Liv Torunn Mydland

Modulation of splenic immune responses to bacterial lipopolysaccharide in rainbow trout (Oncorhynchus mykiss) fed lentinan, a beta-glucan from mushroom Lentinula edodes.

Immunostimulants (IS) are considered a promising approach for improving resistance to pathogens in fish aquaculture. At present, development of IS are complicated due to limited knowledge on the mechanisms of their action. To assess the use of global gene expression analysis for screening of candidate IS we applied lentinan, a beta-glucan from the mushroom Lentinula edodes, as a model. After feeding rainbow trout (Oncorhynchus mykiss) with lentinan-supplemented (L) and control (C) diets for 37 days, fish were injected with bacterial lipopolysaccharide (LPS), a classical inducer of inflammation. Gene expression was analyzed in LPS-challenged compared to saline-injected fish using a salmonid 1.8k cDNA microarray (SFA2.0 immunochip) and real-time qPCR. Spleen was selected for data analyses due to highest magnitude of responses and its key role in the fish immune system. A group of genes implicated in acute inflammatory responses was higher induced in C versus L, including IFN-related and TNF-dependent genes (galectins and receptors, signal transducers and transcription factors), genes involved in MHC class I antigen presentation and leukocyte recruitment. A similar trend was observed in metabolism of iron and xenobiotics, markers of oxidative and cellular stress. Interestingly, differences between C and L were similar to those observed between salmon with low and high resistance to infectious salmon anemia virus. Genes with equal responses to LPS in L and C were related to cell communication (cytokines, chemokines and receptors), signal transduction, activation of immune cells, apoptosis, cellular maintenance and energy metabolism. In conclusion, lentinan decreased the expression of genes involved in acute inflammatory reactions to the inflammatory agent while major parts of the immune response remained unchanged. Such effects are expected for IS, which should modify immunity by enhancing beneficial and reducing detrimental responses.

Postprandial changes in plasma free amino acid levels obtained simultaneously from the hepatic portal vein and the dorsal aorta in rainbow trout (Oncorhynchus mykiss).

SUMMARY For the first time, changes in plasma concentrations of free amino acid (AA) and their metabolites were followed simultaneously in pre- and post-hepatic blood following a single meal in non-anaesthetized and free-swimming fish. Rainbow trout (Oncorhynchus mykiss), kept in 10°C water and fitted with cannulae in the hepatic portal vein (HPV) and the dorsal aorta (DA), were force-fed 1% of their body mass and blood samples were taken from both cannulae at 0, 3, 6, 12, 24 and 48 h postprandially to follow the free AA profile. Almost all free AAs increased rapidly within the first 3 h and only a few free AAs did not change significantly over time. By 6 h, the total free AA concentration had peaked in blood taken from both the DA (7107±369 nmol ml-1) and HPV (9999±572 nmol ml-1). However, individual free AAs showed three main profiles beyond this time: for type I, a peak concentration occurred only at 6 h; for type II, there was a more gradual rise in concentration to a peak at 24 h; and for type III there were two peaks, at 6 h and 24 h. All free AAs returned to or were lower than baseline levels within 48 h, with the exception of threonine and proline. The total free AA concentrations were consistently higher (P<0.05) in the HPV than in the DA at 3 h, 6 h, 12 h and 24 h. Our data provide clear evidence that, during the first pass through the liver, hepatic modification altered individual free AA concentrations as indicated by variable ratios among the simultaneous blood samples. Furthermore, the elevation of ammonium and urea in the HPV indicates intestinal catabolism of ingested free AA before release into the HPV. In conclusion, the dual HPV and DA cannulation shows promise as a useful technique for qualitative and quantitative investigations of absorption and turnover of nutrients, especially if the measurements can be combined with reliable estimates of blood flow and labelled substances.

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

BackgroundUse 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.ResultsDiets 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.ConclusionThe 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.Ethical approvalThe present experiment was approved by the Norwegian Animal Research Authority and conducted according to prevailing animal welfare regulations: FOR-1996-01-15-23 (Norway), European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 18.III.1986) and COUNCIL DIRECTIVE of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes (86/609/EEC).

Bacteria Grown on Natural Gas Prevent Soybean Meal-Induced Enteritis in Atlantic Salmon

Dietary inclusion of solvent extracted soybean meal (SBM) is associated with inflammation in the distal intestine of salmonid fish, commonly referred to as SBM-induced enteritis. The enteritis is linked to alcohol soluble components in SBM, but the mechanisms have not been established. Previous studies show that bacterial meal (BM) containing mainly Methylococcus capsulatus grown on natural gas is a suitable protein source for salmonids. The BM is rich in nucleotides, phospholipids, and small peptides that might be beneficial for intestinal homeostasis. In this study, a fish meal (FM)-based control diet (FM diet) and diets with 200 g/kg SBM (SBM diet), 300 g/kg BM (BM diet), and 300 g/kg BM and 200 g/kg SBM (BM-SBM diet) were fed to juvenile Atlantic salmon (Salmo salar) for 80 d. Dietary inclusion of SBM reduced growth (P = 0.007). Inclusion of BM reduced digestibility of protein (P = 0.002) and lipids (P = 0.011) and increased (P < 0.01) the relative weights (g/kg whole body) of total gut, liver, and stomach, and mid and distal intestine. Fish fed the SBM diet developed enteritis, lacked carbonic anhydrase 12 in the brush border of epithelial cells in distal intestine, and had more epithelial cells reacting for proliferating cell nuclear antigen compared with fish fed the other diets. Fish fed the same amount of SBM combined with BM showed no signs of inflammation in the distal intestine. Our results demonstrate that BM grown on natural gas can be used to prevent SBM-induced enteritis in Atlantic salmon.

Candida utilis and Chlorella vulgaris counteract intestinal inflammation in Atlantic salmon (Salmo salar L.).

Intestinal inflammation, caused by impaired intestinal homeostasis, is a serious condition in both animals and humans. The use of conventional extracted soybean meal (SBM) in diets for Atlantic salmon and several other fish species is known to induce enteropathy in the distal intestine, a condition often referred to as SBM induced enteropathy (SBMIE). In the present study, we investigated the potential of different microbial ingredients to alleviate SBMIE in Atlantic salmon, as a model of feed-induced inflammation. The dietary treatments consisted of a negative control based on fish meal (FM), a positive control based on 20% SBM, and four experimental diets combining 20% SBM with either one of the three yeasts Candida utilis (CU), Kluyveromyces marxianus (KM), Saccharomyces cerevisiae (SC) or the microalgae Chlorella vulgaris (CV). Histopathological examination of the distal intestine showed that all fish fed the SC or SBM diets developed characteristic signs of SBMIE, while those fed the FM, CV or CU diets showed a healthy intestine. Fish fed the KM diet showed intermediate signs of SBMIE. Corroborating results were obtained when measuring the relative length of PCNA positive cells in the crypts of the distal intestine. Gene set enrichment analysis revealed decreased expression of amino acid, fat and drug metabolism pathways as well as increased expression of the pathways for NOD-like receptor signalling and chemokine signalling in both the SC and SBM groups while CV and CU were similar to FM and KM was intermediate. Gene expression of antimicrobial peptides was reduced in the groups showing SBMIE. The characterisation of microbial communities using PCR-DGGE showed a relative increased abundance of Firmicutes bacteria in fish fed the SC or SBM diets. Overall, our results show that both CU and CV were highly effective to counteract SBMIE, while KM had less effect and SC had no functional effects.

Prevention of soya-induced enteritis in Atlantic salmon ( Salmo salar ) by bacteria grown on natural gas is dose dependent and related to epithelial MHC II reactivity and CD8α + intraepithelial lymphocytes

An experiment was carried out to study the preventive effect of bacterial meal (BM) produced from natural gas against plant-induced enteropathy in Atlantic salmon (Salmo salar). Salmon were fed a diet based on fish meal (FM) or seven diets with 200 g/kg solvent-extracted soyabean meal (SBM) to induce enteritis in combination with increasing levels of BM from 0 to 300 g/kg. Salmon fed a SBM-containing diet without BM developed typical SBM-induced enteritis. The enteritis gradually disappeared with increasing inclusion of BM. By morphometry, no significant (P>0.05) differences in the size of stretches stained for proliferating cell nuclear antigen were found with 150 g/kg BM compared with the FM diet. Increasing BM inclusion caused a gradual decline in the number of cluster of differentiation 8 α positive (CD8α+) intraepithelial lymphocytes, and fish fed BM at 200 g/kg or higher revealed no significant difference from the FM diet. Histological sections stained with antibody for MHC class II (MHC II) showed that fish with intestinal inflammation had more MHC II-reactive cells in the lamina propria and submucosa, but less in the epithelium and brush border, compared with fish without inflammation. There were no significant (P>0.05) differences in growth among the diets, but the highest levels of BM slightly reduced protein digestibility and increased the weight of the distal intestine. In conclusion, the prevention of SBM-induced enteritis by BM is dose dependent and related to intestinal levels of MHC II- and CD8α-reactive cells.

Increased broiler muscle carnosine and anserine following histidine supplementation of commercial broiler feed concentrate

Abstract A feeding experiment involving histidine supplementation to broiler feed resulted in increased concentration of the histidine containing dipeptides anserine and carnosine in broiler breast muscle. Supplementation with 1 g histidine per kg feed gave a 64% increase in carnosine, and about 10% increase in anserine in the muscle. The standard broiler feed concentrate now in use in Norway seems to contain less histidine than what may be needed for optimal synthesis of carnosine and anserine. These dipeptides have important roles as antioxidants, pH buffering agents and anti-glycation agents. They may have important roles in meat for increasing its stability, shelf life and antioxidant capacity, and it might be speculated that broiler meat rich in anserine and carnosine in the future will be considered a type of functional food, having possible health-beneficial effects. Histidine supplementation of standard Norwegian broiler feed concentrate should be considered.

Dietary Butyrate Helps to Restore the Intestinal Status of a Marine Teleost (Sparus aurata) Fed Extreme Diets Low in Fish Meal and Fish Oil.

There is a constant need to find feed additives that improve health and nutrition of farmed fish and lessen the intestinal inflammation induced by plant-based ingredients. The objective of this study was to evaluate the effects of adding an organic acid salt to alleviate some of the detrimental effects of extreme plant-ingredient substitution of fish meal (FM) and fish oil (FO) in gilthead sea bream diet. Three experiments were conducted. In a first trial (T1), the best dose (0.4%) of sodium butyrate (BP-70 ®NOREL) was chosen after a short (9-weeks) feeding period. In a second longer trial (T2) (8 months), four diets were used: a control diet containing 25% FM (T2-D1) and three experimental diets containing 5% FM (T2-D2, T2-D3, T2-D4). FO was the only added oil in D1, while a blend of plant oils replaced 58% and 84% of FO in T2-D2, and T2-D3 and T2-D4, respectively. The latter was supplemented with 0.4% BP-70. In a third trial (T3), two groups of fish were fed for 12 and 38 months with D1, D3 and D4 diets of T2. The effects of dietary changes were studied using histochemical, immunohistochemical, molecular and electrophysiological tools. The extreme diet (T2-D3) modified significantly the transcriptomic profile, especially at the anterior intestine, up-regulating the expression of inflammatory markers, in coincidence with a higher presence of granulocytes and lymphocytes in the submucosa, and changing genes involved in antioxidant defences, epithelial permeability and mucus production. Trans-epithelial electrical resistance (Rt) was also decreased (T3-D3). Most of these modifications were returned to control values with the addition of BP-70. None of the experimental diets modified the staining pattern of PCNA, FABP2 or ALPI. These results further confirm the potential of this additive to improve or reverse the detrimental effects of extreme fish diet formulations.

High nutrient availability reduces the diversity and stability of the equine caecal microbiota

Background It is well known that nutrient availability can alter the gut microbiota composition, while the effect on diversity and temporal stability remains largely unknown. Methods Here we address the equine caecal microbiota temporal stability, diversity, and functionality in response to diets with different levels of nutrient availability. Hay (low and slower nutrient availability) versus a mixture of hay and whole oats (high and more rapid nutrient availability) were used as experimental diets. Results We found major effects on the microbiota despite that the caecal pH was far from sub-clinical acidosis. We found that the low nutrient availability diet was associated with a higher level of both diversity and temporal stability of the caecal microbiota than the high nutrient availability diet. These observations concur with general ecological theories, suggesting a stabilising effect of biological diversity and that high nutrient availability has a destabilising effect through reduced diversity. Conclusion Nutrient availability does not only change the composition but also the ecology of the caecal microbiota.

Bacterial translocation and in vivo assessment of intestinal barrier permeability in rainbow trout (Oncorhynchus mykiss) with and without soyabean meal-induced inflammation.

The primary aim of this experiment was to evaluate the intestinal barrier permeability in vivo in rainbow trout (Oncorhynchus mykiss) fed increasing levels of soyabean meal (SBM). The relationship between SBM-induced enteritis (SBMIE) and the permeability markers was also investigated. Our results showed that the mean score of morphological parameters was significantly higher as a result of 37·5 % SBM inclusion in the diet, while the scores of fish fed 25 % SBM or lower were not different from those of the fish meal-fed controls (P < 0·05). SBMIE was found in the distal intestine (DI) in 18 % of the fish (eleven of sixty): ten in the 37·5 % SBM-fed group and one in the 25 % SBM-fed group. Sugar markers in plasma showed large variation among individuals probably due to variation in feed intake. We found, however, a significant linear increase in the level of plasma d-lactate with increasing SBM inclusion level (P < 0·0001). Plasma concentration of endotoxin was not significantly different in groups with or without SBMIE. Some individual fish showed high values of endotoxin in blood, but the same individuals did not show any bacterial translocation. Plasma bacterial DNA was detected in 28 % of the fish with SBMIE, and 8 % of non-SBMIE fish (P = 0·07). Plasma concentration of d-lactate was significantly higher in fish with SBMIE (P < 0·0001). To conclude, SBMIE in the DI of rainbow trout was associated with an increase in bacterial translocation and plasma d-lactate concentration, suggesting that these permeability markers can be used to evaluate intestinal permeability in vivo.