Automatic aa analyzer

Blood samples from coccygeal artery and the subcutaneous abdominal vein were collected from each cow for two consecutive days at approximately 07:00, 15:00, and 22:00. Blood was immediately put on ice until centrifugation (3,000×g) at 4°C for 15 min), and the plasma was stored at −20°C for later analysis. The pooled plasma was analyzed for AA by previously described methods [14 (link)]. Briefly, an aliquot of 1 mL of plasma was deproteinized with 10% sulfosalicylic acid (1:1, plasma to 10% sulfosalicylic acid). Samples were then centrifuged at 10,000×g at 4°C for 15 min. The supernatant was filtered through 0.45 μm and 0.22 μm nylon syringe filter units (Fisher Scientific, Pittsburgh, PA, USA) and placed in microcentrifuge tubes (Fisher Scientific, USA). Before analysis, milk was hydrolyzed by adding 6 N HCl and incubating at 110°C for 24 h [11 ]. The AA concentrations of plasma and milk were analyzed using an automatic AA analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan).

The plasma AA concentration was analyzed with norleucine as an internal standard in an Automatic AA Analyzer (Hitachi High-technologies Corporation, Tokyo, Japan). To measure the plasma glucose levels, plasma was pretreated with ice-cold sulfosalicylic acid (50 g/L) at a ratio of 1:4 (v/v) to precipitate protein^{24 (link)}, followed by centrifugation at 8,320 × g for 30 min at 4 °C. The supernatant was filtered through 0.45-μm and 0.22-μm nylon syringe filter units (Fisher Scientific, Pittsburgh, PA) and placed in microcentrifuge tubes (catalog no. 05-664-34, Fisher Scientific). The plasma glucose concentrations were subsequently measured using an Auto Analyzer 7020 instrument (Hitachi High-technologies) and commercial colorimetric kit (DiaSys Diagnostics Systems GmbH, Frankfurt, Germany)^{25 (link)}.