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ABSTRACT The objective of the present study was to evaluate the effect of feed type, pH, and Cu and Zn concentration on Cu and Zn binding with different feedstuffs after in vitro incubation in water. For this purpose, six feedstuffs (wheat straw, grass hay, corn silage, dried distillers grains with solubles, ground corn, and soybean meal) were incubated in ultrapure water (pH≈6.4) with supplemental Cu concentrations equivalent to 0, 5, 10, 15, 20, 30, and 40 mg Cu/kg DM or supplemental Zn concentrations equivalent to 0, 15, 30, 45, 60, 90, and 120 mg Zn/kg DM for 48 h. This experiment was repeated; however, following the 48-h incubation in ultrapure water (pH≈6.4), samples were incubated for one additional hour at a fitted pH of 2.3. Following incubation, the indigestible residue was analyzed for the presence of Cu and Zn. Copper and zinc bind to other dietary constituents at a pH similar to that of the rumen, regardless of the type of feedstuffs (concentrate or roughage). However, in a pH condition similar to that of the abomasum, part of these bonds is broken. In these pH conditions, there is greater breakdown of bonds in concentrate feedstuffs than in roughages, regardless of the mineral analyzed. Comparing these minerals, zinc forms weaker bonds than copper.