Blood Sampling and Hormone Measurement in Calves

In all calves, blood was sampled during both vaccination visits and then every 4 weeks until slaughter or until the end of the study (4–9 samples/animal). The blood samples were collected by venipuncture from the jugular vein or the V. caudalis mediana into serum tubes (S-Monovetten 9 mL with Clot Activator, Sarstedt, Nümbrecht, Deutschland). After a clotting time of 1–3 h at room temperature, samples were centrifuged (4000× g, 10 min), and serum was then stored at −18 °C for later analysis. Progesterone concentration was measured by chemiluminescence assay on an Immulite 2000 XPi (Siemens Healthcare, Zürich, Switzerland). The analytical sensitivity was 0.1 ng/mL and the measurement range was 0.2–40 ng/mL. The assay was performed according to the manufacturer’s instructions. Cortisol concentration was measured by chemiluminescence assay on an Immulite 2000 XPi (Siemens Healthcare, Zürich, Switzerland). The analytical sensitivity was 5.5 nmol/L and the measurement range was 0.99–1380 nmol/L. The assay was performed according to the manufacturer’s instructions. If serum cortisol levels were >60 nmol/L [28 (link)] the corresponding serum progesterone value was excluded from further analysis.