At the end of the study period (12th week), 24 birds [six birds from each group (1 per replicate)] were selected for sample collection after 12 h fasting. About 5 ml of blood were collected from the jugular veins, then kept in a micro-anticoagulant tube in slanting position for 30 min, centrifuged at 1,500 x g for 15 min (Tang et al., 2017 (link)). The harvested plasma was transferred to Eppendorf tubes (1.5 ml) and stored at –20°C until analysis. The whole blood samples were put together in an ice pack and transported to the laboratory for hematology analysis within 1 h of collection. For hematological indices analysis, an automated hematology analyzer (Model: BC-2800 Vet, Mindray, Shenzhen, China) was used. Red blood cell indices, MCH, MCV and mean cell hemoglobin concentration (MCHC), were calculated (Jain, 1993 ).
Before analysis of biochemical indices, the serum was thawed at 4°C and kept at low temperature during the whole process in order to avoid activation of enzymes. Determination of glutathione peroxidase (GST), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT), and malondialdehyde (MDA) were achieved using an assay kits from ML Bio and Jiancheng Bioengineering Institute (Nanjing, China), and measured spectrophotometrically (Shimadzu, model UV-1800, Tokyo, Japan). Serum concentrations of immunoglobulins A (IgA), immunoglobulins G (IgG), immunoglobulins M (IgM) and complement proteins; C3 and C4 were determined with the corresponding chicken ELISA kits. All standards were tested in duplicate and concentrations of IgA, IgG, IgM, C3, and C4 were determined using standard curves constructed from the standards run on the plate. All the ELISA kits adopted in the study are of high specificity and sensitivity for chickens, and all procedures were done according to the manufacturer’s instructions.
Before analysis of biochemical indices, the serum was thawed at 4°C and kept at low temperature during the whole process in order to avoid activation of enzymes. Determination of glutathione peroxidase (GST), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT), and malondialdehyde (MDA) were achieved using an assay kits from ML Bio and Jiancheng Bioengineering Institute (Nanjing, China), and measured spectrophotometrically (Shimadzu, model UV-1800, Tokyo, Japan). Serum concentrations of immunoglobulins A (IgA), immunoglobulins G (IgG), immunoglobulins M (IgM) and complement proteins; C3 and C4 were determined with the corresponding chicken ELISA kits. All standards were tested in duplicate and concentrations of IgA, IgG, IgM, C3, and C4 were determined using standard curves constructed from the standards run on the plate. All the ELISA kits adopted in the study are of high specificity and sensitivity for chickens, and all procedures were done according to the manufacturer’s instructions.
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