This study investigated the effects of xylo-oligosaccharide supplementation on the production performance, intestinal morphology, cecal short-chain fatty acid levels, and gut microbiota of laying hens. A total of 800 Lohmann pink layers, each 48 wk old, were randomly divided into 5 dietary treatment groups, namely XOS at 0 (CON), 100 (XOS1), 200 (XOS2), 300 (XOS3) and 400 (X0S4) mg/kg. The experimental period was 24 wk. The results revealed that the egg production rate and the number of eggs laid by each layer between 1 to 12 wk increased as the XOS concentration increased (Plinear < 0.05). The sand-shell egg percentage decreased significantly from 1 to 12 wk in the XOS1, XOS2, and XOS3 groups (PANOVA < 0.05). Compared with the CON group, the 4 XOS dosage groups presented significant increases in the villus height and the ratio of villus height to crypt depth in the jejunum (PANOVA < 0.05), whereas a linear decrease in jejunal crypt depth (Plinear < 0.05) was noted. In addition, XOS supplementation significantly increased the concentrations of butyric acid and isovaleric acid in the caeca (PANOVA < 0.05). High-throughput sequencing analysis of bacterial 16S rRNA revealed that dietary XOS supplementation influenced the cecal microbiota. The alpha diversity analysis indicated that the richness of cecal bacteria was greater in the laying hens fed XOS. The modulation of the cecal microbiota composition upon the addition of XOS was characterized by an increased abundance of Firmicutes and Bifidobacteriaceae, and decreased abundance of Bacteroidetes. At the genus level, dietary XOS supplementation resulted in decreases in the abundances of Bacteroides and Rikenellaceae_RC9_gut_group and an increase in the abundance of Lactobacillus. In conclusion, dietary XOS supplementation improved the production performance of laying hens by increasing the production of short-chain fatty acids and improving their intestinal morphology, which was achieved mainly through changes in the composition of the intestinal microbiota. The recommended level of XOS in the diet of laying hens is 200 mg/kg.
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