Eumelanin Detection in Melanized Focal Changes but Not in Red Focal Changes on Atlantic Salmon (Salmo salar) Fillets.

Superficial discolored spots on Atlantic salmon (Salmo salar) fillets are a serious quality problem for commercial seafood farming. Previous reports have proposed that the black spots (called melanized focal changes (MFCs)) may be melanin, but no convincing evidence has been reported. In this study, we performed chemical characterization of MFCs and of red pigment (called red focal changes (RFCs)) from salmon fillets using alkaline hydrogen peroxide oxidation and hydroiodic acid hydrolysis. This revealed that the MFCs contain 3,4-dihydroxyphenylalanine (DOPA)-derived eumelanin, whereas the RFCs contain only trace amounts of eumelanin. Therefore, it is probable that the black color of the MFCs can be explained by the presence of eumelanin from accumulated melanomacrophages. For the red pigment, we could not find a significant signature of either eumelanin or pheomelanin; the red color is probably predominantly hemorrhagic in nature. However, we found that the level of pigmentation in RFCs increased together with some melanogenic metabolites. Comparison with a "mimicking experiment", in which a mixture of a salmon homogenate + DOPA was oxidized with tyrosinase, suggested that the RFCs include conjugations of DOPAquinone and/or DOPAchrome with salmon muscle tissue proteins. In short, the results suggest that melanogenic metabolites in MFCs and RFCs derive from different chemical pathways, which would agree with the two different colorations deriving from distinct cellular origins, namely melanomacrophages and red blood cells, respectively.

Differentiation and traffic of IgM+ B cells between focal dark spots in skeletal muscle of Atlantic salmon, lymphoid and adipose tissues.

Focal dark spots (DS) in farmed Atlantic salmon fillets contain a significant number of B cells as revealed by the high abundance of immunoglobulin (Ig) transcripts in transcriptome data. The immune response in DS remains unknown while they represent a major problem in commercial aquaculture. Here, we characterized the diversity and clonal composition of B cells in DS. Sixteen gene markers of immune cells and antigen presentation were analyzed with RT-qPCR. All genes expression showed a positive correlation with DS area and intensity. The flatter the DS, the higher the expression of cd28, csfr, ctla, igt, and sigm, the lower expression of cd83 and btla, and the larger the cumulative frequency within DS. The expression of most of the analyzed immune genes, including three Ig types and markers of B cells was lower in DS than in the lymphatic organs, head kidney and spleen, but significantly higher compared to skeletal muscle. High levels of ctla4 and cd28 in DS might indicate the recruitment of T cells. Sequencing of IgM repertoire (Ig-seq) assessed migration of B cells by co-occurrence of identical CDR3 sequences in different tissues. The combination of gene expression and Ig-seq revealed the presence of several stages of B cell differentiation in DS. B cells at the earliest stage, with high ratio of membrane to secretory IgM (migm and sigm), showed minor Ig repertoire overlap with other tissues. Further differentiation stage (increased sigm to migm ratio and high expression of pax5 and cd79) was associated with active movement of B cells from DS towards lymphatic organs and visceral fat. Traffic and expression of immune genes decreased at later stages. These B cells could be involved in a response directed against viruses, pathogenic or opportunistic bacteria in DS. Seven of eight fish were positive for salmon alphavirus, and levels were higher in DS than in unstained muscle. PCR with universal primers to the 16S rRNA gene did not detect bacteria in DS. Although the evolution of DS most likely implies local exposure to antigens, neither this nor previous studies have found a necessary association between DS and pathogens or self-antigens.

Increasing dietary levels of the n-3 long-chain PUFA, EPA and DHA, improves the growth, welfare, robustness and fillet quality of Atlantic salmon in sea cages.

The present study evaluated the effects of increasing the dietary levels of EPA and DHA in Atlantic salmon (Salmo salar) reared in sea cages, in terms of growth performance, welfare, robustness and overall quality. Fish with an average starting weight of 275 g were fed one of four different diets containing 10, 13, 16 and 35 g/kg of EPA and DHA (designated as 1·0, 1·3, 1·6 and 3·5 % EPA and DHA) until they reached approximately 5 kg. The 3·5 % EPA and DHA diet showed a significantly beneficial effect on growth performance and fillet quality compared with all other diets, particularly the 1 % EPA and DHA diet. Fish fed the diet containing 3·5 % EPA and DHA showed 400-600 g higher final weights, improved internal organ health scores and external welfare indicators, better fillet quality in terms of higher visual colour score and lower occurrence of dark spots and higher EPA and DHA content in tissues at the end of the feeding trial. Moreover, fish fed the 3·5 % EPA and DHA diet showed lower mortality during a naturally occurring cardiomyopathy syndrome outbreak, although this did not reach statistical significance. Altogether, our findings emphasise the importance of dietary EPA and DHA to maintain good growth, robustness, welfare and fillet quality of Atlantic salmon reared in sea cages.

Dietary inclusion of Antarctic krill meal during the finishing feed period improves health and fillet quality of Atlantic salmon (Salmo salar L.).

There is an urgent need to find alternative feed resources that can further substitute fishmeal in Atlantic salmon diets without compromising health and food quality, in particular during the finishing feeding period when the feed demand is highest and flesh quality effects are most significant. This study investigates efficacy of substituting a isoprotein (35 %) and isolipid (35 %) low fishmeal diet (FM, 15 %) with Antarctic krill meal (KM, 12 %) during 3 months with growing finishing 2·3 kg salmon (quadruplicate sea cages/diet). Final body weight (3·9 (se 0·04) kg) was similar in the dietary groups, but the KM group had more voluminous body shape, leaner hearts and improved fillet integrity, firmness and colour. Ectopic epithelial cells and focal Ca deposits in intestine were only detected in the FM group. Transcriptome profiling by microarray of livers showed dietary effects on several immune genes, and a panel of structural genes were up-regulated in the KM group, including cadherin and connexin. Up-regulation of genes encoding myosin heavy chain proteins was the main finding in skeletal muscle. Morphology examination by scanning electron microscopy and secondary structure by Fourier transform IR spectroscopy revealed more ordered and stable collagen architecture of the KM group. NEFA composition of skeletal muscle indicated altered metabolism of n-3, n-6 and SFA of the KM group. The results demonstrated that improved health and meat quality in Atlantic salmon fed krill meal were associated with up-regulation of immune genes, proteins defining muscle properties and genes involved in cell contacts and adhesion, altered fatty acid metabolism and fat deposition, and improved gut health and collagen structure.

Increased dietary protein-to-lipid ratio improves survival during naturally occurring pancreas disease in Atlantic salmon, Salmo salar L.

This study demonstrated that increased dietary protein-to-lipid ratio (P/L-ratio) improved survival of farmed Atlantic salmon naturally affected by pancreas disease (PD). In addition to diet, body weight (BW) and delousing mortality prior to the PD outbreak also contributed significantly (p < 0.05) to explain the observed variation in PD-associated mortality. Subsequent to the PD outbreak, large amount of fish failed to grow and caused thin fish with poor condition (runts). At the end of the trial, significantly (p < 0.05) lower amounts of runt fish and increased amount of superior graded fish where detected among fish fed increased P/L-ratio and within the fish with the largest BWs prior to PD. Diet, BW and delousing mortality contributed significantly (p < 0.05) to explain the variation in the amount of superior graded fish, whereas BW and diet explained the variation in the amount of runt fish. A significant (p < 0.01) negative linear relationship was observed between the amount of superior graded fish and the total mortality, whereas a positive linear relationship was detected between percentage of fillets with melanin and the total mortality. Thus, increased dietary P/L-ratio seem to reduce the mortality and impaired slaughter quality associated with PD.

Gene expression profiling in melanised sites of Atlantic salmon fillets.

Black spots, which deteriorate quality of Atlantic salmon fillets represent a significant problem for commercial aquaculture. These areas are characterized with accumulation of melanomacrophages, occasional formation of granulomas and substitution of skeletal muscle with connective tissue. A number of possible causative agents have been suggested including vaccination and infection with piscine reovirus (PRV). We report transcriptome profiling of melanised foci with oligonucleotide DNA microarrays. Analyses revealed a multitude of differentially expressed genes associated with melanogenesis, metabolic changes and formation of scar. The immune profile was characterized with inflammation, preferential activation of classical complement pathway, MHCII and helper T cells combined with strong B cells responses and massive induction of immunoglobulins; innate antiviral responses were relatively weak in sharp contrast to PRV-caused heart and skeletal muscle inflammation and other viral infections. A panel of immune genes with specific activation in dark spots was found, most up-regulated were CD209-like lectin (44-fold) and prostaglandin reductase (11-fold). Further, RNA sequencing was performed on the same material to search for the presence of putative pathogens. Transcripts of prokaryotic rRNA with exclusive or preferential location in black spots were found. Results suggest mild chronic inflammation initiated with trauma, bacterial or viral infection followed by sustained immune responses to opportunistic microorganisms as a realistic scenario of dark spots formation.

Effect of sodium bicarbonate and varying concentrations of sodium chloride in brine on the liquid retention of fish (Pollachius virens L.) muscle.

BACKGROUND: Negative health effects associated with excessive sodium (Na) intake have increased the demand for tasty low-Na products (<2% NaCl) rather than traditional heavily salted fish products (∼20% NaCl). This study investigates the causes of improved yield and liquid retention of fish muscle brined with a combination of salt (NaCl) and sodium bicarbonate (NaHCO3 ). RESULTS: Water characteristics and microstructure of saithe (Pollachius virens L.) muscle brined in solutions of NaCl and NaHCO3 or NaCl alone were compared using low-field nuclear magnetic resonance (LF-NMR) T2 relaxometry, microscopy, salt content, liquid retention and colorimetric measurements. Saithe muscle was brined for 92 h in 0, 30, 60, 120 or 240 g kg(-1) NaCl or the respective solutions with added 7.5 g kg(-1) NaHCO3 . NaHCO3 inclusion improved the yield in solutions ranging from 0 to 120 g kg(-1) NaCl, with the most pronounced effect being observed at 30 g kg(-1) NaCl. The changes in yield were reflected in water mobility, with significantly shorter T2 relaxation times in all corresponding brine concentrations. Salt-dependent microstructural changes were revealed by light microscopy, where NaHCO3 supplementation resulted in greater intracellular space at 30 and 60 g kg(-1) NaCl. CONCLUSION: Sodium bicarbonate addition to low-salt solutions can improve yield and flesh quality of fish muscle owing to altered water mobility and wider space between the muscle cells.

Metabolism, health and fillet nutritional quality in Atlantic salmon (Salmo salar) fed diets containing n-3-rich microalgae.

Microalgae, as primary producers of EPA and DHA, are among the most prominent alternative sources to fish oil for n-3 long-chain PUFA in animal and human nutrition. The present study aimed to assess technical, nutritional and fish health aspects of producing n-3-rich Atlantic salmon (Salmo salar) fish fillets by dietary supplementation of increasing levels of a DHA-producing Schizochytrium sp. and reduced or without use of supplemental fish oil. Atlantic salmon smolt were fed diets with graded levels of microalgae for 12 weeks, during which all fish showed high feed intake rates with postprandial plasma leptin levels inversely correlating with final mean fish body weights. Fish performance was optimal in all experimental treatments (thermal growth coefficient about 4·0 and feed conversion ratio 0·8-0·9), protein digestibility was equal in all diets, whereas dietary lipid digestibility inversely correlated with the dietary levels of the SFA 16 : 0. Fillet quality was good and similar to the control in all treatments in terms of n-3 long-chain PUFA content, gaping, texture and liquid losses during thawing. Histological fluorescence staining and immunofluorescence analysis of salmon intestines (midgut: base of intestine and villi) revealed significant effects on slime, goblet cell production and inducible nitric oxide synthase (iNOS) activity with increasing levels of dietary Schizochytrium sp. supplementation. Microarray analysis did not reveal any signs of toxicity, stress, inflammation or any other negative effects from Schizochytrium sp. supplementation in diets for Atlantic salmon.

Soft texture of atlantic salmon fillets is associated with glycogen accumulation.

Atlantic salmon (Salmo salar L.) with soft fillets are not suited for manufacturing high quality products. Therefore fillets with insufficient firmness are downgraded, leading to severe economic losses to the farming and processing industries. In the current study, morphological characteristics of salmon fillets ranging from soft to hard were analysed. Different microscopic techniques were applied, including novel methods in this field of research: morphometric image analysis, periodic acid Schiff staining, immunofluorescence microscopy, transmission electron microscopy and fourier transform infrared microscopy. The results showed that the myocytes of soft muscle had detached cells with mitochondrial dysfunctions, large glycogen aggregates and enlarged inter cellular areas, void of extracellular matrix proteins, including lower amounts of sulfated glycoproteins. Myofibre-myofibre detachment and disappearance of the endomysium in soft muscles coincided with deterioration of important connective tissue constituents such as Collagen type I (Col I), Perlecan and Aggrecan. In summary our investigations show for the first time an association between soft flesh of Atlantic salmon and massive intracellular glycogen accumulation coinciding with degenerated mitochondria, myocyte detachment and altered extracellular matrix protein distribution. The results are important for further understanding the etiology of soft salmon.

Gene expression profiling of soft and firm Atlantic salmon fillet.

Texture of salmon fillets is an important quality trait for consumer acceptance as well as for the suitability for processing. In the present work we measured fillet firmness in a population of farmed Atlantic salmon with known pedigree and investigated the relationship between this trait and gene expression. Transcriptomic analyses performed with a 21 K oligonucleotide microarray revealed strong correlations between firmness and a large number of genes. Highly similar expression profiles were observed in several functional groups. Positive regression was found between firmness and genes encoding proteasome components (41 genes) and mitochondrial proteins (129 genes), proteins involved in stress responses (12 genes), and lipid metabolism (30 genes). Coefficients of determination (R(2)) were in the range of 0.64-0.74. A weaker though highly significant negative regression was seen in sugar metabolism (26 genes, R(2) = 0.66) and myofiber proteins (42 genes, R(2) = 0.54). Among individual genes that showed a strong association with firmness, there were extracellular matrix proteins (negative correlation), immune genes, and intracellular proteases (positive correlation). Several genes can be regarded as candidate markers of flesh quality (coiled-coil transcriptional coactivator b, AMP deaminase 3, and oligopeptide transporter 15) though their functional roles are unclear. To conclude, fillet firmness of Atlantic salmon depends largely on metabolic properties of the skeletal muscle; where aerobic metabolism using lipids as fuel, and the rapid removal of damaged proteins, appear to play a major role.

Pigment-producing granulomatous myopathy in Atlantic salmon: a novel inflammatory response.

Melanin comprises a complex group of pigmented polymers whose primary function is ascribed to dermal solar protection, but may also have an interesting role in innate immunity. In ectothermic vertebrates, melanogenesis is reported in leukocyte populations, but it is not known if this occurs in connection with inflammatory reactions. Melanin accumulations in ectopic locations, in particular muscle, represent a serious quality problem in salmon production. Here, we investigated such changes for the expression of dopachrome tautomerase and tyrosinase as well as some important immune genes and pathogens. Furthermore, the nature of the pathological changes was addressed by morphological methods. Gene transcripts encoding key enzymes in melanogenesis, suggesting a de novo melanin synthesis in pigmented muscle, were found. MHC class II transcripts were up-regulated and there was no indication of bacterial or viral infection. The histological examination revealed granulomatous inflammation with distribution of MHC class II positive cells and T cells, analogous to the pattern found in mammals. Importantly, in contrast to mammals pigmented cells were contributing in the inflammation. We demonstrate that melanin production occurs in granulomatous inflammation in salmon, revealing a close and hitherto unreported link between the pigmentary and immune systems.

The effect of crowding stress on bacterial growth and sensory properties of chilled Atlantic salmon fillets.

UNLABELLED: Atlantic salmon were subjected to minimal preslaughter crowding stress (Control), short-term crowding for 20 min (SS-group), or long-term crowding for 24 h (LS-group). The fish were filleted prerigor, cut into 270 g pieces, and packaged in modified atmosphere (60% CO2 and 40% N2). Fillet quality analyses were determined during 22 d of storage at 0.3 °C. Bacterial growth and unpleasant sensory properties increased earlier in the LS-group. The negative effects of long-term preslaughter stress were more pronounced for raw than cooked samples, and more pronounced for odor than flavor. Sequence analyses of bacterial DNA at the end of storage revealed that 100% of the bacteria were comprised by Photobacterium phosphoreum of the SS- and LS-group, whereas the Control group also contained 21% of Carnobacterium maltaromaticum (lactic acid bacteria, LAB). Counting of LAB, using Man-Rogosa-Sharke agar, similarly showed higher numbers of the Control group after 15 d of storage. A total bacterial count of log 6 CFU/g was observed after 15 d of storage of the LS-group, which was 3 and 7 d earlier compared with the Control and SS-group, respectively. Fillet color, texture, and liquid losses were not negatively affected by preslaughter crowding stress. From the sensory and bacterial analyses, it is concluded that long-term crowding stress accelerates bacterial growth and development of unpleasant sensory properties, hence reduces the shelf life of prerigor modified atmosphere packaged (MAP) salmon. PRACTICAL APPLICATION: Stressful handling of Atlantic salmon before slaughter resulted in faster reduction of fresh taste and smell, faster bacterial growth, and hence shorter shelf life. The deteriorative effects were more pronounced of raw compared to cooked salmon. Therefore, salmon should be handled carefully in connection with slaughter to avoid impaired welfare and fillet quality, in particularly for fish that is consumed raw, such as sushi.

Berry marinades enhance oxidative stability of herring fillets.

Marinating herring fillets in a 50 g/L powder of elderberry, cranberry, or black currant inhibited the oxidation of lipids and proteins and also the degradation of tocopherol. Cranberry and black currant appeared to be more efficient than elderberry in inhibiting the degradation of tocopherol and the formation of ammonium. Elderberry marinades provided the most significant color changes. The injection of fillets with a 5% salt solution resulted in significantly increased levels of carbonyls, ammonium, and biogenic amines, whereas formation of the volatile lipid compounds propanal, hexanal, 2-penten-1-ol, and 1-penten-3-ol was lowest in fillets marinated in black currant following injection of the salt solution. All marinade treatments resulted in a significantly decreased liquid holding ability, coinciding with a lower muscle pH. It is concluded that marinating herring fillets in solutions containing berry powder can enhance the quality and shelf life of the fillets and simultaneously provide the fillets with natural antioxidants beneficial for consumers.

Quality changes of prerigor filleted Atlantic salmon (Salmo salar L.) packaged in modified atmosphere using CO2 emitter, traditional MAP, and vacuum.

Pieces of prerigor salmon fillets were packaged in modified atmosphere (60% CO(2) and 40% N(2)) and in vacuum. The MA packages had a gas to product volume ratio (g/p ratio) of 3/1 (traditional MAP) and 1/1 (packaged with a CO(2) emitter). All the samples were stored at 1.2 degrees C for 25 d. The MA packages had lower bacterial growth during storage compared to vacuum packages. The analyses of 16S rRNA at day 22 indicated a similar bacterial diversity, independent of packaging methods, dominated by Photobacterium phosphoreum. The results therefore suggest that CO(2) inhibited total bacterial count, including, P. phosphoreum. Negative odors and liquid losses were detected earlier for the vacuum-packaged samples (8 d) compared to the MA samples (15 d) and higher levels were detected at the end of the storage period. The breaking strength (firmness) tended to be lower for the MA packaged samples compared with the vacuum samples after 15 d of storage, whereas the redness (a* value) and the yellowness (b* value) were significantly higher for the MA samples. In conclusion, MA packaging preserved the quality better during storage than vacuum packaging. MA packaging with a CO(2) emitter and reduced g/p ratio gave similar or better results compared with traditional MAP, thus CO(2) emitters are well suited for reduction of volume of MA packaged farmed salmon fillet pieces.