Effects of Dietary 5-Aminolevulinic Acid Supplementation on Laying Performance, Immune Function, Intestinal Microbiota, and Serum Metabolomics in Laying Hens

5-Aminolevulinic acid (5-ALA), a naturally occurring non-proteinogenic amino acid and a key precursor in heme biosynthesis, has attracted increasing attention as a potential functional feed additive in animal production. This study investigated the effects of dietary 5-ALA supplementation on 960 Fengda No. 1 laying hens (42 weeks of age) randomly assigned to four dietary treatments with 16 replicates per treatment (15 birds per replicate) and fed diets supplemented with 0, 250, 500, or 1000 mg/kg 5-ALA for 60 days. Results show that 500 mg/kg 5-ALA improved laying performance and enhanced liver antioxidant capacity (T-AOC, SOD, GSH-Px). It also enhanced serum immunoglobulin concentrations and reduced the concentrations of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. Furthermore, 5-ALA supplementation was associated with changes in the abundances of specific cecal bacterial taxa. Metabolomic analysis revealed that vitamin B6 metabolism showed the highest differential abundance score, with increased 4-pyridoxic acid and 4-pyridoxolactone following 5-ALA supplementation. Collectively, these findings suggest that dietary 5-ALA, particularly at 500 mg/kg, has the potential to improve laying performance and physiological homeostasis, potentially through the modulation of antioxidant and immune functions, gut microbial composition, and metabolic pathways.