Low dietary calcium impairs bone quality and induces bone site-specific remodeling in aged laying ducks with minimal alterations in cecal microbiota and metabolite profiles.

Calcium (Ca) is critical for poultry health and production, with deficiency impairing skeletal function and productivity. However, the effects of dietary Ca deficiency on the gut-microbiota-metabolite-bone axis in aged laying ducks remain poorly understood. In this study, 144 healthy 60-week-old Longyan Shanma laying ducks were randomly assigned into two groups: a normal-Ca (3.6%, NC) and low-Ca (1.8%, LC) group, and fed for 5 weeks. The LC diet decreased egg production, egg mass and eggshell quality, while elevating feed conversion ratio (P < 0.05). It also reduced bone strength, mineral density and contents in various bone tissues. Serum levels of 1,25-dihydroxyvitamin D₃and procollagen type I carboxy-terminal propeptide (PICP) were markedly increased. The expression of bone metabolism-related genes, including receptor activator of nuclear factor-κB (RANK) and receptor activator of nuclear factor-κB ligand (RANKL), were upregulated in both tibia and femur. Moreover, dietary low Ca damaged intestinal morphology by reducing intestinal villus height and villus height-to-crypt depth (V/C) ratio in the duodenum, jejunum and ileum (P < 0.05). Furthermore, the LC diet reduced cecal microbial alpha diversity, accompanied by altered microbial composition and functional reprogramming involved in carbohydrate metabolism and ion transport. Low calcium also reshaped cecal metabolite profiles, with obvious changes in ursolic acid, galactosylsphingosine and picolinic acid levels. In summary, dietary low calcium disrupts bone homeostasis and intestinal barrier function in aged laying ducks. Importantly, tibia and femur exhibit distinct transcriptional responses to calcium deficiency, revealing evident bone site-specific regulatory patterns, which provides novel insights into the gut-bone axis mechanism underlying calcium deficiency-induced bone metabolic disorders.