Effects of alternative dietary lipid sources on lipid metabolism and regulation of food intake in larvae and juvenile senegalese sole (Solea senegalensis)

Abstract

The main objective of this thesis was to evaluate the potential impacts of alternative VO–based feed formulations on the regulation of appetite and food intake in a commercial marine aquaculture species (Senegalese sole) by uncovering some of the possible physiological regulatory mechanisms behind these processes in early developmental, as well as juvenile stages. Effects of dietary lipid level and FA composition on lipid absorption, metabolism and sensing, as well as gastrointestinal and central appetite-related peptides were analyzed, both pre– and post–prandially. Apart from gene expression, supporting morphometric, biometric, biochemical, histological and enzymatic data was also collected. During development of molecular tools, numerous transcripts were identified for several genes, most notably for the anorexigenic neuropeptide cart, for which a total of 7 transcripts—more than has been recorded in any vertebrate to date—were characterized. In juvenile sole, hepatic lipid metabolism was affected more by dietary lipid level than source, especially at fasting, where high dietary lipids promoted catabolic and suppressed anabolic pathways. On the other hand, a lower food intake was also observed in fish fed high lipid levels, but only when the lipid source was FO, which may have indicated a higher satiating effect of FO than similar amounts of VO. This could not be related to basal and post–prandial (6 h) expression of anorexigenic gastrointestinal peptides which was only affected by lipid level and lower in fish fed high–lipid diets. The expression of central appetite–regulating genes in the brain was not affected at basal levels by the diets. However, most central neuropeptides (cart1b, cart4, pomca, cckl, npy, agrp2) responded to feeding and showed changes in expression during the post–prandial phase, with only slight differences in relation to lipid level. A system for hypothalamic sensing of circulating FA was discribed in juveniles, for the first time in a marine fish. Results confirmed its sensitivity to oleic acid, previously established in trout, but also, for the first time in a vertebrate, provided evidence of activation by an n-3 PUFA (ALA). A method for quantifying live prey intake in larval fish was developed for this thesis, using fluorescent microspheres as markers. The method also enabled experiments of prey selectivity, by using different coloured microshperes to label prey from different treatmetns. While results showed that no preferance of diet was exhibited when fish were offered a choice, larvae previously grown on Artemia enriched with cod–liver oil (CLO) ingested more of their respective diet (per g body weight) compared to those fed linseed (LSO), soybean or olive oil–based diets. Larvae fed CLO also showed improved growth and survival, faster onset of metamorphosis and maturation of the intestine. Gene expression profiles of both larvae and post–larvae fed CLO stood out most from the other treatments with decreased transcript levels of genes involved in anabolic pathways, and increased of those related to phospholipid metabolism, lipoprotein secretion, lipid transport and hydrolysis. Fish fed CLO were closely followed by those fed LSO, almost catching up in terms of growth by the end of the experiment, and were closely grouped in terms of gene expression to those fed CLO, corroborating the possitive effects of enhanced phospholipid metabolism and apolipoprotein synthesis on larval performance. Peripheral peptides in larvae and post–larvae generally did not mirror their putative function or explain lower food intake in fish fed the VO diets, but fish fed CLO tended to show the most dissimilar pattern of central appetite–regulating gene expression, compared to the other dietary treatments. While this thesis provides generally more descriptive results, it can serve as a basis for future, mechanistic studies.