De protein Crude lipid Crude Ash Gross power (MJ/Kg)aHP ten.00 18.00 18.00 five.00 39.80 four.00 four.ten 1.00 0.ten 100HPBAs 10.00 18.00 18.00 5.00 39.74 four.00 four.10 1.00 0.ten 100a8.24 30.47 7.53 7.25 18.9.11 29.15 7.47 7.31 18.Fish meal: Shandong Chishan Fishmeal Factory, Shandong, China; Soybean meal: Yihai Kerry Investment Co. Ltd., China; CPC: Xinjiang Jinlan Plant Protein Co. Ltd., China. b Nitrocefin Description Vitamin premix (mg g-1 diets): Vitamin A 28; Vitamin B1 12; Vitamin B212; Vitamin B6 16; Vitamin B12 0.2; Vitamin E 300; Vitamin K3 20, Vitamin D 14; Niacinamide 80; Vitamin C 600; Calciumpantothenate 100; Biotin 0.four; Folicacid 3; Corn protein powder 314.four. Mineral premix (mg g-1 diets): FeSO4 2 O 300; ZnSO4 2O 300; MnSO4 2O one hundred; Na2 SeO3 ten; CoCl2 H2 O (ten Co) two.five; Kl 80; Zeolite 1307.five; MgSO4 500. c Bile acids: supplied by Shandong Longchang Animal Overall health Care Co. Ltd. (Dezhou, China), with 8.0 HCA, 70.9 HDCA, and 20.two CDCA. Bile acids were added and well mixed in premix at levels of 0 and 600 mg/kg, respectively.2.3. Plasma Biochemical Parameters Plasma ALT (alanine aminotransferase), AST (aspartate aminotransferase), glucose, and total cholesterol (TC) had been measured by Reagent kits (Nanjing Jiancheng Co., Nanjing, China) following the provided protocols. 2.4. Histopathological Detections of Hepatopancreas Tissues The hepatopancreas tissue fixation, dehydration, embedding, hematoxylin and eosin (H E) and periodic acid Schiff (PAS) staining procedures were performed as described by Yu et al. [32]. Then, the images were visualized using TissueGnostics Fluorescence Calphostin C Biological Activity Imaging Technique (TissueGnostics, Vienna, Austria) and the glycogen granules and efficient nucleus analyzed by the StrataQuest Analysis Software (TissueGnostics, Vienna, Austria). BAs have been extracted and analyzed for the corresponding plasma and bile samples with obvious hepatopancreas damage observed in HP group and no clear abnormalities in the hepatopancreas observed in HPBAs group. The graph abstract is shown in Figure 1. two.five. Bile Acids Quantitative Evaluation Plasma and bile samples had been ready following the earlier report [33]. The eluted substances of UHPLC-TQqQ-MS/MS have been ionized in an electrospray ionization supply in negative mode (ESI-). Each temperatures of ESI- source drying gas and sheath gas had been 300 C. The flow price of ESI- source drying gas and sheath gas have been five and 11 L/min, respectively. The pressure of your nebulizer was 45 psi, and capillary voltage was 4000 V. The dynamic various reaction monitoring (dMRM) was utilized to acquire information in optimized MRM transition (precursor – product), fragment, and collision power (CE) as Table 1. The total scan time per cycle was 300 ms. Chromatographic separation was operated on a UPLC BEH C18 column (one hundred mm 2.1 mm, 1.7). The column temperature was 40 C, and also the flow rate was 0.45 mL/min. The mobile phase consisted of water in 0.1Foods 2021, 10,5 offormic acid (A) and acetonitrile in 0.1 formic acid (B). The chromatographic separation was conducted by a gradient elution plan as follows: 0.five min, 15 B; 1 min, 25 B; 3min, 25 B; 5 min, 34 B; eight min, 40 B; 9 min, 52 B; 10.two min, 58 B; 10.21 min, Foods 2021, ten, x FOR PEER Evaluation 100 B; 11.2 min, 100 B; 11.21 min, 15 B; 12.5 min, 15 B. The gradient elution five of 17 was applied and MS detection proceeded in adverse mode. Standards for all BAs were utilized to determine the distinctive BA metabolites detected by UHPLC-MS/MS. The Agilent Mass Hunter application (version B.08.00)analyzed for the corresponding.