Abstract: A healthy gut microbiota in calves is necessary for optimal performance and development. Considering that probiotics have been shown to improve gut microbiota, the aim of this study was to investigate the effects of Clostridium butyricum on growth performance, blood parameters, and gut microbiota in Hanwoo calves. In total, 92 calves from two farms were randomly assigned to four groups: a control and three treatment groups that received increasing doses of C. butyricum (CB1, CB2, and CB3) during the first 5 days after birth. Independent experiments were conducted at each farm with different measurements, where body weight was monitored and blood, rumen, and fecal samples were collected to assess physiological responses and microbial profiles. Notably, significant differences were observed in blood amylase and acid–base parameters, suggesting that C. butyricum supplementation may enhance metabolic stability and buffering capacity. Microbial profiling revealed preserved alpha diversity and compositional shifts in both the rumen and fecal microbiota. Particularly, there was an increase in the relative abundances of Prevotella and Muribaculaceae and a decrease in the abundances of the pathogenic genera Escherichia and Shigella in calves fed C. butyricum-supplemented diets. These changes, along with a trend toward a reduced frequency and severity of diarrhea, suggest that C. butyricum supplementation may support gut health and promote stable early growth in neonatal calves.
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