum sample from the sled dogs was assayed in duplicate, and a mean value was calculated. To ensure that there was a cytokine response that was measurable, a positive control serum sample (in duplicate) obtained from a dog with active borreliosis (Lyme disease) was included on the two 96-well plates used for the samples from the sled dogs. Results for the positive control serum samples were TNF-αconcentrations of 15.3 and 17.9 pg/mL and IL-6 concentrations of 58.7 and 68.5 pg/mL, respectively, for each of the 96-well plates.
Statistical analysis—Results for almost all serum biochemical analyses were normally distributed, as determined by use of the Lillifors test for normality. Therefore, a Student paired t test was used to compare results in samples obtained before and immediately after racing. Significance was set at values of P ≥0.05. To assess differences among the 3 teams of dogs, all serum variables were subjected to an ANOVA (samples obtained both before and immediately after racing), with the value of ≥set at 0.05; Tukey post hoc analysis was used to determine significant differences between teams. Linear regression analyses were also performed to examine whether decreases in albumin and iron concentrations were directly correlated with increases in CRP concentrations, with ANOVAs used to determine significance of the regression. Significance for that analysis was set at values of P < 0.05.
Results
CRP, albumin, IL-6, and TNF-α concentrations and iron status—Serum CRP concentration increased significantly from a mean ± SD of 22.4 ± 16.3 μg/mL before the race to 263.3 ± 103.8 μg/mL after the race (Table 1). There was a significant decrease in serum albumin and total protein concentrations from before to after the race. When the concentrations of CRP were stratified on the basis of team, there was a significant difference between concentrations for team 3 and concentrations for teams 1 and 2 in samples obtained before the race (Table 2). Additionally, CRP concentrations after the race also differed significantly among teams, with team 1 having a significantly lower CRP concentration, compared with the CRP concentrations for teams 2 and 3.
For all dogs, the serum iron concentration decreased significantly after the race, compared with the concentration before the race (Table 1). Similarly, the serum transferrin concentration also was significantly decreased after the race, as reflected in the total iron binding capacity; however, the percentage saturation of transferrin remained the same before and after the race. Regression analysis to examine the correlation between the decrease in serum albumin concentration from before to after the race and the CRP concentration after the race revealed a significant but modest correlation (r = 0.50; Figure 1). A similar regression analysis was performed to examine the correlation between serum iron and CRP concentrations; that analysis revealed a nonsignificant mild correlation (r = 0.37).
The TNF-α concentrations could not be determined with the assay because all dogs had results for samples obtained before and immediately after the race that were less than the lower limit of detection of the assay system. Mean IL-6 concentrations were detectable in 19 of 25 dogs; mean ± SD concentrations before and immediately after racing were 5.7 ± 6.1 pg/mL and 6.6 ± 7.7 pg/mL, respectively (Figure 2).
Serum biochemical analysis—Significant changes in serum electrolyte concentrations associated with the endurance race included increases in serum concentrations of chloride as well as decreases in serum concentrations of potassium, sodium, bicarbonate, and magnesium (Table 3). Serum creatinine concentrations were significantly lower in samples obtained after the race, compared with concentrations in samples obtained before the race, whereas the SUN concentration had a paradoxical significant increase in samples obtained after the race. Activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and creatine kinase were all significantly higher after the race than before the race. Significant decreases in serum μ-glutamyl transferase and amylase activities after the race, compared with activities before the race, were also detected. Interestingly, there was a mild yet nonsignificant increase in indirect bilirubin concentration after the race.
Mean ± SD values for total protein, albumin, CRR and Iron analyses in serum samples obtained from 25 sled dogs 48 hours before and Immediately after a long-distance endurance race
| Variable | Reference interval | Before | After |
|---|---|---|---|
| Total protein (g/dL) | 5.6-7.1 | 6.1 ± 0.3 | 5.3 ± 0.3* |
| Albumin (g/dL) | 3.1-4.1 | 3.8 ± 0.2 | 3.3 ± 0.2* |
| Iron (μg/dL) | 98-220 | 153.0 ± 51.5 | 129.3 ±32.5* |
| Total iron binding capacity (μg/dL) | 249-496 | 386.4 ± 60.6 | 298.7 ± 40.2* |
| Transferrin saturation (%) | 28-62 | 40.2 ± 12.9 | 44.0 ±12.1 |
| CRP(μg/mL) | < 20 | 22.4 ± 16.3 | 263.3 ± 103.8* |
Within a variable, value differs significantly (P < 0.05) from the value before the race.
Mean ± SD concentrations of CRR sodium, chloride, SUN, and creatinine in serum samples obtained from 25 sled dogs on 3 teams 48 hours before and immediately after a long-distance endurance race.
| Variable | Team 1 | Team 2 | Team 3 |
|---|---|---|---|
| CRP before the race (μg/mL) | 25.2 ± 12.7 | 26.0 ± 16.9 | 7.0 ± 2.6* |
| CRP after the race (μg/mL) | 209.9 ± 98.3* | 315.8 ±73.9 | 326.4 ± 94.0 |
| Sodium after the race (mEq/L) | 144.9 ± 1.9 | 142.2 ± 1.2* | 146.1 ± 1.3 |
| Chloride after the race (mEq/L) | 117.2 ± 1.6 | 113.5 ±2.4* | 118.7 ± 2.0 |
| SUN after the race (mg/dL) | 38.2 ± 5.2* | 16.8 ± 3.2 | 21.4 ± 4.8 |
| Creatinine after the race (mg/dL) | 0.94 ± 0.13 | 0.63 ± 0.05* | 0.89 ± 0.16 |
Of the 25 dogs that completed the race, 12 were from team 1, 6 were from team 2, and 7 were from team 3.
Within a row, value differs significantly (P < 0.05) from the values for the other 2 teams.
Results of regression analysis to compare the decrease in serum albumin concentrations for samples obtained from 25 sled dogs 48 hours before and immediately after a long-distance endurance race with serum CRP concentrations in samples obtained immediately after the race. There was a significant (P = 0.012) correlation (r= 0.50). The equation for the line of best fit is y = 96.922 + 305.84x.
Citation: American Journal of Veterinary Research 71, 10; 10.2460/ajvr.71.10.1207
Results of regression analysis to compare the decrease in serum albumin concentrations for samples obtained from 25 sled dogs 48 hours before and immediately after a long-distance endurance race with serum CRP concentrations in samples obtained immediately after the race. There was a significant (P = 0.012) correlation (r= 0.50). The equation for the line of best fit is y = 96.922 + 305.84x.
Citation: American Journal of Veterinary Research 71, 10; 10.2460/ajvr.71.10.1207
Results of regression analysis to compare the decrease in serum albumin concentrations for samples obtained from 25 sled dogs 48 hours before and immediately after a long-distance endurance race with serum CRP concentrations in samples obtained immediately after the race. There was a significant (P = 0.012) correlation (r= 0.50). The equation for the line of best fit is y = 96.922 + 305.84x.
Citation: American Journal of Veterinary Research 71, 10; 10.2460/ajvr.71.10.1207
—Box-and-whisker plots of serum IL-6 concentrations in samples obtained from 19 sled dogs 48 hours before and immediately after completing a long-distance endurance race. The boxes represent the interquartile range, the horizontal line within each box represents the median value, and the whiskers represent the maximum and minimum values. Outliers are indicated (circles)
Citation: American Journal of Veterinary Research 71, 10; 10.2460/ajvr.71.10.1207
—Box-and-whisker plots of serum IL-6 concentrations in samples obtained from 19 sled dogs 48 hours before and immediately after completing a long-distance endurance race. The boxes represent the interquartile range, the horizontal line within each box represents the median value, and the whiskers represent the maximum and minimum values. Outliers are indicated (circles)
Citation: American Journal of Veterinary Research 71, 10; 10.2460/ajvr.71.10.1207
—Box-and-whisker plots of serum IL-6 concentrations in samples obtained from 19 sled dogs 48 hours before and immediately after completing a long-distance endurance race. The boxes represent the interquartile range, the horizontal line within each box represents the median value, and the whiskers represent the maximum and minimum values. Outliers are indicated (circles)
Citation: American Journal of Veterinary Research 71, 10; 10.2460/ajvr.71.10.1207
Mean ± SD values for serum biochemical analysis in serum samples obtained from 25 sled dogs 48 hours before and immediately after a long-distance endurance race
| Variable | Reference interval | Before | After |
|---|---|---|---|
| Sodium (mEq/L) | 142-151 | 148 ± 3.1 | 144.7 ± 2.2* |
| Potassium (mEq/L) | 2.9-5.3 | 5.2 ± 0.3 | 4.1 ± 0.2* |
| Chloride (mEq/L) | 107-117 | 113.2 ±3.5 | 116.7 ±2.6* |
| Bicarbonate (mEq/L) | 15-25 | 20.8 ±1.9 | 13.6 ±1.7* |
| Anion gap (mEq/L) | 13-25 | 19.8 ±1.4 | 18.5 ±1.2* |
| SUN(mg/dL) | 8-30 | 28.0 ± 10.7 | 40.5 ±11.4* |
| Creatinine (mg/dL) | 0.5-1.3 | 0.9 ± 0.2 | 0.7 ± 0.1* |
| Calcium (mg/dL) | 9.3-11.6 | 9.2 ± 0.7 | 9.1 ± 0.3 |
| Phosphorus (mg/dL) | 2.8-5.3 | 4.5 ± 0.5 | 4.7 ± 0.5 |
| Magnesium (mg/dL) | 1.4-2.0 | 1.9 ±0.2 | 1.7 ±0.1* |
| Total protein (g/dL) | 5.6-7.1 | 6.1 ± 0.3 | 5.3 ± 0.3* |
| Albumin (g/dL) | 3.1-4.1 | 3.8 ± 0.2 | 3.3 ± 0.2* |
| Globulin (g/dL) | 1.9-3.6 | 2.4 ± 0.3 | 2.1 ± 0.2* |
| Glucose (mg/dL) | 60-120 | 95.2 ± 8.4 | 100.2 ± 8.8 |
| Cholesterol (mg/dL) | 124-335 | 259 ± 46 | 254.1 ± 49.7 |
| Alanine aminotransferase (U/L) | 25-106 | 52.0 ±21.0 | 95.6 ±31.2* |
| Aspartate aminotransferase (U/L) | 16-50 | 26.7 ± 8.4 | 140.2 ±120.0* |
| Alkaline phosphatase (U/L) | 12-122 | 50.1 ± 18.1 | 104.3 ±113.7* |
| Creatine kinase (U/L) | 58-241 | 98.0 ± 29.4 | 1,756.1 ± 2,050.6* |
| y-Glutamyl transferase (U/L) | 0-10 | 4.2 ± 0.9 | 3.3 ± 0.7* |
| Amylase(U/L) | 286-1,124 | 388 ± 176 | 315.7 ±95.4 |
| Total bilirubin (mg/dL) | 0-0.3 | 0.1 ± 0.0 | 0.2 ±0.1* |
| Direct bilirubin (mg/dL) | 0-0.1 | 0.0 ± 0.0 | 0.0 ± 0.0* |
| Indirect bilirubin (mg/dL) | 0-0.3 | 0.1 ± 0.0 | 0.2 ±0.1* |
Within a variable, value differs significantly (P < 0.05) from the value before the race.
The ANOVA for other serum biochemical variables before and after the race revealed that team 2 had a significantly lower chloride concentration, a significantly lower serum sodium concentration, and a significantly (P = 0.01) lower serum creatinine concentration after the race, compared with results for teams 1 and 3 (Table 2). In addition, team 1 had a significantly higher SUN concentration after the race, compared with the SUN concentrations for teams 2 and 3 after the race.
Discussion
In endurance athletes, a decrease in albumin concentration is thought to be a part of the APR,11,14 although some have argued that a decrease in albumin concentration may be related to hydration status in exercising sled dogs.20,27 The 1,000% increase in CRP concentration in the study reported here suggests that the mild decrease in albumin concentration over time may be related to the APR, similar to results observed in human ultramarathon athletes.11 This decrease in serum albumin concentration in sled dogs is quite modest in light of the pronounced increase in the CRP concentration. During the APR, hepatic synthesis of albumin typically decreases, whereas synthesis of other acute-phase proteins, such as serum amyloid A, ceruloplasmin, haptoglobin, and CRP, is enhanced. The enhanced synthesis of CRP in sled dogs of the present report is actually much higher than that detected in any humans participating in endurance activities (approx 10-fold higher [30 μg/ml for humans vs 300 μg/ml for sled dogs]), and it is the first evidence of such a response in exercising dogs.
Comparing the concentrations for sled dogs in samples obtained before the race revealed that they were similar to concentrations for resting dogs published in the literature.1-8,24 Interestingly, the sled dogs we evaluated for measurement of CRP concentrations had a modest increase in the samples obtained before the race, compared with concentrations of clinically normal dogs in other studies2,3,5,24 (20 vs 5 μg/ml, respectively). However, caution should be used in the interpretation of these results because some laboratories have reported1,4,6–8,28 CRP concentrations > 10 μg/ml in clinically normal dogs. Our laboratory group has reported that clinically normal dogs, including sedentary nonexercising sled dogs, have CRP concentrations that consistently are < 10 μg/ml. This makes our finding of a higher basal serum CRP concentration in teams 1 and 2 intriguing, and these differences from concentrations of team 3 may have been attributable to the time of their last episode of exercise before collection of the prerace blood sample. Teams 1 and 2 had run approximately 32 km within 24 hours before collection of the prerace blood samples, whereas team 3 had not exercised in 3 days, and dogs of team 3 had CRP concentrations much closer to our laboratory reference intervals and the reference intervals of other North American laboratories.28 Therefore, modest exercise might cause mild increases in CRP concentrations in dogs, similar to those detected in human athletes.9,10,12
The CRP concentrations in the samples obtained after the race in the study reported here appear to be higher than those reported for diseases of chronic inflammation (inflammatory bowel disease and neoplasia) but lower than those detected for acute inflammation attributable to infectious organisms.1–8 Although these mild increases in CRP concentrations before the race may be inconsequential when compared with the concentrations after the race, these increases are well within the range for the lower concentrations observed in dogs with neoplasia or inflammatory bowel disease.7,8 Hence, a history of physical activity is important in the evaluation of baseline CRP concentrations. This also raises a concern regarding the amount of activity that causes an increase in CRP concentrations and whether the intensity of the activity is directly associated with the increase in CRP concentrations. Additional studies in other sporting breeds and companion dogs could shed light on the pattern of exercise that is required to induce this APR.
Stratifying the CRP response to exercise on the basis of team revealed that team 1 had a significantly lower CRP concentration after the race, compared with concentrations after the race for the other 2 teams. Although it could be postulated that this may have been attributable to a difference in ELISA kits, more than half of the samples for team 1 were assayed along with samples from the other 2 teams, which suggested that this was not an artifact attributable to variation in kits or technical error. An interesting observation is that all dogs of team 1 completed the entire 557-km race, whereas 6 dogs of team 2 and 5 dogs of team 3 retired from the race prematurely. It is possible that there may have been an inverse relationship between workload or preparation for racing; this may be a reflection of serum CRP concentrations based on performance, which needs to be elucidated with further investigations in canine athletes.
In humans, the severity of the APR is usually correlated to increases in IL-6 concentrations after exercise.9,11,12,14,16 This is an area of debate because it is also thought that IL-1 is the stimulus for increases in IL-6 or CRP concentrations (or both), particularly because IL-1 has often been associated with increases in CRP concentrations in domestic animals.29 However, investigators in another study30 found that IL-6, and not IL-1, is the stimulus for increases in CRP concentrations in acute muscle damage resulting from IM injection of turpentine oil. The increase in IL-6 concentrations after exercise has been described14 after a single bout of endurance activity; these IL-6 concentrations often return to baseline values within 24 hours, and the effects of repeated bouts of exercise have not been as clearly described.14 Multiple studies conducted to examine effects of repetitive bouts of exercise have yielded contradictory results. In 1 study16 it was suggested that the increase in IL-6 concentration is a transient event, whereas in the other studies,11,14,15 it was suggested that the IL-6 concentration remains higher than the baseline concentration but that the initial postexercise peak is most dramatic within the first 2 to 4 hours after physical activity. Analysis of results for the study reported here suggests that sled dogs do not have a significant increase in IL-6 concentrations from baseline values after completing 5 days of racing. Additional information would have been gained by collecting samples at time points during the race as well as at multiple days after the race, which would have allowed us to monitor the IL-6 response and the response for other acute-phase proteins (eg, haptoglobin and CRP) to determine exactly when the concentrations of these factors increased and whether they would return to within the reference interval after racing. This was a limitation of this study, but it poses further questions regarding the APR of exercise in dogs, whether the lack of an increase in the IL-6 concentration and an excess in the CRP concentration is unique to dogs, and whether critical peaks in serum concentrations were missed.
Endurance activity in humans has been associated with changes in the serum iron status.11,31,32 A mild decrease in serum iron concentration and no decrease in transferrin binding capacity were detected in the dogs of the study reported here, which suggested that iron status was mildly diminished. However, the decrease was modest in comparison with findings for human tri-athletes and ultramarathon participants whose serum iron concentrations decrease by approximately 50% or more,11 whereas the serum iron concentration of the dogs in the present study decreased only approximately 15%. Discrepancies between human and canine athletes may result from the fact that iron consumption in sled dogs is > 70 mg/d, as determined on the basis of information provided by the dog handlers. The iron content in complete and balanced dry food being fed ranged from 248 to 341 mg/kg, with each dog consuming a minimum of 300 g of dry food/d, which provided a minimum of 75 mg of iron/d. Conversely, many human athletes do not even receive an amount of iron in the upper portion of the recommended daily allowance of 18 mg/d, and supplementation of 30 mg of iron/d is enough to maintain serum iron concentrations in active humans.33,34
Indirect bilirubin concentrations were also increased in the dogs after the race, which can be a sign of a decreased life span of RBCs (hemolysis) or of hepatic dysfunction; however, the concentrations for all dogs were well within the reference interval. A mild increase in indirect bilirubin concentrations and increased RBC turnover has been reported in human athletes.15,35 It has been theorized that this increase in indirect bilirubin concentration in athletes is attributable to the repetitive trauma to the plantar surface of the foot causing RBC damage, which may have been contributing to the mild increase in indirect bilirubin concentrations in the sled dogs reported here.
Overall, the effects of endurance exercise on serum biochemical values in the sled dogs of the present study are similar to those in other reports20-23 for dogs racing in Alaska. Hyponatremia and hypokalemia are thought to be part of a pronounced electrolyte shift that may be attributable to the increased metabolic demands of exercise. Although the typical renal response is to conserve sodium and potentially potassium, the enhanced glomerular filtration rate attributable to a high protein intake and excessive water consumption may overwhelm the conservation mechanism, thus inducing a gradual loss of sodium and potassium.22,36 Additionally, the dietary needs of these dogs during exercise (training and racing) requires the use of calorically dense meat products, and all of the teams participating in this study were primarily fed meat to fulfill these additional dietary needs. Meat would contain substantially lower concentrations of sodium and potassium than would be contained in their typical diet of potassium- and sodium-rich commercial dog food, which would further exacerbate the potential loss of sodium and potassium during enhanced glomerular filtration. Furthermore, the mild hypochloremia detected in this and other studies37,38 may be a compensatory mechanism to maintain appropriate anion balance, which can partially be verified by the decrease in the serum bicarbonate concentration.
It is more difficult to explain the modest difference in serum sodium and chloride concentrations among the teams after the race. Teams 1 and 3 had characteristic changes in sodium and chloride concentrations similar to those observed in other studies, whereas team 2 had less hyponatremia and hypochloremia as well as a significantly lower serum creatinine concentration. Nearly all dogs were fed primarily meat rations while racing, but team 2 did not have the same degree of hyperchloremia and hyponatremia as did teams 1 and 3. This may have been attributable to hydration and feeding strategies that alter hormonal control of renal solute load, which has been detected in another study27 on sled dogs. There were no significant differences in hydration status among the teams when they were examined after the race, and team 2 actually had a lower serum creatinine concentration, which ruled out dehydration as a factor involved in the electrolyte differences among the teams. Additionally, team 1 had a marked increase in SUN concentration after the race, which was likely a result of prerenal azotemia attributable to excessive protein consumption, rather than a result of dehydration. Further clarification would require detailed diaries regarding hydration and feed intake to investigate these differences, and that information was not obtained.
Increases in activities of the hepatic enzymes aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were detected in the study reported here, which were similar to results from other investigations. 20-23 Increases in the activity of serum creatine kinase detected in the present study were similar to those detected in other dogs participating in endurance races.20-23 In another report,21 it was suggested that mean creatine kinase activity peaks within the first 320 km of a race and then starts to diminish, although it still remains elevated throughout the race, with values similar to those for the study reported here.
Analysis of our results suggests that dogs participating in endurance races do not have the severe hypoalbuminemia and hypoferritinemia observed in human athletes participating in endurance events, but there were some modest decreases. This may have been associated with the unique physiologic mechanisms or dietary differences in canine athletes versus human endurance athletes. Additionally, the APR of exercise typically observed as an increase in the IL-6 concentration was not detected in the sled dogs of this study; however, the increases in CRP concentrations were much greater than the increases in CRP concentrations observed in human studies (approx 10-fold higher). This suggests that physical activity in dogs can cause variable increases in the CRP concentration, thus confounding its use as an indicator of inflammation, particularly in canine athletes.
Abbreviations
| APR | Acute-phase response |
| CRP | C-reactive protein |
| IL | Interleukin |
| TNF-α | Tumor necrosis factor-α |
Antech Laboratory, Portland, Ore.
Bayer Advia 120, Siemens Corp, New York, NY.
Hitachi 917, Roche Diagnostics, Indianapolis, Ind.
Canine CRP ELISA, Tridelta PLC, Morris Plains, NJ.
Lincoplex canine cytokine multiplex, LINCO Research, St Charles, Mo.
References
- 1
Martinez-Subiela STecles FCeron JJ. Critical differences in acute phase proteins in canine serum samples. Vet J 2003;166:233–237.
- 2
Yamamoto SShida THonda M, et al. Serum C-reactive protein and immune responses in dogs inoculated with Bordatella bronchiseptica. Vet Res Commun 1994;18:347–357.
- 3
Shimada TIshida YShimizy M et al. Monitoring C-reactive protein in Beagle dogs experimentally inoculated with Ehrlichia canis. Vet Res Commun 2002;26:171–177.
- 4↑
Holm JLRozanski EAFreeman LM et al. C-reactive protein concentrations in canine acute pancreatitis. J Vet Emerg Crit Care 2004;14:183–186.
- 5
Yamamoto SShida TMiyaji S et al. Changes in serum C-reactive protein levels in dogs with various disorders and surgical traumas. Vet Res Commun 1993;17:85–93.
- 6
Mastrorilli CDondi FAgnoli C et al. Clinicopathologic features and outcome predictor of Leptospira interrogeais Australis serogroup infection in dogs: a retrospective study of 20 cases (2001-2004). J Vet Intern Med 2007;21:3–10.
- 7↑
Merlo ARezende BCFranchini ML et al. Serum C-reactive protein concentrations in dogs with multicentric lymphoma undergoing chemotherapy. J Am Vet Med Assoc 2007;230:522–526.
- 8↑
Jergens AEShreiner CAFrank DE et al. A scoring index for disease activity in canine inflammatory bowel disease. J Vet Intern Med 2003;17:291–297.
- 9↑
Neubauer OKonig DWagner K. Recovery after an Ironman triathlon: sustained inflammatory responses and muscular stress. Eur J Appl Physiol 2008;104:417–426.
- 10↑
Smith JATelford RDBaker MS, et al. Cytokine immunoreactivity in plasma does not change after moderate endurance exercise. J Appl Physiol 1992;73:1396–1401.
- 11↑
Fallón KE. The acute phase response and exercise: the ultramarathon as prototype exercise. Clin J Sport Med 2001;11:38–43.
- 12↑
Pedersen BK. Special feature for the Olympics: effects of exercise on the immune system: exercise and cytokines. Immunol Cell Biol 2000;78:532–535.
- 13
Mastaloudes AMarrow JDHopkins DW et al. Antioxidant supplementation prevents exercise-induced lipid peroxidation, but not inflammation in ultramarathon runners. Free Radie Biol Med 2004;10:1329–1341.
- 14↑
Stewart LKFlynn MGCampbell WW et al. The influence of exercise on inflammatory cytokines and C-reactive protein. Med Sci Sports Exerc 2007;39:1714–1719.
- 15↑
Suzuki KPeake JNosaka K et al. Changes in markers of muscle damage, inflammation and HSP70 after an Ironman triathlon race. Eur J Appl Physiol 2006;98:525–534.
- 16↑
Robson-Ansley PJBlannin AGleeson M. Elevated plasma interlukin-6 levels in trained male athletes following an acute period of intense interval training. Eur J Appl Physiol 2007;99:353–360.
- 17
Fallon KEFallon SKBoston T. The acute phase response and exercise: court and field sports. Br J Sports Med 2001;35:170–173.
- 18
Smith LL. Cytokine hypothesis of overtraining: a physiological adaptation to excessive stress? Med Sci Sports Exerc 2000;32:317–331.
- 19
Czarkowska-Paczek BBartlomiejczyk IGabryls T et al. Lack of relationship between interleukin-6 and CRP levels in healthy male athletes. Immunol Lett 2005;99:136–140.
- 20↑
Burr JRReinhardt GASwaim SE et al. Serum biochemical values in sled dogs before and after competing in long-distance races. J Am Vet Med Assoc 1997;211:175–179.
- 21↑
Hinchcliff KWOlson JCrusberg C et al. Serum biochemical changes in dogs competing in a long-distance sled race. J Am Vet Med Assoc 1993;202:401–415.
- 22
Hinchcliff KWReinhart GABurr JR et al. Effect of racing on serum sodium and potassium concentrations and acid-base status of Alaskan sled dogs. J Am Vet Med Assoc 1997;210:1615–1618.
- 23
McKenzie ECJose-Cunilleras EHinchcliff KW et al. Serum chemistry alterations in Alaskan sled dogs during five successive days of prolonged endurance exercise. J Am Vet Med Assoc 2007;230:1486–1492.
- 24
Otabe KSugimoto TJinbo T et al. Physiological levels of C-reactive protein in normal canine sera. Vet Res Commun 1998;22:77–85.
- 25
McGrotty YLKnottenbelt CMRamsey IK et al. Evaluation of a rapid assay for canine C-reactive protein. Vet Rec 2004;154:175–176.
- 26↑
Szczepaniak WSZhang YHagerty S et al. Sphingosine l-phosphate rescues canine LPS-induced acute lung injury and alters systemic inflammatory cytokine production in vivo. Transl Res 2008;152:213–224.
- 27↑
Wakshlag JJSnedden KReynolds AJ. Biochemical and metabolic changes due to exercise in sprint-racing sled dogs: implications for post-exercise carbohydrate supplements and hydration management. Vet Ther 2004;5:52–59.
- 28↑
Ceron JJEchersall PDMartinez-Subiela S. Acute phase proteins in dogs and cats: current knowledge and future perspectives. J Vet Clin Pathol 2005;35:85–97.
- 29↑
Yamashita KFujinaga TMiyamoto T et al. Canine acute phase response: relationship between serum cytokine activity and acute phase protein in dogs. J Vet Med Sci 1994;56:487–492.
- 30↑
Petersen HHNielsen JPHeegaard PM. Application of acute phase protein measurements in veterinary medicine. Vet Res 2004;35:163–187.
- 31
Taylor CRogers GGoodman C, et al. Hématologie, iron-related, and acute phase protein responses to sustained strenuous exercise. J Appl Physiol 1987;62:464–469.
- 32
Peeling PDawson BGoodman C et al. Athletic induced iron-deficiency: new insights into the role of inflammation, cytokines and hormones. Eur J Appl Physiol 2008;103:381–391.
- 33
Kim SHYoung HKim WK et al. Nutritional status, iron-deficiency-related indices and immunity of female athletes. Nutrition 2002;18:86–90.
- 34
Hinton PSSinclair LM. Iron supplementation maintains ventilator threshold and improves energetic efficiency in iron-deficient nonanemic athletes. Eur J Clin Nutr 2007;61:30–39.
- 35
Banfi GDiGaetano NLopez RS, et al. Decreased mean sphere cell volume values in top-level rubgy players are related to the intravascular hemolysis induced by exercise. Lab Hematol 2007;13:103–107.
- 36
Hinchcliff KWReinhart GABurr JR et al. Exercise-associated hyponatremia in Alaskan sled dogs: urinary and hormonal responses. J Appl Physiol 1997;83:824–829.
- 37
Steiss JAhmad HACooper P et al. Physiologic response in healthy Labrador Retrievers during field trial training and competition. J Vet Intern Med 2004;18:147–151.
- 38
Matwichuk CLTaylor SMShmon CL et al. Changes in rectal temperature and hematologic, biochemical, blood gas, and acid-base values in healthy Labrador Retrievers before and after strenuous exercise. Am J Vet Res 1999;60:88–92.
