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.

Effects of dietary supplementation with prebiotics and Pediococcus acidilactici on gut health, transcriptome, microbiota, and metabolome in Atlantic salmon (Salmo salar L.) after seawater transfer.

BACKGROUND: Given the importance of gut microbiota for health, growth and performance of the host, the aquaculture industry has taken measures to develop functional fish feeds aiming at modulating gut microbiota and inducing the anticipated beneficial effects. However, present understanding of the impact of such functional feeds on the fish is limited. The study reported herein was conducted to gain knowledge on performance and gut health characteristics in post-smolt Atlantic salmon fed diets varying in content of functional ingredients. Three experimental diets, a diet containing fructo-oligosaccharides (FOS), a diet with a combination of FOS and Pediococcus acidilactici (BC) and a diet containing galacto-oligosaccharides (GOS) and BC, were used in a 10-weeks feeding trial. A commercial diet without functional ingredients was also included as a control/reference. Samples of blood plasma, mucosa and digesta were subjected to microbiota, transcriptome and metabolome profiling for evaluation of the diet effects. RESULTS: No significant growth differences were observed between fish fed the supplemented diets, but FOS-BC fed fish showed significantly faster growth than the control fed fish. The microbiota results showed that the BC was present in both the digesta, and the mucosa samples of fish fed the FOS-BC and GOS-BC diets. Digesta-associated microbiota was altered, while mucosa-associated microbiota was relatively unaffected by diet. Replacing FOS with GOS increased the level of metabolites linked to phospholipid, fatty acid, carnitine and sphingolipid metabolism. Variation in metabolite levels between the treatments closely correlated with genera mainly belonging to Firmicutes and Actinobacteria phyla. The transcriptome analyses indicated diet effects of exchanging FOS with GOS on immune functions, oxidative defense and stress responses. No significant diet effect was observed on intestinal inflammation in the pyloric caeca or in the distal intestine, or on lipid accumulation in the pyloric caeca. CONCLUSIONS: Dietary supplementation with BC induced moderate effects on the microbiota of the digesta, while the effects of replacing FOS with GOS were more marked and was observed also for nutrient metabolism. Our data indicates therefore that the quality of a prebiotic may be of great importance for the effects of a probiotic on gut microbiota, function, and health.

Effects of functional ingredients on gut inflammation in Atlantic salmon (Salmo salar L).

Functional feed ingredients are frequently used in feeds for Atlantic salmon, often claimed to improve immune functions in the intestine and reduce severity of gut inflammation. However, documentation of such effects is, in most cases, only indicative. In the present study effects of two packages of functional feed ingredients commonly used in salmon production, were evaluated employing two inflammation models. One model employed soybean meal (SBM) as inducer of a severe inflammation, the other a mixture of corn gluten and pea meal (CoPea) inducing mild inflammation. The first model was used to evaluate effects of two packages of functional ingredients: P1 containing butyrate and arginine, and P2 containing ß-glucan, butyrate, and nucleotides. In the second model only the P2 package was tested. A high marine diet was included in the study as a control (Contr). The six diets were fed to salmon (average weight of 177g) in saltwater tanks (57 fish per tank), in triplicate, for 69 days (754 ddg). Feed intake was recorded. The growth rate of the fish was high, highest for the Contr (TGC: 3.9), lowest for SBM fed fish (TGC: 3.4). Fish fed the SBM diet showed severe symptoms of inflammation in the distal intestine as indicated by histological, biochemical, molecular, and physiological biomarkers. The number of differently expressed genes (DEG) between the SBM and Contr fed fish was 849 and comprised genes indicating alteration in immune functions, cellular and oxidative stress, and nutrient digestion, and transport functions. Neither P1 nor P2 altered the histological and functional symptoms of inflammation in the SBM fed fish importantly. Inclusion of P1 altered expression of 81 genes, inclusion of P2 altered 121 genes. Fish fed the CoPea diet showed minor signs of inflammation. Supplementation with P2 did not change these signs. Regarding composition of the microbiota in digesta from the distal intestine, clear differences regarding beta-diversity and taxonomy between Contr, SBM, and CoPea fed fish were observed. In the mucosa the microbiota differences were less clear. The two packages of functional ingredients altered microbiota composition of fish fed the SBM and the CoPea diet towards that of fish fed the Contr diet.

Gut immune functions and health in Atlantic salmon (Salmo salar) from late freshwater stage until one year in seawater and effects of functional ingredients: A case study from a commercial sized research site in the Arctic region.

The present study was conducted to strengthen the knowledge on gut immune functions and health in Atlantic salmon under large scale, commercial conditions in the Arctic region of Norway. Two groups of fish were monitored, one fed a series of diets without functional ingredients (Ref) and the other diets with functional ingredients (Test). The nutritional composition of the two diet series varied in parallel according to the nutrient requirements of the fish during the observation time. The content of functional ingredients in the Test diets, i.e. nucleotides, yeast cell walls, a prebiotic and essential fatty acids, varied in accordance with a strategy developed by the feed company. The fish were observed at four sampling time points, the first (FW) in May 2016 two weeks before seawater transfer, the other three throughout the following seawater period until the fish reached a size of about 2 kg, i.e. in June, four weeks after seawater transfer (SW1); in November (SW2), and in April the following year (SW3). Gut health was assessed based on histopathological indicators of lipid malabsorption and gut inflammation, expression of gut immune, barrier and other health related genes, plasma biomarkers, somatic indices of intestinal sections, as well as biomarkers of digestive functions. Seawater transfer of the fish (SW1 compared to FW) caused a marked lowering of expression of genes related to immune and barrier functions in the distal intestine, i.e. cytokines (il1ß, il10, tgfß, ifnγ), T-cell markers (cd3γδ), myd88 and tight junction proteins (zo-1, claudin-15, claudin-25b), indicating suppressed immune and barrier functions. At SW2 and SW3, most of the immune biomarkers showed values similar to those observed at FW. The development of plasma cholesterol and triglyceride levels showed similar picture, with markedly lower levels after seawater transfer. Lipid malabsorption was observed in particular in fish from SW1 and SW2, as indicated by hyper-vacuolation of the pyloric caeca enterocytes with concurrently increased expression levels of plin2. Regarding effects of functional ingredients, significantly lower condition factor and plasma triglyceride level were observed for Test-fed fish at SW2, indicating a metabolic cost of use of a mixture of nucleotides, yeast cell walls and essential fatty acids. No clear effects of functional ingredients on expression of gut immune genes and other health indexes were observed through the observation period. The great, temporary lowering of expression of gut immune and barrier genes at SW1 is suggested to be an important factor underlying the increased vulnerability of the fish at this time point. Our findings regarding supplementation with functional ingredients raise questions whether some of these ingredients overall are beneficial or might come with a metabolic cost. Our results highlight the need for a better understanding of the cause and consequences of the suppression of gut immune functions of farmed Atlantic salmon just after seawater transfer, and the use of functional ingredients under commercial conditions.

Gut health and vaccination response in pre-smolt Atlantic salmon (Salmo salar) fed black soldier fly (Hermetia illucens) larvae meal.

Limited availability of sustainable feed ingredients is a serious concern in salmon aquaculture. Insects may become an important, sustainable resource for expanding the raw material repertoire. Herein, we present data from an 8-week feeding trial with pre-smolt Atlantic salmon (initial body weight 49 ±â€¯1.5 g) fed either a reference diet containing fish meal, soy protein concentrate and wheat gluten as protein sources, or a test diet wherein 85% of the protein was supplied by black soldier fly larvae meal. Possible diet effect on the systemic immune response was evaluated by measuring plasma antibody titers after vaccination against infectious pancreatic necrosis virus (IPNV). The gut health of fish was evaluated using endpoints including organ and tissue indices, histopathological parameters and gene expression. Both diets induced the same level of antibody responses against IPNV. In fish fed the reference diet, the histological examination of the pyloric caeca mucosa showed clear hyper-vacuolization suggestive of lipid accumulation in enterocytes, whereas this was less pronounced in the insect meal fed fish. Expression of genes relevant to lipid metabolism confirmed these histological findings. Immune and barrier-function gene expression profiles were both generally not affected by diet. However, the fish fed insect meal showed increased expression of genes indicative of stress response, immune tolerance and increased detoxification activity. In summary, our results showed no indications that dietary inclusion of insect meal affected the gut health of Atlantic salmon negatively. The insect meal based diet seemed to reduce excessive lipid deposition in the pyloric caeca and stimulate xenobiotic metabolism.

Bile enhances glucose uptake, reduces permeability, and modulates effects of lectins, trypsin inhibitors and saponins on intestinal tissue.

Antinutritional factors (ANFs) can disrupt digestive and other intestinal functions. ANFs in soybean meal (SBM) are implicated in proliferative and inflammatory responses in the intestine of various (functionally) monogastric animals, including Atlantic salmon (Salmo salar L.). The goal of the current study was to investigate the effect of ex vivo exposure of mid and distal intestinal tissue of salmon to soybean saponins (SAP), lectin (LEC) and Kunitz' trypsin inhibitor (KTI), singly and in combination, on epithelial function, as assessed by measuring in vitro glucose uptake pathways along a glucose concentration gradient. As solubilization of SAP in the calcium-containing Ringer's solution was problematic but resolved with the addition of a physiological concentration of bile collected from the gall bladder of salmon, an evaluation of bile effects became an added element. Results indicated that bile increased baseline glucose absorption and possibly transport, and also had a protective effect on the epithelial barrier, at least partially due to taurocholate. Compared to controls, tissues exposed to LEC+bile, KTI+bile and LEC+KTI+bile exhibited increased glucose uptake at the higher glucose concentrations, apparently due to markedly increased tissue permeability. Addition of SAP, however, attenuated the response, possibly by binding bile components. SAP+bile, also in combination with LEC and/or KTI, as well as LEC, KTI and LEC+KTI without bile often reduced transcellular glucose uptake pathways, while maintaining low tissue permeability. SAP+LEC+KTI+bile, LEC and KTI caused the most marked reductions. The distal intestine was more affected, reflecting the restriction of in vivo SBM-induced inflammatory changes to this region.

Intestinal epithelial cell proliferation and migration in Atlantic salmon, Salmo salar L.: effects of temperature and inflammation.

A 28-day feeding trial was carried out to characterise intestinal epithelial cell (IEC) turnover in Atlantic salmon (Salmo salar L.) post-smolts in seawater. Four groups of fish raised at two temperatures of 8°C or 12°C and fed two different diets were investigated. The diets included a reference maize gluten and fishmeal-based diet (FM) and an experimental enteropathy-causing diet containing 20% extracted soybean meal (SBM). IEC proliferation and migration were investigated by labelling cells with the in vivo proliferation marker 5-bromo-2'-deoxyuridine (BrdU). Proliferating cell nuclear antigen (PCNA) labelling was used as a control for identifying proliferating cells. Samples of the proximal (PI), mid (MI) and distal (DI) intestinal regions were collected at five time points (3 h-28 days) over the experimental period. Histologically, FM-fed fish had normal mucosa, whereas the SBM-fed fish developed DI enteropathy. Major zones of cell proliferation were observed in the mucosal fold bases for all intestinal regions. Over time, BrdU-labelled cells migrated up mucosal folds to the tips before being lost. Migration rates were dependent on intestinal region, temperature and diet. Highest migration rates were observed in the PI followed by the MI and DI for FM-fed fish. Diet and temperature barely affected migration in the PI and MI. Migration in the DI was most sensitive to diet and temperature, with both SBM and the higher water temperature increasing proliferation and migration rates. The slow IEC turnover in the DI might help to explain the sensitivity of this region to dietary SBM-induced enteropathy.

Transcriptional regulation of IL-17A and other inflammatory markers during the development of soybean meal-induced enteropathy in the distal intestine of Atlantic salmon (Salmo salar L.).

Progression of soybean meal (SBM)-induced enteropathy in Atlantic salmon (Salmo salar L.) distal intestine (DI) was studied to investigate pathophysiological mechanisms and immune responses. Seawater-adapted salmon were fed an extracted SBM-containing diet (200 g kg(-1)) from day 1-21 and compared with fish fed a fishmeal-based diet (day 0). Histological evaluation of the DI revealed signs of inflammation from day 5, which progressively increased in severity and affected more fish with increasing SBM exposure time. The expression profiles of 16 genes were analyzed by quantitative PCR. The pro-inflammatory cytokines interleukin 17A (IL-17A), IL-1ß, interferon α (IFNα) and IFNγ, as well as IL-17A receptor, T-cell receptor γ (TCRγ), cluster of differentiation 4α (CD4α), CD8ß, transforming growth factor ß (TGFß), trypsin, protease-activated receptor 2 (PAR2) and myeloid differentiation primary response gene 88 (MyD88) were significantly up-regulated during early and/or late inflammation stages, whereas interferon-γ-inducible lysosomal thiol reductase (GILT) was downregulated. Up-regulation of TCRγ from day seven suggests proliferation of intraepithelial γδ T cells. IL-17A, up-regulated by 218-fold during early inflammation, indicates involvement of T helper 17 cells in the pathogenesis of the SBM-induced inflammatory response.

Interaction of soyasaponins with plant ingredients in diets for Atlantic salmon, Salmo salar L.

The effects of combining soyasaponins with plant ingredients on intestinal function and fish health were investigated in an 80 d study with Atlantic salmon (270 g) distributed thirty each into twenty-four tanks with seawater. Soyasaponins were supplemented (2 g/kg) to diets with maize gluten (MG), pea protein concentrate (PPC) and sunflower (SFM), rapeseed (RSM) or horsebean meals. A diet with soyabean meal (SBM) and another with wheat gluten and soyasaponins served as reference diets. Marked soyasaponin effects were observed when combined with PPC. This combination induced inflammation in the distal intestine (DI) similar to SBM, reduced feed intake, apparent digestibility of lipid, most amino acids and ash, decreased bile salt levels in intestinal chyme and decreased leucine aminopeptidase (LAP) activity but increased trypsin activity in the DI. No enteritis was observed in other diet groups, but small consistent negative soyasaponin effects were seen on lipid and fatty acid digestibility, faecal DM and LAP activity of the DI. Soyasaponin combination with RSM reduced digestibility of all nutrients including minerals. The mineral effect was also seen for SFM, whereas with MG and SFM a positive soyasaponin effect on feed intake was observed. Caution should be exercised to avoid ingredient combinations giving high saponin levels, a condition that appears to be a key factor in diet-induced enteritis together with certain plant ingredients.