C9377

As described previously, an animal feeding experiment was conducted (31 (link)). In brief, Fish oil (FO) and soybean oil (SO) diets, as well as soybean oil (SO) diets that contain CDCA (C9377, Sigma) at 300 mg or 900 mg/kg (SO-CDCA 300 or SO-CDCA 900) were developed (31 (link)) (Supplementary Table 1). Randomly, each diet was assigned to a cage (60 fish/cage, 10.03 ± 0.02 g) in triplicate. Fish were fed two times a day at 05:00 and 17:00 until satiation with under appropriate conditions for 10 weeks. After fasting for 24 hours, the fish were anesthetized with eugenol (1:10,000) at the end of the feeding trial. Then, six individuals in one cage were sampled for head kidney tissues and immediately frozen in liquid nitrogen.

The bile acid profiles of the aspirated gastric juice samples were measured using a mass spectrometer API 4000Q TRAP (AB Sciex, Redwood City, CA, U.S.A.). Initially, the gastric juice was diluted 20– 200-fold using distilled water. Then, 100 µL of the diluted gastric juice was mixed with an internal standard solution (CA-d5 ng/mL in 50% methanol). After mixing the solution by vortexing for 3 s, 200 µL acetonitrile was added, and the mixture was centrifuged at 20,000 × g for 2 min. Subsequently, 20 µL of the diluted supernatant, which was diluted with 180 µL of 20 mM ammonium acetate, was injected for analysis. Standard components of Sigma-Aldrich C9377, G0759, C1129, T4009, L6250, D2510 were used to measure the cholic acid (CA), chenodeoxycholic acid (CDCA), taurocholic acid (TCA), glycochenodexoycholic acid (GCDCA), lithocholic acid (LCA), and deoxycholic acid (DCA), respectively (Sigma-Aldrich Co. LLC, St.Louis, USA). LC–MS/MS data for each bile acid were analyzed using Analyst software version 1.6.3 (AB Sciex Pte. Ltd. Redwood City, CA, USA).