The study was conducted to evaluate the effects of increasing inclusion levels of the mixture of Bacillus subtilis and essential oils (BSEO) on production, hatching performance, egg quality, serum antioxidant capacity, immune response, and hormone levels of laying hens. A total of 768 24-wk-old layers were randomly allotted into 4 treatments with 6 replicates of 32 birds each replicate. The experiment lasted for 84 d, and the birds were fed a basal diet (CON) or diets with BSEO at 300 (BSEO-1), 600 (BSEO-2), and 900 mg/kg (BSEO-3) in the other 3 groups, respectively. As the BSEO level increased, egg production (linear, p < .05), yolk index (linear, p < .01), glutathione peroxidase activity (linear, p < .01), total antioxidant capacity (linear, p < .01), oestradiol level (linear, p < .01), the value luteinizing hormone/follicle-stimulating hormone (linear, p < .05) increased in a linear manner. Hatchability (linear and quadratic, p < .01), hatchability of fertile of eggs (linear and quadratic, p < .05), avian influenza virus antibody level (linear, p < .01; quadratic, p < .05), parathyroid hormone level (linear and quadratic, p < .01) in the serum increased linearly and quadratically. Taken together, dietary with BSEO in laying hens could significantly improve egg production, hatchability, and hatchability of fertile eggs, which were associated with an enhancement of antioxidant capacity and the level of AIV-Ab in the serum. This study provided evidence of using BSEO as a potential feed additive for laying hens. Highlights Dietary supplementation of BSEO decreased the feed conversion ratio and eggshell thickness of laying hens. Dietary supplementation of BSEO improved the egg weight, fertility, hatchability, hatchability of fertile eggs, and yolk index of laying hens. Dietary supplementation of BSEO enhanced the antioxidant capacity and the level of AIV-Ab in the serum.
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