Dietary crude protein levels modulate rumen microbiota composition and network stability in Hanwoo steers during late fattening

Optimizing dietary protein is critical for improving ruminant productivity while minimizing environmental nitrogen losses in sustainable beef production. This study investigated how dietary crude protein (CP) affects rumen microbiota composition, predicted functional profiles, and microbial co-occurrence networks in late-fattening Hanwoo steers. Twenty-four steers (707 ± 50.1 kg) were fed tall fescue hay and one of four concentrate mixes containing 15.0%, 16.2%, 17.5%, or 18.5% CP (dry matter basis) over a 12-week trial, and rumen samples were analyzed using full-length 16S rRNA gene sequencing. Unweighted UniFrac-based beta diversity analysis revealed distinct microbial community separation between high and low CP treatments. Bacteroidota and Bacillota dominated across all diets, while Spirochaetes abundance declined linearly with increasing CP levels. Functional predictions indicated differential enrichment of amino acid and vitamin biosynthesis pathways among treatments. Network analysis showed similar complexity in low and moderate CP groups, but the highest CP diet caused a marked reduction in nodes, edges, modularity, and clustering, indicating disrupted microbial interactions. These findings suggest that excessive dietary CP supply may destabilize rumen microbial networks and impair growth performance, whereas moderate CP levels better support microbial ecosystem stability and productive efficiency and productive efficiency in sustainable Hanwoo beef production.