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.

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.

Intestinal fatty acid binding protein (fabp2) in Atlantic salmon (Salmo salar): Localization and alteration of expression during development of diet induced enteritis.

In the present study full-length cDNAs corresponding to three isoforms of intestinal fatty acid binding protein (fabp2) in Atlantic salmon were cloned and characterized. Gene expression of fabp2 was observed in all tissues investigated, although differences were observed between isoforms. The highest fabp2a1, fabp2a2, and fabp2b expression was in the intestine. A 15kDa protein, corresponding to putative Fabp2 protein, was identified by immunoblotting using anti-human Fabp2 antibody. Immunoblotting and immunohistochemistry confirmed that Fabp2 protein was present in most Atlantic salmon tissues. Similar to gene expression, intestinal tissues had the highest Fabp2 protein levels, decreasing gradually from proximal to distal intestine. During development of distal intestinal inflammation caused by dietary soybean meal from 0 to 21days, Fabp2 decreased significantly on both transcriptional and protein levels. The reduction in Fabp2 was preceded by a down regulation of peroxisome proliferator activated receptor (ppar) alpha and gamma, fabp2's presumed regulatory proteins, and followed by a progressive increase in proliferating cell nuclear antigen (Pcna) staining. Results illustrate that the early decline of distal intestinal fabp2 was likely caused by a down regulation of their regulatory proteins, but at later time points reduced Fabp2 may largely be due to a less mature enterocyte population resulting from rapid cell turnover.

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.

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.

Decreased expression of TGF-beta, GILT and T-cell markers in the early stages of soybean enteropathy in Atlantic salmon (Salmo salar L.).

This study investigated the early expression of T-cell markers and genes potentially involved in the induction of soybean meal (SBM) enteropathy in the distal intestine (DI) of Atlantic salmon (Salmo salar L.). Quantitative PCR was used to study the expression of CD3, CD8beta, transforming growth factor beta (TGF-beta), interferon-gamma-inducible lysosomal thiol reductase (GILT) and interleukin-1beta (IL-1beta) in salmon fed SBM for 1, 3 and 7 days using fish fed fishmeal as controls. In the same tissue, the morphological development of SBM enteropathy was evaluated by routine histology and the presence of T cells was mapped by immunohistochemistry. TGF-beta was significantly down-regulated on all days of feeding SBM. GILT was significantly down-regulated on days 3 and 7 compared to day 1. A depression in the expression of T-cell markers was observed on day 3 whereas increased densities of T cells were observed at the base of mucosal folds after 7 days of feeding SBM. Down-regulation of GILT and TGF-beta may lead to sensitization of intraepithelial lymphocytes and failure to maintain normal mucosal integrity in the DI. These responses are implicated in the pathogenesis of SBM enteropathy in Atlantic salmon.

Effects of dietary soyabean meal, inulin and oxytetracycline on intestinal microbiota and epithelial cell stress, apoptosis and proliferation in the teleost Atlantic salmon (Salmo salar L.).

Soyabean meal (SBM)-induced enteritis in the distal intestine of the teleost Atlantic salmon (Salmo salar L.) and other salmonids may be considered a model for diet-related mucosal disorders in other animals and man. The role of the intestinal microbiota in its pathogenesis was explored. Compared to diets containing fishmeal (FM) as the sole protein source, responses to extracted SBM or the prebiotic inulin, with or without oxytetracycline (OTC) inclusion, were studied following a 3-week feeding trial. Intestinal microbiota, organosomatic indices and histology, as well as immunohistochemical detection of proliferating cell nuclear antigen (PCNA), heat shock protein 70 (HSP70) and caspase-3-positive cells in the distal intestine, were studied. Distal intestine somatic indices (DISI) were higher in inulin and lower in SBM compared to FM-fed fish. The low DISI caused by SBM corresponded with histological changes, neither of which was affected by OTC, despite a significant decrease in adherent bacteria count. Image analysis of PCNA-stained sections showed a significant increase in the proliferative compartment length in SBM-fed fish, accompanied by apparent increases in reactivity to HSP70 and caspase-3 along the mucosal folds, indicating induction of cellular repair and apoptosis, respectively. Fish fed the SBM diet had higher total number as well as a more diverse population composition of adherent bacteria in the distal intestine. Thus SBM-induced enteritis is accompanied by induction of distal intestinal epithelial cell protective responses and changes in microbiota. Putative involvement of bacteria in the inflammatory response merits further investigation.