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Effects of the investigational methane (CH₄) inhibitor 3-nitrooxypropanol (3-NOP) on animal performance, health and enteric CH₄ production of beef cattle were evaluated in a commercial feedlot. Two concurrent studies were conducted: a large pen study (4,048 cattle, eight pen replicates per experimental group) to measure animal performance and health and a small pen study (a subset of 50 cattle from the large pen study, n = 25 per experimental group) to measure enteric CH₄ emissions. Within the study, animals (body weight ± SD, 282 ± 8 kg) were assigned in a completely randomized design to one of two groups: control, fed a backgrounding diet (70% corn or barley silage, 30% steam-flaked barley grain concentrate; dry matter (DM) basis) and 3-NOP, fed the backgrounding diet containing 3-NOP. The treatment group in the large pen study was adapted to 3-NOP (12 ± 3 d) before receiving the final target level of 200 mg/kg of DM, which was fed for 108 ± 8 d. Animals in the small pen CH₄ emissions study received a basal diet or a basal diet with 3-NOP, with the dose increased every 28 d: low (150 mg/kg DM; 1.27 g/d), medium (175 mg/kg DM; 2.25 g/d), and high (200 mg/kg DM; 2.75 g/d). Intake in the small pens was monitored by electronic feeding bunks and CH₄ was measured using the GreenFeed system. In the large pen study, total weight gained, average daily gain, and animal health variables were not affected by 3-NOP, but DM intake (DMI) tended to decrease (P = 0.06) by 2.6% relative to control (8.07 kg/d), while gain:feed ratio tended to be improved (P = 0.06) by 2.5% relative to control (0.161 kg weight gain/kg DMI). In the small …