Control serum 2

Blood samples obtained from jugular venipuncture were collected in 10 ml Vacutainer tubes without anticoagulant. Sera were obtained by centrifugation at 1,500 g for 10 min and transferred to new tubes to be stored in aliquots at −80°C until further analysis. At the day of analysis, aliquots were thawed. Measurement of serum clinical biochemistry analytes was performed on the Olympus AU400 analyser except cortisol and TNF-alpha. The protocols for the use of Olympus System Reagents (OSRs) and other commercial reagents were in reference of previous studies (16 (link), 17 (link)). Quality control protocols were based on the daily quantification of two control sera of low and high concentration (Control serum I and Control serum II, Beckman Coulter). For cortisol and TNF-alpha in piglets, two commercial ELISA kits were used: Cortisol ELISA Kit (DRG, Marburg, Germany) and Porcine TNF-alpha Quantikine ELISA Kit (R&D Systems, Abingdon, UK). The analytes, methods, reagents, and coefficients of variation (CV%) are shown in Table S3.

Serum biochemistry analytes were analysed with the Olympus AU400 analyser with following techniques and OSR reagents (Olympus System Reagent)^{35 (link),36 (link)}: glucose (HK method), urea (GLDH method), creatinine (Jaffé method), cholesterol (CHOP-PAP-method), triglyceride (GPO-PAP method), total protein (Biuret method), ALT (International Federation of Clinical Chemistry (IFCC) method), AST (IFCC method) and GGT (IFCC method). NEFAs were determined with NEFA-C reagent (Wako Chemicals GmbH, Neuss, Germany). Quality control protocols were based on the daily quantification of two control sera of low and high concentration (Control serum I and Control serum II, Beckman Coulter) Haptoglobin was determined by a colorimetric method (Tridelta, Ireland). Insulin and IGF-I were determined by ELISA (Bovine Insulin ELISA from Mercodia, Uppsala, Sweden and Human IGF-I ELISA E20 from Mediagnost, Reutlingen, Germany). This reagent is suitable for bovine samples, as declared by the manufacturer, but the cross-reactivity was not assessed by the authors.
Intra-assay Coefficients of Variation (CV) were: glucose (1.7%), urea (2.1%), creatinine (1.4%), cholesterol (1.1%), triglycerides (3.0%), total protein (0.9), ALT (1.7%), AST (1.0%), GGT (0.6%), NEFAs (2.7%), Haptoglobin (4.1%), insulin (4.1%) and IGF-1 (4.4%).