Pgo enzyme product no p7119

On d -12 and 12 relative to expected calving date, 10 cows in the control group and 10 in the Met group received a glucose tolerance test (GTT) while restrained in a squeeze chute, approximately 2 h after feeding. A solution of 50% glucose (Dextrose 50%, Agri Laboratories Ltd., St. Joseph, IL) was administrated at 0.25 g/kg of BW with an intravenous set (PBS Animal Health, Massillon, OH) and a disposable 14-gauge × 3.8 cm needle (Tyco Healthcare Group LP, Mansfield, MA) for jugular vein access. Cows were restrained in a squeeze chute and blood samples (20 mL) were obtained by puncture of the coccygeal vein or artery with 20-gauge × 2.5-cm needles (Becton Dickinson and Company, Franklin Lakes, NJ) at -15, -10, -5, 5, 10, 15, 30, 60, and 120 min relative to administration of glucose. Glucose concentration was measured using a glucose oxidase method (PGO Enzyme Product No. P7119; Sigma Chemical Co., St. Louis, MO). Intra-and interassay coefficients of variation were 5.1 and 8.5%, respectively.

Feed and TMR samples were processed and analyzed for DM, NDF, CP, and starch contents. All samples were dried in a 55°C forced-air oven for 72 h and analyzed for DM concentration. Samples were then ground in a Wiley mill (1-mm screen; Arthur H. Thomas Co., Philadelphia, PA) and analyzed for NDF, CP, and starch. Concentration of NDF was determined according to Mertens (2002) . Crude protein was determined according to Hach et al. (1987) . Starch was gelatinized with sodium hydroxide and hydrolyzed using an enzymatic method (Karkalas, 1985) ; glucose was then measured using a glucose oxidase method (PGO Enzyme Product No. P7119; Sigma Chemical Co., St. Louis, MO).
Individual milk samples were analyzed for fat, true protein, and lactose concentration by mid-infrared spectroscopy (AOAC, 1990; method 972.160 ) by the NorthStar Michigan Laboratory (Grand Ledge, MI). Yields of milk components were calculated using milk yield and component concentrations for each milking.
Plasma samples were analyzed in duplicate. Plasma concentration of nonesterified fatty acids (NEFA) was determined using a commercial kit [NEFA-HR (2) kit, Wako Chemicals USA Inc., Richmond, VA]. Plasma glucose concentration was analyzed using the glucose oxidase method (PGO Enzyme P7119, Sigma-Aldrich, St. Louis, MO). Plasma and liver Pi concentrations were analyzed using a commercial kit (MAK030, Sigma-Aldrich).

Samples of TMR and orts were processed and analyzed for DM, NDF, CP, and starch contents as described in Piantoni et al. (2013) . Plasma samples were analyzed in duplicate. Commercial kits were used to determine plasma concentrations of insulin (bovine insulin ELISA, #10-1201-01, Mercodia, Uppsala, Sweden; intraassay CV: 2.6%), NEFA [NEFA-HR (2) kit, Wako Chemicals USA Inc., Richmond, VA; intraassay CV: 2.9%], and betahydroxybutyrate (kit no. 2240, Stanbio Laboratory, Boerne, TX; intraassay CV: 4.8%). Plasma glucose concentration was analyzed using a glucose oxidase method (PGO Enzyme Product No. P7119, Sigma Chemical Co.; intraassay CV: 1.8%). Liver samples were analyzed for AcCoA content as described in Stocks and Allen (2012) . Liver AcCoA content is expressed per weight of wet liver.