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The role of supplementary dietary antioxidants on immune response in puppies

Authors:

Abstract

The objective of this study was to evaluate the effect of supplementary antioxidants (AOX) and whey protein on the immune function health of puppies. Four groups of 10 puppies were fed a control and 3 different test foods (control + antioxidants (AOX), control + AOX + 1% whey protein, and a grocery brand (low AOX)) for 6 weeks. A standard vaccination protocol with a combination canine parvovirus (CPV) and distemper (CDV) vaccine was carried out at 2 and 4 weeks. The results showed that animals on high AOX foods had significantly increased titers, memory cells and serum E concentrations compared to the control and groc groups respectively.
In this issue:
Edited by: Phil Roudebush, DVM, DACVIM
Kenn Temple, DVM, MBA
Antioxidants and Immunity
Volume 5 • Issue 2
A condensed study review:
The role of supplementary dietary
antioxidants on immune response
in puppies
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 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.
Background
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 evidence-based clinical nutrition
Improving Outcomes
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
Cognitive, motor, and physiologic development
improve when beagle puppies are fed an antioxidant-
supplemented food from weaning to 12 months.
Page 3
Specific nutrients are shown to promote healthy
development and antioxidant status in large breed
puppies.
Page 5
8
Volume 5 • Issue 2
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Kansas City, MO 64141-0476
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In this issue:
Antioxidants and Immunity
Improving Outcomes
Giant Strides
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7
References from Antioxidants and Immunity
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.
References from Growing Beagle Puppies
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;1960-
1966.
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.
References from Large Breed Puppies
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.
®/™ 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
Serum measures are affected by food in growing large breed puppies
Continued from page 5
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).
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.
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
Biomarker Start 3 Months 5 Months 12 Months
EPA+DHA, μg/L (mean)
Control* 10.6 18.7 17.2 17.3
Test** 10.6 30.8 33.4 32
Vitamin E, μg/ml (mean)
Control* 24.5 34.5 37.1 34.8
Test** 28.8 47.7 54.1 46.1
Taurine μmol/L (mean)
Control* 242 320 286 271
Test** 247 369 346 288
2
The role of supplementary dietary antioxidants on immune response in puppies.
Continued from page 1
Study Details
Healthy male and female puppies with no prior infections were
chosen for the study. Four groups of 10 puppies each were fed a
control and 3 different test foods [Control (Ctrl), control +
antioxidants (AOX), control + AOX + 1% whey protein (WPI), and a
grocery brand (GROC)]. All foods were complete and balanced for
puppies, with vitamin E levels exceeding AAFCO recommendations
(50 IU/kg). The AOX combination consisted of vitamin E [500 IU/kg
dry matter (DM)], vitamin C (70 mg/kg DM), -carotene (0.4 mg/kg
DM) and selenium (0.8 mg/kg DM). A total of 40 puppies,
7 weeks old, were selected for the 6-week intervention period, with
10 puppies for each dietary intervention. Meals were fed twice a
day (morning and afternoon) for a 1-hour period and food intake
data collected. Body weights were recorded weekly and blood was
drawn on days 1 and 42 of the study.
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.
AOX
CTRL
GROC
booster
0
0
1000
2000
3000
4000
5000
6000
1 2 3 4
WPI
vaccination
Time after vaccination (week)
Titer
AOX
CTRL
GROC
booster
0
0
1000
2000
3000
4000
5000
6000
1 2 3 4
WPI
vaccination
Time after vaccination (week)
Titer
Figure 1. Response of canine distemper neutralizing antibodies to vaccination with a
product containing canine distemper, adenovirus type 2, parainfluenza and parvovirus
modified live virus, and Leptospira bacterin (Proguard®-7, Intervet). Note that the group
consuming an antioxidant enhanced food (AOX) tended to have higher titers at all time
points and had significantly higher titers (p<0.05) four weeks after the first vaccination.
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 Hills
®
Science Diet
®
Puppy pet foods contain superior
antioxidant formulas to help ensure that puppies develop a healthy
immune system.
Serum Vitamin
0.0
10.0
20.0
30.0
40.0
50.0
60.0
Pre
Post
AOX CTRL GROCWPI
µg/ml
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).
3
A condensed study review:
Improved learning, psychomotor and physiologic functions may be
attributed to post-weaning dietary fortification in growing beagle
puppies
Zicker SC, Jewell DE, Yamka RM, et al. Unpublished data. Hill’s Pet Nutrition Center, Topeka, Kansas 2008.
Key points:
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.
The test food contained high levels of DHA, vitamin E
and taurine.
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
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, health-
related 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.
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
Nutrient (dmb) Grocery
*
Premium
**
Test Food
***
Protein, % 32.5 31.6 31.3
Fat, % 16.2 17.6 18.7
Calcium, % 1.47 1.45 1.52
Total n-3 fatty acids, % 0.13 0.38 1.49
DHA, % <0.01 0.10 0.20
EPA, % <0.01 0.14 0.33
Total n-6 fatty acids, % 1.86 3.05 3.94
Carnitine, ppm <30 <30 332
Vitamin E, IU/kg 65 335 868
Taurine, % 0.08 0.15 0.14
5
A condensed study review:
Serum measures are affected by food in growing large breed puppies
Schoenherr WD, Friesen KG, Yamka RM. Unpublished data. Hill’s Pet Nutrition Center, Topeka, Kansas 2008.
Key points:
Many large and giant breed dogs are at risk for
developmental orthopedic disease (DOD).
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 puppieslives, 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.
Study Details
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).
Table 1. Analyzed nutrient levels of foods fed to large breed puppies.
Significantly different
*EukanubLarge Breed Puppy
**Nutritional profile of Science Diet® Puppy Large Breed Dry
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
4
Improved learning, psychomotor and physiologic functions may be attributed to
post-weaning dietary fortification in growing beagle puppies
Continued from page 3
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 Die
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).
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
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.
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
Biomarker Start 3 Months 4 Months 6 Months 9 Months 12 Months
DHA, μg/L (mean)
Grocery* 2.3 3.4 4.7 4.3 3.8 4.3
Premium** 2.4 13.6 15.4 15.0 12.8 12.9
Test Food*** 2.3 19.0 24.2 24.4 21.9 20.3
Vitamin E, ng/ml (mean)
Grocery* 14.5 10.8 13.2 14.5 15.7 16.8
Premium** 14.4 19.7 21.0 22.3 20.6 21.9
Test Food*** 14.4 26.1 32.1 36.9 33.3 33.4
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... Since immune cell homeostasis and metabolism are stringently correlated, this interdependence could open to innovative immune-modulating therapeutic approaches for the treatment of infectious diseases [44,45]. In this context, the use of plant-derived nutraceuticals may regulate immune responses [46] and improve the clinical outcome of infectious diseases in both human and dog models [47][48][49][50]. Cortese et al. [15] reported that the combination of nutraceutical pet food with conventional therapy may modulate immune responses in CL. ...
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The use of nutraceuticals as immunomodulators in the treatment of visceral leishmaniasis has generated interest in the current approaches to treat the disease. In this clinical and immunological study, we investigated whether the administration of a nutritional supplement mediates the immune-modulatory response in canine leishmaniosis (CL) and improves the clinical outcome of the disease. With this purpose, we analysed T lymphocyte subsets in peripheral blood (PB) of 12 dogs naturally infected by Leishmania infantum, following treatment with a nutritional supplement. The regulatory T (Treg) cells and the T helper (Th) 1 population were specifically evaluated. The animals underwent complete clinical examination and blood sample collection for haematological, biochemical, serological and immunological analysis before treatment (T0), one month (T30) and 3 months (T90) after the onset of the nutraceutical supplementation. We observed that nutraceutical supplementation was associated with immunomodulation of Th1 response and significant clinical improvement of the animals. No side effects were observed. Therefore, a potential supportive role for the nutraceutical supplement during canine leishmaniasis is proposed.
... It is of some relevance that the role of some biological principles -mainly derived from plants and usually referred as nutraceuticals -appears to be of some relevance in modulating the immune system homeostasis [37] as well as their use is of efficacy in dogs [38][39][40][41]. ...
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