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This research was designed to conduct a deeper exploration of the impacts of dietary supplementation with BA on the apparent digestibility of nutrients and the composition of rectal microbiota in weaned Holstein dairy calves. Sixteen Holstein heifer calves with similar body weights (91.2 \u00b1 0.7 kg) were selected and randomly allocated into two groups, each comprising eight calves. Calves in the control group (CON group) were fed with a basal diet, while those in the benzoic acid group (BA group) were fed with the basal diet supplemented with 0.50% benzoic acid (on a dry matter basis). The experimental period started at 60 days of age and ended at 102 days of age, lasting for a total of 42 days. The calves were weaned at 60 days of age, with a transition period of 7 days. Feed samples were collected every two weeks, fecal samples were collected from 99 to 101 days of age, and blood samples were collected at 102 days of age. The results showed that supplementation with BA did not influence the digestibility of dry matter, crude protein, ether extract, neutral detergent fiber, acid detergent fiber, calcium, and phosphorus between the two groups. Compared with the CON group, BA supplementation tended to decrease the total cholesterol (TC) in the serum of the calves (p = 0.067). Supplementation with BA increased the relative abundances of the two beneficial bacteria, Bifidobacterium and Bifidobacterium pseudolongum (p &lt; 0.05, LDA &gt; 2), but decreased that of the harmful bacterium, Clostridium sensu stricto 1, in the rectum of dairy calves. The microbial functional prediction revealed that the fecal microbial metabolism involved in primary bile acid biosynthesis was higher in the calves from the BA group. In conclusion, the present study demonstrated that adding 0.50% BA to the diet did not influence the apparent nutrient digestibility, but improved rectal microbiota health, which finally promoted the growth performance in weaned Holstein dairy calves.<\/jats:p>","DOI":"10.3390\/ani15142080","type":"journal-article","created":{"date-parts":[[2025,7,15]],"date-time":"2025-07-15T08:04:41Z","timestamp":1752566681000},"page":"2080","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Effect of Benzoic Acid on Nutrient Digestibility and Rectal Microbiota of Weaned Holstein Dairy Calves"],"prefix":"10.3390","volume":"15","author":[{"given":"Haonan","family":"Dai","sequence":"first","affiliation":[{"name":"State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dewei","family":"Du","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qi","family":"Huang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia","family":"Guo","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shujing","family":"Li","sequence":"additional","affiliation":[{"name":"Shijiazhuang Tianquan Elite Dairy Ltd., Shijiazhuang 050200, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenli","family":"Yu","sequence":"additional","affiliation":[{"name":"Shijiazhuang Tianquan Elite Dairy Ltd., Shijiazhuang 050200, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zengyuan","family":"Zhao","sequence":"additional","affiliation":[{"name":"Shijiazhuang Tianquan Elite Dairy Ltd., Shijiazhuang 050200, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2370-265X","authenticated-orcid":false,"given":"Peng","family":"Sun","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Wang, S., Wang, F., Kong, F., Cao, Z., Wang, W., Yang, H., Wang, Y., Bi, Y., and Li, S. 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