Influence of dietary menadione nicotinamide bisulphite (vitamin K3) and phylloquinone (vitamin K1) on Atlantic salmon (Salmo salar L.) tissue levels,determined by high‐performance liquid chromatography with fluorescence detection

The aim of the present study was to investigate the retention of menadione nicotinamide bisulphite (MNB; vitamin K₃) and phylloquinone (vitamin K₁) in Atlantic salmon (Salmo salar L.). Another objective was to find a reliable method for determination of menadione in fish feed, and to include and validate more matrices in the methods for phylloquinone and menaquinones (vitamin K₂). Duplicate tanks of Atlantic salmon (～93 g) were fed four levels (0–1000 mg menadione kg^?1 feed) of MNB for 9 weeks. The concentration of menadione and phylloquinone in the feed and the concentration of phylloquinone and menaquinone‐4 (MK‐4) in the tissues were determined. The analysed concentration of dietary menadione found in feed indicated a substantial loss of MNB during feed production. This assumption was supported by screening 15 commercial fish feed samples which also revealed menadione concentrations far below the recommended level. MNB fed salmon showed only a minor increase in liver MK‐4 concentration, compared to salmon fed phylloquinone which had a considerably higher level of liver phylloquinone, indicating a higher retention of phylloquinone compared to menadione in Atlantic salmon. Due to highly varying stability and bioavailability of the different vitamin K derivatives, vitamin K supplementation in fish feed needs a revision.     

A multivariate study on the effects of dietary vitamin K, vitamin D3 and calcium, and dissolved carbon dioxide on growth, bone minerals, vitamin status and health performance in smolting Atlantic salmon Salmo salar L.

Bone deformities represent an increasing, unsolved problem for intensive farming of Atlantic salmon, Salmo salar L. To increase insight into nutritional and environmental impacts on bone mineralization, 16 groups of Atlantic salmon parr were fed diets with different levels of menadione sodium bisulphite (MSB), vitamin D₃, and calcium (Ca), and exposed to different levels of dissolved carbon dioxide (CO₂) based on a multivariate factorial design (2⁴). The composition of the experimental diets was either a high or low level of each of the design variables. In addition, four groups (centre points) were fed a diet with intermediate levels of all dietary parameters and exposed to an intermediate level of dissolved CO₂ to record the variation among tanks. After 6 weeks in fresh water, fish were marked according to group, transferred to a common net pen in sea water and fed commercial feed for another 11 weeks. High levels of CO₂ decreased fish growth both in fresh water and sea water. No visible bone deformities were recorded at any time. Dry matter, protein and ash compositions of bone and concentrations of Ca, phosphorus (P) and magnesium (Mg) in bone were not significantly affected by any of the design variables. Minor effects were observed in clinical parameters recording health performance, stress and plasma Ca homeostasis. The mean level of vitamin D₃ measured as cholecalciferol in fish organs increased significantly during the freshwater period and reflected dietary vitamin D₃, whereas the level decreased when fish were fed commercial feed in sea water. The level of menaquinone in liver increased significantly in response to high dietary levels of MSB, indicating that dietary MSB was converted to menaquinone in salmon.     

Three different levels of dietary vitamin D3 fed to first-feeding fry of Atlantic salmon (Salmo salar L.): effect on growth, mortality, calcium content and bone formation

Atlantic salmon ( Salmo salar L.) fry were reared on a fishmeal-based diet with three different levels of vitamin D₃ (0.2, 5 and 57 mg vitamin D₃ kg^–1 feed, ww) from first-feeding for 14 weeks. No significant differences were recorded in weight, length, specific growth rate, mortality, or kidney calcium concentration between the different dietary groups. No skeletal malformations or histopathological changes were recorded in any of the dietary groups. These results suggest Atlantic salmon fry to be highly tolerant of megadoses of vitamin D₃ over a limited period of time.     

The effect of dietary vitamin A on the immunocompetence of Atlantic salmon (Salmo salar L.)

Atlantic salmon, Salmo salar L. were maintained on diets containing low (0.37 mg kg^–1 diet), normal (1.95 mg kg^–1 diet) and high (15 mg kg^–1 diet) levels of vitamin A fed at 1.5% body weight per day. After 4 months, liver vitamin A levels reflected dietary intake and growth rates of all three groups were similar. Kidney leucocyte migration and serum bactericidal activity were found to be significantly reduced in fish fed low levels of vitamin A. On the other hand, high levels of vitamin A in the diet were found to augment serum antiprotease activity relative to the levels found in the other dietary groups. However, phagocyte respiratory burst activity, bactericidal activity and eicosanoid production were unaffected by the dietary vitamin A regime, as were lymphocyte functions (lymphokine and antibody production) and both serum lysozyme and classical complement activity. That the overall immunomodulatory effect of vitamin A was small was reflected in the resistance to Aeromonas salmonicida. No significant differences were found between the different vitamin A intake groups despite a trend to decreased resistance in the low vitamin A diet group.     

Hypervitaminosis A in first‐feeding fry of the Atlantic salmon (Salmo salar L.)

Atlantic salmon fry were reared on a fishmeal based diet with increasing levels of vitamin A (VA) (6, 122 and 938 mg retinol kg^–1 dry feed) from startfeeding and for 14 weeks. Signs of VA stress, such as reduced fat stores, liver size and growth, were found for groups receiving 122 and 938 mg retinol kg^–1. Signs of vitamin A toxicity, such as increased mortality, abnormal vertebral growth, and reduced growth, were found for groups receiving 938 mg retinol kg^–1. These results suggest that excess VA in the early life stages of Atlantic salmon is deleterious for normal development.     

Dietary vitamin C,immunity and disease resistance in Atlantic salmon (Salmo salar)

Presmolt Atlantic salmon were fed a fish meal based experimental diet supplemented with graded levels of ascorbate-2-monophosphate (AP), equivalent to 40, 400, 2000 and 4000 mg ascorbic acid (AA)/kg for 6 months prior to a bacterial challenge experiment. The liver AA concentration reflected the dietary intake of AP, but not linearly. Growth, hematology and acid phosphatase activity in zymosan stimulated macrophages were not affected by dietary AP. Serum hemolytic complement activity was higher in fish fed the highest AP level, but the variation was not significant. Production of specific antibodies was significantly higher in fish fed the highest AP level 11 and 17 weeks after vaccination. Bacterial challenge with Aeromonas salmonicida showed increased survival in the 4000 AP group. Lysozyme activity in headkidney and serum complement activity and serum iron in fish surviving the challenge were higher in the 4000 AP group, indicating important roles of vitamin C on lysozyme, complement and iron in non-specific disease resistance. The results indicate that high dietary levels of AP favourably affect health in Atlantic salmon. Disease resistance was, however, not correlated with the AA status in the liver.     

Establishing an upper level of intake for vitamin A in Atlantic salmon (Salmo salar L.) postsmolts

The vitamin A (VA) concentration in salmon aquaculture feeds is varying and may lead to sublethal adverse effects. In this study, 135 g Atlantic salmon postsmolts were given eight diets in duplicates with 6, 12, 26, 55, 82, 112, 360 and 749 mg retinol (ROL) kg⁻¹ for 116 days. Subsequently, fish given 6, 82 and 749 mg ROL kg⁻¹ were transferred to a common net pen and given a standard commercial diet for further 28 weeks. Feed conversion rate, liver functionality and markers of VA homoeostasis were not negatively affected by dietary VA level, but chronic high VA intakes led to adverse effects on growth and bone health. In plasma, there was an antagonistic effect of dietary ROL on circulating 1,25 (OH)₂ vitamin D₃ (calcitriol). Moreover, a dose–response of VA on craniofacial deformities, condition factor and vertebral morphometry and mechanical strength was observed. Vertebral deformities were observed after 28 weeks on a standard diet and not immediately after the 116 days on the experimental diet. Elevated VA is a risk factor for bone deformities, and the dietary intake of VA should not exceed 37 mg ROL kg⁻¹ body weight day⁻¹ in Atlantic salmon postsmolts.     

Determination of the threonine requirement for maintenance in Atlantic salmon (Salmo salar L.) fry with the diet dilution procedure

A study was conducted to evaluate the threonine (Thr) requirement for maintenance in Atlantic salmon using the diet dilution procedure (DDP). Fourteen groups of 60 salmon fry [1.46 g initial body weight (BW)] were fed on seven semi‐purified diets containing graded levels of N (3–64 g kg⁻¹ dry matter) and L‐Thr (1–39 g kg⁻¹ dry matter). Seven doses of Thr represented 1–31% of its ideal level for optimum protein deposition. Indispensable amino acids (AA) other than Thr were included in the same proportion as in the Atlantic salmon fry whole‐body carcass. Protein deposition and Thr accretion were linear functions of Thr intake. At zero Thr intake fry lost 4.4 mg Thr kg BW^−0.75 day⁻¹ and the Thr requirement for maintenance was 5.8 mg kg BW^−0.75 day⁻¹, which represented 11% of the total need for Thr. Increasing doses of Thr did not show any effect on AA concentrations in whole‐body protein, except for cystine. The linear relationship between Thr gain and Thr intake indicates a constant Thr cumulative efficiency (77%) at marginal Thr intake. Finally, our results suggest that (1) both the DDP and the graded supplementation technique can be used in Atlantic salmon fry for the determination of AA maintenance requirement and utilization efficiency, at least for Thr; (2) the dietary protein level has only a minor effect on the Thr maintenance requirement determination.     

Tissue distribution of vitamin D3 in Atlantic salmon Salmo salar: effect of dietary level

Atlantic salmon Salmo salar were given three dietary doses of vitamin D₃ for a period of 11 weeks: diet groups I, II and III received a 0.04, 2.21 and 28.68 μg g⁻¹ diet of vitamin D₃, respectively. The tissue distribution of vitamin D₃ was investigated, and between diet groups II and III, the level of vitamin D₃ found in the tissues was increased by a factor similar to the increased level in the feed. No effects were observed on weight gain, survival, plasma level of calcium, red blood cell count or haematocrit, relative to the dietary levels of vitamin D₃. The results showed that while the plasma concentration of 25(OH)D₃ increased between diet groups II and III, the concentration of 1,25(OH)₂D₃ decreased.     

Effect of the arachidonic acid/vitamin E interaction on the immune response of juvenile Atlantic salmon (Salmo salar) challenged against Piscirickettsia salmonis

Atlantic salmon (Salmo salar) were fed 6 experimental diets containing three levels of arachidonic acid (ARA) (0.18 g kg^?1, 0.28 g kg^?1 and 0.63 g kg^?1 for low, medium and high levels, respectively) and two levels of vitamin E (150 and 730 mg kg^?1 for low and high levels, respectively). At the end of the experimental period, fatty acids in the liver and immunity markers (lysozyme activity, respiratory burst and phagocytic activity) were determined and fish subjected to a challenge test against the salmonid rickettsial syndrome (SRS) pathogen. ARA, vitamin E or their interaction did not exert an effect on fish performance, whereas ARA alone clearly increased the deposition of ARA. Dietary vitamin E only enhanced liver vitamin E deposition, while the interaction of ARA and vitamin E influenced lysozyme activity and EPA/ARA ratio pointing out the effect of both nutrients on the fish immune system and metabolism. Only the medium concentration contributed to reducing mortality when the fish were exposed to the SRS pathogen. In conclusion, different levels of supplementation with ARA and vitamin E in the diet had no effect on productivity, but did have effects on immune markers and cumulative mortality when fish were exposed to the SRS pathogen.     

Growth, feed conversion and chemical composition of Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.) fed diets supplemented with krill or amphipods

The effects of partial replacement of fish meal (FM) with meal made from northern krill (Thysanoessa inermis), Antarctic krill (Euphausia superba) or Arctic amphipod (Themsto libellula) as protein source in the diets for Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.) on growth, feed conversion, macro‐nutrient utilization, muscle chemical composition and fish welfare were studied. Six experimental diets were prepared using a low‐temperature FM diet as control. The other diets included northern krill where 20, 40 or 60% of the dietary FM protein was replaced with protein from northern krill, and two diets where the FM protein was replaced with protein from Antarctic krill or Arctic amphipod at 40% protein replacement level. All diets were iso‐nitrogenous and iso‐caloric. Atlantic salmon grew from 410 g to approximately 1500 g during the 160 day experiment, and Atlantic halibut grew from 345 g to 500–600 g during the 150 day experiment. Inclusion of krill in the diets enhanced specific growth rate in salmon, especially during the first 100 days (P < 0.01), and in a dose–response manner in halibut for over the 150 day feeding period (P < 0.05). Feed conversion ratio did not differ between dietary treatments, and no difference was found in dry matter digestibility, protein digestibility and fish muscle composition. Good growth rates, blood parameters within normal ranges and low mortalities in all experimental treatments indicted that fish health was not affected either Atlantic salmon or Atlantic halibut fed the various zooplankton diets.     

In vitro hydroxylation of vitamin D3 and 25-hydroxy vitamin D3 in tissues of Atlantic salmon Salmo salar, Atlantic mackerel Scomber scombrus, Atlantic halibut Hippoglossus hippoglossus and Atlantic cod Gadus morhua

The in vitro metabolism of ¹⁴CD₃ and ³H25OHD₃ was investigated in different tissues from Atlantic salmon Salmo salar , Atlantic mackerel Scomber scombrus , Atlantic halibut Hippoglossus hippoglossus and Atlantic cod Gadus morhua . The tissues analysed were liver, kidney, head kidney, gills, spleen and intestine. The metabolites were extracted in methanol–chloroform and separated by normal-phase high-pressure liquid chromatography (HPLC) followed by scintillation counting. Identification of the metabolites was by comigration with standards on normal and reversed-phase HPLC systems and by protein-binding assays. All tissues from all species analysed produced hydroxylated derivatives identified as 25OHD₃, 24,25(OH)₂D₃ and 1,25(OH)₂D₃. In addition, some unidentified derivatives were recorded, one probably being 25,26(OH)₂D₃. Organs producing great amounts of one metabolite also produced considerable amounts of the other possible derivatives, suggesting a lower degree of specificity in fish organs than in human organs. The predominating metabolite was 24,25(OH)₂D₃ in all organs from salmon and mackerel during incubation with ¹⁴CD₃ and within most organs from all species during ³H25OHD₃ incubation. The latter observation probably results from the need for decreasing rather than increasing the calcium absorption in these species, which live at least some periods of life in a marine environment.     

Temperature modulates liver lipid accumulation in Atlantic salmon (Salmo salar L.) fed low dietary levels of long‐chain n‐3 fatty acids

Atlantic salmon (Salmo salar) were fed five graded levels of eicosapentaenoic acid (EPA, 20:5n‐3) and docosahexaenoic acid (DHA, 22:6n‐3), from 1.4 to 5.2% of total fatty acids (FA, 5–17 mg kg^?1 feed), and grew from ~160 g to ~3000 g, with the period from 1450 g onwards conducted both at 6 °C and at 12 °C. All fish appeared healthy, and there were no diet‐related differences in haematological or plasma parameters, as well as intestinal histological or gut microbiota analysis. Fish reared at 6 °C had higher accumulation of storage lipids in the liver compared to fish reared at 12 °C. Liver lipids also increased with decreasing dietary EPA + DHA at 6 °C, while there was no such relationship at 12 °C. Gene expression of SREBP1 and 2, LXR, FAS and CPT1 could not explain the differences in liver lipid accumulation. In liver polar lipids, DHA was found to be reduced when dietary EPA + DHA was <2.7% of FAs, while the level of EPA in the membranes was not affected. In conclusion, reducing dietary EPA + DHA from 5.2 to 1.4% of total FAs had a minor impact on fish health. Temperature was the factor that most affected the liver lipid accumulation, but there was also an interaction with dietary components.     

Dietary phosphorus does not reduce the risk for spinal deformities in a model of adjuvant‐induced inflammation in Atlantic salmon (Salmo salar) postsmolts

Inflammation is a non‐specific protective mechanism towards injury known to affect bone remodelling. This study aimed to investigate the effect of Freund’s complete adjuvant (FCA) induced‐inflammation on the prevalence of spinal deformities of Atlantic salmon postsmolts fed with two different dietary P levels. Sextuple groups of salmon postsmolts were fed with either a low‐phosphorous (6 g kg^?1 available P, LP) or a high‐phosphorous (9 g kg^?1 available P) diet for a period of 101 days. On Day 102, individually tagged fish were subjected to (i) single injection with FCA (0.125 mg kg^?1 BW) dissolved in phosphate‐buffered saline (PBS) (ii) placebo injection with PBS or (iii) sham injection (insertion of needle only) or (iv) remained untreated. On Day 103, fish were given a common diet for 174 days in seawater. No significant differences in body weight were observed. Injected fish, particularly the FCA group, had more compressions in the injection site than untreated fish. No effect of diet and no interaction between treatment and diet were observed. Severe scoliosis was observed in ～7% of FCA‐injected individuals, corresponding to a mixture of bone malformations in the tail region. In conclusion, experimentally induced inflammation may be an independent risk factor for bone deformities in Atlantic salmon.     

Effect of gelatinized wheat and maize in diets for large Atlantic salmon (Salmo salar L.) on glycogen retention, plasma glucose and fish health

This publication reports on analytical data from a large-scale experiment, using 3360 Atlantic salmon, Salmo salar L., distributed into 12 sea cages. Salmon were grown from an average 600 g to an average 3.5 kg. Samples for analyses were taken when fish were in the range of 1.8 and 3.5 kg. Dietary changes between groups were increased starch from 24 to 230 g kg^?1 and balanced with protein. The diets were isolipic. All salmon showed small stores of glycogen in all analysed organs, and only in heart, gills and kidney of large fish (3.5 kg) were the levels correlated with dietary starch. Minor differences between groups were found in liver NADPH production, but with substantially decreased NADPH production per g protein as the fish grew from 1.8 to 3.5 kg, indicating that increasing dietary starch did not lead to induction of liver hexokinase, and that the activity of this enzyme may decline as fish size increases. An increase in plasma glucose concentrations was found as dietary starch increased, but all levels were moderate and ranged within reference values. Plasma total protein concentrations did not, however, vary according to decreased dietary protein, but increased substantially in all groups as the fish grew from 1.8 to 3.5 kg. Dietary treatments had no influence on haematological parameters, except for decreased haemoglobin concentrations as dietary starch increased in large fish (3.5 kg). No impared liver function was detected, evaluated by activities of ASAT, ALAT and LDH, and by histological analyses. Low serum lysozyme activities were recorded in all groups, and were not correlated with plasma glucose or liver glycogen concentrations.     

Organs development,gene expression and health of Atlantic salmon (Salmo salar L.) fed genetically modified soybeans compared to the near‐isogenic non‐modified parental line

The present experiment was conducted to study the possible effects of genetically modified (GM), full‐fat soybean meal (FFSBM) from Round‐up Ready^® soybeans compared to its parental, and closest near‐isogenic, non‐modified (nGM) soybean variety, added at moderate (150 g kg^?1) and high (300 g kg^?1) inclusion levels. The fish showed a high specific growth rate (SGR 1.27–1.52), and nearly doubled their body weight (BW), with final weights varying from 1009 to 1110 g. Increased levels of dietary FFSBM, independent of the soy being GM or not, significantly decreased mean values of SGR, thermal growth rate, condition factor, final BW, liver somatic index, lipid efficiency ratio, apparent digestibility coefficients (ADC) of protein and gross energy, liver lipid content and plasma cholesterol, and significantly increased ADC of starch and muscle fatty acid levels of 18:3n‐3, 20:4n‐6, 20:5n‐3 and total n‐3. Increasing dietary GM FFSBM significantly increased feed conversion ratio, and significantly decreased protein efficiency ratio, ADC of lipid and dry matter and plasma triacylglycerol (TAG) levels. Spleen somatic index was significantly larger in fish groups fed GM FFSBM compared to groups fed nGM FFSBM, which might indicate a possible immune response exerted by the GM soybeans. Mean normalized expression of heat shock protein 70 mRNA in distal intestine was significantly up‐regulated while normalized expression of catalase in liver was down‐regulated, in fish fed FFSBM compared to fish fed FM. In conclusion, substituting moderate to high levels of GM Round‐up Ready^® FFSBM in diets for Atlantic salmon and compared to the closest near‐isogenic counterpart available, resulted in many effects independent of the soy being GM or not, but with the notable exceptions of enlarged spleen and lowered plasma TAG.     

High dietary 18:2n‐6/18:3n‐3 ratio does not inhibit elongation and desaturation of 18:3n‐3 to EPA and DHA in Atlantic salmon (Salmo salar L.)

This study investigated whether retention of n‐3 fatty acids (FAs) is influenced by the levels of dietary monounsaturated (MUFA)‐, saturated (SFA)‐ and n‐6 FAs. The feeding trial used a mixture design, with rapeseed‐, palm (PO)‐ and soybean oil (SO), providing high levels of MUFA, SFA and n‐6 FAs, respectively, while 18:3n‐3, eicosapentaenoic acid (20:5n‐3) and docosahexaenoic acid (DHA, 22:6n‐3) were kept constant. Furthermore, a diet group with high SO and reduced 18:3n‐3 was included in addition to the mixture design. There were no differences in growth or proximate composition, but the PO diet gave reduced FA digestibility and increased feed conversion ratio. All diet groups had a net production of DHA, shown by retention values >100% (133%–177%). High dietary n‐6 FAs of up to 43% of FAs (18:2n‐6/18:3n‐3 ratio 8.6) did not negatively affect DHA retention, but rather had a small positive impact. High levels of substrate (dietary 18:3n‐3 at ~10% of FAs) did not enhance DHA retention compared to 5% 18:3n‐3. This is a highly significant finding for the aquaculture industry, allowing for greater flexibility in the choice of lipid sources to replace fish oil in salmon feeds.     

Effects of substitution of dietary fish oil with a blend of vegetable oils on liver and peripheral blood leucocyte fatty acid composition, plasma prostaglandin E2 and immune parameters in three strains of Atlantic salmon (Salmo salar)

Duplicate groups of three genetic strains of Atlantic salmon smolts were cultured on diets containing either fish oil (FO) or a blend of vegetable oils (VO). Fatty acid compositions of liver and peripheral blood leucocytes were compared. The effect of different strains and diets on innate immune parameters and plasma prostaglandin E₂ were also measured. Two strains were selected as being either 'fat' or 'lean' in terms of muscle adiposity. The third strain was a commercial stock (MH). Total replacement of dietary FO with VO resulted in reduced docosahexaenoic (DHA; 22:6 n -3) and eicosapentaenoic acids (EPA; 20:5 n -3) in liver, while oleic (18:1 n -9), linoleic (18:2 n -6) and α-linolenic (18:3 n -3) acids were all increased in VO-fed fish. Fatty acid compositions of blood leucocytes showed similar changes. Evaluation of innate immune function showed that in the fat strain, circulating leucocytes were significantly lower in VO fish. The lean strain also had significantly higher serum lysozyme activity than MH fish. Reduced haematocrit was seen in VO lean fish compared with FO lean fish. This study provides evidence of strain-induced differences in liver and leucocyte fatty acid compositions and innate immunity in Atlantic salmon fed either FO- or VO-based diets.