Abstract: 【Objective】 The aim of this study was to investigate the colonization ability of Clostridium butyricum in the digestive tract of broilers and the effects of Clostridium butyricum colonization on intestinal short-chain fatty acid content and microflora diversity of broilers.【Method】 90 one-day-old Abaric broilers with good health were randomly divided into blank group and Clostridium butyricum group with 3 replicates per group and 15 broilers per replicate. The blank group was fed with normal saline, while the Clostridium butyrate group was given fluorescently labeled Clostridium butyrate. At 3 h and 16 days of age, ileum epithelial cells were dissected and collected to observe the fluorescence. Then 30 one-day-old chicks with good health were randomly divided into control and experimental groups. The experimental group was fed the diet containing Clostridium butyricum from 1 to 6 days, followed by the basal diet, while the control group was fed the basal diet throughout the whole period, the period was 21 days. At 16 and 21 days of age, the contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid and total short-chain fatty acid in ileum and cecum were determined. The difference analysis of microbial species, species abundance ratio, bacterial species and protein functional abundance were carried out.【Result】 There was fluorescence around the ileum epithelial cells in Clostridium butyricum group at 3 h and 16 days of age, indicating that Clostridium butyricum could colonize gastrointestinal ileum epithelial cells. The results of short-chain fatty acid content determination showed that compared with control group, the contents of acetic acid, propionic acid, isobutyric acid, iso-valeric acid and valeric acid in ileum of broilers at 16 days of age in experimental group were extremely significantly increased (P<0.01). The contents of propionic acid, iso-valeric acid and valeric acid in ileum of broilers at 21 days of age were extremely significantly increased (P<0.01), while the contents of acetic acid、isobutyric acid and total short-chain fatty acid were significantly increased (P<0.05). The contents of butyric acid and valeric acid in cecal of broilers at 16 days of age were extremely significantly increased (P<0.01), while the contents of iso-valeric acid and caproic acid were significantly increased (P<0.05). Compared with control group, the contents of butyric acid and isovaleric acid in cecum of broilers at 21 days of age were extremely significantly increased (P<0.01), while the contents of acetic acid, isobutyric acid, caproic acid and total short-chain fatty acid were significantly increased (P<0.05). The results of microbial species difference analysis at phylum level showed that Firmicutes at 16 days of age were the dominant flora with the highest abundance, and the number of microbial species was increased at 21 days of age samples, and Proteobacteria, Firmicutes and Bacteroidetes were the dominant bacterial groups with high abundance, and the dominant bacteria at 16 and 21 days of age experimental groups were higher than those in control group. The results of species abundance ratio analysis showed that compared with control group, the species abundance ratio of Anaerolineaceae at 16 days of age experimental group was significantly increased (P<0.05), while the proportions of 7 genera, such as Defluviicoccus, Comamonas, Enterobacter were significantly decreased (P<0.05). At 21 days of age in experimental group, the species abundance ratio of 6 genera, such as Ferribacterium, Trichococcus were increased significantly (P<0.05), while Proteobacteria, Plancyomycetes, and Armatimonadetes GP5 were significantly decreased (P<0.05). The results of genus difference analysis showed that the most important microorganisms in experimental group at 16 days of age were Blautia, while the most important microorganisms in control group were Clostridium and unclassified Lachnospiraceae. At 21 days of age, the most important microorganisms in experimental group were unclassified Bacteroidetes, Trichococcus, Lactobacillales and Lactococcus, while the most important microorganisms in control group were norank Acidobacteria GP4 and unclassified Comamonadaceae. The results of functional abundance analysis of flora proteins showed that the abundance of methyl-accepting chemotaxis protein, glutathione S-transferase and amino acidotransferase subunit A at 16 days of age experimental group were higher than those in control group, and the abundance of ECF subfamily RNA polymerases δ-70 factor and alanine at 21 days of age experimental group were higher than those in control group.【Conclusion】 Clostridium butyricum could colonize the ileum epithelial cells of broilers. Clostridium butyricum could increase the content of short-chain fatty acids, microbial species and beneficial bacteria genera in intestinal contents, and improve the abundance of species and functional proteins, so as to promote intestinal function.

Key words: Clostridium butyricum; colonization; broilers; short chain fatty acids; intestinal microflora