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Serum measures are affected by food in growing large breed puppies References from Antioxidants and Immunity Continued from page 5 1.VanSteenhouse JL. Free radicals: relation to tissue damage – a review. Vet Clin Pathol. 1987;16:29-35. 2.Jewell DE, Toll PW, Wedekind KJ, et al. Effect of increasing dietary antioxidants on concentrations of vitamin E and total alkenals in serum of dogs and cats. Vet Ther. 2000;1:264-272. 3.Wedekind KJ, Zicker S, Lowry S, et al. Antioxidant status of adult beagles is affected by dietary antioxidant intake. J Nutr. 2002;132:1658S-1660S. 4.Chew BS, Park JS, Wong TS, et al. Dietary -carotene stimulates cell-mediated and humoral immune response in dogs. J Nutr. 2000;130:1910-1913. 5.Heaton P, Reed CF, Mann SJ, et al. Role of dietary antioxidants to protect against DNA damage in adult dogs. J Nutr. 2002;132:1720S-1724S. Food was offered in portions in accordance with feeding guides recommended by the manufacturers. No other foods or treats were allowed to be fed for the duration of the study. Food offerings were adjusted to maintain body condition score close to 3 (5-point scale) but were not allowed to be above or below feeding guidelines on the bag. All puppies received standard vaccinations and were spayed or neutered at six months of age by their local veterinarian. During the test period, blood samples were drawn at baseline, 3, 5 and 12 months of age for analysis of health biomarkers including serum chemistry, complete blood count, vitamin E and fatty acid analysis. Dogs were scanned by dual X-ray absorptiometry (DXA) at 2, 5 and 12 months of age to document changes in body composition and bone density. All hemogram and blood chemistry values were within normal ranges for all groups at the beginning and end of the study. Concentration of omega-3 fatty acids in the blood (alpha-linoleic acid [ALA], eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) was not significantly different between groups prior to dietary intervention but puppies fed the test food with the nutritional profile of Science Diet® Puppy Large Breed Dry pet food formula had significantly greater blood concentration of omega-3 fatty acids than puppies fed the control food at all subsequent time points (Table 2). Biomarker Start There were no significant differences in body weight or growth hormone concentrations between groups throughout the entire study. Feeding the test food (Science Diet® Puppy Large Breed formula) resulted in ideal body weight when the puppies reached adulthood. Clinical Importance This study supports the hypothesis that fortification of a large breed puppy food with specific nutrients can enhance physiologic outcomes beneficial to their health when fed in appropriate amounts. Large breed puppies fed the test food with a nutritional profile of Science Diet® Puppy Large Breed formula had significantly higher blood concentrations of omega-3 fatty acids, vitamin E and taurine when compared with another commercially available food as administered by the owner. Higher vitamin E levels in puppies fed the test food have been shown in other studies to support immune function health, and higher omega-3 fatty acid levels in puppies fed the test food have been shown in other studies to support healthy brain and eye development. 3 Months 5 Months 12 Months EPA+DHA, μg/L (mean) 18.7 17.2 17.3 References from Large Breed Puppies Test** 10.6 30.8 33.4 32 Control* 24.5 34.5 37.1 34.8 Test** 28.8 47.7 54.1 46.1 Control* 242 320 286 271 Test** 247 369 346 288 1.Richardson DC, Toll PW. Relationship of nutrition to developmental skeletal disease in young dogs. Vet Clin Nutr. 1996;3:1-8. 2.Hedhammer A, Wu F, Krook L, et al. Overnutrition and skeletal disease: an experimental study in growing Great Dane dogs. Cornell Vet. 1974;64 (Suppl 5):1-160. 3.Kasström J. Nutrition, weight gain and development of hip dysplasia. Acta Radiol Suppl 1975;344:135-179. 4.Kealy RD, Olsson SE, Monti KL, et al. Effects of limited food consumption on the incidence of hip dysplasia in growing dogs. J Am Vet Med Assoc. 1992;210:857-863. Table 2. Serum biomarkers in puppies fed two different foods during growth. Significantly different *Eukanuba® Large Breed Puppy **Nutritional profile of Science Diet® Puppy Large Breed Original Dry formula P.O. Box 410476 Kansas City, MO 64141-0476 Volume 5 • Issue 2 A condensed study review: The role of supplementary dietary antioxidants on immune response in puppies In this issue: Antioxidants and Immunity In this issue: Improving Outcomes Antioxidants and Immunity 1.Head E. Combining an antioxidant-fortified diet with behavioral enrichment leads to cognitive improvement and reduced brain pathology in aging canines: strategies for healthy aging. Ann N Y Acad Sci. 2007;1114:398-406. 2.Milgram NW, Head E, Zicker SC, et al. Learning ability in aged beagle dogs is preserved by behavioural enrichment and dietary fortification: a two-year longitudinal study. Neurobiol Aging. 2005;26:77-90. 3.Heinemann KM, Waldron MK, Bigley KE, et al. Long-chain (n-3) polyunsaturated fatty acids are more efficient than alpha-linolenic acid in improving electroretinogram responses of puppies exposed during gestation, lactation, and weaning. J Nutr. 2005:135;19601966. 4.Heinemann KM, Bauer JE. Docosahexaenoic acid and neurologic development in animals. J Am Vet Med Assoc. 2006:228;700-705. 5.Kelley RL, Lepine AJ, Burr JR, et al. Effect of dietary fish oil on puppy trainability (abstr). Preconf Workshop 6th Intl Soc Study Fatty Acids Lipids Cong 2004;51. 10.6 Taurine μmol/L (mean) Volume 5 • Issue 2 References from Growing Beagle Puppies Control* Vitamin E, μg/ml (mean) PRESORT STD. U.S. POSTAGE PAID KANSAS CITY, MO PERMIT NO. 1710 Giant Strides A clinical study finds that an antioxidant-enhanced food (including 500 IU/kg vitamin E, as fed) improves immune response in puppies. Page 1 Improving Outcomes Hill’s evidence-based clinical nutrition™ HillsVet.com • 1-800-548-VETS (8387) Cognitive, motor, and physiologic development improve when beagle puppies are fed an antioxidantsupplemented food from weaning to 12 months. Khoo C, Cunnick J, Friesen K, et al. Vet Therapeutics 2005;6:43-56. Key points: • A combination CDV and CPV vaccine was used to assess immune function health in puppies. • Puppies were fed high-antioxidant and low-antioxidant foods during the study. • Puppies consuming the high-antioxidant foods showed improved vaccination response and increased memory T-cells, which may help to provide longerlived protection against infections. Page 3 • The minimum AAFCO recommendation for dietary vitamin E (50 IU/kg) may not be sufficient to protect cells during periods of immune stress. Giant Strides Background Specific nutrients are shown to promote healthy development and antioxidant status in large breed puppies. Page 5 During periods of immune stimulation such as vaccination, immune cells generate free radicals. These free radicals have the potential to damage cells and tissues in the body.1 The body’s antioxidant defense system as well as exogenous antioxidants in the food may help to promote formation and maintenance of healthy immune cells by reducing oxidative stress in the body and protecting the cells from harmful free radicals.2,3 Previous studies have demonstrated a beneficial effect of administering a single antioxidant nutrient on immune response.4,5 In this study, the standard vaccination protocol for puppies with a canine distemper (CDV) and parvovirus (CPV) combination vaccine was used as a measure of immune function stimulation to determine the effect of an antioxidant-enhanced food on the immune function health of the puppies. Continued on page 2 ®/™ Hill’s and Science Diet are registered trademarks owned by Hill’s Pet Nutrition, Inc.; Prograd is a registered trademark owned by Intervet Inc.; Purina and Puppy Chow are registered trademarks owned by Société des Produits Nestlé S.A. Eukanuba is a trademark owned by The Iams Company. ©2009 Hill’s Pet Nutrition, Inc. 6 7 Edited by: Phil Roudebush, DVM, DACVIM Kenn Temple, DVM, MBA Hill’s evidence-based clinical nutrition™ Hill’s evidence-based clinical nutrition™ HillsVet.com • 1-800-548-VETS (8387) 8 TD-516 The role of supplementary dietary antioxidants on immune response in puppies. A condensed study review: Continued from page 1 Study Details 6000 6000 vaccination booster booster vaccination A standard vaccination protocol was used during the study. The puppies had no prior vaccinations. A combination vaccine, which included canine distemper (CDV) and parvovirus (CPV) antigens (Progard®-7, Intervet Inc., Millsboro, DE) was given on day 14 and a booster was given on day 28. Blood samples were obtained weekly and serum was sent for measurement of titers from day 14 to day 42. The titers were measured using the canine parvovirus-2 hemagglutinan inhibition test and the canine distemper virus serum neutralization test. Leukocytes were isolated from peripheral blood to measure lymphocyte proliferation and natural killer cell activity. Results showed that food intake and body weight gain were similar among groups. Puppies consuming high AOX foods without whey protein had significantly higher CPV titers at weeks 2 and 3 (4096 vs. 2560 and 3072 vs. 1920, respectively [P<0.05]), and CDV titer at week 4, than puppies receiving the control food (5529 vs 2816 [P<0.05]) (Figure 1). Puppies consuming both high AOX foods had significantly increased memory CD4+ cells and serum vitamin E concentrations compared with the control and grocery brand groups (Figure 2). Serum E concentrations were significantly decreased from pre-feeding values for the group on the grocery brand food (Figure 2). This suggests that immune stress adversely affects serum E concentrations and that this is prevented by supplementing vitamin E. There were no significant differences in the natural killer cell activity among the groups. Titer AOX CTRL GROC WPI • Puppies fed the test food showed improved learning ability and mobility. Background In aged beagle dogs, complex mixtures of antioxidants added to adult maintenance-type foods have been shown to slow the onset of cognitive decline and associated neuropathology.1,2 Supplementation of growth foods with docosahexaenoic acid (DHA) and fed to the bitch during gestation and lactation has been reported to improve trainability and electroretinogram activity in puppies.3,4,5 Post 40.0 30.0 20.0 10.0 0.0 AOX WPI CTRL Key points: • The test food contained high levels of DHA, vitamin E and taurine. Pre 50.0 Zicker SC, Jewell DE, Yamka RM, et al. Unpublished data. Hill’s Pet Nutrition Center, Topeka, Kansas 2008. • Three groups of puppies were fed either a grocery brand, a premium brand or a test food with the nutritional profile of Science Diet® Puppy Healthy Development Original Dry formula. Serum Vitamin 60.0 µg/ml Titer 5000 Healthy male and female puppies with no prior infections were 5000 chosen for the study. Four groups of 10 puppies each were fed a AOX 4000 4000 control and 3 different test foods [Control (Ctrl), control + CTRL GROC antioxidants (AOX), control + AOX + 1% whey protein (WPI), and a 3000 3000 WPI grocery brand (GROC)]. All foods were complete and balanced for 2000 2000 puppies, with vitamin E levels exceeding AAFCO recommendations (50 IU/kg). The AOX combination consisted of vitamin E [500 IU/kg 1000 1000 dry matter (DM)], vitamin C (70 mg/kg DM), -carotene (0.4 mg/kg 0 DM) and selenium (0.8 mg/kg DM).0A total of 40 puppies, 1 2 3 4 1 period, with 2 3 40 7 weeks old, were selected for the 6-week0intervention Time after vaccination (week) Time after vaccination (week) 10 puppies for each dietary intervention. Meals were fed twice a Figure 1. Response of canine distemper neutralizing antibodies to vaccination with a day (morning and afternoon) for a 1-hour period and food intake product containing canine distemper, adenovirus type 2, parainfluenza and parvovirus modified live virus, and Leptospira bacterin (Proguard®-7, Intervet). Note that the group data collected. Body weights were recorded weekly and blood was consuming an antioxidant enhanced food (AOX) tended to have higher titers at all time drawn on days 1 and 42 of the study. points and had significantly higher titers (p<0.05) four weeks after the first vaccination. Improved learning, psychomotor and physiologic functions may be attributed to post-weaning dietary fortification in growing beagle puppies GROC Figure 2. Serum vitamin E concentrations before (Pre) and after (Post) feeding puppies a control food (CTRL), a low vitamin E grocery brand food (GROC), the control food plus enhanced levels of antioxidants, including vitamin E (AOX), and the control food plus enhanced levels of antioxidants and whey protein (WPI). Serum E concentrations were all significantly different from each other after dietary intervention (p <0.05). Note that serum vitamin E concentrations were highest in puppies consuming antioxidant enhanced foods (AOX, WPI) and declined in puppies consuming a grocery brand food (GROC). Clinical Importance Dietary antioxidants are thought to improve immune health by protecting immune and other cells from free radical damage. The combination of antioxidants in this study, vitamin E (500 IU/kg DM), vitamin C (70 mg/kg DM), -carotene (0.4 mg/kg DM) and selenium (0.8 mg/kg DM), was found to improve the response to CDV and CPV vaccination and to increase the number of memory T cells, which may help to provide longer lived protection against infections. The minimum AAFCO recommendation for dietary vitamin E (50 IU/kg) may not be sufficient to protect cells during periods of immune stress. All Hill’s® Science Diet® Puppy pet foods contain superior antioxidant formulas to help ensure that puppies develop a healthy immune system. 2 The present investigation sought to provide more definitive evidence of the effectiveness of dietary supplementation on cognitive, motor, and physiologic development when fed after weaning to puppies until 12 months of age. Throughout the first year of the puppies’ lives, body composition, healthrelated measures, sensory, cognitive, motor and neurological development were assessed at regular time points to determine which foods would provide the maximum benefit to their natural growth and development. Study Details Following weaning, 48 beagle puppies were assigned to three groups of 16 with equal representation from each litter in each group. Sixteen puppies each were assigned to a grocery brand food (Purina® Puppy Chow,® Société des Produits Nestlé S.A., Vevey, Switzerland), a premium brand food (Eukanuba® Puppy Growth – Medium Breed, Procter and Gamble Pet Care, Lewisburg, OH), and to a test food with the nutritional profile of Hill’s® Science Diet® Puppy Healthy Development Original Dry (Hill’s Pet Nutrition, Inc., Topeka, KS). The rationale for the selection of foods was to compare commercially available foods with similar proximate analysis while assessing gradations of other assayable nutrients implicated in neurocognitive development such as vitamin E, taurine, DHA and carnitine (Table 1). All puppies were group housed, and food was offered in portions in accordance with feeding guides recommended on the labels. Portions were adjusted to maintain body condition score close to 3 (5-point scale) but portions were not allowed to be above or below feeding guidelines on the label. All puppies received standard vaccinations and at 16 weeks of age all dogs were vaccinated for rabies. Puppies were neutered or spayed at approximately 6 months of age. During the 10-month test period, blood samples were drawn for analysis of health biomarkers including serum chemistry, complete blood count, response to rabies vaccination, vitamin E and fatty acid analysis. Dogs were scanned by dual X-ray absorptiometry at 2, 4, 6, 9 and 12 months of age to document changes in body composition. Throughout the duration of the study, body weights were recorded weekly and food intake of the group-housed puppies were recorded daily. Nutrient (dmb) Improved learning, psychomotor and physiologic functions may be attributed to post-weaning dietary fortification in growing beagle puppies A condensed study review: Serum measures are affected by food in growing large breed puppies Continued from page 3 Schoenherr WD, Friesen KG, Yamka RM. Unpublished data. Hill’s Pet Nutrition Center, Topeka, Kansas 2008. Finally, a schedule of cognitive and motor skill assessment protocols was followed over the entire study to assess learning, memory and psychomotor development. A battery of learning and memory tests were included that had previously been developed for use in studying cognitive decline associated with canine aging. The cognitive assessment battery included discrimination and reversal learning, an oddity discrimination learning task, a working memory test (delayed-non-matching-to-position test) and a landmark discrimination learning test. There were no significant differences in weight, body condition score or routine blood values between the groups of puppies at the beginning or by the end of the study. Concentration of DHA in the blood was not significantly different between groups prior to dietary intervention. Blood concentrations of DHA in puppies fed the test food with the nutritional profile of Science Diet® Puppy Healthy Development Original Dry pet food formula were significantly greater than either the grocery or premium food at all subsequent time points (Table 2). Following weaning, the test food group had significantly greater concentrations of vitamin E and taurine in blood compared to puppies consuming the grocery or premium brand foods at all time points measured (Table 2). discrimination testing. Improved mobility in the group fed the food with the nutritional profile of Science Diet Puppy Healthy Development Original Dry was noted between 3 and 6 months of age as measured by tasks that assessed psychomotor development (a hoops mobility course). Clinical Importance The results of this study support the hypothesis that foods rich in nutrients to support neurologic development (DHA, vitamin E, taurine), immune function (vitamin E) and combat oxidative stress (vitamin E and taurine) resulted in improved outcomes as measured by immune function, agility and cognitive tests. These findings are clinically relevant as they show that modification of food even following weaning may improve desired outcomes for young dogs. Puppies consuming both foods containing added sources of DHA showed significantly better learning ability as measured on the reversal portion of the T-maze when compared to the grocery brand group. Cognitive learning differences were maintained to 1 year of age as shown in contrast discrimination and landmark Grocery* Premium** Test Food*** Protein, % 32.5 31.6 31.3 Fat, % 16.2 17.6 18.7 DHA, μg/L (mean) Calcium, % 1.47 1.45 1.52 Grocery* 2.3 3.4 4.7 4.3 3.8 4.3 2.4 13.6 15.4 15.0 12.8 12.9 2.3 19.0 24.2 24.4 21.9 20.3 Biomarker Start 3 Months 4 Months 6 Months 9 Months 12 Months Total n-3 fatty acids, % 0.13 0.38 1.49 Premium** DHA, % <0.01 0.10 0.20 Test Food*** EPA, % <0.01 0.14 0.33 Vitamin E, ng/ml (mean) Total n-6 fatty acids, % 1.86 3.05 3.94 Grocery* 14.5 10.8 13.2 14.5 15.7 16.8 Carnitine, ppm <30 <30 332 Premium** 14.4 19.7 21.0 22.3 20.6 21.9 Vitamin E, IU/kg 65 335 868 Test Food*** 14.4 26.1 32.1 36.9 33.3 33.4 0.08 0.15 0.14 Taurine, % Table 1. Key nutrient levels in foods fed to puppies to assess cognitive, immune and agility outcomes. *Purina® Puppy Chow,® Société des Produits Nestlé S.A., Vevey, Switzerland **Eukanuba® Puppy Growth – Medium Breed, Procter and Gamble Pet Care, Lewisburg, OH ***Nutritional profile of Science Diet® Puppy Healthy Development Original Dry 3 Key points: Study Details • Many large and giant breed dogs are at risk for developmental orthopedic disease (DOD). Large breed puppies (Labrador retrievers, n=19, 11 males, 8 females; or golden retrievers, n=24, 13 males, 11 females) were purchased from breeders or bred by a specialty dog service and were reared individually in homes of puppy-raisers for the duration of the study. The dogs were assigned to one of two food treatments at weaning and remained on that food through 12 months of age. The puppy-raisers were masked regarding the food treatment assigned to their dog. Eighteen puppies were assigned to a positive control food (Eukanuba® Large Breed Puppy, Procter and Gamble Pet Care, Lewisburg, OH) and 25 puppies were assigned to a test food with the nutritional profile of Hill’s® Science Diet® Puppy Large Breed Dry pet food (Hill’s Pet Nutrition, Inc., Topeka, KS). The rationale for the selection of foods was to compare commercially available foods with similar proximate analysis, while assessing gradations of other assayable nutrients implicated in growth, body composition and antioxidant status such as amino acids, vitamin E, vitamin C, fatty acids, and carnitine (Table 1). • Nutrient excesses such as calcium and energy (fat) are known risk factors. • Two groups of large breed puppies were fed either a premium puppy food or a test food with the nutritional profile of Science Diet® Puppy Large Breed Dry. • The test food supported healthy development with improved fatty acid and antioxidant status. Background Many large and giant breed dogs are at risk for developmental orthopedic disease (DOD).1 Current research indicates that rate of growth, specific nutrients, food consumption, and feeding methods influence the pathogenesis of DOD. Specifically, nutrient excesses, such as calcium and energy, and rapid growth related to overfeeding and excess energy intake are known risk factors.1 To this end, studies have demonstrated beneficial effects of limiting specific nutrients, calcium or energy, on the expression of skeletal disease.2,3,4 The present investigation sought to provide more definitive evidence of the effectiveness of dietary supplementation early in development by comparing food designed for the large breed puppy on growth and body composition when fed after weaning. Throughout the first 12 months of the puppies’ lives, body composition and other health-related measures were assessed at regular time points to determine which foods fed to the large breed puppies would provide the maximum benefit to the natural growth and development. Nutrient (dmb) Control* Test** Protein, % 28.8 32.7 Fat, % 15.5 16.8 Calcium, % 0.87 1.22 Total n-3 fatty acids, % 0.37 1.97 EPA + DHA, % 0.23 0.62 Total n-6 fatty acids, % 2.9 3.9 Carnitine, ppm <30 326 Vitamin E, IU/kg 377 842 Vitamin C, ppm <10 153 Table 1. Analyzed nutrient levels of foods fed to large breed puppies. Significantly different *Eukanuba® Large Breed Puppy **Nutritional profile of Science Diet® Puppy Large Breed Dry Table 2. Serum biomarkers in puppies fed three different foods during growth Significantly different *Purina® Puppy Chow®, Société des Produits Nestlé S.A., Vevey, Switzerland **Eukanuba® Puppy Growth – Medium Breed, Procter and Gamble Pet Care, Lewisburg, OH ***Nutritional profile of Science Diet® Puppy Healthy Development Original Dry 4 5