assessing-mineral-availability-fish-feeds-using-complementary-methods

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 ...

Institute of Marine Research

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).

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

The effects of low marine ingredient diets supplemented with graded levels (L1, L2, L3) of a micronutrient package (NP) on growth and metabolic responses were studied in diploid and triploid salmon parr. Diploids fed L2 showed significantly improved growth and reduced liver, hepatic steatosis, and viscerosomatic indices, while fish fed L3 showed suppressed growth rate 14 weeks post feeding. In contrast, dietary NP level had no effect on triploid performance. Whole body mineral composition, with exception of copper, did not differ between diet or ploidy. Whole fish total AAs and N-metabolites showed no variation by diet or ploidy. Free circulating AAs and white muscle N-metabolites were higher in triploids than diploids, while branch-chained amino acids were higher in diploids than triploids. Diploids had higher whole body α-tocopherol and hepatic vitamins K and K than triploids. Increased tissue B-vitamins for niacin and whole-body folate with dietary NP supplementation were observed in diploids but not triploids, while whole body riboflavin was higher in diploids than triploids. Hepatic transcriptome profiles showed that diploids fed diet L2 was more similar to that observed in triploids fed diet L3. In particular, sterol biosynthesis pathways were down-regulated, whereas cytochrome P450 metabolism was up-regulated. One‑carbon metabolism was also affected by increasing levels of supplementation in both ploidies. Collectively, results suggested that, for optimised growth and liver function, micronutrient levels be supplemented above current National Research Council (2011) recommendations for Atlantic salmon when fed low marine ingredient diets. The study also suggested differences in nutritional requirements between ploidy.

The effect of micronutrient supplementation on growth and hepatic metabolism in diploid and triploid Atlantic salmon (Salmo salar) parr fed a low marine ingredient diet.

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 ± 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.

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

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   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   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.

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

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  (8),  (5),  (3) and  (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.

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

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 (IM, 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, IM and IM, 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-1β, IL-8, IL-10 and TNF-α were elevated in LPS treated leukocytes isolated from salmon fed the three dietary groups (IM, IM and IM). 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-β were down-regulated by the LPS treatment in the cells isolated from salmon fed insect-based diets (IM and IM) compared to fish fed the IM. Similarly, the leukocytes challenged with LPS and isolated from fish fed with IM and IM down-regulated the expression of Mn-SOD, GPx1, HSP27 and HSP70 compared to salmon fed IM. 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.

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.

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 () 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 () larvae fed media containing 60% seaweed (). 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.

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

Atlantic salmon () feeds have changed drastically in their composition from being predominantly marine-based to plant-based. This has altered the dietary supply and availability of micro-nutrients to Atlantic salmon. The impact of graded inclusion levels of a nutrient package (NP) comprising of 25 different micro-nutrients were studied in Atlantic salmon parr in freshwater (Trial 1) and post-smolts in seawater (Trial 2). In brief, the NP was included from 0 to 400%, where 100% corresponded to the recommendation by the National Research Council, 2011. Micro-nutrients, namely Zn, Mn, Se, Cu, Fe, Co, I and vitamin D3 were included in the NP with the objective of (re)evaluating the dietary need to meet the requirement of Atlantic salmon parr and post-smolt, when fed low fish meal, plant ingredient-based diets. Responses in apparent availability coefficient (AAC), whole body and vertebrae mineral concentrations, and retention were analysed. AAC of Cu, Mn, Se and Zn responded in a quadratic fashion with an increase in NP from 0 to 400% in freshwater parr; AAC could not be measured in post-smolt salmon. The whole-body concentration of Zn, Se, Co and I in Atlantic salmon parr were significantly affected by increasing NP inclusion; the same was observed for Zn, Se and Co in post-smolt Atlantic salmon. Vertebrae mineral concentration as the response criterion was non-responsive in parr; whereas, in post-smolt, Co had a linear increase, while Zn and Se showed a non-linear increase upon 0 to 400 NP inclusion. Zinc concentration and activities of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) in vertebrae indicated increased bone resorption in post-smolt Atlantic salmon; TRAP activity increased linearly with NP inclusion in post-smolt, but not in parr. Significant correlations between Zn and Se were observed in AAC and vertebral concentrations, indicating an interaction in intestinal uptake and vertebral deposition. Overall, Atlantic salmon parr held in freshwater were able to satisfy the requirement for the trace minerals Zn, Mn, Se, Cu, and Fe through supply from 100-150 NP, corresponding to 101-132, 47-63, 0.6-0.8, 12-16 and 150-166 mg kg , respectively; for iodine, dietary supply from 150-200 NP, corresponding to 0.7-1.6 mg kg, was required. In the seawater, Atlantic salmon post-smolt, in general, required micro-minerals and vitamin D3 levels as supplied through 150-200 NP, corresponding to 140-177, Zn; 61-67, Mn; 0.9-1, Se; 14-16, Cu; and vitamin D3, 0.06-0.09 mg kg  to fulfil the requirement, except for Cu which was satisfied at 100-150 NP, equivalent to 13-14 mg kg  diet.

Recommendations for dietary level of micro-minerals and vitamin D to Atlantic salmon () parr and post-smolt when fed low fish meal diets.

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.

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.

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.

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.).

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.

Article	Year
Lower levels of Persistent Organic Pollutants, metals and the marine omega 3-fatty acid DHA in farmed compared to wild Atlantic salmon (Salmo salar). Environmental research\| PubMed ID: 28189073	2017
The effect of micronutrient supplementation on growth and hepatic metabolism in diploid and triploid Atlantic salmon (Salmo salar) parr fed a low marine ingredient diet. Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology\| PubMed ID: 30367964	2019
Gut health and vaccination response in pre-smolt Atlantic salmon (Salmo salar) fed black soldier fly (Hermetia illucens) larvae meal. Fish & shellfish immunology\| PubMed ID: 30590165	2019
Growth and Safety Assessment of Feed Streams for Black Soldier Fly Larvae: A Case Study with Aquaculture Sludge. Animals : an open access journal from MDPI\| PubMed ID: 31018596	2019
Species-Specific Discrimination of Insect Meals for Aquafeeds by Direct Comparison of Tandem Mass Spectra. Animals : an open access journal from MDPI\| PubMed ID: 31067722	2019
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. Fish & shellfish immunology\| PubMed ID: 31121289	2019
Replacing fish meal with insect meal in the diet of Atlantic salmon () does not impact the amount of contaminants in the feed and it lowers accumulation of arsenic in the fillet. Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment\| PubMed ID: 31161892	2019
Recommendations for dietary level of micro-minerals and vitamin D to Atlantic salmon () parr and post-smolt when fed low fish meal diets. PeerJ\| PubMed ID: 31183254	2019
Enhanced micronutrient supplementation in low marine diets reduced vertebral malformation in diploid and triploid Atlantic salmon (Salmo salar) parr, and increased vertebral expression of bo... Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology\| PubMed ID: 31461683	2019
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.)... Journal of the science of food and agriculture\| PubMed ID: 31650558	2020
Polycyclic aromatic hydrocarbons modulate the activity of Atlantic cod (Gadus morhua) vitamin D receptor paralogs in vitro. Aquatic toxicology (Amsterdam, Netherlands)\| PubMed ID: 34304057	2021

Erik-Jan Lock

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