Ruminal Fermentation, Methanogenic Archaeal Diversity, and Metabolomic Responses to <em>Urochloa brizantha</em> Extracts in Sheep

<jats:p>Background: Phytogenic feed additives are being explored as alternatives to conventional rumen modulators, yet their effects on the ruminal ecosystem remain insufficiently characterized when assessed only through conventional fermentation parameters. This study evaluated the effects of ethanolic and hydroalcoholic extracts of Urochloa brizantha on ruminal fermentation, methanogenic archaeal diversity, and the ruminal metabolome of sheep. Methods: Eight rumen-cannulated wethers were assigned to a replicated 4 × 4 Latin square design with four 28-day periods. Treatments consisted of no additive (CTL), ethanolic extract of U. brizantha (EE; 50 mL/day), hydroalcoholic extract (HE; 80 mL/day), and monensin (MON; 25 mg/day). Ruminal fluid was evaluated for fermentation traits, archaeal 16S rRNA gene diversity, and untargeted metabolomic profiles. Results: Conventional fermentation parameters and dry matter intake were largely unaffected by treatment, with only marginal shifts detected by orthogonal contrasts. Archaeal alpha diversity remained stable, although Methanimicrococcus was selectively enriched in the EE group (P = 0.012). Untargeted metabolomics revealed the clearest response: despite substantial overlap in global PCA profiles, PLS-DA showed significant temporal separation between day 0 and day 21 only in EE-treated sheep (P = 0.027). This EE-specific response was characterized by lower levels of aspartate, succinate, glutamate, phenylalanine, leucine, and choline, with an increased relative contribution of carboxylic acids. No comparable temporal discrimination was observed in CTL, HE, or MON. Conclusions: Urochloa brizantha extracts produced limited effects on conventional ruminal fermentation and methanogenic archaeal diversity. However, the ethanolic extract induced a distinct temporal metabolomic shift, suggesting a selective metabolic response that warrants further investigation.</jats:p>