AbstractThe objective of Trial 1 was to determine the effects of condensed tannins (CT) from sericea lespedeza [SL; Lespedeza cuneata (Dum. Cours.) G. Don] on in vitro digestible organic matter (IVDOM), total gas production (GP), methane (CH₄) emission, and ruminal fluid parameters after fermentation. Substrates used in four 48-h in vitro fermentations were 100% bermudagrass [(Cynodon dactylon (L.) Pers.] hay (0SL), 100% SL hay (100SL), and a mix of both hays (50SL). Linear reductions were observed for all parameters (P < 0.05) with the inclusion of SL, except for CH₄ in relation to GP, that presented a quadratic effect (P = 0.005). In Trial 2, SL plants were enriched with ¹³C–CO₂ to obtain pure enriched CT to identify the destination of fermentation end products of CT degradation. The enrichment of CT through the SL was successful (P < 0.001), and carbon originated from CT was detected in the fermentation end products [microbial mass, clarified rumen fluid, and in the CH₄ produced (P < 0.001)]. Therefore, inclusion of SL was effective in reducing in vitro CH₄ production and compound-specific tracing of δ¹³C abundance provided better quantitative understanding of the mechanisms of partitioning CT during ruminal fermentation processes.