Six ruminally cannulated steers (345 +/- 20 kg initial BW) were used in a 6 x 6 Latin square to evaluate effects of diet and antibiotics on ruminal protein metabolism. Two diets and three antibiotic treatments were arranged factorially. One diet contained (DM basis) 72% dry-rolled corn, 12% soybean meal, 10% alfalfa hay, and 4% molasses (SBM), and the other contained 63% dry-rolled corn, 30% wet corn gluten feed, and 5% alfalfa hay (WCGF). Antibiotic treatments included control, virginiamycin (175 mg/d; VM), and monensin/tylosin (250 and 100 mg/d, respectively; MT). Steers were fed at 12-h intervals at a rate of 2.4% of empty BW daily. Each period included 18 d of adaptation and 3 d of ruminal fluid collections. Samples were collected at 0, 2, 4, 6, 8, and 10 h after the morning feeding on d 19 and 20. On d 21, rumens were dosed 2 h after the morning feeding with 350 g of solubilized casein to evaluate in vivo ruminal protease and deaminase activities. Ruminal fluid samples were collected 1, 2, 3, 4, and 6 h after the casein dose. On d 19 and 20, antibiotics had no effect on ruminal pH or concentrations of VFA, lactate, ammonia, ciliated protozoa, alpha-amino nitrogen (AAN), or peptide N, but VM reduced (P < 0.01) the concentration of isovalerate compared to MT and control. After casein dosing (d 21), peptide N concentration was unaffected by antibiotics, but AAN were higher (P < 0.01) for VM than MT and control. Relative to MT and control, VM reduced ruminal isovalerate (P = 0.05) and increased ruminal propionate (P < 0.01) on d 21. Ruminal pH was lower (P < 0.01) in steers fed SBM than in steers fed WCGF, but lactate concentrations were unaffected by diet. Steers fed SBM had higher (P < 0.05) ruminal concentrations of total VFA and propionate. Ammonia concentrations were lower before feeding and higher after feeding for steers fed WCGF (P < 0.01). Steers fed WCGF had higher counts of total ciliated protozoa than steers fed SBM (P < 0.05) due to greater Entodinium sp. (P < 0.05). Steers fed WCGF had higher (P < 0.01) ruminal AAN and peptide N concentrations than those fed SBM on d 19 and 20. After casein dosing, ruminal peptide N concentrations were similar, but AAN were lower (P < 0.01) for WCGF than SBM. Overall, VM appeared to depress ruminal deaminase activity, and MT had minimal effects on ruminal fermentation products. The protein in WCGF appeared to be more readily degradable than that in SBM.

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