Low Omega-3 Levels in the Diet Disturbs Intestinal Barrier and Transporting Functions of Atlantic Salmon Freshwater and Seawater Smolts

Due to a limited access to marine raw materials from capture fisheries, Atlantic salmon feeds are currently based on mainly plant ingredients (75%) while only 25% come from traditional marine ingredients including marine fish meal and fish oil. Thus, current feeds contain less of the essential omega-3 fatty acids. The aim of the study was to assess the impact of different omega-3 levels in fish feed on intestinal barrier and transporting functions of Atlantic salmon freshwater and seawater smolts. Atlantic salmon were fed three levels of omega-3 (2, 1 and 0.5%) and fish performance was followed through smoltification and the subsequent seawater acclimation. Intestinal barrier and transporting functions were assessed using Ussing chamber methodology and combined with transcript analysis of tight junction related proteins and ion transporters. A linear decrease in growth was observed with decreasing omega-3 levels. Low (0.5%) inclusion of omega-3 impaired the barrier function of the proximal intestine compared to 2% inclusion. Further, low levels of omega-3 decrease the transepithelial electrical potential across the epithelium indicating disturbed ion transport. It can be concluded that low dietary levels of omega-3 impair somatic growth and intestinal function of Atlantic salmon.publishedVersio

Haematological and intestinal health parameters of rainbow trout are influenced by dietary live yeast and increased water temperature

Live yeast may be a sustainable protein source in salmonid diets while exhibiting a probiotic effect to counteract environmental stressors, such as increased water temperature that is being exacerbated by climate change. The objective of this study was to evaluate the effects of feeding a high dietary inclusion of live yeast and increased water temperature on growth, haematological and intestinal physiology of rainbow trout. For six weeks, 129 g fish in 16 tanks (n = 4) were fed either a diet based on fishmeal or based on live yeast (214 g kg−1 of diet or 7.6 log CFU g−1 of Saccharomyces cerevisiae) that replaced 40% of fishmeal protein while fish were reared in water temperatures of either 11 °C (cold) or 18 °C (warm). Fish weights, caudal blood and proximal and distal intestines were collected and analysed. Fish fed live yeast resulted in reduced growth (SGR and WG) and higher FCR, while growth in cold and warm water was similar despite differences in TGC. However, increased mortality, plasma cortisol, and intestinal oedema and villous damage indicated fish reared in warm water were subjected to chronic stress. Temperature had a significant effect on haematocrit and red blood cell counts that resulted in significantly higher haemoglobin levels in fish kept in warm water attributed to an elevated oxygen demand. In the proximal intestine, increased temperature resulted in reduced expression of pro-inflammatory cytokines, e.g. TNFα and IL8, that were further reduced in fish fed live yeast. In addition, feeding live yeast reduced gene expression of CLD6 involved in gut barrier function, which suggests that the level of yeast was too high and masked any beneficial effects on fish health. In conclusion, feeding a high inclusion of live yeast reduced fish growth and expression of intestinal genes, while increasing the temperature from 11 to 18 °C subjected fish to chronic stress that restricted growth, suppressed innate immunity and induced intestinal damage. Replacing 40% of fishmeal protein with live yeast did not counteract negative effects caused by increased temperature, thus alternative strategies need to be explored and implemented to protect the growth and health of rainbow trout from seasonal and long-term rises in water temperature

Low intestinal inflammation model (HP48) in Atlantic salmon (Salmo salar) and inflammatory mitigation by Bactocell

Moderate levels of intestinal damage and inflammation are often seen in intensive fish aquaculture. The causes may be due to antinutrients from plant meals, stress or other causes. There is currently a lack of good models to explore these effects and so how to mitigate the consequences. Most studies have used full-fat soy or other compounds that cause intestinal damage that are likely not reversible. In this study we have explored the possibility of using soybean HP48, made from solvent extracted peeled soybeans, as a low-inflammation model in post-smolt Atlantic salmon, and then investigated whether supplementation of the probiotic Pediococcus acidilactici CNCM I-4622 – MA 18/5 M (Bactocell) could diminish this effect. The fish were fed triplicate diets. A Control diet containing 18.08% soy protein concentrate (SPC), a HP48 diet where most of the SPC was replaced by HP48 (5.00% SPC and 17.68% HP48), and a Bactocell diet that was identical to the HP48 diet but contained 0.03% Bactocell. After 10 weeks of feeding, the mid- and hind-intestinal health were assessed by histology, integrity (Ussing chamber) and gene expression (RNAseq). Transcriptomic and integrity data suggests that HP48 led to a disturbed mid-intestinal homeostasis with impaired cellular integrity and increased inflammation and cell turnover. Most of the transcriptomic effects were reversed with Bactocell including downregulation of immune genes and upregulation of transmembrane proteins such as type IV collagen, which is important in restoring epithelial homeostasis. In the hind-intestine, the HP48 diet led to deleterious morphological changes such as widening of lamina propria and stratum granulosum, disrupted mucosal folds, loss of absorptive vacuoles, and upregulation of several immune regulated genes and downregulation of genes involved in solute- and water transport. The intestinal integrity assessed by Ussing chamber was not affected. Bactocell supplementation did alleviate several of the morphological effects. However, it was not able to completely reverse the expression of immune- or transport related genes, suggesting a higher effect of probiotic supplement in the mid-intestine compared to the hind-intestine. This study demonstrates that the level of HP48 used here is sufficient to create low-level intestinal changes in Atlantic salmon, which is within range for functional feed ingredients to reverse.publishedVersio

Intestinal barrier function of Atlantic salmon (Salmo salar L.) post smolts is reduced by common sea cage environments and suggested as a possible physiological welfare indicator

<p>Abstract</p> <p>Background</p> <p>Fish farmed under high intensity aquaculture conditions are subjected to unnatural environments that may cause stress. Therefore awareness of how to maintain good health and welfare of farmed fish is important. For Atlantic salmon held in sea cages, water flow, dissolved oxygen (DO) levels and temperature will fluctuate over time and the fish can at times be exposed to detrimentally low DO levels and high temperatures. This experimental study investigates primary and secondary stress responses of Atlantic salmon post smolts to long-term exposure to reduced and fluctuating DO levels and high water temperatures, mimicking situations in the sea cages. Plasma cortisol levels and cortisol release to the water were assessed as indicators of the primary stress response and intestinal barrier integrity and physiological functions as indicators of secondary responses to changes in environmental conditions.</p> <p>Results</p> <p>Plasma cortisol levels were elevated in fish exposed to low (50% and 60% saturation) DO levels and low temperature (9°C), at days 9, 29 and 48. The intestinal barrier function, measured as electrical resistance (TER) and permeability of mannitol at the end of the experiment, were reduced at 50% DO, in both proximal and distal intestine. When low DO levels were combined with high temperature (16°C), plasma cortisol levels were elevated in the cyclic 1:5 h at 85%:50% DO group and fixed 50% DO group compared to the control (85% DO) group at day 10 but not at later time points. The intestinal barrier function was clearly disturbed in the 50% DO group; TER was reduced in both intestinal regions concomitant with increased paracellular permeability in the distal region.</p> <p>Conclusions</p> <p>This study reveals that adverse environmental conditions (low water flow, low DO levels at low and high temperature), that can occur in sea cages, elicits primary and secondary stress responses in Atlantic salmon post smolts. The intestinal barrier function was significantly affected by prolonged hypoxic stress even when no primary stress response was observed. This suggests that intestinal barrier function is a good experimental marker for evaluation of chronic stress and that it can be a valuable tool to study the impact of various husbandry conditions on health and welfare of farmed Atlantic salmon.</p

Marine yeast (Candida sake) cultured on herring brine side streams is a promising feed ingredient and omega-3 source for rainbow trout (Oncorhynchus mykiss)

A major challenge for the aquaculture industry is the supply of sustainable feeds. A promising model to achieve this is to utilize circular flows where feed ingredients, such as single cell protein, are cultivated using side streams of the food industry. The aim of this study was to evaluate the marine yeast Candida sake, produced on herring brine side streams, as a source of protein and immune stimulant in feed for salmonid fish. The dry C. sake product contained 54% protein (3.3% lysine and 0.8% methionine) and 13% lipids (1.1% eicosapentaenoic, EPA, and 1% docosahexaenoic acid, DHA). Four experimental diets were designed and tested in a 9-week feeding trial using juvenile rainbow trout (Oncorhynchus mykiss). A control diet containing both fish and plant-based ingredients constituted the base feed to which 20% (to evaluate effects on digestibility, growth and intestinal physiology), 20% heat-treated (to evaluate effects of downstream processing) and 3% (to evaluate immune stimulatory properties, replacing 3% soy protein concentrate) C. sake was added. The apparent digestibility coefficient of C. sake for protein, fat and gross energy was above 80%, and for amino acids above 90% regardless of treatment, suggesting a high bioavailability of C. sake. All three yeast containing diets performed equally to the control regarding specific growth rate, feed conversion ratio and functional intestinal health. These results suggest that C. sake is a promising alternative protein source for circular feeds in the salmonid industry. The presence of EPA and DHA represents an added value. The heat treatment increased the apparent digestibility coefficient of dry matter by 8% but decreased amino acid digestibility by on average 3%, indicating that heat treatment may not be the optimal downstream processing technique. Furthermore, the inclusion of 3% C. sake increased the intestinal lamina propria width and TGF-β transcription, indicating an immune stimulating effect. Future research is needed to understand these immune modulatory effects of C. sake supplementation

Intestinal health in Atlantic salmon post-smolt (Salmo salar) when fed low- and high HUFA diets

It is well established that farmed Atlantic salmon (Salmo salar) need n3-highly unsaturated fatty acids (HUFA) in their diet to thrive and grow. However, the biological functions to the individual HUFAs may differ, implying that future supplementation could require fixed ratios for maximum benefit. The intestinal barrier is essential to f ish health, and any disruption of the barrier can have detrimental effects. The current experiment was designed to examine the response of the intestinal hindgut when fed a low HUFA diet with 8.5 g/kg EPA + DHA (4.5% total fatty acid) or two high HUFA diets, with either high DHA (28.7 g/kg and 5.9% total fatty acid) or high EPA (25 g/kg and 14.2% total fatty acid). The diets were fed to Atlantic salmon post-smolt over 10 weeks and thereafter exposed to 3 weeks of chronic stress. After 10 weeks of feeding there were no differences in intestinal permeability and integrity, but intestinal morphology indicated increased intestinal health in the high EPA group. Gene expression also suggest that fish fed the high EPA diet had more regulation of pathways related to protein turnover compared to the high DHA fed fish. There was also indication of lower energy utilization in the low HUFA fed fish than high HUFA. Subjecting fish to 3 weeks of chronic stress led to a reduction in transepithelial resistance, increased ion flux and active L-lysine transport across the intestinal barrier in addition to a decrease in mucosal fold, enterocyte height and supranuclear vacuole density and an increase in thickness of the intestinal muscularis. After stress, the low HUFA group showed signs of inflammation with increased infiltration of MHCII positive cells. Gene expression also showed that low HUFA fed fish had a lower response to chronic stress compared to the high HUFA groups. Comparing fish fed either high DHA or EPA exposed to chronic stress showed few physical effects, but a lower density of supranuclear vacuoles and upregulation in immune-related gene expression indicate inflammation in the high DHA group.publishedVersio

Marine yeast (Candida sake) cultured on herring brine side streams is a promising feed ingredient and omega-3 source for rainbow trout (Oncorhynchus mykiss)

A major challenge for the aquaculture industry is the supply of sustainable feeds. A promising model to achieve this is to utilize circular flows where feed ingredients, such as single cell protein, are cultivated using side streams of the food industry. The aim of this study was to evaluate the marine yeast Candida sake, produced on herring brine side streams, as a source of protein and immune stimulant in feed for salmonid fish. The dry C. sake product contained 54% protein (3.3% lysine and 0.8% methionine) and 13% lipids (1.1% eicosapentaenoic, EPA, and 1% docosahexaenoic acid, DHA). Four experimental diets were designed and tested in a 9-week feeding trial using juvenile rainbow trout (Oncorhynchus mykiss). A control diet containing both fish and plant-based ingredients constituted the base feed to which 20% (to evaluate effects on digestibility, growth and intestinal physiology), 20% heat-treated (to evaluate effects of downstream processing) and 3% (to evaluate immune stimulatory properties, replacing 3% soy protein concentrate) C. sake was added. The apparent digestibility coefficient of C. sake for protein, fat and gross energy was above 80%, and for amino acids above 90% regardless of treatment, suggesting a high bioavailability of C. sake. All three yeast containing diets performed equally to the control regarding specific growth rate, feed conversion ratio and functional intestinal health. These results suggest that C. sake is a promising alternative protein source for circular feeds in the salmonid industry. The presence of EPA and DHA represents an added value. The heat treatment increased the apparent digestibility coefficient of dry matter by 8% but decreased amino acid digestibility by on average 3%, indicating that heat treatment may not be the optimal downstream processing technique. Furthermore, the inclusion of 3% C. sake increased the intestinal lamina propria width and TGF-beta transcription, indicating an immune stimulating effect. Future research is needed to understand these immune modulatory effects of C. sake supplementation

Health of farmed fish: its relation to fish welfare and its utility as welfare indicator

This brief review focuses on health and biological function as cornerstones of fish welfare. From the function-based point of view, good welfare is reflected in the ability of the animal to cope with infectious and non-infectious stressors, thereby maintaining homeostasis and good health, whereas stressful husbandry conditions and protracted suffering will lead to the loss of the coping ability and, thus, to impaired health. In the first part of the review, the physiological processes through which stressful husbandry conditions modulate health of farmed fish are examined. If fish are subjected to unfavourable husbandry conditions, the resulting disruption of internal homeostasis necessitates energy-demanding physiological adjustments (allostasis/acclimation). The ensuing energy drain leads to trade-offs with other energy-demanding processes such as the functioning of the primary epithelial barriers (gut, skin, gills) and the immune system. Understanding of the relation between husbandry conditions, allostatic responses and fish health provides the basis for the second theme developed in this review, the potential use of biological function and health parameters as operational welfare indicators (OWIs). Advantages of function- and health-related parameters are that they are relatively straightforward to recognize and to measure and are routinely monitored in most aquaculture units, thereby providing feasible tools to assess fish welfare under practical farming conditions. As the efforts to improve fish welfare and environmental sustainability lead to increasingly diverse solutions, in particular integrated production, it is imperative that we have objective OWIs to compare with other production forms, such as high-density aquaculture. However, to receive the necessary acceptance for legislation, more robust scientific backing of the health- and function-related OWIs is urgently neede

Stress Impairs Skin Barrier Function and Induces α2-3 Linked N-Acetylneuraminic Acid and Core 1 O-Glycans on Skin Mucins in Atlantic Salmon, Salmo salar

The skin barrier consists of mucus, primarily comprising highly glycosylated mucins, and the epithelium. Host mucin glycosylation governs interactions with pathogens and stress is associated with impaired epithelial barrier function. We characterized Atlantic salmon skin barrier function during chronic stress (high density) and mucin O-glycosylation changes in response to acute and chronic stress. Fish held at low (LD: 14–30 kg/m3) and high densities (HD: 50-80 kg/m3) were subjected to acute stress 24 h before sampling at 17 and 21 weeks after start of the experiment. Blood parameters indicated primary and secondary stress responses at both sampling points. At the second sampling, skin barrier function towards molecules was reduced in the HD compared to the LD group (Papp mannitol; p < 0.01). Liquid chromatography–mass spectrometry revealed 81 O-glycan structures from the skin. Fish subjected to both chronic and acute stress had an increased proportion of large O-glycan structures. Overall, four of the O-glycan changes have potential as indicators of stress, especially for the combined chronic and acute stress. Stress thus impairs skin barrier function and induces glycosylation changes, which have potential to both affect interactions with pathogens and serve as stress indicators. View Full-TextpublishedVersio

Dietary replacement of fishmeal with marine proteins recovered from shrimp and herring process waters promising in Atlantic salmon aquaculture

There is a general agreement that fish meal (FM) and fish oil (FO) are valuable resources for aquafeed, but that the production cannot keep the same pace as the current growth of the aquaculture industry. Therefore, there is a need to find alternative sources for lipids and protein. This study examines the possibility of using proteins recovered from seafood industry side stream waters as a complement to FM in feed for Atlantic salmon. To recover the proteins, herring and shrimp process side streams waters were flocculated then treated with dissolved air flotation (DAF), which is considered a gentle technology. Shrimp steam water was used to obtain shrimp protein (SP) and herring salt brine to obtain herring protein (HP). The recovered semi-solid protein fraction was spray dried and formulated into experimental diets at inclusion levels of 1.9–10% (dw/dw). Two feeding trials were conducted to investigate; 1) the properties of different flocculants to recover SP from shrimp steam water, alginate (Alg; 1.9% inclusion), carrageenan (Carr; 2.0% inclusion) and a synthetic flocculant from Kemira (Kem; 3.8% inclusion). 2) total or partial replacement of FM with SP-Alg (10% and no FM) and HP-Alg (3% and 8% FM). For both feeding trials each diet was provided to triplicate tanks (n = 31 and 30/tank) of Atlantic salmon with a start weight of 193 g (duration 10 weeks) and 304 g (duration 7 weeks) respectively. All fish showed similar feed intake, feed conversion ratio (on tank basis) and weight gain. The inclusion of SP or HP as total or partial replacement of FM did neither influence adiposity of the fish, as measured by condition factor, nor heposomatic index (HSI). No negative effect of the alternative protein could be found through histological examination of the intestine. In feeding trial two, diets did not affect the adaptive immune indicators CD8α and MHC II. The SP-Alg diet did not affect intestinal barrier and transporting functions, assessed using Ussing-chamber technology. However, HP-Alg affected the trans-epithelial resistance, which indicate that the intestinal barrier function could be affected by low inclusions. We conclude that from a biological perspective, SP recovered from shrimp steaming waters using Alg and DAF technology represents a new marine biomass with potential as a replacement for FM in Atlantic salmon feed. To diversify the possibility of using flocculants to retrieve proteins we also suggest further investigation of the potential to use Carr in larger inclusions