Effects of dietary lipid level and environmental temperature on lipid metabolism in the intestine and liver, and choline requirement in Atlantic salmon (Salmo salar L) parr.

Choline was recently established as an essential nutrient for Atlantic salmon at all life stages. Choline deficiency is manifested as an excessive accumulation of dietary fat within the intestinal enterocytes, a condition known as steatosis. Most of today's plant-based salmon feeds will be choline-deficient unless choline is supplemented. Choline's role in lipid transport suggests that choline requirement may depend on factors such as dietary lipid level and environmental temperature. The present study was therefore conducted to investigate whether lipid level and water temperature can affect steatosis symptoms, and thereby choline requirement in Atlantic salmon. Four choline-deficient plant-based diets were formulated differing in lipid level of 16, 20, 25 and 28 % and fed to salmon of 25 g initial weight in duplicate tanks per diet at two different environmental temperatures: 8 and 15 °C. After 8 weeks of feeding, samples of blood, tissue and gut content from six fish per tank were collected, for analyses of histomorphological, biochemical and molecular biomarkers of steatosis and choline requirement. Increasing lipid level did not affect growth rate but increased relative weight and lipid content of the pyloric caeca and histological symptoms of intestinal steatosis and decreased fish yield. Elevation of the water temperature from 8 to 15 °C, increased growth rate, relative weight of the pyloric caeca, and the histological symptoms of steatosis seemed to become more severe. We conclude that dietary lipid level, as well as environmental temperature, affect choline requirement to a magnitude of importance for fish biology and health, and for fish yield.

Dietary Fish Meal Level and a Package of Choline, ß-Glucan, and Nucleotides Modulate Gut Function, Microbiota, and Health in Atlantic Salmon (Salmo salar, L.).

Steatosis and inflammation have been common gut symptoms in Atlantic salmon fed plant rich diets. Choline has recently been identified as essential for salmon in seawater, and ß-glucan and nucleotides are frequently used to prevent inflammation. The study is aimed at documenting whether increased fishmeal (FM) levels (8 levels from 0 to 40%) and supplementation (Suppl) with a mixture of choline (3.0 g/kg), ß-glucan (0.5 g/kg), and nucleotides (0.5 g/kg) might reduce the symptoms. Salmon (186 g) were fed for 62 days in 16 saltwater tanks before samples were taken from 12 fish per tank for observation of biochemical, molecular, metabolome, and microbiome indicators of function and health. Steatosis but no inflammation was observed. Lipid digestibility increased and steatosis decreased with increasing FM levels and supplementation, seemingly related to choline level. Blood metabolites confirmed this picture. Genes in intestinal tissue affected by FM levels are mainly involved in metabolic and structural functions. Only a few are immune genes. The supplement reduced these FM effects. In gut digesta, increasing FM levels increased microbial richness and diversity, and changed the composition, but only for unsupplemented diets. An average choline requirement of 3.5 g/kg was indicated for Atlantic salmon at the present life stage and under the present condition.

Dose-response relationship between dietary choline and lipid accumulation in pyloric enterocytes of Atlantic salmon (Salmo salar L.) in seawater.

Foamy, whitish appearance of the pyloric caeca, reflecting elevated lipid content, histologically visible as hypervacuolation, is frequently observed in Atlantic salmon fed high-plant diets. Lipid malabsorption syndrome (LMS) is suggested as term for the phenomenon. Earlier studies have shown that insufficient supply of phospholipids may cause similar symptoms. The objective of the present study was to strengthen knowledge on the role of choline, the key component of phosphatidylcholine, in development of LMS as well as finding the dietary required choline level in Atlantic salmon. A regression design was chosen to be able to estimate the dietary requirement level of choline, if found essential for the prevention of LMS. Atlantic salmon (456 g) were fed diets supplemented with 0, 392, 785, 1177, 1569, 1962, 2354, 2746 and 3139 mg/kg choline chloride. Fish fed the lowest-choline diet had pyloric caeca with whitish foamy surface, elevated relative weight, and the enterocytes were hypervacuolated. These characteristics diminished with increasing choline level and levelled off at levels of 2850, 3593 and 2310 mg/kg, respectively. The concomitant alterations in expression of genes related to phosphatidylcholine synthesis, cholesterol biosynthesis, lipid transport and storage confirmed the importance of choline in lipid turnover in the intestine and ability to prevent LMS. Based on the observations of the present study, the lowest level of choline which prevents LMS and intestinal lipid hypervacuolation in post-smolt Atlantic salmon is 3·4 g/kg. However, the optimal level most likely depends on the feed intake and dietary lipid level.

Choline supplementation prevents diet induced gut mucosa lipid accumulation in post-smolt Atlantic salmon (Salmo salar L.).

BACKGROUND: Various intestinal morphological alterations have been reported in cultured fish fed diets with high contents of plant ingredients. Since 2000, salmon farmers have reported symptoms indicating an intestinal problem, which we suggest calling lipid malabsorption syndrome (LMS), characterized by pale and foamy appearance of the enterocytes of the pyloric caeca, the result of lipid accumulation. The objective of the present study was to investigate if insufficient dietary choline may be a key component in development of the LMS. RESULTS: The results showed that Atlantic salmon (Salmo salar), average weight 362 g, fed a plant based diet for 79 days developed signs of LMS. In fish fed a similar diet supplemented with 0.4% choline chloride no signs of LMS were seen. The relative weight of the pyloric caeca was 40% lower, reflecting 65% less triacylglycerol content and histologically normal gut mucosa. Choline supplementation further increased specific fish growth by 18%. The concomitant alterations in intestinal gene expression related to phosphatidylcholine synthesis (chk and pcyt1a), cholesterol transport (abcg5 and npc1l1), lipid metabolism and transport (mgat2a and fabp2) and lipoprotein formation (apoA1 and apoAIV) confirmed the importance of choline in lipid turnover in the intestine and its ability to prevent LMS. Another important observation was the apparent correlation between plin2 expression and degree of enterocyte hyper-vacuolation observed in the current study, which suggests that plin2 may serve as a marker for intestinal lipid accumulation and steatosis in fish. Future research should be conducted to strengthen the knowledge of choline's critical role in lipid transport, phospholipid synthesis and lipoprotein secretion to improve formulations of plant based diets for larger fish and to prevent LMS. CONCLUSIONS: Choline prevents excessive lipid accumulation in the proximal intestine and is essential for Atlantic salmon in seawater.

Effects of dietary sodium selenite and organic selenium sources on immune and inflammatory responses and selenium deposition in growing pigs.

The study was conducted to compare effects of different dietary Se sources (sodium selenite [NaSe], Se-enriched yeast [Se yeast] or L-selenomethionine [SeMet]) and one Se-deficient control diet on the expression of selected genes, hematological and clinical biochemical parameters, and muscle morphology in two parallel trials with finisher pigs. Se concentrations in blood plasma and tissues were also monitored. From the pigs in one of the parallel groups, muscle samples obtained from Musculus longissimus dorsi (LD) before and during the trial were examined. The pigs in the other parallel group were challenged once with lipopolysaccharide (LPS) intravenously. Transcriptional analyses of LD showed that selenogenes SelenoW and H were higher expressed in pigs fed Se-supplemented diets compared with control. Furthermore, the expression of interferon gamma and cyclooxygenase 2 was lower in the Se-supplemented pigs versus control. In whole blood samples prior to LPS, SelenoN, SelenoS and thioredoxin reductase 1 were higher expressed in pigs fed NaSe supplemented feed compared with the other groups, possibly indicating a higher level of oxidative stress. After LPS exposure glutathione peroxidase 1 and SelenoN were more reduced in pigs fed NaSe compared with pigs fed organic Se. Products of most above-mentioned genes are intertwined with the oxidant-antioxidant system. No significant effects of Se-source were found on hematologic parameters or microscopic anatomy. The Se-concentrations in various skeletal muscles and heart muscle were significantly different between the groups, with highest concentrations in pigs fed SeMet, followed by those fed Se yeast, NaSe, and control diet. Consistent with previous reports our results indicate that dietary Se at adequate levels can support the body's antioxidant system. Our results indicate that muscle fibers of pigs fed organic Se are less vulnerable to oxidative stress compared with the other groups.

Bile components and lecithin supplemented to plant based diets do not diminish diet related intestinal inflammation in Atlantic salmon.

BACKGROUND: The present study was undertaken to gain knowledge on the role of bile components and lecithin on development of aberrations in digestive functions which seemingly have increased in Atlantic salmon in parallel with the increased use of plant ingredients in fish feed. Post smolt Atlantic salmon were fed for 77 days one of three basal diets: a high fish meal diet (HFM), a low fishmeal diet (LFM), or a diet with high protein soybean meal (HPS). Five additional diets were made from the LFM diet by supplementing with: purified taurocholate (1.8 %), bovine bile salt (1.8 %), taurine (0.4 %), lecithin (1.5 %), or a mix of supplements (suppl mix) containing taurocholate (1.8 %), cholesterol (1.5 %) and lecithin (0.4 %). Two additional diets were made from the HPS diet by supplementing with: bovine bile salt (1.8 %) or the suppl mix. Body and intestinal weights were recorded, and blood, bile, intestinal tissues and digesta were sampled for evaluation of growth, nutrient metabolism and intestinal structure and function. RESULTS: In comparison with fish fed the HFM diet fish fed the LFM and HPS diets grew less and showed reduced plasma bile salt and cholesterol levels. Histological examination of the distal intestine showed signs of enteritis in both LFM and HPS diet groups, though more pronounced in the HPS diet group. The HPS diet reduced digesta dry matter and capacity of leucine amino peptidase in the distal intestine. None of the dietary supplements improved endpoints regarding fish performance, gut function or inflammation in the distal intestine. Some endpoints rather indicated negative effects. CONCLUSIONS: Dietary supplementation with bile components or lecithin in general did not improve endpoints regarding performance or gut health in Atlantic salmon, in clear contrast to what has been previously reported for rainbow trout. Follow-up studies are needed to clarify if lower levels of bile salts and cholesterol may give different and beneficial effects, or if other supplements, and other combinations of supplements might prevent or ameliorate inflammation in the distal intestine.

Effects of dietary plant meal and soya-saponin supplementation on intestinal and hepatic lipid droplet accumulation and lipoprotein and sterol metabolism in Atlantic salmon (Salmo salar L.).

Altered lipid metabolism has been shown in fish fed plant protein sources. The present study aimed to gain further insights into how intestinal and hepatic lipid absorption and metabolism are modulated by plant meal (PM) and soya-saponin (SA) inclusion in salmon feed. Post-smolt Atlantic salmon were fed for 10 weeks one of four diets based on fishmeal or PM, with or without 10 g/kg SA. PM inclusion resulted in decreased growth performance, excessive lipid droplet accumulation in the pyloric caeca and liver, and reduced plasma cholesterol levels. Intestinal and hepatic gene expression profiling revealed an up-regulation of the expression of genes involved in lipid absorption and lipoprotein (LP) synthesis (apo, fatty acid transporters, microsomal TAG transfer protein, acyl-CoA cholesterol acyltransferase, choline kinase and choline-phosphate cytidylyltransferase A), cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase) and associated transcription factors (sterol regulatory element-binding protein 2 and PPARγ). SA inclusion resulted in reduced body pools of cholesterol and bile salts. The hepatic gene expression of the rate-limiting enzyme in bile acid biosynthesis (cytochrome P450 7A1 (cyp7a1)) as well as the transcription factor liver X receptor and the bile acid transporter abcb11 (ATP-binding cassette B11) was down-regulated by SA inclusion. A significant interaction was observed between PM inclusion and SA inclusion for plasma cholesterol levels. In conclusion, gene expression profiling suggested that the capacity for LP assembly and cholesterol synthesis was up-regulated by PM exposure, probably as a compensatory mechanism for excessive lipid droplet accumulation and reduced plasma cholesterol levels. SA inclusion had hypocholesterolaemic effects on Atlantic salmon, accompanied by decreased bile salt metabolism.

Dietary soyasaponin supplementation to pea protein concentrate reveals nutrigenomic interactions underlying enteropathy in Atlantic salmon (Salmo salar).

BACKGROUND: Use of plant ingredients in aquaculture feeds is impeded by high contents of antinutritional factors such as saponins, which may cause various pharmacological and biological effects. In this study, transcriptome changes were analyzed using a 21 k oligonucleotide microarray and qPCR in the distal intestine of Atlantic salmon fed diets based on five plant protein sources combined with soybean saponins. RESULTS: Diets with corn gluten, sunflower, rapeseed or horsebean produced minor effects while the combination of saponins with pea protein concentrate caused enteritis and major transcriptome changes. Acute inflammation was characterised by up-regulation of cytokines, NFkB and TNFalpha related genes and regulators of T-cell function, while the IFN-axis was suppressed. Induction of lectins, complement, metalloproteinases and the respiratory burst complex parallelled a down-regulation of genes for free radical scavengers and iron binding proteins. Marked down-regulation of xenobiotic metabolism was also observed, possibly increasing vulnerability of the intestinal tissue. A hallmark of metabolic changes was dramatic down-regulation of lipid, bile and steroid metabolism. Impairment of digestion was further suggested by expression changes of nutrient transporters and regulators of water balance (e.g. aquaporin, guanylin). On the other hand, microarray profiling revealed activation of multiple mucosal defence processes. Annexin-1, with important anti-inflammatory and gastroprotective properties, was markedly up-regulated. Furthermore, augmented synthesis of polyamines needed for cellular proliferation (up-regulation of arginase and ornithine decarboxylase) and increased mucus production (down-regulation of glycan turnover and goblet cell hyperplasia) could participate in mucosal healing and restoration of normal tissue function. CONCLUSION: The current study promoted understanding of salmon intestinal pathology and establishment of a model for feed induced enteritis. Multiple gene expression profiling further characterised the inflammation and described the intestinal pathology at the molecular level.

Modulation of steroidogenesis and xenobiotic biotransformation responses in zebrafish (Danio rerio) exposed to water-soluble fraction of crude oil.

The induction of CYP enzyme activities, particularly CYP1A1, through the aryl hydrocarbon receptor (AhR) in most vertebrate species is among the most studied biochemical response to planar and aromatic organic contaminant exposure. Since P450 families play central roles in the oxidative metabolism of a wide range of exogenous and endogenous compounds, interactions between the biotransformation processes and reproductive physiological responses are inevitable. Steroidogenesis is the process by which specialized cells in specific tissues, such as the gonad, brain (neurosteroids) and kidney, synthesize steroid hormones. In the present study, we evaluated the effects of water-soluble fraction (WSF) of crude oil on the xenobiotic biotransformation and steroidogenic processes in the head (brain) and whole-body tissue of a model species by transcript analysis using quantitative (real-time) polymerase chain reaction (qPCR), enzyme activities and steroid hormone (testosterone: T and 17beta-estradiol: E2) levels using enzyme immune assay (EIA). Our data showed that exposure of fish to WSF produced an apparent concentration-specific increase of AhR1, CYP1A1 and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNA levels, and decrease of AhR2. On the activity level, WSF produced concentration-specific increase of ethoxyresorufin O-deethylase (EROD), benzyloxyresorufin (BROD) methoxyresorufin (MROD) and pentoxyresorufin (PROD) activities in whole-body tissue. In the steroidogenic pathway, WSF exposure produced apparent concentration-specific decrease of ER* and ERbeta, steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage (P450scc), P450aromA and P450aromB mRNA expression. For steroid hormones, while T levels decreased, E2 levels increased in an apparent WSF concentration-specific manner. In general, the xenobiotic biotransformation and estrogenic responses showed negative relationship after exposure of zebrafish to WSF, suggesting an interaction between these physiological pathways. The relationship between WSF mediated changes in brain StAR, P450scc, 3beta-HSD, ER*alpha, ERbeta, P450aromA, P450aromB and whole-body steroid hormone levels suggests that the experimental animals might be experiencing altered neurosteroidogenesis probably through increased activity level of the biotransformation system. Thus, these responses might represent sensitive diagnostic tools for short-term and acute exposure of fish or other aquatic organisms to WSF.