Abstract: In this study, we tested the effects of the inclusion in silages of bioactive legumes containing condensed tannins (CT) or polyphenol oxidase (PPO), ensiled alone or in mixture with one grass species, on in vitro rumen fermentations. Six mini-silos were prepared in triplicate as follows: pure sainfoin (SF), pure red clover (RC), pure timothy (T, control without bioactive compounds); binary mixtures T–SF and T–RC (in g/kg on a DM basis, 500:500); ternary mixture T–SF–RC (in g/kg on a DM basis, 500:250:250). Samples from each mini-silo were incubated under anaerobic conditions in culture bottles containing buffered rumen fluid from sheep. Rumen fermentation parameters, namely DM disappearance, volatile fatty acids (VFA), ammonia (NH3), gas production and methane (CH4) were determined. The presence of RC in mixtures with T or T–SF results in fermentation similar to pure T and a positive associative effect between T and RC on total VFA production was observed (P = 0.029). The NH3: total N ratio was similar among the treatments, but lower values for NH3: insoluble N ratio in silages containing SF or RC (P< 0.05, except for T–SF) indicates that the bioactive compounds can limit silage protein degradation in the rumen. The CH4 production per g of DM only tended to differ among treatments (P = 0.096), being slightly lower for pure SF than for pure T and RC, probably due to differences in the fermentation pathways towards less acetate and butyrate productions. Overall, the benefits of including RC in forage mixtures previously observed on silage quality are also present on ruminal digestion efficiency
