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Background The aquaculture sector now accounts for almost 50% of all fish for human consumption and is anticipated to provide 62% by 2030. Innovative strategies are being sought to improve fish feeds and feed additives to enhance fish performance, welfare, and the environmental sustainability of the aquaculture industry. There is still a lack of knowledge surrounding the importance and functionality of the teleost gut microbiome in fish nutrition. In vitro gut model systems might prove a valuable tool to study the effect of feed, and additives, on the host’s microbial communities. Several in vitro gut models targeted at monogastric vertebrates are now in operation. Here, we report the development of an Atlantic salmon gut model, SalmoSim, to simulate three gut compartments (stomach, pyloric caecum, and midgut) and associated microbial communities. Results The gut model was established in a series of linked bioreactors seeded with biological material derived from farmed adult marine-phase salmon. We first aimed to achieve a stable microbiome composition representative of founding microbial communities derived from Atlantic salmon. Then, in biological triplicate, the response of the in vitro system to two distinct dietary formulations (fishmeal and fishmeal free) was compared to a parallel in vivo trial over 40 days. Metabarcoding based on 16S rDNA sequencing qPCR, ammoniacal nitrogen, and volatile fatty acid measurements were undertaken to survey the microbial community dynamics and function. SalmoSim microbiomes were indistinguishable (p = 0.230) from their founding inocula at 20 days and the most abundant genera (e.g., Psycrobacter, Staphylococcus, Pseudomonas) proliferated within SalmoSim (OTUs accounting for 98% of all reads shared with founding communities). Real salmon and SalmoSim responded similarly to the introduction of novel feed, with majority of the taxa (96% Salmon, 97% SalmoSim) unaffected, while a subset of taxa (e.g., a small fraction of Psychrobacter) was differentially affected across both systems. Consistent with a low impact of the novel feed on microbial fermentative activity, volatile fatty acid profiles were not significantly different in SalmoSim pre- and post-feed switch. Conclusion By establishing stable and representative salmon gut communities, this study represents an important step in the development of an in vitro gut system as a tool for the improvement of fish nutrition and welfare. The steps of the system development described in this paper can be used as guidelines to develop various other systems representing other fish species. These systems, including SalmoSim, aim to be utilised as a prescreening tool for new feed ingredients and additives, as well as being used to study antimicrobial resistance and transfer and fundamental ecological processes that underpin microbiome dynamics and assembly.

In this paper we report the results of the impact of a prebiotic (alpha-MOS supplementation) on microbial communities, using anin vitrosimulator of the gut microbial environment of the Atlantic salmon. Our data suggest that Bio-Mos may be of value in salmonid production as it enhances volatile fatty acid production by the microbiota from salmon pyloric ceca and correlates with a significant shift in microbial community composition with observed increase in lactic acid producingCarnobacterium.

Alpha mannose-oligosaccharide (MOS) prebiotics are widely deployed in animal agriculture as immunomodulators as well as to enhance growth and gut health. Their mode of action is thought to be mediated through their impact on host microbial communities and their associated metabolism. Bio-Mos is a commercially available prebiotic currently used in the agri-feed industry, but studies show contrasting results of its effect on fish performance and feed efficiency. Thus, detailed studies are needed to investigate the effect of MOS supplements on the fish microbiome to enhance our understanding of the link between MOS and gut health. To assess Bio-Mos for potential use as a prebiotic growth promoter in salmonid aquaculture, we have modified an established Atlantic salmon

A good understanding of the utilisation of energy in fish diets is important for accurate feed formulation in aquaculture. One of the primary reasons for differences in the utilisation of digestible energy between fish species are differences that exist in the composition of growth (fat versus protein gain). However, it has also been observed that composition of growth can differ between genetic strains within a single fish species. The main focus of the current experiment was to investigate whether the genetic background of different strains of rainbow trout affects the relationship between digestible energy intake and retained energy. To test this, two different commercial trout strains were selected based on their differences in body fat content (a Lean-Strain and Fat-Strain) and therefore expected differences in composition of growth. Furthermore, this research investigated whether such a potential strain difference in the relationship between digestible energy and retained energy was dependent on the type of non-protein energy in the diet (a Carb-Diet versus a Fat-Diet). Three feeding levels were used in order to estimate the utilisation efficiency of digestible energy for retained energy, leading to a 2 by 2 by 3 factorial design. The results of this study showed that the relationship between digestible energy and retained energy was affected by both strain and diet, but not by an interaction effect between these two factors. Firstly, it was observed that the utilisation efficiency of digestible energy for growth (kgDE) was higher in the Fat-Strain (72% in the Lean-Strain versus 87% in the Fat-Strain) which may be related to a higher potential for fat deposition. This higher kgDE in the Fat-Strain was however balanced by a higher maintenance requirement (47 kJ/kg0.8 per day versus 28 kJ/kg0.8 per day) leading to a similar retained energy between strains in the current trial. Secondly, it was shown that the exchange of dietary carbohydrates for dietary fat on an isoenergetic basis also increased kgDE (74% for the Carb-Diet versus 85% for the Fat-Diet). The lack of an interaction effect between strain and diet showed that kgDE in both strains was affected by the exchange of carbohydrates for fat on an isoenergetic basis in a similar way. The results of the current trial demonstrated that both dietary macronutrient composition and the composition of growth of specific trout strains should be accounted for in calculating the true net available energy for fish in feed formulation.

Mannose-oligosaccharide (MOS) pre-biotics are widely deployed in animal agriculture as immunomodulators as well as to enhance growth and gut health. Their mode of action is thought to be mediated through their impact on host microbial communities and the associated metabolism. Bio-Mos is a commercially available pre-biotic currently used in the agri-feed industry. To assess Bio-Mos for potential use as a prebiotic growth promotor in salmonid aquaculture, we have modified an established Atlantic salmonin vitrogut model, SalmoSim, to evaluate its impact on the host microbial communities. Inoculated from biological triplicates of adult farmed salmon pyloric caeca compartments, the microbial communities were stabilised in SalmoSim followed by a twenty-day exposure to the prebiotic and in turn followed by an eight day ‘wash out’ period. Dietary inclusion of MOS resulted in a significant increase in formate (p=0.001), propionate (p=0.037) and isovalerate (p=0.024) levels, correlated with increased abundances of several, principally, anaerobic microbial genera (Fusobacterium,Agarivorans,Pseudoalteromonas). DNA metabarcoding with the 16S rDNA marker confirmed a significant shift in microbial community composition in response to MOS supplementation with observed increase in lactic acid producingCarnobacterium. In conjunction with previousin vivostudies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggests that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role ofin vitrogut models to augmentin vivotrials of microbiome modulators.ImportanceIn this paper we report the results of the impact of prebiotic (MOS supplementation) on microbial communities within recently developed Atlantic salmon gut microbiomein vitrosimulator. Our data suggest that Bio-Mos may be of value in salmonid production as it enhances volatile fatty acid production in the Atlantic salmon gut and correlates with a significant shift in microbial community composition with observed increase in lactic acid producingCarnobacterium. In conjunction with previousin vivostudies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggest that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role ofin vitrogut models to augmentin vivotrials of microbiome modulators.

Accurate feed evaluation is important for feed formulation in aquaculture. In this study a net energy (NE) evaluation method was tested which recognises differences in the utilisation of the different macro-nutrients (protein, fat and carbohydrates) for growth. Therefore, a feeding trial was performed with feeds that differed in energy level and with a wide contrast in macronutrient composition, reflecting those found in practical rainbow trout diets. The trial was set up in a 2 by 4 factorial design which included two protein levels (CP400 and CP500) and four different fat levels (CF140, CF190, CF240, CF290) coinciding in a wide range in carbohydrate levels. These differences were achieved by starting from a basal high protein and fat diet where protein and or fat were replaced by carbohydrates. These 8 diets were then used to assess the prediction precision of growth performance by a NE approach as compared to a digestible energy (DE) approach in feed evaluation for rainbow trout. The results of the current study showed that replacing fat by carbohydrates reduced the utilisation of energy for growth. This effect was stronger at the highest carbohydrates levels which indicated a curvilinear response in the utilisation of carbohydrates. However in terms of the prediction precision of growth rate an improvement was only shown when using a NE approach using linear efficiencies of digestible macronutrients (R2 = 0.890 compared to R2 = 0.848 for the DE approach). A NE approach assuming a curvilinear effect of carbohydrates did not lead to an improved prediction of growth rate in the current study (R2 = 0.617).

The first part of this study evaluates the occurrence of mycotoxin patterns in feedstuffs and fish feeds. Results were extrapolated from a large data pool derived from wheat (n = 857), corn (n = 725), soybean meal (n = 139) and fish feed (n = 44) samples in European countries and based on sample analyses by liquid chromatography/tandem mass spectrometry (LC-MS/MS) in the period between 2012–2019. Deoxynivalenol (DON) was readily present in corn (in 47% of the samples) > wheat (41%) > soybean meal (11%), and in aquafeeds (48%). Co-occurrence of mycotoxins was frequently observed in feedstuffs and aquafeed samples. For example, in corn, multi-mycotoxin occurrence was investigated by Spearman’s correlations and odd ratios, and both showed co-occurrence of DON with its acetylated forms (3-AcDON, 15-AcDON) as well as with zearalenone (ZEN). The second part of this study summarizes the existing knowledge on the effects of DON on farmed fish species and evaluates the risk of DON exposure in fish, based on data from in vivo studies. A meta-analytical approach aimed to estimate to which extent DON affects feed intake and growth performance in fish. Corn was identified as the ingredient with the highest risk of contamination with DON and its acetylated forms, which often cannot be detected by commonly used rapid detection methods in feed mills. Periodical state-of-the-art mycotoxin analyses are essential to detect the full spectrum of mycotoxins in fish feeds aimed to prevent detrimental effects on farmed fish and subsequent economic losses for fish farmers. Because levels below the stated regulatory limits can reduce feed intake and growth performance, our results show that the risk of DON contamination is underestimated in the aquaculture industry.

Atlantic salmon are a species of major economic importance. Intense innovation is underway to improve salmon feeds and feed additives to enhance fish performance, welfare, and the environmental sustainability of the industry. Several gut models targeted at monogastric vertebrates are now in operation. Here we report progress in the development of an Atlantic salmonin vitrogut model, SalmoSim, to simulate three gut compartments (stomach, pyloric caecum and mid gut) and associated microbial communities. The artificial gut model was established in a series of linked bioreactors seeded with biological material derived for adult marine phase salmon. In biological triplicate, the response of thein vitrosystem to two distinct dietary formulations (fish meal and fish meal free) was compared to a parallelin vivotrial over forty days. 16S rDNA sequencing, qPCR, ammoniacal nitrogen and volatile fatty acid measurements were undertaken to survey microbial community dynamics and function. SalmoSim communities were indistinguishable (p=0.230) from their founding inocula at 20 days and most abundant genera (e.g.Psycrobacter, Staphylococcus, Pseudomonas) proliferated thein vitrosystem. Real salmon and SalmoSim responded similarly to the introduction of the novel feed, with most taxa (96% Salmon, 97% SalmoSim) unaffected, while a subset of taxa was affected non-identically across both systems. Consistent with a low impact of the novel feed on microbial community function, VFA profiles were not significantly different in SalmoSim pre and post the switch feed. This study represents an important first-step in the development of anin vitrogut system as a tool for the improvement of salmon nutrition.

Porous films are used nowadays as dielectrics with low dielectric constant (so-called low-k dielectrics). Knowing the porous structure of such films is important for successful integration in the semiconductor manufacturing technology. We developed a simple characterization method based on diffusion of solvents inside a porous film. In our experiments toluene as a non-polar solvent and ethanol as a polar solvent were used. A porous film is covered with a barrier impermeable for solvent so the solvent only can enter the film from the side. The barrier is transparent that allows observation of diffusion as a color change of the film. Measuring diffused distance as a function of diffusion time, diffusion coefficients can be calculated. It was found that the diffusion coefficients strongly depend on the porous structure of the films. Small pores (with the size comparable with the solvent molecule size) show low diffusion coefficients with high activation energies comparable with the enthalpy of evaporation. Bigger pores exhibit high diffusion coefficients with low activation energies corresponding to the activation energy of the solvent viscosity. Measuring diffusion coefficients as functions of porosity/pore size it is possible to evaluate the interconnectivity of the porous structure of a film in question.

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