Protein quantification kit

Total protein concentration was determined using a protein quantification kit (Jiancheng, Nanjing, China). The protein concentration, total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), glutathione (GSH), and catalase (CAT) activities or concentrations were measured according to the manufacturer's instruction (Jiancheng, Nanjing, China).

All sampling and growth performance measuring procedures used were similar to those previously described for exp. 1.
Serum glucose, cholesterol, low-density lipoprotein, high-density lipoprotein, urea nitrogen, phosphorus and calcium levels as well as aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activities were assayed using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the catalog numbers of which were F006-1-1, A111-1-1, A113-1-1, A112-1-1, C013-2-1, C006-1-1, C004-2-1, C010-2-1, C009-1-1, and A059-2-2, respectively. The jejunal digesta samples, were weighed, and then broken with an ultrasonic cell disruptor with the addition of certain saline in the ice bath. After centrifugation, the supernatant protein concentration was assayed using a protein quantification kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) as the protein standard, and the catalog number of which was A045-2-2. Subsequently, activities of trypsin and chymotrypsin in the supernatant solution were analyzed using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instruction, and the catalog numbers of which were A080-2-2 and A080-3-1, respectively.

Approximately 1 g of frozen SF, AF, and liver samples were respectively weighed and homogenized with nine times the volume (wt/vol) of pre-cooled physiological saline. The mixture was centrifuged at 4,000×g for 10 min at 4°C, to collect the supernatant solution. The supernatant protein concentration was assayed using a protein quantification kit (Nanjing Jiancheng Bioengineering Institute, China) as the protein standard. Subsequently, in the supernatant solution, activities of LPL in SF and AF as well as fatty acid synthetase (FAS), malic enzyme (MLE), and glucose-6-phosphate dehydrogenase (G6PDH) in liver were analyzed using commercial kits (Nanjing Jiancheng Bioengineering Institute, China) combined with a UV-VIS Spectrophotometer (UV1100; MAPADA, Shanghai, China) according to the manufacturer’s instructions. All measurements were conducted in triplicate at minimum according to the manufacturer’s instructions.