Dietary Chlorella, Spirulina, and acidifier modulate jejunal cytokine-related gene expression in broiler chickens

Dietary microalgae and organic acidifiers are increasingly evaluated as non-antibiotic feed additives in poultry nutrition, but their effects on intestinal immune-related transcription remain incompletely defined. This study investigated the effects of dietary Chlorella vulgaris, Spirulina platensis, an organic-acid-based acidifier, and their combinations on jejunal cytokine-related mRNA expression in broiler chickens. Broilers were assigned to eight dietary treatments with three replicate pens per treatment and six birds per pen: basal diet control, acidifier, Chlorella, Spirulina, acidifier + Chlorella, acidifier + Spirulina, Chlorella + Spirulina, and acidifier + Chlorella + Spirulina. The acidifier was supplied at 0.1%, whereas Chlorella and Spirulina were included at 0.2%. At day 28, one bird was sampled from each replicate pen, yielding three independent pen-level biological replicates per treatment for RT-qPCR analysis. The relative expression of IFN-β, IFN-γ, IL-6, and IL-12 was quantified by USI-based TaqMan RT-qPCR and normalized to GAPDH. The treatments produced gene-specific and combination-dependent transcriptional responses. Chlorella alone reduced IL-6, IL-12, and IFN-γ expression, whereas the acidifier reduced IL-6 but increased IL-12 and IFN-β. The Chlorella + Spirulina and triple-combination groups showed broader upregulation across the measured cytokine-related transcripts. Because the study was limited to jejunal mRNA expression, used three pen-level biological replicates per treatment, and did not include cytokine protein quantification, intestinal histomorphometry, or microbiota profiling, the findings should be interpreted as preliminary transcriptional evidence rather than proof of improved gut health or functional immune modulation.