A transcriptomic analysis of diploid and triploid Atlantic salmon lenses with and without cataracts.

To avoid negative environmental impacts of escapees and potential inter-breeding with wild populations, the Atlantic salmon farming industry has and continues to extensively test triploid fish that are sterile. However, they often show differences in performance, physiology, behavior and morphology compared to diploid fish, with increased prevalence of vertebral deformities and ocular cataracts as two of the most severe disorders. Here, we investigated the mechanisms behind the higher prevalence of cataracts in triploid salmon, by comparing the transcriptional patterns in lenses of diploid and triploid Atlantic salmon, with and without cataracts. We assembled and characterized the Atlantic salmon lens transcriptome and used RNA-seq to search for the molecular basis for cataract development in triploid fish. Transcriptional screening showed only modest differences in lens mRNA levels in diploid and triploid fish, with few uniquely expressed genes. In total, there were 165 differentially expressed genes (DEGs) between the cataractous diploid and triploid lens. Of these, most were expressed at lower levels in triploid fish. Differential expression was observed for genes encoding proteins with known function in the retina (phototransduction) and proteins associated with repair and compensation mechanisms. The results suggest a higher susceptibility to oxidative stress in triploid lenses, and that mechanisms connected to the ability to handle damaged proteins are differentially affected in cataractous lenses from diploid and triploid salmon.

A comparative study: Difference in omega-6/omega-3 balance and saturated fat in diets for Atlantic salmon (Salmo salar) affect immune-, fat metabolism-, oxidative and apoptotic-gene expression, and eicosanoid secretion in head kidney leukocytes.

The aim of this study was to compare how different dietary vegetable oil n-6/n-3 ratios affect gene responses involved in inflammation, signaling pathways, fatty acid synthesis and oxidation, oxidation and apoptosis as well as eicosanoid production in salmon head kidney tissues and isolated head kidney leukocytes. Salmon smolts (200 g) were fed four different diets where the main lipid components were palm oil (n-6/n-3 ratio = 0.7), rapeseed oil (n-6/n-3 ratio = 0.9), and soybean oil (n-6/n-3 ratio = 2.4) and a high soybean oil diet with an n-6/n-3 ratio = 4. Both head kidney tissue and leukocytes isolated from head kidneys were sampled from the four diets, but from different fish. Leukocytes isolated from the head kidneys were seeded into culture wells and added lipopolysaccharide (LPS) to induce inflammatory responses. Controls without LPS were included. Head kidney leukocytes and the tissues should have the same phenotype reflecting the different diets. Interleukin 1ß (IL-1ß) transcription was elevated in head kidney tissue and especially in LPS treated leukocytes isolated from soybean oil (n-6/n-3 = 2.4) fed salmon, which confirmed the suitability of the in vitro model in this experiment. Leukocytes, treated with LPS, and isolated from salmon fed the soybean oil diet (n-6/n-3 = 2.4) also upregulated tumor necrosis factor alpha (tnf-α), cyclooxygenase (cox2), prostaglandin D and E synthase (ptgds, ptges), fatty acyl synthase (fas), 5 and 6 desaturases (5des, 6 des) and a fatty acid translocase protein (cd36) when compared to the other diets. The results suggest that diets with a specific n-6/n-3 ratio influence the transcription of pro-inflammatory genes and may be cross-linked to transcription of selected fatty acid metabolism genes. Salmon fed the palm oil diet (n-6/n-3 = 0.7) showed a lower expression of inflammatory genes. Instead, peroxisome proliferator activated receptor ß1 (pparß1), acyl coenzyme A (aco), apoptosis regulator (bax) and superoxide dismutase (sod) were upregulated in leukocytes in vitro, while head kidney tissue transcription of a dendritic marker (cd83) was lower than measured in tissues from fish fed the other diets. The concentration of LTB4 (10-20 ng/mL) were relatively constant in leukocyte supernatants, all diets. Head kidney leukocytes from soybean oil (n-6/n-3 = 2.4) fed fish produced LPS induced PGE2 (mean 0.5 ng/mL) while leukocytes isolated from palm oil diet (n-6/n-3 = 0.7) secreted very high amounts of LTB5 (50-70 ng/mL). In addition, equal amounts of LPS induced PGE2 and PGE3 (mean 0, 5 ng/mL) were produced, indicating that the n-6/n-3 ratio of this saturated fatty acid may have a specific impact on eicosanoid production in the head kidney of salmon.

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.

The metabolic response in fish to mildly elevated water temperature relates to species-dependent muscular concentrations of imidazole compounds and free amino acids.

Fish species show distinct differences in their muscular concentrations of imidazoles and free amino acids (FAA). This study was conducted to investigate whether metabolic response to mildly elevated water temperature (MEWT) relates to species-dependent muscular concentrations of imidazoles and FAA. Thirteen carp and 17 Nile tilapia, housed one per aquarium, were randomly assigned to either acclimation (25°C) or MEWT (30°C) for 14 days. Main muscular concentrations were histidine (HIS; P<0.001) in carp versus N-α-acetylhistidine (NAH; P<0.001) and taurine (TAU; P=0.001) in tilapia. Although the sum of imidazole (HIS+NAH) and TAU in muscle remained constant over species and temperatures (P>0.05), (NAH+HIS)/TAU ratio was markedly higher in carp versus tilapia, and decreased with MEWT only in carp (P<0.05). Many of the muscular FAA concentrations were higher in carp than in tilapia (P<0.05). Plasma acylcarnitine profile suggested a higher use of AA and fatty acids in carp metabolism (P<0.05). On the contrary, the concentration of 3-hydroxyisovalerylcarnitine, a sink of leucine catabolism, (P=0.009) pointed to avoidance of leucine use in tilapia metabolism. Despite a further increase of plasma longer-chain acylcarnitines in tilapia at MEWT (P=0.009), their corresponding beta-oxidation products (3-hydroxy-longer-chain acylcarnitines) remained constant. Together with higher plasma non-esterified fatty acids (NEFA) in carp (P=0.001), the latter shows that carp, being a fatter fish, more readily mobilises fat than tilapia at MEWT, which coincides with more intensive muscular mobilization of imidazoles. This study demonstrates that fish species differ in their metabolic response to MEWT, which is associated with species-dependent changes in muscle imidazole to taurine ratio.

Fish pre-acclimation temperature only modestly affects cadmium toxicity in Atlantic salmon hepatocytes.

An emerging focus in environmental toxicology is how climate change will alter bioavailability and uptake of contaminants in organisms. Ectothermic animals unable to adjust their temperature by local migration, such as farmed fish kept in net pens, may become more vulnerable to contaminants in warmer seas. The aim of this work was to study cadmium (Cd) toxicity in cells obtained from fish acclimated to sub-optimal growth temperature. Atlantic salmon hepatocytes, harvested from fish pre-acclimated either at 15°C (optimal growth temperature) or 20°C (heat-stressed), were exposed in vitro to two concentrations of Cd (control, 1 and 100µM Cd) for 48h. Cd-induced cytotoxicity, determined with the xCELLigence system, was more pronounced in cells from fish pre-acclimated to a high temperature than in cells from fish grown at optimal temperature. A feed spiked with antioxidants could not ameliorate the Cd-induced cytotoxicity in cells from temperature-stressed fish. At the transcriptional level, Cd exposure affected 11 out of 20 examined genes, of which most are linked to oxidative stress. The transcriptional levels of a majority of the altered genes were changed in cells harvested from fish grown at sub-optimal temperature. Interaction effects between Cd exposure and fish pre-acclimation temperature were seen for four transcripts, hmox1, mapk1, fth1 and mmp13. Overall, this study shows that cells from temperature-stressed fish are modestly more vulnerable to Cd stress, and indicate that mechanisms linked to oxidative stress may be differentially affected in temperature-stressed cells.

Role of the GH-IGF-1 system in Atlantic salmon and rainbow trout postsmolts at elevated water temperature.

A comparative experiment with Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) postsmolts was conducted over 35 days to provide insight into how growth, respiration, energy metabolism and the growth hormone (GH) and insulin-like growth factor 1 (IGF-1) system are regulated at elevated sea temperatures. Rainbow trout grew better than Atlantic salmon, and did not show reduced growth at 19 °C. Rainbow trout kept at 19 °C had increased blood hemoglobin concentration compared to rainbow trout kept at 13 °C, while salmon did not show the same hemoglobin response due to increased temperature. Both species showed reduced length growth and decreased muscle glycogen stores at 19 °C. Circulating IGF-1 concentration was higher in rainbow trout than in Atlantic salmon, but was not affected by temperature in either species. Plasma IGF-binding protein 1b (IGFBP-1b) concentration was reduced in Atlantic salmon reared at 19 °C after 15 days but increased in rainbow trout at 19 °C after 35 days. The igfbp1b mRNA level in liver showed a positive correlation to plasma concentrations of glucose and IGFBP-1b, suggesting involvement of this binding protein in carbohydrate metabolism at 19 °C. At this temperature muscle igfbp1a mRNA was down-regulated in both species. The muscle expression of this binding protein correlated negatively with muscle igf1 and length growth. The plasma IGFBP-1b concentration and igfbp1b and igfbp1a expression suggests reduced muscle igf1 signaling at elevated temperature leading to glucose allostasis, and that time course is species specific due to higher thermal tolerance in rainbow trout.

Effect of temperature and diet on wound healing in Atlantic salmon (Salmo salar L.).

Compromised skin integrity of farmed Atlantic salmon, commonly occurring under low temperature and stressful conditions, has major impacts on animal welfare and economic productivity. Even fish with minimal scale loss and minor wounds can suffer from secondary infections, causing downgrading and mortalities. Wound healing is a complex process, where water temperature and nutrition play key roles. In this study, Atlantic salmon (260 g) were held at different water temperatures (4 or 12 °C) and fed three different diets for 10 weeks, before artificial wounds were inflicted and the wound healing process monitored for 2 weeks. The fish were fed either a control diet, a diet supplemented with zinc (Zn) or a diet containing a combination of functional ingredients in addition to Zn. The effect of diet was assessed through subjective and quantitative skin histology and the transcription of skin-associated chemokines. Histology confirmed that wound healing was faster at 12 °C. The epidermis was more organised, and image analyses of digitised skin slides showed that fish fed diets with added Zn had a significantly larger area of the epidermis covered by mucous cells in the deeper layers after 2 weeks, representing more advanced healing progression. Constitutive levels of the newly described chemokines, herein named CK 11A, B and C, confirmed their preferential expression in skin compared to other tissues. Contrasting modulation profiles at 4 and 12 °C were seen for all three chemokines during the wound healing time course, while the Zn-supplemented diets significantly increased the expression of CK 11A and B during the first 24 h of the healing phase.

High levels of dietary phytosterols affect lipid metabolism and increase liver and plasma TAG in Atlantic salmon (Salmo salar L.).

Replacing dietary fishmeal (FM) and fish oil (FO) with plant ingredients in Atlantic salmon (Salmo salar L.) diets decreases dietary cholesterol and introduces phytosterols. The aim of the present study was to assess the effect of dietary sterol composition on cholesterol metabolism in Atlantic salmon. For this purpose, two dietary trials were performed, in which Atlantic salmon were fed either 100 % FM and FO (FM-FO) diet or one of the three diets with either high (80 %) or medium (40 %) plant protein (PP) and a high (70 %) or medium (35 %) vegetable oil (VO) blend (trial 1); or 70 % PP with either 100 % FO or 80 % of the FO replaced with olive, rapeseed or soyabean oil (trial 2). Replacing ≥ 70 % of FM with PP and ≥ 70 % of FO with either a VO blend or rapeseed oil increased plasma and liver TAG concentrations. These diets contained high levels of phytosterols and low levels of cholesterol. Fish fed low-cholesterol diets, but with less phytosterols, exhibited an increased expression of genes encoding proteins involved in cholesterol uptake and synthesis. The expression of these genes was, however, partially inhibited in rapeseed oil-fed fish possibly due to the high dietary and tissue phytosterol:cholesterol ratio. Atlantic salmon tissue and plasma cholesterol concentrations were maintained stable independent of the dietary sterol content.

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.

Transcriptional effects of dietary exposure of oil-contaminated Calanus finmarchicus in Atlantic herring (Clupea harengus).

Suppression subtractive hybridization (SSH) cDNA library construction and characterization was used to identify differentially regulated transcripts from oil exposure in liver of male Atlantic herring (Clupea harengus) fed a diet containing 900 mg crude oil/kg for 2 mo. In total, 439 expressed sequence tags (EST) were sequenced, 223 from the forward subtracted library (enriched for genes putatively upregulated by oil exposure) and 216 from the reverse subtracted library (enriched for genes putatively downregulated by oil exposure). Follow-up reverse-transcription (RT) quantitative polymerase chain reaction (qPCR) analyses of gene transcription were conducted on additional herring exposed to food containing 9 (low), 90 (medium), and 900 (high) mg crude oil/kg feed for 2 mo. Chronic exposure of Atlantic herring to an oil-contaminated diet mediated upregulation of transcripts encoding antifreeze proteins, proteins in the classical complement pathway (innate immunity), and iron-metabolism proteins. Gene ontology (GO) analysis showed that "cellular response to stress," "regulation to biological quality," "response to abiotic stimuli," and "temperature homeostasis" were the most affected go at the biological processes level, and "carbohydrate binding," "water binding," and "ion binding" at the molecular function level. Of the genes examined with RT-qPCR, CYP1A, antifreeze protein, retinol binding protein 1, deleted in malignant brain tumor 1, and ovary-specific C1q-like factor demonstrated a significant upregulation. Myeloid protein 1, microfibrillar-associated protein 4, WAP65, and pentraxin were downregulated in liver of fish from the high exposure group. In conclusion, this study suggests that 2 mo of oil exposure affected genes encoding proteins involved in temperature homeostasis and possible membrane stability in addition to immune-responsive proteins in Atlantic herring.

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.

N-acetylhistidine, a novel osmolyte in the lens of Atlantic salmon (Salmo salar L.).

Volume homeostasis is essential for the preservation of lens transparency and this is of particular significance to anadromous fish species where migration from freshwater to seawater presents severe osmotic challenges. In Atlantic salmon (Salmo salar L.), aqueous humor (AH) osmolality is greater in fish acclimated to seawater compared with young freshwater fish, and levels of lens N-acetylhistidine (NAH) are much higher in seawater fish. Here we investigate NAH as an osmolyte in the lenses of salmon receiving diets either with or without histidine supplementation. In the histidine-supplemented diet (HD) histidine content was 14.2 g/kg, and in the control diet (CD) histidine content was 8.9 g/kg. A transient increase in AH osmolality of 20 mmol/kg was observed in fish transferred from freshwater to seawater. In a lens culture model, temporary decreases in volume and transparency were observed when lenses were exposed to hyperosmotic conditions. A positive linear relationship between extracellular osmolality and lens NAH content was also observed, whereas there was no change in lens histidine content. Hypoosmotic exposure stimulated [(14)C]-histidine efflux by 9.2- and 2.6-fold in CD and HD lenses, respectively. NAH efflux, measured by HPLC, was stimulated by hypoosmotic exposure to a much greater extent in HD lenses. In vivo, lens NAH increased in response to elevated AH osmolality in HD but not CD fish. In conclusion, NAH has an important and novel role as a compatible osmolyte in salmon lens. Furthermore, it is the major osmolyte that balances increases in AH osmolality when fish move from freshwater to seawater. A deficiency in NAH would lead to a dysfunction of the normal osmoregulatory processes in the lens, and we propose that this would contribute to cataract formation in fish deficient in histidine.

Effects of dietary lipid, vitamins and minerals on total amounts and redox status of glutathione and ubiquinone in tissues of Atlantic salmon (Salmo salar): a multivariate approach.

The hypothesis of the present study was that Atlantic salmon (Salmo salar) would respond to large variations in supplementation of dietary pro- and antioxidants, and marine lipid, with adjustment of the endogenously synthesised antioxidants, glutathione (GSH) and ubiquinone (UQ). An experiment with 2(7-3) reduced factorial design (the number of cases reduced systematically from 2(7) (full design) to 2(4) (reduced design)) was conducted, where vitamins, minerals and lipid were supplemented in the diet at high and low levels. For the vitamins and minerals the high levels were chosen to be just below anticipated toxic levels and the low levels were just above the requirement (vitamin C, 30 and 1000 mg/kg; vitamin E, 70 and 430 mg/kg; Fe, 70 and 1200 mg/kg; Cu, 8 and 110 mg/kg; Mn, 12 and 200 mg/kg). For astaxanthin, the dietary levels were 10 and 50 mg/kg and for lipid, 150 and 330 g/kg. The experiment was started with post-smolts (148 (sd 17 g)) and lasted for 5 months. The only effect on GSH was a minor increase ( < 10 %) in total concentration in the liver in response to high dietary lipid. GSH redox state was not affected. UQ responded to dietary lipid, astaxanthin and vitamin E, both with regard to total concentration and redox state. Except for an effect of Fe on plasma GSH, the trace elements and vitamin C had no effect on tissue levels and oxidation state of GSH and UQ. This shows that the endogenous redox state is quite robust with regard to variation of dietary pro- and antioxidants in Atlantic salmon.

Optimisation of gene expression analysis in Atlantic salmon lenses by refining sampling strategy and tissue storage.

Analysis of gene expression in the lens is one of the analytical tools employed to investigate cataract formation in Atlantic salmon (Salmo salar L.). High quality RNA preparations are an essential prerequisite for gene expression analysis. The first aim of the present study was to investigate the possible effects of two methods of tissue preservation on the quality of RNA extracted from Atlantic salmon lenses. RNA was extracted from lenses either stored in RNAlater or flash-frozen in liquid nitrogen. Both tissue preservation methods yielded RNA of similarly high quality. The second aim was to examine if stress related to fish handling and the choice of anaesthesia during the sampling procedure affected gene expression in the lens. Six sampling procedures were tested on groups of sea water adapted Atlantic salmon smolt. Fish were either killed instantaneously (control group) or sampled after 30 min anaesthetised with isoeugenol, after 30 min without anaesthesia, after 120 min anaesthetised with isoeugenol, after 120 min with 15 min anaesthesia with metacaine or after 120 min without anaesthesia. The expression levels of specific genes, of special interest in the study of molecular mechanisms of cataractogenesis, were analysed in lenses by real-time RT-PCR. Fish not anaesthetised had significantly increased levels of heat shock protein 70 (HSP70) mRNA after 30 min compared to the control group. Glutathione reductase (GR) and manganese superoxide dismutase (Mn-SOD) were expressed at significantly lower levels in groups of Atlantic salmon sampled after 120 min anaesthetised with isoeugenol or metacaine, and anaesthetised with isoeugenol, metacaine or without anaesthesia, respectively. The same expression patterns were found in corresponding gill tissues for these two antioxidant genes. In conclusion, preservation in liquid nitrogen instead of RNAlater is recommended due to practical conditions in RNA extraction. A quick sampling protocol with the use of anaesthetics and not exceeding 30 min should be preferred to avoid effects of the sampling procedure on lens gene expression in Atlantic salmon.

Genome-wide transcription analysis of histidine-related cataract in Atlantic salmon (Salmo salar L).

PURPOSE: Elevated levels of dietary histidine have previously been shown to prevent or mitigate cataract formation in farmed Atlantic salmon (Salmo salar L). The aim of this study was to shed light on the mechanisms by which histidine acts. Applying microarray analysis to the lens transcriptome, we screened for differentially expressed genes in search for a model explaining cataract development in Atlantic salmon and possible markers for early cataract diagnosis. METHODS: Adult Atlantic salmon (1.7 kg) were fed three standard commercial salmon diets only differing in the histidine content (9, 13, and 17 g histidine/kg diet) for four months. Individual cataract scores for both eyes were assessed by slit-lamp biomicroscopy. Lens N-acetyl histidine contents were measured by high performance liquid chromatography (HPLC). Total RNA extracted from whole lenses was analyzed using the GRASP 16K salmonid microarray. The microarray data were analyzed using J-Express Pro 2.7 and validated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Fish developed cataracts with different severity in response to dietary histidine levels. Lens N-acetyl histidine contents reflected the dietary histidine levels and were negatively correlated to cataract scores. Significance analysis of microarrays (SAM) revealed 248 significantly up-regulated transcripts and 266 significantly down-regulated transcripts in fish that were fed a low level of histidine compared to fish fed a higher histidine level. Among the differentially expressed transcripts were metallothionein A and B as well as transcripts involved in lipid metabolism, carbohydrate metabolism, regulation of ion homeostasis, and protein degradation. Hierarchical clustering and correspondence analysis plot confirmed differences in gene expression between the feeding groups. The differentially expressed genes could be categorized as "early" and "late" responsive according to their expression pattern relative to progression in cataract formation. CONCLUSIONS: Dietary histidine regimes affected cataract formation and lens gene expression in adult Atlantic salmon. Regulated transcripts selected from the results of this genome-wide transcription analysis might be used as possible biological markers for cataract development in Atlantic salmon.

Recommendations for dietary level of micro-minerals and vitamin D3 to Atlantic salmon (Salmo salar) parr and post-smolt when fed low fish meal diets.

Atlantic salmon (Salmo salar) 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 -1, respectively; for iodine, dietary supply from 150-200 NP, corresponding to 0.7-1.6 mg kg-1, 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 -1 to fulfil the requirement, except for Cu which was satisfied at 100-150 NP, equivalent to 13-14 mg kg -1 diet.

The impact of different water gas levels on cataract formation, muscle and lens free amino acids, and lens antioxidant enzymes and heat shock protein mRNA abundance in smolting Atlantic salmon, Salmo salar L.

The present experiment was conducted to examine if freshwater (FW) oxygen and carbon dioxide regimes cause physiological responses that lead to cataract formation in Atlantic salmon (Salmo salar L.) smolt. Duplicate groups of 50 g Atlantic salmon smolts were exposed to three freshwater oxygen saturation regimes (95, 112 or 125% saturation), with or without addition of carbon dioxide (measured 17-18 and 2-3 mg L(-1), respectively), for six weeks before transfer to seawater (SW). The FW exposure groups were followed up for another six weeks under a common SW regime. Fish were screened for cataract and sampled accordingly, at start, after 6 weeks in FW and after 6 weeks in SW. Increased growth related cataract incidences and severities were recorded in SW, mainly in the groups previously exposed to normoxic and hyperoxic conditions in FW, as compared to the respective groups added carbon dioxide. The concentration of histidine compounds (imidazoles) in muscle and lens tissue, used as quantitative risk markers of cataract, were lower than observed in earlier studies, however, neither were affected by the present water gas regimes in FW nor after follow up in SW. Independently of water oxygenation in FW, muscle free amino acid profiles in salmon groups concomitantly exposed to elevated carbon dioxide indicated use of selected free amino acids for energy purposes. Significantly lower abundance of heat shock protein 70 mRNA and trends towards stepwise reduction of antioxidant enzymes mRNA in the lens from fish exposed to increased water oxygenation were recorded, probably linked to increased growth and/or external stress during smoltification. This represents a first communication on using early molecular markers to express reduced protection of the fish lens against external stress to explain cataract development.

Effect of storage time, temperature, antioxidant and thawing on fatty acid composition of plasma, serum and red blood cells - A pilot biobank study.

BACKGROUND: It studies on the factors that affect the stability of fatty acid profiles from human blood specimens are generally performed by evaluating the effect of a single factor on an individual fatty acid and excluding a considerable amount of data from the total fatty acid profiles. METHODS: The stability of fatty acids from plasma, serum and red blood cells (RBC) was evaluated in terms of time, temperature, antioxidant and thawing. The fatty acids were methylated and analyzed by gas chromatography. The large volume of data is evaluated simultaneously and automatically by observing an Excel-based colour scale that indicates whether the fatty acid profiles have changed significantly as a result of the storage time (0-52weeks), temperature (-20°C/-80°C), butylated hydroxytoluene (BHT) antioxidant (presence/absence) or thawing (single/multiple). RESULTS: Fatty acids from plasma were stable at both temperatures (-20°C/-80°C) regardless of BHT. Fatty acids from serum without BHT degrades faster at -80°C than -20°C and fatty acids from RBC without BHT degrades faster at -20°C than -80°C. Addition of BHT inhibits this effect in serum and RBC. Multiple thawing of RBC without BHT demonstrated that polyunsaturated fatty acids were generally more susceptible for changes at -80°C than at -20°C while BHT prevents partially this effect. CONCLUSIONS: This study draws attention to the importance of pre-analytical considerations when storing blood samples in biobanks and the need of careful judgments when analyzing fatty acids profiles.