In ovo injection of vitamin D₃ improves intestinal development of chicks via activating vitamin D receptor

Abstract

Vitamin D₃ (VD₃) is an essential fat-soluble micronutrient that regulates diverse biological processes. However, its long-term impact on avian intestinal development remains largely unknown. In this study, we detected that the VD₃ content was significantly lower in late-cycle production eggs (68 weeks old, termed “LP eggs”) compared to the peak-cycle production eggs (44 weeks old, named “PP eggs”) (P < 0.05). Notably, the intestine index was reduced (P < 0.05), and the intestinal expression of tight junction (TJ) proteins (Occludin and ZO-1) was decreased for chicks hatched from LP eggs versus those from PP eggs (P < 0.05). Thus serial dosages of VD₃ (2.5, 5.0, or 7.5 μg VD₃/egg) were injected into the amniotic cavity of embryos hatched from LP eggs on embryonic day 17 (E17). This intervention effectively improved hatchability, promoted intestinal development that persisted through 14-days post-hatch, and showed immuno-modulatory effects manifesting as Interleukin-6 (IL-6) downregulation and Interleukin-10 (IL-10) upregulation. Further in vivo and in vitro analysis revealed that VD₃ improved intestinal development by activating TJ-related genes (P < 0.05), with vitamin D receptor (VDR) mediating these effects. Chromatin immunoprecipitation (ChIP) assays confirmed that 1,25-(OH)₂D₃ strengthened VDR binding to promoter regions of Occludin and ZO-1 but decreased the interaction degree of VDR with the IL-6 promoter (P < 0.05). These findings establish that in ovo injection of VD₃ is an efficacious perinatal intervention strategy, which significantly mitigates developmental constraints in late-phase poultry production through VDR-dependent intestinal barrier potentiation and immuno-homeostasis modulation.

Abbreviation

VD₃

Vitamin D₃

LP

Late-cycle production

PP

Peak-cycle production

TJ

Tight junction

IOI-VD₃

In ovo injection of VD₃

E17

Embryonic day 17

VDR

Vitamin D receptor

HPLC

High performance liquid chromatography

H&E

Hematoxylin and eosin

RT-qPCR

Real-time quantitative PCR

WB

Western Blotting

VDRE

Vitamin D response element

Introduction

Breeder hens, serving as the parental stock for commercial chickens, exhibit a progressive decline in reproductive performance after 60 weeks of age. This decline is characterized by reduced weekly egg production and diminished nutrient deposition in eggs (Gu et al., 2021; Chang et al., 2024). The senescence-associated reduction in egg nutrient components directly correlates with suboptimal embryonic development, as evidenced by lower hatchability and reduced neonatal body mass (Araújo et al., 2016; Alo et al., 2024). Given the compromised nutrient absorption efficiency in aging layers, nutritional interventions, such as dietary inclusion of vitamins (Gan et al., 2020; Lim and Ryu, 2022) and micronutrients (Hajjarmanesh et al., 2023; Eltahan et al., 2023), could effectively mitigate these negative effects. While dietary modulation requires 6–8 weeks to produce measurable outcomes (Lim and Ryu, 2022), in ovo supplementation demonstrates immediate potential for embryonic nutrient reprogramming (Peebles, 2018; Das et al., 2021).

As an essential fat-soluble steroid hormone, vitamin D₃ (VD₃) not only regulates calcium–phosphorus homeostasis (Voiculescu et al., 2025), but also plays critical roles in modulating both intestinal and systemic immunity (Sassi et al., 2018; Cantorna et al., 2019). The biological activity of VD₃ depends largely on its active metabolite, 1,25(OH)₂D₃, which is generated through sequential enzymatic hydroxylations, first by hepatic 25-hydroxylase and subsequently by renal 1α-hydroxylase (Saponaro et al., 2020). Acting primarily through the vitamin D receptor (VDR), 1,25(OH)₂D₃ exerts pleiotropic effects across multiple physiological systems (Bakke et al., 2018). Although dietary VD₃ supplementation has been shown to improve eggshell quality and hatchability in aged laying hens (Wen et al., 2019), the impacts of embryonic VD₃ exposure on organogenesis, hatchability, and postnatal growth remain insufficiently explored.

The intestine is not only a key organ for digestion and nutrient absorption, but also is the largest immune organ in poultry (Duangnumsawang et al., 2021). Accordingly, this study primarily investigates the effects of in ovo injection of vitamin D₃ (IOI-VD₃) on intestinal development. We first assessed egg quality across different laying stages and compared intestinal length and weight in chicks hatched from late-cycle production eggs (LP eggs) versus those from peak-cycle production eggs (PP eggs). Subsequently, we evaluated the impact of IOI-VD₃ administered to LP eggs on post-hatch growth performance and intestinal development. Furthermore, the role of the vitamin D receptor (VDR) in mediating VD₃-regulated intestinal barrier function was elucidated using complementary in vivo and in vitro models. Collectively, these findings provide a scientific basis for optimizing late-cycle chick production through micronutrient-driven embryonic programming.

Materials and methods

Measurement of egg quality

Fertile eggs were collected from Hy-Line Gray laying hens (Huayu Agricultural Technology Co., Ltd., Handan, China) at two distinct physiological stages, 44 weeks (peak production, PP) and 68 weeks (late production, LP), with 20 eggs per group. Each egg was individually labeled and weighed. Egg quality parameters, including Haugh unit and yolk color, were assessed using an Egg Multi-Tester (EMT-5200, Robotmation, Japan). The yolk was carefully separated from the albumen and weighed, while the eggshell was rinsed, air-dried at room temperature for 24 hours, and then weighed. The proportion of each egg component (yolk, albumen, and shell) relative to total egg weight was subsequently calculated.

Analysis of VD₃ content in egg yolks

Egg yolk samples were dehydrated in an electric thermostatic drying oven and homogenized using a high-speed mixer. Two grams of the dried yolk were subjected to saponification, followed by extraction with petroleum ether. The extract was concentrated using a rotary evaporator and reconstituted in methanol to a final volume of 2 mL. After filtration through a 0.22 μm organic-phase membrane filter, the solution was analyzed for VD₃ concentration by high-performance liquid chromatography (HPLC), following the Chinese National Food Safety Standard GB 5009.82–2016. Separation was achieved on an Agilent C18 reversed-phase column (100 × 4.6 mm, 5.0 μm) with isocratic elution using a mobile phase composed of 95% methanol in water. Detection was performed with a diode array detector set at 264 nm. Chromatographic conditions were maintained as follows: flow rate of 1.0 mL min⁻¹, column temperature of 25°C, and injection volume of 10 μL.

Animal experiments and sample collection

All animal procedures were approved by the Animal Care and Use Committee of Shandong Agricultural University (Tai’an, Shandong, China). Vitamin D₃ (VD₃; ≥98% purity) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) and initially dissolved in ethanol to a stock concentration of 25 mg/mL. This stock solution was serially diluted with phosphate-buffered saline (PBS) to achieve the desired working concentrations and filtered through a 0.22-μm syringe filter prior to use. Fertile eggs from Hy-Line Gray laying hens were used throughout the study. In Experiment 1, 40 peak-production (PP, designated Con1) and 40 late-production (LP, designated Con2) eggs of comparable weight were incubated in an automated incubator (Haijiang Incubation Equipment Co., Beijing, China). In Experiment 2, 160 LP eggs were randomly assigned to one of four groups (n = 40 per group): a control group receiving PBS containing 0.1% ethanol, and three VD₃ treatment groups receiving 2.5 μg, 5.0 μg, or 7.5 μg of VD₃ dissolved in PBS with 0.1% ethanol.

On embryonic day 17 (E17), in ovo injection was performed as follows. The eggshell at the sharp end was disinfected with a 75% alcohol-soaked cotton swab, and a small hole was made using a sterile needle. A volume of 0.2 mL of the respective solution (with or without VD₃) was then injected into the amniotic cavity using a 1-mL sterile syringe. Eggs were turned automatically once every hour for 3 min until E17. From E18 onward, they remained stationary to facilitate hatching. Hatchability and incubation duration were recorded. Chicks were weighed immediately after hatch, and female chicks were reared for 14 days under controlled environmental conditions. The breeding temperature was maintained at 32–33 °C during days 1–3 and 30–32 °C during days 4–7, with a relative humidity of 60–70%. Thereafter, the temperature was gradually reduced by 2 °C per week until it reached a stable 21 °C.

On day 14 post-hatch, six chicks per group were fasted for 12 hours, weighed, and humanely euthanized by cervical dislocation. Intestinal tracts were rapidly excised, and the duodenum, jejunum, and ileum were measured, photographed, and processed for further analysis. Segments for histomorphology were fixed in 4% paraformaldehyde, while those intended for molecular assays were snap-frozen in liquid nitrogen and stored at −80 °C.

Hematoxylin and eosin (H&E) staining

Duodenal and ileal specimens were fixed in 4% paraformaldehyde more than 24 h at 4°C, followed by routine dehydration using a graded ethanol series (70%, 80%, 90%, 95% and 100% ethanol). After dehydration, the samples were cleared with xylene, embedded in paraffin, and sectioned at a thickness of 5 μm. Then, the sections were stained with hematoxylin for 5–10 min, differentiated with 1% hydrochloric acid ethanol, and blued in running tap water. Subsequently, the sections were counterstained with eosin for 1–3 min, dehydrated again with graded ethanol, cleared with xylene, and finally mounted with neutral balsam for microscopic observation. The stained slices were photographed using a light microscope (Ti2-U, Nikon, Tokyo, Japan), with villus height being quantified via the Image-Pro Plus software.

Isolation, cultivation and treatment of intestinal epithelial cells

The intestinal epithelial cells was isolated using E18 SPF (Specific pathogen free) embryos, as described by Ghiselli et al. (2021). The duodenal tissue was harvested from the embryo, which was dissected to remove adipose tissue, pancreas, and mesentery in PBS. Subsequently, the tissue was sectioned into fragments smaller than 1 mm³, washed in PBS, and centrifuged at 1,000 × g for 8 min, which was enzymatically digested using 0.5 mg/mL collagenase I at 37°C for 30 min. The digested cells were suspended in DMEM/F12 medium supplemented with 7% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Solarbio, China), which were sequentially filtered through 100 μm and 50 μm cell strainers. The obtained suspension was transferred into culture plates for microscopic verification of predominant intestinal crypt clusters and was cutivated at 37°C with 5% CO₂. The intestinal epithelial cells were treated with 10⁻⁹ M, 10⁻⁸ M, or 10⁻⁷ M 1,25(OH)₂D₃ (≥97% purity, Aladdin, China) for 72 hours based on our pre-experiment.

Real-time quantitative PCR (RT-qPCR)

Total RNA was isolated from intestinal tissues or treated cells using Trizol reagent (AG21101, Accyrate Biology, Changsha, China), with subsequent cDNA synthesis via HiScript II Q RT SuperMix (R223-01, Vazyme, Nanjing, China). RT-qPCR was conducted with one cycle at 95°C for 10 s, followed by 40 cycles at 95°C for 30 s and 60°C for 10 s by using a 7500 Fast Real-Time PCR System (Applied Biosystems, USA). The relative mRNA expression levels of target genes were calculated using the 2^−ΔΔCt method, with β-actin as the reference gene. The primer sequences are listed in detail in Table 1.

Table 1.

The primers used in this study.

Primers	Sequences (5′−3′)
β-actin	F-CAGCCAGCCATGGATGATGA
	R-CATACCAACCATCACACCCTGA
Claudin-1	F-ATGACCAGGTGAAGAAGATGC
	R-TGCCCAGCCAATGAAGAG
Occludin	F-CTGCTGTCTGTGGGTTCCT
	R-CCAGTAGATGTTGGCTTTGC
ZO-1	F-CGTAGTTCTGGCATTATTCGT
	R-TGGGCACAGCCTCATTCT
VDR	F-CCGGATTCAGGGATCTGACG
	R-AAGTCATTGCTTCCGCAGGT
Il-6	F-CTCCTCGCCAATCTGAAGTC
	R-AGGCACTGAAACTCCTGGTC
I-10	F-AGGAGACGTTCGAGAAGATGGA
	R-TCAGCAGGTACTCCTCGATGT
Occludin-promoter	F-TGGATGTTGCATTTGGACCC
	R-GCGGTCGTACTCATGGATCT
ZO-1-promoter	F-AAAAGTACCACCCACTGCCT
	R-TTGTTGCCGTGTGACAGATG
IL-6-promoter	F-TGGGCAGAACGGGACATTAT
	R-GCTTTTGGAGTGTCACCCTG

Western blotting (WB)

Intestinal tissues and cells were lysed using RIPA lysis buffer supplemented with a protease inhibitor cocktail (NCM Biotech, Suzhou, China). The lysates were subjected to centrifugation at 12,000 × g for 10 min, followed by protein quantification using a BCA assay kit. Protein extracts were mixed with SDS loading buffer and denatured by heating at 100°C for 15 min. Subsequently, proteins were seperated by SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with primary antibodies: Occludin (CY5997, Abways, China), ZO-1 (AF8394, Beyotime, China), IL-6 (AF7236, Beyotime, China), and VDR (CY6787, Abways, China) overnight at 4°C. Species-matched secondary antibodies were then incubated for another 2 hours at room temperature, followed by detection using an ECL kit (P0018M, Beyotime, China).

VDR shRNA-lentivirus production and infection

A shRNA targeted VDR mRNA (5′-GATCCGGATAGTGTAGCACAGTTACTCGA GTAACTGTGCTACACTATCCTTTTTG-3′) and a control shRNA (5′-GATCCCTC GAGTAACTGTGCTACACTATCCTTTTTG-3′) were cloned into the pSIH1-H1-copGFP vector for lentiviral production via HEK-293T cell packaging. Lentiviruses encoding VDR shRNA (sh-VDR) or control (sh-Con) were infected with polybrene into intestinal epithelial cells at 5 MOI when reaching to 50% confluence. The medium was replaced with DMEM/F-12 medium containing 10% FBS 24 hours after infection. Subsequently, the cells were treated with 10^-7 1,25(OH)₂D₃ for 72 hours.

Chromatin immunoprecipitation (ChIP) assay

To investigate the binding of VDR to promoters of Occludin, ZO-1, and IL-6, ChIP analysis was conducted by using a ChIP assay kit (P2078, Beyotime, Shanghai, China). Intestinal epithelial cells were subjected to formaldehyde-mediated protein-DNA crosslinking before complete membrane disruption with SDS lysis buffer. The chromatin was sonicated into fragments ranging from 200 to 1000 bp. Immunoprecipitation with a VDR antibody (CY6787, Abways, Shangai, China) was used to isolate the target protein-DNA complexes. Subsequently, the DNA-protein complexes were extracted, purified, and eluted. VDR-binding sequences were identified via qPCR by using promoter-specific primers for Occludin, ZO-1, and IL-6, respectively.

Statistical analysis

All statistical analyses were performed using data from at least 3 independent experiments. Data were presented as the mean ± standard error of the mean (SEM). Statistical processing was carried out with GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). The Shapiro-Wilk test was used to assess data normality, and appropriate parametric or non-parametric tests were performed accordingly. A two-tailed Student’s t-test was applied to compare the difference between two groups, while one-way ANOVA with post-hoc test was used for comparison among three or more groups. P < 0.05 was defined as statistical significant, and P < 0.01 was set as extremely significant

Results

LP eggs exhibited reduced overall quality and lower VD₃ content

This study compared the quality of eggs from Hy-Line Gray hens at peak production (PP) and late-cycle (LP) stages. Quantitative analysis revealed no significant differences between PP and LP eggs in total egg weight, yolk mass, or albumen mass (Fig. 1A-E). However, the yolk color and Haugh unit showed marked reduction in LP eggs (P < 0.01) (Fig. 1F-G). Notably, micronutrient profiling revealed a 39% depletion in VD₃ content within LP yolks (PP: 14.67 μg/egg vs LP: 10.56 μg/egg, P < 0.01) (Fig. 1H). These results indicate that LP eggs exhibit poorer quality, evidenced by lower yolk color scores and reduced Haugh units, as well as decreased VD₃ content.

Fig. 1.

The egg quality and the VD₃ content in eggs at different laying period. A Egg weight (n = 20). B Yolk weight (n = 20). C Yolk percentage (n = 20). D Albumen weight (n = 20). E Albumen percentage (n = 20). F Yolk color (n = 10). G Haugh unit (n = 10). H VD₃ content in egg yolk (n = 10). Data are presented as means±SEM. Statistical analysis was performed by the student’s t-test. **P < 0.01.

The intestinal development of chicks hatched from LP eggs is inferior to that of PP eggs-hatched chicks

Given the differences between PP and LP eggs, we incubated 40 eggs from each group to assess the impact of maternal age on hatchability and post-hatch intestinal development. Chicks hatched from LP eggs (Con2) exhibited a 7.9% reduction in hatchability and delayed hatching compared to those from PP eggs (Con1) (Fig. 2A, Table 2). Neonatal morphometric analysis revealed no significant difference in body weight at hatch (Table 2), and both groups showed similar growth rates through post-hatch day 14 (Fig. 2B). By day 14, total intestinal length and mass remained comparable between groups (Fig. 2C, Table 2). Histomorphometric evaluation indicated non-significant trends toward duodenal villous hypotrophy and disorganized ileal architecture in Con2 chicks (Fig. 2D and E, Table 2). However, Con2 chicks displayed significantly lower intestinal index and jejunal mass (P < 0.05) (Table 2). Collectively, these findings demonstrate that compromised egg quality, particularly suboptimal maternal VD₃ deposition, might adversely affect early intestinal development in offspring.

Fig. 2.

The hatchability and intestinal development of 2-week-old chicks hatched from breeding eggs at different laying stages. A The accumulated hatching rate during incubation. B The growth curves of chicks. C Representative intestine images of 2-week-old chicks. D and E Representative images of H&E staining for duodenum and ileum sections. Data are presented as means±SEM. Statistical analysis was performed by the student’s t-test.

Table 2.

The hatchability and intestinal development of 2-week-old chicks hatched from breeding eggs at different laying stages.

Items	Treatments		P-values
	Con1	Con2
Hatchablity (%)	92.90	85.00	/
Hatched weight (g)	40.63±3.63	38±4.06	0.051
Duodenum villus height (mm)	860±45.83	793.33±30.55	0.057
Ileum villus height (mm)	456.67±30.55	425±18.03	0.106
Body weight at day 14 (g)	113.17±8.67	113.82±10.49	0.912
Intestine weight (g)	7.8 ± 0.76	7.27±0.52	0.106
Intestine /body weight (%)	6.88±0.31	6.41±0.51	0.045
Duodenum weight (g)	1.9 ± 0.15	1.81±0.19	0.211
Duodenum/body weight (%)	1.68±0.12	1.6 ± 0.21	0.224
Jejunum weight (g)	2.7 ± 0.37	2.37±0.29	0.070
Jejunum/body weight (%)	2.38±0.15	2.09±0.26	0.033
Ileum weight (g)	1.36±0.28	1.17±0.08	0.090
Ileum/body weight (%)	1.19±0.2	1.04±0.14	0.095

Chicks hatched from LP eggs exhibit a compromised intestinal barrier integrity compared to those from PP eggs

In light of the well-established role of intestinal TJ complexes in maintaining epithelial polarity and paracellular selectivity (Otani and Furuse, 2020), we performed compartment-specific analyses of key junctional proteins in the intestines of chicks hatched from PP (Con1) and LP (Con2) eggs. In the duodenum of 2-week-old chicks, protein expression levels of Occludin and ZO-1 were significantly lower in the Con2 group compared to Con1 (P < 0.05) (Fig. 3B, C), despite no significant differences in mRNA levels of Claudin-1, Occludin, or ZO-1 between groups (Fig. 3A). Some post-transcriptional regulation might lead to the discrepancy between mRNA and protein levels for duodenal TJ proteins. In contrast, in the ileum, both Claudin-1 mRNA abundance and Occludin/ZO-1 protein levels were markedly reduced in Con2 chicks relative to Con1 (P < 0.05) (Fig. 3D-F). These results indicate that chicks hatched from LP eggs exhibit a compromised intestinal barrier integrity compared to those from PP eggs.

Fig. 3.

The mRNA and protein expression of tight junction-related genes in the intestine of chicks hatched from breeding eggs at different laying periods. A and D The relative mRNA levels of Claudin-1, Occludin, and ZO-1 in the duodenum (A) and ileum (D) of chicks. (B and E) The protein contents of Occludin and ZO-1 in the duodenum (B) and ileum (E) determined by WB. β-actin was used as an internal control. (C and F) Relative quantification of the Occludin and ZO-1 bands in the duodenum (C) and ileum (F) by using the Image J software. Data are presented as means±SEM. Statistical analysis was performed by the student’s t-test. *P < 0.05.

IOI-VD₃ could improve the growth and intestinal development of chicks hatched from LP eggs

Given the VD₃ deficiency in LP eggs and the associated intestinal hypoplasia in their offspring, we administered VD₃ via in ovo injection into LP eggs at doses of 2.5, 5.0, or 7.5 μg per egg. Surprisingly, IOI-VD₃ enhanced hatchability compared to the vehicle control, hatch rates were 80.00% (control), 84.62% (2.5 μg), 91.67% (5 μg), and 90.91% (7.5 μg), respectively (Fig. 4A, Table 3). Although IOI-VD₃ did not affect body weight at hatch (Table 3), administration of 5 μg VD₃ significantly increased chick body weight by post-hatch day 13 (P < 0.05) (Fig. 4B). Furthermore, in ovo injection of 5 or 7.5 μg VD₃ markedly increased total intestinal length, weight, and index relative to controls (P < 0.01) (Fig. 4C, Table 3). Specifically, 5 μg IOI-VD₃ significantly elevated both duodenal weight and duodenal index (P < 0.05) (Table 3), while doses of 5 or 7.5 μg enhanced jejunal weight (P < 0.05) (Table 3). Notably, the 7.5 μg dose also increased the jejunal index (P < 0.05) (Table 3). These findings demonstrate that IOI-VD₃, particularly at 5 μg, effectively ameliorates growth deficits and promotes intestinal development in chicks hatched from LP eggs.

Fig. 4.

IOI-VD₃ promotes the growth and intestinal development of chicks hatched from LP eggs. A The accumulated hatching rate during incubation. B The growth curves of chicks. C Representative intestine images of 2-week-old chicks. Data are presented as means±SEM. Statistical analysis was performed by one-way ANOVA with post-hoc test. *P < 0.05.

Table 3.

IOI-VD₃ promotes the growth and intestinal development of chicks hatched from LP eggs.

Items	Treatments				P-values vs PBS
	PBS	2.5μgVD3	5.0μgVD3	7.5 μg VD3	2.5 μg VD3	5.0 μg VD3	7.5 μg VD3
Hatchablity (%)	80.00	84.62	91.67	90.91	/
Hatched weight (g)	36.5 ± 1.5	36.77±3.61	38.38±2.4	37.28±2.56	0.435	0.065	0.256
Body weight at day 14 (g)	107.48±10.22	110.03±5.76	111.76±8.32	111.83±10.33	0.303	0.080	0.227
Intestine weight (g)	7.01±0.63	7.69±0.99	7.98±1.09	8.81±1.11	0.092	0.002	0.003
Intestine /body weight (%)	6.47±0.58	6.99±0.8	7.15±0.72	7.9 ± 0.91	0.103	0.007	0.004
Duodenum weight (g)	1.68±0.15	1.67±0.16	1.91±0.23	1.76±0.2	0.477	0.003	0.191
Duodenum/body weight (%)	1.57±0.1	1.52±0.13	1.7 ± 0.12	1.57±0.09	0.262	0.013	0.430
Jejunum weight (g)	2.35±0.2	2.62±0.38	2.55±0.43	2.85±0.55	0.079	0.021	0.011
Jejunum/body weight (%)	2.2 ± 0.18	2.38±0.34	2.27±0.26	2.54±0.43	0.203	0.180	0.020
Ileum weight (g)	1.24±0.25	1.48±0.34	1.35±0.27	1.56±0.31	0.100	0.154	0.051
Ileum/body weight(%)	1.15±0.16	1.34±0.29	1.21±0.2	1.39±0.22	0.090	0.277	0.052

IOI-VD₃ improves the intestinal barrier function and activates VDR

In view of the observed improvement in intestinal development following IOI-VD₃, we further evaluated its effects on the expression of TJ- and inflammation-related genes. In both the duodenum and ileum of 2-week-old chicks, IOI-VD₃ across all tested doses significantly upregulated mRNA levels of Claudin-1 and Occludin, as well as protein expression of Occludin and ZO-1, key components of TJ complexes (P < 0.01) (Fig. 5A-C, J-L). Intestinal homeostasis relies on a balanced interplay between pro- and anti-inflammatory mediators (Luissint et al., 2016). We therefore assessed the expression of IL-6 (a typical pro-inflammatory cytokine) and IL-10 (a major anti-inflammatory cytokine) in intestinal segments after IOI-VD₃ treatment. IOI-VD₃ significantly reduced both transcript (P < 0.05) and protein levels of IL-6 in the duodenum and ileum, whereas IL-10 expression remained unchanged (Fig. 5D-F, M-O). Moreover, IOI-VD₃ markedly enhanced the expression of VDR, the primary nuclear receptor mediating VD₃ signaling, at both the mRNA (P < 0.05) and protein levels (P < 0.01) (Fig. 5G-I, P-R). Collectively, these results indicate that IOI-VD₃ strengthens intestinal barrier function, an effect likely mediated through VDR activation and accompanied by attenuation of intestinal inflammation.

Fig. 5.

IOI-VD₃ enhances the intestinal barrier function and VDR expression of chicks. (A and J) The relative mRNA levels of Claudin-1, Occludin, and ZO-1 in the duodenum (A) and ileum (J) of chicks under PBS or IOI-VD₃ treatments. (B and K) The protein contents of Occludin and ZO-1 in the duodenum (B) and ileum (K) determined by WB. β-actin was used as an internal control. (C and L) Relative quantification of the Occludin and ZO-1 bands in the duodenum (C) and ileum (L) by using the Image J software. (D and M) The relative mRNA levels of IL-6, and IL-10 in the duodenum (D) and ileum (M) of chicks under PBS or IOI-VD₃ treatments. (E-F and N—O) The protein contents and relative quantification of IL-6 in the duodenum (E-F) and ileum (N—O) of chicks under PBS or IOI-VD₃ treatments.(G and P) The relative mRNA levels of VDR in the duodenum (G) and ileum (P) of chicks under PBS or IOI-VD₃ treatments. (H-I and Q-R) The protein contents and relative quantification of IL-6 in the duodenum (H-I) and ileum (Q-R) of chicks under PBS or IOI-VD₃ treatments. Data are presented as means±SEM. Statistical analysis was performed by one-way ANOVA with post-hoc test. *P < 0.05, **P < 0.01.

1,25(OH)2D3. induces TJ protein and reduces IL-6 expression in chicken intestinal epithelial cells, associating with VDR activation

To further define the effect of VD₃ on intestinal barrier function, we treated primary chicken intestinal epithelial cells with increasing concentrations of 1,25(OH)₂D₃ (10⁻⁹ M, 10⁻⁸ M, or 10⁻⁷ M). Treatment with 10⁻⁸ M or 10⁻⁷ M 1,25(OH)₂D₃ significantly upregulated mRNA levels of Claudin-1 (P < 0.01) and Occludin (P < 0.05) and enhanced protein expression of Occludin and ZO-1 (P < 0.01) (Fig. 6A, D, E). Moreover, 1,25(OH)₂D₃ markedly suppressed both mRNA and protein levels of the pro-inflammatory cytokine IL-6 (P < 0.01), while simultaneously increasing IL-10 transcription (P < 0.05) (Fig. 6B-F). Notably, higher concentrations of 1,25(OH)₂D₃ induced a significant increase in VDR protein abundance (P < 0.01), despite no detectable change in VDR mRNA levels (Fig. 6C, D, F). These results demonstrate that 1,25(OH)₂D₃ enhances tight junction integrity and attenuates inflammation in chicken intestinal epithelial cells, effects that are closely linked to VDR activation.

Fig. 6.

1,25(OH)₂D₃ regulates the expression of Occludin, ZO-1, and IL-6 in chicken intestinal epithelial cells via activating VDR. Intestinal epithelial cells were treated with 10^-9, 10^-8 and 10^-7 M 1,25(OH)₂D₃. (A-B) Relative mRNA levels of TJ-related genes (A), inflammation-associated genes (B), and VDR (C) in chicken intestinal epithelial cells treated with 0, 10^-9, 10^-8, or 10^-7 M 1,25(OH)₂D₃. (D-F) The protein contents and relative quantification of Occludin, ZO-1, IL-6, and VDR in cells treated with 0, 10^-9, 10^-8, or 10^-7 M 1,25(OH)₂D₃. β-actin was used as an internal control. Data are presented as means±SEM. Statistical analysis was performed by one-way ANOVA with post-hoc test. *P < 0.05, **P < 0.01.

VDR is involved in 1,25(OH)₂D₃ regulation of TJ protein and IL-6 expression in chicken intestinal epithelial cells

To determine the role of the VDR in 1,25(OH)₂D₃-mediated regulation of tight TJ proteins and inflammatory factors, we used VDR-targeting shRNA (sh-VDR) to knock down VDR expression in chicken intestinal epithelial cells and evaluated its impact on 1,25(OH)₂D₃ responsiveness. sh-VDR effectively suppressed VDR protein levels (P < 0.05) without significantly altering VDR mRNA abundance (Fig. 7A, D, F). VDR knockdown markedly attenuated 1,25(OH)₂D₃-induced upregulation of Occludin and ZO-1 and abolished its suppressive effect on IL-6 expression (P < 0.05) (Fig. 7B-F), indicating that VDR is essential for these regulatory actions. We therefore hypothesized that VDR directly modulates transcription of Occludin, ZO-1, and IL-6. Using the JASPAR database (https://jaspar2022.genereg.net/), we identified putative VDREs in the promoter regions of these genes. ChIP assays with primers spanning the predicted VDR-binding sites confirmed direct binding of VDR to the Occludin promoter at positions −1568 to −1554 bp (P < 0.01), to the ZO-1 promoter at −299 to −285 bp (P < 0.05), and to the IL-6 promoter at −1083 to −1069 bp (P < 0.05) (Fig. 7G-I). Additionally, qPCR analysis of ChIP-enriched DNA revealed that 1,25(OH)₂D₃ treatment enhanced VDR occupancy at the Occludin and ZO-1 promoters, while reducing its binding to the IL-6 promoter (Fig. 7G-I). Collectively, these findings demonstrate that 1,25(OH)₂D₃ enhances TJ protein expression and suppresses IL-6 primarily through VDR-dependent transcriptional regulation.

Fig. 7.

VDR is involved in 1,25(OH)₂D₃ regulation of TJ protein and IL-6 expression in chicken intestinal epithelial cells. (A-C) Relative mRNA levels of VDR (A), TJ-related genes (B), and inflammation-associated genes (C) in cells treated with vehicle or 10^-7 M 1,25(OH)₂D₃ in the presence of sh-Con or sh-VDR. (D-E) The protein contents and relative quantification of Occludin, ZO-1, IL-6, and VDR in cells treated with vehicle or 10^-7 M 1,25(OH)₂D₃ in the presence of sh-Con or sh-VDR. β-actin was used as an internal control. (G-H) ChIP-PCR amplification indicated the increased abundance of VDR binding to the promoter regions of Occludin and ZO-1 following 1,25(OH)₂D₃ treatment. (I) ChIP-PCR amplification showed the decreased abundance of VDR binding to the promoter region of IL-6 following 1,25(OH)₂D₃ treatment. Data are presented as means±SEM. Statistical analysis was performed by one-way ANOVA with post-hoc test. *P < 0.05, **P < 0.01.

Discussion

This study investigates the impact of breeder hen age on egg quality, hatchability, and post-hatch intestinal development in offspring chicks. We demonstrate that eggs from LP hens exhibit reduced yolk color, Haugh unit, and VD₃ content compared to those from PP hens. Given this VD₃ deficiency, we administered in ovo amniotic injection of VD₃ at E17 and found that it significantly enhanced intestinal development over the first 14 days post-hatch. This improvement was associated with upregulation of TJ proteins and downregulation of the pro-inflammatory cytokine IL-6. Furthermore, in vitro experiments confirmed that these effects are mediated by the VDR, which directly regulates the transcription of TJ- and inflammation-related genes.

Egg quality and nutrient deposition are profoundly influenced by breeder age (Ulmer-Franco et al., 2010; Kowalska et al., 2021). Previous studies report marked declines in eggshell strength and thickness, albumen height, and yolk pigmentation (Gu et al., 2021; Zhao et al., 2024; Chang et al., 2024) in eggs from aged hens. Nutrient levels, including amino acids (Heflin et al., 2018), minerals (Santos et al., 2022), and vitamins (Ko et al., 2020; Guo et al., 2021), are also significantly reduced during the late laying period. Consistent with these findings, our data confirm lower yolk color scores, Haugh units, and VD₃ concentrations in LP eggs. Importantly, egg quality and maternal nutrient deposition directly influence embryonic development and post-hatch performance (Torres and Korver, 2018; Neto et al., 2024). For example, insufficient trace minerals in eggs impair skeletal mineralization in chicks (Torres and Korver, 2018). Similarly, we observed reduced hatchability in LP eggs, aligning with prior reports (Araújo et al., 2016). The development of the intestinal tract from late incubation through early post-hatch is critical for lifelong poultry performance, including growth, immune competence, and sexual maturation (Chen et al., 2021; Ayalew et al., 2025). In this study, chicks hatched from LP eggs exhibited significantly lower intestinal organ indices and shorter total intestinal length compared to PP-derived chicks, likely attributable to compromised egg quality and diminished VD₃ availability.

Extensive evidence supports a beneficial role of VD₃ in intestinal health. In mammals, VD₃ alleviates colitis by promoting colonic epithelial proliferation (Chen et al., 2023). In poultry, dietary VD₃ supplementation enhances duodenal and ileal villus height, reduces crypt depth, and improves overall mucosal architecture (Wei et al., 2024). Our findings extend these observations by showing that in ovo VD₃ delivery not only improves hatchability but also promotes long-term intestinal growth, effects closely linked to enhanced expression of TJ proteins (Claudin-1, Occludin, and ZO-1). These proteins are essential for maintaining intestinal barrier integrity, which is vital for host defense and homeostasis (Otani and Furuse, 2020; Frazer and Good, 2022). Notably, VD₃ treatment in cirrhotic rats reduced intestinal permeability and bacterial translocation via upregulation of occludin and Claudin-1 (Lee et al., 2021), and vitamin d-deficient mice exhibit impaired barrier function in colitis models (Vernia et al., 2022). Our results confirm that VD₃ exerts similar protective effects on the intestinal barrier in avian species.

Intestinal immune homeostasis depends on a balanced interplay between pro- and anti-inflammatory mediators. VD₃ modulates both innate and adaptive immunity by regulating T cells, B cells, and macrophages (Sassi et al., 2018; Wang et al., 2024; Artusa and White;, 2025). Dysregulation characterized by excessive pro-inflammatory signaling and inadequate anti-inflammatory responses compromises mucosal immunity (Otani and Furuse, 2020). IL-6 is a pivotal cytokine in intestinal inflammation, its overactivation via the IL-6/STAT3 pathway drives colitis progression (Kang et al., 2020; Wu et al., 2022). Conversely, IL-10 is a potent anti-inflammatory factor, and its absence leads to spontaneous colitis in mice (Wang et al., 2022). In our study, IOI-VD₃ significantly suppressed intestinal IL-6 expression in vivo, while in vitro treatment with 10⁻⁸ M or 10⁻⁷ M 1,25(OH)₂D₃ increased IL-10 mRNA levels in intestinal epithelial cells, supporting a dual immunomodulatory role of VD₃.

1,25(OH)₂D₃, the biologically active metabolite of VD₃, exerts its effects by binding to the nuclear VDR (Christakos et al., 2016; Saponaro et al., 2020). The resulting 1,25(OH)₂D₃–VDR complex binds to VDREs in target gene promoters to regulate transcription (Hu et al., 2020; Chen et al., 2020; Voltan et al., 2023). For instance, VDR directly controls Claudin-2 expression in mouse intestine (Zhang et al., 2019) and activates Claudin-5, with VDR deficiency promoting tumorigenesis (Zhang et al., 2022). Here, we identified functional VDREs in the promoters of Occludin, ZO-1, and IL-6. ChIP assays confirmed that VDR directly binds these regions, and 1,25(OH)₂D₃ treatment enhanced VDR occupancy at Occludin and ZO-1 promoters while reducing its binding to the IL-6 promoter. These data indicate that 1,25(OH)₂D₃ promotes TJ protein expression and suppresses IL-6 transcription in a VDR-dependent manner. Although deeper mechanistic validation is warranted, our loss-of-function experiments using sh-VDR establish VDR as the central mediator of VD₃’s effects on intestinal barrier and immune regulation.

In conclusion, using a chick embryo model, we demonstrate that in ovo VD₃ administration, particularly at a dose of 5 μg per egg, significantly enhances long-term intestinal development by reinforcing tight junction integrity and attenuating inflammation, primarily through VDR activation. These findings provide novel insights into the developmental programming of intestinal health by maternal VD₃ status and suggest that targeted in ovo VD₃ supplementation could mitigate the adverse effects of advanced breeder age on offspring performance. However, large-scale animal trials are needed to evaluate the practical applicability of this strategy. Moreover, the precise crosstalk between IL-6, IL-10, and TJ proteins in the context of VD₃ signaling warrants further investigation.

Data availability

Data will be available on reasonable request.

CRediT authorship contribution statement

Guangxiao Zhang: Writing – original draft, Investigation, Formal analysis, Data curation. Yawen Sun: Methodology, Conceptualization. Ling He: Validation. Bowen Zhao: Validation. Hongchao Jiao: Methodology. Jingpeng Zhao: Methodology. Xiaojuan Wang: Methodology. Min Liu: Methodology. Kelin Li: Methodology. Hai Lin: Supervision, Methodology, Funding acquisition, Conceptualization. Haifang Li: Writing – review & editing, Supervision, Methodology, Conceptualization.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.