Growth Performance, Nutrient Digestibility, and Retention in Atlantic Salmon, Salmo salar L., Fed Diets with Fermented Sugar Kelp, Saccharina latissima.

Using low trophic marine resources such as sugar kelp (Saccharina latissimi) is of great interest to increase the circular food production in the ocean. Sugar kelp does, however, contain high levels of carbohydrates and iodine and does not have considerable levels of protein and lipids, which may make it less suitable as a feeding ingredient. A 10-week feeding trial was done to investigate the effect of graded dietary inclusion levels of fermented sugar kelp (FSK), on growth performance, digestibility, retention of nutrients, and mineral composition in postsmolt Atlantic salmon (Salmo salar L.). The experimental diets were made to simulate a standard grower feed for salmon postsmolts in SW with ∼63% plant-based ingredients vs ∼34% marine ingredients and increasing concentrations of FSK between 0% (control feed) and 4% of the diet. During the feeding trial, the weight gain and specific growth rate (SGR) decreased linearly with increasing dietary FSK levels, where the SGR was slightly reduced from 1.2% for the fish given the control feed to 1.1% in the fish given feeds containing 3% and 4% FSK. This resulted in a lower weight gain of up to 9% in the fish given 4% FSK compared to the control. Feed intake and feed conversion ratio were, however, similar in all diet groups, and FSK inclusion did not influence the digestibility of macronutrients or minerals, except for lipid. The reduced growth is likely related to a lower digestible energy level in the diets, and the retention of both lipids and energy was affected by FSK inclusion. Inclusion of FSK also influenced iodine availability and retention, as well as increasing iodine status in whole body and muscle in a dose-dependent manner until reaching a plateau, which corresponds to 124 mg I kg-1 WW (135 mg I kg-1 DW), at 3% FSK inclusion.

Total replacement of dietary fish meal with black soldier fly (Hermetia illucens) larvae does not impair physical, chemical or volatile composition of farmed Atlantic salmon (Salmo salar L.).

BACKGROUND: The aquafeed sector has been replacing conventional dietary ingredients with more economic and eco-friendly ingredients. Insects embody a promising alternative as a result of being highly nutritious and showing traits leading to a circular bioeconomy. Atlantic salmon (Salmo salar L.) at the sea-water stage were fed diets with a partial or complete substitution of fishmeal with meal of Hermetia illucens larvae reared on a media containing Ascophyllum nodosum mixed with organic wastes (60:40). The present study aimed to assess the quality of fillets by characterizing its physico-chemical traits with conventional and innovative methods, such as the proton transfer reaction-time of flight-mass spectrometer technique, allowing the analysis of samples at room temperature. Finally, steamed fillets underwent a consumer test to investigate the liking of consumers and their intention of re-consumption. RESULTS: The main findings showed that a complete dietary substitution of fishmeal with H. illucens larvae meal did not impair the physico-chemical quality of A. salmon fillets. Notably, neutral n-3 polyunsaturated fatty acids (PUFA) slightly but significantly increased in the fillets of A. salmon fed H. illucens, also as a result of the additional fish oil present in the diets containing insect. The volatile organic profile was not altered by the different diets. The consumer-liking test revealed that Italian consumers appreciated the tested salmon irrespective of the administered feed. CONCLUSION: Tailoring the insect fatty acid profile by rearing the larvae on a PUFA-rich substrate, coupled with a dietary modulation of the oily source, can successfully maintain or even increase the cardioprotective characteristics of fillets. © 2019 Society of Chemical Industry.

Effect of dietary replacement of fish meal with insect meal on in vitro bacterial and viral induced gene response in Atlantic salmon (Salmo salar) head kidney leukocytes.

With the fast growth of today's aquaculture industry, the demand for aquafeeds is expanding dramatically. Insects, which are part of the natural diet of salmonids, could represent a sustainable ingredient for aquaculture feed. The aim of the current study was to test how a partial or total replacement of dietary fishmeal with insect meal affect gene responses involved in inflammation, the eicosanoid pathway and stress response in Atlantic salmon (Salmo salar L.) in isolated head kidney leukocytes after exposure to bacterial or viral mimic. Insect meal (IM) was produced from black soldier fly (BSF, Hermetia illucens) larvae. Seawater Atlantic salmon were fed three different diets for 8 weeks; a control diet (IM0, protein from fishmeal and plant based ingredients (25:75) and lipid from fish oil and vegetable oil (33:66); and two insect-meal containing diets, IM66 and IM100, where 66 and 100% of the fishmeal protein was replaced with IM, respectively. Leukocytes were isolated from the head kidney of fish (n = 6) from each of the three dietary groups. Isolated leukocytes were seeded into culture wells and added either a bacterial mimic (lipopolysaccharide, LPS) or a viral mimic (polyinosinic acid: polycytidylic acid, poly I: C) to induce an inflammatory response. Controls (Ctl) without LPS and poly I: C were included. The transcription of interleukins IL-1ß, IL-8, IL-10 and TNF-α were elevated in LPS treated leukocytes isolated from salmon fed the three dietary groups (IM0, IM66 and IM100). The inflammatory-related gene expression in head kidney cells were, however, not affected by the pre-fed substitution of fish meal with IM in the diet of salmon. Gene transcriptions of PTGDS and PTGES were neither affected by LPS, poly I: C or the experimental diets fed prior to cell isolation, while salmon fed with IM showed a lower expression of LOX5. The gene expression of TLR22 and C/EBP-ß were down-regulated by the LPS treatment in the cells isolated from salmon fed insect-based diets (IM66 and IM100) compared to fish fed the IM0. Similarly, the leukocytes challenged with LPS and isolated from fish fed with IM66 and IM100 down-regulated the expression of Mn-SOD, GPx1, HSP27 and HSP70 compared to salmon fed IM0. In general, these results suggested that replacement of fishmeal with IM in the diets of Atlantic salmon had no effect on the transcription of pro-inflammatory genes in the head kidney cells. There was, however, an effect of dietary IM on the transcription of antioxidant and stress related genes in the leukocytes.

Gut health and vaccination response in pre-smolt Atlantic salmon (Salmo salar) fed black soldier fly (Hermetia illucens) larvae meal.

Limited availability of sustainable feed ingredients is a serious concern in salmon aquaculture. Insects may become an important, sustainable resource for expanding the raw material repertoire. Herein, we present data from an 8-week feeding trial with pre-smolt Atlantic salmon (initial body weight 49 ±â€¯1.5 g) fed either a reference diet containing fish meal, soy protein concentrate and wheat gluten as protein sources, or a test diet wherein 85% of the protein was supplied by black soldier fly larvae meal. Possible diet effect on the systemic immune response was evaluated by measuring plasma antibody titers after vaccination against infectious pancreatic necrosis virus (IPNV). The gut health of fish was evaluated using endpoints including organ and tissue indices, histopathological parameters and gene expression. Both diets induced the same level of antibody responses against IPNV. In fish fed the reference diet, the histological examination of the pyloric caeca mucosa showed clear hyper-vacuolization suggestive of lipid accumulation in enterocytes, whereas this was less pronounced in the insect meal fed fish. Expression of genes relevant to lipid metabolism confirmed these histological findings. Immune and barrier-function gene expression profiles were both generally not affected by diet. However, the fish fed insect meal showed increased expression of genes indicative of stress response, immune tolerance and increased detoxification activity. In summary, our results showed no indications that dietary inclusion of insect meal affected the gut health of Atlantic salmon negatively. The insect meal based diet seemed to reduce excessive lipid deposition in the pyloric caeca and stimulate xenobiotic metabolism.

Chemical characterization of 21 species of marine macroalgae common in Norwegian waters: benefits of and limitations to their potential use in food and feed.

BACKGROUND: In the past few years, much effort has been invested into developing a new blue economy based on harvesting, cultivating and processing marine macroalgae in Norway. Macroalgae have high potential for a wide range of applications, e.g. as source of pharmaceuticals, production of biofuels or as food and feed. However, data on the chemical composition of macroalgae from Norwegian waters are scant. This study was designed to characterize the chemical composition of 21 algal species. Both macro- and micronutrients were analysed. Concentrations of heavy metals and the metalloid arsenic in the algae were also quantified. RESULTS: The results confirm that marine macroalgae contain nutrients which are relevant for both human and animal nutrition, the concentrations whereof are highly dependent on species. Although heavy metals and arsenic were detected in the algae studied, concentrations were mostly below maximum allowed levels set by food and feed legislation in the EU. CONCLUSION: This study provides chemical data on a wide range of algal species covering the three taxonomic groups (brown, red and green algae) and discusses both benefits of and potential limitations to their use for food and feed purposes. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Uptake of heavy metals and arsenic in black soldier fly (Hermetia illucens) larvae grown on seaweed-enriched media.

BACKGROUND: The black soldier fly (Hermetia illucens) is one of the most promising insect species for use in animal feed. However, studies investigating feed and food safety aspects of using black soldier fly as feed are scarce. In this study, we fed black soldier fly larvae feeding media enriched with seaweed, which contains naturally high concentrations of heavy metals and arsenic. The aim of this study was to investigate the potential transfer of such undesirable substances from the feeding media to the larvae. RESULTS: The larvae accumulated cadmium, lead, mercury and arsenic. Concentrations of these elements in the larvae increased when more seaweed was added to the feeding media. The highest retention was seen for cadmium (up to 93%) and the lowest for total arsenic (up to 22%). When seaweed inclusion exceeded 20% in the media, this resulted in larval concentrations of cadmium and total arsenic above the current European Union maximum levels for these elements in complete feed. CONCLUSION: Our results confirm that insect larvae can accumulate heavy metals and arsenic when present in the feeding media. A broader understanding of the occurrence of these undesirable substances in processed larvae products is needed to assess feed and food safety. © 2017 Society of Chemical Industry.

Lower levels of Persistent Organic Pollutants, metals and the marine omega 3-fatty acid DHA in farmed compared to wild Atlantic salmon (Salmo salar).

Contaminants and fatty acid levels in farmed- versus wild Atlantic salmon have been a hot topic of debate in terms of food safety. The present study determined dioxins (polychlorinated dibenzo-p-dioxin and dibenzofuran), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), organochlorine pesticides (OCPs), metals and fatty acids in wild and farmed Atlantic salmon. Contaminant levels of dioxins, PCBs, OCPs (DDT, dieldrin, lindane, chlordane, Mirex, and toxaphene), and mercury were higher in wild salmon than in farmed salmon, as were the concentrations of the essential elements selenium, copper, zinc and iron, and the marine omega-3 fatty acid docosahexaenoic acid (DHA). PBDE, endosulfan, pentachlorobenzene, hexachlorobenzene, cadmium and lead levels were low and comparable in both wild and farmed fish, and there was no significant difference in the marine omega-3 fatty acid eicosapentaenoic acid (EPA) concentration. The total fat content was significantly higher in farmed than wild salmon due to a higher content of both saturated and monounsaturated fatty acids, as well as a higher content of omega-6 fatty acids. The omega-3 to omega-6 fatty acid ratio was considerably lower in farmed than wild salmon due to the high level of omega-6 fatty acids. Contaminant concentrations in Atlantic salmon were well below maximum levels applicable in the European Union. Atlantic salmon, both farmed and wild, is a good source of EPA and DHA with a 200g portion per week contributing 3.2g or 2.8g respectively, being almost twice the intake considered adequate for adults by the European Food Safety Authority (i.e. 250mg/day or 1.75g/week).

Dietary eicosapentaenoic acid supplementation accentuates hepatic triglyceride accumulation in mice with impaired fatty acid oxidation capacity.

Reduced mitochondrial fatty acid (FA) ß-oxidation can cause accumulation of triglyceride in liver, while intake of eicosapentaenoic acid (EPA) has been recommended as a promising novel therapy to decrease hepatic triglyceride content. However, reduced mitochondrial FA ß-oxidation also facilitates accumulation of EPA. To investigate the interplay between EPA administration, mitochondrial activity and hepatic triglyceride accumulation, we investigated the effects of EPA administration to carnitine-deficient mice with impaired mitochondrial FA ß-oxidation. C57BL/6J mice received a high-fat diet supplemented or not with 3% EPA in the presence or absence of 500 mg mildronate/kg/day for 10 days. Liver mitochondrial and peroxisomal oxidation, lipid classes and FA composition were determined. Histological staining was performed and mRNA level of genes related to lipid metabolism and inflammation in liver and adipose tissue was determined. Levels of pro-inflammatory eicosanoids and cytokines were measured in plasma. The results showed that mildronate treatment decreased hepatic carnitine concentration and mitochondrial FA ß-oxidation and induced severe triglyceride accumulation accompanied by elevated systemic inflammation. Surprisingly, inclusion of EPA in the diet exacerbated the mildronate-induced triglyceride accumulation. This was accompanied by a considerable increase of EPA accumulation while decreased total n-3/n-6 ratio in liver. However, inclusion of EPA in the diet attenuated the mildronate-induced mRNA expression of inflammatory genes in adipose tissue. Taken together, dietary supplementation with EPA exacerbated the triglyceride accumulation induced by impaired mitochondrial FA ß-oxidation. Thus, further thorough evaluation of the potential risk of EPA supplementation as a therapy for NAFLD associated with impaired mitochondrial FA oxidation is warranted.

Dietary linoleic acid elevates endogenous 2-arachidonoylglycerol and anandamide in Atlantic salmon (Salmo salar L.) and mice, and induces weight gain and inflammation in mice.

Dietary intake of linoleic acid (LA) has increased dramatically during the twentieth century and is associated with a greater prevalence of obesity. Vegetable oils are recognised as suitable alternatives to fish oil (FO) in feed for Atlantic salmon (Salmo salar L.) but introduce high amounts of LA in the salmon fillet. The effect on fish consumers of such a replacement remains to be elucidated. Here, we investigate the effect of excessive dietary LA from soyabean oil (SO) on endocannabinoid levels in Atlantic salmon and mice, and study the metabolic effects in mice when SO replaces FO in feed for Atlantic salmon. Atlantic salmon were fed FO and SO for 6 months, and the salmon fillet was used to produce feed for mice. Male C57BL/6J mice were fed diets of 35% of energy as fat based on FO- and SO-enriched salmon for 16 weeks. We found that replacing FO with SO in feed for Atlantic salmon increased LA, arachidonic acid (AA), decreased EPA and DHA, elevated the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA), and increased TAG accumulation in the salmon liver. In mice, the SO salmon diet increased LA and AA and decreased EPA and DHA in the liver and erythrocyte phospholipids, and elevated 2-AG and AEA associated with increased feed efficiency, weight gain and adipose tissue inflammation compared with mice fed the FO salmon diet. In conclusion, excessive dietary LA elevates endocannabinoids in the liver of salmon and mice, and increases weight gain and counteracts the anti-inflammatory properties of EPA and DHA in mice.

Co-diversification of an intestinal Mycoplasma and its salmonid host.

Understanding the evolutionary relationships between a host and its intestinal resident bacteria can transform how we understand adaptive phenotypic traits. The interplay between hosts and their resident bacteria inevitably affects the intestinal environment and, thereby, the living conditions of both the host and the microbiota. Thereby this co-existence likely influences the fitness of both bacteria and host. Whether this co-existence leads to evolutionary co-diversification in animals is largely unexplored, mainly due to the complexity of the environment and microbial communities and the often low host selection. We present the gut metagenome from wild Atlantic salmon (Salmo salar), a new wild organism model with an intestinal microbiota of low complexity and a well-described population structure, making it well-suited for investigating co-evolution. Our data reveal a strong host selection of a core gut microbiota dominated by a single Mycoplasma species. We found a clear co-diversification between the population structure of Atlantic salmon and nucleotide variability of the intestinal Mycoplasma populations conforming to expectations from co-evolution between host and resident bacteria. Our results show that the stable microbiota of Atlantic salmon has evolved with its salmonid host populations while potentially providing adaptive traits to the salmon host populations, including defence mechanisms, biosynthesis of essential amino acids, and metabolism of B vitamins. We highlight Atlantic salmon as a novel model for studying co-evolution between vertebrate hosts and their resident bacteria.

Nutritional regulation of bile acid metabolism is associated with improved pathological characteristics of the metabolic syndrome.

Bile acids (BAs) are powerful regulators of metabolism, and mice treated orally with cholic acid are protected from diet-induced obesity, hepatic lipid accumulation, and increased plasma triacylglycerol (TAG) and glucose levels. Here, we show that plasma BA concentration in rats was elevated by exchanging the dietary protein source from casein to salmon protein hydrolysate (SPH). Importantly, the SPH-treated rats were resistant to diet-induced obesity. SPH-treated rats had reduced fed state plasma glucose and TAG levels and lower TAG in liver. The elevated plasma BA concentration was associated with induction of genes involved in energy metabolism and uncoupling, Dio2, Pgc-1α, and Ucp1, in interscapular brown adipose tissue. Interestingly, the same transcriptional pattern was found in white adipose tissue depots of both abdominal and subcutaneous origin. Accordingly, rats fed SPH-based diet exhibited increased whole body energy expenditure and heat dissipation. In skeletal muscle, expressions of the peroxisome proliferator-activated receptor ß/δ target genes (Cpt-1b, Angptl4, Adrp, and Ucp3) were induced. Pharmacological removal of BAs by inclusion of 0.5 weight % cholestyramine to the high fat SPH diet attenuated the reduction in abdominal obesity, the reduction in liver TAG, and the decrease in nonfasted plasma TAG and glucose levels. Induction of Ucp3 gene expression in muscle by SPH treatment was completely abolished by cholestyramine inclusion. Taken together, our data provide evidence that bile acid metabolism can be modulated by diet and that such modulation may prevent/ameliorate the characteristic features of the metabolic syndrome.

A rapid acid hydrolysis method for the determination of chitin in fish feed supplemented with black soldier fly (Hermetia illucens) larvae.

Insects are a natural source of feed for fish and have received more attention as a potential source of sustainable high-quality protein. However, contrasting results in different feeding trials have been ascribed to the chitin contained in the exoskeleton of insects and highlighted the importance of developing reliable methods for the quantification of chitin to draw meaningful conclusions about its effect on fish health. A rapid method based on the hydrolysis of chitin into glucosamine and further quantification by liquid chromatography tandem mass spectrometry is evaluated. The method offers good selectivity, linearity, limit of detection (1.08 × 10-5 % w/v or 5.38 × 10-4 % w/w), limit of quantification (3.26 × 10-5 % w/v or 1.63 × 10-3 % w/w), trueness (88.39-109.29 %) and precision (2.24-10.72 %). The quantitative method was successfully applied to real samples of fish feed supplemented with chitin from black soldier fly (Hermetia illucens) larvae.

Assessing amino acid solubility of black soldier fly larvae meal in Atlantic salmon (Salmo salar) in vivo and in vitro.

In vitro and in vivo methods were used to evaluate amino acids solubility of black soldier fly (BSF) larvae meal and two experimental diets (reference and test diets) for Atlantic salmon. The current study used in vitro method such as pH stat to compare and standardise the salmon extracted enzyme (SE), and commercial enzyme (CE) based on their hydrolytic capacity on a purified protein substrate. Further, an in vitro amino acid solubility of feed ingredients and diets were measured using the standardised enzyme volume from SE and CE. Results showed that SE and CE exhibit similar protein hydrolytic capacity upon standardisation on purified substrates. However, when using the two-stage hydrolysis (acidic and alkaline steps), significantly higher amino acid solubility was observed with CE except for glycine, and proline which were equally solubilised by both SE, and CE. No significant difference was observed between reference and test diet using the SE except for tyrosine, valine, leucine, and phenylalanine, which were significantly higher solubilised in reference diet than test diet. Whereas higher solubility of valine, isoleucine, aspartic acid, and glutamic acid was observed in test diet using CE than SE. Similarly, the solubility of valine, isoleucine, and glutamic acid were higher in BSF larvae meal when CE was used. The in vivo true protein digestibility of BSF larvae meal was 99%, and 81% for the test diet containing BSF larvae meal. The results demonstrated a positive correlation (r = 0.91; p < 0.01) between salmon and commercial enzymes but overall, no significant correlation was observed for amino acid solubility between in vivo and in vitro. However, there was a strong positive correlation for protein solubility using SE (r = 0.98) than CE (r = 0.74) with the in vivo true protein digestibility. The efficiency of SE, and CE can be compared, and standardised based on DH%, and hence correlates better with the in vivo protein digestibility but not with amino acid solubilities.

Polycyclic aromatic hydrocarbons modulate the activity of Atlantic cod (Gadus morhua) vitamin D receptor paralogs in vitro.

Vitamin D receptor (VDR) mediates the biological function of the steroid hormone calcitriol, which is the metabolically active version of vitamin D. Calcitriol is important for a wide array of physiological functions, including calcium and phosphate homeostasis. In contrast to mammals, which harbor one VDR encoding gene, teleosts possess two orthologous vdr genes encoding Vdr alpha (Vdra) and Vdr beta (Vdrb). Genome mining identified the vdra and vdrb paralogs in the Atlantic cod (Gadus morhua) genome, which were further characterized regarding their phylogeny, tissue-specific expression, and transactivational properties induced by calcitriol. In addition, a selected set of polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene, fluorene, pyrene, chrysene, benzo[a]pyrene (BaP), and 7-methylbenzo[a]pyrene, were assessed for their ability to modulate the transcriptional activity of gmVdra and gmVdrb in vitro. Both gmVdra and gmVdrb were activated by calcitriol with similar potencies, but gmVdra produced significantly higher maximal fold activation. Notably, none of the tested PAHs showed agonistic properties towards the Atlantic cod Vdrs. However, binary exposures of calcitriol together with phenanthrene, fluorene, or pyrene, antagonized the activation of gmVdra, while chrysene and BaP significantly potentiated the calcitriol-mediated activity of both receptors. Homology modeling, solvent mapping, and docking analyses complemented the experimental data, and revealed a putative secondary binding site in addition to the canonical ligand-binding pocket (LBP). Calcitriol was predicted to interact with both binding sites, whereas PAHs docked primarily to the LBP. Importantly, our in vitro data suggest that PAHs can interact with the paralogous gmVdrs and interfere with their transcriptional activities, and thus potentially modulate the vitamin D signaling pathway and contribute to adverse effects of crude oil and PAH exposures on cardiac development and bone deformities in fish.

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon.

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a systematic approach combining different methodologies to assess the availability of zinc (Zn) in Atlantic salmon (Salmo salar). Considering that several Zn chemical species can be present in an Atlantic salmon feed, it was hypothesised that Zn availability is influenced by the Zn chemical species present in the feed. Thus, in this study, the first protocol is about how to extract the different Zn chemical species from the feed and to analyze them by a size exclusion chromatography-inductively coupled plasma mass spectroscopy (SEC-ICP-MS) method. Subsequently, an in vitro method was developed to evaluate the solubility of dietary Zn in Atlantic salmon feeds. The third protocol describes the method to study the impact of changing Zn chemical species composition on the uptake of Zn in a fish intestinal epithelial model using a rainbow trout gut cell line (RTgutGC). Together, the findings from the in vitro methods were compared with an in vivo study examining the apparent availability of inorganic and organic sources of Zn supplemented to Atlantic salmon feeds. The results showed that several Zn chemical species can be found in feeds and the efficiency of an organic Zn source depends very much on the amino acid ligand used to chelate Zn. The findings of the in vitro methods had less correlation with that outcome of the in vivo study. Nevertheless, in vitro protocols described in this article provided crucial information regarding Zn availability and its assessment in fish feeds.

Vitamin K-dependent gamma-glutamylcarboxylase in Atlantic salmon (Salmo salar L.).

Due to problems with bone deformities in farmed Atlantic salmon, there is a growing interest in the possible involvement of vitamin K in normal bone development, and sensitive biomarkers for evaluating vitamin K status are therefore needed. The vitamin K-dependent (VKD) enzyme gamma-glutamylcarboxylase (GGCX, EC 6.4.x.x) requires vitamin K as a cofactor for its post-translational modification of glutamic acid (Glu) residues to gamma-carboxyglutamic acid (Gla) residues in VKD proteins, and is required for their function in haemostasis and bone metabolism. The present study was designed to evaluate the enzyme assay for GGCX activity in isolated liver microsomes and its distribution in the tissues of Atlantic salmon. The effect of KH(2) and menadione on the GGCX activity in salmon liver was also compared. Results from the present study show a widespread tissue distribution and expression of GGCX in Atlantic salmon. The GGCX activity and ggcx expression in all bony tissues examined imply the presence of vitamin K, and suggest the involvement of vitamin K in bone metabolism of Atlantic salmon. We propose the GGCX assay as a sensitive marker for vitamin K status, and confirm that menadione does not work as a cofactor for GGCX in Atlantic salmon liver.

Replacing fish meal with insect meal in the diet of Atlantic salmon (Salmo salar) does not impact the amount of contaminants in the feed and it lowers accumulation of arsenic in the fillet.

Insects are promising sources of protein and lipid in feeds for farmed animals. In the European Union, the use of insect meal (IM) and insect oil is permitted in fish feed. However, the European Food Safety Authority has highlighted the lack of data regarding the chemical safety of insects and products thereof. In this study, Atlantic salmon (Salmo salar) were fed diets in which fish meal (FM) was partially or fully substituted with IM, resulting in four diets with an FM replacement of 0%, 33%, 66% and 100% by IM. The IM was produced from Black soldier fly (Hermetia illucens) larvae fed media containing 60% seaweed (Ascophyllum nodosum). After 16 weeks of feeding, fish fillet samples were collected. The concentrations of undesirable substances, e.g., heavy metals, arsenic, dioxins, mycotoxins, pesticides, in the IM, the diets and fillets were determined. The concentrations of the analysed compounds in the IM were all below EU maximum levels for feed ingredients, except for arsenic. However, for complete feeds the concentrations of these compounds in the feeds, including arsenic, were all below EU MLs. Arsenic was transferred from seaweed to IM, resulting in arsenic levels in IM similar to what has been documented for FM. Transfer of arsenic from feed to fillet was observed; however, total arsenic concentrations in the fillet significantly decreased when fish were fed diets with more IM and less FM. Arsenic speciation analysis of the diets showed that although total arsenic levels were similar, the arsenic species were different. Arsenobetaine was the major organoarsenic species in the diets containing FM, while in diets containing IM several unidentified arsenic species were detected. The results suggest that the lower feed-to-fillet transfer of arsenic when FM is replaced by IM may be due to the presence of arsenic species with low bioavailability in the IM.

Enhanced micronutrient supplementation in low marine diets reduced vertebral malformation in diploid and triploid Atlantic salmon (Salmo salar) parr, and increased vertebral expression of bone biomarker genes in diploids.

Previously we showed that, for optimum growth, micronutrient levels should be supplemented above current National Research Council (2011) recommendations for Atlantic salmon when they are fed diets formulated with low levels of marine ingredients. In the present study, the impact of graded levels (100, 200, 400%) of a micronutrient package (NP) on vertebral deformities and bone gene expression were determined in diploid and triploid salmon parr fed low marine diets. The prevalence of radiologically detectable spinal deformities decreased with increasing micronutrient supplementation in both ploidy. On average, triploids had a higher incidence of spinal deformity than diploids within a given diet. Micronutrient supplementation particularly reduced prevalence of fusion deformities in diploids and compression and reduced spacing deformities in triploids. Prevalence of affected vertebrae within each spinal region (cranial, caudal, tail and tail fin) varied significantly between diet and ploidy, and there was interaction. Prevalence of deformities was greatest in the caudal region of triploids and the impact of graded micronutrient supplementation in reducing deformities also greatest in triploids. Diet affected vertebral morphology with length:height (L:H) ratio generally increasing with level of micronutrient supplementation in both ploidy with no difference between ploidy. Increased dietary micronutrients level in diploid salmon increased the vertebral expression of several bone biomarker genes including bone morphogenetic protein 2 (bmp2), osteocalcin (ostcn), alkaline phosphatase (alp), matrix metallopeptidase 13 (mmp13), osteopontin (opn) and insulin-like growth factor 1 receptor (igf1r). In contrast, although some genes showed similar trends in triploids, vertebral gene expression was not significantly affected by dietary micronutrients level. The study confirmed earlier indications that dietary micronutrient levels should be increased in salmon fed diets with low marine ingredients and that there are differences in nutritional requirements between ploidies.

Growth and Safety Assessment of Feed Streams for Black Soldier Fly Larvae: A Case Study with Aquaculture Sludge.

The production of food is an intensive source of environmental impact. In aquaculture, one source of impact is solid waste, which contains high concentrations of minerals, other nutrients, and metals. The larvae of Hermetia illucens are capable of consuming this material, but applying technology that is based on these larvae for managing waste streams, like those from aquaculture, requires careful examination of safety risks. A study is performed examining the growth performance of larvae that were fed on solid aquaculture waste. Subsequently, a thorough analysis of safety risks from inorganics, with detailed the results on microelements that have previously received little attention in the literature, is performed to serve as a guideline for how to assess the safety of waste streams such as these. Findings confirm existing results in the literature that Cd is bioaccumulative, but also that other elements, including Hg, Mn, and especially K, are bioaccumulative. To the authors' knowledge, this is the first research where the accumulation of Ag is also tested. The results of these tests are explained within the context of regulations in various countries where Hermetia illucens is cultivated, serving as a reference for practitioners to rigorously screen out high risk feed streams that they may consider using as feed sources. It is intended that these references and the demonstrated accumulation of a range of elements motivate comprehensive industry safety practices when evaluating new feed sources.

Species-Specific Discrimination of Insect Meals for Aquafeeds by Direct Comparison of Tandem Mass Spectra.

Insect protein has the potential to become a sustainable feed ingredient for the rapidly growing aquaculture industry. In the European Union, insect derived protein is placed under the same legislation as processed animal proteins (PAP). It is therefore of interest to develop methods for regulatory use, which unambiguously identify the species origin of insect-based ingredients. We performed (i) total protein quantification of insect samples using the traditional nitrogen-to-protein conversion factor of 6.25 and the sum of anhydrous amino acids, (ii) quantitative amino acid profiling and (iii) high-throughput tandem mass spectrometry to describe and differentiate 18 different commercial-grade insect meal samples derived from Hermetia illucens (8), Tenebrio molitor (5), Alphitobius diaperinus (3) and Acheta domesticus (2). In addition, we investigated and compared different protein extraction and digestion protocols for proteomic analysis. We found that irrespective of sample preparation, shotgun proteomics in combination with direct spectral comparison were able to differentiate insect meal according to their taxonomic classification. The insect specific spectral libraries created in the present work can in future be used to develop more sensitive targeted methods of insect PAP identification and quantification in commercial feed mixtures.