Ontogeny of the Digestive System of Atlantic Salmon (Salmo salar L.) and Effects of Soybean Meal from Start-Feeding

Despite a long history of rearing Atlantic salmon in hatcheries in Norway, knowledge of molecular and physiological aspects of juvenile development is still limited. To facilitate introduction of alternative feed ingredients and feed additives during early phases, increased knowledge regarding the ontogeny of the digestive apparatus in salmon is needed. In this study, we characterized the development of the gastrointestinal tract and accessory digestive organs for five months following hatch by using histological, biochemical and molecular methods. Furthermore, the effects of a diet containing 16.7% soybean meal (SBM) introduced at start-feeding were investigated, as compared to a fishmeal based control diet. Salmon yolk sac alevins and fry were sampled at 18 time points from hatch until 144 days post hatch (dph). Histomorphological development was investigated at 7, 27, 46, 54 and 144 dph. Ontogenetic expression patterns of genes encoding key digestive enzymes, nutrient transporters, gastrointestinal peptide hormones and T-cell markers were analyzed from 13 time points by qPCR. At 7 dph, the digestive system of Atlantic salmon alevins was morphologically distinct with an early stomach, liver, pancreas, anterior and posterior intestine. About one week before the yolk sac was internalized and exogenous feeding was started, gastric glands and developing pyloric caeca were observed, which coincided with an increase in gene expression of gastric and pancreatic enzymes and nutrient transporters. Thus, the observed organs seemed ready to digest external feed well before the yolk sac was absorbed into the abdominal cavity. In contrast to post-smolt Atlantic salmon, inclusion of SBM did not induce intestinal inflammation in the juveniles. This indicates that SBM can be used in compound feeds for salmon fry from start-feeding to at least 144 dph and/or 4-5 g body weight.     

Effects of diets containing soybean meal on trypsin mRNA expression and activity in Atlantic salmon (Salmo salar L)

Atlantic salmon develop subacute enteritis in the distal intestine (DI) when fed diets containing soybean meal (SBM) at high levels, a condition accompanied by increased trypsin activity in the DI intestinal content compared to fish fed conventional fishmeal (FM) based diets. To further investigate the responses of Atlantic salmon to dietary SBM, we measured trypsin activity in intestinal contents, quantified pancreatic trypsin mRNA expression, surveyed trypsin mRNA expression in selected tissues and characterized active forms of trypsin in the intestinal wall and brain. Enzyme measurements showed that trypsin activity in the intestinal content of SBM fed fish was lower in the proximal segments of the intestine, but higher in the DI compared to FM fed fish. The difference in enzyme activity was not reflected in a differential expression of pancreatic trypsin mRNA between fish fed the different diets (FM or SBM). Trypsin mRNA was expressed in 18 different tissues (esophagus, stomach, pancreas, pyloric tissue, midintestine, distal intestine, liver, head kidney, kidney, heart, spleen, thymus, brain, eye, gills, gonads, muscle and skin) but was most prominently expressed in tissues of the gastrointestinal (GI) tract and brain. We report for the first time an upregulation of trypsin-like activity in the DI wall using an in-gel trypsin activity assay, as well as modulated activity in the brain of fish fed SBM. The increased activity in the DI wall may contribute to disease severity and higher trypsin activity in the intestinal content.     

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.     

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 (200gkg(-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.     

Fermented Soybean Meal Increases Lactic Acid Bacteria in Gut Microbiota of Atlantic Salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

The main goal of the present study was to address the effect of feeding fermented soybean meal-based diet to Atlantic salmon on gut microbiota. Further, expression of genes of interest, including cathelicidin antimicrobial peptide (cath), mucin 2 (muc2), aquaporin (aqp8ab), and proliferating cell nuclear antigen (pcna), in proximal intestine of fish fed either experimental diet was analyzed. Three experimental diets, including a control fishmeal (30% FM), soybean meal (30% SBM), or fermented soybean meal diet (30% FSBM) were randomly assigned to triplicate tanks during a 50-day trial. The PCR-TTGE showed microbiota composition was influenced by experimental diets. Bands corresponding to genus Lactobacillus and Pediococcus were characteristic in fish fed the FSBM-based diet. On the other hand, bands corresponding to Isoptericola, Cellulomonas, and Clostridium sensu stricto were only observed in fish FM-based diet, while Acinetobacter and Altererythrobacter were detected in fish fed SBM-based diet. The expression of muc2 and aqp8ab were significantly greater in fish fed the FSBM-based diet compared with the control group. Our results suggest feeding FSBM to Atlantic salmon may (1) boost health and growth physiology in fish by promoting intestinal lactic acid bacteria growth, having a prebiotic-like effect, (2) promote proximal intestine health by increasing mucin production, and (3) boost intestinal trans-cellular uptake of water. Further research to better understands the effects of bioactive compounds derived from the fermentation process of plant feedstuff on gut microbiota and the effects on health and growth in fish is required.     

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.     

Effects of soybean meal and salinity on intestinal transport of nutrients in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss)

Groups of fresh- and seawater-adapted Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss) were fed diets with (SBM diet) or without (control diet) extracted soybean meal (30% of protein substituted with SBM) for 3 weeks. Average fish size per group ranged from 597 to 1763 g. One tank or net pen per species, dietary group and water salinity was used. In vitro nutrient transport (D-glucose, the L-amino acids aspartate, lysine, methionine, phenylalanine and proline, and the dipeptide glycyl-sarcosine) was measured using intact tissue (everted sleeve method) from the different postgastric intestinal regions. The dimensions of the different intestinal regions were also measured for each treatment group. Results indicate that SBM causes decreased carrier-mediated transport and increased permeability of distal intestinal epithelium for the nutrients, and the capacity of this region to absorb nutrient was diminished. Salinity may also affect the relative contribution of carrier-mediated and independent uptake to total nutrient absorption.     

Effects of dietary soybean meal and photoperiod cycle on osmoregulation following seawater exposure in Atlantic salmon smolts

Atlantic salmon Salmo salar juveniles were fed either fishmeal-based diets (FM) or diets in which soybean meal (SBM) partly replaced the FM from first feeding on. The fish were kept at continuous daylight during the juvenile stage. During the last 3 weeks before reaching 100 g body mass, all fish were subjected to 12L:12D. Starting at 100 g body mass, groups of 60 fish from each feeding background were subjected to continuous light for 12 weeks (short winter), or a square-wave photoperiod cycle to stimulate parr to smolt transformation with 8L:16D during the first 6 weeks, and then continuous light during the last 6 weeks (long winter). After the 12 weeks, 20 fish from each treatment were subjected to 0, 24 or 96 h seawater exposure at a water salinity of 34. Hypo-osmoregulatory ability at seawater exposure was assessed by mortality, intestinal pathology, plasma ion concentrations and osmolality, gill Na⁺/K⁺-ATPase activity and element concentrations in the cytoplasm of distal intestinal enterocytes using X-ray microanalysis. The hypo-osmoregulatory capacity was higher in fish kept at short winter than at long winter, apparently due to more rapid development of gill Na⁺/K⁺-ATPase activity. Fish fed SBM suffered typical soybean meal-induced histological alterations of the distal intestine and apparent reductions in digestive function in the more proximal gastrointestinal regions. The net osmoregulatory capacity of these fish was maintained, as indicated by higher gill Na⁺/K⁺-ATPase activity and lower plasma Na⁺, Ca²⁺ and osmolality compared to the FM-fed fish. Thus, feeding SBM did not impair the hypo-osmoregulatory ability of the Atlantic salmon following seawater exposure.     

Haemato-immunological and growth response of mirror carp (Cyprinus carpio) fed a tropical earthworm meal in experimental diets

An investigation was conducted to evaluate the effect of feeding a tropical earthworm meal (Perionyx escavatus) on the haemato-immunological response and growth performance of mirror carp (Cyprinus carpio). Fish were fed diets for a total of 88 days, fishmeal served as the main protein source in the control diet. Two remaining diets consisted of fishmeal fixed at 33.65% provision of protein and the remaining 66.35% protein was provided by soybean meal (SBM diet) or P. excavatus meal (EW diet). Compared to control and SBM fed fish (7.69 ± 0.28 and 5.92 ± 0.31 g/dl, respectively), a significant increase in haemoglobin was measured in EW fed fish (9.57 ± 0.24 g/dl). Consequently significant elevations were also observed in mean corpuscular haemoglobin (MCH; 79.13 ± 4.59 pg) and mean corpuscular haemoglobin concentration (MCHC; 22.69 ± 0.54 pg) in EW fed fish. On the contrary, compared to control and SBM fed carp total leukocyte levels (2.72 ± 0.17 and 3.10 ± 0.17 × 10(4)/mm(3), respectively) were significantly decreased in the EW group (2.15 ± 0.14 × 10(4)/mm(3)). Moreover at day 14 and 21 post immunisation with bacterin isolated from Aeromonas hydrophila fish fed the EW diet displayed a significant reduction in respiratory burst activity (RBA) compared to control and SBM fed fish. After 60 days of feeding, fish fed EW diet showed a significant elevation in final body weight compared to fish fed a fishmeal based diet (control treatment) and fish fed a soybean meal based diet. Similar improvements were observed in feed utilisation efficiency. The present study shows that feeding P. excavatus meal to mirror carp decreases some aspects of the innate immune response, but at the same time gives rise to significant enhancement of growth and feed utilisation efficiency.     

Growth performance and gut function of piglets weaned at four weeks of age and fed protease-treated soya-bean meal

Two experiments were carried out to investigate the effects of the protease-treatment of soya-bean meal (SBM) on its composition and nutritive value when fed to piglets weaned at 4 weeks of age in cereal-based diets. In experiment 1, two proteases, active at either alkaline (protease P1) or acid pH (protease P2), were compared with alkali or acid treatment alone. In experiment 2, treatment with protease P2 at acid pH was compared with untreated-SBM, acid-treated SBM, a soya protein concentrate or skim milk powder+fishmeal. Protease treatment increased SBM alpha amino nitrogen concentrations and reduced in vitro SBM antigenicity. Protease P1 reduced in vitro antigenicity to a greater extent than protease P2. However, protease P1 hydrolysed SBM polypeptides non-specifically whereas protease P2 preferentially hydrolysed a high molecular weight polypeptide. In experiment 1, piglets (12 diet⁻¹) fed alkali-treated SBM gained more weight than piglets fed acid-treated SBM in the first 7 days after weaning. Protease P1 did not improve live-weight gain compared with alkali-treatment whereas protease P2 did improve live-weight gain compared with acid treatment. In experiment 2, piglets (24 diet⁻¹) fed protease P2-treated SBM or skim milk powder+fishmeal gained significantly more weight over the first 7 days post-weaning than piglets fed untreated SBM. In neither experiment were there significant differences between diets in piglet serum anti-SBM specific antibodies. There were no consistent changes in small intestine enzyme specific activities in response to the diets fed. Histopathological examination of the small intestine of piglets in experiment 2 revealed no evidence of enteric disease. Neither were there any differences between diets in villus height or crypt depth.     

Breeding salmonids for feed efficiency in current fishmeal and future plant-based diet environments

The aquaculture industry is increasingly replacing fishmeal in feeds for carnivorous fish with soybean meal (SBM). This diet change presents a potential for genotype-environment (G × E) interactions. We tested whether current salmonid breeding programmes that evaluate and select within fishmeal diets also improve growth and efficiency on potential future SBM diets. A total of 1680 European whitefish from 70 families were reared with either fishmeal- or SBM-based diets in a split-family design. Individual daily gain (DG), daily feed intake (DFI) and feed efficiency (FE) were recorded. Traits displayed only weak G × E interactions as variances and heritabilities did not differ substantially between the diets, and cross-diet genetic correlations were near unity. In both diets, DFI exhibited moderate heritability and had very high genetic correlation with DG whereas FE had low heritability. Predicted genetic responses demonstrated that selection to increase DG and FE on the fishmeal diet lead to favourable responses on the SBM diet. Selection for FE based on an index including DG and DFI achieved at least double FE gain versus selection on DG alone. Therefore, current breeding programmes are improving the biological ability of salmonids to use novel plant-based diets, and aiding the aquaculture industry to reduce fishmeal use.     

Effects of Cortisol on the Intestinal Mucosal Immune Response during Cohabitant Challenge with IPNV in Atlantic Salmon (Salmo salar)

Infectious pancreatic necrosis virus (IPNV) causes high incidence of disease in salmonids during the first period after SW transfer. During this period as well as during periods of stress, cortisol levels increase and indications of a relationship between IPNV susceptibility and cortisol have been suggested. The intestine is an entry route and a target tissue for IPNV displaying severe enteritis and sloughing of the mucosa in infected fish. The mechanisms behind effects of the virus on the intestinal tissue and the impact of cortisol on the effect remain unclear. In the present study, Atlantic salmon post smolts treated with or without slow release cortisol implants were subjected to a cohabitant IPNV challenge. Analysis of genes and proteins related to the innate and acquired immune responses against virus was performed 6 days post-challenge using qPCR and immunohistochemistry. An increased mRNA expression of anti-viral cytokine interferon type I was observed in the proximal intestine and head kidney as a response to the viral challenge and this effect was suppressed by cortisol. No effect was seen in the distal intestine. T-cell marker CD3 as well as MHC-I in both intestinal regions and in the head kidney was down regulated at the mRNA level. Number of CD8α lymphocytes decreased in the proximal intestine in response to cortisol. On the other hand, mRNA expression of Mx and IL-1β increased in the proximal intestine and head kidney in IPNV challenged fish in the presence of cortisol suggesting that the immune activation shifts in timing and response pathway during simulated stress. The present study clearly demonstrates that IPNV infection results in a differentiated epithelial immune response in the different intestinal regions of the Atlantic salmon. It also reveals that the epithelial immune response differs from the systemic, but that both are modulated by the stress hormone cortisol.     

Nutrient digestibility in Atlantic salmon and broiler chickens related to viscosity and non-starch polysaccharide content in different soyabean products

Total tract digestibility in Atlantic salmon and ileal digestibility in chicken were assessed from diets with different soyabean products (hulled, toasted, extracted, SBM; reduced oligosaccharide content, ROM; ethanol-extracted protein concentrate, SPC; isolated protein, ISP). The concentration of dietary fibre was highest in SBM and ROM, while it was low in ISP. In vitro viscosity was also higher in SBM than in the other soyabean products. The diets for the salmon and chickens were based on the same feed ingredients, with the exception that fish meal provided half the crude protein in the salmon diets. For each species, the diets were isonitrogenous, contained similar amounts of fat (fish oil), and were balanced with dextrin, thus substituting soyabean non-starch polysaccharides (NSP) and other non-proteinous components by dextrin.In the salmon, total tract digestibility of nitrogen and dry matter were lower (p < 0.05) with the SBM and ROM diets than with the ISP diet. In the chickens, ileal digestibility of dry matter differed among all treatments (p < 0.05). Digestibility of nitrogen, starch, phosphorus and calcium in the chickens was lower (p < 0.05) with the SBM diet than with the ISP and SPC diets, and fat digestibility was lower (p < 0.05) with the SBM and ROM diets than with the SPC diet. Also, the SBM diet gave lower (p < 0.05) digestibility of nitrogen, phosphorus and calcium than the ROM diet, while the ROM diets gave lower (p < 0.05) digestibilities of nitrogen, fat and starch than the ISP and SPC diets. Intestinal viscosity was higher (p < 0.05) and cholesterol content in the blood was lower (p < 0.05) for the chickens fed the SBM-based diet than those fed the other diets. There was a similar response to the different soy products in salmon and chickens with regard to digestibility of nitrogen and fat (p < 0.05). The negative effects of soyabean meals with a low protein content could indicate antinutritive effects of NSP fractions in soyabean meal, as indicated by the elevated viscosity and the decrease in blood cholesterol content.     

Effects of dietary supplementation of keratinase on growth performance, nitrogen retention and intestinal morphology of broiler chickens fed diets with soybean and cottonseed meals

A preparation of keratinase (KE, 400,000 enzyme units/g) was evaluated as a feed additive for its effects on growth performance, nutrient digestibility and intestinal development in broiler chickens. Four experimental diets were arranged as a 2 × 2 factorial with two sources of protein, either soybean meal (SBM) alone or a combination of SBM and cottonseed meal (1:1 on crude protein basis) and with or without 1.0 g KE/kg diet. Dietary supplementation of KE increased body weight gain from 1 to 21 days of age and improved feed conversion ratio from 1 to 42 days of age. Also, KE supplementation enhanced nitrogen retention (P<0.001) and starch digestibility (P<0.001), reflecting the high level of KE and some amylase activities in the enzyme preparation. Microscopic examination of the small intestine on day 21 indicated that KE increased the ratio of villus height to crypt depth (P<0.05). The results suggest that KE has positive effects on growth performance, dietary component digestibility and intestinal development of broilers.     

Soybean meal‐induced uptake block in Atlantic salmon Salmo salar distal enterocytes

Soybean meal‐induced enteritis was characterized by the disappearance of the supranuclear vacuoles (SNV) in the enterocytes of the distal intestine of Atlantic salmon Salmo salar. The loss of SNV was correlated with an endocytosis uptake block as shown with ferritin as a marker molecule.