The transcriptomics-related mechanisms of supplementing laying

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The transcriptomics-related mechanisms of supplementing laying broiler breeder hens with dietary daidzein to improve the immune function and growth performance of offspring Hao Fan, Zengpeng Lv, Liping Gan, and Yuming Guo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06069 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 19, 2018

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Journal of Agricultural and Food Chemistry

The transcriptomics-related mechanisms of supplementing laying broiler breeder hens with dietary daidzein to improve the immune function and growth performance of offspring

Hao Fan+, Zengpeng Lv+, Liping Gan, Yuming Guo* State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, 2 Yuanmingyuan West Road, Beijing, 100193, PR China Hao Fan: [email protected] Zengpeng Lv: [email protected] Liping Gan: [email protected] Yuming Guo: [email protected] *Corresponding author: Yuming Guo; E-mail: [email protected]; Tel.: +86 10 62733900; Fax: +86 10 62733900; Mailing address: China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, China +

These authors contributed equally to this work.

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ACS Paragon Plus Environment


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ABSTRACT: Daidzein (DA) is an isoflavone that is primarily extracted from soy

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plants. This study evaluated the effects of supplementing laying broiler breeder hens

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with dietary DA on the immune function and growth performance of their offspring

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and the underlying mechanism. A total of 720 breeders were divided into 3 treatment

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groups that were fed either a control diet (CON), a DA-low-supplemented diet (DLS,

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CON+20 mg/kg DA) or a DA-high-supplemented diet (DHS, CON+100 mg/kg DA)

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for 8 weeks, eggs were collected for hatching during the final week. The broiler

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offspring received a basal diet for 42 days, and blood, livers and immune organs were

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collected at 21 and 42 days of age. DLS treatment promoted embryonic development,

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and increased growth hormone levels, body weight, feed intake and carcass traits on

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days 21 and 42 of broilers. Additionally, the IgA and IgG concentrations, antibody

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titers and antioxidant capacity of broilers were increased at 21 days of age, and B

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lymphocyte differentiation was increased at 42 days. Besides, DLS treatment

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upregulated the expression of genes related to embryonic and muscle development in

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offspring and regulated mitogen-activated protein kinase (MAPK), transforming

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growth factor beta (TGF-β), nuclear factor kappa-light-chain-enhancer of activated B

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cells (NF-κB) and Toll-like receptor signaling. DHS treatment decreased the

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percentage of abdominal fat in the broilers at 42 days, but it did not significantly

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affect embryonic development, growth performance, IgA and IgG concentrations. In

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summary, providing dietary DA supplementation at 20 mg/kg to broiler breeders can

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improve their immune function and growth performance.

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KEYWORDS: Daidzein, Breeders, Offspring broilers, Transcriptome, Embryonic 2


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development, Growth performance, Immune function

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Introduction

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Daidzein (DA, 4′,7-dihydroxyisoflavone) is a natural isoflavonic phytoestrogen

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belonging to the non-steroidal estrogens that is primarily derived from leguminous

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plants.1 DA is also a major bioactive ingredient in many traditional Chinese

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medicines. The chemical structure of DA is similar to that of estrogens, and it exerts

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dual-directional protective effects against certain diseases that are linked to estrogen

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regulation, such as breast cancer, osteoporosis, and diabetes.2 DA also has a number

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of other biological activities independent of the estrogen receptor (ER), such as

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anti-inflammation, anticancer and oxidative damage inhibition.3

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In recent years, the growth-promoting effect of DA has been widely reported. In

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addition to promoting animal growth4, supplemental isoflavones have been shown to

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have beneficial effects on the performance and digestive function of broilers.5

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Furthermore, dietary DA at 200 ppm has been shown to be a weak enhancer of body

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growth in virally challenged pigs,6 and DA supplementation can promote dietary

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protein digestion and improve the productive performance of bull calves.7 It has also

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been demonstrated that feeding soy isoflavone increases the weight gain, feed intake

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and meat quality of broilers.8 However, excessive DA can have detrimental effects

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on animals; 400 mg/kg dietary DA supplements negatively affected weight gain and

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splenic morphology of pigs.9

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DA has been shown to precisely orchestrate processes related to immune

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function. Isoflavones can modulate immune function in postmenopausal women,10

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and DA in infant formula has a positive effect on the immune system.11 In addition, 4


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DA has been effective in the treatment of lipopolysaccharide-induced inflammation12,

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and

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(TPA)-induced skin inflammation by reducing the activation of nuclear factor

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kappa-light-chain-enhancer of activated B cells (NF-κB).13 In vitro, DA has been

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shown to suppress macrophage infiltration and decrease production of reactive

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oxygen species (ROS), expression of pro-inflammatory mediators such as inducible

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isoform (iNOS) and cyclooxygenase-2 (COX-2) and pro-inflammatory factors such

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as tumor necrosis factor alpha (TNF-α) by inhibiting the mitogen-activated protein

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kinase (MAPK) signaling pathway.14

it

has

been

used

to

suppress

12-O-tetradecanoylphorbol-13-acetate

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In animals, early nutrition are extremely important; maternal nutrition can affect

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the early developmental status of offspring through metabolism or gene

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modification.15 Consumption of DA by the mother will positively affect the growth

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and immune functions of the offspring. Studies have revealed that legume

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consumption by pregnant women can play a favorable regulatory role in fetal

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development,16 and feeding isoflavones to adult mice induces DNA modification and

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affects the metabolic status of their offspring.17 Nevertheless, there is a lack of

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understanding of the effects of DA supplementation to broiler breeder hens on the

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immune function and growth performance of their offspring as well as the

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underlying molecular mechanisms. Therefore, the present study was conducted to

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determine whether supplementing broiler breeder hens with dietary DA can affect

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embryonic development and improve the growth performance and immune function

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of broiler chicks. 5



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Materials and Methods

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Materials. The DA used in this study was synthetically produced by the Kai Meng

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Co. (Xi An, Shanxi, China) Chemical Plant with a purity of 99.9%.

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Feeding Experiment Design and Bird Management. The experimental animal

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procedures were approved by the China Agricultural University Animal Care and

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Use Committee, Beijing, China. The experiment was carried out with laying broiler

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breeder hens and broilers housed at a commercial farm (Zhuozhou, China) under

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standard conditions. After a 2-week acclimation period, a total of 720 57-week-old

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Ross 308 laying broiler breeder hens were allocated to three treatment groups:

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control (CON), DA-low-supplemented (DLS) and DA-high-supplemented (DHS).

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Each treatment was replicated 8 times; each replicate included 30 broiler breeder

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hens. Broiler breeder roosters were housed at a ratio of 30 hens to 1 male. Artificial

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insemination was applied at a ratio of 30 hens to 1 male, and it was conducted once

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every five days. Breeder hens were assigned to the CON, DLS, and DHS treatment

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groups and fed a nutritionally balanced corn-miscellaneous meal (CSCM) with DA

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added at 0, 20, and 100 mg/kg, respectively, for 8 weeks. The CSCM diets were

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formulated to meet the nutrient requirements of laying broiler breeders according to

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the NRC guidelines (1994) (TABLE 1); male breeders were caged and given a

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commercial diet (New Hope Group, Qingdao, Shandong, China). Hatching eggs

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(200 eggs per treatment group, 8 replicates per treatment, 25 eggs per replicate) from

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66-week-old hens from the three groups were divided into the same three treatment

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groups as their mothers (CON, DLS and DHS). Eggs were incubated under standard 6


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conditions of 70%–80% humidity at 37.8 °C with intermittent rotation. After

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hatching, chicks from each group were divided into 8 replicates of 10 birds each, and

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they were housed in wire cages under a standard, gradually decreasing temperature

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regimen that ranged from 35 °C to 26 °C. The experiment continued for 6 weeks,

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during which diets and water were provided ad libitum. The broiler offspring were

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fed a basal corn–soybean meal diet (TABLE 1) formulated based on NRC guidelines

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(1994).

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Sample Collection and Chemical Analysis. At 11, 13, 15, 17, and 19 embryonic

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days of age, one offspring chicken embryo per replicate was selected to measure the

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relative embryo length (embryo length/egg weight), and all 200 eggs in each

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treatment were used to measure incubation parameters during hatching. At 21 and 42

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days of age, one chick per replicate, with a body weight close to the average of its

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replicate (10 broilers), was selected after 8 h of feed deprivation. One blood sample

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was collected from the wing vein of each replicate chick into vacuum blood

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collection tubes, and the serum was centrifuged at 3000× g for 15 min and stored at

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–20 °C until use to detect hormones, antibodies and immunoglobulins (Igs). Another

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blood sample was collected from the wing vein of each replicate chick into vacuum

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blood collection tubes (with heparin sodium) to detect lymphocyte proliferation and

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percentages. Then, one chicken from each replicate was slaughtered. One liver

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sample was immediately collected from each replicate to measure gene expression

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and antioxidative indices, and samples were frozen in liquid nitrogen and stored in a

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freezer (−80 °C). Body weight gain (BWG), feed intake (FI) and the feed conversion 7



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ratio (FCR) were determined for all broilers in each replicate, and the average values

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were calculated for each replicate. At 42 days of age, 1 broiler from each replicate,

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close to the average live bodyweight in each replicate, were selected for

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measurements of carcass traits.

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Radioimmunoassay for Serum Hormone Concentrations. The serum levels of

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triiodothyronine (T3), tetraiodothyronine (T4), growth hormone (GH) and estradiol

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(E2) were measured using commercial double-antibody radioimmunoassay kits

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purchased from the Shanghai Institute of Biological Products (Shanghai, China). The

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inter-assay coefficient of variation was 10%.

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Serum Antibody and Immunoglobulin Levels. The serum antibody titers against

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Newcastle disease (ND) and infectious bursal disease (IBD) viruses and IgM, IgG

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and IgA levels were determined using a commercial ELISA kit (IDEXX laboratories

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Inc., Westbrook, Maine, USA) according to the manufacturer’s protocol.

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Lymphocyte Classification and Proliferation. Peripheral blood mononuclear cells

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(PBMCs) were isolated using a Ficoll density centrifugation.18 Briefly, heparinized

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blood was diluted with Hank’s balanced salt solution at a ratio of 1:1 (no calcium

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and no magnesium, Life Technologies, Burlington, Vermont, USA) and carefully

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layered on top of Histopaque 1077 (Sigma-Aldrich Corporation, Burlington,

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Vermont, USA) in a 10-mL centrifuge tube at a 2:1 ratio. After centrifugation for 30

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min at 3,000 rpm (20 °C), the PBMCs at the plasma-Ficoll interface were collected

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and then washed three times with cold RPMI-1640 medium (containing 5.0%

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inactivated fetal bovine serum, 0.0599 mg/mL penicillin, 100 µg/mL streptomycin 8


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and 24 mM of HEPES) by centrifugation at 1,800 rpm for 10 min (4 °C). Cell counts

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and viability were evaluated using trypan blue staining. Lymphocytes were then

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mixed with CD3 (SPRD), CD4 (FITC) and CD8 (RPE) antibodies or with Bu-1

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(RPE) antibodies, and the cells were then incubated in a water bath for 30 min

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(37 °C), washed twice with Hanks solution and fixed with 3% paraformaldehyde.

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The results are expressed as percentages. The proliferative responses of T cells and B

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cells after stimulation with concanavalin A (ConA, 45 µg/mL) and LPS (25 µg/mL),

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respectively, were determined by MTT assay.19 ConA from Canavalia ensiformis

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(C2010) and LPS from Escherichia coli (L2880) were both obtained from

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Sigma-Aldrich Corporation. The results are expressed as stimulation index (SI)

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values.

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Antioxidant Index Measurements. Livers were homogenized with saline to make a

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10% homogenate. The malondialdehyde (MDA) levels, total superoxide dismutase

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(T-SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities and total

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antioxidant capacity (T-AOC) of the 10% homogenate were determined using a kit

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(Nanjing Jiancheng Inc., Nanjing, China) according to the manufacturer’s protocol.

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Next-Generation Sequencing (NGS). Total RNA samples for sequencing were

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purified from 20 mg of tissue samples from 12 chickens (4 replicates in each

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treatment group) using the RNeasy Fibrous Tissue Mini messenger RNA (mRNA)

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extraction

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recommendations. The concentration and purity of total RNA were determined using

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a UV/Vis spectrophotometer (Actgene, New Jersey, USA) at 260 nm, and sample

kit

(Qiagen,

Hilden,

Germany)

following

9


the

manufacturer’s


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integrity was evaluated using a microfluidic assay on a Bioanalyzer system (Agilent

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Technologies, Inc., Santa Clara, CA, USA). Only high-quality RNA extracts (RNA

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integrity number (RIN)≥8) were used for pooling within each treatment group using

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equal amounts of RNA per chicken, and NGS data were obtained from pooled RNA

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samples within each group to ensure the most robust transcriptome. Complementary

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DNA (cDNA) libraries for RNA sequencing (RNA-Seq) were constructed using a

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TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA), and RNA-Seq

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analysis was performed to identify transcriptional changes using a MiSeq instrument

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(Illumina) using paired-end libraries (CapitalBio, http://cn.capitalbio.com/).20 Four

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replicates from each treatment were analyzed independently for library synthesis and

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sequencing, and the quality of the raw reads was assessed using FastQC (Version

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0.10.1).21 Adapters, low-quality reads at the 3ʹ end, reads with fuzzy N bases,

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ribosomal RNA (rRNA), sequences shorter than 20 nt and low-quality reads (those

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with a Q

The transcriptomics-related mechanisms of supplementing laying

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