Fatty acid isotopic composition in Atlantic pollock is not influenced by environmentally relevant dietary fat concentrations.

The application of fatty acid (FA) isotopic analysis has great potential in elucidating food web structure, but it has not experienced the same wide-spread use as amino acid isotopic analyses. The failure to adopt FA isotopic methods is almost certainly linked to a lack of reliable information on trophic fractionation of FA, particularly in higher predators. In this work, we attempt to address this shortfall, through comparison of FA δ13C values in captive Atlantic pollock (Pollachius virens) liver and their known diets. Since catabolism is likely the main cause of fractionation and it may vary with dietary fat content, we investigated the impact of dietary fat concentration on isotopic discrimination in FA. We fed Atlantic pollock three formulated diets with similar FA isotopic compositions but different fat concentrations (5-9% of diet), representative of the range found in natural prey, for 20 weeks. At the conclusion of the study, δ13C values of liver FA were very similar to the FA within the corresponding diets, with most discrimination factors < 1. For all FA except 22:6n-3, dietary fat had no effect on discrimination factors. Only for 22:6n-3 did fish fed the highest fat diet have lower δ13C values than the diet consumed. Thus, these FA-specific discrimination factors can be applied to evaluate diets in marine fish consuming natural diets and will serve as additional and valuable biomarkers in fish feeding ecology.

Correction: Lall, S.P.; Kaushik, S.J. Nutrition and Metabolism of Minerals in Fish. Animals 2021, 11, 2711.

The authors found some omissions and errors in the original paper [...].

Nutrition and Metabolism of Minerals in Fish.

Aquatic animals have unique physiological mechanisms to absorb and retain minerals from their diets and water. Research and development in the area of mineral nutrition of farmed fish and crustaceans have been relatively slow and major gaps exist in the knowledge of trace element requirements, physiological functions and bioavailability from feed ingredients. Quantitative dietary requirements have been reported for three macroelements (calcium, phosphorus and magnesium) and six trace minerals (zinc, iron, copper, manganese, iodine and selenium) for selected fish species. Mineral deficiency signs in fish include reduced bone mineralization, anorexia, lens cataracts (zinc), skeletal deformities (phosphorus, magnesium, zinc), fin erosion (copper, zinc), nephrocalcinosis (magnesium deficiency, selenium toxicity), thyroid hyperplasia (iodine), muscular dystrophy (selenium) and hypochromic microcytic anemia (iron). An excessive intake of minerals from either diet or gill uptake causes toxicity and therefore a fine balance between mineral deficiency and toxicity is vital for aquatic organisms to maintain their homeostasis, either through increased absorption or excretion. Release of minerals from uneaten or undigested feed and from urinary excretion can cause eutrophication of natural waters, which requires additional consideration in feed formulation. The current knowledge in mineral nutrition of fish is briefly reviewed.

Molecular Mechanism Involved in Carotenoid Metabolism in Post-Smolt Atlantic Salmon: Astaxanthin Metabolism During Flesh Pigmentation and Its Antioxidant Properties.

A better understanding of carotenoid dynamics (transport, absorption, metabolism, and deposition) is essential to develop a better strategy to improve astaxanthin (Ax) retention in muscle of Atlantic salmon. To achieve that, a comparison of post-smolt salmon with (+ Ax) or without (- Ax) dietary Ax supplementation was established based on a transcriptomic approach targeting pyloric, hepatic, and muscular tissues. Results in post-smolts showed that the pyloric caeca transcriptome is more sensitive to dietary Ax supplementation compared to the other tissues. Key genes sensitive to Ax supplementation could be identified, such as cd36 in pylorus, agr2 in liver, or fbp1 in muscle. The most modulated genes in pylorus were related to absorption but also metabolism of Ax. Additionally, genes linked to upstream regulation of the ferroptosis pathway were significantly modulated in liver, evoking the involvement of Ax as an antioxidant in this process. Finally, the muscle seemed to be less impacted by dietary Ax supplementation, except for genes related to actin remodelling and glucose homeostasis. In conclusion, the transcriptome data generated from this study showed that Ax dynamics in Atlantic salmon is characterized by a high metabolism during absorption at pyloric caeca level. In liver, a link with a potential of ferroptosis process appears likely via cellular lipid peroxidation. Our data provide insights into a better understanding of molecular mechanisms involved in dietary Ax supplementation, as well as its beneficial effects in preventing oxidative stress and related inflammation in muscle.

Dietary fat concentrations influence fatty acid assimilation patterns in Atlantic pollock (Pollachius virens).

A key aspect in the use of fatty acids (FA) to estimate predator diets using quantitative FA signature analysis (QFASA) is the ability to account for FA assimilation through the use of calibration coefficients (CC). Here, we tested the assumption that CC are independent of dietary fat concentrations by feeding Atlantic pollock (Pollachius virens) three formulated diets with very similar FA proportions but different fat concentrations (5-9% of diet) for 20 weeks. CC calculated using FA profiles of diet and triacylglycerols in pollock liver were significantly different for the three diets. To test the robustness of diet estimates to these differences, we used the CC set derived from feeding the diet with the lowest fat concentration, published prey FA profiles and realistic diet estimates of pollock to construct 'pseudo-predators'. Application of QFASA to each pseudo-predator using the three sets of CC and the same prey FA profiles resulted in diet estimate biases of twofold for major prey items and approximately fivefold for minor prey items. This work illustrates the importance of incorporating diets with fat concentrations that are similar to natural prey when conducting feeding experiments to calculate CC. This article is part of the theme 'The next horizons for lipids as 'trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'.

Red and White Chinook Salmon (Oncorhynchus tshawytscha): Differences in the Transcriptome Profile of Muscle, Liver, and Pylorus.

Astaxanthin (Ax), the main carotenoid responsible for the distinct red flesh color in salmonids (Oncorhynchus, Salvelinus, Salmo, and Parahucho), is added to the diet of farmed fish at a substantial cost. Despite the great economical value for the salmon industry, the key molecular mechanisms involved in the regulation of muscle coloration are poorly understood. Chinook salmon (Oncorhynchus tshawytscha) represent an ideal model to study flesh coloration because they exhibit a distinct color polymorphism responsible for two color morphs, white and red flesh pigmented fish. This study was designed to identify the molecular basis for the development of red and white coloration of fish reared under the same experimental conditions and to better understand the absorption mechanism of Ax in salmonids. Pyloric caeca, liver, and muscle of both groups (n = 6 each) were selected as the most likely critical target organs to be involved respectively in the intestinal uptake, metabolism, and retention of Ax. Difference in the transcriptome profile of each tissue using next-generation sequencing technology was conducted. Ten KEGG pathways were significantly enriched for differentially expressed genes between red and white salmon pylorus tissue, while none for the transcriptome profile in the other two tissues. Differential expressed gene (DE) analyses showed that there were relatively few differences in muscle (31 DE genes, p < 0.05) and liver (43 DE genes, p < 0.05) of white and red Chinook salmon compared approximately 1125 DE genes characterized in the pylorus tissue, with several linked to Ax binding ability, absorption, and metabolism.

Reproductive impacts and physiological adaptations of zebrafish to elevated dietary nickel.

Nickel (Ni) concentrations in the environment can rise due to human industrial activities. The toxicity of waterborne Ni to aquatic animals has been examined in a number of previous studies; however, little is known about the impacts of elevated dietary Ni. In the present study, zebrafish were chronically fed diets containing two concentrations of Ni [3.7 (control) and 116 µg Ni/g diet]. Ni-exposed males, but not females, were significantly smaller (26%) compared to controls at 80 days. In addition, total egg production was decreased by 65% in the Ni treatment at 75-78 days of the experiment. Ni was ubiquitously distributed in control animals (similar to previous studies), and concentrations varied between tissues by 15-fold. Ni exposure resulted in modest but significant Ni accumulation in some tissues (increases were highest in brain, vertebrae and gut; 44%, 34% and 25%, respectively), an effect observed only at 80 days. The limited Ni accumulation may be due to (1) the lack of an acidified stomach in zebrafish and/or (2) the efficient upregulation of Ni transport and excretion mechanisms, as indicated by the 4.5-fold increase in waterborne (63)Ni uptake by Ni-exposed fish. Eggs from Ni-exposed adults had Ni concentrations that were 5.2-fold higher than controls. However, by 4 days post fertilization, larvae had similar Ni concentrations as controls, demonstrating a capacity for rapid Ni depuration. Larvae from Ni-exposed adults were also more resistant to waterborne Ni (35% increase in the 96-h LC50 over controls). In conclusion, elevated dietary Ni significantly affected zebrafish reproduction despite only modest tissue Ni accumulation. There were also indications of adaptation, including increased Ni uptake rates and increased Ni tolerance of offspring from Ni-exposed adults. Ni concentrations were particularly elevated in the brain with exposure; possible relations to growth and reproductive impacts require further study.

Polar lipids from the marine macroalga Palmaria palmata inhibit lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophage cells.

The EtOAc soluble fraction of a MeOH/CHCl3 extract of Palmaria palmata showed strong nitric oxide (NO) inhibitory activity against lipopolysaccharide (LPS)-induced NO production in murine RAW264.7 cells. NO inhibition-guided isolation led to identification of three new polar lipids including a sulfoquinovosyl diacylglycerol (SQDG) (2S)-1-O-eicosapentaenoyl-2-O-myristoyl-3-O-(6-sulfo-α-D-quinovopyranosyl)-glycerol (1) and two phosphatidylglycerols, 1-O-eicosapentaenoyl-2-O-trans-3-hexadecenoyl-3-phospho-(1'-glycerol)-glycerol (3) and 1-O-eicosapentaenoyl-2-O-palmitoyl-3-phospho-(1'-glycerol)-glycerol (4) from the EtOAc fraction. Seven known lipids were also isolated including a SQDG (2), a phospholipid (5) and five galactolipids (6-10). Structures of the isolated lipids were elucidated by spectral analyses. The isolated SQDGs, phosphatidylglycerols and phospholipid possessed strong and dose-dependent NO inhibitory activity compared to N(G)-methyl-L-arginine acetate salt (L-NMMA), a well-known NO inhibitor used as a positive control. Further study suggested that these polar lipids suppressed NO production through down-regulation of inducible nitric oxide synthase (iNOS).

Tissue-specific differential induction of duplicated fatty acid-binding protein genes by the peroxisome proliferator, clofibrate, in zebrafish (Danio rerio).

BACKGROUND: Force, Lynch and Conery proposed the duplication-degeneration-complementation (DDC) model in which partitioning of ancestral functions (subfunctionalization) and acquisition of novel functions (neofunctionalization) were the two primary mechanisms for the retention of duplicated genes. The DDC model was tested by analyzing the transcriptional induction of the duplicated fatty acid-binding protein (fabp) genes by clofibrate in zebrafish. Clofibrate is a specific ligand of the peroxisome proliferator-activated receptor (PPAR); it activates PPAR which then binds to a peroxisome proliferator response element (PPRE) to induce the transcriptional initiation of genes primarily involved in lipid homeostasis. Zebrafish was chosen as our model organism as it has many duplicated genes owing to a whole genome duplication (WGD) event that occurred ~230-400 million years ago in the teleost fish lineage. We assayed the steady-state levels of fabp mRNA and heterogeneous nuclear RNA (hnRNA) transcripts in liver, intestine, muscle, brain and heart for four sets of duplicated fabp genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, fabp10a/fabp10b and fabp11a/fabp11b in zebrafish fed different concentrations of clofibrate. RESULT: Electron microscopy showed an increase in the number of peroxisomes and mitochondria in liver and heart, respectively, in zebrafish fed clofibrate. Clofibrate also increased the steady-state level of acox1 mRNA and hnRNA transcripts in different tissues, a gene with a functional PPRE. These results demonstrate that zebrafish is responsive to clofibrate, unlike some other fishes. The levels of fabp mRNA and hnRNA transcripts for the four sets of duplicated fabp genes was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR). The level of hnRNA coded by a gene is an indirect estimate of the rate of transcriptional initiation of that gene. Clofibrate increased the steady-state level of fabp mRNAs and hnRNAs for both the duplicated copies of fabp1a/fabp1b.1, and fabp7a/fabp7b, but in different tissues. Clofibrate also increased the steady-state level of fabp10a and fabp11a mRNAs and hnRNAs in liver, but not for fabp10b and fabp11b. CONCLUSION: Some duplicated fabp genes have, most likely, retained PPREs, but induction by clofibrate is over-ridden by an, as yet, unknown tissue-specific mechanism(s). Regardless of the tissue-specific mechanism(s), transcriptional control of duplicated zebrafish fabp genes by clofibrate has markedly diverged since the WGD event.

Effect of hormone implantation on cryopreservation of Atlantic halibut (Hippoglossus hippoglossus L.) sperm.

Hormone implantation is widely applied in halibut (Hippoglossus hippoglossus L.) aquaculture to extend the sperm production season of broodstock males. The ability to combine this technique with cryopreservation would increase sperm availability, thereby improving reproduction success and facilitating gene management. In this paper, the cryopreservation ability of sperm from hormone-treated males was examined at three times post-implantation and compared with that of sperm from males that were not hormone-treated. All sperm samples were cryopreserved using the same method. The effectiveness of these techniques was assessed by examining the fertilization rate and motility of thawed sperm. The spermotocrit and concentration of fresh sperm samples were measured to reveal the effect of hormone implantation on sperm characteristics. The reported results indicate that hormone implantation did not affect cryopreservation efficiency. The fertilization rate resulting from thawed sperm of hormone-treated males showed no significant difference from that of untreated males or from fresh sperm. A significant positive relationship was demonstrated between the spermatocrit and concentration of sperm; and a significant decrease of spermatocrit was found in sperm collected from hormone-treated males 14days post-implantation. No significant linear relationship between spermotocrit and fertilization rate of thawed sperm was shown.

Cryopreservation of sperm from Atlantic halibut (Hippoglossus hippoglossus, L.) for commercial application.

Development of Atlantic halibut (Hippoglossus hippoglossus) aquaculture will be enhanced with cryopreservation of halibut sperm by ensuring a reliable supply of sperm of desired quality and quantity. To assist in its commercial application, the cryopreservation of large volumes of halibut sperm was investigated. Three cryoprotectants were compared: dimethyl sulfoxide (DMSO), polyethylene glycol (PG) and glycerol (GLY) at two concentrations (10% or 15%). Two salt solutions, Hanks' balanced salt solution (HBSS) and 0.1M KHCO(3) with 0.125 M sucrose solution (KS) were tested as diluents. Both factors were examined in 1.6 mL volumes. A cryopreservation volume of 4 mL and a low dilution ratio (1:1) were examined separately. Based on motility and fertilization rate, 10% and 15% DMSO diluted with HBSS or KS solution proved to be effective extenders with mean fertilization rates ranging from 52.2 ± 27.2% to 65.8 ± 26.1%; none of which were significantly different from that of the control. Four other extenders, 10% PG or 10% GLY with HBSS or KS, resulted in significantly lower fertilization rates. Use of a 4 mL cryopreservation volume did not exhibit a significant effect on fertilization rate or motility of post-thawed sperm compared to a 1.6 mL volume (P>0.05); while the use of a dilution ratio of one part sperm with three parts cryopreservation solution (1:3 v/v with sperm concentration of 0.51± 0.11×10(10)cells/ml) had a significantly better preservation effect than using a ratio of 1:1 with sperm concentration of 1.02 ± 0.21×10(10)cells/ml (P<0.05). From these results, an optimized protocol for the cryopreservation of Atlantic halibut sperm using a volume as large as 4mL has been established.

Are apparent negative effects of feeding GM MON810 maize to Atlantic salmon, Salmo salar, caused by confounding factors?

The present study was conducted to follow up on apparent differences in growth, relative organ sizes, cellular stress and immune function in Atlantic salmon fed feed containing GM Bacillus thuringiensis maize compared with feed containing the non-modified parental maize line. Gene expression profiling on the distal intestinal segment and liver was performed by microarray, and selected genes were followed up by quantitative PCR (qPCR). In the liver, qPCR revealed some differentially regulated genes, including up-regulation of gelsolin precursor, down-regulation of ferritin heavy subunit and a tendency towards down-regulation of metallothionein (MT)-B. This, combined with the up-regulation of anti-apoptotic protein NR13 and similar tendencies for ferritin heavy chain and MT-A and -B in the distal intestine, suggests changes in cellular stress/antioxidant status. This corresponds well with and strengthens previous findings in these fish. To exclude possible confounding factors, the maize ingredients were analysed for mycotoxins and metabolites. The GM maize contained 90 µg/kg of deoxynivalenol (DON), while the non-GM maize was below the detection limit. Differences were also observed in the metabolite profiles of the two maize varieties, some of which seemed connected to the mycotoxin level. The effects on salmon observed in the present and previous studies correspond relatively well with the effects of DON as reported in the literature for other production animals, but knowledge regarding effects and harmful dose levels in fish is scarce. Thus, it is difficult to conclude whether the observed effects are caused by the DON level or by some other aspect of the GM maize ingredient.

Differential transcriptional modulation of duplicated fatty acid-binding protein genes by dietary fatty acids in zebrafish (Danio rerio): evidence for subfunctionalization or neofunctionalization of duplicated genes.

BACKGROUND: In the Duplication-Degeneration-Complementation (DDC) model, subfunctionalization and neofunctionalization have been proposed as important processes driving the retention of duplicated genes in the genome. These processes are thought to occur by gain or loss of regulatory elements in the promoters of duplicated genes. We tested the DDC model by determining the transcriptional induction of fatty acid-binding proteins (Fabps) genes by dietary fatty acids (FAs) in zebrafish. We chose zebrafish for this study for two reasons: extensive bioinformatics resources are available for zebrafish at zfin.org and zebrafish contains many duplicated genes owing to a whole genome duplication event that occurred early in the ray-finned fish lineage approximately 230-400 million years ago. Adult zebrafish were fed diets containing either fish oil (12% lipid, rich in highly unsaturated fatty acid), sunflower oil (12% lipid, rich in linoleic acid), linseed oil (12% lipid, rich in linolenic acid), or low fat (4% lipid, low fat diet) for 10 weeks. FA profiles and the steady-state levels of fabp mRNA and heterogeneous nuclear RNA in intestine, liver, muscle and brain of zebrafish were determined. RESULT: FA profiles assayed by gas chromatography differed in the intestine, brain, muscle and liver depending on diet. The steady-state level of mRNA for three sets of duplicated genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, and fabp11a/fabp11b, was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR). In brain, the steady-state level of fabp7b mRNAs was induced in fish fed the linoleic acid-rich diet; in intestine, the transcript level of fabp1b.1 and fabp7b were elevated in fish fed the linolenic acid-rich diet; in liver, the level of fabp7a mRNAs was elevated in fish fed the low fat diet; and in muscle, the level of fabp7a and fabp11a mRNAs were elevated in fish fed the linolenic acid-rich or the low fat diets. In all cases, induction of the steady-state level of fabp mRNAs by dietary FAs correlated with induced levels of hnRNA for a given fabp gene. As such, up-regulation of the steady-state level of fabp mRNAs by FAs occurred at the level of initiation of transcription. None of the sister duplicates of these fabp genes exhibited an increase in their steady-state transcript levels in a specific tissue following feeding zebrafish any of the four experimental diets. CONCLUSION: Differential induction of only one of the sister pair of duplicated fabp genes by FAs provides evidence to support the DDC model for retention of duplicated genes in the zebrafish genome by either subfunctionalization or neofunctionalization.

Nutrition, feeding, and behavior of fish.

Nutrition and feeding influence growth, reproduction, and health of fish and their response to physiologic and environmental stressors and pathogens. The basics of fish metabolism are similar to those of warm-blooded animals in that they involve food intake, digestion, absorption, and transport of nutrients to the various tissues. Fish, however, being the most primitive form of vertebrates, possess some distinguishing features which will be discussed. Unlike warm-blooded animals, which are homoeothermic, fish are poikilothermic, so their body temperature and metabolic rate depends on the water temperature and this has practical implications for the nutrition, feeding and health of fish. Several behavioral responses have been linked to methods of feeding, feeding habits, frequency of feeding, mechanisms of food detection, and food preferences. Fish are also unique among vertebrates in their ability to absorb minerals not only from their diets but also from water through their gills and skin.

Vitamin K deficiency inhibits mineralization and enhances deformity in vertebrae of haddock (Melanogrammus aeglefinus L.).

Vitamin K has been known to regulate bone formation through osteocalcin synthesis by osteoblasts, which is important for mineralization and bone structure. The mechanism underlying the relationship of vitamin K with the changes of microanatomy is not fully understood, and our goal is to test whether bone deformities develop in association with vitamin K deficiency. Fish were fed a semi-purified diet containing either devoid (0.00 mg/kg diet) or adequate (40.0 mg/kg diet supplemented but 20.8 mg/kg analyzed) levels of vitamin K (menadione sodium bisulphite) for 20 weeks. At the end of 8 and 20 weeks, fish were subjected to gross examination and X-ray, and mineral content of the vertebrae was measured. The vertebrae were also subjected to histological, histomorphometric and enzyme histochemical examinations to determine the bone formation and resorption. Vitamin K deficiency primarily decreased bone mineralization and subsequently a decrease in bone mass thus resulted in an increased susceptibility to bone deformity. The occurrence of bone deformities coincided with an increased amount of osteoid tissue and decreased bone mineral content. Number of osteoblasts and osteoclasts were not affected by dietary vitamin K. In conclusion, vitamin K deficiency can impair bone mineralization and enhances bone deformities.

Effects of oxidized dietary oil and vitamin E supplementation on lipid profile and oxidation of muscle and liver of juvenile atlantic cod (Gadus morhua).

The effects of oxidized dietary lipid and the role of vitamin E on lipid profile, retained tocopherol levels, and lipid oxidation of juvenile Atlantic cod (Gadus morhua) were evaluated following a 9-week feeding trial. Four isonitrogenous experimental diets containing fresh or oxidized (peroxide value of 94 mequiv/kg) fish oil with or without added vitamin E (alpha-tocopherol or mixed tocopherols) were fed to juvenile cod in duplicate tanks. There was no significant (P > 0.05) influence on major lipid classes of cod liver and muscle by diet with the exception of sterols. Sterols content was increased in liver but decreased in muscle by oxidized dietary oil in the absence of vitamin E. Dietary vitamin E supplementation decreased the sterols level in cod liver but with no significant (P > 0.05) effect on their level in the muscle. Fatty acid composition varied between lipid fractions in muscle tissue and was affected by the diet. Oxidized oil significantly (P < 0.05) decreased the deposition of alpha-tocopherol in liver but not in muscle. gamma- and delta-Tocopherols from dietary tocopherol mixtures were retained at very low levels in liver, but higher retention was observed in muscle tissue. The oxidative state of both liver and muscle, as measured by the 2-thiobarbituric acid reactive substances (TBARS) and headspace propanal, negatively correlated with tissue vitamin E levels. It is suggested that oxidized oil affected juvenile Atlantic cod by causing vitamin E deficiency in certain tissues and that these effects could be alleviated by supplementation of a sufficient amount of dietary vitamin E. The results also indicate that mixed tocopherols were good antioxidants for Atlantic cod, although less effective than alpha-tocopherol alone in many tissues with the exception of muscle, where gamma- and delta-tocopherols were deposited at relatively high levels.

Astaxanthin binding to solubilized muscle proteins of Atlantic salmon (Salmo salar L.), haddock (Melanogrammus aeglefinus L.) and Atlantic halibut (Hippoglossus hippoglossus L.).

A study was conducted to compare astaxanthin binding ability of solubilized muscle proteins of Atlantic salmon (Salmo salar L.), haddock (Melanogrammus aeglefinus L.) and Atlantic halibut (Hippoglossus hippoglossus L.). Muscle proteins of juvenile Atlantic salmon, haddock and halibut were solubilized by sequential extraction of muscle tissue using low ionic strength solutions. Electrophoretic protein profiles of the six solubilized fractions from these species were similar. Each solubilized fraction from the three species was examined for its relative astaxanthin binding capacity. The amount of bound astaxanthin was significantly different (P<0.05) among the six fractions of each species. Significant differences in astaxanthin binding were only found for fractions A and E among the species. The amount of bound astaxanthin in various fractions of each species showed a good correlation (R2=0.80-0.92) with the ANS (8-anilino-1-naphthalenesulfonate) fluorescence intensity of those fractions. The pattern and extent of astaxanthin binding to the muscle proteins of juvenile salmon, haddock and halibut is comparable to that reported previously for adult Atlantic salmon (Saha, M.R., Ross, N.W., Gill, T.A., Olsen, R.E., Lall, S.P., 2005. Development of a method to assess binding of astaxanthin to Atlantic salmon S. salar L. muscle proteins. Aquacult. Res. 36, 336-343.). These combined observations suggest that the carotenoid binding capacity of the muscle proteins of salmon is not the limiting factor in the deposition of carotenoid in their flesh.

Astaxanthin binding protein in Atlantic salmon.

The rubicund pigmentation in salmon and trout flesh is unique and is due to the deposition of dietary carotenoids, astaxanthin and canthaxanthin in the muscle. The present study was undertaken to determine which protein was responsible for pigment binding. Salmon muscle proteins were solubilized by sequential extractions with non-denaturing, low ionic strength aqueous solutions and segregated as such into six different fractions. Approximately 91% of the salmon myofibrillar proteins were solubilized under non-denaturing conditions using a protocol modified from a method described by Krishnamurthy et al. [Krishnamurthy, G., Chang, H.S., Hultin, H.O., Feng, Y., Srinivasan, S., Kelleher. S.D., 1996. Solubility of chicken breast muscle proteins in solutions of low ionic strength. J. Agric. Food Chem. 44: 408-415.] for the dissolution of avian muscle. To our knowledge, this is the first time this solubilization approach has been applied to the study of molecular interactions in myofibrillar proteins. Astaxanthin binding in each fraction was determined using an in vitro binding assay. In addition, SDS-PAGE and quantitative densitometry were used to separate and determine the relative amounts of each of the proteins in the six fractions. The results showed that alpha-actinin was the only myofibrillar protein correlating significantly (P<0.05) with astaxanthin binding. Alpha-actinin was positively identified using electrophoretic techniques and confirmed by tandem mass spectroscopy. Purified salmon alpha-actinin bound synthetic astaxanthin in a molar ratio of 1.11:1.00. The study was repeated using halibut alpha-actinin, which was found to have a molar binding ratio of astaxanthin to alpha-actinin of 0.893:1. These results suggest that the difference in pigmentation between white fish and Atlantic salmon is not due to binding capacity in the muscle, but rather differences in the metabolism or transport of pigment.

Dietary rapeseed oil affects the expression of genes involved in hepatic lipid metabolism in Atlantic salmon (Salmo salar L.).

Supplies of marine fish oils (FO) are limited, and sustainable production in aquaculture dictates that alternatives that do not compromise fish health and product quality, such as vegetable oils, must be found. Nutrigenomics will increase our understanding of how nutrition influences metabolic pathways and homeostatic control, and may be used to measure and validate subtle changes in organ-specific, metabolic gene expression signatures. We compared 2 groups of Atlantic salmon fed diets containing 100% FO or 75% rapeseed oil (RO) for 42 wk. A small-scale cDNA microarray was constructed to screen for changes in the expression of lipid metabolism genes in the liver resulting from this partial substitution of RO for FO. Delta5 fatty acid desaturase gene expression was significantly greater in fish fed 75% RO than in fish fed the control diet; this was confirmed by quantitative real time PCR analysis. In addition, several genes, among these mitochondrial proteins, peroxisome proliferator-activated receptor gamma, as well as other transcription factors, coactivators, and signal transducers, showed significant differential regulation. This partially validated microarray may be used for further gene expression profiling using other dietary comparisons, and for further characterization of selected genes.