Colorimetric kit

The total antioxidant capacity (TAOC), total superoxide dismutase (TSOD), reduced glutathione (rGSH), and catalase (CAT) were evaluated in the collected plasma samples following the protocols of colorimetric kits (K136, K019, K030, and K031, respectively) obtained from Elabscience Biotechnology Inc. (Houston, TX, USA). The TAOC assay was performed according to Uwikor et al. [44 (link)]. The method is based on reducing ferric salt reagent by antioxidant enzymes and molecules in the body system, then converting this reaction into a colored product by phenanthroline and measuring it at 520 nm. The TSOD was assayed based on its inhibitory effect on the nitrite formation from hydroxylamine oxidation by the xanthine-xanthine oxidase reaction system. The nitrite could be turned purple by adding a chromogenic reagent and finally measured calorimetrically at 550 nm [45 (link)]. The rGSH was determined indirectly through its reaction with dinitrobenzoic acid (DNBT) and releasing a yellow complex which can be measured by colorimetric assay at 405 nm [46 (link)]. The CAT activity was also assayed based on the principle that CAT decomposes H2O2. Then, the residual H2O2 reacts with ammonium molybdate, forming a yellowish complex that can be measured at 405 nm [47 (link)].

The total antioxidant capacity (TAOC), total superoxide dismutase (TSOD), reduced glutathione (rGSH), and catalase (CAT) were evaluated in the collected plasma samples following the protocols of colorimetric kits (K136, K019, K030, and K031, respectively) obtained from Elabscience Biotechnology Inc. (Houston, TX, USA). The TAOC assay was performed according to Uwikor et al. [44 (link)]. The method is based on reducing ferric salt reagent by antioxidant enzymes and molecules in the body system, then converting this reaction into a colored product by phenanthroline and measuring it at 520 nm. The TSOD was assayed based on its inhibitory effect on the nitrite formation from hydroxylamine oxidation by the xanthine-xanthine oxidase reaction system. The nitrite could be turned purple by adding a chromogenic reagent and finally measured calorimetrically at 550 nm [45 (link)]. The rGSH was determined indirectly through its reaction with dinitrobenzoic acid (DNBT) and releasing a yellow complex which can be measured by colorimetric assay at 405 nm [46 (link)]. The CAT activity was also assayed based on the principle that CAT decomposes H2O2. Then, the residual H2O2 reacts with ammonium molybdate, forming a yellowish complex that can be measured at 405 nm [47 (link)].

At the end of the trial (the 42nd day of age), blood samples were collected from the brachial vein of a bird in each replicate per treatment group (n = 10) and immediately put into tubes containing heparin. Plasma was separated by cooling centrifugation at 2000× g for 10 min and later used to analyze the antioxidant biomarkers. The glutathione reduced (GSH), the superoxide dismutase (SOD), and the malondialdehyde (MDA) reactions were assayed by colorimetric kits (Elabscience Biotechnology Inc., Houston, TX, USA), following a previous study [25 (link)]. The ceruloplasmin (CP) assay was performed using ELISA protocol [36 (link)] with specific kits for chicken (MyBioSource Inc., San Diego, CA, USA).