Phytogenic supplementation mitigates necrotic enteritis in broilers through modulation of gut microbiota, short-chain fatty acids, and nutrient-related gene expression.

With the withdrawal of antibiotic growth promoters, phytogenic compounds have gained attention as potential alternatives for enhancing gut health and modulating the intestinal microbiota. This study investigated the effects of a phytogenic blend (PHB) containing thymol, oleic acid, capric acid, and refined sunflower fat on cecal short-chain fatty acids (SCFA), jejunal gene expression, and cecal microbiota composition in broiler chickens under necrotic enteritis (NE) challenge. A total of 450 d-old mixed-sex Cobb 500 broiler chicks were randomly allocated to three treatments: non-challenged control without additives (NC), NE-challenged control without additives (CC), and NE-challenged supplemented with 0.01% phytogenic blend (PHB). Birds in challenge groups were orally inoculated with Eimeria spp. on d9 and Clostridium perfringens on d14 to induce NE. Data were analyzed using one-way ANOVA, and mean separation was performed by Tukey's test (P < 0.05). Microbiota composition was assessed through 16S rRNA sequencing, and downstream microbiome analyses and visualization were performed using R version 4.6.1 (R Core Team, 2026) in RStudio. The NE challenge upregulated interferon-gamma (IFN-γ; P < 0.05), downregulated claudin 5 (CLDN5; P < 0.05) and immunoglobulin M (IgM; P < 0.05) genes, and significantly increased iso-butyric, iso-valeric, and valeric acid concentrations compared to the NC group (P < 0.05). Moreover, it reduced microbial diversity (P < 0.05) and increased the abundance of potentially pathogenic genera, including Clostridium sensu stricto 1, and Escherichia Shigella. The dietary PHB significantly reduced the concentrations of iso-butyric, iso-valeric, and valeric acids, and increased lactic and succinic acids (P < 0.05), and upregulated APN (P < 0.05). l-type amino acid transporter 1 (LAT1) showed an intermediate response in the PHB group and did not reach statistical significance compared to CC (P > 0.05). Furthermore, PHB did not significantly affect IFN-γ, CLDN5, or IgM levels (P > 0.05). Microbiota analysis revealed distinct β-diversity clustering between CC and PHB groups and increased abundance of beneficial bacterial taxa in the PHB group, including Tyzzerella, Lactobacillus, Anaerostipes, Ruminococcus_torques_group, Butyricicoccus pullicaecorum, with an elevated Firmicutes-to-Bacteroidetes ratio. These findings suggest that PHB supplementation alleviated NE-induced gut dysbiosis and metabolic disruption by promoting beneficial bacteria, improving SCFA profiles, and enhancing mucosal gene expression related to digestion.