Protective Effect of Vitamin E on laying performance, antioxidant capacity, and immunity in laying hens challenged with Salmonella Enteritidis

Protective Effect of Vitamin E on laying performance, antioxidant capacity, and immunity in laying hens challenged with Salmonella Enteritidis 2

∗

State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; and † Institute of Animal Science, University of Hohenheim, 70593 Stuttgart, Germany received the same volume of PS. The egg mass of VE0 treatment decreased (P < 0.05) in contrast to VE treatment after challenge. The serum concentrations of interleukins (IL-1β and IL-6) and malondialdehyde (MDA) levels of SE treatments increased (P < 0.05) at week 44 and week 46, respectively. In both VE30 treatments, the decrease (P < 0.05) in birds’ mortality was associated with higher IgA, IgG, IgM concentrations at week 44, and higher IgA, IgM concentrations at week 46. There is an interaction (P < 0.05) between SE challenge and VE levels with regard to feed conversion, daily egg mass, and serum MDA, IgA, and IgM levels. It can be concluded that supplemental VE (30 IU/kg) in diets for laying hens may alleviate oxidative and immune stress due to SE challenge.

ABSTRACT Vitamin E (VE) has proven to function as potent lipid-soluble antioxidant, a signaling molecule, and a regulator of the immune system. The objective of the study was to assess the protective effect of VE on laying performance, antioxidant capacity, and immunity in laying hens exposed to Salmonella Enteritidis (SE). A total of 80 32-week-old salmonellafree double negative Hy-Line brown laying hens were randomly assigned to 4 treatments with 20 replicates each (1 bird per replicate) according to a 2 × 2 factorial design with 2 VE supplementation levels [0 IU/kg (VE0) vs. 30 IU/kg (VE30)], and 2 challenge treatments [SE vs. physiological saline solution (PS)]. During the last 3 D of week 43 of age, birds were orally challenged with 1.0 mL suspension of 109 cfu/mL S. Enteritidis daily, whereas the birds of negative treatments (VE0)

Key words: vitamin E, Salmonella Enteritidis, laying hen, immunity 2019 Poultry Science 0:1–8 http://dx.doi.org/10.3382/ps/pez227

INTRODUCTION

immune homeostasis will cause an increased vulnerability to diseases and depress animals’ production performance (Yang et al., 2011). Efforts aiming to maintain birds’ health and immune homeostasis comprise the use of dietary supplements (Liu et al., 2014) including vitamin E (VE). Besides its function as antioxidant (Burton et al., 1983; Kiefer et al., 2004; Amara et al., 2011; Surai et al., 2019) and as essential nutrient for reproduction, VE has also been described as immune response modulating factor (Zhang et al., 2010; Zhao et al., 2011; Kaiser et al., 2012; Liu et al., 2015). According to recommendations of both the National Research Council (NRC, 1994) and the Chinese Feeding Standard of Chicken (Ministry of Agriculture of People’s Republic of China, 2004), diets for healthy laying hens should be supplemented with 4 to 6 IU VE/kg. However, under practical conditions, both feed industry and producer often prefer to supplement diets for laying hens with more than 25 IU VE/kg to improve laying performance or to alleviate the negative effects of heat stress (Puthpongsiriporn et al. 2001; Jiang et al., 2013). Results of some studies point towards beneficial effects of additional VE supplementation for broilers or un-matured layer chickens on birds’

Salmonella can be held responsible for foodborne salmonellosis, which has a negative impact on poultry production worldwide (Brenner et al., 2000). Poultry products such as meat, eggs, and feather have been identified as important transmission vehicles of Salmonella infections (Humphrey, 2004). Most frequently, Salmonella Enteritidis (SE) can be found in humans with gastronenteritis (Smith et al., 2014), pathogenic laying hens (Howard et al., 2012) and even in asymptomatic poultry flocks (Van Immerseel et al., 2005; Balan and Babu, 2017). Salmonella can colonize the gut of poultry and invade the tissue (Barrow and Lovell, 1991). Infections with SE can activate both humoral and cell mediated immune responses (Desmidt et al., 1998; Babu et al., 2004; Luoma et al., 2017), and can trigger the production of pro-inflammatory cytokines (Van Immerseel et al., 2002). As a result, the loss of  C 2019 Poultry Science Association Inc. Received November 23, 2018. Accepted March 26, 2019. 1 These authors contributed equally to this study. 2 Corresponding author: [email protected]

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Y. J. Liu,∗,1 L. H. Zhao,∗,1 R. Mosenthin,∗,† J. Y. Zhang,∗ C. Ji,∗ and Q. G. Ma∗,

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Table 1. Ingredient composition and nutrient content of basal diet (% DM). Ingredient Corn Soybean meal Limestone Dicalcium phosphate Sodium chloride DL-Met Choline chloride Vitamin premix1 Mineral premix2 Total

(%)

Nutrient3

%

66.45 22.80 8.20 1.70 0.30 0.12 0.10 0.30 0.03 100

CP AME VE Lys Met Met+Cys Thr Ca Total P Available P

15.52% 2700 (Kcal/kg) 23.0 (IU/kg) 0.75 % 0.37 % 0.64 % 0.57 % 3.60 % 0.65% 0.39%

1 Vitamin premix supplied (per kg of diet): Vitamin A, 6000 IU; Vitamin D3 , 1500 IU; Vitamin K3 , 4.2 mg; Vitamin B1 , 3 mg; Vitamin B2 , 10.2 mg; Folic acid, 0.9 mg; Calcium pantothenate, 15 mg; Niacin 45 mg; Vitamin B6, 5.4 mg; Vitamin B12, 24 μ g; Biotin 150 μ g. 2 Mineral premix provided (per kg of diet): Cu (CuSO4·5H2O), 6.8 mg; Fe (FeSO4·7H2O), 66th mg; Zn (ZnSO4·7H2O), 83 mg; Mn (MnSO4·H2O), 80 mg; I (KI), 1 mg; Se (Na2SeO3), 0.3 mg. 3 Values of VE, CP, and Ca are based on chemical analysis. Contents of other nutrients are calculated based on Feeding Standard of Chickens (Ministry of Agriculture of People’s Republic of China, 2004).

immune-competence. For example, supplementation of 150 to 300 mg VE/kg to diets for chickens resulted in a 2- to 3-fold increase in log2 antibody titer and increased protection against Escherichia coli infection (Heinzerling et al., 1974). Similarly, dietary supplementation of 300 mg VE/kg diet increased antibody production and phagocytosis, and reduced mortality of 6-week-old chickens exposed to E. coli infection (Tengerdy and Brown, 1977). The addition of 100 IU VE/kg to the diet of broilers challenged with 150,000 oocysts of Eimeria tenella increased both body weight gain and feed intake, and there was also a tendency for lower feed conversion ratio compared to birds fed the un-supplemented diet (Colnago et al., 1984). Moreover, broilers receiving a diet supplemented with 250 mg VE/kg had higher antiSalmonella Typhimurium immunoglobulin A (IgA) antibody titres in serum, intestinal scrapings and bile, compared to birds receiving the basal diet (Muir et al, 2001). However, there are no reports so far aiming to assess the effect of supplemental VE in laying hens challenged with SE, which has been frequently associated

with the outbreak of foodborne salmonellosis. The objective of this study was to assess the protective effect of VE on laying performance, antioxidant status, serum cytokines, and immunoglobulin status of laying hens under SE challenge conditions.

MATERIALS AND METHODS Experimental Design and Treatments A total of 80 32-week-old double negative salmonellafree brown-egg laying hens were randomly assigned to 4 treatments with 20 replicates each (1 bird per replicate) according to a 2 × 2 factorial design with 2 dietary VE supplementation levels [0 IU/kg (VE0) vs. 30 IU/kg (VE30)] and 2 challenge treatments [SE vs. physiological saline solution (PS)]. The 4 treatments were as follows (Figure 1): VE0-PS (basal diet + 0 IU/kg VE + PS), VE0-SE (basal diet + 0 IU/kg VE + SE), VE30PS (basal diet + 30 IU/kg VE + PS), and VE30-SE (basal diet + 30 IU/kg VE +SE).

Salmonella Enteritidis Inoculum and Challenge Salmonella enterica ssp. enterica serovar Enteritidis (preservation number CVCC3377) were obtained from the China Institute of Veterinary Drug Control (Beijing, China). The frozen culture was thawed and 10 μL were inoculated into sterile tubes containing 10 mL of sterile tryptone soy broth. The inoculated broth was incubated at 37◦ C with orbital shaking for 24 h. Subsequently, 5 mL of Salmonella Enteritidis pre-culture were transferred to 100 mL of tryptone soy broth and incubated with orbital shaking at 37◦ C for 16 to 18 h. To determine the concentration of viable Salmonella Enteritidis in the culture, the inoculum was diluted with sterile PBS (pH = 7.2) and plated on Xylose Lysine Deoxycholate (XLD). Black colonies were counted after incubating the plate for 24 h at 37◦ C. The stock culture was prepared in sterile PBS and adjusted to 1 × 109 cfu/mL of Salmonella Enteritidis to be used

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Figure 1. Experimental treatments and timeline of vitamin E (VE) supply and birds challenged. : Take blood; : challenge Salmonella Enteritidis (SE); : challenge physiological saline solution (PS); : dietary supplement with 0 IU/kg VE; : dietary supplement with 30 IU/kg VE.

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Table 2. Effect of dietary vitamin E (VE) supplementation and Salmonella Enteritidis (SE) challenge on laying performance of laying hens (n = 20). VE (IU/kg)

SE1

Laying rate (%) Before challenge

– + – + SEM2

0 30

P-values3 VE(IU/kg) 0 0 30 30

SEM2 – + SEM2 VE SE VE×SE SE2 – + – + SEM2

0 30

P-values3

SEM2 – + SEM2 VE SE VE×SE

93.44 92.05 1.51 – – – – – – 0.533 – –

Before challenge

93.12 82.83 88.53 89.88 2.73 87.98 89.20 1.93 90.83 86.35 1.93 0.662 0.128 0.055

55.66 55.55 0.96 – – – – – – 0.937 – –

Daily feed intake (g/d) Before challenge 113.23 114.29 2.51 – – – – – – 0.772 – –

After challenged 106.65 105.51 109.91 104.43 1.70 105.35 107.17 1.20 108.28 104.97 1.20 0.533 0.075 0.444

Egg weight(g)

After challenge a

57.56 50.03b 55.14a 54.86a,b 1.58 53.79 55.00 1.11 56.35a 52.44b 1.11 0.459 0.029 0.040

Before challenge

After challenge

59.57

61.11 60.43 62.26 61.82 0.59 61.13 61.68 0.42 62.04 60.77 0.42 0.369 0.054 0.847

60.34 0.29 – – – – – – 0.089 – –

Feed conversion (g/g) Before challenge 2.04 2.06 0.05 – – – – – – 0.743 – –

After challenge 1.85 2.12 2.00 1.91 0.07 1.99 1.95 0.05 1.93 2.01 0.05 0.649 0.233 0.024

Mortality (%) Before challenge 10.71 12.96 4.64 – – – – – – 0.736 – –

After challenge 22.50 27.50 10.00 10.00 2.70 25.00a 10.00b 1.91 16.25 18.75 1.91 < 0.001 0.373 0.373

Means within a row with no common superscripts differ (P < 0.05). SE = challenged with Salmonella Enteritidis; –, without SE challenge; +, with SE challenge. 2 SEM, pooled standard error of the mean. 3 P-values for main effect of VE, the main effect of SE challenge, and the interaction between the VE treatments and SE challenge. a,b 1

as inoculum. During the last 3 D of week 43 of age, birds were orally challenged with 1.0 mL suspension of 109 cfu/mL Salmonella Enteritidis daily, whereas negative treatments (VE0) received the same volume of PS. A syringe with an attached flexible tube was used for the administration of the suspension and the PS.

Birds The protocol was reviewed and approved by the Animal Care and Use Committee of China Agricultural University. All procedures were carried out in strict accordance with the recommendations of the Guide for Guidelines for Experimental Animals of the Ministry of Science and Technology (Beijing, China). Fecal samples were taken by cloacal swabs and examined for the presence of Salmonella by the selective enrichment method to assure that the experimental hens were salmonella-free (Bai et al., 2014). Thereafter, blood samples of salmonella-free birds were drawn from the wing vein to determine whole blood flat agglutination as described by Dailey et al (2017).

In total, 80 30-week-old double negative Jinghong brown-egg laying hens (Beijing Yukou Poultry Co., Ltd., China) with similar laying rate (93.0 ± 4.0%) and body weight (1.51 ± 0.13 kg) were randomly assigned to 4 treatments with 20 replicates each. Challenged and non-challenged birds were separately housed in 2 closed, mechanically ventilated henhouses under the same environmental conditions. Layers were caged individually. The cage size was 45 cm in width, depth, and height. Birds were maintained on a 16-h-light schedule and allowed ad libitum access to the experimental diets and water. Room temperature was kept at 23 ± 2◦ C. Disinfection and vaccination procedures were conducted according to the Jinghong brown commercial management manual. The whole experiment comprised 16 wk (from 210 to 322 D of age) including 2 wk adaptation period and 14 wk experimental period. All laying hens were fed with a corn-soybean meal-based diet (Table 1), which was formulated to meet the Chinese Feeding Standard of Chickens (Ministry of Agriculture of People’s Republic of China, 2004), except for VE, which was added to the basal diet according to the experimental design. It was assured that all diets and water were devoid of salmonella before and during this experiment.

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0 0 30 30

After challenge

Daily egg mass(g)

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Sample Collection and Procedure Laying Performance Egg weight and egg production were recorded daily in replicates. Feed consumption and feed conversion were calculated weekly.

Samples of feed ingredients and the basal diet were analyzed for crude protein (CP), Calcium (Ca), and VE. Blood samples were collected at 12: 00 h on the last day of week 44 and 46 via a bronchial vein from 6 fasted laying hen of each treatment. Their BW corresponded to average BW of the treatment. Blood samples were centrifuged at 3000 rpm for 10 min at 4◦ C for separation of serum, and stored at -20◦ C for further analyses. Antioxidant Capacity The superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and total antioxidant capacity (T-AOC) content in serum were determined by using an assay kit from Jiancheng Bioengineering Institute (Nanjing, China) according to the kit instructions (Fan et al., 2015; Liu et al., 2016). Malondialdehyde (MDA) was determined using the thiobarbituric acid colorimetric method as described by Mak et al. (1983). Immunological Assays Serum immunoglobulin M (IgM), IgA, immunoglobulin G (IgG) were measured by double antibody sandwich ELISA method using commercial kits from Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s instructions (Geng et al., 2018). Serum Cytokines Serum concentrations of interleukins (IL-1β and IL-6), interferon-γ (IFN-γ ), and tumor necrosis factor-α (TNF-α) were determined using commercial kits (Beijing North Institute of Biological Technology) by means of an immunoenzymtic ELISA method (Geng et al., 2018).

Statistical Analysis Experimental data were analyzed using SAS statistical software (2010, version 9.2, SAS Institute Inc., Cary, NC, USA). All data were analyzed as a 2-way ANOVA (2 factorial arrangement) using the General Linear Model (GLM) procedure. The model included the main effects of VE supplementation levels and SE challenge status as well as their interaction. Tukey’s multiple comparisons test was used to separate means when interactive effects differed significantly. Data of hen mortality were transformed to arc sine values prior to statistical analysis. Differences among means were tested using the LSD method, and statistical significance was set at P < 0.05.

RESULTS Laying Performance The production performance of laying hens is presented in Table 2. Before challenged with SE, no

Serum Antioxidant Status The serum antioxidant indices are listed in Table 3. Prior to the challenge with SE, supplementation of VE increased (P < 0.05) the SOD activity, but decreased (P < 0.05) the GSH-Px activity. There were no differences (P > 0.05) in T-AOC and MDA concentrations. Upon SE challenge, MDA concentrations were elevated (P < 0.05) at week 46. There was an interaction (P < 0.05) between VE supplementation and SE challenge for serum MDA activity at week 46. However, challenged hens without supplemental VE had a higher (P < 0.05) serum MDA content at week 46 compared to the PS treatment, whereas there was no difference (P > 0.05) between treatments supplemented with 30 IU/kg VE.

Serum Immunoglobulin The serum immunoglobulin concentrations are shown in Table 4. Prior to the challenge with SE, no difference (P > 0.05) in serum immunoglobulin levels between treatment groups was observed. Following challenge with SE, the supplementation of VE increased (P < 0.05) concentrations of IgA, IgM, and IgG at week 44. There were interactions (P < 0.05) between VE supplementation and SE challenge for IgA concentration at week 44 and for IgM concentration at week 46. Compared to PS treatment, SE challenged hens without supplemental VE had a higher (P < 0.05) IgA concentration at week 44; however, there was no difference (P > 0.05) between treatments supplemented with 30 IU/kg VE. The supplementation of VE increased (P < 0.05) the IgM concentrations in birds treated with PS treatment at week 46, but not in SE challenged laying hens (P > 0.05).

Serum Cytokines The serum cytokines concentrations are shown in Table 5. Before challenged with SE, there were no differences (P > 0.05) in serum cytokines between treatment groups. Due to SE challenge, levels of IL-1β , and IL-6 levels were higher (P < 0.01) at week 44.

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Sample Collections and Preparations

difference (P > 0.05) was observed in laying performance between treatments. After challenged with SE, hens fed the VE30 diet had a lower (P < 0.01) mortality compared to the PS treatment, and in addition, SE challenge decreased (P < 0.05) daily egg mass. There was an interaction (P < 0.05) between VE and SE on feed conversion and daily egg mass. No difference between VE treatments was observed for the laying rate of hens treated with PS. However, challenged hens without supplemental VE had a reduced (P < 0.05) egg mass and an increased (P < 0.05) feed conversion compared to the PS treatment, whereas there was no difference (P > 0.05) between treatments supplemented with 30 IU/kg VE.

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Table 3. Effect of dietary vitamin E (VE) supplementation and Salmonella Enteritidis (SE) challenge on serum antioxidant status of laying hens (n = 6). SOD2 (NU/ml) VE (IU/kg)

SE

42 wk b

– + – + SEM6

795.48

842.18a 8.03 – – – – – – 0.006 – –

0 30

P-values7

SEM6 – + SEM6 VE SE VE × SE

44 wk 850 788 751 839 42.9 819 789 29.3 794 814 29.3 0.582 0.760 0.104

T-AOC3 (U/ml) 46 wk 586 660 590 606 36.83 617 597 28.14 588 633 28.06 0.544 0.284 0.482

42 wk 6.60 6.94 0.74 – – – – – – 0.756 – –

44 wk 9.28 6.11 8.98 5.40 1.81 7.70 7.19 1.28 9.13 5.75 1.28 0.784 0.087 0.913

GSH-Px4 (U/ml)

46 wk

42 wk

4.60 7.93 7.09 5.89 1.24 6.26 6.49 0.88 5.84 6.91 0.88 0.856 0.408 0.092

a

740.75

614.38b 19.95 – – – – – – 0.004 – –

MDA5 (nmol/ml)

44 wk

46 wk

42 wk

44 wk

46 wk

765 546 750 732 57.1 671 742 43.6 757 639 43.5 0.196 0.085 0.133

790 675 638 625 43.6 732 631 33.2 703 646 33.3 0.058 0.206 0.301

6.02

3.69 11.77 5.33 8.21 3.89 7.73 7.00 2.86 4.42 9.99 2.86 0.821 0.205 0.530

2.98b 5.82a 3.42b 3.40b 0.59 4.40 3.41 0.41 3.20b 4.61a 0.41 0.118 0.033 0.031

5.30 0.58 – – – – – – 0.415 – –

Means within a row with no common superscripts differ (P < 0.05). SE = challenged with Salmonella Enteritidis; –, without SE challenge; +, with SE challenge. 2 SOD = superoxide dismutase. 3 T-AOC = total antioxidant capacity. 4 GSH-Px = glutathione peroxidase. 5 MDA = malondialdehyde. 6 SEM, pooled standard error of the mean. 7 P-values for main effect of VE, the main effect of SE challenge, and the interaction between the VE treatments and SE challenge. a,b 1

Table 4 . Effect of dietary vitamin E (VE) supplementation and Salmonella Enteritidis (SE) challenge on serum immune of laying hens (n = 6). IgA2 (mg/ml) VE (IU/kg) 0 0 30 30

1

IgM4 (mg/ml)

SE

42 wk

44 wk

46 wk

42 wk

44 wk

46 wk

42 wk

44 wk

46 wk

– + – + SEM5

1.15

0.93b 1.36a 1.53a 1.40a 0.10 1.18b 1.46a 0.07 1.23 1.38 0.07 0.012 0.179 0.023

0.71 0.82 1.06 0.99 0.10 0.77b 1.02a 0.08 0.89 0.91 0.08 0.039 0.851 0.412

6.37

5.15 5.67 6.74 7.28 0.33 5.45b 7.01a 0.23 5.95 6.36 0.23 < 0.001 0.136 0.970

4.97 4.97 7.02 6.66 0.91 4.97 6.84 0.70 5.85 5.70 0.70 0.088 0.863 0.861

0.73

0.68 0.82 1.06 0.95 0.10 0.75b 1.01a 0.07 0.87 0.89 0.07 0.035 0.885 0.236

0.55b 0.66a,b 0.80a 0.66a,b 0.05 0.62b 0.72a 0.04 0.68 0.66 0.04 0.047 0.782 0.040

0 30

P-values6

IgG3 (mg/ml)

SEM5 – + SEM5 VE SE VE×SE

1.08 0.14 – – – – – – 0.740 – –

5.96 0.52 – – – – – – 0.600 – –

0.77 0.07 – – – – – – 0.701 – –

Means within a row with no common superscripts differ (P < 0.05). SE = challenged with Salmonella Enteritidis; –, without SE challenge; +, with SE challenge. 2 IgA = immunoglobulin A. 3 IgG = immunoglobulin G. 4 IgM = immunoglobulin M. 5 SEM, pooled standard error of the mean. 6 P-values for main effect of VE, the main effect of SE challenge, and the interaction between the VE treatments and SE challenge. a,b 1

Supplementation with VE had no effect (P > 0.05) on cytokine concentrations, and there was no interaction between VE supplementation and SE challenge for TNF-α, IFN-γ , IL-1β , and IL-6 levels at week 44 and 46.

DISCUSSION The results of this study confirm the protective effect of VE on the prevalence of SE in laying hens as supplementation of 30 IU VE/kg to the diet of laying hens improved both laying performance and immunity in SE challenged birds.

There are no reports so far on the effect of SE challenge on performance of laying hens without VE supplementation. According to the results of a previous study by Fan et al. (2014), SE challenge at week 22 did not affect laying rate and egg weight of White Leghorns hens fed on a commercial diet supplemented with 25 IU VE/kg according to manufacturer’s information (http://www.cpbz.gov.cn/standardProduct/showDetail. do?innerCode=Q-9112011660050259XW-201,609,030, 902–0001&token=f0098f5f-836b-4d1b-8d3b-76eb2d0264 d4) . These findings are in support of the present results, as SE challenge did also not affect production performance of laying hens fed diet supplemented

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0 0 30 30

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Table 5 . Effect of dietary vitamin E (VE) supplementation and Salmonella Enteritidis (SE) challenge on serum cytokine concentrations of laying hens (n = 6). TNF-α 2 (ng/L) VE (IU/kg)

IL-1β 4 (ng/L)

IL-65 (ng/L)

SE

42 wk

44 wk

46 wk

42 wk

44 wk

46 wk

42 wk

44 wk

46 wk

42 wk

44 wk

46 wk

– + – + SEM6

71.36

230 418 227 351 159 337 289 108 228 389 108 0.827 0.338 0.839

489 294 518 673 133 377 606 102 503 483 102 0.187 0.894 0.252

49.43

547 522 380 1070 299 535 774 204 464 835 204 0.528 0.279 0.247

870 389 605 997 219 629 801 155 737 693 155 0.449 0.844 0.070

26.05

8.25 33.63 14.31 29.50 3.24 20.94 21.90 2.29 11.28b 31.56a 2.29 0.770 < 0.0001 0.142

23.50 14.19 21.63 25.56 4.31 18.84 23.59 3.05 22.56 19.88 3.05 0.293 0.545 0.151

125.52

142.50 380.58 132.17 232.56 53.32 261.54 189.54 36.48 137.33b 296.00a 36.48 0.160 0.010 0.215

200.42 131.50 131.92 176.58 41.05 161.03 154.25 28.09 166.16 150.82 28.09 0.775 0.767 0.187

0 30

P-values7

IFN-γ 3 (ng/L)

SEM6 – + SEM6 VE SE VE×SE

80.08 7.89 – – – – – – 0.465 – –

48.64 6.75 – – – – – – 0.937 – –

18.13 3.24 – – – – – – 0.135 – –

134.50 25.57 – – – – – – 0.812 – –

Means within a row with no common superscripts differ (P < 0.05). SE = challenged with Salmonella Enteritidis; –, without SE challenge; +, with SE challenge. TNF-α = tumor necrosis factor-α . 3 IFN-γ = interferon-γ . 4 IL-1β = interleukin-1β . 5 IL-6 = interleukin-6. 6 SEM, pooled standard error of the mean. 7 P-values for main effect of VE, the main effect of SE challenge, and the interaction between the VE treatments and SE challenge. a,b 1 2

with 30 IU VE/kg. On the other hand, according to the result of a study by Kulshreshtha et al. (2017), SE challenge of Lohmann Lite laying hens at week 81 of age, and fed a diet supplemented with 20 IU VE/kg decreased birds’ feed intake, body weight, and laying rate. It cannot be ruled out that differences in birds’ age, SE origin, and administration of SE may have contributed to these conflicting results. For example, flocks used in the study of Kulshreshtha et al. (2017) were older than those in the study of Fan et al. (2014) and the present study, which, in turn, may have resulted in a general decline in health conditions (Gan et al., 2018) and higher sensitivity to pathogens (Mazaheri et al., 2005). Moreover, Kulshreshtha et al. (2017) used a SE field strain isolated from a clinical case of salmonellosis in laying hens, which might have exhibited a greater toxicity than the standard strains (Kasatskaia, 1975) used in the present study and that by Fan et al. (2014). Finally, birds were orally gavaged with SE culture at a dosage of 2 × 109 cfu SE for 7 D and 109 cfu/mL SE for 3 D in the study by Kulshreshtha et al. (2017) and in the present study, respectively, whereas in the experiment performed by Fan et al. (2014) 9.8 × 108 cfu were administered for 1 D only. It is well known that T-AOC, GSH-Px, and SOD are important enzymes indicating organ damage due to exposure to oxidative stress (Gutteridge, 1995; Th´erond et al., 2000). In the present study, VE supplementation increased the SOD activity and decreased the GSH-Px activity prior to the birds’ challenge with SE. Following SE challenge, higher levels of MDA and TAOC point towards the presence of severe oxidative stress as MDA is the final metabolic product of lipid

oxidation, and it is associated with the development of oxidative stress (Bakar et al., 2015). After 3 wk, the serum MDA content of challenged birds was similar to that of un-challenged birds fed diet supplemented with 30 IU VE/kg, while it was still higher than that of unchallenged birds fed the diet without VE supplementation. There is some evidence that VE may have bound to peroxyl radicals, thereby inhibiting the chain reaction of lipid peroxidation (Burton and Traber, 1990). Also, in association with other antioxidants, such as SOD and GSH-Px, VE might have exerted synergistic effects on the efficiency of these antioxidants. The synergistic antioxidation of VE and SOD may reduce burden of GSH-Px and other antioxidant systems (Khan et al., 2012) under oxidative stress conditions induced by SE challenge as described herein. Consequently, the lower mortality in challenged laying hens due to VE supplementation might reflect improved balance between antioxidants such as SOD activites and oxidants such as serum MDA content. Prior to SE challenge, the supplementation of 30 IU VE/kg diet had no effect on serum immunoglobulin concentrations during a 10-wk-feeding period. This is in agreement with results of a study by Muir et al. (2002), where dietary supplementation of 25 IU VE/kg had also no effect on serum IgA antibody of broilers at 21 D and 42 D of age. In the same study, however, dietary supplementation of 250 IU VE/kg resulted in elevated serum IgA antibody titres, which indicates beneficial effects of supplemental VE on birds’ immune system. Similarly, Niu et al. (2009) determined higher titers of total antibody, IgM and IgG in broilers in response to increasing dietary VE supplementation up to 200 IU VE/kg;

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Thus, further studies are warranted to evaluate potential dose-effects of VE immunomodulation, and to further elaborate the protective mechanisms of VE against SE exposure.

CONCLUSION In the present study, the reduced laying performance of laying hens upon SE exposure can be ascribed to the development of oxidative and immune stress. Dietary supplementation of 30 IU/kg VE may alleviate the observed stress symptoms caused by SE challenge and can be used as dietary intervention for control of Salmonella infection in laying hens, thus improving poultry health and production performance.

ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (contract number: 31772621), China Agricultural Research System program (CARS-40-K08), National Key Research and Development Program of China (2017YFD0500500), and Public Sector (Agriculture) Scientific Research of China (Grant No.201403047).

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comparable results were also reported in rats (Kaku et al., 1999) and male mice (El-Shenawy et al., 2015). Higher pathogenic exposure is often associated with higher immune response. According to Desmidt et al. (1998), serum levels of IgA, IgG, and IgM increased in chickens 2 wk after being infected with SE. In part, this observation is in accordance with the results of the present study, where SE challenge increased serum IgA levels in laying hens fed a diet devoid of supplemental VE after the week 1 succeeding challenge, whereas there was no effect on IgA after the week 3 and serum IgG and IgM after week 1 and 3. In the present study, the supplementation of VE increased IgA, IgG, and IgM concentrations compared to PS treatment. However, supplemental VE did not affect these serum immunoglobulin concentrations in laying hens challenged with SE. There are no reports on potential protective effects of dietary VE on immune system of laying hens challenged with SE; however, there is evidence that dietary VE may support the immune system of growing pullets against E. coli infection (Heinzerling et al., 1974). These authors determined in chickens a 2- to 3-fold increase in log2 antibody titer upon supplementation with 150 to 300 mg VE/kg. Similarly, according to Tengerdy and Brown (1977), dietary supplementation of 300 mg VE/kg diet increased antibody production and phagocytosis, and reduced mortality of 6-week-old chickens exposed to E. coli infection. It needs to be emphasized, however, that the dietary inclusion of supplemental VE used in these studies was considerably higher than in commercial diets. The innate immune system plays an essential role in host defense against infection. Cytokines are a group of proteins or peptides secreted by cells that could affect other cells, thus playing an important role in immune and inflammatory response (Giansanti et al., 2006). In the present study, SE challenge increased serum levels of pro-inflammatory cytokines IL-1β and IL-6, suggesting SE may induce inflammatory response. These proinflammatory cytokines are produced in defense against pathogen challenge, thereby also inducing metabolic changes such as inhibiting growth performance (Klasing and Jonasone, 1991). This may explain, in part, why in the present study SE challenge increased feed conversion of hens fed diet without VE supplementation. Although there is evidence that VE may enhance both humoral and cell immune response (Haq et al., 1996; Kaku et al., 1999), no interactions between VE supplementation and SE challenge were observed in the present study for TNF-α, IFN-γ , IL-1β , and IL-6 levels at week 44 and 46. It can be speculated, if these differences in immune response to dietary VE supplementation have to be attributed to varying supplementation levels of VE (Friedman et al., 1998), the use of different animal species (Kaku et al., 1999; El-Shenawy et al., 2015), differences in stress level among studies and/or to the extent of pathogenic exposure (Fan et al., 2014; Kulshreshtha et al. 2017).

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