We aimed to investigate the effects of adding Bacillus subtilis to milk replacers on the incidence of diarrhea, growth performance, intestinal microbiota composition, and short-chain fatty acid (SCFA) production in newborn Hu lambs. Thirty newborn lambs with similar genetic backgrounds and initial body weights were selected and nursed by their dams for 4 days before transitioning to a milk replacer. At age 7 days, the lambs were randomly assigned to two experimental groups of 15 lambs each: CON (basic milk replacer feeding) and BS (basic milk replacer feeding supplemented with 0.2% B. subtilis). At age 28 days, eight lambs in each group were randomly euthanized, and their colonic and cecal contents were collected for microbiological and SCFA analyses. The lambs were monitored daily to determine the incidence of diarrhea. The BS group showed significantly increased average daily feed intake and gain and significantly reduced incidence of diarrhea. Furthermore, dry matter digestibility and crude protein and ether extract content increased in the BS group compared with those in the CON group. Increased acetate and total SCFA concentrations were noted in the colon and cecum, with elevated propionate levels specifically in the cecum. Microbiota analysis revealed that B. subtilis supplementation enhanced microbial diversity and beneficially modulated bacterial communities. Potentially harmful bacteria showed significantly decreased abundance in the colon (Campylobacterota by 11.09% and Fusobacteriota by 2.98%), whereas beneficial bacteria exhibited increased abundance (Firmicutes by 8.9%, Actinobacteriota by 3.59%, Lactobacillus by 5.92%, and Bifidobacterium by 3.18%). Similar trends were observed in the cecum, with decreases in Campylobacterota (4.82%) and Fusobacteriota (3.12%) and increases in Bacteroidota (4.11%), Lactobacillus (3.75%), and Prevotella (3.59%). These findings indicate that adding 0.2% B. subtilis to the milk replacer is an effective approach for enhancing lamb growth performance, maintaining intestinal health, and improving the intestinal microbiota.
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