Dietary calcium citrate enhances nutrient digestibility and modulates cecal microbiota function in pre-laying hens

Background and Aim: Calcium source and bioavailability are critical determinants of nutrient utilization, gut microbial ecology, and future productivity in laying hens, particularly during the pre-laying period. Organic calcium salts may exert additional functional effects through microbiota modulation beyond mineral supply alone. This study evaluated the effects of replacing calcium carbonate with calcium citrate on nutrient digestibility, cecal microbiota composition, short-chain fatty acid (SCFA) production, and predicted microbial metabolic pathways in pre-laying hens. Materials and Methods: Sixty Hisex Brown pre-laying hens (13–20 weeks of age) were allocated to two dietary treatments: a control diet containing calcium carbonate and an experimental diet in which calcium carbonate was fully replaced with calcium citrate. Diets were formulated to be isocaloric, isonitrogenous, and equal in total calcium content. Nutrient digestibility coefficients were determined using a physiological balance trial. Cecal SCFA concentrations were quantified by gas chromatography. Cecal microbiota composition was analyzed by 16S rRNA gene sequencing, and functional pathway prediction was performed using Kyoto Encyclopedia of Genes and Genomes-based bioinformatic analysis. Statistical significance was set at p < 0.05. Results: Replacement of calcium carbonate with calcium citrate significantly increased the digestibility of crude fat (+28.7%, p ≤ 0.001), crude protein (+7.29%, p ≤ 0.001), calcium (+7.56%, p ≤ 0.05), and phosphorus (+2.92%, p ≤ 0.05). Cecal concentrations of propionic, butyric, and valeric acids were significantly higher in the calcium citrate group (p ≤ 0.001). Microbiota analysis revealed a higher relative abundance of Bacillota, particularly Lactobacillaceae and Oscillospiraceae, and a reduced proportion of Bacteroidota, including Alistipes. Alpha diversity indices were higher in the experimental group. Functional prediction indicated enrichment of microbial genes associated with carbohydrate, amino acid (phenylalanine, tyrosine, tryptophan), and fatty acid metabolism, alongside reduced methane metabolism. Conclusion: Dietary calcium citrate markedly improves nutrient digestibility and beneficially reshapes cecal microbiota composition and function in pre-laying hens. These findings highlight calcium citrate as a promising nutritional strategy to enhance gut health, mineral utilization, and feed efficiency, with potential implications for subsequent egg production and sustainable poultry systems. Keywords: calcium citrate, cecal microbiota, feed efficiency, gut health, nutrient digestibility, poultry nutrition, pre-laying hens, 16S rRNA sequencing.