Simple SummaryIn some fish farming practices, cholesterol has been used as a feed additive for growth stimulation. Therefore, it is important to increase our knowledge and experience about dietary cholesterol effects on fish. This study provides useful information for the application of cholesterol in fish diets by analyzing the muscle lipidomics changes in turbot and tiger puffer in response to dietary cholesterol supplementation.AbstractExogenous cholesterol has been supplemented into aqua-feeds due to the reduced proportions of fishmeal and fish oil. This study aimed to investigate the effects of dietary cholesterol supplementation on the muscle lipidomics of two marine fish species, turbot and tiger puffer. A 70-day feeding trial was conducted, where two low-fishmeal diets supplemented with 0 or 1% cholesterol were used. The lipidomic analysis with targeted tandem mass spectrometry showed that, in turbot, a total of 49 individual lipids exhibited significant differences in their abundance in response to dietary cholesterol, whereas the number was 30 for tiger puffer. Dietary cholesterol up-regulated the abundance of cholesterol and cholesterol ester in both species. In turbot, the dietary cholesterol also increased the abundance of triacylglycerol and acylcarnitine, whereas in tiger puffer, it primarily regulated the abundance of phospholipids and BMP. This was the first time the responses of marine fish muscle lipidomics to dietary cholesterol supplementation have been investigated.