The two-stage Tilley and Terry technique (incubation with rumen fluid followed by an acid-pepsin digest), used to estimate dry matter (DM) digestibility of forages in vitro, was evaluated with oven-dried corn silage as a substrate. The effect of weight of substrate (100–3000 mg), continuous shaking of incubations for the period of incubation with rumen fluid, number of bacteria present in the inoculum, and the contribution of bacterial dry matter to residual feed DM was measured. Percent dry matter digested decreased linearly as weight of substrate per incubation tube increased. Continuous shaking, as opposed to intermittent mixing (twice daily) during incubation with rumen fluid, increased the rate of DM disappearance and resulted in higher digestibility coefficients. Both the volume of inoculum and the number of bacteria present in that volume of inoculum influenced the percent DM digested. Bacteria contribute weight to residual feed DM unless steps are taken to remove them by centrifugation or solubilization.
