SummaryThis study evaluated the effects of Quebracho tannin extract (QTE) on in vitro ruminal fermentation, chemical composition of rumen microbes, ruminal degradation and intestinal digestibility of crude protein (iCPd). Three treatments were tested, the control (basal diet without QTE), the basal diet with 15 g QTE/kg dry matter (DM) and the basal diet with 30 g QTE/kg DM. The basal diet contained (g/kg DM): 339 grass silage, 317 maize silage and 344 concentrate. In vitro gas production kinetic was determined using the Hohenheim gas test (Experiment 1). The Ankom RF technique, a batch system with automatic gas pressure recordings, was used to determine in vitro production of shortchain fatty acids (SCFA) and ammonia-nitrogen concentration (NH 3 -N), as well as nitrogen and purine bases content in liquid-associated microbes (LAM) and in a residue of undegraded feed and solid-associated microbes (Feed+SAM) (Experiment 2). Ruminal degradation and iCPd were determined using the nylon bag technique and the mobile nylon bag technique, respectively (Experiment 3). Gas production (Experiment 1), total SCFA and NH 3 -N (Experiment 2) decreased with increasing QTE levels. Microbial mass and composition of LAM were not affected by QTE, but total mass of Feed+SAM linearly increased, likely due to decreased substrate degradation with increasing QTE levels.The total amount of N in microbial mass and undegraded feed after the in vitro incubation increased with increasing QTE levels, suggesting a potential greater N flow from the rumen to the duodenum. In contrast to in vivo studies with the same QTE, no effects were detected on ruminal effective degradability and iCPd, when using the nylon bag techniques. Based on the in vitro procedures, QTE increased the supply of N postrumen; however, some evidence of a decreased fibre degradation were also observed.Therefore, the benefit of adding QTE to diets of cattle is still questionable. disputed. In line with this, particularly about the risk that tannin may actually decrease nutrients absorption in the small intestine (Frutos, Hervás, Giráldez, & Mantecón, 2004) by failing to dissociate from the bound nutrients under the intestinal conditions. On the other hand, by (re-)binding to digestive and microbial enzymes and to microbial protein (Waghorn, 2008). Moreover, CT may also bind to microbes or to their enzymes in the rumen, thus inhibiting microbial growth and reducing microbial CP (MCP) supply to the animal.Recent studies of Ahnert, Dickhoefer, Schulz, and Susenbeth (2015) and Henke et al. (2017)