Total protein assay kit

Approximately 300 mg of frozen liver samples was placed in a 1:9 (wt/vol) saline solution and then homogenized with a high-speed benchtop homogenizer (Tekmar, Cincinnati, OH, USA). The supernatant was collected by centrifugation and then used for the analysis of superoxide dismutase (SOD), glutathione peroxidase (GPx), and amount of reduced glutathione (GSH) and malondialdehyde (MDA) using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The total protein concentration in the homogenate was measured using a total protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Hepatic mitochondria were extracted using a commercial mitochondrial extraction kit (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) following the manufacturer’s instructions. The activities of the hepatic mitochondrial complex and citrate synthase (CS) and the amount of nicotinamide adenine dinucleotide (NAD⁺), reduce nicotinamide adenine dinucleotide (NADH), and adenosine triphosphate (ATP) were measured with commercial kits (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The total protein concentration in each sample was measured using a total protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The content of total carbohydrate was determined by the phenol-sulfuric acid method using fucose as the standard [64 (link)] since fucose was the most predominant monosaccharide in S. fusiforme polysaccharides [21 (link),35 (link)]. The content of protein was measured using the Total Protein Assay Kit (A045-4, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocol. The amount of reducing sugar was determined by the dinitrosalicylic acid method with glucose as the standard [65 (link)]. The content of uronic acids was measured using the carbazole-sulfuric acid method with glucuronic acid as the reference [66 (link)], since glucuronic acid was the most abundant uronic acid in S. fusiforme polysaccharides [67 (link)]. The content of sulfate was measured by the barium sulfate turbidimetry method using potassium sulfate as the standard [68 (link)].

Roots of wild-type and rtcs were harvested at 12 h, 24 h, 48 h and 96 h after two N treatments. The length of primary root (PRL) and total lateral root on primary root (LRL) were measured with the root scanner (EPSON EXPRESSION) and WinRHIZO Pro 5.0 (Quebec City, Canada). Three replicate pots with three seedlings per pot were selected randomly and sampled. The total protein content and glutamine synthetase (GS) activity were selected as representative indicators of primary nitrogen assimilation in maize roots. There were three replicates with a random design, and each biological replicate constituted a pool of three seedlings. The total protein content and GS activity were measured by total protein assay kit (Nanjing Jiancheng, China) and glutamine synthetase assay kit (Nanjing Jiancheng, China), respectively, following the manufacturer’s protocol. Statistical analyses were performed using SAS statistical software.

PO (R27A7X13777) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Uric acid assay kit (20170316), total protein assay kit (20170321), triglyceride assay kit (20170316), and creatinine kit were purchased from Nanjing Jiancheng Bioengineering Institute (China). HPLC-grad solvents (Formic acid, Isopropanol, acetonitrile) were purchased from Fisher (USA). Chloroform and methanol are all analytical regents.

In this study, the enzymatic activities of pepsin, lipase, and α-amylase in the intestine and stomach were detected using a Pepsin Assay Kit, a Lipase Assay Kit, and an α-Amylase Assay Kit (Nanjing Jiancheng, Bioengineering Institute, China). The enzymatic activities of ACP, AKP, LDH, SOD, CAT, GPT, and GOT in the gill, brain, intestine, stomach, kidney, liver, and plasma were detected using an Acid Phosphatase Assay Kit, an Alkaline Phosphatase Assay Kit, a Lactate Dehydrogenase Assay Kit, a Superoxide Dismutase Assay Kit, a Catalase (CAT) Assay Kit, an Alanine Aminotransferase Assay Kit, and an Aspartate Aminotransferase Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). Total protein was determined with a Total Protein Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). The operation steps were carried out according to the operation guide of the reagent kit, and the operation guide of the corresponding reagent kit can be searched for on the website (http://www.njjcbio.com/, accessed on 8 June 2023). In addition, the calculation formulas for these enzyme activities are detailed in the Supplemental Material.

At 3 days to the end of the experiment, 2 fresh eggs per replicate (8 eggs per treatment) were randomly selected and weighed for the determination of egg yolk characteristics. The yolk ratio was calculated using the formula: yolk weight/egg weight. Thereafter, the yolks from each replicate were mixed and stored at −20°C for subsequent analysis of antioxidant indices. Briefly, the yolk samples (0.4 g) were homogenized in 3.6 ml of ethanol for the determination of malondialdehyde (MDA) and in 1.6 ml of physiological saline for the determination of glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), catalase (CAT), and total superoxide dismutase (T-SOD). Thereafter, the homogenates were centrifuged (at 3,000 rpm for approximately 10 min at 4°C) to obtain the supernatant. Furthermore, the protein concentration in the supernatants was determined using a Total Protein Assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Then, the GSH-Px concentration, T-SOD activity, T-AOC, CAT, and MDA contents were determined using commercial biochemistry assay kits following the instructions provided by the manufacturer (Bioengineering Institute of Nanjing Jiancheng, China).