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Regarding docosahexaenoic acid (DHA), currently the regulating effects were mostly evaluated in female fish rather than males. This study aimed to investigate the DHA effects on the sex steroid hormone production in male Chinese tongue sole. Three experimental diets were prepared, containing different DHA levels: a control (C) group (5.16% DHA of total fatty acids [TFA]), a low DHA group (DHA\u2010L, 8.93%), and a high DHA group (DHA\u2010H, 16.47%). A 58\u2010day feeding experiment was conducted, and each diet was fed to triplicate tanks of fish. Additionally, an in vitro study with Leydig\u2019s cells of this species was conducted to validate the in vivo results. The concentration of testosterone (T) and 11\u2010ketotestosterone (11\u2010KT) in the serum increased with increasing levels of dietary DHA. Dietary DHA significantly upregulated the expression of steroid hormone biosynthetic genes\n                    <jats:italic>p450c17<\/jats:italic>\n                    ,\n                    <jats:italic>hsd17b1<\/jats:italic>\n                    ,\n                    <jats:italic>hsd3b1<\/jats:italic>\n                    ,\n                    <jats:italic>aromatase<\/jats:italic>\n                    ,\n                    <jats:italic>hsd11b2<\/jats:italic>\n                    , and\n                    <jats:italic>p45011b<\/jats:italic>\n                    in the testis. The protein expression of Hsd17b1 in the testis of the DHA\u2010H group was significantly higher compared to the other two groups, while the expression level of P450c17 showed an increasing trend with increasing dietary DHA levels. However, the in vitro results confirmed that the final concentration of DHA at 50 \u03bcmol\/L could significantly increase the gene expression of\n                    <jats:italic>p450c17<\/jats:italic>\n                    in Leydig\u2019s cells. In conclusion, dietary DHA may promote the synthesis of T and 11\u2010KT through the regulation of protein (Hsd17b1 and P450c17) and gene (\n                    <jats:italic>p450c17<\/jats:italic>\n                    ,\n                    <jats:italic>hsd17b1<\/jats:italic>\n                    ,\n                    <jats:italic>hsd3b1<\/jats:italic>\n                    ,\n                    <jats:italic>aromatase<\/jats:italic>\n                    ,\n                    <jats:italic>hsd11b2<\/jats:italic>\n                    , and\n                    <jats:italic>p45011b<\/jats:italic>\n                    ) expression of a series of key steroid hormone biosynthetic enzymes in male Chinese tongue sole.\n                  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