Dietary 25-hydroxyvitamin D improves productive performance and intestinal health of laying hens under Escherichia coli lipopolysaccharide challenge

Abstract

The effect of 25-hydroxyvitamin D (25OHD) on the immune response of laying hens is not well elucidated. This study investigated the effects of 25OHD on egg production, egg quality, immune response, and intestinal health of laying hens challenged with Escherichia coli lipopolysaccharide (LPS). One hundred and sixty laying hens at 45 wk of age were randomly divided into 4 dietary treatments with 10 replicates of 4 birds. Hens were fed the corn-soybean based diets contained either 0 or 80 µg/kg 25OHD for 8 wks. At wk of 53 wk, birds of each dietary treatment were injected into the abdomen with 1.5 mg/kg body weight of either LPS or saline a day at 24-h intervals for continuous 7 d. LPS injection significantly decreased (P_LPS < 0.05) egg laying rate, feed intake and feed efficiency; while the supplementation of 25OHD increased (P_Interaction < 0.05) egg laying rate, feed efficiency and decreased (P_Interaction < 0.05) the broken egg rate in layers under LPS injection. LPS challenge decreased (P_LPS < 0.05) eggshell strength, eggshell thickness, albumen height and Haugh unit, while dietary 25OHD supplementation increased eggshell strength and eggshell thickness (P_25OHD < 0.05). The serum proinflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6)], endotoxin and diamine oxidase (DAO) levels were higher in layers under LPS challenge (P_LPS < 0.05); whereas the dietary addition of 25OHD were shown to decrease (P_25OHD < 0.05) serum IL-1β and IL-6 concentration irrespective of LPS challenge and led to a higher serum 25OHD level and a reduction in endotoxin concentration in layers under LPS challenge (P_Interaction < 0.05). The layers under LPS challenge had higher crypt depth and lower villus height/crypt depth (V/C) ratio in duodenum and jejunum (P_LPS < 0.05), while feeding 25OHD were shown to have decreasing effect on crypt depth and increasing effect V/C ratio in layers under LPS challenge (P_Interaction < 0.05). Layers under LPS challenge had lower mRNA expression of intestinal barrier associated proteins (claudin-1 and mucin-1) (P_LPS < 0.05), while the addition of 25OHD up-regulated claudin-1 and mucin-1 expression (P_interaction < 0.05). Lower antioxidant enzymes activities, including superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), glutathione peroxidase (GPx) and higher malondialdehyde (MDA) content in jejunum were found in layers challenged with LPS (P_25OHD < 0.05). The effect of 25OHD reversed the effect of LPS on SOD, T-AOC, and MDA content (P_Interaction< 0.05). These results suggest that supplementing 80 µg/kg 25OHD in diets may elevate laying performance and egg quality through the improvement of intestinal barrier function, antioxidant capacity, and decreased the proinflammatory cytokines levels in laying hens with Escherichia coli LPS challenge.

INTRODUCTION

Large number of stressors often threated the health of the modern laying hens, such as pathogens associated disease, high flocking density, heat stress, other harmful substance derived from feed (Nawab et al., 2018). Existing analyses show that total economic loss and cost associated with mortality and disease control are above 20% of the gross value of income from poultry production. Avian pathogenic Escherichia coli causes systemic disease that is highly lethal in laying hens worldwide (Vandekerchove et al., 2004). Lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria and a highly efficient proinflammatory substance, has been widely used as a model of pathogen infection experimentally in poultry and livestock ( Geng et al., 2018; Redweik et al., 2020). Inhalation or contamination of environmental gram-negative bacteria (their endotoxins LPS in particular) has been suggested to be a major poultry health problem in practice.

Vitamin D₃ (cholecalciferol), is an essential nutrient and plays a pivotal role in maintaining calcium and phosphorus homeostasis, skeletal health, and muscle development (Overbergh et al., 2000; Gorman et al., 2017). Vitamin D₃ is also involved in our immune system to maintain the animal health through lowering inflammation and stress response ( Mora et al., 2008,Fakhoury et al., 2020; Wang et al., 2021). Geng et al. (2018) found that VD₃ supplementation could protect laying hens from immunological stress caused by LPS challenge (8 mg/kg body weight). The 25-hydroxyvitamin D (25OHD) is an active metabolite of vitamin D_3, which were shown to have better bioactivity than vitamin D₃ (Lou et al., 2003;Wang et al., 2020, Wang et al., 2021 ). Literatures have shown that 25OHD improve livability and alleviate inflammation in breeders and hens (Lin et al., 2019; Chou et al., 2020). In our previous study, we found that dietary administration of 69 µg/kg 25OHD improved the egg quality and tibia quality of layers under high stocking density (Wang et al., 2020; Wang et al., 2021). However, it remains unknown whether supplemental 25OHD may provide additional benefits when laying hens are challenged with LPS.

Therefore, the aim of the present study was to investigate the effects of 25OHD on productive performance, egg quality and immune response of laying hens under LPS challenge.

MATERIALS AND METHODS

Birds, Diets, Management, and Sampling

The Animal Care and Use Committee of the Sichuan Agricultural University (Chengdu, Sichuan, China) approved all the experimental protocol of the current study. At 45 wk of age, 160 Lohman pink-shell laying hens were randomly divided into 4 treatments with 10 replicates per treatment. Two 25OHD levels (0 and 80 µg/kg, the dosage of 25OHD was settled according to our previous study) and LPS challenge (injection with LPS or saline) were designed by 2 × 2 factorial experiment. Laying hens were fed a complete feeding mixture in a mash form (Supplementary Table 1), and a premix with 80 µg/kg 25OHD and 1 kg corn were made prior to supplementation. The birds were maintained on the dietary treatments for 8 wk after which all hens were injected intravenously with 1.5 mg/kg body weight of either LPS (serotype 0111:B4, Sigma Aldrich Inc., St. Louis, MO) or saline at 24-h intervals for continuous 7 d. Layers in each replicate were raised individually in 4 adjacent cages (45 cm width × 50 cm length × 45 cm height) with the room temperature was maintained at 22 to 24°C and relative humility of 55 to 65% by a daily lighting schedule of 16 h light and 8 h dark. Birds were allowed ad libitum access to water and feed during the whole experimental period.

Measurements and Sample Collection

Laying rate (egg number), egg weight, feed intake, broken eggs, and unqualified eggs (egg weight <50 g or >75 g, misshaped egg, dirty egg, and sand-shelled egg) were recorded daily for each replicate. Feed conversion ratio (FCR) was calculated as the amount of feed consumed required to produce a unit (g) of egg mass (feed conversion = g feed/g egg). At end of 9 wk, a total of 30 eggs except for unqualified eggs and broken eggs were randomly collected from each treatment and assessed for egg quality traits. At the end of 9 wk (4 h after last LPS injection), 20 hens (2 layers/replicate, 10 replicates/treatment) were individually weighted and blood samples were collected from the wing vein into a sterile syringe and then transferred in vacuum tube with K₃ EDTA. Blood samples were then centrifuged at 3000 × g for 15 min after clotting, and then serum was stored at −20°C pending analysis. The same hens were then sacrificed with an overdose intravenous injection of sodium pentobarbital and the small intestine (middle section of each duodenum, jejunal, and ileal) was taken and stored in 4% polyformaldehyde for morphology and histopathology.

Egg Quality

Egg yolk color, albumen height and Haugh unit were determined using an egg multi tester (EMT-7300, Robotmation Co., Ltd., Tokyo, Japan). Eggshell strength was evaluated using an eggshell force gauge model II (Robotmation Co., Ltd., Tokyo, Japan). Eggshell thickness was measured using an eggshell thickness gauge (Robotmation Co., Ltd., Tokyo, Japan), in three distinct regions of the shell (large end, equatorial region, and small end). The eggshell, albumen and yolk were separated, and the relative weight were calculated by each section divided by total egg weight.

Serum Cytokine Concentration

The concentration of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), 25OHD, endotoxin, and diamine oxidase (DAO) in serum were assessed by enzyme-linked immunosorbent assay (ELISA) kits obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer's instructions.

Morphology of Intestinal Mucosa

Duodenal and jejunal mucosa (1 layer/replicate, 10 replicates/treatment) morphology were analyzed as described previously (Gong et al., 2021; Wang et al., 2021). Briefly, the intestinal segments were fixed in 4% paraformaldehyde, then embedded in paraffin, and stained with hematoxylin-eosin. Villus height and crypt depth were calculated at 40 × magnification with a digital camera microscope (BA400Digital, McAudi Industrial, Group Co., Ltd). A total of 10 intact villi and crypts were randomly selected in each sample. The villus height was measured from the tip of the villus to the villus-crypt junction. The crypt depth was defined as the depth of the invagination between adjacent villi. Then, the data included villus height, crypt depth and their ratio (V/C) was calculated.

Real-Time Quantitative PCR for Intestinal Barrier Related Genes

Total RNA of jejunal mucosa (1 layer/replicate, 10 replicates/treatment) was isolated from jejunum (20 mg issue/sample; n = 10 hens per group) using TRIzol reagent (TaKaRa Biotechnology (Dalian Co., Ltd, Dalian, China) on basis of the manufacturer's instructions. The RNA quality and concentration were determined using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA) at 260 and 280 nm. Reverse transcription to synthesize the complementary DNA library was performed using the PrimeScriptTM RT reagent kit (TaKaRa, Kusatsu, Japan). The mRNA levels of the genes were analyzed using a q-PCR machine (CFX384; Bio-Rad, Hercules, CA) with SYBR Green Dye (Bio-Rad), following the manufacturer's instructions. The primers of target genes (Supplementary Table 2), including claudin-1, occludin, zonula occluden-1 (ZO-1), zonula occluden-2 (ZO-2), mucin-1, mucin-2 and the reference gene β-actin were designed using Primer Express 3.0 (Applied Biosystems, Foster City, CA) were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd (Dalian, China). The real-time quantitative PCR with SYBR Premix Ex Taq reagents (TaKaRa Biotechnology, Ltd, Dalian, China) and a CFX-96 Real-Time PCR detection System (Bio-Rad Laboratories, Richmond, CA) were performed. The 2^−ddCt method was used for the quantification with β-actin as a reference gene, and the relative abundance was normalized to the control group.

Antioxidant Enzyme Activity of Jejunum

The jejunal tissue was then suspended by vortexing before analysis. ELISA kits obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) were used to measure the enzymatic activities of superoxide dismutase (SOD) activity, catalase (CAT), total antioxidant capacity (T-AOC), glutathione peroxidase (GPx), glutathione S-transferase (GST) and concentration of malondialdehyde (MDA) in jejunum (1 layer/replicate, 10 replicates/treatment) according to the manufacturer's instructions.

Statistical Analysis

A 2 × 2 factorial arrangement of treatments by 2-way ANOVA analysis of variance was performed using GLM procedure of SAS 9.2 software (SAS Institute Inc., Cary, NC). The model included the main effects of LPS and 25OHD, as well as their interaction. Contrasts between treatments were evaluated by Tukey's range test at a significance level of 0.05.

RESULTS

Laying Performance

Laying performance of laying hens before and after challenges were shown in Tables 1 and 2. As shown in Tables 1 and 2, dietary 25OHD supplementation didn't influence layers’ performance, but it decreased the broken egg rate before challenge (P < 0.05). LPS injection significantly decreased egg laying rate, feed intake and feed efficiency (P_LPS < 0.05; Table 2); while the supplementation of 25OHD increased (P_Interaction < 0.05) egg laying rate, feed efficiency, and decreased (P_Interaction < 0.05) the broken egg rate under LPS injection.

Table 1.

Effect of lipopolysaccharide and 25-hydroxyvitamin D on productive performance of laying hens during 1 to 6 wk (before challenge, 45 to 53 wk of age).¹

Treatment		Egg laying rate, %	Egg weight, g	FCR	Feed intake, g	Broken egg rate, %
LPS	25OHD
−	-	92.34	61.23	2.05	116.00	3.43a
−	+	93.11	61.59	2.04	117.00	1.55b
+	−	92.44	62.10	2.05	118.00	3.98a
+	+	93.67	61.72	2.02	117.00	2.14b
SEM		1.27	1.07	0.29	1.14	0.17
P-Value		0.78	0.74	0.34	0.31	0.04

^a,bMeans with different superscripts within a column differ significantly (P ≤ 0.05).

¹Each mean represents 10 replicates per treatment, with 4 layers per replicate.

Abbreviations: FCR, feed conversation ratio; LPS, lipopolysaccharide; SEM, standard error of mean; 25OHD, 25-hydroxyvitamin D.

Table 2.

Effect of lipopolysaccharide and 25-hydroxyvitamin D on productive performance of laying hens after challenge (53 to 54 wk of age).¹

Treatment		Egg laying rate, %	Egg weight, g	FCR	Feed intake, g	Broken egg rate, %
LPS	25OHD
−	-	92.27a	61.23	2.09b	118a	2.33b
−	+	93.90a	61.19	2.04b	117a	1.55b
+	-	55.23c	60.30	2.94a	98b	3.98a
+	+	59.88a	60.82	2.85a	104a	2.14b
SEM		1.44	1.78	0.02	1.14	0.17
P-Value		0.01	0.74	0.04	0.05	0.04
P-Value
LPS		<0.01	0.44	0.02	<0.01	<0.01
25OHD		0.19	0.69	0.13	0.44	<0.01
LPS × 25OHD		0.01	0.78	0.03	0.04	0.03

^a,bMeans with different superscripts within a column differ significantly (P ≤ 0.05).

¹Each mean represents 10 replicates per treatment, with 4 layers per replicate.

Abbreviations: FCR, feed conversation ratio; LPS, lipopolysaccharide; SEM, standard error of mean; 25OHD, 25-hydroxyvitamin D.

Egg Quality

As shown in Table 3, LPS challenge decreased (P_LPS < 0.05) eggshell strength, eggshell thickness, albumen height, and Haugh unit, while dietary 25OHD supplementation increased eggshell strength and eggshell thickness (P_25OHD < 0.05). Moreover, the increasing effect of 25OHD on eggshell quality (eggshell strength, eggshell thickness) were more pronounced in layers under LPS challenge (P_Interaction < 0.05). There were no differences in the relative weight of eggshell, yolk, and yolk color among treatments (P > 0.05).

Table 3.

Effect of lipopolysaccharide and 25-hydroxyvitamin D on egg quality after 1-wk challenge (9th wk) of laying hens (53 to 54 wk of age).^a

Treatment		Eggshell strength, kg/cm3	Eggshell thickness, mm−2	Eggshell relative weight, %	Yolk relative weight, %	Yolk color	Albumen height, mm	Haugh unit	Albumen relative weight, %
LPS	25OHD
−	-	4.25a	41.25a	10.98	28.98	8.74	8.54	88.44	38.96
−	+	4.34a	40.44a	10.71	28.41	8.42	8.38	89.21	38.12
+	-	3.67c	36.18c	10.87	28.07	7.54	6.45	65.45	37.94
+	+	4.01b	37.89b	10.24	28.66	7.69	7.14	70.14	37.90
SEM		0.14	0.12	0.24	0.44	0.18	0.89	3.22	0.47
P-Value		0.01	0.02	0.39	0.57	0.37	<0.01	<0.01	0.34
P-Value
LPS		<0.01	0.01	0.24	0.55	0.54	0.04	<0.01	0.58
25OHD		0.02	0.04	0.69	0.98	0.41	0.15	0.32	0.18
LPS × 25OHD		<0.01	0.04	0.29	0.77	0.64	0.47	0.17	0.73

^a,bMeans with different superscripts within a column differ significantly (P ≤ 0.05).

¹Each mean represents 10 replicates per treatment, with 3 eggs per replicate.

Abbreviations: LPS, lipopolysaccharide; SEM, standard error of mean; 25OHD, 25-hydroxyvitamin D.

Serum Inflammatory Associated Cytokine Levels

The serum proinflammatory factors (TNF-α, IL-1β, IL-6) were higher in layers under LPS challenge (P_LPS < 0.05; Table 4), while the dietary addition of 25OHD were shown to decrease serum IL-1β and IL-6 concentration irrespective of LPS challenge. The decreasing effect of 25OHD on IL-1β and IL-6 concentration were more pronounced in layers under LPS challenge (P_Interaction < 0.05). No effect of dietary treatments was observed on serum IL-8 levels in current study (P > 0.05).

Table 4.

Effect of lipopolysaccharide and 25-hydroxyvitamin D on serum cytokine levels of laying hens.¹

Treatment		TNF-α	IL-1β	IL-6	IL-8
LPS	25OHD	ng/L	ng/L	ng/L	ng/L
-	-	50.33	100.88b	34.88c	150.46
-	+	52.98	109.69b	35.34c	153.72
+	-	58.61	117.66a	42.36a	158.71
+	+	51.53	101.69b	37.89b	157.23
SEM		3.48	2.17	0.67	4.20
P-Value		0.56	<0.01	<0.01	0.39
P-Value
LPS		0.21	<0.01	<0.01	<0.01
25OHD		0.19	<0.01	<0.01	0.04
LPS × 25OHD		0.56	<0.01	<0.01	0.03

^a,b,cMeans with different superscripts within a column differ significantly (P ≤ 0.05).

¹Each mean represents 10 replicates per treatment, with 2 layers per replicate.

Abbreviations: IL-1β, interleukin 1β; IL-6, interleukin 6; IL-8, interleukin 8; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; SEM, standard error of mean; 25OHD, 25-hydroxyvitamin D.

As shown in Table 5, the serum endotoxin and DAO concentration were also increased by LPS challenge (P_LPS < 0.05), whereas the dietary supplementation with 25OHD led to a higher serum 25OHD level and a reduction in endotoxin and DAO concentration in layers under LPS challenge (P_Interaction < 0.05).

Table 5.

Effect of lipopolysaccharide and 25-hydroxyvitamin D on serum characteristics of laying hens.¹

Treatment		25-OH-D3	Endotoxin	DAO
LPS	25OHD	ng/ml	ng/L	ng/L
−	−	8.00b	169.03c	104.39c
−	+	8.66b	193.85b	89.45d
+	−	7.38c	212.44a	122.72a
+	+	9.01a	201.66ab	112.41b
SEM		0.14	3.61	2.48
P-Value		<0.01	<0.01	<0.01
P-Value
LPS		0.65	<0.01	<0.01
25OHD		<0.01	0.10	0.03
LPS × 25OHD		<0.01	<0.01	<0.01

^a,b,c,dMeans with different superscripts within a column differ significantly (P ≤ 0.05).

¹Each mean represents 10 replicates per treatment, with 1 layer per replicate.

Abbreviations: DAO, diamine oxidase; SEM, standard error of mean; 25OHD, 25-hydroxyvitamin D.

Intestinal Morphology

The effect of LPS and 25OHD on intestinal morphology were presented in Table 6, the layers under LPS challenge had higher crypt depth and lower V/C ratio in duodenum and jejunum (P_LPS < 0.05), while feeding 25OHD were shown to have decreasing effect on crypt depth and increasing effect V/C ratio in layers under LPS challenge (P_Interaction < 0.05). There was no significant different in villus height of duodenum and jejunal mucosa (P > 0.05).

Table 6.

Effect of lipopolysaccharide and 25-hydroxyvitamin D on intestinal morphology of laying hens.¹

Treatment		Duodenum			Jejunum
LPS	25OHD	Villus height, μm	Crypt depth, μm	V/C	Villus height, μm	Crypt depth, μm	V/C
−	-	1099.78	168.77b	6.52a	1208.38a	166.75b	7.25a
−	+	1102.75	177.42b	6.22a	1204.05a	173.47b	6.94a
+	-	1134.52	208.05a	5.45b	1048.40b	210.92a	4.97c
+	+	1104.71	175.25b	6.30a	1152.58a	189.92b	6.07b
SEM		19.48	8.39	0.08	21.72	2.03	0.09
P-Value		0.54	<0.01	<0.05	<0.01	<0.01	<0.01
P-Value
LPS		0.15	0.02	<0.01	0.03	0.74	0.15
25OHD		0.34	0.18	0.78	0.31	0.12	<0.01
LPS × 25OHD		0.19	<0.01	0.03	<0.01	<0.01	0.56

^a,bMeans with different superscripts within a column differ significantly (P ≤ 0.05).

¹Each mean represents 10 replicates per treatment, with 1 layer per replicate.

Abbreviations: LPS, lipopolysaccharide; , SEM, standard error of mean; V/C, ratio of villus height to crypt depth; 25OHD, 25-hydroxyvitamin D.

The mRNA Expression Levels of Gut-Barrier Related Gene in Jejunal Mucosa

Compared with the control treatment, the layers challenged with LPS had lower mRNA expression of intestinal barrier associated protein (claudin-1 and mucin-1) (Table 7; P_LPS < 0.05), while the addition of 25OHD up-regulated the mRNA expression of claudin-1 and mucin-1 (P_25OHD<0.05).

Table 7.

Effect of lipopolysaccharide and 25-hydroxyvitamin D on jejunal barrier related gene expression of laying hens.¹

Treatment		Claudin-1	Occludin	ZO-1	ZO-2	Mucin-1	Mucin-2
LPS	25OHD
−	−	1.00a	1.00	1.00	1.00	1.00a	1.00
−	+	1.40a	1.46	1.01	1.07	1.21a	0.94
+	−	0.47b	1.21	1.14	1.02	0.56c	0.89
+	+	1.04a	1.00	1.24	0.83	0.97b	0.88
SEM		0.08	0.11	0.07	0.07	0.26	0.10
P-Value		0.04	0.28	0.45	0.51	0.02	0.69
P-Value
LPS		0.03	0.88	0.64	0.51	<0.01	0.53
25OHD		0.04	0.91	0.27	0.44	0.04	0.43
LPS × 25OHD		0.04	0.14	0.13	0.86	0.02	0.75

^a,bMeans with different superscripts within a column differ significantly (P ≤ 0.05).

¹Each mean represents 10 replicates per treatment, with 1 layer per replicate.

Abbreviations: LPS, lipopolysaccharide; SEM, standard error of mean; ZO-1, zonula occluden-1; ZO-2, zonula occluden-2; 25OHD, 25-hydroxyvitamin D.

Antioxidative Capacity of Jejunum

As shown in Table 8, lower antioxidant enzymes activity, including SOD, CAT, T-AOC, GPx and higher MDA content in jejunum were found in layers challenged with LPS (P_(Density) < 0.05). Layers treated with 25OHD led to an enhanced T-AOC (P_(25-OH-D3) < 0.05), and also the effect of 25OHD reversed the effect of LPS on T-AOC, CAT and MDA content (P_{(Interaction)} < 0.05).

Table 8.

Effect of lipopolysaccharide and 25-hydroxyvitamin D on antioxidant enzyme activity in jejunum of laying hens.¹

Treatment		SOD	CAT	T-AOC	MDA	GPx	GST
LPS	25OHD	U/mg prot	U/mg prot	U/mg prot	mg prot/mL	U/mg prot	mg prot/mL
-	-	140.40a	257.62a	3.45a	0.27b	122.60a	895.34
-	+	150.32a	243.31a	3.67a	0.24b	119.65a	902.11
+	-	89.45b	144.32b	2.14b	0.74a	78.42b	913.44
+	+	94.54b	194.28a	3.17a	0.49b	83.42b	920.53
SEM		24.22	27.98	0.34	0.02	17.23	36.89
P-Value		<0.01	<0.01	0.04	0.04	0.01	0.67
P-Value
LPS		<0.01	<0.01	0.01	0.02	<0.01	0.21
25OHD		0.45	0.37	0.04	0.04	0.43	0.65
LPS × 25OHD		0.78	0.04	0.05	0.04	0.29	0.44

^a,bMeans with different superscripts within a column differ significantly (P ≤ 0.05).

¹Each mean represents 10 replicates per treatment, with 1 layer per replicate.

Abbreviations: CAT, catalase; GPx, glutathione peroxidase; GST, glutathione S-transferase; LPS, lipopolysaccharide; MDA, malondialdehyde; SOD, superoxidase; T-AOC, total antioxidative capacity; SEM, standard error of mean; 25OHD, 25-hydroxyvitamin D.

DISCUSSION

Lipopolysaccharide (LPS), is a cell wall component of Gram-negative bacteria and is a potent immune stimulator, has been widely used to model bacterial infection experimentally in poultry and livestock (Buyse et al., 2007). In current study, the LPS challenge led to a reduction in laying performance (lower egg production rate, feed intake and feed efficiency). Similarly, it has been demonstrated that the immunological stress reduced feed intake and daily gain following LPS injection in pigs and broilers (Geng et al., 2018; Nie et al., 2018). Furthermore, we also found laying hens challenged with LPS had higher broken egg rate than those received saline. It has been suggested that the immunological stress induced by LPS may reduce the intake and absorption of nutrients (vitamins and minerals) to affect the calcium and phosphorus metabolism (Han et al., 2016; Nie et al., 2018), which may be the reason to explain the lower eggshell quality (higher eggshell strength and thickness) of layers under LPS challenge. It has been demonstrated that both 25OHD and its active hormonal form 1,25(OH)₂D₃ are essential for physiological functions, including immunomodulatory, antioxidant, anti-inflammatory, antibacterial and antiviral properties ( Nakai et al., 2014; Wimalawansa, 2019). Previous studies also reported that 25OHD and vitamin D₃ can alleviate the immune response and improve performance of birds against LPS or coccidia-induced immunological stress (Morris et al., 2014, 2015; Geng et al., 2018). On the other hand, the dietary supplementation of 25OHD had no significant effect on production performance while it can alleviate egg laying rate and feed efficiency under LPS challenge (interactive effect) in current study, suggesting effect of 25OHD can be more effective when layers are physiologically challenged (under stress or aging). This is also in agreement with previous observation, in which found that the 25OHD improve production performance and eggshell quality under high-stoking density (Wang et al., 2020). Also, 25OHD were found to improve eggshell quality (as indicated by lower broken egg rate, higher eggshell strength ad thickness) irrespective of LPS challenge. Similarly, the 25OHD and vitamin D₃ were also found to increase the eggshell thickness, and this increasing effect were more pronounced when layers are aged or under stress in previous studies (Keshavarz, 2003; Wen et al., 2019; Wang et al., 2020).

Intestinal morphology changes with nutritional variations, stress, aging, and (or) disease and it determines the nutrient absorption capacity and are also closely related to the immune response . An increase in villus height indicated a larger absorption area, while deeper crept health indicates that the villi in the small intestinal mucosa are atrophied and their absorptive capacity is decreased . The intestinal barrier includes the mucus layer, epithelial cells, and plasmocytic cells. Intestinal barrier function is one of the important components that maintain gut health and function, and it is generally determined by tight junction integrity of epithelial cells and mucus gel layer (Camilleri, 2019). In general, intestinal permeability-related genes, including tight junction protein, that is, occludin, claudin, and zonula occludens (Ulluwishewa et al., 2011 ) play important roles in the intestinal barrier function. Mucins are the main constituent of mucus gel layer on intestinal mucosa. The decreased expression of tight junction and mucus layer related genes and proteins has been considered a molecular evidence for impaired intestinal health (Moretó and Pérez-Bosque, 2009; Gilani et al., 2018). In this study, we found that LPS led to down-regulation of intestinal barrier associated protein (claudin-1, mucin-1, and mucin 2). This result agrees with previous researches that found LPS induced a redistribution of tight junction proteins expression (occluding, claudin-1, and ZO-1) and increased the concentration of proinflammatory cytokine (TNF-α, IL-1β, and IL-6) of model animals (Sheth et al., 2007; Song et al., 2009; Andrzejczak et al., 2016; Han et al., 2016). Also, LPS were found to increase the endotoxin concentration and led to a lower DAO levels in serum. In mammals, VD₃ deficiency result in not only dysfunction of the innate and adaptive immune systems but also promotes microinflammation, as well as an increased risk of viral or bacterial infections (Di Rosa et al., 2011; Greiller and Martineau, 2015). We also observed that 25OHD supplementation modified intestinal morphology (lower crypt depth and higher villi height) and improved the intestinal barrier function (higher gene expression of claudin-1and mucin-1) of small intestine of layers regardless of LPS. Vitamin D plays a key role in immunity and in intestinal mucosa barrier homeostasis. It ensures appropriate levels of mucosal antimicrobial peptides and maintains epithelial integrity by reinforcing intercellular junctions (Liu et al., 2013; Fakhoury et al., 2020). Also, vitamin D and its receptor exhibited protective effect on intestinal structure and barrier function in colitis model (Liu et al., 2013; Fakhoury et al., 2020 ). Previous studies also reported that 25OHD and vitamin D3 can alleviate the immune response and improve performance of layers against lipopolysaccharide-induced immunological stress (Morris et al., 2014; Geng et al., 2018). As an indicator of intestine integrity and damage, DAO plays an important role in breaking down excess histamine in your body. Vitamin D acts as a fat-soluble hormone that facilitates intestinal absorption of calcium, iron, magnesium, and zinc. The latter two are essential components needed by the body to produce DAO, which may explain that the 25OHD can increased the DAO levels in serum at present study. It has been evident that vitamin D ensures an appropriate level of antimicrobial peptides in the mucus and maintains epithelial integrity (Fakhoury et al., 2020). It has been demonstrated that both 25OHD and its active hormonal form (1,25(OH)₂D₃) are essential for physiological functions, including damping down inflammation and oxidative stress (Nakai et al., 2014; Wimalawansa, 2019). Although, literatures about supplementation 25OHD in layers exposed to LPS is limited, previous studies also reported that 25OHD and vitamin D₃ can alleviate the immune response and improve performance of layers against lipopolysaccharide or coccidia-induced immunological stress (Morris et al., 2014, 2015; Geng et al., 2018).

As an endotoxin, LPS is known to induce oxidative stress by generating reactive oxidative species (ROS) in cells (Simon and Fernández, 2009 ). Oxidative stress resulted from LPS or other stressors were proved to impair the health and production performance of layers (Wang et al., 2019; Wang et al., 2020). In the current study, we found LPS decreased the antioxidative capacity of small intestine, as demonstrated by lower activities of antioxidant enzymes, SOD, CAT, GPx, and T-AOC, and higher corresponding levels of MDA. MDA is generally used as an indicator of membrane lipid peroxidation and its elevated level is considered as marker of oxidative stress. In previous study, injection of LPS led to a reduction in activities of T-AOC, SOD, and GPx and higher MDA in serum of broilers (Jiang et al., 2019). We also found that 25OHD supplementation improved T-AOC, SOD, and CAT activity of jejunum of layers, and the protective effect of 25OHD on CAT, T-AOC activities and MDA were more pronounced in layers suffered from LPS. It has been demonstrated that vitamin D insufficiency is associated with increased oxidative stress or reduced antioxidant capacity in vivo and in vitro (Kim et al., 2020). This is also agreed with the result of our previous study, in which we found that 25OHD alleviate the oxidative stress of layers induced by high stocking density (Wang et al., 2021). This may indicate that 25OHD exert protective effect against LPS as indicated by intestinal morphology, barrier function and antioxidant capacity.

CONCLUSION

These results suggest that dietary supplementing of 80 μg/kg 25-hydroxyvitamin D in diets may alleviate the laying performance and intestinal health through the improvement of intestinal morphology, barrier function and antioxidant capacity in laying hens challenged with LPS.