Ruminal Fermentation, Methanogenic Archaeal Diversity, and Metabolomic Responses to Urochloa brizantha Extracts in Sheep

Abstract

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 hydroethanolic 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), hydroethanolic extract (HE; 80 mL/day), and monensin (MON; 25 mg/kg of DMI). Ruminal fluid was evaluated for fermentation traits, archaeal 16S rRNA gene diversity, and untargeted metabolomic profiles. Results: Dry matter intake and fermentation parameters were unaffected by treatment. Archaeal alpha diversity showed a contrast-dependent response, with lower Shannon diversity in sheep receiving plant extracts than in those receiving monensin (p = 0.034), and 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 indicated treatment-specific temporal separation between day 0 and day 21 only in EE-treated sheep, supported by permutation testing based on classification error rate (p = 0.027), although predictive performance should be interpreted cautiously. This EE-specific response was characterized by a significant shift in the carboxylic acid subclass (FDR = 0.022). Top VIP-ranked discriminants (aspartate, succinate, glutamate, phenylalanine, leucine, and choline) decreased nominally but failed multiple-testing correction (FDR = 0.0602–0.2580). CTL, HE, or MON showed no temporal separation. Conclusions: Urochloa brizantha extracts produced limited effects on conventional ruminal fermentation parameters but were associated with selective archaeal responses. In particular, the ethanolic extract was associated with a treatment-specific temporal metabolomic pattern, including a significant shift in the carboxylic acid subclass, while individual metabolite changes remained exploratory and warrant further investigation.