Effects of Vitamin A on Immune Responses and Vitamin A Metabolism in Broiler Chickens Challenged with Necrotic Enteritis

**Background:** Necrotic enteritis (NE) is an economically significant enteric inflammatory disease in poultry caused by Clostridium perfringens, often predisposed by coccidial infection. Vitamin A (VitA) is known as an anti-inflammatory micronutrient that modulates immune function through retinoic acid signaling via RAR/RXR nuclear receptors. Previous work suggested that phospholipase C from C. perfringens might stimulate prostaglandin E2 (PGE2) production, potentially interfering with the conversion of vitamin A to retinoic acid. This study aimed to determine the effects of high-dose dietary VitA supplementation on immune responses and VitA metabolism in NE-challenged broilers, and to explore the involvement of JAK/STAT and RAR/RXR signaling pathways.

**Methods:** Using a 2 × 2 factorial design, 336 one-day-old Ross 308 broiler chicks were randomly assigned to 4 groups (7 replicates of 12 birds each): Ctrl (basal diet without extra VitA), VitA (basal diet + 12,000 IU/kg VitA), NE (basal diet + NE challenge), and VitA+NE (VitA diet + NE challenge). NE was induced by oral inoculation with a 20-fold dose of a trivalent coccidian vaccine (Eimeria maxima, E. tenella, E. acervulina) on day 14, followed by oral gavage of C. perfringens type A (3 × 10^8 CFU/mL) on days 18–20. On day 28, blood, jejunum, spleen, and liver samples were collected. Jejunal NE lesion scores (0–6 scale), serum biochemical indices, serum IgA and IgG, CD3+ T cell density in jejunal villi and crypts, hepatic VitA content (by HPLC), serum PGE2 (ELISA), and mRNA expression of cytokines (IFN-γ, IL-13, IL-17, TGF-β4), JAK/STAT pathway genes (JAK1, JAK2, STAT1, STAT5, STAT6), retinoic acid synthesis genes (Adh-1, RALDH-2, RALDH-3), and retinoic acid receptor genes (RAR-α, RAR-β, RAR-γ, RXR-α, RXR-γ) in jejunum and spleen were measured. Data were analyzed by two-factorial ANOVA with Duncan's multiple comparison test (p < 0.05).

**Key Results:** NE challenge significantly increased jejunal lesion scores (p < 0.05) and decreased serum glucose, total glyceride, calcium, phosphorus, and uric acid levels (p < 0.05). VitA supplementation reduced serum phosphorus and uric acid and increased LDL, AST, and creatine kinase (p < 0.05). An interaction was found for alkaline phosphatase: VitA decreased its activity in NE-challenged birds (p < 0.05). Serum IgA, IgG, total protein, albumin, and globulin were not significantly affected. CD3+ T cell density in jejunal villi and crypts was unchanged. NE challenge upregulated jejunal IL-13 and TGF-β4 mRNA expression (p < 0.05). VitA supplementation increased jejunal IL-13 (p < 0.05) but downregulated splenic IL-13 (p < 0.05). An interaction was found for jejunal IFN-γ: VitA increased it in unchallenged birds (p < 0.05) but not in challenged birds. VitA downregulated splenic STAT5 and STAT6 (p < 0.05). Interactions were found for JAK1 and STAT1 in spleen: VitA downregulated splenic JAK1 in unchallenged birds and STAT1 in challenged birds (p < 0.05). Hepatic VitA content was significantly higher in VitA-supplemented groups (p < 0.001). An interaction was found for serum PGE2: VitA increased PGE2 in NE-challenged birds (p < 0.05). NE challenge upregulated jejunal RALDH-2 and RALDH-3 (p < 0.05). VitA downregulated splenic RALDH-3 in unchallenged birds (interaction, p < 0.05). NE challenge upregulated jejunal RAR-β and RXR-α and splenic RAR-α and RAR-β (p < 0.05). VitA upregulated jejunal RAR-β but downregulated splenic RXR-α and RXR-γ (p < 0.05).

**Clinical Implications:** High-dose vitamin A supplementation (12,000 IU/kg) did not alleviate jejunal injury or reduce Th2-related cytokine expression in NE-challenged broilers, indicating it is not a simple therapeutic agent for NE. However, VitA increased hepatic vitamin A storage and showed tissue-specific immunomodulatory effects—enhancing Th2 cytokine expression in the jejunum while suppressing it in the spleen, and inhibiting JAK/STAT signaling and RXR expression in the spleen. The upregulation of jejunal RALDH enzymes during NE challenge suggests a host-driven mechanism to produce retinoic acid for anti-pathogen immune responses, contrary to the hypothesis that C. perfringens would suppress RALDH via PGE2. The elevated PGE2 in VitA+NE birds warrants further investigation. These findings highlight the complexity of vitamin A–pathogen interactions and suggest that vitamin A's effects depend on tissue, infection status, and immune context, which has implications for designing nutritional strategies in poultry production, particularly in the context of antibiotic-free production systems.