Stage-dependent effects of composite probiotics on gut mineral transport, Skeletal health, and egg quality in laying hens

Two hundred and forty one-day-old Lohmann Brown laying hens were used to investigate the stage-dependent effects of dietary supplementation with a composite probiotic on production performance, egg quality, calcium and phosphorus metabolism, skeletal characteristics, and the expression of mineral transport- and bone metabolism-related genes from 1 to 52 weeks of age. Hens were randomly allocated to four dietary treatments with six replicates of ten birds per replicate. The treatments differed in the timing of probiotic supplementation relative to the onset of lay, including continuous supplementation, early-life supplementation only, late-phase supplementation only, and no supplementation throughout the experiment. The composite probiotic consisted of Bacillus subtilis, lactic acid bacteria, and yeast. During the peak laying period, hens receiving probiotics showed higher egg production rates and lower eggshell breakage (P < 0.05). In the late laying period, continuous probiotic supplementation further reduced eggshell breakage and maintained higher laying performance compared with non-supplemented hens (P < 0.05). Egg quality was improved by probiotic inclusion, as indicated by increased eggshell strength and yolk height during peak and late lay (P < 0.05), although eggshell thickness was slightly reduced (P < 0.05).Probiotic supplementation exerted marked effects on skeletal traits and mineral metabolism in a stage-dependent manner. Continuous supplementation increased tibial calcium and phosphorus content during the peak and late laying periods (P < 0.05) and alleviated age-related reductions in bone mineral density. At the molecular level, probiotic-fed hens exhibited increased intestinal expression of calcium and phosphorus transport-related genes, including TRPV6, PMCA1b and NaPi-IIb, particularly during peak and late laying stages (P < 0.05). In tibial tissue, continuous probiotic supplementation enhanced the expression of osteogenic markers, including RUNX2 (P < 0.05).In conclusion, composite probiotic supplementation produces stage-dependent improvements in mineral transport, bone health, and egg quality in laying hens. Continuous supplementation throughout the production cycle is most effective for maintaining skeletal mineral reserves and reducing egg breakage, providing a practical nutritional strategy for long-cycle commercial laying hens.