Optimisation of dietary n-3 and n-6 fatty acids for a robust Atlantic salmon (Salmo salar)
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An optimal diet for Atlantic salmon (Salmo salar) should promote a healthy fish that is robust to changes in its environmental conditions and can withstand the handling it will encounter under farming conditions, all while promoting good and rapid growth. The plant ingredients commonly used in aquafeeds do not have an ideal FA composition for salmon. In particular, they are lacking the n-3 fatty acids (FA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are essential nutrients for salmon. Further, they are rich in the n-s6 FA linoleic acid (LA) and the n-9 FA oleic acid (OA), which are not common in the natural diet of salmon. The exact requirement of EPA and DHA for Atlantic salmon is, however, still not known. There are also indications that a higher inclusion of dietary n-6 FA can increase the requirement for EPA and DHA. Many previous trials investigating these nutrients have been short-term, land-based trials where the fish have been shielded from difficult situations. Though such trials can define minimum requirements, the practical requirements need to be determined in a challenging environment. The focus of this thesis has been to investigate the effect of dietary EPA, DHA and n-6 FA on the robustness of Atlantic salmon exposed to challenging environmental conditions. It has been suspected that dietary n-6 can affect the requirement of EPA and DHA, and that it may have an effect on the response to chronic and acute stress. To investigate this, a feeding experiment was conducted with three diets containing equal absolute amounts of n-3 FA and increasing n-6 FA (n-6/n-3 ratios of 1, 2 and 6), as well as a final diet with double absolute n-3 FA content and an n-6/n-3 ratio of 1. This allowed for a separation between effects of ratio and absolute amounts on tissue FA levels. These diets were used in a 13-week growth trial and a 4-week stress trial. In the stress trial, half the fish were exposed to a repeated stressor (hypoxia) three times weekly, while the other half were undisturbed controls. At the end of the experiment, all fish were exposed to a confinement stressor. These trials confirmed that dietary n-6 FA increases the requirement for EPA, but not DHA. Despite equal level of dietary EPA + DHA, an increasing level of dietary n-6 FA (a higher n-6/n-3 ratio) resulted in lower levels of EPA in polar lipids (liver polar lipids, red blood cells, skin phospholipids, brain polar lipids). A higher feed n-6/n-3 ratio increased the level of all n-6 FA (including the longer-chain n-6 FA). However, maintaining a low n-6/n-3 ratio inhibited the increased incorporation of n-6 into polar lipids despite higher dietary absolute levels of n-6 FA. Generally, polar lipids reflected the relative n-6 and n-3 level of the feed, while in neutral lipids the FA composition was more related to the absolute contents. These results indicate a competition between n-3 and n-6 FA for incorporation into polar lipids. When investigating the effect of these diets on the stress response, it was found that all fish appeared phenotypically healthy, and were able to mount an acute stress response. There were hardly any significant effects of the repeated hypoxia stressor, possibly indicating an adaptation. Hepatic production of prostaglandin D2 (PGD2) and leukotriene B4 (LTB4) responded differently to acute stress depending on the feed n-6/n-3 FA ratio, which suggests a dietary impact on the acute stress response. Based on still declining rather than recovering PGD2 levels 24 hours after exposure to the acute stressor, fish fed an n-6/n-3 ratio of 6 recovered more slowly from the stress compared to fish fed a ratio of 1. Furthermore, the n-6/n-3 ratio of 6 resulted in rising levels of LTB4 in the fish liver one hour after acute stress compared to a decline seen in fish fed a ratio of 1. A general increase of the arachidonic acid (ARA) derived prostaglandins PGE2 and PGD2 was further seen in fish fed the high n-6/n-3 ratio. Eicosanoids are highly potent molecules, particularly the ones derived from ARA. Upsetting the balance between their n-3 and n-6 FA precursors, and hence their production, might lead to overly strong responses when salmon are exposed to stressors. A low dietary n-6/n-3 ratio will therefore likely be beneficial for the stress coping ability of salmon. The final study examined how graded levels of EPA and DHA from 10 to 35 g/kg feed affected liver lipid metabolism in salmon kept in open sea cages during a full production cycle. Changes to the hepatic energy metabolism when reducing dietary EPA + DHA were discovered using a metabolomics approach. An inhibition of hepatic β‐oxidation likely occurred in the fish fed the lowest levels of EPA + DHA (10 g/kg), as evidenced by less tricarboxylic acid cycle intermediates originating from β‐oxidation. Additionally, in the low EPA + DHA groups, other pathways providing metabolic energy, such as the pentose phosphate pathway, branched chain amino acid catabolism and creatine metabolism were activated in the liver. Increases in various acylcarnitines in the liver of the same fish, in particular accumulation of 3-hydroxyacrnintines (intermediates in mitochondrial β‐oxidation), supported this and indicated disturbances in the hepatic lipid metabolism. Elevated liver lipids were furthermore observed in fish fed lower levels of EPA and DHA, aligning well with the metabolite data. The study showed that diets containing 10 and 13 g/kg EPA and DHA were insufficient for maintaining good liver metabolic health in Atlantic salmon. However, 35 g/kg dietary EPA and DHA was also significantly better than 16 g/kg, indicating that 16 g/kg might be suboptimal as well.
Består avPaper I: Hundal BK, Liland NS, Rosenlund G, Bou M, Stubhaug I & Sissener NH. (2021a) Increasing dietary n-6 fatty acids while keeping n-3 fatty acids stable decreases EPA in polar lipids of farmed Atlantic salmon (Salmo salar). British Journal of Nutrition, vol. 125, issue 1, pp. 10-25. The article is available in the thesis file. The article is also available at: https://doi.org/10.1017/S0007114520002494
Paper II: Hundal BK, Liland NS, Rosenlund G, Höglund E, Araujo P, Stubhaug I & Sissener NH. (2021b). Increasing the dietary n-6/n-3 ratio alters the hepatic eicosanoid production after acute stress in Atlantic salmon (Salmo salar). Aquaculture, vol. 534, article number 736272. The article is available in the thesis file. The article is also available at: https://doi.org/10.1016/j.aquaculture.2020.736272
Paper III: Hundal BK, Lutfi E, Sigholt T, Rosenlund G, Liland NS, Glencross B, Sissener NH. (2022). A Piece of the Puzzle—Possible mechanisms for why low dietary EPA and DHA cause hepatic lipid accumulation in Atlantic salmon (Salmo salar). Metabolites, 12, issue 2, article number 159. The article is available in the thesis file. The article is also available at: https://doi.org/10.3390/metabo12020159