Dogs involved in intense or endurance-related activity such as hunting, competing, playing, or working undergo several physiologic changes during and after exercise, including depletion of energy stores (muscle glycogen and triglycerides),1–4 fluctuation of hematologic and biochemical variables within and beyond reference limits,5–7 and an increase in protein catabolism and amino acid oxidation.8–10 The ability of a dog to replenish its muscle and liver energy stores, rebuild protein, and manage its oxidative stress load contributes to how well it will perform during subsequent bouts of exercise.
In human athletes, carbohydrate consumption immediately after exercise significantly increases blood glucose concentration and enhances glycogen replenishment in muscle.11–13 Results of studies1,2 involving aerobically conditioned dogs indicate that consumption of rapidly digestible carbohydrates (glucose polymers or maltodextrin) immediately after exercise also enhances replenishment of muscle glycogen stores within the first 4 to 24 hours after exercise. Dogs fed glucose polymers immediately after exercise had enhanced muscle glycogen replenishment and increased blood glucose concentrations within 30 minutes, compared with those of dogs that consumed only water.2 A rapid increase in blood glucose concentration is important during the immediate postexercise period because the capacity of muscle to absorb glucose is enhanced during the initial 60 minutes after cessation of exercise, which increases glycogenesis.11 However, dogs in the studies1,2 conducted to evaluate glycogen replenishment following exercise were administered only 1.5 g of maltodextrin/kg in a liquid form. The effect of the ingestion of different formulations or doses of carbohydrate on the blood glucose concentration and glycogen replenishment in dogs following exercise is unknown.
Aside from glycogen depletion, muscle10,14 and liver15 protein turnover and amino acid metabolism are affected during and after exercise. Results of 1 study10 indicate that muscle protein synthesis in dogs was stimulated when an infusion of amino acids with glucose was administered immediately after exercise but was reduced when the infusion was initiated 2 hours after exercise. In dogs, data regarding exercise-induced changes in venous amino acid concentrations and the effect of consumption of a protein-enriched food immediately after exercise on postprandial aminoacidemia are lacking.
Besides macronutrients like carbohydrates and protein, other nutrients ingested as purified supplements, such as vitamin E, β-carotene, and lutein,16 or natural ingredients, such as blueberries,17 may provide nutritional ergonomic benefit for canine athletes. Astaxanthin, a carotenoid naturally found in marine algae (Haematococcus pluvialis) and invertebrates like krill and shrimp, has antioxidant and anti-inflammatory properties in a variety of applications.18 Astaxanthin had a positive influence on glucose and lipid metabolism in rodent exercise studies19–21 but not in studies22,23 that involved human athletes.
The effect of feeding H pluvialis as a naturally enriched source of astaxanthin on the glucose and fat metabolism of exercise-conditioned dogs is unknown. Although it is accepted that canine athletes benefit from nutrition-based glycogen replenishment strategies following exercise, data regarding postprandial changes in blood glucose and amino acid concentrations and biochemical variables associated with the feeding of additional and complementary supplemental nutrients in the immediate postexercise period are lacking. The purpose of the study reported here was to assess the effects of feeding a supplemental carbohydrate and protein bar with or without astaxanthin from H pluvialis to exercise-conditioned dogs immediately after exercise. We hypothesized that a supplemental food containing dextrose, maltodextrin, and starches fed to dogs immediately after exercise would result in a rapid postprandial blood glycemic response similar to that reported by investigators of another study1 in which dogs received only maltodextrin for enhancement of glycogen recovery. Additionally, we hypothesized that a supplemental food containing whey protein, which stimulates muscle protein synthesis in human athletes, would cause a rapid postprandial increase in blood BCAA concentration and aminoacidemia in dogs when fed immediately after exercise.
Materials and Methods
The study consisted of 2 phases. All study protocols were designed and followed in strict accordance with the guidelines established by the Nestlé Purina PetCare Animal Care and Use Advisory Committee. For both phases of the study, all dogs were privately owned and were enrolled only after owner consent was obtained.
Percentage of supplemental carbohydrate necessary to significantly increase postexercise plasma glucose concentration from the pre-exercise concentration (phase 1)
The objective of phase 1 was to determine the percentage of supplemental carbohydrate necessary to significantly increase postexercise plasma glucose concentration from the pre-exercise concentration. It consisted of 3 exercise trials that were performed in the morning when the ambient temperature ranged from 7.2° to 18.3°C during July and August.
Animals, care, and conditioning—Ten adult Huskey-Pointer crossbred dogs that ranged in age from 1 to 7 years with a mean ± SD body weight of 21.4 ± 3.4 kg were enrolled in phase 1. All dogs selected for phase 1 were fed the same basal dieta prior to and throughout the duration of the observation period and had similar exercise training (ie, physical conditioning and experience running on an exercise wheel). Each dog was individually fed to maintain an optimal body condition score of 4 on the scale (range, 1 to 9) developed and validated for dogs by Laflamme.24 Dogs were fed once daily in the afternoon after exercise, and fresh water was available ad libitum before and after exercise.
Each dog had undergone exercise conditioning for at least 1 month prior to initiation of phase 1. The training regimen consisted of 1.5 to 2 hours of moderate-paced trotting (approx 15 km/h) on an exercise wheel every other day for 3 days a week. The exercise wheel used allowed 10 dogs to be exercised simultaneously with approximately 3 m between dogs.
Experimental design—Phase 1 consisted of 3 trials (A, B, and C) in which the supplemental nutrients (bar or dry food) were fed immediately after exercise. For trial A, the 10 study dogs were randomly allocated by means of a computer-generated programb to 1 of 2 treatment groups. Five dogs were fed a supplemental carbohydrate and protein bar that contained 13.5% maltodextrin and 5% dextrose as rapidly digestible carbohydrates (ie, rapidly digestible carbohydrate content, 18.5%) and 15% whey protein as rapidly digestible protein, and 5 dogs were not fed the supplemental bar and served as untreated controls. Trials B and C were designed as crossover trials, and as such, consisted of 2 exercise sessions. In the same manner as that used for trial A, each dog was randomly assigned to receive 1 of 2 treatments (treatment, n = 5; control, 5) after the first exercise session and then, after a 1-week washout period, received the opposite treatment after the second exercise session. Thus, all 10 dogs received the supplemental nutrients administered during trials B and C. The treatment for trial B consisted of a supplemental carbohydrate and protein bar that was similar to that used in trial A, except the rapidly digestible carbohydrate fraction consisted of 32.4% maltodextrin and 5% dextrose (ie, rapidly digestible carbohydrate content, 37.4%). The treatment for trial C consisted of 60 g of a dry kibble ration of extruded chicken and rice made with only cereal grain starches and formulated to be representative of a maintenance food without any supplemental maltodextrin, dextrose (ie, rapidly digestible carbohydrate content, 0%), or whey protein. The components of the treatment supplements were summarized (Appendix). For each of the supplemental nutrient bars assessed in trials A and B, the ingredients were blended and heat treated to at least 79.4°C. The cooked product was then cut into 60-g bars. For each batch of treatment bars, a proximate analysis, which included determination of moisture, crude protein, crude fat, crude fiber, and ash content, was performed by use of standard methods.c The amount of test food fed to dogs during all 3 trials of phase 1 was set at 60 g and was not standardized to the body weight of each dog.
Each exercise session consisted of a 2-hour run on an exercise wheel, in a manner similar to that described for exercise conditioning. During each exercise session, the dogs assigned to the treatment and control groups were exercised simultaneously.
Sample collection and analysis—From each dog during each trial, blood samples (2 mL) collected into tubes containing sodium fluoride were obtained by cephalic venipuncture immediately after (0 minutes) exercise before the treatment food was administered and at 15, 20, and 60 minutes after treatment administration. The sample collected at 0 minutes was obtained while the dogs were tethered to the exercise wheel, whereas all other samples were collected while the dogs were housed in their kennels. The blood samples were processed in a routine manner and plasma was harvested for glucose determination by means of an automated biochemical assayd that was performed in accordance with the manufacturer's instructions.
Postprandial nutrient profiles among exercise-conditioned dogs that were and were not fed a supplemental carbohydrate and protein bar with or without astaxanthin from H pluvialis (phase 2)
The objective of phase 2 was to compare the postprandial nutrient profiles among exercise-conditioned dogs that were or were not fed a supplemental carbohydrate and protein bar with or without astaxanthin from H pluvialis immediately after exercise. It was conducted outside when the ambient daily temperature ranged between −8.9° and 1.7°C.
Animals, care, and conditioning—Twenty-four adult Husky-Pointer crossbred dogs that were not involved in phase 1 and that ranged in age from 1 to 7 years with a mean ± SD weight of 22.1 ± 3.2 kg were enrolled in phase 2. All dogs were selected, fed, and cared for in the same manner as that described for the dogs of phase 1.
Each dog had undergone exercise conditioning for at least 2 months prior to initiation of phase 2. The training regimen for the dogs in phase 2 was the same as that for the dogs in phase 1. Additionally, once a week, dogs were harnessed as a group in front of an all-terrain vehicle and trotted at a speed of 27.4 km/h for 11.3 km.
Experimental design—A computer-generated programb was used to randomly assign each dog to 1 of 3 treatment groups. Dogs in the control group (n = 8) were not fed any supplemental nutrients. Dogs in the CP group (n = 8) were fed the same supplemental bar as that fed during trial B of phase 1 (ie, 32.4% maltodextrin and 5% dextrose for a rapidly digestible carbohydrate content of 37.4% plus 15% whey protein). Dogs in the CPA group (n = 8) were fed the same supplemental bars as the CP group, except each bar also contained astaxanthin (29.6 μg/g) from H pluvialise (Appendix). The astaxanthin content in the bars fed to the CPA group was determined by reverse phase high-performance liquid chromatography.f All treatments were administered immediately after exercise on 3 consecutive days. Dogs were fed their basal diet 2 to 3 hours after exercise, and the amount fed was not adjusted for the caloric intake from the postexercise supplemental nutrition.
The exercise session on each of 3 consecutive days consisted of the dogs running in a sled harness. On days 1 and 2, dogs ran for 11.3 km at a rate of 27.4 km/h; thus, each exercise session lasted approximately 25 minutes. On day 3, dogs ran for 16.1 km at a rate of 24.1 km/h so the session lasted approximately 40 minutes. On each day, dogs were exercised in 2 groups of 12 dogs, and each exercise group consisted of 4 dogs from each of the 3 treatment (CP, CPA, and control) groups. Because of the exercise conditioning, all dogs were able to complete all the exercise sessions of phase 2.
Sample collection and analysis—From each dog during each sample collection, a blood sample (6 mL) was obtained via cephalic venipuncture and divided among 3 blood tubes (1 serum separator tube, 1 tube containing sodium fluoride, and 1 tube containing heparin). On day 1, blood samples were obtained 20 minutes before the start of the exercise session while the dogs were tethered at their kennels and immediately after the exercise session prior to treatment administration while the dogs were still harnessed to the sled. On day 3, blood samples were obtained 20 minutes before the start of the exercise session (pre-exercise) and at 0 (immediately after the exercise session prior to treatment administration), 15, 30, and 60 minutes and 24 hours after the exercise session. All blood samples were collected while the dogs were tethered at their kennels with the exception of the 0-minute sample, which was obtained while the dogs were still harnessed to the sled. On both days 1 and 3, treatment was administered within 1 minute after the 0-minute blood sample was collected.
The blood samples were processed in a routine manner, and plasma was harvested for analysis. Plasma samples obtained from the blood samples collected in the tubes containing sodium fluoride were used to determine plasma glucose and lactate concentrations and those obtained from the blood samples collected in the tubes containing heparin were used to determine the concentrations of other biochemical analytes by means of an automated biochemical system.d Serum samples were used to determine the amino acid profiles by use of gas chromatography with a flame ionization detectorg in accordance with the manufacturer's instructions.
Statistical analysis
For each trial within phase 1, the plasma glucose concentration over time was assessed by use of a 2-way ANOVA. The model included fixed effects for blood collection time, treatment group (treatment or control), and the interaction between time and treatment group and a random effect to account for repeated measures within each dog.
Outcomes of interest for phase 2 were plasma concentrations of glucose and each biochemical analyte as well as serum amino acids. To assess whether exercise had a similar effect on all analytes (especially glucose) among dogs of all 3 treatment groups (CP, CPA, and control) before treatment initiation, a 2-way ANOVA was used to evaluate the plasma or serum concentration of each respective analyte on day 1 before and after exercise prior to treatment administration. The model included fixed effects for blood collection time (pre-exercise or postexercise), treatment group, and the interaction between time and treatment group and a random effect to account for repeated measures within each dog. To assess the effect of postexercise treatment for 3 consecutive days on all analytes among the 3 treatment groups, a repeated-measures ANOVA was used to evaluate the plasma concentration of each respective analyte over time on day 3. An unstructured covariance matrix was used, and the model included fixed effects for blood collection time (pre-exercise; 0, 15, 30, and 60 minutes; and 24 hours), treatment group, and the interaction between time and group. A random effect was also included in the model to account for repeated measures within individual dogs. A 1-way ANOVA was used to perform post hoc analyses, and the Bonferroni method was used to compare means among treatment groups. Linear regression was used to generate regression coefficients. All analyses were performed with statistical software,h and values of P ≤ 0.05 were considered significant.
Results
Phase 1
Mean ± SE plasma glucose concentrations for the treatment and control groups during trials A, B, and C were summarized (Table 1). Results indicated that the plasma glucose concentration did not change significantly over time between dogs fed no supplemental nutrients immediately after exercise (controls) and dogs fed a supplemental bar that contained 18.5% rapidly digestible carbohydrates (trial A) or dogs fed 60 g of a dry food without additional rapidly digestible carbohydrates (trial C). However, the plasma glucose concentration of dogs fed a supplemental bar that contained 37.4% rapidly digestible carbohydrates immediately after exercise (trial B) increased significantly, compared with the pre-exercise concentration (0 minutes; baseline), and was significantly higher than that for control dogs at 15, 30, and 60 minutes after treatment administration. During trial B, the mean plasma glucose concentration did not differ between the control and treatment groups at baseline. The mean plasma glucose concentration for the dogs in the treatment group increased from baseline by 9% and 14% at 15 and 30 minutes, respectively, after treatment administration, whereas that for the control dogs varied less than 1% from baseline during the same period. On the basis of these results, we chose to use the supplemental bar with 37.4% rapidly digestible carbohydrates in phase 2.
Mean ± SE plasma glucose concentrations (mg/dL) at various times after an exercise session for 10 adult Husky-Pointer crossbred dogs during trials A, B, and C of phase 1.
| Time after exercise (min) | |||||
|---|---|---|---|---|---|
| Trial | Treatment group | 0 | 15 | 30 | 60 |
| A | 18.5% RDC | 83.4 ± 4.2 | 79.4 ± 1.0 | 83.4 ± 3.8 | 88.8 ± 2.5 |
| Control | 90.4 ± 5.0 | 83.6 ± 2.7 | 87.4 ± 3.3 | 85.8 ± 3.8 | |
| B | 37.4% RDC | 96.0 ± 3.7 | 104.3 ± 6.1*† | 109.6 ± 5.0*† | 100.8 ± 5.4*† |
| Control | 97.7 ± 3.2 | 96.6 ± 3.3 | 97.2 ± 3.1 | 93.8 ± 3.2 | |
| C | 0% RDC | 94.9 ± 3.8 | 95.1 ± 3.2 | 97.4 ± 3.3 | 88.7 ± 2.5 |
| Control | 92.5 ± 2.7 | 96.5 ± 2.5 | 95.7 ± 2.1 | 90.1 ± 2.2 | |
Each exercise session consisted of a 2-hour run (rate, 15 km/h) on an exercise wheel with 3 m between dogs. All 10 dogs were exercised simultaneously during each session, and the same dogs participated in all 3 trials. Trial A was a randomized trial in which 5 dogs were fed a 60-g supplemental carbohydrate and protein bar that contained 13.5% maltodextrin and 5% dextrose (RDC content, 18.5%) and 15% whey protein and the other 5 dogs were not fed any supplemental nutrients (control) immediately after an exercise session. Trials B and C were randomized crossover trials, in which 5 dogs were randomly assigned to be controls while the other 5 dogs received supplemental nutrients immediately after 1 exercise session, then after a 1-week washout period, the dogs received the opposite treatment following a second exercise session. The treatment for trial B was a 60-g supplemental carbohydrate and protein bar that contained 32.4% maltodextrin and 5% dextrose (RDC content, 37.4%), whereas that for trial C was 60 g of a dry kibble ration of extruded chicken and rice that did not contain any additional maltodextrin or glucose (RDC content, 0%). Thus, for each treatment group, the values for trial A represent the mean ± SE for 5 dogs, whereas the values for trials B and C represent the mean ± SE for 10 dogs.
Within a treatment group, value differs significantly (P ≤ 0.05) from that at 0 minutes.
Within a trial, value for treatment group differs significantly (P ≤ 0.05) from the corresponding value for the control group.
RDC = Rapidly digestible carbohydrate.
Phase 2
Because the blood samples obtained on day 1 were collected prior to treatment administration, day 1 data were analyzed separately from day 3 data. Therefore, the data for day 1 represented the effect of a single exercise session on the measured analytes in the absence of treatment, whereas the data for day 3 represented the cumulative effect of 3 days of postexercise treatment administration on the measured analytes.
Day 1—The pre- and postexercise least squares means for the plasma analyte concentrations measured on day 1 were summarized (Table 2). For all biochemical analytes except cholesterol, the postexercise concentrations differed significantly from the pre-exercise concentrations; however, the interaction between sample collection time and treatment group was not significant for any biochemical analyte. This suggested that the changes in concentration were the result of the effect of exercise and not the effect of treatment group assignment; thus, prior to treatment initiation, the dogs in all 3 treatment groups were similar.
Least squares mean values for various plasma biochemical analyte and serum amino acid concentrations for 24 adult Husky-Pointer crossbred dogs before and immediately after the exercise session on day 1 of phase 2.
| Variable | Pre-exercise | Postexercise | SE | P value |
|---|---|---|---|---|
| Electrolytes | ||||
| Calcium (mg/dL) | 10.38 | 10.26 | 0.07 | 0.050 |
| Chloride (mg/dL) | 113.0 | 117.5 | 0.8 | < 0.001 |
| Magnesium (mg/dL) | 2.36 | 2.17 | 0.04 | < 0.001 |
| Phosphorus (mg/dL) | 4.6 | 3.0 | 0.1 | < 0.001 |
| Potassium (mg/dL) | 4.69 | 4.48 | 0.05 | 0.006 |
| Sodium (mg/dL) | 151.2 | 158.3 | 0.7 | < 0.001 |
| Enzymes and proteins | ||||
| Alanine aminotransferase (U/L) | 52.3 | 58.5 | 7.3 | < 0.001 |
| Alkaline phosphatase (U/L) | 34.5 | 45.2 | 5.1 | < 0.001 |
| Aspartate aminotransferase (U/L) | 35.0 | 56.3 | 4.9 | < 0.001 |
| Albumin (g/dL) | 3.85 | 4.04 | 0.04 | < 0.001 |
| Creatine kinase (U/L) | 128.6 | 226.4 | 11.5 | < 0.001 |
| C-reactive protein (μg/mL) | 6.4 | — | — | — |
| γ-Glutamyltransferase (U/L) | 5.1 | 4.6 | 1.3 | 0.008 |
| Globulin (g/dL) | 2.24 | 2.35 | 0.06 | < 0.001 |
| Lactate dehydrogenase (U/L) | 55.3 | 129.7 | 6.6 | < 0.001 |
| Total protein (g/dL) | 6.1 | 6.4 | 0.1 | < 0.001 |
| Metabolites | ||||
| Cholesterol (mg/dL) | 197.0 | 199.6 | 12.8 | 0.170 |
| Creatinine (mg/dL) | 0.84 | 1.22 | 0.03 | < 0.001 |
| Glucose (mg/dL) | 86.7 | 114.0 | 4.8 | 0.002 |
| Lactate (mg/dL) | 1.0 | 4.8 | 0.1 | < 0.001 |
| Total bilirubin (mg/dL) | 0.11 | 0.20 | 0.01 | < 0.001 |
| Triglycerides (mg/dL) | 32.2 | 62.8 | 3.5 | < 0.001 |
| Urea nitrogen (mg/dL) | 18.9 | 20.5 | 0.6 | 0.002 |
| Amino acids | ||||
| BCAAs (nmol/mL) | 475.9 | 530.5 | 36.1 | 0.04 |
| Histidine (nmol/mL) | 310.0 | 269.7 | 191.2 | 0.67 |
| Isoleucine (nmol/mL) | 89.3 | 89.1 | 6.4 | 0.95 |
| Leucine (nmol/mL) | 163.8 | 183.9 | 12.4 | 0.04 |
| Lysine (nmol/mL) | 118.9 | 139.4 | 20.3 | 0.23 |
| Methionine (nmol/mL)* | 56.7 | 79.9 | 12.7 | < 0.001 |
| Phenylalanine (nmol/mL) | 73.3 | 123.3 | 8.0 | < 0.001 |
| Threonine (nmol/mL) | 229.0 | 276.2 | 38.5 | 0.07 |
| Tryptophan (nmol/mL) | 49.7 | 47.0 | 5.9 | 0.60 |
| Tyrosine (nmol/mL) | 50.9 | 88.1 | 7.1 | < 0.001 |
| Valine (nmol/mL) | 223.1 | 257.8 | 19.4 | 0.02 |
| Tyrosine-to-tryptophan ratio | 1.1 | 2.1 | 0.2 | < 0.001 |
The phase 2 study population consisted of different dogs than those that participated in phase 1. Each dog was randomly assigned to 1 of 3 treatment groups, and treatments were administered immediately after an exercise session on each of 3 consecutive days. Dogs in the control group (n = 8) were not fed any supplemental nutrients. Dogs in the CP group (n = 8) were fed the same supplemental bar as that fed during trial B of phase 1. Dogs in the CPA group (n = 8) were fed the same supplemental bar as the CP group, except each bar also contained astaxanthin (29.6 μg/g) from Haematococcus pluvialis. Postexercise values were determined before the assigned treatment was administered. The exercise session on day 1 consisted of the dogs running in a sled harness for 11.3 km at a rate of 27.4 km/h. Pre-exercise and postexercise concentrations for all electrolytes, enzymes, proteins, and metabolites analyzed except cholesterol differed significantly (P ≤ 0.05), which indicated that the dogs of the 3 treatment groups responded similarly to exercise; thus, the data for all dogs were pooled for reporting purposes on day 1.
The interaction between sample collection time and treatment group was significant (P = 0.006) for this analyte.
— = Not determined.
See Table 1 for information about phase 1 of the study.
The pre- and postexercise least squares means for the plasma amino acid concentrations measured on day 1 were also summarized (Table 2). The postexercise concentrations of BCAAs, leucine, methionine, phenylalanine, tyrosine, and valine, and the tryosine-to-tryptophan ratio were all significantly higher than the corresponding pre-exercise measurements. Results of the ANOVA indicated that the fixed effects for treatment group and the interaction between treatment group and sample collection time were not significantly associated with the plasma concentrations of any of those amino acids, except methionine The mean ± SE postexercise plasma methionine concentration for dogs in the control group (93.6 ± 5.9 nmol/mL) was significantly (P = 0.005) higher than that for the dogs in the CPA group (60.3 ± 7.3 nmol/mL) but was not significantly (P = 0.200) different from that for the dogs in the CP group (79.0 ± 5.8 nmol/mL).
Day 3—On day 3, the mean plasma lactate concentration did not differ significantly among the 3 treatment groups at any sample collection time. Results of the ANOVA indicated that sample collection time was significantly (P < 0.001) associated with plasma lactate concentration. The mean ± SE plasma lactate concentration immediately after exercise and prior to treatment administration (0 minutes; 1.7 ± 0.5 mmol/L) was significantly greater than that pre-exercise (baseline; 1.1 ± 0.3 mmol/L), and no dog in the control group had a plasma lactate concentration > 1.9 mg/dL. The mean plasma lactate concentration for all treatment groups returned to near baseline concentrations by 15 minutes after exercise and did not differ significantly from the baseline concentrations for the remainder of the observation period (60 minutes; data not shown).
The mean day 3 plasma glucose concentration was significantly (P < 0.001) associated with the interaction between sample collection time and treatment group (Figure 1). At baseline, the mean plasma glucose concentration for dogs in the CP group did not differ significantly from that for dogs in the CPA or the control groups; however, the mean plasma glucose concentration for dogs in the control group was significantly higher, compared with that for dogs in the CPA group. For dogs in the CPA group, the mean plasma glucose concentration was decreased from the baseline concentration by only 4.1% immediately after exercise and did not differ significantly from the baseline concentration at any time throughout the observation period. Conversely, the mean plasma glucose concentration for dogs in the CP and control groups was significantly decreased from the baseline concentrations by 34.1% and 30.5%, respectively, immediately after exercise. For dogs in the CP group, the mean plasma glucose concentration at 15 minutes after exercise was increased by 21.6%, compared with that immediately after exercise (P = 0.10) and remained 15.5% less than that at baseline (P = 0.06). By 30 minutes after exercise, the mean plasma glucose concentration for the dogs in the CP group increased 28.8% from that at 15 minutes after exercise and did not differ significantly from the baseline concentration. The mean plasma glucose concentration for the control dogs was significantly decreased from the baseline concentration throughout the 60 minutes immediately after exercise. At its nadir 15 minutes after exercise, the mean plasma glucose concentration for the control dogs was decreased by 45%, compared with the baseline concentration, and it was still decreased 40% from the baseline concentration at 60 minutes after exercise. Mean plasma glucose concentrations for all 3 treatment groups had returned to baseline concentrations and did not differ significantly at 24 hours after exercise on day 3 (data not shown).
Mean ± SE plasma concentrations of glucose (A) and triglyceride (B) over time on day 3 of phase 2 for dogs that were fed a 60-g supplemental carbohydrate and protein bar that contained 32.4% maltodextrin and 5% dextrose as rapidly digestible carbohydrates and 15% whey protein as rapidly digestible protein with (CPA group; n = 8; solid black line with black squares) or without (CP group; 8; solid black line with white squares) astaxanthin (29.6 μg/g) from Haematococcus pluvialis or were not fed any supplemental nutrients (control group; 8; dashed line with black circles) immediately after an exercise session (time, 0) on each of 3 consecutive days. On days 1 and 2, dogs ran for 11.3 km at a rate of 27.4 km/h; thus, each exercise session lasted approximately 25 minutes. On day 3, dogs ran for 16.1 km at a rate of 24.1 km/h so the session lasted approximately 40 minutes. On each day, dogs were exercised in 2 groups of 12 dogs, and each exercise group consisted of 4 dogs from each of the 3 treatment (CP, CPA, and control) groups. For dogs in the CPA and CP groups, the blood sample obtained at 0 minutes was collected immediately prior to administration of the supplemental bar. The black box on the x-axis represents the time the dogs spent exercising on day 3. Notice the scale of the y-axis varies between panels. *Within a treatment group, mean differs significantly (P ≤ 0.05) from the mean 20 minutes prior to exercise (–60 minutes; baseline). a,bWithin a sample collection time, means with different letters differ significantly (P ≤ 0.05).
Citation: American Journal of Veterinary Research 76, 4; 10.2460/ajvr.76.4.338
Mean ± SE plasma concentrations of glucose (A) and triglyceride (B) over time on day 3 of phase 2 for dogs that were fed a 60-g supplemental carbohydrate and protein bar that contained 32.4% maltodextrin and 5% dextrose as rapidly digestible carbohydrates and 15% whey protein as rapidly digestible protein with (CPA group; n = 8; solid black line with black squares) or without (CP group; 8; solid black line with white squares) astaxanthin (29.6 μg/g) from Haematococcus pluvialis or were not fed any supplemental nutrients (control group; 8; dashed line with black circles) immediately after an exercise session (time, 0) on each of 3 consecutive days. On days 1 and 2, dogs ran for 11.3 km at a rate of 27.4 km/h; thus, each exercise session lasted approximately 25 minutes. On day 3, dogs ran for 16.1 km at a rate of 24.1 km/h so the session lasted approximately 40 minutes. On each day, dogs were exercised in 2 groups of 12 dogs, and each exercise group consisted of 4 dogs from each of the 3 treatment (CP, CPA, and control) groups. For dogs in the CPA and CP groups, the blood sample obtained at 0 minutes was collected immediately prior to administration of the supplemental bar. The black box on the x-axis represents the time the dogs spent exercising on day 3. Notice the scale of the y-axis varies between panels. *Within a treatment group, mean differs significantly (P ≤ 0.05) from the mean 20 minutes prior to exercise (–60 minutes; baseline). a,bWithin a sample collection time, means with different letters differ significantly (P ≤ 0.05).
Citation: American Journal of Veterinary Research 76, 4; 10.2460/ajvr.76.4.338
Mean ± SE plasma concentrations of glucose (A) and triglyceride (B) over time on day 3 of phase 2 for dogs that were fed a 60-g supplemental carbohydrate and protein bar that contained 32.4% maltodextrin and 5% dextrose as rapidly digestible carbohydrates and 15% whey protein as rapidly digestible protein with (CPA group; n = 8; solid black line with black squares) or without (CP group; 8; solid black line with white squares) astaxanthin (29.6 μg/g) from Haematococcus pluvialis or were not fed any supplemental nutrients (control group; 8; dashed line with black circles) immediately after an exercise session (time, 0) on each of 3 consecutive days. On days 1 and 2, dogs ran for 11.3 km at a rate of 27.4 km/h; thus, each exercise session lasted approximately 25 minutes. On day 3, dogs ran for 16.1 km at a rate of 24.1 km/h so the session lasted approximately 40 minutes. On each day, dogs were exercised in 2 groups of 12 dogs, and each exercise group consisted of 4 dogs from each of the 3 treatment (CP, CPA, and control) groups. For dogs in the CPA and CP groups, the blood sample obtained at 0 minutes was collected immediately prior to administration of the supplemental bar. The black box on the x-axis represents the time the dogs spent exercising on day 3. Notice the scale of the y-axis varies between panels. *Within a treatment group, mean differs significantly (P ≤ 0.05) from the mean 20 minutes prior to exercise (–60 minutes; baseline). a,bWithin a sample collection time, means with different letters differ significantly (P ≤ 0.05).
Citation: American Journal of Veterinary Research 76, 4; 10.2460/ajvr.76.4.338
The mean baseline plasma triglyceride concentration for dogs in all 3 treatment groups on day 3 was 2.5 to 3 times that of the mean baseline plasma triglyceride concentration for those dogs on day 1. Although results of the ANOVA failed to indicate a significant association between plasma triglyceride concentration and the interaction between sample collection time and treatment group, the mean day 3 baseline plasma triglyceride concentration for the control dogs was lowest and was significantly lower (approx 31%) than that for the dogs in CPA group, which was the highest (Figure 1). However, the mean plasma triglyceride concentration did not differ significantly among the treatment groups at any other time during the postexercise observation period. Plasma triglyceride concentration was significantly (P < 0.001) associated with sample collection time. For each treatment group, the mean plasma triglyceride concentration was significantly lower than the baseline concentration at 15, 30, and 60 minutes after exercise. Results of linear regression indicated that the baseline plasma triglyceride concentration on day 3 was inversely correlated with the baseline plasma glucose concentration on day 3 (R2 = 0.51; P < 0.001; Figure 2). Although other plasma biochemical parameters varied among the treatment groups over time, none of the changes were considered clinically relevant, but were reported as supplementary data for characterization of acute postexercise variation influenced by dietary treatment.i
Scatterplot of the baseline plasma triglyceride concentration versus the baseline plasma glucose concentration on day 3 of phase 2 for 21 of the 24 dogs of Figure 1 (CPA group, black squares [n = 8]; CP group, white circles [7]; control group, white triangles [6]). The solid black line represents the regression line of best fit and has an equation of y = −2.1154x + 265.11 and R2 = 0.5075. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 76, 4; 10.2460/ajvr.76.4.338
Scatterplot of the baseline plasma triglyceride concentration versus the baseline plasma glucose concentration on day 3 of phase 2 for 21 of the 24 dogs of Figure 1 (CPA group, black squares [n = 8]; CP group, white circles [7]; control group, white triangles [6]). The solid black line represents the regression line of best fit and has an equation of y = −2.1154x + 265.11 and R2 = 0.5075. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 76, 4; 10.2460/ajvr.76.4.338
Scatterplot of the baseline plasma triglyceride concentration versus the baseline plasma glucose concentration on day 3 of phase 2 for 21 of the 24 dogs of Figure 1 (CPA group, black squares [n = 8]; CP group, white circles [7]; control group, white triangles [6]). The solid black line represents the regression line of best fit and has an equation of y = −2.1154x + 265.11 and R2 = 0.5075. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 76, 4; 10.2460/ajvr.76.4.338
Results of the ANOVA indicated that the interaction between sample collection time and treatment group was significantly associated with the serum concentrations of all amino acids measured except histidine. Serum concentrations of leucine, BCAAs, threonine, phenylalanine, tyrosine, tryptophan, and lysine, and the tyrosine-to-tryptophan ratio prior to and throughout the first 60 minutes after exercise on day 3 were plotted (Figure 3).
Mean ± SE serum concentrations of leucine (A), BCAAs (B), threonine (C), phenylalanine (D), tyrosine (E), tryptophan (F), and lysine (G), and the tyrosine-to-tryptophan ratio (H) on day 3 of phase 2 for the dogs of Figure 1. †Within a treatment group, mean differs significantly (P ≤ 0.05) from the mean immediately after exercise (0 minutes). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 76, 4; 10.2460/ajvr.76.4.338
Mean ± SE serum concentrations of leucine (A), BCAAs (B), threonine (C), phenylalanine (D), tyrosine (E), tryptophan (F), and lysine (G), and the tyrosine-to-tryptophan ratio (H) on day 3 of phase 2 for the dogs of Figure 1. †Within a treatment group, mean differs significantly (P ≤ 0.05) from the mean immediately after exercise (0 minutes). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 76, 4; 10.2460/ajvr.76.4.338
Mean ± SE serum concentrations of leucine (A), BCAAs (B), threonine (C), phenylalanine (D), tyrosine (E), tryptophan (F), and lysine (G), and the tyrosine-to-tryptophan ratio (H) on day 3 of phase 2 for the dogs of Figure 1. †Within a treatment group, mean differs significantly (P ≤ 0.05) from the mean immediately after exercise (0 minutes). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 76, 4; 10.2460/ajvr.76.4.338
Compared with baseline concentrations, the mean serum tryptophan concentration for the control dogs was decreased 50%, whereas the mean serum phenylalanine and tyrosine concentrations and the tyrosine-to-tryptophan ratio were increased by 50%, 65%, and 212%, respectively. The mean concentrations for the rest of the amino acids assessed for the control group immediately after exercise did not differ significantly from baseline concentrations.
The mean concentrations of leucine, isoleucine, and tryptophan immediately after exercise for the dogs in the CP and CPA groups were significantly decreased from the corresponding baseline concentrations. Similarly, the histidine concentration immediately after exercise for dogs in the CPA group was significantly decreased from the baseline concentration. Conversely, the tyrosine-to-tryptophan ratio immediately after exercise for the dogs in the CP and control groups was significantly increased from the baseline ratio.
For dogs in both the CP and CPA groups, the mean serum leucine concentration at 15 minutes after exercise was increased 41%, compared with that immediately after exercise, and it continued to increase for both groups until 60 minutes after exercise (Figure 3). Conversely, the mean serum leucine concentration for the control dogs decreased from the baseline concentration and reached its nadir at 15 minutes after exercise. At 30 minutes after exercise, the mean serum leucine concentration for dogs in the CP and CPA groups was significantly higher than that for the control dogs. At 60 minutes after exercise, the mean serum leucine concentration for dogs in the CP group was significantly higher than that for the dogs in the CPA and control groups.
Immediately after exercise, the mean serum phenylalanine and tyrosine concentrations did not differ significantly from the baseline concentrations for dogs in the CP and CPA groups but were increased 50% and 65%, respectively, from the baseline concentrations for the control dogs. The mean serum isoleucine and valine concentrations responded similarly among the 3 treatment groups for the first 60 minutes after exercise.
Discussion
Results of the present study indicated that feeding exercise-conditioned dogs a 60-g supplemental carbohydrate and protein bar that contained 32.4% maltodextrin and 5% dextrose as rapidly digestible carbohydrates and 15% whey protein as rapidly digestible protein immediately after exercise resulted in rapid postprandial increases in plasma concentrations of glucose as well as some amino acids such as BCAAs, leucine, threonine, and tryptophan within 15 minutes. Interestingly, the addition of astaxanthin from H pluvialis to the supplemental carbohydrate and protein bar increased the pre-exercise (baseline) plasma triglyceride concentration and prevented an exercise-induced decrease in plasma glucose concentration.
The prototype supplemental carbohydrate and protein bars administered to the dogs in the CPA group were the same as those administered to the dogs of the CP group except for the addition of H pluvialis for enrichment of astaxanthin. Given the mean weight of the study dogs and the supplemental bar fed, the dogs ingested 1.7 g of carbohydrates/kg, of which 1.0 g/kg was rapidly digestible carbohydrates (maltodextrin and dextrin), and 0.65 g of protein/kg, of which 0.33 g/kg was rapidly digestible protein (whey). Thus, the amount of supplemental carbohydrates and protein fed to the dogs of this study was similar to the amount of supplemental carbohydrates and protein (carbohydrate, 0.8 g/kg; protein, 0.2 g/kg) consumed by human endurance athletes,25 and which was beneficial for increasing synthesis of muscle glycogen after exercise.11,12,26–28 The amount of supplemental carbohydrates (1.7 g/kg) fed to the dogs of the present study was slightly more than that (1.5 g/kg) fed to dogs of other studies1,2 in which no supplemental protein was fed and exceeded the optimal amount (1.2 g/kg) recommended for human athletes.11,12 The supplemental bars fed in the present study contained multiple carbohydrate ingredients that provided polysaccharide chains of various lengths and were especially selected to provide a rapid and sustained increase in plasma glucose concentration to promote replenishment of glycogen stores.
In dogs and humans, glucose absorption capacity in the gastrointestinal tract29,30 and muscle28,31 is temporarily enhanced following exercise because of an increase in the number of glucose transporters (GLUT4) in the plasma membrane of muscle cells32,33 and a concurrent increase in both the active and passive absorption of glucose in the small intestine,33 which are maximally maintained for 60 minutes after the cessation of exercise. Results of multiple studies11,28 indicate that optimal recovery of muscle glycogen stores following exercise is time dependent; therefore, ingestion of rapidly digestible carbohydrates as soon as possible within the 60-minute period of maximum glucose absorption and glycogenesis after exercise will facilitate a rapid increase in blood glucose concentration. In the present study, the feeding of a supplemental carbohydrate and protein bar to exercise-conditioned dogs immediately after exercise resulted in an increase in plasma glucose concentration within 15 minutes, and feeding the bar with astaxanthin resulted in a plasma glucose concentration that was fairly stable from before to after exercise and was significantly higher after exercise than that for dogs that were not administered any supplemental nutrients (controls). Although muscle glycogen content was not measured in the dogs of the present study, we suspect that it would have mirrored the plasma glucose concentration because ingestion of a recovery food immediately after exercise in canine1,2 and human11–13 athletes causes a rapid increase in blood glucose concentration that facilitates replenishment of muscle glycogen stores. Moreover, the design of the present study did not allow us to assess fatigue or performance of the dogs. A decrease in fatigue or an improvement in performance of canine athletes fed the supplemental bars used in the present study immediately after exercise would provide additional support for our speculation that glycogen replenishment was enhanced.
In a study12 that involved adult human athletes, ingestion of approximately 20 g of protein in addition to rapidly digestible carbohydrates below the recommended optimum of 1.2 g/kg/h stimulated muscle glycogenesis. Results of another study34 that involved human athletes indicate that the magnitude of postexercise glycogenesis following ingestion of protein at a rate of 0.4 g/kg/h in the form of leucine combined with a protein hydrolysate in addition to carbohydrates was significantly greater than that following ingestion of carbohydrates only. Further research is necessary to determine whether consumption of foods or supplemental nutrients that contain proteins enriched with specific amino acids in combination with rapidly digestible carbohydrates might similarly improve postexercise glycogen replenishment in canine athletes. The amount of leucine and protein ingested by the dogs of the present study met or exceeded the amount of leucine and protein proven to be beneficial for glycogen replenishment in human athletes.
One of the most interesting findings of the present study was that the dogs fed the supplemental carbohydrate and protein bars with astaxanthin had a significantly higher baseline (pre-exercise) plasma triglyceride concentration on day 3 than did the control dogs and did not have a significant decrease in plasma glucose concentration after exercise. Results of multiple studies18,35–37 suggest that astaxanthin has potent antioxidant and anti-inflammatory properties. The metabolism of mice fed astaxanthin (1.2 to 30 mg/kg) for at least 3 weeks differed (endurance capacity was improved, and postexercise blood lactate concentration was decreased) from that of mice that were not fed astaxanthin.19 In another study,20 mice fed astaxanthin for 4 weeks had increased lipid and decreased glucose utilization, compared with mice that were not fed astaxanthin, which suggested that astaxanthin contributes to the sparing of endogenous glycogen stores. In rodents, the mechanism by which astaxanthin influences lipid metabolism appears to be mediated by peroxisome proliferator-activated receptor-γ coactivator-1α.21 However, astaxanthin ingestion has yielded conflicting results for human endurance athletes. The performance of human athletes who ingested astaxanthin was improved in 1 study22 and unchanged in another,23 compared with the performance of athletes who did not ingest astaxanthin. The amount of astaxanthin administered to the dogs of the present study (0.08 mg/kg or 1.8 mg) was considerably lower than that administered to rodents19 (1.2 to 30 mg/kg) and higher than that administered to human athletes22 (0.05 mg/kg). Because the mean plasma triglyceride and glucose concentrations for the dogs administered the astaxanthin-enriched bar differed significantly from those of the control dogs, we suspect that the effect of astaxanthin on the energy metabolism of dogs is similar to that of rodents; however, we cannot determine that conclusively because we did not measure plasma free fatty acid concentrations. Further research is necessary to more fully characterize the effect of astaxanthin ingestion on the metabolism of dogs.
Catabolism of muscle protein occurs both during and after exercise38 and is characterized by an increase in the oxidation of BCAAs because they are an energy source for exercising muscles.39–41 Oxidation of BCAAs causes a decrease in muscle protein synthesis and an increase in muscle protein degradation.40 In human athletes, the leucine concentration in blood and skeletal muscles decreases significantly following exercise.42 Replenishment of BCAAs, particularly leucine, activates protein synthesis, promotes glycogen replenishment, and increases insulin secretion, all of which are necessary for postexercise muscle recovery.43–48
In humans, the ingestion of approximately 0.026 to 0.04 g of leucine/kg, which is equivalent to 0.25 to 0.31 g of whey protein/kg, after exercise stimulates muscle protein synthesis.49–51 The ingestion of leucine immediately after exercise results in a significant and rapid increase in blood leucine and total essential amino acid concentrations within 30 to 80 minutes, which in turn benefits muscle protein synthesis.49–51 The supplemental carbohydrate and protein bars fed in the present study were composed of multiple protein sources and contained approximately 14% leucine and 24.5% BCAAs to rapidly promote the delivery of specific amino acids critical for muscles during and after exercise. For the dogs that were fed these supplemental bars, plasma leucine concentration was significantly increased from that immediately prior to ingestion at 30 and 60 minutes. Given that the mean weight of the dogs in this study was 22.7 kg, consumption of a 60-g supplemental bar resulted in an intake of approximately 0.09 g of leucine/kg, which was substantially higher than the leucine intake required to increase blood amino acid concentration and postexercise protein synthesis in human athletes.49–51
Another interesting finding of the present study was that the plasma phenylalanine and tyrosine concentrations of the control dogs were significantly increased from baseline immediately after exercise but rapidly returned to baseline concentrations by 15 minutes after exercise, whereas those for the dogs fed the supplemental bars did not vary significantly from baseline concentrations at any time during the first 60 minutes after exercise. Exercise induces catabolism of muscle proteins and results in a net increase in blood phenylalanine concentration,10,52 which could cause a subsequent increase in blood tyrosine concentration.
We suspect that moderately intense exercise sessions on 3 consecutive days increased protein catabolism in dogs because the mean baseline plasma concentration of leucine for the control group on day 3 (282 nmol/mL) was 1.7 times as great as that on day 1 (169 nmol/mL). It is possible that this increase in the baseline plasma leucine concentration was the result of leucine released from enteric sources during and after exercise52,53 The exercise-induced increase in plasma leucine concentration observed in the dogs of this study is contrary to the decrease in plasma leucine concentration observed in human athletes involved in intensive training.42 These conflicting results for human and canine athletes may be a consequence of the intensity and duration of the exercise training, which was shorter for dogs (25 to 40 minutes), compared with that for the human athletes (50 minutes).42 Unpublished data recently obtained by our laboratory group for exercise-conditioned dogs that ran at a moderate speed (12.9 km/h) for 120 minutes (ie, similar exercise conditioning as that used in phase 1) suggest that plasma leucine concentration decreases by 20% to 30% between 60 and 120 minutes of exercise regardless of whether the dogs had run only 1 day or on 3 consecutive days. The mean pre-exercise or baseline serum leucine concentration for the dogs in that study was similar, compared with that on day 1 for the dogs of phase 2 in the present study, which was expected because the exercise regimens were the same. Most of the published literature8–11 about protein and amino acid metabolism in canine athletes was generated from dogs that underwent a single exercise session that involved running on a treadmill. Studies that involve dogs that undergo multiple or consecutive-day high-intensity exercise sessions are necessary to better elucidate the energy (glucose and triglyceride) and amino acid and protein metabolism of canine athletes.
In the present study, ingestion of the supplemental carbohydrate and protein bar resulted in a rapid postprandial increase in the mean serum tryptophan concentration, whereas the mean serum tryptophan concentration for the control dogs continued to decrease, reached a nadir at 15 minutes after exercise, and varied little from that nadir at 30 and 60 minutes after exercise. Conversely, ingestion of the supplemental bar had no effect on the mean serum tyrosine concentration until 60 minutes after exercise, whereas the mean serum tyrosine concentration for the control dogs was significantly increased from baseline immediately after exercise and then rapidly decreased by 15 minutes. Tryptophan and tyrosine are required for the production of the neurotransmitters dopamine and serotonin, the concentrations of which were not measured in this study. Additional research is necessary to determine how dopamine and serotonin concentrations respond to fluctuating concentrations of amino acid precursors in canine athletes.
Postexercise nutritional intervention is necessary to optimize the performance of dogs during exercise events, particularly those that occur over multiple days. The use of a postexercise nutritional intervention such as the supplemental carbohydrate and protein bar fed to the dogs of this study might aid in the replenishment and recovery of muscle energy and protein stores and delay or diminish the effects of exercise-induced fatigue. The benefits of the supplemental carbohydrate and protein bar evaluated in this study are best realized when it is fed to dogs after the exercise regimen for the day is complete and a prolonged period of rest is expected. Intermittent feeding of supplemental nutrients with a different composition or portion size than that of supplemental bar fed in this study to dogs before or between exercise sessions within a given day might further optimize the performance of canine athletes, but additional research is warranted to confirm or refute this. Given the current issue of obesity in companion animals, various types and doses of supplemental nutritional interventions should be investigated in conjunction with the type and duration of exercise performed and the exercise-conditioning status of the dogs receiving the intervention. Some investigators might advocate the administration of a low dose of supplemental nutrients to minimize consumption of extra calories, whereas others might advise that less conditioned or sedentary dogs be fed a high dose of supplemental nutrients to offset exercise-induced glycogen depletion. Further research is necessary to better characterize the appropriate use of supplemental nutrient interventions in dogs.
In the present study, feeding exercise-conditioned dogs a 60-g supplemental carbohydrate and protein bar that contained 32.4% maltodextrin and 5% dextrose as rapidly digestible carbohydrates and 15% whey protein as rapidly digestible protein immediately after exercise for 3 consecutive days facilitated the delivery of dietary glucose and amino acids (particularly leucine) into the circulation. Thus, this carbohydrate and protein formula provides dogs with the macronutrients necessary for replenishment of muscle glycogen and protein stores and facilitates postexercise recovery. The addition of astaxanthin (29.6 μg/g) from H pluvialis to the supplemental bar maximized pre-exercise plasma triglyceride concentration and prevented the exercise-induced decrease in plasma glucose concentration observed in the dogs fed the supplemental bar without astaxanthin and control dogs. However, the effect of astaxanthin on the energy and protein metabolism of canine athletes warrants further research.
Acknowledgments
Supported by Nestlé Purina PetCare.
None of the authors have any conflict of interest or affiliation to disclose related to this study.
The authors thank Wendell Kerr for assistance with statistical analyses.
ABBREVIATION
| BCAA | Branched-chain amino acid |
Footnotes
Purina ProPlan Performance chicken and rice dry dog food, Nestlé Purina PetCare Co, St Louis, Mo.
RAND function, Excel 2003, version 11.0, Microsoft Corp, Redmond, Wash.
NP Analytical Laboratories, St Louis, Mo.
Cobas, model c311, Roche Diagnostics, Indianapolis, Ind.
NatuRose, Cyanotech Corp, Kona, Hawaii.
Astaxanthin in concentrates and pet foods, NP Analytical Laboratories, St Louis, Mo.
EZ FAAST kit, GC-FID Free (Physiological) Amino Acid Analysis Kit, Phenomenex, Torrance, Calif.
SAS, version 9.1.2., SAS Institute Inc, Cary, NC.
A supplemental table of the least squares means for plasma biochemical analytes over time on day 3 for each treatment group is posted with the article at avmajournals.avma.org.
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Appendix
Ingredients and nutritional composition of the supplemental nutrients fed to exercise-conditioned adult Husky-Pointer dogs during phases 1 and 2.
| Phase 1 | Phase 2 | ||||
|---|---|---|---|---|---|
| Trial A | Trial B | Trial C | CP | CPA | |
| Ingredient (% of formulation) | |||||
| Dextrose | 5.0 | 5.0 | — | 5.0 | 5.0 |
| Maltodextrin | 13.5 | 32.4 | — | 32.4 | 32.4 |
| Cereal flours* | 42.9 | 28.6 | 33.1 | 28.6 | 28.6 |
| Corn gluten meal | 14.9 | 14.7 | 21.2 | 14.7 | 14.7 |
| Whey protein concentrate | 14.9 | 14.7 | — | 14.7 | 14.7 |
| Animal protein† | — | — | 42.3 | — | — |
| Tallow | 3.1 | 2.9 | 13.3 | 2.9 | 2.9 |
| Dry digest | 0.5 | 0.5 | 1.5 | 0.5 | 0.5 |
| Vitamins and minerals | 0.95‡ | 0.95‡ | 3.16§ | 0.95‡ | 0.95‡ |
| Emulsifier | 0.06 | 0.15 | — | 0.15 | 0.15 |
| Haematococcus pluvialis‖ | — | — | — | — | 0.48 |
| Proximate analysis (% as fed) | |||||
| Dry matter | 91.0 | 91.2 | 92.5 | 91.2 | 92.4 |
| Ash | 1.6 | 1.56 | 5.52 | 1.56 | 1.83 |
| Crude protein | 24.0 | 24.8 | 30.6 | 24.8 | 25.1 |
| Crude fat | 5.0 | 5.1 | 20.6 | 5.1 | 5.5 |
| Crude fiber | 0.20 | 0.23 | 1.53 | 0.23 | 0.50 |
| Astaxanthin (ppm) | 0.0 | 0.0 | 0.0 | 0.0 | 29.6 |
Cereal flours include rice, corn, and wheat flours.
Animal protein includes poultry, fish, and eggs.
Vitamins and minerals include sodium chloride, vitamin E, niacin, vitamin A, vitamin B12, zinc sulfate, calcium pantothenate, thiamine mononitrate, riboflavin, pyridoxine hydrochloride, menadione sodium bisulfite complex for vitamin K activity, biotin, folic acid, and vitamin D3.
Vitamins and minerals include sodium chloride, calcium carbonate, potassium chloride, calcium phosphate, vitamin E supplement, choline chloride, l-lysine monohydrochloride, l-ascorbyl-2-polyphosphate (source of vitamin C), zinc sulfate, ferrous sulfate, manganese sulfate, niacin, vitamin A supplement, calcium pantothenate, thiamine mononitrate, copper sulfate, riboflavin supplement, vitamin B12 supplement, pyridoxine hydrochloride, folic acid, vitamin D3 supplement, calcium iodate, biotin, menadione sodium bisulfite complex (source of vitamin K activity), and sodium selenite.
From NatuRose.e