Simple SummaryEfficiency of nitrogen utilization (ENU) for productive purposes is low (20–30%), partially due to post-absorptive amino acid (AA) catabolism. Catabolism can be reduced by increasing mammary AA removal from the blood, allowing less AA to reach non-mammary tissues. Reducing the AA supply did not decrease milk protein yield and increased ENU, while altering AA profile did not affect ENU. Infusing AA into a low protein diet increased energy corrected milk due to changes in mammary metabolism. Such response seemed to be not only due to substrate effect but also to the regulatory role of the infused AA.AbstractTo evaluate the effects of amount and profile of amino acid (AA) on milk protein yield (MPY), mammary metabolism, and efficiency of nitrogen use (ENU), ten cows were used in 5 × 5 replicated Latin squares and fed a positive control (16.1% crude protein-CP) or two lower CP diets (14.6 and 13.2%) with or without essential AA (EAA) infusion. The EAA solutions provided predicted limiting EAA in each treatment and were continuously infused into the abomasum of the cows. Milk production and MPY were not affected by treatment (mean 35.4 kg/d and 1.03 kg/d, respectively). Efficiency of nitrogen utilization was increased as dietary CP decreased but was not affected by EAA infusion (p< 0.01). Energy-corrected milk production was increased by EAA infusion into 13.2% CP, but not into 14.6% CP diet (p= 0.09), reaching the positive control value. Infusions increased mammary affinity for non-infused EAA (Ile, Phe, Thr, and Trp), allowing the same MPY despite lower arterial concentrations of these AA. Higher arterial concentrations of infused EAA did not increase their mammary uptake and MPY (p= 0.40;p= 0.85). Mammary metabolism did not fully explain changes in N efficiency, suggesting that it might be driven by less extramammary catabolism as AA supply was reduced.