Comment[ArrayExpressAccession]	E-MTAB-3268
MAGE-TAB Version	1.1
Investigation Title	Evaluation of a high-EPA oil from transgenic Camelina sativa in feeds for Atlantic salmon (Salmo salar L.): Effects on tissue fatty acid composition, histology and gene expression
Comment[Submitted Name]	Evaluation of a high-EPA oil from transgenic Camelina sativa in feeds for Atlantic salmon (Salmo salar L.): Effects on tissue fatty acid composition, histology and gene expression
Experiment Description	Currently, the only sustainable alternatives for dietary fish oil (FO) in aquafeeds are vegetable oils (VO) that are devoid of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA). Entirely new sources of n-3 LC-PUFA such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids through de novo production is a potential solution to fill the gap between supply and demand of these important nutrients. Camelina sativa,was metabolically engineered to produce a seed oil (ECO) with > 20 % EPA and its potential to substitute for FO in Atlantic salmon feeds was tested. Fish were fed one of three experimental diets containing FO, wild-type camelina oil (WCO) or ECO as the sole lipid sources for 7-weeks. Inclusion of ECO did not affect any of the performance parameters studied and enhanced apparent digestibility of individual n-6 and n-3 PUFA compared to dietary WCO. High levels of EPA were maintained in brain, liver and intestine (pyloric caeca), and levels of DPA and DHA were increased in liver and intestine of fish fed ECO compared to fish fed WCO likely due to increased LC-PUFA biosynthesis based on up-regulation of the genes. Fish fed WCO and ECO showed slight lipid accumulation within hepatocytes similar to that with WCO, although not significantly different to fish fed FO. The regulation of a small number of genes could be attributed to the specific effect of ECO (311 features) with metabolism being the most affected category. The EPA oil from transgenic Camelina (ECO) could be used as a substitute for FO, however it is a hybrid oil containing both FO (EPA) and VO (18:2n-6) fatty acid signatures that resulted in similarly mixed metabolic and physiological responses.
Experimental Design	reference design
Experimental Design Term Source REF	EFO
Experimental Design Term Accession Number	EFO_0001775
Experimental Factor Name	diet
Experimental Factor Type	diet
Experimental Factor Term Source REF	
Experimental Factor Term Accession Number	
Person Last Name	Betancor
Person First Name	Mónica
Person Mid Initials	
Person Email	m.b.betancor@stir.ac.uk
Person Phone	
Person Fax	
Person Address	
Person Affiliation	
Person Roles	submitter
Date of Experiment	2012-06-29
Public Release Date	2017-10-30
Protocol Name	P-MTAB-43049	P-MTAB-43050	P-MTAB-43051	P-MTAB-43052	P-MTAB-43053	P-MTAB-43054	P-MTAB-43055
Protocol Type	growth protocol	treatment protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning and feature extraction protocol	normalization data transformation protocol
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO	EFO	EFO
Protocol Term Accession Number	EFO_0003789	EFO_0003969	EFO_0002944	EFO_0003813	EFO_0003815	EFO_0003814	EFO_0003816
Protocol Description	A total of 405 post-smolt Atlantic salmon with an average body weight of 82.5 ± 8.1 g (mean ± S.D.) were distributed into 9 seawater tanks (45 per tank) and fed one of the three experimental feeds in triplicate for 7 weeks. Prior to the start of the experimental period, during a 1 week acclimation period, fish were fed the WCO diet. The experimental system comprised 1 m2, 500 L tanks supplied by flow-through seawater (15 L min-1) at ambient temperature that averaged 10.2 ± 0.6 °C. Experimental feeds were delivered in excess by automatic disc feeders with an automated uneaten feed collection system in order to determine accurate feed efficiency.	At the end of the trial, fish were not fed for 48 h prior to being anaesthetised and killed by overdose with metacaine sulphonate (MS222). Three whole fish per tank (9 per treatment) were frozen for analyses of proximate composition. A further 9 fish per tank were used for biometric measurements (hepato-somatic and viscera-somatic indices) and tissue analyses. Samples of flesh (Norwegian quality cut; NQC) and liver from 3 fish per tank were immediately frozen in liquid nitrogen and stored at – 70 oC prior to total lipid and fatty acid analyses. Further samples of liver were collected from six fish per treatment (two per tank) and stabilised in RNAlater® (Sigma, Poole, UK) prior to RNA extraction	Pyloric caeca from six individual fish per dietary treatment were homogenised in 1 ml of TriReagent® (Sigma-Aldrich, Dorset, UK) RNA extraction buffer using a bead tissue disruptor (Bio Spec, Bartlesville, Oklahoma, USA). Total RNA was isolated following manufacturer’s instructions and quantity and quality determined by spectrophotometry using a Nanodrop ND-1000 (Labtech Int., East Sussex, UK) and electrophoresis using 500 ng of total RNA in a 1 % agarose gel. 	Each RNA sample was amplified using TargetAmpTM 1-Round Aminoallyl-aRNA Amplification Kit according to the manufacturer’s instructions and aaRNA quality was assessed via Nanodrop quantification and agarose gel electrophoresis. aaRNA sample was indirectly labelled and purified. Briefly, Cy dye suspensions (Cy3 and Cy5) in sufficient quantity for all labelling reactions were prepared by adding 40 uL high purity dimethyl sulphoxide per tube of Cy dye (PA23001 or PA25001; GE HealthCare). Each sample (2.5 ug aRNA) was denatured at 75°C for 5 min and then 3 uL 0.5 M NaHCO3 pH8.5 and 1.5 uL Cy3 or 1.0 uL Cy5 dye was added achieving a total volume of 15 uL per reaction. Samples were incubated for an hour at 25oC in the dark, purified using Illustra AutoSeq G-50 Dye Terminator Removal Kit and concentration, dye incorporation and purity were assessed via spectrophotometer (NanoDrop) with products also visualised on a fluorescent scanner (Typhoon Trio, GE Healthcare).	Hybridisation was performed over two consecutive days using the Agilent Gene Expression Hybridisation Kit (Agilent Technologies) as per manufacturer’s instructions. For each reaction, 825ng Cy5 labelled reference pool and 825 ng Cy3 labelled individual samples were combined in 35 uL nuclease free water and then 20 uL fragmentation master mix added (consisting of 11 uL of 10X blocking agent, 2uL 25x fragmentation buffer and 7uL nuclease free water). The reactions were then incubated at 60oC in the dark for 30 mins, chilled on ice, and mixed with 57 uL 2x GEx Hybridisation buffer (pre heated to 37oC), Following centrifugation (18000 x g for 1 min) the samples were kept on ice until loaded (103 uL) in a semi randomised order onto the microarray slides. Samples from the six biological replicates were spread across different slides, Cy3 fluorescence content (dye incorporation rate x volume) was also taken into consideration. To aid scanning, samples with the most similar amounts of Cy3 were grouped on the same slide. Hybridisation was carried out in a rotating rack oven (Agilent Technologies) at 65oC, 10 rpm over 17 hours. Following hybridisation, slides were subject to a number of washing steps performed in Easy-DipTM slide staining containers (Canemco Inc., Quebec, Canada). First, each microarray and backing gasket was disassembled in Agilent Wash Buffer 1 and microarray slides were transferred to an Easy Dip Rack submersed in Wash Buffer 1. Following 1 min incubation at room temperature (c. 20°C) and 150 rpm (Stuart Orbital Incubator), slides were briefly dipped into Wash Buffer 1 pre-heated to 31oC, then placed into Wash Buffer 2 (31oC) for 1 min at 150rpm. Finally, the slides were transferred to acetonitrile for 10 s and then Agilent Stabilization and Drying Solution for 30 s. The slides were then air dried in the dark and scanned within two hours.	Scanning was carried out at 5um resolution on an Axon GenePix Pro scanner at 40% laser power. The “auto PMT” function was enabled to adjust PMT for each channel such that less than 0.1% of features were saturated and so that the mean intensity ratio of Cy3:Cy5 signal was close to one.	Agilent Feature Extraction Software (v 9.5) was used to identify features and extract background subtracted raw intensity values that were then transferred to GeneSpring GX (v.12) software where the quality filtering and normalisation steps took place. Intensity values ? 1 were adjusted to 1 and a Lowess normalisation undertaken. Stringent quality filtering ensured that features that represented technical controls, saturated probes, probe population outliers or probes which were not significantly different from the background were removed. This left 33,688 of the original 43,466 probes available for downstream analysis. A single array was excluded from the analysis as it was flagged as sub-standard by the feature extraction software and also appeared as a clear outlier on a Principal Component Analysis performed within Genespring in order to compare arrays. Thus 35 of the 36 arrays were statistically analysed.
Protocol Hardware							
Protocol Software							
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/
Term Source Version		
Comment[AEExperimentType]	transcription profiling by array
SDRF File	E-MTAB-3268.sdrf.txt