Histological, digestive, metabolic, hormonal and some immune factor responses in Atlantic salmon, Salmo salar L., fed genetically modified soybeans

The paper reports the second and final part of an experiment aiming to study physiological and health-related effects of genetically modified (GM) soybean meal (SBM) type Roundup Ready soybean (RRS) in diets for post-smolt Atlantic salmon. For 3 months salmon were fed diets containing 172 g kg(-1) full-fat SBM from RRS (GM-soy) or an unmodified, non-isogenic line (nGM-soy), or a reference diet with fishmeal as the sole protein source (FM). Slight differences in anti-nutrient levels were observed between the GM and nGM-soy. Histological changes were observed only in the distal intestine of the soy-fed fish. The incidence of moderate inflammation was higher in the GM-soy group (9 of 10 sampled fish) compared with the nGM-soy group (7 of 10). However, no differences in the concomitant decreases in activities of digestive enzymes located in the brush border (leucine aminopeptidase and maltase) and apical cytoplasm (acid phosphatase) of enterocytes or in the number of major histocompatibility complex class II+ cells, lysozyme activity, or total IgM of the distal intestine were observed. GM compared with nGM-soy fed fish had higher head kidney lysozyme (11,856 vs. 10,456 units g(-1) tissue) and a tendency towards higher acid phosphatase (0.45 vs. 0.39 micromol h(-1) kg(-1) body mass in whole tissue) activities, respectively. Plasma insulin and thyroxin levels, and hepatic fructose-1,6-bisphosphatase and ethoxyresorufin-O-deethylase activities were not significantly affected. It is not possible, however, to conclude whether the differences in responses to GM-soy were due to the genetic modification or to differences in soy cultivars in the soy-containing diets. Results from studies using non-modified, parental line soybeans as the control group are necessary to evaluate whether genetic modification of soybeans in diets poses any risk to farmed Atlantic salmon.     

Characterisation of the microbiota associated with intestine of Atlantic cod (Gadus morhua L.): The effect of fish meal, standard soybean meal and a bioprocessed soybean meal

Populations of aerobic heterotrophic bacteria present in the gastrointestinal (GI) tract of healthy Atlantic cod (Gadus morhua L.) fed three different diets, fish meal, standard or a bioprocessed soybean meal (BPSBM), were estimated using the dilution plate technique. A total of 944 isolates were characterised by biochemical and physiological properties and 425 isolates were identified further by sequencing the 16S rRNA genes. Our results showed that gut microbiota were affected by dietary manipulation. The GI tract of fish fed fish meal was dominated by Gram-positive bacteria of the genera Brochothrix and Carnobacterium. The Gram-negative bacteria Chryseobacterium spp. and Psychrobacter glacincola, and Gram-positive bacteria belonging to Carnobacterium, dominated in the digestive tract of fish fed soybean meal. In contrast to these results, genus Psychrobacter dominated in the GI tract when fish were fed BPSBM.Until recently, it was generally suggested that the gut microbiota of fish were less diverse than in homoeothermic animals. However, the present study identified several “new” bacterial species isolated from the alimentary tract of Atlantic cod. These “new” bacterial species are not normally isolated from the GI tract of fish. Based on our finding we suggest that the GI tract microbiota of fish might not be as simple as believed.Antagonistic activity of carnobacteria regarding inhibition of growth of two fish pathogens (Aeromonas salmonicida ssp. salmonicida and Vibrio anguillarum) was observed. However, some difference in the antibacterial activity of Carnobacterium spp. was observed. Whether this antagonistic activity has any effect in challenge studies will be discussed, especially in relation to the finding that the digestive tract is one of the major infection routes in fish.     

Nutritive Value of Four Soybean Products in Diets for Atlantic Salmon (Salmo salar,L.)

Abstract

Four processed soybean products were evaluated as protein sources for Atlantic salmon: solvent-extracted soybean meal (SBM44), dehulled and solvent-extracted soybean meal (SBM50), dehulled full-fat soybean meal (FFSBM) and soybean concentrate (SBC). The soybean products replaced high-quality fish meal at levels corresponding to 0, 14, 28, 42 and 56% soybean protein of total protein.

Dietary inclusion of SBC, at the expense of fish meal, did not affect weight gain, carcass lipids, fecal excretion of nutrients or fecal dry matter content significantly. The nutritive value of the SBC protein appeared comparable to that of the fish meal. The three other soybean products impaired performance increasingly with increasing levels of inclusion, indicating lower nutritive value than for the fish meal. The favourable results seen with SBC indicated a great potential of soybean to become an important protein source for Atlantic salmon through improved processing.     

Evaluation of the effect of commercially available plant and animal protein sources in diets for Atlantic salmon (Salmo salar L.): digestive and metabolic investigations

The present study investigated the effect of various alternative diet ingredients partially replacing fishmeal (FM) on digestive and metabolic parameters in Atlantic salmon (Salmo salar L.) post-smolts (initial body mass 305 ± 69 g) following 12 weeks of feeding. Experimental diets containing 20 % extracted sunflower (ESF), pea protein concentrate (PPC), soy protein concentrate (SPC), feather meal (FeM) and poultry by-product (PBY) were compared to a reference diet containing FM as the main protein source. For the different intestinal compartments trypsin, lipase, bile salts, dry matter and chyme-associated leucine aminopeptidase (LAP) were measured from the content and LAP was measured in the tissue. Selected metabolites were measured in plasma samples. In general, use of plant proteins resulted in low C-LAP activity, low plasma cholesterol and high plasma magnesium. The plasma levels of cholesterol and Mg reflecting were most likely reflections of the composition of the diet, while the LAP activity in chyme may indicate lower epithelial cell turnover. Other responses varied depending on the plant protein source. Results from the animal protein substitution also varied both between diets and compartments; however, both materials increased lipase activity in DI. FeM resulted in a significant increase in both total and specific LAP activities suggesting an attempt to increase the digestive capacity in response to low digestibility of the diet while PBY showed very little difference from the FM-fed control fish. The present trial indicates that 20 % PPC, SPC and PBY can partially replace FM in diets for Atlantic salmon. The qualities of ESF and FeM used in this trial show little promise as FM replacement at 20 % inclusion level.     

Effect of dietary components on the gut microbiota of aquatic animals. A never‐ending story?

It is well known that healthy gut microbiota is essential to promote host health and well‐being. The intestinal microbiota of endothermic animals as well as fish are classified as autochthonous or indigenous, when they are able to colonize the host's epithelial surface or are associated with the microvilli, or as allochthonous or transient (associated with digesta or are present in the lumen). Furthermore, the gut microbiota of aquatic animals is more fluidic than that of terrestrial vertebrates and is highly sensitive to dietary changes. In fish, it is demonstrated that [a] dietary form (live feeds or pelleted diets), [b] dietary lipid (lipid levels, lipid sources and polyunsaturated fatty acids), [c] protein sources (soybean meal, krill meal and other meal products), [d] functional glycomic ingredients (chitin and cellulose), [e] nutraceuticals (probiotics, prebiotics, synbiotics and immunostimulants), [f] antibiotics, [g] dietary iron and [h] chromic oxide affect the gut microbiota. Furthermore, some information is available on bacterial colonization of the gut enterocyte surface as a result of dietary manipulation which indicates that changes in indigenous microbial populations may have repercussion on secondary host–microbe interactions. The effect of dietary components on the gut microbiota is important to investigate, as the gastrointestinal tract has been suggested as one of the major routes of infection in fish. Possible interactions between dietary components and the protective microbiota colonizing the digestive tract are discussed.     

Causes for improvement in nutritive value of broiler chicken diets with whole wheat instead of ground wheat

1. Two experiments were conducted with broiler chicks to compare nutritive values of ground and whole wheat diets containing titanium oxide as indigestible marker. 2. In Experiment 1, a wheat and soy isolate based cold-pelleted diet, in which all the wheat was ground in a hammer mill to pass through a 3 mm sieve, was compared with a diet in which about half (375 g/kg) of the ground wheat was taken out of the diet and supplied as whole wheat mixed into the diet after pelleting. 3. In Experiment 2, a diet based on ground wheat and soy isolate which was pelleted after preconditioning (final temperature 81 degrees C) was compared with a diet in which 500 g of the ground wheat was replaced by whole wheat mixed into the diet before pelleting. 4. In Experiment 1, replacement of ground wheat with whole wheat increased apparent metabolisable energy (AME) content and starch digestibility measured at ileal as well as faecal level, while weight gain and feed conversion efficiency (FCE) were not affected. Jejunal chyme showed increased amylase activity and bile salt concentration. 5. In Experiment 2, replacement of ground wheat with whole wheat increased dry matter content as well as bile content of gizzard, and stimulated an increase in gizzard as well as pancreas weight. Jejunal amylase and bile concentrations were not significantly affected and neither were growth, FCE or AME. 6. Results from this experiment indicate that the improved feed value sometimes observed with whole wheat may be associated with modulation of digestive processes resulting in increased pancreas and liver secretions.     

Response to soy: T-cell-like reactivity in the intestine of Atlantic salmon, Salmo salar L

T-cell-mediated hypersensitivity could be central in soybean meal (SBM)-induced intestinal changes in salmon. However, tools for immunohistochemical detection of T cells have been lacking in teleosts, including Atlantic salmon. Application of a specific histochemical protocol allowed demonstration of T-cell-like reactivities in formalin-fixed, paraffin-embedded tissues using an antibody reacting to a conserved region of human CD3epsilon (Dako A0452). Characteristic staining was observed in cells of the thymus as well as distal intestine, skin, gills and spleen. These cells were negative for immunoglobulin M (IgM). Intestinal intraepithelial leucocytes were CD3epsilon positive. During the SBM-induced enteropathy, the mixed inflammatory infiltrate in the lamina propria of the distal intestine included many lymphocytes with a T-cell-like reactivity. Real-time polymerase chain reaction revealed significantly increased expression of a complex polypeptide (CD3pp), CD4 and CD8beta (P < 0.05) in the distal intestine of SBM-fed fish compared to fish meal-fed reference fish. Increased reactivity for extracellular IgM in the lamina propria and a positive material between the epithelial cells at the tips of the folds was observed, possibly due to leakage of IgM through an abrogated epithelial barrier. In conclusion, a T-cell-like response appears to be involved in this example of a food-sensitive enteropathy.     

Use of 16S rRNA gene sequencing analysis to characterize culturable intestinal bacteria in Atlantic salmon (Salmo salar) fed diets with cellulose or non-starch polysaccharides from soy

Duplicate groups of Atlantic salmon (Salmo salar L.), kept in seawater, were fed fish meal‐based cold‐pelleted diets. Diets with non‐starch polysaccharides (NSP), either cellulose, purified soybean NSP or extruded purified soybean NSP at a dietary level of 100 g kg^?1, were compared with a diet without supplemental NSP and a diet with soybean meal in a 28‐day feeding trial. Isolation and characterizations were limited to culturable bacteria and population levels of aerobic and facultative aerobic heterotrophic autochthonous (adherent) and allochthonous (transient) bacteria present in the mid and distal intestines of Atlantic salmon fed the five different diets estimated using traditional bacteriological techniques. The presence of an autochthonous microbiota was demonstrated using transmission electron microscopy. No significant effects of diet composition were observed on total population levels of culturable bacteria present in the digestive tract, but the study showed that the composition of the gut microbiota (autochthonous or allochthonous) was sensitive to dietary changes. A total of 752 culturable isolates from the intestines were characterized by biochemical and physiological properties. Of these, 188 isolates were further characterized by partial sequencing the 16S rRNA genes. Among these, 146 isolates belonged to 31 phylotypes that were >94% identical to previously described species. However, 42 isolates showed similarity <94% to species available at the National Center of Biotechnology Information. Several of the phylotypes identified in the present study have not been reported previously in the gastrointestinal (GI) tract of fish, including the Gram‐negative bacteria Gelidibacter salicanalis, Pseudoalteromonas elyakovii, Psychrobacter aquimaris, Psychrobacter cibarius, Psychrobacter fozii, Psychrobacter maritimus, Psychrobacter okhotskensis and Psychrobacter psychrophilus. Among the Gram‐positive bacteria identified were Arthrobacter bergeri, Arthrobacter psychrolactophilus, Arthrobacter rhombi, Bacillus pumilus, Bacillus subtilis, Exiguobacterium spp., Microbacterium oxydans, Planococcus maritimus, Sporosarcina ginsengisoli and several bacteria that have been described as unculturable previously. In addition, we identified Carnobacterium inhibens, a lactic acid bacterium that is not frequently isolated from the GI tract of fish. Psychrobacter cibarius was the dominant bacterial species and was isolated from the digestive tract of all fish investigated.     

Alcohol-soluble components in soybeans cause morphological changes in the distal intestine of Atlantic salmon, Salmo salar L.

Standard soybean meal, heat treated to differing degrees and containing varying levels of soybean lectin and trypsin inhibitor, was used to study the effect of soybean-containing diets on the morphology of the distal intestine in Atlantic salmon, Salmo salar L. A reference diet based on fish meal and a fish meal diet mixed with an alcohol extract of soybean meal were used. The fish (145 g) were kept in 27 m³ net pens (200 fish per pen) in sea water with an average temperature of 11 °C for 12 weeks. Whereas a normal morphology of the distal intestine was seen in the fish fed the reference diet, all soybean diets caused alterations in the distal intestine irrespective of heat treatment. Similar changes were observed with the diet which had added alcohol solubles. It is concluded that alcohol-soluble components in the soybeans are responsible for the observed changes. It cannot be determined which of the soybean antinutrients is causing the alterations from the present study. Oligosaccharides and saponines are possible candidates. However, the possibility that unknown antinutrients are affecting the salmon intestine should not be overlooked.     

Development and regression of soybean meal induced enteritis in Atlantic salmon, Salmo salar L., distal intestine: a comparison with the intestines of fasted fish

The development of a pathological condition in the distal intestine of Atlantic salmon, Salmo salar L., induced by dietary soybean meal, was studied in a 6-week feeding experiment. The fully developed condition, as observed after 3 weeks on the experimental diets, was characterized by: (1) a shortening of heights of the mucosal foldings; (2) a loss of the normal supranuclear vacuolization of the absorptive cells in the intestinal epithelium; (3) a widening of the central stroma within the mucosal foldings, with increased amounts of connective tissue; and (4) a profound infiltration of inflammatory cells in the lamina propria. The first signs of morphological changes were observed after 2 days on a diet containing a solvent extracted soybean meal, and within 7 days, all the above mentioned signs were observed. When the fish were subsequently transferred to a control diet, the mucosal folds were rebuilt from the base, resulting in an apparently functional epithelium after 3 weeks. Starved fish also exhibited characteristic changes of the mucosa, including a finely granular cytoplasm replacing the supranuclear vacuoles seen in the epithelial cells of normal fish. In addition, a pattern of irregularly spaced indentations developed in the epithelium of the simple folds. The condition induced by dietary soybean meal was classified as a no n-infectious subacute enteritis, and a pathogenesis involving immunological mechanisms is suggested.