Saturated sodium chloride

Muscle and liver samples were harvested from cyp17a1-/- and cyp17a1+/+ fish for free fatty acid composition analysis, as described in previous research (Tan et al., 2020 (link); Ubhayasekera et al., 2013 (link)). The samples (50 mg) were mixed with 150 µL of methanol (Darmstadt, Germany), 200 μL of methyl tert-butyl ether (Darmstadt, Germany), and 50 μL of 36% phosphoric acid (Sigma-Aldrich, USA)/water (Millipore, USA), then vortexed for 3 min at 580 ×g, followed by centrifugation at 13 500 ×g for 5 min at 4°C. The collected supernatant (200 μL) was transferred to a new centrifuge tube, evaporated to dryness, reconstituted with 300 μL of a 15% boron trifluoride (RHAWN, China) methanol solution, vortexed for 3 min at 580 ×g, and incubated in an oven at 60°C for 30 min. After cooling to room temperature, 500 μL of n-hexane (Darmstadt, Germany) and 200 μL of saturated sodium chloride (Sigma-Aldrich, USA) solution were added, after which the solution was vortexed for 3 min and centrifuged at 13 500 ×g for 5 min at 4°C, with 100 μL of n-hexane layer solution then taken for subsequent GC-MS analysis using the Agilent 7890B-7000D GC-MS/MS platform (Agilent, USA). Fatty acids were identified by retention times of standard mixtures (Personal Bio, China). Details on GC-EI-MS/MS procedures and assays are provided in the Supplementary Materials.