Rumen microbial community and functional response to high- and low-starch diets: Metagenomic evidence from original Brown Swiss cows

This study aimed to explore how dietary starch levels influence rumen microbial communities and their metabolic functions using a metagenomic approach, as part of a broader multi-omics integration project. A switchback design was employed over three 9-day periods (7 days of adaptation followed by 2 days of sampling) using four rumen-cannulated Original Brown Swiss cows. Animals were alternately fed lowstarch (LS; 6.31% DM) and high-starch (HS; 16.2% DM) total mixed rations in an ABA/BAB sequence. Rumen contents (both solid and fluid fraction) were sampled 2 h post-feeding and subjected to shotgun metagenomic sequencing. Taxonomic composition was estimated directly from metagenomic raw reads using SingleM, while the differential abundance of bacterial and archaeal taxa was evaluated via MetaCoder, with statistical comparisons conducted using the Wilcoxon rank sum test (adjusted P ≤ 0.10). For functional profiling, metagenomic assemblies (>1000 base pairs) were processed with Prodigal to predict open reading frames, and annotations were assigned via Hidden Markov Model searches against the KOfam database. The KEGG orthologs were further categorized into 11 major metabolic functions, and feature-level differential abundance was assessed using MetagenomeSeq (adjusted P ≤ 0.10; log2 fold-change with cumulative-sum scaling). Functional pathway enrichment analysis was conducted with reference to the KEGG database. Taxonomic profiling indicated a significant dominance of Bacteroidia in the LS diet and Clostridia in the HS diet (P < 0.05), consistent with known fiber- versus starch-degrading microbial preferences. Functional annotation revealed similar overall distributions of carbohydrate and glycan biosynthesis pathways in both diet groups. Although no individual KEGG orthologs showed significant differential abundance, pathway enrichment analysis highlighted consistent enrichment (P < 0.05) of carbohydrate-related metabolic routes, including glycolysis, pyruvate metabolism, galactose metabolism, and starch and sucrose metabolism across both dietary treatments. In summary, dietary starch levels induced clear shifts in the structure of the rumen microbial community, particularly in taxa associated with fiber and starch metabolism. However, the metagenomic functional profiles based on predicted gene content remained largely unchanged between diets. This suggests that while the microbial composition adapts to dietary changes, the overall functional potential encoded in the metagenome is relatively stable in the short term. Ongoing metatranscriptomic analyses will help clarify whether these structural changes are accompanied by shifts in gene expression and active microbial metabolism under varying starch inputs.