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Effects of dietary supplementation with quercetin on broiler immunological characteristics
Accepted Manuscript Title: Effects of dietary supplementation with quercetin on broiler immunological characteristics Author: A.L. Hager-Theodorides M. Goliomytis S. Delis S. Deligeorgis PII: DOI: Reference:
S0377-8401(14)00304-6 http://dx.doi.org/doi:10.1016/j.anifeedsci.2014.09.021 ANIFEE 13161
To appear in:
Animal
Received date: Revised date: Accepted date:
24-3-2014 16-9-2014 22-9-2014
Feed
Science
and
Technology
Please cite this article as: Hager-Theodorides, A.L., Goliomytis, M., Delis, S., Deligeorgis, S.,Effects of dietary supplementation with quercetin on broiler immunological characteristics, Animal Feed Science and Technology (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.09.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of dietary supplementation with quercetin on broiler immunological
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characteristics
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A. L. Hager-Theodorides*, M. Goliomytis, S. Delis and S. Deligeorgis
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Laboratory of Animal Breeding and Husbandry, Department of Animal Science and
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Aquaculture, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece
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*Corresponding author:
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Telephone number: +30 210 5294453
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e-mail: [email protected]
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Fax +30 210 5294442
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Highlights
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• Quercetin did not affect broilers' cellular immune response to phytohaemagglutinin. • It did not affect the birds’ thymus cell subset composition and maturity. • It did not affect lymphoid organ weight or cellularity. • Quercetin significantly affected humoral responses to SRBC
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immunization.
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• IgY antibody titers significantly increased with increasing quercetin
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levels.
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Abstract
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Quercetin, among other flavonoids, is under investigation as feed additive for poultry
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due to its antioxidative properties. Quercetin is also known to possess
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immunomodulatory
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uncompromised immune function is important for poultry productivity and welfare
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this study investigates possible effects of dietary supplementation of broilers with
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quercetin on their immune function and immune status. We assessed aspects of the
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chickens cell-mediated and humoral immune response and immune status when fed
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with diets supplemented with quercetin at 0.5 and 1 g/kg feed compared to control
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diets. We measured their cellular immune response to the phytohaemagglutinin skin
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test, their humoral immune response to sheep red blood cells and assessed the status
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of the birds’ lymphoid organs and the cell subset composition of the thymus. We
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and
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anti-inflammatory
properties.
As
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found no differences between dietary treatment groups in cellular immune response or
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immune status (P>0.05). Interestingly, while total and IgM antibody titers did not
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differ among treatment groups (P>0.05), IgY antibody titers significantly increased
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with increasing quercetin supplementation (P-linear<0.05). Therefore, dietary
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quercetin did not compromise any of the immune parameters tested but on the
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contrary enhanced IgY antibody production.
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1. Introduction
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Flavonoids, naturally occurring plant secondary metabolites, are currently under
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investigation as feed additives in poultry for their antioxidative properties (Rice-Evans,
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2001; Williams et al., 2004). Antioxidative agents are important for poultry nutrition as
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they reduce lipid peroxidation, a major concern for the organoleptic characteristics and
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nutritional value of meat and eggs, and can prolong poultry product shelf life (Fellenberg
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and Speisky, 2006). Naturally occurring polyphenols, including flavonoids, are attractive
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antioxidative feed additive candidates as they are safer alternatives to synthetic ones
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(Balasundram et al., 2006).
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Apart from potent antioxidants, flavonoids have been shown to possess
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immunomodulatory and in particular anti-inflammatory properties (González-Gallego et
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al., 2010; Serafini et al., 2010). Animal model and human in vitro studies have shown
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that flavonoids reduce inflammatory immune function often by modulating the
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production of pro- and anti-inflammatory molecules by cells of the innate and adaptive
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immune system (e.g. macrophages and T cells) in response to stimuli that upregulate
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inflammatory processes (Kumazawa et al., 2006; González-Gallego et al., 2010).
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Quercetin, a flavonoid belonging to the class of flavonols, is present in several
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fruits, vegetables and herbs (Manach et al., 2004). It has been shown to improve
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oxidative status of broiler meat when added to the birds’ feed and was detectable in the
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plasma and in several tissues of the animals (Rupasinghe et al., 2010). Quercetin has been
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shown to act as an anti-inflammatory agent in mice (Comalada et al., 2006; Hamalainen
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et al., 2007; Huang et al., 2010) and in humans (Sternberg et al., 2008).
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A functional immune system is essential for the maintenance of good health,
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especially in the case of broilers’ crowded rearing conditions that increase physical
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contact and therefore exposure to pathogens (Korver, 2012). Possible immunomodulatory
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properties of flavonoids should be carefully investigated to ascertain that they will not
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compromise the immune defense of the birds. Immunomodulatory compounds tend to
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differentially influence different immune functions, so several parameters of innate and
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acquired immunity should be investigated, including cellular and humoral responses.
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So far studies on flavonoids in poultry examine, in addition to meat oxidative
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status, effects on growth performance and other productive traits, including meat (Batista
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et al., 2007; Jiang et al., 2007; Peña et al., 2008; Simitzis et al., 2011; Kamboh and Zhu,
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2013) and egg (Lien et al., 2008; Ting et al., 2011; Goliomytis et al., 2014a) quality
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parameters. The effect of quercetin on broiler meat quality parameters was tested by
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(Goliomytis et al., 2014b).
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This study investigated possible effects of dietary quercetin on parameters of
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broiler immune function and status. We examined parameters of cellular and humoral
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immunity, lymphoid organs size and cellularity and thymus subset composition to
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determine whether the immunomodulatory properties of quercetin exhibited in other
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animals are manifested in broilers when it is included in their feed.
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2. Materials and methods
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2.1 Animals, diets and experimental design
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One hundred and fifty day-old Cobb 500 broiler chickens, obtained from a commercial
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hatchery, as hatched, were housed in a controlled environment at the facilities of the
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Agricultural University of Athens. The birds were reared in 12 pens, 2 m2 each (stocking
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density of 6.25 birds/m2), for 51 days. Each chicken was individually wing tagged upon
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arrival. Lighting program, environmental conditions and management practices were in
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accordance to standard Cobb guidelines. In brief, the lighting program consisted of
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23L:1D on arrival, and was gradually decreased to 18L:6D by day 24, remained constant
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until day 37, and thereafter increased to 23L:1D till slaughter. Feed, in mash form, and
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water were provided ad libitum. The composition and the chemical analysis of the three
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diets offered, throughout the rearing period (starter, grower and finisher), are presented in
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Table 1.
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Chickens were randomly assigned to three treatment groups, control (C),
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quercetin-0.5 (Q-0.5) and quercetin-1 (Q-1), each replicated in four pens. The diet of
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treatment groups C, Q-0.5 and Q-1 were supplemented for the duration of the experiment
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with 0, 0.5 and 1 g quercetin (MP Biochemicals, France) per kg feed, respectively. Feed
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enrichment with quercetin was performed daily.
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Animal handling and all procedures performed in the present experiment were in
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accordance with the national legislation and the guidelines of the Research Ethics
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Committee of the Department of Animal Science and Aquaculture of the Agricultural
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University of Athens.
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2.2 Phytohaemagglutinin skin test
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The phytohaemagglutinin (PHA) skin test was adapted from (Corrier and DeLoach,
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1990) and (Lavoie et al., 2007). In brief, 30 l containing 100g PHA (PHA-P, Sigma-
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Aldrich L1668, diluted in phosphate buffered saline (PBS) to a final concentration of
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3333 g/ml PHA-P) were injected intradermally with 30-gauge needles into the
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interdigital web space between the second and third toes of the left foot of 15 days old
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birds. Thirty microliters of PBS were administered into the corresponding interdigital
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web of the right foot of each bird. Thickness of the web in both feet was measured before
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injection and 24 hours after injection using a thickness gauge with 0.01mm precision
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(Peacock, Ozaki MFG. CO. Ltd, Tokyo, Japan). To calculate the PHA stimulation index
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the change of thickness in the control foot web was subtracted from the corresponding
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change in the PHA-treated foot web. Sixteen birds per treatment chosen at random (four
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from each replicate pen) were subjected to the PHA skin test.
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The PHA skin test was repeated to the same birds at 48 days of age with a lower
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dose of PHA, to avoid any possible saturating effects of a high stimulus on the response
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of the birds, that would possibly mask differences between groups. In the second test 30
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l containing 20g PHA were injected into the interdigital web space between the first
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and second toes of the left foot and same volume of PBS was injected in the
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corresponding area of the right foot. Skin thickness measurements and stimulation index
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were performed as above.
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2.3 Immunization with sheep red blood cells (SRBC)
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SRBC were prepared from total sheep blood from a healthy individual, collected with
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EDTA anticoagulant (2mg EDTA/ml of blood) spun at 400xg, washed three times with
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phosphate buffered saline (PBS) and resuspended in Alsevier’s solution to a final
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haematocrit (Ht) of 10%. SRBC was stored in the fridge and diluted with PBS at 1% Ht
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for immunization of the birds (adapted from (Olsson and Oldenborg, 2008). Sixteen 21d
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old birds per treatment, the same submitted to the PHA skin treatment, were injected
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intravenously (into the brachial vein) with 100l 1% SRBC in PBS. Six days post SRBC
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challenge blood samples were collected from the immunized birds to determine plasma
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anti-SRBC antibody titer with the haemagglutination test (protocol adapted from (Lavoie
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et al., 2007). Briefly, blood samples, collected with EDTA anticoagulant (2mg EDTA/ml
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of blood), were centrifuged at 800xg for 20 minutes and supernatant was collected and
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complement was heat inactivated at 56oC for 30 minutes. To determine total antibody
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titers, 25 l of plasma were mixed with 25 l PBS in the first well of a 96-well U bottom
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microtiter plate. Plasma was serially diluted across a plate row, 25 l 1% SRBC was
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added to each well and the plate was incubated at 37oC for one hour. Anti-SRBC total
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antibody titer was determined as the logarithm to base two of the dilution factor of the
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highest dilution showing haemagglutination. To determine IgY (bird equivalent to
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mammalian IgG) antibody titer (-mercaptoethanol, 2-ME, resistant) 25 l of plasma
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were incubated with 25 l of 0.2 M 2-ME in the first column of a 96-well U bottom
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microtiter plate at 37oC for 1 hour and then haemagglutination test was performed as for
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total antibody titer. To determine IgM antibody titer IgY titer was subtracted from total
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antibody titer. All plasma samples were tested in duplicates.
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2.4 Lymphoid organ weights and cell counts
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At 51 days of age the sixteen birds per treatment that were subjected to PHA skin test and
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were immunized with SRBC were weighed, electrically stunned and slaughtered. Bursa
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of Fabricius, spleen and one thymus lobe (second lobe from the right side) were dissected
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and weighed. Organ indexes were calculated as the percentage of live body weight (organ
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weight/live body weight x 100). Bursa of Fabricius cell suspensions from 6 birds per
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treatment and thymus lobe cell suspensions from 16 birds per treatment were prepared by
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crushing tissue aliquots (of known weight) between two pieces of ground glass. Cells
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were counted on a haematocytometer and total cell count was calculated for the whole
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organ or lobe.
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2.5 Thymus subset composition
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Thymocyte suspensions were prepared as described above and cells were stained using
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the following combination of directly conjugated chicken reactive antibodies obtained
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from Abcam (UK): anti-CD8-Fluorescein isothiocyanate (FITC), anti-CD4-phycoerythrin
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(PE), anti-CD3-PE/Cyanine (Cy)5. Briefly, approximately 5x106 cells were spun and
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resuspended in 50l staining medium (PBS supplemented with 2% FCS and 0.01%
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sodium azide) containing 0.5g/ml of each antibody and were incubated for 30 minutes
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on ice. Cells were washed in staining medium, spun at 300xg and resuspended in 100l
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1% paraformaldehyde (PFA). Cells were fixed in PFA for 20 minutes on ice, washed and
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spun as above, resuspended in staining medium and stored in the fridge until analyzed on
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the flow cytometer (FC-500, Beckman- Coulter, USA). 5x105 events were collected per
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sample, in list mode, using CXP software and data were analyzed using FlowJo software.
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Live lymphocytes were gated according to their forward scatter (FSC) and side scatter
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(SSC) profiles.
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2.6 Statistical analysis
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Data were subjected to ANOVA using the general linear models, of SAS software (SAS
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Institute, Cary NC, USA, version 9.0, 2002), with quercetin level as the fixed effect. The
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individual bird was considered as the experimental unit for all traits measured except for
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BW at slaughter for which the experimental unit was the pen. Alpha for determination of
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significance was 0.05. The linear dose response of quercetin was determined with
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contrasts among means. Means are presented as least square means.
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3. Results
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3.1 Immune function
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3.1.1 Phytohaemagglutinin skin test
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To assess the effect of quercetin dietary supplementation on the birds’ localized
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inflammatory response and cell-mediated immunocompetence we performed the
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phytohaemagglutinin (PHA) skin test that elicits a cell-mediated cutaneous basophil
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hypersensitivity immune reaction and is often used to assess cellular immunity in birds
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(Stadecker et al., 1977; Fairbrother et al., 2004; Martin et al., 2006). Response to the
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PHA skin test is widely interpreted as an index of birds’ cell-mediated
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immunocompetence (Corrier, 1990; Fairbrother et al., 2004). Large responses to the PHA
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skin test are also considered to be an indicator of unregulated local inflammation or
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allergy (Elgert, 2009).
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As shown in Table 2 the PHA stimulation index, was not affected by quercetin dietary
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supplementation at either 15 or 48 days of age (P-linear>0.05)
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3.1.2 Humoral immunity
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To investigate possible effects of quercetin on the chickens’ humoral immune response
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we immunized them with sheep red blood cells (SRBC) and measured the antibody titers
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in their blood plasma six days later (Table 3). Immunization with SRBC is a commonly
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used in vivo assay for humoral immunity in birds and other species. It elicits a T helper
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dependent activation of B cells and production of anti-SRBC antibodies (Fairbrother et
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al., 2004). Means for total and IgM antibody titers for treatment groups were not
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statistically different from control (P>0.05). Interestingly, a linear dose response of
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quercetin on IgY titers (P-linear<0.05) was observed.
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quercetin resulted in increasing anti-SRBC IgY antibody production.
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Increasing levels of dietary
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3.2 Immune status
At 51 days of age thymus homeostasis was assessed by flowcytometric analysis of
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thymocyte developmental subset composition based on the expression of cell surface
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proteins CD4, CD8 and CD3. Thymus homeostasis is often assessed by the analysis of
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the ratios of the developmental stages of the thymocytes present in the organ, as
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thymocytes develop from lymphoid progenitor cells to mature and functional T cells that
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migrate to sites of lymphoid activity. Thymocyte developmental stages are
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phenotypically determined by the expression of cell surface markersCD4 and CD8
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proteins. Immature thymocytes that do not express either CD4 or CD8 (double negative
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stage, DN) progress to a CD4+CD8+ double positive stage (DP) and the most mature
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thymocytes are either CD4+ or CD8+ single positive (SP) cells. Cell surface expression of
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CD3 further denotes thymocyte maturation, it is upregulated at the DP compared to the
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DN stage and finally mature and functional CD4+SP and CD8+SP thymocytes, express
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high levels of CD3 (Cooper et al., 1991).
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We found no statistically significant differences (P>0.05) in mean DN, DP,
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CD4SP or CD8SP cell percentages between treatment groups (Table 4). In addition, we
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found no differences (P>0.05) between groups in the mean percentages of CD4 or CD8
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single positive cells that expressed CD3 on their surface neither in the mean relative level
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of CD3 protein cell surface expression on double positive or single positive cells, as
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evident by the mean fluorescence intensity of the cells for the PE/Cy5 fluorochrome
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conjugated to the anti-CD3 antibody, used to detect CD3 expression (p>0.05, data not
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shown).
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To further assess immune status of the animals at day 51, spleen, bursa of
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Fabricius and thymus of the chickens were weighed and thymus and bursa of Fabricius
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cell numbers were counted to assess the immune status of the animals. We found no
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differences among treatment groups in organ weights or organ weights as a percentage of
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live body weight (P>0.05) (Table 5). In addition, means for bursa and thymus cell counts
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were not statistically different for different treatments (P>0.05) (Table 5).
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4. Discussion
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Quercetin, among other flavonoids, is under investigation as a putative antioxidant
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supplement for broiler feed. There is no published data on the effects of quercetin or
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other flavonoids on chicken cell-mediated immune response. Nevertheless, it is well
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documented that quercetin exhibits immunomodulatory properties in other species. A
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large number of in vitro and in vivo studies support an immunomodulatory and anti-
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inflammatory role for quercetin and other flavonoids in man and in rodents (Boots et al.,
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2008; García-Lafuente et al., 2009; González-Gallego et al., 2010; Serafini et al., 2010).
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Studies on human lymphocytes showed that flavonoids reduced immune cell proliferation
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and pro-inflammatory cytokine production in response to PHA stimulation in vitro
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(Pandey et al., 2005). Quercetin, administered intraperitoneally (75mg quercetin/ kg body
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weight), was shown to reduce inflammation in vivo in mice in experimental models of
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acute and chronic inflammatory responses (Rotelli et al., 2003). In contrast to these data
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from humans and mice, our experiments did not reveal an in vivo effect of dietary
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quercetin on the cellular immune response of broilers as assessed by the PHA skin test.
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This can be attributed to species differences, to quercetin dosage and route of
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administration and/or to differences in the in vivo challenges employed to assess
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inflammatory responses.
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Effects on humoral immunity have not been reported for quercetin in chickens or
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other species. Other antioxidant compounds have been shown to affect antibody
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production in chickens. In broilers, supplementation with vitamin E increased humoral
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immune responses to SRBC (Boa-Amponsem et al., 2000; Niu et al., 2009), infectious
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bronchitis virus (Leshchinsky and Klasing, 2001) and Newcastle disease virus (Vakili
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and Daliri, 2010) immunizations. In this study although no effects of quercetin were
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detected on total and IgM antibody titers, IgY titers increased with increasing levels of
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quercetin supplementation in the primary response to SRBC immunization. In the
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primary response IgM immunoglobulins are produced before and are more abundant than
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IgY. This is reversed in additional immunizations where IgG is the most abundant
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immunoglobulin circulating in the blood plasma. Our finding suggests that dietary
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quercetin enhances humoral immune response in a dose-dependent manner. Nutrients that
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were shown to increase both humoral responses and growth performance in non-
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immunized broilers were reported to lose the growth performance positive effect when
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birds were immune challenged (Buyse et al., 2009). Here quercetin did not affect mean
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body weight of immune challenged birds at 51 days of age (p>0.05, overall mean
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3049±56 g) but it remains to be investigated if quercetin affects growth performance
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parameters differently in immune challenged compared to naïve birds.
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Size and cellularity of immune organs and thymocyte developmental stage were
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analyzed to assess immune status and thymus homeostasis respectively. We found no
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effect of quercetin on any of the parameters tested.
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There is evidence that fast growing chicken strains such as modern broilers have
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reduced immune function compared to slower growing strains (Qureshi and Havenstein,
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1994; Yunis et al., 2000; Cheema et al., 2003). It is argued that selection for growth traits
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may have unintentionally caused indirect selection in favor of decreased immune
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function, both cellular and humoral (Van Der Most et al., 2011; Korver, 2012). A
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possible cause for this could be that pro-inflammatory responses and cytokines produced
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decrease the birds’ metabolic activity and appetite thus negatively influencing feed intake
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and growth (Lochmiller and Deerenberg, 2000). Improved growth performance of
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modern broiler chickens may have been partly accomplished at the expense of immune
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function. In the case of subclinical infections or non-infectious microorganisms or agents,
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reduced inflammatory responses can redirect nutrients and energy towards growth
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without having adverse effects on animal health. But decreased responses can lead to
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increased morbidity and/or mortality in the case of infections that rely mainly on
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inflammatory immune responses to be cleared and result in deterioration of animal
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welfare and substantial economic loss. An anti-inflammatory or other immunomodulatory
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effect of dietary supplements would therefore not be beneficial for broilers if it
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compromised their resistance to infections.
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5. Conclusion
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Here we assessed aspects of cellular and humoral immune responses, thymus
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subset composition and immune status of broilers fed with quercetin, supplemented at 0,
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0.5 and 1 g/kg feed. We did not detect any effects of quercetin on broiler chickens
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cellular immunity, thymus subset composition or immune status parameters studied.
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Interestingly, quercetin was found to impact with a linear dose-dependent manner on IgY
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antibody production in response to SRBC immunization. Therefore, dietary quercetin did
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not adversely affect any of the immune parameters tested but on the contrary enhanced
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primary IgY antibody production. These findings suggest that quercetin (a) would not
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compromise the birds immunocompetence, if used as a feed additive for its antioxidant
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properties, and (b) could potentially enhance broilers’ humoral immune responses.
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Acknowledgements
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The authors wish to thank Dr Nikos Demiris for useful discussions on the statistical
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analysis and Drs Panagiotis Simitzis and Maria Charismiadou for discussions on the
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experimental design. They also wish to thank Jenny Biniari and Dimitra Tsoureki for
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technical assistance.
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disease. Inflammation Research 58, 537-552.
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STAT-1 and NF-kappa B activations, whereas flavone, isorhamnetin, naringenin, and
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pelargonidin inhibit only NF-kappa B activation along with their inhibitory effect on
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Immunosuppressive Effect of Quercetin on Dendritic Cell Activation and Function.
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2007. Effect of in ovo exposure to an organochlorine mixture extracted from double
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vitamin E and the immune response of broiler chickens. Poultry Science 80, 1590-1599.
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Martin, L., Han, P., Lewittes, J., Kuhlman, J., Klasing, K., Wikelski, M., 2006.
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immunoecological technique. Functional Ecology 20, 290-299.
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Niu, Z., Liu, F., Yan, Q., Li, W., 2009. Effects of different levels of vitamin E on growth
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performance and immune responses of broilers under heat stress. Poultry Science 88,
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2101-2107.
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Olsson, M., Oldenborg, P.A., 2008. CD47 on experimentally senescent murine RBCs
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inhibits phagocytosis following Fcγ receptor-mediated but not scavenger receptor-
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mediated recognition by macrophages. Blood 112, 4259-4267.
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Pandey, R., Maurya, R., Singh, G., Sathiamoorthy, B., Naik, S., 2005. Immuno
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suppressive properties of flavonoids isolated from Boerhaavia diffusa Linn. International
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Immunopharmacology 5, 541-553.
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Peña, J.E.M., Vieira, S.L., López, J., Reis, R.N., Barros, R., Furtado, F.V.F., Silva, P.X.,
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2008. Ascorbic acid and citric flavonoids for broilers under heat stress: Effects on
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performance and meat quality. Revista Brasileira de Ciencia Avicola 10, 125-130.
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Qureshi, M.A., Havenstein, G.B., 1994. A comparison of the immune performance of a
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1991 commercial broiler with a 1957 randombred strain when fed "typical" 1957 and
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1991 broiler diets. Poultry science 73, 1805-1812.
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Rice-Evans, C., 2001. Flavonoid antioxidants. Current Medicinal Chemistry 8, 797-807.
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Rotelli, A.E., Guardia, T., Juárez, A.O., De La Rocha, N.E., Pelzer, L.E., 2003.
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Comparative
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Pharmacological Research 48, 601-606.
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Rupasinghe, H.P.V., Ronalds, C.M., Rathgeber, B., Robinson, R.A., 2010. Absorption
415
and tissue distribution of dietary quercetin and quercetin glycosides of apple skin in
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broiler chickens. Journal of the Science of Food and Agriculture 90, 1172-1178.
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Serafini, M., Peluso, I., Raguzzini, A., 2010. Session 1: Antioxidants and the immune
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system Flavonoids as anti-inflammatory agents. P Nutr Soc 69, 273-278.
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Simitzis, P.E., Symeon, G.K., Charismiadou, M.A., Ayoutanti, A.G., Deligeorgis, S.G.,
420
2011. The effects of dietary hesperidin supplementation on broiler performance and
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chicken meat characteristics. Canadian Journal of Animal Science 91, 275-282.
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Stadecker, M.J., Lukic, M., Dvorak, A., Leskowitz, S., 1977. The cutaneous basophil
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response to phytohemagglutinin in chickens. Journal of Immunology 118, 1564-1568.
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Sternberg, Z., Chadha, K., Lieberman, A., Hojnacki, D., Drake, A., Zamboni, P., Rocco,
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P., Grazioli, E., Weinstock-Guttman, B., Munschauer, F., 2008. Quercetin and interferon-
426
beta modulate immune response(s) in peripheral blood mononuclear cells isolated from
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multiple sclerosis patients. Journal of Neuroimmunology 205, 142-147.
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Ting, S., Yeh, H.S., Lien, T.F., 2011. Effects of supplemental levels of hesperetin and
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naringenin on egg quality, serum traits and antioxidant activity of laying hens. Animal
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Feed Science and Technology 163, 59-66.
431
Vakili, R., Daliri, R., 2010. The effectofdifferentlevels of vitamin e on humoralimmunity,
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and performance in broiler chicks. Journal of Veterinary Research 65, 239-244.
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flavonoids
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study
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Van Der Most, P.J., De Jong, B., Parmentier, H.K., Verhulst, S., 2011. Trade-off between
434
growth and immune function: A meta-analysis of selection experiments. Functional
435
Ecology 25, 74-80.
436
Williams, R.J., Spencer, J.P.E., Rice-Evans, C., 2004. Flavonoids: Antioxidants or
437
signalling molecules? Free Radical Bio Med 36, 838-849.
438
Yunis, R., Ben-David, A., Heller, E.D., Cahaner, A., 2000. Immunocompetence and
439
viability under commercial conditions of broiler groups differing in growth rate and in
440
antibody response to Escherichia coli vaccine. Poultry Science 79, 810-816.
an
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cr
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433
441
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442
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442
Table 1
444
Ingredients and chemical composition of the diets used
cr
us
M
Maize Soybean meal, 450 g crude protein/kg Corn gluten meal, 60 g crude protein/kg Soybean oil Sodium chloride Monocalcium phosphate Limestone Methionine Lysine Vitamin premix1 Mineral premix2
Starter, Grower, Finisher, 1 to 10 day 11 to 22 day 23 to 42 day 558 629 652 336 264 230 50 50 50 16 17 26 5 4 4 15 14 13 16 16 15 0.3 0.5 0.6 0.1 0.1 0.2 2 2 2 2 2 2
an
Ingredients g/kg
ip t
443
445 446 447 448 449 450 451
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Chemical composition g/kg Metabolizable energy (Mj/kg) 3 13.62 13.92 14.25 Dry matter 888 888 900 Ash 62 55 53 Crude protein 227 208 193 Fat 37 42 53 Fiber 26 23 25 3 Lysine 12 11 10.5 8.3 7.7 7.4 Methionine + cystine3 3 Calcium 10 9.6 9 Available phosphorus3 5 4.8 4.5 1 The vitamin premix provided per kg of diet: Vitamin A (retinyl acetate), 12,000 IU; vitamin D3, 75 μg; vitamin E, 50 IU; vitamin K3, 7 mg; vitamin B1, 3 mg; vitamin B2, 6 mg; vitamin B6, 6 mg; vitamin B12, 25 μg; nicotinic acid, 40 mg; pantothenic acid, 12 mg; folic acid, 1.2 mg; biotin, 150 μg. 2 The mineral premix provided per kg of diet: 400 mg of choline chloride, 250 μg of Co, 1.5 mg of I, 300 μg of Se, 50 mg of Fe, 130 mg of Mn, 20 mg of Cu, and 100 mg of Zn. 3 Calculated
452 453
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Table 2
454
Effect of dietary quercetin on cutaneous basophilic hypersensitivity response to
455
phytohaemagglutinin-P (PHA-P) of broiler chickens
456 457
Quercetin level, g/kg
458
feed
459
0
0.511
460
0.5
0.412
us
PHA index
ip t
453
461
1
0.466
0.673
462
SEM
0.038
0.076
463
P-linear
0.406
0.421
cr
0.759
M
an
0.608
n=16
466
Test 1, at 15 d with 100 g PHA-P
467
Test 2, at 48 d with 20 g PHA-P
Ac ce p
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465
468
Test 2
d
464
Test 1
Page 24 of 27
25
Table 3
469
Effect of dietary quercetin on anti-SRBC antibody titer of broiler chickens (least square
470
means ± se)
471
475 476 477
Total
level, g/kg feed
IgY
IgM
cr
474
n
antibody
0
15
6.17 ± 0.61
0.50 ± 0.11 5.67 ± 0.59
0.5
16
6.59 ± 0.59
0.53 ± 0.11 6.06 ± 0.57
1
16
7.13 ± 0.59
P-linear
0.266
0.81 ± 0.11 6.31 ± 0.57 0.044
0.435
M
478
us
473
Quercetin
an
472
Anti-SRBC antibody titter
ip t
468
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479
Page 25 of 27
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479
Table 4
481
Percentages of thymocytes in the four developmental stages based on the cell surface
482
expression of CD4 and CD8 proteins: from most immature to mature T, CD4-CD8-
483
Double Negative (DN) CD4+CD8+ Double Positive (DP) CD8+ Single Positive
484
(SP) or CD4+SP (least square means ± se)
cr
ip t
480
% Cells expressing
us
% in Thymocyte Subset
n
CD4-D8- CD4+D8+ CD8+SP DN DP
M
Quercetin level,
an
high levels of CD3
% of
17.5±1.3 68.7±1.9 11.3±1.2 2.48±0.28
0.5
13
17.8±1.3 68.7±2.0 11.4±1.3 2.13±0.28
60.6±4.4 57.5±5.6
1
13
17.6±1.3 69.7±2.0 10.7±1.3 2.11±0.28
58.2±4.4 53.9±5.6
te
14
Ac ce p
486
% of
0
P-linear 485
(% of subset)
CD8+SP CD4+SP
d
g/kg feed
CD4+SP
on their cell surface
0.969
0.726
0.705
0.354
59.4±4.3 60.2±5.4
0.814
0.424
Page 26 of 27
27
Table 5
488
Effect of dietary quercetin on lymphoid organ weights and on bursa and thymus cell
489
numbers of broiler chickens
ip t
486 487
Organ* Spleen,
g
%
level,
Bursa, Bursa, Thymus, g
%
g
g/kg feed 3.05
1.104
1.75
0.058
0.5
2.92
1.096
1.70
0.066
1
2.91
1.093
1.96
0.062
SEM
0.167
0.006
0.34
P-linear
0.551
0.205
0.659
491
%, organ index (g/100g BW)
(one lobe)
8.09
2.88
7.51
3.39
0.93
0.030
4.68
3.00
0.012
0.075
0.003
3.77
0.42
0.810
0.701
0.952
0.531
0.837
M
0.034
Ac ce p
492
0.030
x 109
1.03
d
*n=16, **n=6
0.89
x 108
te
490
%
an
0
Thymus, Bursa** Thymus*
cr
Spleen,
us
Quercetin
Cell number
Page 27 of 27
S0377-8401(14)00304-6 http://dx.doi.org/doi:10.1016/j.anifeedsci.2014.09.021 ANIFEE 13161
To appear in:
Animal
Received date: Revised date: Accepted date:
24-3-2014 16-9-2014 22-9-2014
Feed
Science
and
Technology
Please cite this article as: Hager-Theodorides, A.L., Goliomytis, M., Delis, S., Deligeorgis, S.,Effects of dietary supplementation with quercetin on broiler immunological characteristics, Animal Feed Science and Technology (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.09.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Effects of dietary supplementation with quercetin on broiler immunological
2
characteristics
3
A. L. Hager-Theodorides*, M. Goliomytis, S. Delis and S. Deligeorgis
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Laboratory of Animal Breeding and Husbandry, Department of Animal Science and
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Aquaculture, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece
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*Corresponding author:
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Telephone number: +30 210 5294453
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e-mail: [email protected]
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Fax +30 210 5294442
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Highlights
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• Quercetin did not affect broilers' cellular immune response to phytohaemagglutinin. • It did not affect the birds’ thymus cell subset composition and maturity. • It did not affect lymphoid organ weight or cellularity. • Quercetin significantly affected humoral responses to SRBC
21
immunization.
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• IgY antibody titers significantly increased with increasing quercetin
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levels.
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Abstract
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Quercetin, among other flavonoids, is under investigation as feed additive for poultry
27
due to its antioxidative properties. Quercetin is also known to possess
28
immunomodulatory
29
uncompromised immune function is important for poultry productivity and welfare
30
this study investigates possible effects of dietary supplementation of broilers with
31
quercetin on their immune function and immune status. We assessed aspects of the
32
chickens cell-mediated and humoral immune response and immune status when fed
33
with diets supplemented with quercetin at 0.5 and 1 g/kg feed compared to control
34
diets. We measured their cellular immune response to the phytohaemagglutinin skin
35
test, their humoral immune response to sheep red blood cells and assessed the status
36
of the birds’ lymphoid organs and the cell subset composition of the thymus. We
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and
in
particular
anti-inflammatory
properties.
As
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found no differences between dietary treatment groups in cellular immune response or
38
immune status (P>0.05). Interestingly, while total and IgM antibody titers did not
39
differ among treatment groups (P>0.05), IgY antibody titers significantly increased
40
with increasing quercetin supplementation (P-linear<0.05). Therefore, dietary
41
quercetin did not compromise any of the immune parameters tested but on the
42
contrary enhanced IgY antibody production.
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1. Introduction
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Flavonoids, naturally occurring plant secondary metabolites, are currently under
45
investigation as feed additives in poultry for their antioxidative properties (Rice-Evans,
46
2001; Williams et al., 2004). Antioxidative agents are important for poultry nutrition as
47
they reduce lipid peroxidation, a major concern for the organoleptic characteristics and
48
nutritional value of meat and eggs, and can prolong poultry product shelf life (Fellenberg
49
and Speisky, 2006). Naturally occurring polyphenols, including flavonoids, are attractive
50
antioxidative feed additive candidates as they are safer alternatives to synthetic ones
51
(Balasundram et al., 2006).
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Apart from potent antioxidants, flavonoids have been shown to possess
53
immunomodulatory and in particular anti-inflammatory properties (González-Gallego et
54
al., 2010; Serafini et al., 2010). Animal model and human in vitro studies have shown
55
that flavonoids reduce inflammatory immune function often by modulating the
56
production of pro- and anti-inflammatory molecules by cells of the innate and adaptive
57
immune system (e.g. macrophages and T cells) in response to stimuli that upregulate
58
inflammatory processes (Kumazawa et al., 2006; González-Gallego et al., 2010).
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Quercetin, a flavonoid belonging to the class of flavonols, is present in several
60
fruits, vegetables and herbs (Manach et al., 2004). It has been shown to improve
61
oxidative status of broiler meat when added to the birds’ feed and was detectable in the
62
plasma and in several tissues of the animals (Rupasinghe et al., 2010). Quercetin has been
63
shown to act as an anti-inflammatory agent in mice (Comalada et al., 2006; Hamalainen
64
et al., 2007; Huang et al., 2010) and in humans (Sternberg et al., 2008).
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A functional immune system is essential for the maintenance of good health,
66
especially in the case of broilers’ crowded rearing conditions that increase physical
67
contact and therefore exposure to pathogens (Korver, 2012). Possible immunomodulatory
68
properties of flavonoids should be carefully investigated to ascertain that they will not
69
compromise the immune defense of the birds. Immunomodulatory compounds tend to
70
differentially influence different immune functions, so several parameters of innate and
71
acquired immunity should be investigated, including cellular and humoral responses.
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So far studies on flavonoids in poultry examine, in addition to meat oxidative
73
status, effects on growth performance and other productive traits, including meat (Batista
74
et al., 2007; Jiang et al., 2007; Peña et al., 2008; Simitzis et al., 2011; Kamboh and Zhu,
75
2013) and egg (Lien et al., 2008; Ting et al., 2011; Goliomytis et al., 2014a) quality
76
parameters. The effect of quercetin on broiler meat quality parameters was tested by
77
(Goliomytis et al., 2014b).
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This study investigated possible effects of dietary quercetin on parameters of
79
broiler immune function and status. We examined parameters of cellular and humoral
80
immunity, lymphoid organs size and cellularity and thymus subset composition to
81
determine whether the immunomodulatory properties of quercetin exhibited in other
82
animals are manifested in broilers when it is included in their feed.
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2. Materials and methods
85
2.1 Animals, diets and experimental design
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One hundred and fifty day-old Cobb 500 broiler chickens, obtained from a commercial
87
hatchery, as hatched, were housed in a controlled environment at the facilities of the
88
Agricultural University of Athens. The birds were reared in 12 pens, 2 m2 each (stocking
89
density of 6.25 birds/m2), for 51 days. Each chicken was individually wing tagged upon
90
arrival. Lighting program, environmental conditions and management practices were in
91
accordance to standard Cobb guidelines. In brief, the lighting program consisted of
92
23L:1D on arrival, and was gradually decreased to 18L:6D by day 24, remained constant
93
until day 37, and thereafter increased to 23L:1D till slaughter. Feed, in mash form, and
94
water were provided ad libitum. The composition and the chemical analysis of the three
95
diets offered, throughout the rearing period (starter, grower and finisher), are presented in
96
Table 1.
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Chickens were randomly assigned to three treatment groups, control (C),
98
quercetin-0.5 (Q-0.5) and quercetin-1 (Q-1), each replicated in four pens. The diet of
99
treatment groups C, Q-0.5 and Q-1 were supplemented for the duration of the experiment
100
with 0, 0.5 and 1 g quercetin (MP Biochemicals, France) per kg feed, respectively. Feed
101
enrichment with quercetin was performed daily.
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Animal handling and all procedures performed in the present experiment were in
103
accordance with the national legislation and the guidelines of the Research Ethics
104
Committee of the Department of Animal Science and Aquaculture of the Agricultural
105
University of Athens.
106
2.2 Phytohaemagglutinin skin test
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The phytohaemagglutinin (PHA) skin test was adapted from (Corrier and DeLoach,
108
1990) and (Lavoie et al., 2007). In brief, 30 l containing 100g PHA (PHA-P, Sigma-
109
Aldrich L1668, diluted in phosphate buffered saline (PBS) to a final concentration of
110
3333 g/ml PHA-P) were injected intradermally with 30-gauge needles into the
111
interdigital web space between the second and third toes of the left foot of 15 days old
112
birds. Thirty microliters of PBS were administered into the corresponding interdigital
113
web of the right foot of each bird. Thickness of the web in both feet was measured before
114
injection and 24 hours after injection using a thickness gauge with 0.01mm precision
115
(Peacock, Ozaki MFG. CO. Ltd, Tokyo, Japan). To calculate the PHA stimulation index
116
the change of thickness in the control foot web was subtracted from the corresponding
117
change in the PHA-treated foot web. Sixteen birds per treatment chosen at random (four
118
from each replicate pen) were subjected to the PHA skin test.
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The PHA skin test was repeated to the same birds at 48 days of age with a lower
120
dose of PHA, to avoid any possible saturating effects of a high stimulus on the response
121
of the birds, that would possibly mask differences between groups. In the second test 30
122
l containing 20g PHA were injected into the interdigital web space between the first
123
and second toes of the left foot and same volume of PBS was injected in the
124
corresponding area of the right foot. Skin thickness measurements and stimulation index
125
were performed as above.
126
2.3 Immunization with sheep red blood cells (SRBC)
127
SRBC were prepared from total sheep blood from a healthy individual, collected with
128
EDTA anticoagulant (2mg EDTA/ml of blood) spun at 400xg, washed three times with
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Page 7 of 27
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phosphate buffered saline (PBS) and resuspended in Alsevier’s solution to a final
130
haematocrit (Ht) of 10%. SRBC was stored in the fridge and diluted with PBS at 1% Ht
131
for immunization of the birds (adapted from (Olsson and Oldenborg, 2008). Sixteen 21d
132
old birds per treatment, the same submitted to the PHA skin treatment, were injected
133
intravenously (into the brachial vein) with 100l 1% SRBC in PBS. Six days post SRBC
134
challenge blood samples were collected from the immunized birds to determine plasma
135
anti-SRBC antibody titer with the haemagglutination test (protocol adapted from (Lavoie
136
et al., 2007). Briefly, blood samples, collected with EDTA anticoagulant (2mg EDTA/ml
137
of blood), were centrifuged at 800xg for 20 minutes and supernatant was collected and
138
complement was heat inactivated at 56oC for 30 minutes. To determine total antibody
139
titers, 25 l of plasma were mixed with 25 l PBS in the first well of a 96-well U bottom
140
microtiter plate. Plasma was serially diluted across a plate row, 25 l 1% SRBC was
141
added to each well and the plate was incubated at 37oC for one hour. Anti-SRBC total
142
antibody titer was determined as the logarithm to base two of the dilution factor of the
143
highest dilution showing haemagglutination. To determine IgY (bird equivalent to
144
mammalian IgG) antibody titer (-mercaptoethanol, 2-ME, resistant) 25 l of plasma
145
were incubated with 25 l of 0.2 M 2-ME in the first column of a 96-well U bottom
146
microtiter plate at 37oC for 1 hour and then haemagglutination test was performed as for
147
total antibody titer. To determine IgM antibody titer IgY titer was subtracted from total
148
antibody titer. All plasma samples were tested in duplicates.
149
2.4 Lymphoid organ weights and cell counts
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At 51 days of age the sixteen birds per treatment that were subjected to PHA skin test and
151
were immunized with SRBC were weighed, electrically stunned and slaughtered. Bursa
152
of Fabricius, spleen and one thymus lobe (second lobe from the right side) were dissected
153
and weighed. Organ indexes were calculated as the percentage of live body weight (organ
154
weight/live body weight x 100). Bursa of Fabricius cell suspensions from 6 birds per
155
treatment and thymus lobe cell suspensions from 16 birds per treatment were prepared by
156
crushing tissue aliquots (of known weight) between two pieces of ground glass. Cells
157
were counted on a haematocytometer and total cell count was calculated for the whole
158
organ or lobe.
159
2.5 Thymus subset composition
160
Thymocyte suspensions were prepared as described above and cells were stained using
161
the following combination of directly conjugated chicken reactive antibodies obtained
162
from Abcam (UK): anti-CD8-Fluorescein isothiocyanate (FITC), anti-CD4-phycoerythrin
163
(PE), anti-CD3-PE/Cyanine (Cy)5. Briefly, approximately 5x106 cells were spun and
164
resuspended in 50l staining medium (PBS supplemented with 2% FCS and 0.01%
165
sodium azide) containing 0.5g/ml of each antibody and were incubated for 30 minutes
166
on ice. Cells were washed in staining medium, spun at 300xg and resuspended in 100l
167
1% paraformaldehyde (PFA). Cells were fixed in PFA for 20 minutes on ice, washed and
168
spun as above, resuspended in staining medium and stored in the fridge until analyzed on
169
the flow cytometer (FC-500, Beckman- Coulter, USA). 5x105 events were collected per
170
sample, in list mode, using CXP software and data were analyzed using FlowJo software.
171
Live lymphocytes were gated according to their forward scatter (FSC) and side scatter
172
(SSC) profiles.
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2.6 Statistical analysis
174
Data were subjected to ANOVA using the general linear models, of SAS software (SAS
175
Institute, Cary NC, USA, version 9.0, 2002), with quercetin level as the fixed effect. The
176
individual bird was considered as the experimental unit for all traits measured except for
177
BW at slaughter for which the experimental unit was the pen. Alpha for determination of
178
significance was 0.05. The linear dose response of quercetin was determined with
179
contrasts among means. Means are presented as least square means.
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an
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3. Results
182
3.1 Immune function
183
3.1.1 Phytohaemagglutinin skin test
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To assess the effect of quercetin dietary supplementation on the birds’ localized
185
inflammatory response and cell-mediated immunocompetence we performed the
186
phytohaemagglutinin (PHA) skin test that elicits a cell-mediated cutaneous basophil
187
hypersensitivity immune reaction and is often used to assess cellular immunity in birds
188
(Stadecker et al., 1977; Fairbrother et al., 2004; Martin et al., 2006). Response to the
189
PHA skin test is widely interpreted as an index of birds’ cell-mediated
190
immunocompetence (Corrier, 1990; Fairbrother et al., 2004). Large responses to the PHA
191
skin test are also considered to be an indicator of unregulated local inflammation or
192
allergy (Elgert, 2009).
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193
As shown in Table 2 the PHA stimulation index, was not affected by quercetin dietary
194
supplementation at either 15 or 48 days of age (P-linear>0.05)
ip t
195
3.1.2 Humoral immunity
197
To investigate possible effects of quercetin on the chickens’ humoral immune response
198
we immunized them with sheep red blood cells (SRBC) and measured the antibody titers
199
in their blood plasma six days later (Table 3). Immunization with SRBC is a commonly
200
used in vivo assay for humoral immunity in birds and other species. It elicits a T helper
201
dependent activation of B cells and production of anti-SRBC antibodies (Fairbrother et
202
al., 2004). Means for total and IgM antibody titers for treatment groups were not
203
statistically different from control (P>0.05). Interestingly, a linear dose response of
204
quercetin on IgY titers (P-linear<0.05) was observed.
205
quercetin resulted in increasing anti-SRBC IgY antibody production.
207 208
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Increasing levels of dietary
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206
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3.2 Immune status
At 51 days of age thymus homeostasis was assessed by flowcytometric analysis of
209
thymocyte developmental subset composition based on the expression of cell surface
210
proteins CD4, CD8 and CD3. Thymus homeostasis is often assessed by the analysis of
211
the ratios of the developmental stages of the thymocytes present in the organ, as
212
thymocytes develop from lymphoid progenitor cells to mature and functional T cells that
213
migrate to sites of lymphoid activity. Thymocyte developmental stages are
Page 11 of 27
12
phenotypically determined by the expression of cell surface markersCD4 and CD8
215
proteins. Immature thymocytes that do not express either CD4 or CD8 (double negative
216
stage, DN) progress to a CD4+CD8+ double positive stage (DP) and the most mature
217
thymocytes are either CD4+ or CD8+ single positive (SP) cells. Cell surface expression of
218
CD3 further denotes thymocyte maturation, it is upregulated at the DP compared to the
219
DN stage and finally mature and functional CD4+SP and CD8+SP thymocytes, express
220
high levels of CD3 (Cooper et al., 1991).
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214
We found no statistically significant differences (P>0.05) in mean DN, DP,
222
CD4SP or CD8SP cell percentages between treatment groups (Table 4). In addition, we
223
found no differences (P>0.05) between groups in the mean percentages of CD4 or CD8
224
single positive cells that expressed CD3 on their surface neither in the mean relative level
225
of CD3 protein cell surface expression on double positive or single positive cells, as
226
evident by the mean fluorescence intensity of the cells for the PE/Cy5 fluorochrome
227
conjugated to the anti-CD3 antibody, used to detect CD3 expression (p>0.05, data not
228
shown).
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To further assess immune status of the animals at day 51, spleen, bursa of
230
Fabricius and thymus of the chickens were weighed and thymus and bursa of Fabricius
231
cell numbers were counted to assess the immune status of the animals. We found no
232
differences among treatment groups in organ weights or organ weights as a percentage of
233
live body weight (P>0.05) (Table 5). In addition, means for bursa and thymus cell counts
234
were not statistically different for different treatments (P>0.05) (Table 5).
235
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4. Discussion
237
Quercetin, among other flavonoids, is under investigation as a putative antioxidant
238
supplement for broiler feed. There is no published data on the effects of quercetin or
239
other flavonoids on chicken cell-mediated immune response. Nevertheless, it is well
240
documented that quercetin exhibits immunomodulatory properties in other species. A
241
large number of in vitro and in vivo studies support an immunomodulatory and anti-
242
inflammatory role for quercetin and other flavonoids in man and in rodents (Boots et al.,
243
2008; García-Lafuente et al., 2009; González-Gallego et al., 2010; Serafini et al., 2010).
244
Studies on human lymphocytes showed that flavonoids reduced immune cell proliferation
245
and pro-inflammatory cytokine production in response to PHA stimulation in vitro
246
(Pandey et al., 2005). Quercetin, administered intraperitoneally (75mg quercetin/ kg body
247
weight), was shown to reduce inflammation in vivo in mice in experimental models of
248
acute and chronic inflammatory responses (Rotelli et al., 2003). In contrast to these data
249
from humans and mice, our experiments did not reveal an in vivo effect of dietary
250
quercetin on the cellular immune response of broilers as assessed by the PHA skin test.
251
This can be attributed to species differences, to quercetin dosage and route of
252
administration and/or to differences in the in vivo challenges employed to assess
253
inflammatory responses.
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254
ip t
236
Effects on humoral immunity have not been reported for quercetin in chickens or
255
other species. Other antioxidant compounds have been shown to affect antibody
256
production in chickens. In broilers, supplementation with vitamin E increased humoral
257
immune responses to SRBC (Boa-Amponsem et al., 2000; Niu et al., 2009), infectious
258
bronchitis virus (Leshchinsky and Klasing, 2001) and Newcastle disease virus (Vakili
Page 13 of 27
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and Daliri, 2010) immunizations. In this study although no effects of quercetin were
260
detected on total and IgM antibody titers, IgY titers increased with increasing levels of
261
quercetin supplementation in the primary response to SRBC immunization. In the
262
primary response IgM immunoglobulins are produced before and are more abundant than
263
IgY. This is reversed in additional immunizations where IgG is the most abundant
264
immunoglobulin circulating in the blood plasma. Our finding suggests that dietary
265
quercetin enhances humoral immune response in a dose-dependent manner. Nutrients that
266
were shown to increase both humoral responses and growth performance in non-
267
immunized broilers were reported to lose the growth performance positive effect when
268
birds were immune challenged (Buyse et al., 2009). Here quercetin did not affect mean
269
body weight of immune challenged birds at 51 days of age (p>0.05, overall mean
270
3049±56 g) but it remains to be investigated if quercetin affects growth performance
271
parameters differently in immune challenged compared to naïve birds.
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Size and cellularity of immune organs and thymocyte developmental stage were
273
analyzed to assess immune status and thymus homeostasis respectively. We found no
274
effect of quercetin on any of the parameters tested.
275
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There is evidence that fast growing chicken strains such as modern broilers have
276
reduced immune function compared to slower growing strains (Qureshi and Havenstein,
277
1994; Yunis et al., 2000; Cheema et al., 2003). It is argued that selection for growth traits
278
may have unintentionally caused indirect selection in favor of decreased immune
279
function, both cellular and humoral (Van Der Most et al., 2011; Korver, 2012). A
280
possible cause for this could be that pro-inflammatory responses and cytokines produced
281
decrease the birds’ metabolic activity and appetite thus negatively influencing feed intake
Page 14 of 27
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and growth (Lochmiller and Deerenberg, 2000). Improved growth performance of
283
modern broiler chickens may have been partly accomplished at the expense of immune
284
function. In the case of subclinical infections or non-infectious microorganisms or agents,
285
reduced inflammatory responses can redirect nutrients and energy towards growth
286
without having adverse effects on animal health. But decreased responses can lead to
287
increased morbidity and/or mortality in the case of infections that rely mainly on
288
inflammatory immune responses to be cleared and result in deterioration of animal
289
welfare and substantial economic loss. An anti-inflammatory or other immunomodulatory
290
effect of dietary supplements would therefore not be beneficial for broilers if it
291
compromised their resistance to infections.
292
5. Conclusion
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Here we assessed aspects of cellular and humoral immune responses, thymus
294
subset composition and immune status of broilers fed with quercetin, supplemented at 0,
295
0.5 and 1 g/kg feed. We did not detect any effects of quercetin on broiler chickens
296
cellular immunity, thymus subset composition or immune status parameters studied.
297
Interestingly, quercetin was found to impact with a linear dose-dependent manner on IgY
298
antibody production in response to SRBC immunization. Therefore, dietary quercetin did
299
not adversely affect any of the immune parameters tested but on the contrary enhanced
300
primary IgY antibody production. These findings suggest that quercetin (a) would not
301
compromise the birds immunocompetence, if used as a feed additive for its antioxidant
302
properties, and (b) could potentially enhance broilers’ humoral immune responses.
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Acknowledgements
305
The authors wish to thank Dr Nikos Demiris for useful discussions on the statistical
306
analysis and Drs Panagiotis Simitzis and Maria Charismiadou for discussions on the
307
experimental design. They also wish to thank Jenny Biniari and Dimitra Tsoureki for
308
technical assistance.
309
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439
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an
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441
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442
Table 1
444
Ingredients and chemical composition of the diets used
cr
us
M
Maize Soybean meal, 450 g crude protein/kg Corn gluten meal, 60 g crude protein/kg Soybean oil Sodium chloride Monocalcium phosphate Limestone Methionine Lysine Vitamin premix1 Mineral premix2
Starter, Grower, Finisher, 1 to 10 day 11 to 22 day 23 to 42 day 558 629 652 336 264 230 50 50 50 16 17 26 5 4 4 15 14 13 16 16 15 0.3 0.5 0.6 0.1 0.1 0.2 2 2 2 2 2 2
an
Ingredients g/kg
ip t
443
445 446 447 448 449 450 451
Ac ce p
te
d
Chemical composition g/kg Metabolizable energy (Mj/kg) 3 13.62 13.92 14.25 Dry matter 888 888 900 Ash 62 55 53 Crude protein 227 208 193 Fat 37 42 53 Fiber 26 23 25 3 Lysine 12 11 10.5 8.3 7.7 7.4 Methionine + cystine3 3 Calcium 10 9.6 9 Available phosphorus3 5 4.8 4.5 1 The vitamin premix provided per kg of diet: Vitamin A (retinyl acetate), 12,000 IU; vitamin D3, 75 μg; vitamin E, 50 IU; vitamin K3, 7 mg; vitamin B1, 3 mg; vitamin B2, 6 mg; vitamin B6, 6 mg; vitamin B12, 25 μg; nicotinic acid, 40 mg; pantothenic acid, 12 mg; folic acid, 1.2 mg; biotin, 150 μg. 2 The mineral premix provided per kg of diet: 400 mg of choline chloride, 250 μg of Co, 1.5 mg of I, 300 μg of Se, 50 mg of Fe, 130 mg of Mn, 20 mg of Cu, and 100 mg of Zn. 3 Calculated
452 453
Page 23 of 27
24
Table 2
454
Effect of dietary quercetin on cutaneous basophilic hypersensitivity response to
455
phytohaemagglutinin-P (PHA-P) of broiler chickens
456 457
Quercetin level, g/kg
458
feed
459
0
0.511
460
0.5
0.412
us
PHA index
ip t
453
461
1
0.466
0.673
462
SEM
0.038
0.076
463
P-linear
0.406
0.421
cr
0.759
M
an
0.608
n=16
466
Test 1, at 15 d with 100 g PHA-P
467
Test 2, at 48 d with 20 g PHA-P
Ac ce p
te
465
468
Test 2
d
464
Test 1
Page 24 of 27
25
Table 3
469
Effect of dietary quercetin on anti-SRBC antibody titer of broiler chickens (least square
470
means ± se)
471
475 476 477
Total
level, g/kg feed
IgY
IgM
cr
474
n
antibody
0
15
6.17 ± 0.61
0.50 ± 0.11 5.67 ± 0.59
0.5
16
6.59 ± 0.59
0.53 ± 0.11 6.06 ± 0.57
1
16
7.13 ± 0.59
P-linear
0.266
0.81 ± 0.11 6.31 ± 0.57 0.044
0.435
M
478
us
473
Quercetin
an
472
Anti-SRBC antibody titter
ip t
468
Ac ce p
te
d
479
Page 25 of 27
26
479
Table 4
481
Percentages of thymocytes in the four developmental stages based on the cell surface
482
expression of CD4 and CD8 proteins: from most immature to mature T, CD4-CD8-
483
Double Negative (DN) CD4+CD8+ Double Positive (DP) CD8+ Single Positive
484
(SP) or CD4+SP (least square means ± se)
cr
ip t
480
% Cells expressing
us
% in Thymocyte Subset
n
CD4-D8- CD4+D8+ CD8+SP DN DP
M
Quercetin level,
an
high levels of CD3
% of
17.5±1.3 68.7±1.9 11.3±1.2 2.48±0.28
0.5
13
17.8±1.3 68.7±2.0 11.4±1.3 2.13±0.28
60.6±4.4 57.5±5.6
1
13
17.6±1.3 69.7±2.0 10.7±1.3 2.11±0.28
58.2±4.4 53.9±5.6
te
14
Ac ce p
486
% of
0
P-linear 485
(% of subset)
CD8+SP CD4+SP
d
g/kg feed
CD4+SP
on their cell surface
0.969
0.726
0.705
0.354
59.4±4.3 60.2±5.4
0.814
0.424
Page 26 of 27
27
Table 5
488
Effect of dietary quercetin on lymphoid organ weights and on bursa and thymus cell
489
numbers of broiler chickens
ip t
486 487
Organ* Spleen,
g
%
level,
Bursa, Bursa, Thymus, g
%
g
g/kg feed 3.05
1.104
1.75
0.058
0.5
2.92
1.096
1.70
0.066
1
2.91
1.093
1.96
0.062
SEM
0.167
0.006
0.34
P-linear
0.551
0.205
0.659
491
%, organ index (g/100g BW)
(one lobe)
8.09
2.88
7.51
3.39
0.93
0.030
4.68
3.00
0.012
0.075
0.003
3.77
0.42
0.810
0.701
0.952
0.531
0.837
M
0.034
Ac ce p
492
0.030
x 109
1.03
d
*n=16, **n=6
0.89
x 108
te
490
%
an
0
Thymus, Bursa** Thymus*
cr
Spleen,
us
Quercetin
Cell number
Page 27 of 27