Acid citrate dextrose

Whole blood (~40 mL) was collected into EDTA tubes (Becton Dickinson Vacutainer System, Franklin Lakes, NJ) for analysis of respiratory burst activity. Blood collected in Vacutainer tubes containing EDTA was analyzed for leukocyte population within 1 h of collection using a ProCyte Dx hematology analyzer (Idexx Laboratories Inc., Westbrook, ME). Plasma for analysis of metabolites, insulin, and tocopherol concentration was collected after centrifugation at 1,300 × g for 15 min at 4°C and stored in 1.5-mL tubes at -20°C for future analysis. For gene expression analysis, jugular blood [~200 mL; i.e., sampling on d 0 (-Tmix) and d 7 (+Tmix)] was collected from each cow into 15-mL tubes containing 1 mL of acid-citrate dextrose (Thermo Fisher Scientific Inc., Pittsburgh, PA), gently inverted to mix, and placed on ice. Blood samples were processed for PMN isolation within 1 h after collection as described below.

Fourteen primiparous and 7 multiparous (≥2 lactations) Holstein cows in early (n = 10; DIM = 17 ± 3.1; 34.2 kg of milk/d) and mid-lactation (n = 10; DIM = 168 ± 14.8; 37.8 kg of milk/d) were used for this study. All cows were free from clinical signs of disease before sample collection for this study with median composite milk SCC of 107,000 cells/mL. All cows were housed and fed in free stalls, had free access to water, and were milked twice daily at 0600 and 1800 h. At 0730, 1400, and 1730 h, cows were fed ad libitum daily a TMR formulated to provide the NRC ( 2001 Jugular blood (~150 mL) was collected from each cow after the morning milking and before the morning feeding. Blood was collected into vacutainer tubes containing acid-citrate dextrose (Fisherbrand, Thermo Fisher Scientific Inc., Pittsburgh, PA) , inverted to mix and placed on ice. Blood samples were processed within 1 h after collection. In addition, blood (~10 mL) was collected without anticoagulant (Fisherbrand, Thermo Fisher Scientific Inc.) . Blood containing no anticoagulant was allowed to clot at room temperature for ~2 h. Tubes were centrifuged at 2,000 × g for 15 min at 4°C (model 5810R, Eppendorf, Hauppauge, NY) and media were frozen at -20°C for metabolite analysis.