A015 2 1

Contents of moisture, crude protein, IMF, ash, and inosinic acid in LT were measured by freeze-dryer (ALPHA 2–4 LSC, Christ Martin GmbH), automatic nitrogen analyzer (8400, FOSS, Denmark), fat analyzer (2055 SOXTEC, FOSS, Denmark), box resistance furnace (SX2-4-10N, Yiheng, Shanghai, China), and high performance liquid chromatography (HPLC, LC-20AD, Shimazu, Japan), respectively, as described previously (16 (link)). Briefly, the contents of moisture, crude protein, IMF, and ash were analyzed by the freeze-drying method, Kjeldahl method, Soxhlet extraction, and burning method, respectively.
The sample preparation procedure comprised homogenization of LT (0.1 g) and 0.9% saline (0.9 mL), and 1,500 r/min for 2 min, followed by centrifugation at 3,500 × g for 15 min at 4°C to collect the supernatant. Then, contents of cholesterol, triglyceride, and malondialdehyde (MDA) and activities of total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) were determined according to the instructions (F001-1-1, F002-1-1, A003-2-2, A015-1-2, A005-1-2, A001-1-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The plate was read by a multi-functional enzyme labeling instrument (Spectra Max M5, Molecular Devices, USA) at 532 nm (MDA), 520 nm (T-AOC), 412 nm (GSH-Px), and 450 nm (SOD).

The total Se concentrations in the feeds and in the fresh tissues were measured by means of a hydride generation atomic fluorescence spectrometer (AF-610B; Beijing Rayleigh Analytical Instrument Corporation) [26 (link)]. The pectoral muscle and jejunum activities of the total antioxidant capacity (T-AOC) as well as the glutathione peroxidase (GPX) and lipopolysaccharide (LPS), malondialdehyde (MDA) and protein carbonyl (PC) concentrations were measured by means of a colorimetric method, using specific assay kits (H255, A015-1-2, A005, A006-1, A061-1, A003 and A087-1-2) from the Nanjing Jiancheng Bioengineering Institute of China. The thioredoxin reductase (TXNRD) activity was measured by considering the NADPH-dependent reduction of 5,5-dithiobis-(2-nitrobenzoic acid), using a specific assay kit (BW11) from the Suzhou Comin Biotechnology Co., Ltd. of China [26 (link)]. The protein concentrations were analyzed through a bicinchoninic acid assay [26 (link)].

Total antioxidant capacity (T-AOC), glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) activities and malondialdehyde (MDA) and protein carbonyl (PC) concentrations were determined by their specific assay kits (A015-1-2, A005-1-2, A007-1-1, A001-3-2, A003-1-2, and A087-1-2) from the Nanjing Jiancheng Bioengineering Institute of China. Protein concentration was analyzed by the bicinchoninic acid assay [19 (link)]. Jejunal chyme lipase, α-amylase, and neutral protease activities were measured by their specific assay kits (A054-1, C016-1, A080-3-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) [20 (link)]. Small intestinal histopathology and morphology were analyzed as previously described [18 (link)]. Briefly, the duodenum, jejunum, and ileum tissues were washed with saline, fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm, and then stained with hematoxylin and eosin. All slides were examined by a microscope.