| Summary: | This study was conducted to evaluate the potential of hydrolysable tannin (chestnut tannin, CHT) without or with condensed tannin (quebracho tannin, QT) for modulating alfalfa silage fermentation characteristics and in vitro ruminal methane (CH<sub>4</sub>) production, fermentation profile, and microbiota. Alfalfa (235 g/kg fresh weight) was ensiled with no tannins (control), 2% CHT (CHT2), 5% CHT (CHT5), the combination of CHT and QT at 1% each (CHQ2), and CHT and QT at 2.5% each (CHQ5) of forage dry matter (DM). The CHQ2 treatment was more effective in reducing DM losses, pH, and ammonia–nitrogen to total nitrogen ratios of alfalfa silage than CHT2 and CHT5 treatments. All tannin treatments decreased ruminal CH<sub>4</sub> production, and the magnitude of the decrease was greater for the combinations than the individual ones. Total volatile fatty acid (VFA) concentrations and DM degradation decreased by tannin treatments, but microbial protein (MCP) synthesis increased. The total VFA concentrations and DM degradation were lower with CHQ2 treatment than with CHT5 and CHQ5 treatments, but the MCP concentrations were comparable among these treatments. Tannin inclusion decreased the abundance of the anaerobic fungi <i>Ruminococcus albus</i> and <i>Ruminococcus flavefaciens</i>, but enhanced <i>Fibrobacter succinogenes</i>. The combination of CHT and QT alleviated the inhibition of CHT supply alone in <i>Butyrivibrio fibrisolvens</i>, <i>Ruminobacer amylophilus</i>, and <i>Prevotella ruminicola</i> as well as protease. The results revealed that a combination of HT from CHT and CT from QT at a low level can reduce proteolysis and CH<sub>4</sub> production of alfalfa silage without impairing ruminal fermentation and microbiota.
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