Optimisation of dietary n-3 and n-6 fatty acids for a robust Atlantic salmon (Salmo salar)

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.Doktorgradsavhandlin

Increasing the dietary n-6/n-3 ratio alters the hepatic eicosanoid production after acute stress in Atlantic salmon (Salmo salar)

Earlier studies have indicated that a high inclusion of n-6 fatty acids (FA) in feeds for Atlantic salmon can affect the stress response. To test this hypothesis, Atlantic salmon (Salmo salar) were fed diets containing varying dietary n-6/n-3 FA ratios and different absolute levels of n-6 and n-3 FAs. The fish were divided into two different stress challenge groups, where one group was exposed to three weekly hypoxia challenges for 4 weeks (repeated stress), while one group was left undisturbed. At the end of the experiment, both groups were exposed to an acute stressor (lowering of water level). Thus, effects of the diets on acute stress, repeated stress and the combined effect of these could be investigated. In general, there were few effects of the repeated stress, while fish in all diet groups responded strongly to the acute stress based on several stress markers. Dietary n-6/n-3 ratio did not affect growth, all fish appeared phenotypically healthy, and all groups were able to mount an acute stress response. However, there was an interaction between diet and repeated stress on cortisol response after acute stress, possibly indicating altered hypothalamic-pituitary adrenal axis reactivity in fish fed high n-6/n-3 FA ratio. Hepatic levels of prostaglandin D2 (PGD2) and leukotriene B4 responded differently to acute stress depending on the dietary n-6/n3 FA ratio, indicating an altered acute stress response. Additionally, increasing the dietary n-6/n-3 FA content led to higher levels of PGD2 and PGE2 as well as higher liver triacylglycerol. In summary, the results suggest that increasing the dietary n-6/n-3 FA ratio in salmon feeds can affect the way they respond to stressors in an aquaculture setting, possibly affecting the fish robustness.publishedVersio

Optimisation of dietary n-3 and n-6 fatty acids for a robust Atlantic salmon (Salmo salar)

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

The impact of micro-mineral sources and their availability on hepatic lipid metabolism in Atlantic salmon (Salmo salar)

The increased use of plant-based ingredients in aquafeeds for Atlantic salmon has led to an increase in phytate, an antinutrient binding micro minerals and reducing their bioavailability. It has been suggested that the chemical form of the minerals (organic/inorganic) can alter their bioavailability, especially in feeds with high phytate content. The functional role of minerals in hepatic intermediary metabolism is poorly understood in fish, though studies have shown that dietary mineral levels can affect hepatic lipid metabolism. However, this effect has not been examined in the nutritionally relevant context of dietary mineral availability in plant ingredient based diets. The aim of this study was to investigate whether availability and chemical form of zinc, selenium and manganese affected liver lipid metabolism of Atlantic salmon. A feeding trial involving five different diets was performed. The two control diets contained inorganic Zn, Se and Mn with different phytate contents. Unfortunately, the difference in phytate turned out to be too small to have any effect on the mineral digestibility. The three other diets all had the higher phytate content and in each diet one of the inorganic minerals Zn, Se and Mn were exchanged with chelate of Zn, selenium methionine or chelate of Mn, respectively. The mineral content of the liver was investigated to see if there had been any changes to the mineral status. No significant differences were found. Genes involved in β-oxidation (PPARα, CPT1), lipogenesis (LXR, SREBP1, FAS), bioconversion into LC-PUFA (Δ5Fad, Δ6Fad) and transport out of the liver (ApoB100) were examined to see if there were any effects on hepatic lipid metabolism. There were no significant effects on LXR, FAS, PPARα, CPT1, Δ5Fad or Δ6Fad. ApoB100 and SREBP1 were significantly reduced in the higher phytate control group compared to the lower phytate control. However, these two groups had the same chemical form of all the minerals, no impact of phytate on mineral digestibility was detected and there was similar hepatic content of all three examined minerals. Thus, these effects are probably random effects rather than induced by the diets. As the minerals and phytate likely were not the cause of differences between the two control diets, the organic Mn is probably not the cause of the difference between the higher phytate control diet and the diet containing organic Mn either. Finally, a lipid class distribution analysis was also performed, but no significant effects of minerals on the hepatic lipid composition was discovered. Thus it was concluded that the chemical form of the micro-minerals Zn, Se and Mn probably have no effect on the hepatic lipid metabolism of Atlantic salmon

Increasing dietary n-6 FA while keeping n-3 FA stable decreases EPA in polar lipids of farmed Atlantic salmon (Salmo salar)

There is an increased use of vegetable oils containing n-6 fatty acids (FA) in aquafeeds, and several trials indicate that there might be an increased requirement of EPA and DHA for Atlantic salmon when they are fed higher dietary n-6 FA. With a limited supply of EPA and DHA for production of aquafeeds, it is important to know how to efficiently use these FA to maintain growth and health of the fish. In the present trial, three diets containing equal amounts of n-3 FA (about 7·7 % of total FA) and different n-6:n-3 FA ratios (about 1, 2 and 6), as well as one diet with n-6:n-3 FA ratio at about 1 but twice as much n-3 FA, were fed to Atlantic salmon. Despite constant dietary n-3, increasing dietary n-6 led to significantly reduced n-3 in tissue polar lipids. Interestingly, EPA was significantly reduced while DHA was not. Maintaining a stable n-3 content in the polar lipids when increasing dietary n-6 FA was only obtained by simultaneously increasing the dietary n-3 content and with this maintaining the same n-6:n-3 FA ratio. Polar lipid n-6 FA in tissues thus primarily reflected the dietary n-6:n-3 FA ratio and not the absolute dietary n-6 FA content. Neutral lipids, on the other hand, reflected the dietary absolute levels of both n-3 and n-6 FA. This study indicates that a better use of dietary EPA is achieved by keeping the dietary n-6:n-3 FA ratio low

Increasing dietary n-6 FA while keeping n-3 FA stable decreases EPA in polar lipids of farmed Atlantic salmon (Salmo salar)

There is an increased use of vegetable oils containing n-6 fatty acids (FA) in aquafeeds, and several trials indicate that there might be an increased requirement of EPA and DHA for Atlantic salmon when they are fed higher dietary n-6 FA. With a limited supply of EPA and DHA for production of aquafeeds, it is important to know how to efficiently use these FA to maintain growth and health of the fish. In the present trial, three diets containing equal amounts of n-3 FA (about 7·7 % of total FA) and different n-6:n-3 FA ratios (about 1, 2 and 6), as well as one diet with n-6:n-3 FA ratio at about 1 but twice as much n-3 FA, were fed to Atlantic salmon. Despite constant dietary n-3, increasing dietary n-6 led to significantly reduced n-3 in tissue polar lipids. Interestingly, EPA was significantly reduced while DHA was not. Maintaining a stable n-3 content in the polar lipids when increasing dietary n-6 FA was only obtained by simultaneously increasing the dietary n-3 content and with this maintaining the same n-6:n-3 FA ratio. Polar lipid n-6 FA in tissues thus primarily reflected the dietary n-6:n-3 FA ratio and not the absolute dietary n-6 FA content. Neutral lipids, on the other hand, reflected the dietary absolute levels of both n-3 and n-6 FA. This study indicates that a better use of dietary EPA is achieved by keeping the dietary n-6:n-3 FA ratio low.publishedVersio

A Piece of the Puzzle—Possible Mechanisms for Why Low Dietary EPA and DHA Cause Hepatic Lipid Accumulation in Atlantic Salmon (Salmo salar)

The present study aimed at elucidating the effects of graded levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the hepatic metabolic health of Atlantic salmon reared in sea cages. Diets containing 10, 13, 16 and 35 g/kg EPA + DHA (designated diets 1.0, 1.3, 1.6 and 3.5, respectively) were fed in triplicate through a full production cycle from an average starting weight of 275 g to slaughter size (~5 kg). Feeding low dietary EPA + DHA altered the hepatic energy metabolism, evidenced by reductions in tricarboxylic acid cycle intermediates originating from β-oxidation, which was compensated by elevated activity in alternative energy pathways (pentose phosphate pathway, branched chain amino acid catabolism and creatine metabolism). Increases in various acylcarnitines in the liver supported this and indicates issues with lipid metabolism (mitochondrial β-oxidation). Problems using lipids for energy in the lower EPA + DHA groups line up well with observed increases in liver lipids in these fish. It also aligns with the growth data, where fish fed the highest EPA + DHA grew better than the other groups. The study showed that diets 1.0 and 1.3 were insufficient for maintaining good liver metabolic health. However, diet 3.5 was significantly better than diet 1.6, indicating that diet 1.6 might also be suboptimal