The replacement of fish oil (FO) with vegetable oil (VO) in feed formulations reduces 24 the availability of n-3 long-chain polyunsaturated fatty acids (LC-PUFA) to marine fish 25 such as gilthead seabream. The aim of this study was to examine compositional and 26 physiological responses to a dietary gradient of n-3 LC-PUFA. Six isoenergetic and 27 isonitrogenous diets (D1-D6) were fed to seabream, with the added oil being a blend of 28 FO and VO to achieve a dietary gradient of n-3 LC-PUFA. Fish were sampled after four 29 months feeding, to determine biochemical composition, tissue fatty acid concentrations 30 and lipid metabolic gene expression. The results indicated a disturbance to lipid 31 metabolism, with fat in the liver increased and fat deposits in the viscera reduced. 32Tissue fatty acid profiles were altered towards the fatty acid compositions of the diets. 33There was evidence of endogenous modification of dietary PUFA in the liver which 34 correlated with the expression of fatty acid desaturase 2 (fads2). Expression of sterol 35 regulatory element-binding protein 1 (srebp1), fads2 and fatty acid synthase increased 36 in the liver, while peroxisome proliferator-activated receptor alpha 1 pathways appeared 37 to be supressed by dietary VO in a concentration-dependent manner. The effects in 38 lipogenic genes appear to become measurable in D1-D3, which agrees with the weight 39 gain data suggesting that disturbances to energy metabolism and lipogenesis may be 40 related to performance differences. These findings suggested that suppression of beta-41 oxidation and stimulation of srebp1-mediated lipogenesis may play a role in 42 contributing toward steatosis in fish fed n-3 LC-PUFA deficient diets. 43 44 3
Introduction 45Sustainable expansion of aquaculture requires reduction in the use of fishmeal (FM) and 46 fish oil (FO) in aquafeed formulations (1)(2)(3)(4) . Both raw materials, particularly FO, are rich 47 in the two key n-3 (or omega-3) long-chain (≥ C 20 ) polyunsaturated fatty acids (LC-48 PUFA), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 3), recognised as essential fatty acids (EFA) for the majority of marine fish species (5) . 50 DHA is an essential component of neural and retinal membranes (6) and both EPA and 51 DHA are precursors for an extensive range of autocrine signalling molecules (e.g. 52 eicosanoids, resolvins, protectins, etc.) (7) . Dietary deficiency of n-3 LC-PUFA has 53 impacts on the health (8) , metabolism (9, 10) , composition (11, 12) and growth (13) of marine 54 fish. 55Typically, an aquafeed for a given marine fish species contains a combination of 56 FO to supply essential n-3 LC-PUFA and vegetable oils (VO) that, while devoid of LC-57 PUFA, supply dietary energy (14, 15) . Marine fish lack sufficient activity of the LC-PUFA 58 biosynthesis pathway to satisfy requirements (5) . In terms of fatty acid composition, the 59 key effects of high inclusion levels of VO are an increase in C 18 unsaturated fatty acids 60 (α-linolenic acid, linoleic...