Edta plasma tubes

Blood samples were taken by jugular vein puncture from 36 calves seven time points, i.e., 36–48 h after birth, from week 8 until week 16 of life in two-week intervals (week 8, 10, 12, 14, 16), and in week 20 of life. Out of a total of 37 calves, blood samples were taken from only 36, because one calf failed to complete the sampling schedule and was therefore excluded. This calf was neither diseased nor dead. Blood samples were also collected from the respective dams by coccygeal vein puncture 36–48 h after calving. Blood was collected in 10 mL serum and EDTA-plasma tubes (Sarstedt, Nümbrecht, Germany). The EDTA-plasma samples were centrifuged at 4 °C for 20 min at 3000× g, whereas serum tubes were incubated for 45 min at room temperature (RT) for clotting before centrifugation for 10 min at 3000× g at room temperature. Afterwards, serum and plasma were aliquoted and stored at −20 °C until analysis. The total protein content in the first serum samples (36–48 h after birth/post natum (p.n.)) was determined by an optical refractometer (Euromex MicroscopenB.V., Arnhem, Netherlands). Haptoglobin (Hp), leptin and adiponectin in serum were measured using in-house developed ELISAs [22 (link),23 (link),24 (link)]. The mean intra and interassay coefficients of variation (CV) were 9.9% and 12.1% for Hp, 6.7% and 11.3% for leptin, and 9.6% and 11.2% for adiponectin, respectively.

Approximately 9 mL of venous blood was collected into EDTA plasma tubes (Sarstedt Leicester UK). The tubes were centrifuged at 1200×g at 4°C for 30 minutes, and the plasma collected and stored in aliquots at –80°C until analysis. All samples were promptly processed and frozen within 60 minutes of collection.