Crude protein levels adjusted by Distiller’s dried grains with solubles reshape rumen microbiota in Hanwoo steers during early fattening period

Distiller’s dried grains with solubles (DDGS), a by-product typically used in cattle diets as a protein source, is a promising feed ingredient for CH₄ mitigation because of its high ether extract level. This study investigated how increasing levels of crude protein (CP) adjusted by DDGS affect rumen microbiota in relation to CH₄ mitigation and animal productivity. A total of 24 Hanwoo steers in the early fattening period (16 months old, 504 ± 33.0 kg) were studied over 16 weeks, including a 2-week adaptation period. Tall fescue hay and four concentrate mixes were used as experimental diets: 15% CP in dry matter (DM) (low CP [LCP]), 18% CP (lower middle CP [LMCP]), 19% CP (higher middle CP [HMCP]), and 21% CP (high CP [HCP]). The rumen microbiota was analyzed using full-length 16S rRNA sequencing. Dietary CP levels did not significantly alter alpha diversity, whereas beta diversity, based on the weighted UniFrac distance, showed clear separation between the LCP and HCP groups. Among the major taxa, 18 bacterial taxa were differentially abundant among the treatments, and 22 showed linear associations with dietary CP levels. Notably, Selenomonas dextrinosolvens, the unclassified species Gammaproteobacteria, Ruminobacter amylophilus, and Lentimicrobium saccharophilum exhibited a negative linear association with dietary CP levels, whereas the unclassified species Bacteroidales and Eubacterium ventriosum showed a positive linear association. The predicted KEGG modules related to amino acid biosynthesis were enriched in LCP compared to HCP and were negatively associated with increasing CP levels. The unclassified species Gammaproteobacteria and L. saccharophilum were negatively correlated with one or more CH₄ measurements. Dietary CP levels adjusted by DDGS supplementation influenced shifts in rumen bacterial composition and had functional implications in Hanwoo steers during the early fattening period. Specifically, certain bacterial taxa related to lipid metabolism and predicted metabolic functions related to amino acid biosynthesis were linearly associated with dietary CP levels as adjusted by DDGS. This study provides a valuable insight into how dietary CP levels adjusted by DDGS shape rumen microbial communities and their predicted functional profiles, with potential implications for optimizing CP utilization in Hanwoo steers.