Blood was collected via the coccygeal vein using lithium–heparin vacutainer tubes (BD Vacutainer, BD and CO., Franklin Lakes, NJ) in the morning before feeding on d 1, 30, 63, 64, 65, 66, 67, 68, and 69 of the study. Tubes were placed on ice and transported to the laboratory. For plasma, tubes were centrifuged at 2,500 × g at 4 °C for 15 min. Samples were then aliquoted into microcentrifuge tubes and stored at −80 °C until analysis. Plasma non-esterified fatty acids (NEFAs), β-hydroxybutyrate (BHB), glucose, total cholesterol, total bilirubin, creatinine, urea, aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), total plasma reactive oxygen metabolites (ROMs), ferric reducing ability of plasma (FRAP), haptoglobin, ceruloplasmin, paraoxonase (PON) activity, and magnesium (Mg) were analyzed as described previously (Trevisi et al., 2013 (link)). Myeloperoxidase (MPO) was measured as described by Bionaz et al. (2007) (link). In addition, blood samples collected on d 1, 30, 64, 66, and 68 were used to analyze monocyte and neutrophil oxidative burst activity and phagocytosis capacity via a flow cytometry-based assay as described previously by Zhou et al. (2018) (link).
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