Anne Marie Bakke

2 - Feeding, digestion and absorption of nutrients

Publisher Summary The main function of the alimentary tract of any animal is the acquisition of food with subsequent assimilation of vital nutrients. The natural diet of fishes varies tremendously between fish species and their natural habitats. This chapter summarizes the current state of knowledge, or lack thereof, highlighting the similarities and differences of fish digestive physiology relative to that of the more well studied mammalian systems. Important references, especially review articles, are discusses in the chapter to aid in finding more detailed information as needed. Feeding strategies and development of digestive processes in fish larvae and juveniles, as well as physiological responses to changes in ingredient composition of formulated feeds for cultured fish species have been reviewed.

Early response of gene expression in the distal intestine of Atlantic salmon (Salmo salar L.) during the development of soybean meal induced enteritis.

Plant products in general and soybeans in particular can challenge the function and health of the intestinal tract. Salmonids develop an intestinal inflammation when fed diets containing soybean meal (SBM) and certain other legume ingredients. In the present study a 44K oligonucleotide salmonid microarray, qPCR and histology were used to investigate early response mechanisms in the distal intestine of Atlantic salmon (Salmo salar L.) during the first week of oral exposure to a diet containing 20% extracted SBM. The distal intestine transcriptome was profiled on days 1, 2, 3, 5 and 7 and compared to a control group fed fishmeal as the sole protein source. Histological evaluation of the distal intestine revealed the first signs of inflammation on day 5. The most prominent gene expression changes were seen on days 3 and 5. Up-regulation in immune-related genes was observed during the first 5 days, including GTPase IMAP family members, NF-kB-related genes and regulators of T cell and B cell function. Many functional genes involved in lipid metabolism, proteolysis, transport, metabolism and detoxification were initially up-regulated on days 1-3, possibly as an attempt by the tissue to compensate for the initiating immune response. Cell repair and extracellular matrix remodeling genes were up-regulated (heparanase, collagenase) on days 3 and 5. Down regulation of genes related to endocytosis, exocytosis, detoxification, transporters and metabolic processes from day 5 indicated initiation of dysfunction of digestive and metabolic functions that may occur as a result of inflammation or as a response to the introduction of soybean meal in the diet. This is the first study conducting transcriptomic profiling to characterize early responses during the development of SBMIE. Switching Atlantic salmon from a fishmeal to a 20% SBM diet resulted in rapid changes to the intestinal transcriptome, indicating an immune reaction with subsequent impaired epithelial barrier function and other vital intestinal functions.

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-1β, interferon α (IFNα) and IFNγ, as well as IL-17A receptor, T-cell receptor γ (TCRγ), cluster of differentiation 4α (CD4α), CD8β, transforming growth factor β (TGFβ), 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.

A high-resolution map of the gut microbiota in Atlantic salmon (Salmo salar): A basis for comparative gut microbial research

Gut health challenges, possibly related to alterations in gut microbiota, caused by plant ingredients in the diets, cause losses in Atlantic salmon production. To investigate the role of the microbiota for gut function and health, detailed characterization of the gut microbiota is needed. We present the first in-depth characterization of salmon gut microbiota based on high-throughput sequencing of the 16S rRNA gene’s V1-V2 region. Samples were taken from five intestinal compartments: digesta from proximal, mid and distal intestine and of mucosa from mid and distal intestine of 67.3 g salmon kept in seawater (12–14 °C) and fed a commercial diet for 4 weeks. Microbial richness and diversity differed significantly and were higher in the digesta than the mucosa. In mucosa, Proteobacteria dominated the microbiota (90%), whereas in digesta both Proteobacteria (47%) and Firmicutes (38%) showed high abundance. Future studies of diet and environmental impacts on gut microbiota should therefore differentiate between effects on mucosa and digesta in the proximal, mid and the distal intestine. A core microbiota, represented by 22 OTUs, was found in 80% of the samples. The gut microbiota of Atlantic salmon showed similarities with that of mammals.

Transcriptional regulation of cholesterol and bile acid metabolism after dietary soyabean meal treatment in Atlantic salmon (Salmo salar L.).

Inclusion of plant protein sources such as soyabean meal (SBM) in aquafeeds is associated with decreased lipid digestibility, reduced bile acid levels and hypocholesterolaemia. The mechanism for these metabolic abnormalities is unknown. The present study aimed at gaining further insight into how cholesterol and bile acid metabolism is modulated by SBM feeding by quantifying a number of mRNA species corresponding to key proteins involved in cholesterol and bile acid metabolism using quantitative real-time PCR. A 21 d feeding trial with sequential sampling at ten time points following initiation of 20% SBM exposure was conducted on Atlantic salmon. A histological evaluation confirmed distal intestinal enteritis after 5 d of dietary exposure to the SBM, whereas diminished glycogen/lipid deposition was the only relevant finding observed in the liver. SBM inclusion resulted in reduced body pools of cholesterol and bile acids. Hepatic gene expression profiles revealed up-regulation of genes encoding rate-limiting enzymes in cholesterol (3-hydroxy-3-methyl-glutaryl-CoA reductase; HMGCR) and bile acid (cytochrome P4507A1 (CYP7A1)) biosynthesis, as well as up-regulation of their associated transcription factors (sterol regulatory element binding proteins 1 and 2, liver X receptor, farnesoid X receptor and PPAR isoforms). Hepatic gene expressions of cholesterol (ATP binding cassette G5 (ABCG5)) and bile acid (ATP binding cassette B11 (ABCB11)) transporters were, by and large, not influenced by the SBM, but distal intestinal expression patterns of ABCG5 and apical Na-dependent bile acid transporter indicated impaired cholesterol and bile acid reabsorption. In conclusion, hepatic gene expression profiles indicated that the capacity for cholesterol and bile acid synthesis was up-regulated, whereas the indicated impaired cholesterol and bile acid reabsorption probably occurred as a direct result of distal intestinal inflammation.

Decreased expression of TGF-β, 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.

Soya Saponins Induce Enteritis in Atlantic Salmon (Salmo salar L.).

Soybean meal-induced enteritis (SBMIE) is a well-described condition in the distal intestine of salmonids, and saponins have been implicated as the causal agent. However, the question remains whether saponins alone cause SBMIE. Moreover, the dose-response relationship has not been described. In a 10 week feeding trial with Atlantic salmon, a highly purified (95%) soya saponin preparation was supplemented (0, 2, 4, 6, or 10 g/kg) to two basal diets, one containing fishmeal as the major protein source (FM) and the other 25% lupin meal (LP). Saponins caused dose-dependent increases in the severity of inflammation independent of the basal diet, with concomitant alterations in digestive functions and immunological marker expression. Thus, saponins induced inflammation whether the diet contained other legume components or not. However, responses were often the same or stronger in fish fed the corresponding saponin-supplemented LP diets despite lower saponin exposure, suggesting potentiation by other legume component(s).

Effects of oral Bt-maize (MON810) exposure on growth and health parameters in normal and sensitised Atlantic salmon, Salmo salar L.

Responses to GM maize Bt-maize, MON810) expressing Cry1Ab protein from the soil bacterium Bacillus thuringiensis (Bt) in diets for both normal and immune-sensitised (with soyabean meal (SBM)-induced enteropathy) post-smolt Atlantic salmon were investigated following 33 and 97 d of exposure. Triplicate tanks of salmon were fed one of four diets, all containing 20% whole-kernel meal maize, either Bt-maize or its near-isogenic maternal line, without or with 15% extracted SBM inclusion. The fish fed Bt-maize utilised the feed less efficiently, as revealed by lower protein and mineral digestibilities and lower lipid and energy retention efficiencies. Higher intestinal weight, as well as increased interferon-γ and decreased sodium-glucose co-transporter mRNA expression, and a transient increase in T-helper cell presence, as measured by cluster of differentiation 4 (CD4) protein in the distal intestine (DI), may partly explain the lower nutrient digestibilities and retentions. The Bt-maize seemed to potentiate oxidative cellular stress in the DI of immune-sensitised fish, as indicated by increases in superoxide dismutase and heat shock protein 70 mRNA expression. The data suggest that Cry1Ab protein or other antigens in Bt-maize have local immunogenic effects in salmon DI. No systemic immune responses could be detected, as indicated by haematology, differential leucocyte counts, plasma clinical chemistry, as well as absence of Cry1Ab-specific antibodies and Cry1Ab protein in plasma. The responses to Bt-maize observed in the present study differed from results from earlier studies in salmon and other animals fed the same event Bt-maize. Longer-term experiments and more in-depth studies on intestinal physiology and immune responses are needed to evaluate health implications.

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, fabp2s 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.

Alternative Protein Sources in the Diet Modulate Microbiota and Functionality in the Distal Intestine of Atlantic Salmon (Salmo salar)

ABSTRACT The present study aimed to investigate whether alternative dietary protein sources modulate the microbial communities in the distal intestine (DI) of Atlantic salmon, and whether alterations in microbiota profiles are reflected in modifications in host intestinal function and health status. A 48-day feeding trial was conducted, in which groups of fish received one of five diets: a reference diet in which fishmeal (diet FM) was the only protein source and four experimental diets with commercially relevant compositions containing alternative ingredients as partial replacements of fishmeal, i.e., poultry meal (diet PM), a mix of soybean meal and wheat gluten (diet SBMWG), a mix of soy protein concentrate and poultry meal (diet SPCPM), and guar meal and wheat gluten (diet GMWG). Samples were taken of DI digesta and mucosa for microbial profiling using high-throughput sequencing and from DI whole tissue for immunohistochemistry and expression profiling of marker genes for gut health. Regardless of diet, there were significant differences between the microbial populations in the digesta and the mucosa in the salmon DI. Microbial richness was higher in the digesta than the mucosa. The digesta-associated bacterial communities were more affected by the diet than the mucosa-associated microbiota. Interestingly, both legume-based diets (SBMWG and GMWG) presented high relative abundance of lactic acid bacteria in addition to alteration in the expression of a salmon gene related to cell proliferation (pcna). It was, however, not possible to ascertain the cause-effect relationship between changes in bacterial communities and the hosts intestinal responses to the diets. IMPORTANCE The intestine of cultivated Atlantic salmon shows symptoms of compromised function, which are most likely caused by imbalances related to the use of new feed ingredients. Intestinal microbiota profiling may become in the future a valuable endpoint measurement in order to assess fish intestinal health status and effects of diet. The present study aimed to gain information about whether alternative dietary protein sources modulate the microbial communities in the Atlantic salmon intestine and whether alterations in microbiota profiles are reflected in alterations in host intestinal function and health status. We demonstrate here that there are substantial differences between the intestinal digesta and mucosa in the presence and abundance of bacteria. The digesta-associated microbiota showed clear dependence on the diet composition, whereas mucosa-associated microbiota appeared to be less affected by diet composition. Most important, the study identified bacterial groups associated with diet-induced gut dysfunction that may be utilized as microbial markers of gut health status in fish.