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The aim of this study was to evaluate the fermentation activity of ruminal inoculum from cattle fed fresh pasture and supplemented or not with corn grain at different daily frequencies. Twenty heifers with ruminal catheters were randomly assigned to four treatments. Animals were fed pasture ad libitum and non-supplemented (T0) or supplemented with corn grain at 1% of bodyweight offered in one (T1), two (T2) or eight (T8) meals per day. After 20 days of adaptation, ruminal inoculum of each heifer was used to evaluate fermentation activity by the in vitro gas-production technique, using alfalfa, white clover or ryegrass as substrates. Gas production was measured at 2, 4, 6, 8, 10, 12, 18, 24, 48, 72 and 96 h from the beginning of incubation. Data were fitted to an exponential model and potential gas volume, fractional rate of gas production and lag time were analysed by PROC MIXED, considering the effect of treatment and substrate, and their interaction. The three parameters were affected by the substrate. Supplementation frequency did not affect the potential gas volume or the fractional rate of gas production. An interaction between treatment and substrate was detected (P < 0.01) on lag time, but only when ryegrass was used as the substrate. In conclusion, increasing the frequency of supplementation did not show benefits for the fermentation activity of ruminal inoculum, at least when the type of pasture used in this experiment was supplemented with corn at 1% of bodyweight. This study evaluated rumen fermentation activity of heifers fed pasture supplemented with corn at different daily frequencies. Increasing supplementation frequency did not show benefits, at least with this type of pasture (9% of crude protein, 60% of neutral detergent fiber) at this supplementation rate (1% BW) with corn. [ABSTRACT FROM AUTHOR]