The sample preparation was carried out as previously described (32 (link), 47 (link)). Individual livers from each fish were surgically removed and quickly rinsed with cold saline buffer to remove blood. Then, cold methanol was added at 800 μl/100 mg. The whole liver was homogenized in methanol, followed by sonication for 5 min at a 10-W power setting. A total of 30 C. carassius accommodated at 26°C were used in this experiment (n = 10 for each group). The liver from individual C. carassius was treated as one biological sample. Samples were then centrifuged to remove unresolved matter at 12,000 × g, 4°C for 10 min. The supernatant was collected, and 10 μl 0.1 mg/ml ribitol (Sigma-Aldrich, St. Louis, MO, USA) was added as an internal standard. Afterward, the aqueous sample was concentrated in a rotary vacuum centrifuge device (Labconco, Kansas City, MO, USA) for 4 h, and the resultant dried extracts were used for gas chromatography–mass spectrometry (GC-MS) analysis.
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