Evaluation of the jejunal microbiota and microbial markers in laying hens fed diets with black soldier fly larval fat as a partial or total replacement for soybean oil.

In this study, the effects of partial or complete replacement of soybean oil with Hermetia illucens larval (BSF) fat on the jejunal microbial ecosystem and microbial activity in laying hens were evaluated. Three diets were used: a soybean oil-based diet (SO) and two diets in which 50% (BSF50) or 100% (BSF100) of soybean oil was replaced with insect fat. Jejunal digesta were analyzed for physicochemical parameters, short-chain fatty acids (SCFAs), and microbial composition using 16S rRNA gene sequencing. Functional metabolic potential was predicted using PICRUSt2. Dietary treatments did not influence (P > 0.05) digesta weight, dry matter, viscosity, pH, total SCFA levels, or microbial alpha diversity. However, the inclusion of BSF fat significantly reduced putrefactive short-chain fatty acids, mainly through a decrease (P < 0.001) in isobutyric acid content, while the iso-valeric acid content increased (P = 0.011) under complete replacement. The relative proportion of acetic acid increased (P = 0.002), whereas that of propionic acid decreased (P < 0.001) compared with that in the group fed the soybean oil diet. The complete replacement of soybean oil promoted shifts (P < 0.05) toward Firmicutes-associated fermentative and spore-forming taxa, including Bacillus, Paenibacillus, and several Clostridia-related lineages, whereas the presence of soybean oil favored (P < 0.05) the growth of Lactobacillus reuteri. Biomarker analysis (LEfSe) supported these patterns, revealing the enrichment of Clostridia-related taxa under partial substitution and Bacillales- and Paenibacillales-associated taxa under complete substitution. Functional prediction using PICRUSt2 indicated that BSF fat inclusion was associated with a higher (P < 0.05) predicted representation of pathways related to bile acid biosynthesis and carbohydrate metabolism, and a lower (P < 0.05) predicted representation of pathways associated with oxidative phosphorylation and certain lipid and amino acid metabolic processes. Overall, replacement of soybean oil with H. illucens larval fat modulated the jejunal microbiota and its predicted metabolic potential without disrupting overall microbial diversity or fermentation intensity, indicating that insect-derived lipids may serve as alternative dietary fat sources capable of shaping the jejunal microbial ecosystem of laying hens.