Phosphorus alleviates age-associated osteoporosis in laying hens by reshaping bone metabolism and modulating cecal microbial communities

Osteoporosis in aged laying hens poses a major constraint on long-term egg production system. This study investigated the efficacy and underlying mechanisms of dietary phosphorus (P) in alleviating age-associated osteoporosis. A completely randomized design was employed, with 300 Hy-Line Brown laying hens (58 weeks of age) randomly assigned to one of three dietary treatments: a low-phosphorus diet (LP, 0.12% non-phytate phosphorus [NPP]), a regular-phosphorus diet (RP, 0.32% NPP), or the LP diet supplemented with 10,000 FTU/kg phytase (PHY, 0.12% NPP). Each treatment comprised 10 replicates of 10 hens. Following a 2-week adaptation period, the experiment lasted for 12 weeks. The results showed that dietary P reduction or phytase supplementation had no significant effect (P > 0.05) on egg production performance or egg quality throughout the experimental phase. However, at the end of the trial, hens fed the LP diet exhibited significantly lower serum P concentration and alkaline phosphatase (ALP) activity, along with reduced tibial P and magnesium (Mg) contents (P < 0.05), compared with the RP group. Conversely, serum calcium (Ca) level and tartrate-resistant acid phosphatase (TRACP) activity were significantly elevated in the LP group (P < 0.05). In contrast, phytase supplementation in the PHY group significantly increased serum ALP activity and tibial P and Mg contents relative to the LP group (P < 0.05). Notably, the PHY group also showed higher serum Ca concentration than the RP group (P < 0.05). Histomorphological examination revealed progressive cortical bone thinning accompanied by irregular erosion and increased demineralized areas in the tibiae of LP-fed hens, whereas only mild pathological changes were observed in the PHY group. Analysis of cecal microbiota indicated that dietary P modulation significantly altered microbial community structure: principal coordinate analysis (PCoA) showed distinct clustering for the LP and PHY group at week 8 and week 12, respectively (P < 0.05). Linear discriminant analysis effect size (LEfSe) further revealed that the LP group was enriched with pro-inflammatory taxa and microbes associated with nutrient metabolism stress. The RP group harbored a more complex and diverse microbial community, while the PHY group was characterized by an enrichment of beneficial bacteria involved in short-chain fatty acid (SCFA) production. Spearman correlation analysis demonstrated significant associations between specific bacterial genera and tibial health parameters. Genera such as Phascolarctobacterium, Rikenellaceae_RC9_gut_group, and Bacteroides were positively correlated with tibial mineral content and structural integrity, whereas Lactobacillus, Olsenella, and Prevotellaceae_Ga6A1_group showed negative correlations with these bone traits (P < 0.05). Collectively, these findings indicate that chronic P deficiency induces severe skeletal deterioration and gut microbiota dysbiosis in aged laying hens. Phytase supplementation partially counteracts the adverse effects of low-P diet by improving P utilization and reshaping the cecal microbiota toward a more beneficial composition. Nevertheless, long-term feeding of P-deficient diet even with phytase remains insufficient to fully preserve skeletal health. Therefore, ensuring adequate dietary P supply is essential for alleviating age-associated osteoporosis in aged laying hens, likely through integrated regulation of bone metabolic homeostasis and gut microbial ecology.