Metagenomic and metabolomic analyses of rumen fiber digestion in Mongolian cattle fed fresh grass versus hay

ABSTRACT Mongolian cattle exhibit exceptional roughage tolerance due to their rumen microbiome’s robust fiber-degrading capacity, enabling efficient utilization of low-quality forage under the Mongolian Plateau’s seasonal fluctuations. This study compared rumen microbial composition, CAZyme profiles, fermentation parameters, and metabolic pathways in cattle fed fresh grass (FG) versus hay to elucidate microbe-metabolite interactions underlying fiber digestion. Thirty non-pregnant female Mongolian cattle (460 ± 35 kg, 3–4 years old) were randomly divided into two groups ( n = 15/group): one grazed on FG, the other housed and fed autumn-harvested hay (HG). Six animals per group were subsampled for rumen fluid collection and multi-omics analyses ( n = 6/group, total n = 12). Compared with the FG group, the HG group showed an increased molar proportion of acetate and a higher acetate-to-propionate ratio, along with reduced molar proportions of propionate and butyrate in rumen fermentation parameters. Metagenomic analysis revealed a higher abundance of Bacteroidales bacteria and anaerobic fungi (including Neocallimastix sp. JGI-2020a and Piromyces sp. E2) in the HG group. Functional annotation further indicated enriched carbohydrate metabolism pathways in the HG group, along with a greater diversity of CAZymes, particularly those involved in hemicellulose and pectin degradation. Metabolomics identified 13 differentially abundant carbohydrate metabolites, with gluconolactone upregulated in the HG group. Additionally, carbohydrate metabolism pathways identified in the metabolome corroborated the reliability of the metagenomic functional annotations. Correlation network analysis revealed positive associations of Bacteroidaceae bacteria, Neocallimastix sp. JGI-2020a, and Piromyces sp. E2 with acetate, hemicellulose-degrading GHs, and carbohydrate metabolic pathways. In conclusion, hay feeding enhanced ruminal fiber degradation in Mongolian cattle through increased Bacteroidales and anaerobic fungi, diversified CAZymes (especially hemicellulases/pectinases), and upregulated carbohydrate metabolism, reflecting microbial adaptation to low-quality forage. IMPORTANCE Mongolian cattle’s superior roughage tolerance depends on a specialized rumen microbiome that degrades fibrous substrates via diverse CAZymes. However, microbe-metabolite interactions driving fiber digestion in this breed remain poorly understood. This study revealed an increased abundance of bacteria and fungi involved in rumen fiber degradation, which may be responsible for secreting enzymes associated with hemicellulose and pectin breakdown. Furthermore, the upregulation of key metabolites, including gluconolactone, indirectly promotes acetate production through pathways such as glycolysis and the pentose phosphate pathway. These findings reveal microbial adaptations enhancing low-quality forage utilization, offering new strategies for improving ruminant efficiency in seasonal or resource-limited grazing systems.