Effect on lipid metabolism of vegetable lipid interaction with carbohydrate; a comparative study with two important marine aquaculture species, Gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax)

A sustainable development of aquaculture requires reducing fish meal (FM) and fish oil (FO) use in aquafeeds, by replacing it with eco-friendly ingredients such as vegetable protein and oil sources. Vegetable ingredients are usually rich in carbohydrates (CH) and, contrarily to FO, are abundant in 18-carbon polyunsaturated fatty acids (C18 PUFA) but lack n-3 long-chain polyunsaturated fatty acids (LC-PUFA). These nutritional drawbacks may limit the use of vegetable ingredients in aquafeeds, especially for carnivorous marine fish species, which have specific requirements of n-3 LC-PUFA, as they are not able to efficiently elongate C18 PUFA to n-3 LC-PUFA, and are metabolically adapted to diets almost devoid of CH. For an increased incorporation of vegetable ingredients in aquafeeds at the expense of FM and FO, more knowledge is needed on the effects of dietary vegetable oil (VO) and CH per se, and the possible interactive effects between dietary VO and CH on lipid and CH related metabolic pathways and on fish oxidative status. For this purpose, using as models two important Mediterranean aquaculture species - European seabass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) - the experimental work applied in thesis relied on an integrative approach combining classical zootechnical parameters, plasma metabolites, activity of key enzymes and expression of selected genes involved in key metabolic pathways. In Chapters 2 and 4, diets with FO or a blend of VO as main lipid sources, and with 0 or 20% gelatinized starch as CH source were used to evaluate processes involved in nutrient digestion, absorption and transport in European sea bass and gilthead sea bream juveniles. Moreover, in European sea bass the effects of dietary lipid source in intestinal digestive enzyme activities and in liver and intestine histomorphology were also evaluated (Chapter 3). Afterwards, the effects of the same diets on growth performance, tissue fatty acid (FA) composition, glucose and lipid metabolism (Chapters 5 and 6), and on fish oxidative status (Chapters 7 and 8) were also evaluated for both species. Replacement of FO by VO in the diets slightly but significantly reduced apparent lipid digestibility in both species. However, this decreased lipid digestibility did not induce marked changes in lipid serum profile, liver and intestine transcriptional mechanisms involved in lipid transport and absorption. In European sea bass, no alterations in digestive enzyme activities and in liver and intestine histomorphology were also noticed. In gilthead sea beam, dietary CH inclusion did not affect lipid digestibility but modulated the transcriptional mechanisms involved in lipid absorption and transport at intestinal level. As expected, replacement of FO by VO and inclusion of dietary CH led to small effects on growth performance and feed efficiency in both species. In gilthead sea bream, interactions between dietary CH and lipid source were observed in liver lipids and in muscle glycogen deposition. However, lipid and glycogen deposition in muscle and liver was mainly positively related to the dietary CH content in the two target species. Muscle and liver FA profiles markedly reflected the FA composition of dietary lipids, although dietary starch increased tissue saturated FA content. Dietary CH, but not dietary VO, enhanced liver lipogenic activity in both species and promoted a moderated liver transcriptional regulation of the glycolytic (only up-regulation of glucokinase gene expression) and the gluconeogenic (only down-regulation of phosphoenolpyruvate carboxykinase gene expression) pathways. No increase in liver and muscle n-3 LC-PUFA content were observed in both species, but enhanced hepatic transcript levels of FADS2 - the key enzyme involved LC-PUFA biosynthesis – was observed with dietary VO at 6 hours after feeding in European sea bass and at 18 hours after feeding in gilthead sea bream. In gilthead sea bream fed the dietary CH-rich- diets, enhanced hepatic transcript levels of FADS2 was also observed at 18 hours after feeding. In European sea bass, but not in gilthead sea bream, dietary CH inclusion enhanced plasma cholesterolemia and, at molecular level, the liver cholesterol biosynthesis capacity (HMGCR, the rate-limiting enzyme of cholesteroliosynthesis). In European sea bass dietary VO increased transcript levels of key enzymes involved in cholesterol biosynthesis (HMGCR and CYP51A1) liver but did not affect plasma and liver cholesterol levels; whereas in gilthead sea bream no adaptive response in cholesterol biosynthesis pathway was observed at a molecular level, although it was observed a decrease of plasma cholesterol but not of hepatic cholesterol content. In both species CH-rich diets promoted a reduction of lipid oxidative damage in the liver but not in the intestine. The mechanism by which dietary CH exerted their protective role against oxidative stress in the liver seems to be linked to an increased activity of glucose-6-phosphate dehydrogenase to generate reducing equivalents for the regeneration of reduced glutathione. In gilthead sea bream, dietary VO improved liver and intestine oxidative status, while in European sea bass such effect was only observed at intestinal level. The positive antioxidant effect of dietary VO may be related to the lower degree of unsaturation of the dietary FA and to an enhanced activity of glutathione reductase and glutathione peroxidase. Overall, in this study no substantial interactions between dietary CH and lipid source were observed. However, we provided for the first time evidence of a transcriptional induction of FADS2, a key enzyme of LC-PUFA biosynthesis pathway, by dietary CH, in addition to VO, in gilthead sea bream. This may open new perspectives for the use of nutritional strategies to induce LC-PUFA biosynthesis in marine fish species. We also highlighted important aspects on the regulation of cholesterol biosynthesis pathway and fish oxidative status that are important in the context of VO and CH rich aquafeeds. In addition, species-specific differences in the regulation of cholesterol and LC-PUFA biosynthesis pathways in fish fed CH and VO were also highlighted, and require further elucidation in future studies