Functional Genomic Analysis of the Impact of Camel

Authors: Tyler D Brown Tiago S Hori Xi Xue Chang Lin Ye Derek M Anderson Matthew L Rise

Publish Date: 2016/06/02

Volume: 18, Issue: 3, Pages: 418-435

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Abstract

The inclusion of plant meals in diets of farmed Atlantic salmon can elicit inflammatory responses in the distal intestine DI For the present work fish were fed a standard fish meal FM diet or a diet with partial replacement of FM with solventextracted camelina meal CM 8 16 or 24  CM inclusion during a 16week feeding trial A significant decrease in growth performance was seen in fish fed all CM inclusion diets Hixson et al in Aquacult Nutr 22615–630 2016 A 4x44K oligonucleotide microarray experiment was carried out and significance analysis of microarrays SAM and rank products RP methods were used to identify differentially expressed genes between the DIs of fish fed the 24  CM diet and those fed the FM diet Twelve features representing six known transcripts and two unknowns were identified as CM responsive by both SAM and RP The six known transcripts including thioredoxin and ependymin in addition to tgfb mmp13 and GILT were studied using qPCR with RNA templates from all four experimental diet groups All six microarrayidentified genes were confirmed to be CM responsive as was tgfb and mmp13 Histopathological analyses identified signs of inflammation in the DI of salmon fed CMcontaining diets including lamina propria and subepithelial mucosa thickening infiltration of eosinophilic granule cells increased goblet cells and decreased enterocyte vacuolization All of these were significantly altered in 24  CM compared to all other diets with the latter two also altered in 16  CM compared with 8  CM and control diet groups Significant correlation was seen between histological parameters as well as between five of the qPCR analyzed genes and histological parameters These molecular biomarkers of inflammation arising from longterm dietary CM exposure will be useful in the development of CMcontaining diets that do not have deleterious effects on salmon growth or physiologyFish products are an important component of many human diets and the demand for marine products is increasing as the global population has grown past seven billion FAO 2009 The increasing demand for fish products for human consumption has fueled a rapid increase in the production of the aquaculture industry and its continued growth is required to meet the global demand In 2003 the global production from aquaculture of aquatic animals was approximately 39 million tonnes while in 2012 this production had reached approximately 67 million tonnes in this same time the global production from capture fisheries remained relatively unchanged at around 90 million tonnes per year FAO 2012Atlantic salmon Salmo salar is a carnivorous species and as such aquaculture feeds for farmed Atlantic salmon typically include marine animalbased protein and oil sources such as fish and crustacean that rely heavily on the inputs of unsustainable fishery resources Tacon et al 2009 The supplies of these raw marine products are limited and include fish oil FO and fish meal FM from natural stocks of lower trophic level fish species such as anchovy and herring Tacon et al 2009 The global output of FO and FM is restricted as the populations of some natural fish stocks are declining due to overexploitation Tacon and Metian 2009 Therefore sustainable costeffective substitutes for FM are currently being sought with many plantbased alternatives being investigated Tacon and Metian 2008 Plantbased products are not typically part of the diet of wild Atlantic salmon and the supplementation of feeds for Atlantic salmon with plant meals PM may lead to inflammation of the distal intestine DI enteritis Chikwati et al 2013 Marjara et al 2012 Moldal et al 2014 Overland et al 2009 Sahlmann et al 2013 This inflammation is typically characterized by the shortening of intestinal villi a decrease in supranuclear vacuoles SNV in the intestinal epithelial cells significant infiltration of inflammatory cells in the lamina propria LP an increase in the number of goblet cells GC and thickening of the subepithelial mucosa SEM Baeverfjord and Krogdahl 1996 The presence of inflammation is preceded by changes in the expression of genes that are responsible for mediating inflammatory responses This inflammatory response paired with many other factors leads to decreased digestibility of essential nutrients and a decrease in the overall growth performance of the fish Chikwati et al 2013 Marjara et al 2012 Moldal et al 2014 Overland et al 2009 Sahlmann et al 2013 The specific components of PMs that are responsible for this inflammatory response are not known for certain However many plant products contain antinutritional factors Sahlmann et al 2013 and these components may be responsible for the inflammatory response seen in the intestine In camelina the main antinutrients are glucosinolates phytic acid sinapine and tannins Russo and Reggiani 2012 Glucosinolates and their metabolites are known to cause impairment of growth and irritation of the gastrointestinal tract Phytic acid and tannins have been shown to render essential minerals insoluble and inhibit digestive enzymes respectively which may also inhibit growth due to decreased utilization of vitamins and minerals Amarowicz et al 2010 Schlemmer et al 2009 In Atlantic salmon the hindgut is the main site of amino acid absorption in the digestive tract and as such any loss of function that may be caused by inflammation can lead to a decline in the growth performance of the fish Lokka et al 2013 Sire et al 1981A novel source of plantbased protein that is currently being considered as a supplementation to FM is the meal from camelina Camelina sativa Camelina is a flowering oilseed crop of the Brassicaceae family with many characteristics that are favorable to agriculture These include low fertilizer and nitrogen requirements and high resistance to biotic and abiotic stress Ghamkhar et al 2010 CM contains up to 45  crude protein and is comparable in this regard to other oilseed crops such as canola and rapeseed Acamovic et al 1999 Frame et al 2007 Hixson et al 2016 CM also contains adequate levels of at least 18 amino acids with nine of them being essential and only requires supplementation of some limiting amino acids such as methionine and/or lysine Cherian 2012 Zubr 2003Recently a feeding trial was conducted to examine the growth performance of Atlantic salmonfed diets with partial replacement of FM with CM The current study uses some of the same diets in the same feeding trial as Hixson et al 2016 which focused on tissue lipid and amino acid analyses The current study examines the effect of CMsupplemented diets on DI transcript expression and histology Functional genomics analyses of hindgut tissue samples were carried out using DNA microarrays and realtime quantitative polymerase chain reaction qPCR These techniques have been used in previous studies for nutrigenomic analyses to assess the impact of other PM inclusion on the hindgut of Atlantic salmon Chikwati et al 2013 Marjara et al 2012 Moldal et al 2014 Overland et al 2009 Sahlmann et al 2013 The current functional genomics study aimed to identify and validate putative inflammatory biomarker genes that are responsive to longterm exposure to CMcontaining aquafeeds The gene expression data were paired with histological data of the hindgut tissue to allow for a more thorough understanding of the effect of CM on the Atlantic salmon hindgut Molecular biomarkers of chronic inflammation arising from this study will be useful in future research aimed at determining the maximum inclusion of CM that does not cause deleterious effects in the Atlantic salmon DIA study Ye 2014 analyzing the effect of high oil residue CM rather than solventextracted CM SECM which was used in the current study on growth performance carcass composition and DI histology of Atlantic salmon smolts was recently carried out in addition to using a different CM product Ye 2014 is different from the current study in that it did not include gene expression assays and the two studies used distinct methods for histological analyses Another recent study Ye et al 2016 evaluated the effects of SECMcontaining diets on Atlantic salmon parr using carcass composition analysis and the same semiquantitative histological methods as in Ye 2014Camelina Calena cultivar was grown and harvested by the staff of the Dalhousie Agricultural Campus in Canning NS Canada The seeds were cold pressed using a KEK 0500 screw press at Atlantic Oilseed Processing Summerside PEI Canada The resulting meal was ground with a hammer mill screen size 8 mm into a prepressed meal cake at Atlantic Oilseed Processing and solvent extracted with petroleum ether at a concentration of 3 mL g−1 at the Faculty of Agriculture Campus Dalhousie University Truro NS Canada The solventextracted CM used in this study contained 39  crude protein and had a gross energy content of 4320 kcal/kg Hixson et al 2016 Ye 2014

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