Amino acid supply is an important determinant of milk production. In addition, insulin is a hormone that indirectly regulates protein synthesis. It has been previously observed that hyperinsulinemia stimulates amino acid utilization. The aim of this study was to investigate mammary gland efficiency (MGE) of use of essential amino acids (EAA) in response to dietary protein and hyperinsulinemia. Six lactating Holstein (590 kg BW; 33.0 kg milk/day) cows were randomly assigned in a 2 × 2 factorial arrangement crossover design. The diet was formulated based on NRC requirements for milking cows to be protein sufficient (17.5%; SCP) or deficient (14.0%; DCP). The diets were composed of corn silage, mixed grass and legume silage, and a concentrate mix. Milk samples were collected daily. Arterial and venous blood were sampled via catheters once an hour for 8 h without infused insulin (NI), and again after 4 d of an intravenous infusion of 1 µg of insulin/kg of BW per h plus varying glucose to achieve hyperinsulinemic-euglycemic status (HI). Plasma was prepared by centrifugation, and the supernatant analyzed for EAA concentrations by isotope dilution. MGE was calculated as: Milk_EAA_(g/d)/(AV_EAA_(g/L) × Plasma Flow_(L/d)). Statistical analyses were performed using R Studio. Milk yield output was not affected by dietary protein and insulin. There was an interaction between dietary CP and insulin for MGE of Leu, Lys, and Phe. Hyperinsulinemia decreased the efficiency of use of Thr, but did not affect others. MGE for Ile and Val tended to increase and for Met and Thr tended to decrease with decreasing dietary CP. Table 1 (Abstr. 118). Amino acid net uptake in the mammary gland

	NI			HI		SE	P-value
SCP	DCP	SCP	DCP	CP	Insulin	CP × I
Milk yield, kg/8 h	11.4	11.7		12.7	10.9	1.11	0.22	0.66	0.06
Essential AA, g/g
Arg	0.20	0.20		0.19	0.27	0.04	0.44	0.60	0.41
Ile	0.34	0.36		0.32	0.33	0.03	0.12	0.59	0.27
Leu	0.41a	0.40a		0.22b	0.49a	0.06	<0.01	0.22	<0.01
Lys	0.65b	0.51bc		0.41c	0.89a	0.11	0.06	0.37	<0.01
Met	1.02	0.95		1.14	1.02	0.08	0.17	0.17	0.66
Phe	0.56a	0.50ab		0.39b	0.58a	0.06	0.26	0.42	0.03
Thr	0.63a	0.44a		0.32b	0.18b	0.07	0.09	<0.01	0.78
Val	0.27	0.32		0.25	0.40	0.08	0.12	0.62	0.48