We investigated the efficacy of a single bacterium strain, Bacillus subtilis (B. subtilis) YW1, on the performance, morphology, cecal microbiota, and intestinal barrier function of laying hens. A total of 216 28-week-old Hy-line Brown laying hens were divided into three dietary treatment groups, with six replicates of 12 birds each for 4 weeks. The control group (Ctr) was fed a basal diet and the treatment groups, T1 and T2, were fed a basal diet supplemented with B. subtilis at a dose rate of 5 × 10⁸ CFU/kg and 2.5 × 10⁹ CFU/kg, respectively. Dietary supplementation with B. subtilis did not significantly affect overall egg production in both groups, with no obvious changes in average egg weight and intestine morphology. B. subtilis administration also improved the physical barrier function of the intestine by inducing significantly greater expression levels of the tight junction protein occludin in T1 (p = 0.07) and T2 (p < 0.05). Further, supplementation with B. subtilis effectively modulated the cecal microbiota, increasing the relative level of beneficial bacteria at the genus level (e.g., Bifidobacterium p < 0.05, Lactobacillus p = 0.298, Bacillus p = 0.550) and decreasing the level of potential pathogens (e.g., Fusobacterium p < 0.05, Staphylococcus p < 0.05, Campylobacter p = 0.298). Overall, B. subtilis YW1 supplementation cannot significantly improve the egg production; however, it modulated the cecal microbiota towards a healthier pattern and promoted the mRNA expression of the tight junction protein occludin in laying hens, making B. subtilis YW1 a good probiotic candidate for application in the poultry industry, and further expanding the resources of strains of animal probiotics.

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