This experiment is designed to pinpoint where in the metabolic network there are differences between salmon of different genetic families and on different diets. Analyses of this material will help inform feeding and breeding strategies.
Salmon will be reared on feeds with contrasting levels of very-long-chain polyunsaturated fatty acids. Then some fish will be crossed over to the other diet while others remain as controls. This perturbation of diet should provoke changes in omega-3 metabolism that are evident in RNA expression, protein expression and eventually in the fatty acid composition of tissues. We also hypothesize that gut microbiota may be affected and/or modulate the effect of the feed switch.
Activities: ● Generate two fish groups that differ genetically and in EPA/DHA filet content for feeding trial ● Carry out feeding trial to promote differences in FA metabolism and EPA/DHA phenotype ● Supply downstream WPs with tissue samples for RNA sequencing and proteomics with known FA phenotype. Rationale: A dietary switch experiment in fresh- and seawater will provide data for a rich phenotypic characterization of the metabolic steady states under different diets. Although not directly relevant for aquaculture, we have chosen to include a diet switch at the parr stage (in fresh water) for two reasons: Firstly, to test whether restriction in feed fish oil content early in life affects FA metabolism at later life stages, with implications for aquacultural feeding strategies. Secondly, EPA/DHA synthesis is more active in the freshwater phase, ensuring high contrast between the diet groups. The outcome of WP2 (the effects of diet, genetics, and gene-by-environment interactions) will form the fundamental basis for systems biology analyses in WP5 and represent a valuable metabolic reference database for future studies. Operationalization: To ensure informative contrasts in genetics and EPA/DHA-related metabolic patterns, we will rear two genetically distinct groups of fish on contrasting diets in a crossover study design. AquaGen will select parent groups with high vs low filet EPA/DHA from ongoing studies, creating two groups of approx. 500 offspring each. Half of each group will be reared together in one of two fish tanks, being fed on a low-fish-oil or a standard diet, respectively. When parr are large enough to visually tag, fish of each group will cross over (i.e. be moved) to the other tank and diet. This perturbation will bring into play regulatory mechanisms of metabolism, provoking the genetic variation into revealing itself through phenotypic variation in the synthesis, transport and EPA/DHA retention in muscle. After 2-3 weeks of metabolic acclimatization on the new feed, the transferred fish will be sacrificed, as will an identical number of fish that have not crossed over in diet. Liver and filet samples will be sampled for phenotyping by RNA sequencing and filet fatty acid profiling by gas chromatography (GC). The GC analyses will be performed with an added standard to be able to estimate absolute FA levels. A subset of samples with highly distinct FA metabolism inferred from gene expression and lipid measurements will be selected for proteomic analyses using a shotgun method at the proteomics core facility at NMBU (https://www.nmbu.no/en/faculty/kbm/research/ms_proteomics). The above described experiment will be repeated in seawater after smoltification, when the FA metabolism resembles that of slaughter sized fish which is of relevance to the industry and nutritional quality. The gut microbiota samples for both seawater and freshwater samples were mostly taken by Yang (except a few by Jacob and Tom). The sampling procedure involved squeezing out the gut content by using tweezers.
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Created at: 20th Aug 2021 at 11:03
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Created: 20th Aug 2021 at 11:03
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