Novel Omega-3 Sources in Feeds and Impacts on Salmon Health

Lead Research Organisation: University of Stirling
Department Name: Institute of Aquaculture

Abstract

Dietary omega-3 fatty acids are key nutrients that have beneficial health effects playing a critical role in the immune system as well as contributing to the normal function of the heart and development of the brain. The human body is unable to produce omega-3, which is why it is important to eat these essential fats. Fish and seafood are unique sources of omega-3 in the human diet, but decline in traditional fisheries means that more than half of all fish consumed is now farmed. Until recently the only way to ensure high levels of omega-3 in farmed fish and seafood was to deliver these nutrients in feeds by using the marine ingredients, fishmeal and fish oil. However, paradoxically, they too are derived from marine fisheries that are at their sustainable limit making them finite and limited resources. The rapid expansion of fish farming in the last two decades has ony been possible with increasing dilution of the traditional marine ingredients with more economical alternatives, primarily plant meals and vegetable oils that lack the omega-3 fatty acids found in fish. Therefore, the development of these alternative feeds in Atlantic salmon (Salmo salar) farming has seen levels of omega-3 in farmed salmon halved in recent years.
Dietary omega-3 are equally essential for the health of fish, just as they are for humans. Several negative issues related to farmed fish performance and health have paralleled the development of alternative, low marine feeds with reduced levels of the beneficial omega-3, including higher incidence and severity of inflammatory diseases. However, in addition, inflammation is a key component of the immune response to all pathogens, including parasitic, bacterial and viral infections and so the current low levels of omega-3 in feeds for farmed fish has had consequences for fish health and welfare. Thus, the dual impacts of reduced dietary omega-3 levels on fish health, and the nutritional quality of farmed fish products for human consumers has meant that fish farming, especially of salmon, has spearheaded efforts to both highlight and close the gap between supply and demand for the key omega-3 nutrients. Specifically, the Global Salmon Initiative announced a tender for commercial organisations to supply up to 200,000 tons annually of novel omega-3-rich oils. In addition, a consortium including The World Bank, announced the F3 (Fish Free Feed) Fish Oil Challenge to create a fish-free 'fish oil' substitute. Consequently, new sources of omega-3-rich oils from marine microalgae and genetically-modified oilseed crops have been developed and tested as feed ingredients for farmed fish, including salmon. While these studies have proved the effectiveness of these new oils in increasing omega-3 content of farmed fish, the impact on fish health has been neglected.
This project will directly investigate the important health impacts of entirely novel omega-3 rich oils as feed ingredients for farmed Atlantic salmon. The studies will focus on determining not only impact of the new omega-3 sources on the response of salmon to specific disease and parasite challenges, but also on defining the biochemical and molecular mechanisms underpinning fish health, quantifying the potential of these new dietary oils for use in UK salmon farming. The proposal is timely and highly relevant and appropriate as it responds to current needs with cutting edge research to improve the quality and effectiveness of modern alternative feeds in fish farming, enhancing production and feed efficiency, while maintaining the health and improving the nutritional quality of farmed fish, delivering greater sustainability and food security.

Technical Summary

We will investigate whether novel omega-3 oils supplying de novo eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) will impact the health of fish fed these oils. Our contention is that these new dietary oils will have effects on lipid and fatty acid metabolism of fish that will, in turn, modulate production of crucial lipid mediators affecting inflammatory responses and immune functions with consequent impacts on resistance to pathogens and parasites. To test this hypothesis, a feeding trial with Atlantic salmon (Salmo salar) will investigate four novel omega-3 oils from macroalgae and genetically-modified oilseed (Camelina sativa) crops supplying DHA alone, EPA alone, or combinations of EPA+DHA, in comparison with traditional fish oils as reference feeds, and omega-3-rich krill oil and omega-6-rich sunflower oil as positive and negative health controls, respectively. Salmon in both freshwater and seawater phases will be subjected to specific pathogen challenges including Aeromonas samonicida, sea lice and amoebic gill disease. The project will provide detailed data on the influence of the novel omega-3 oils on lipid class and fatty acid composition of salmon tissues, and determine the consequences this has on critical metabolic and immune pathways. The insight that these data will provide will, in turn, enable us to elucidate the impact the novel omega-3 oils have on the production of lipid mediators that are crucial in mediating the molecular, mechanisms involved in the inflammatory responses and immune function. The results will enable us to define the positive and negative impacts of novel omega-3 oils on salmon health and welfare, and provide definitive advice on the use of these oils as feed ingredients for farmed salmon.

Planned Impact

Outcomes will impact at many levels including the salmon feed and production industries, consumers and the general public, in addition to academia. Primary exploitation will be through development and production of novel sustainable feed formulations utilising entirely new de novo sources of the omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), EPA and DHA, as replacements for the dwindling and expensive marine ingredients, fishmeal and especially fish oil, currently used in UK aquafeeds. The project will therefore provide industry with a new strategy with huge potential to improve the nutritional quality of farmed fish and, in turn, the economic viability and sustainability of UK aquaculture. Tangible outcomes will be the development of more sustainable feeds formulated with very low marine ingredients, but without the negative impacts on n-3 LC-PUFA levels in farmed salmon, expandable to all sectors of the aquaculture industry. However, our exploitation strategy will be far wider than just the feed sector, as we will target fish production industries as a whole, and increase awareness of the potential of new ingredients supplying high levels of EPA and/or DHA to mitigate fish health and product quality issues currently associated with existing sustainable feed formulations.
There are two main beneficiaries, the UK salmonid farming, and aquaculture feed industries. The aquafeed industry itself will play a key central role in the commercial application and uptake of the omega-3 sources with our industrial project partner, BioMar taking a lead role. All partners, Universities of Stirling (UoS), Aberdeen (UoA), Highlands and Islands (UHI) and Rothamsted Research (RRES) have worked extensively with BioMar for more than a decade, collaborating on several research studies including BBSRC IPA, BBSRC-NERC and BBSRC Super Follow-on-Funding projects, and PhD CASE studentships. Moreover, UoS and UoA have long established histories as centres of excellence for fish nutrition and health research and are recognised as trusted sources of independent research. All four partners have extensive links with all sectors and key stakeholders in the UK aquaculture industry that involve regular direct contact, engagement and discussion. Thus, we will use these well-established links, along with the support of the industry-led Scottish Aquaculture Innovation Centre (SAIC), to engage with potential end-users including major fish farming companies, to ensure industry-wide dissemination and engagement.
Currently, all partners have joint projects with industrial partners at all levels of the industry and these direct links are ideal for a two-way flow of information, and actively involving and engaging end users, stakeholders and beneficiaries, seeking their input throughout the project. UoS, UoA and UHI membership of ARCH-UK (BBSRC-NERC aquaculture Research Hub-UK) involves both Government and HEIs, knowledge will be rapidly disseminated to relevant stakeholders and used to develop further collaborations and strategic initiatives. Tocher and Martin lead ARCH-UK Working Group 1 on finfish nutrition. Communication of issues relating to human health, such as farmed fish and dietary omega-3 will be effected through existing contacts directly in Government (e.g. Food Standards Agency, the Scottish Executive and UK Government), and through various pan-Governmental food security initiatives that we are involved with. Results will be published as usual in scientific peer-reviewed literature (conference proceedings and papers in high impact journals), but also disseminated widely to industry through contributions to trade and professional magazine articles and online sites such as FISHupdate, and the public via Institutional web pages and social media, as well as mainstream media articles and interviews in local and national press.
 
Description NOSIFISH aims to investigate the impact of diets formulated with non conventional oils (GM-derive oils, microalgal oil, krill oil) on performance and health of Atlantic salmon. The trial started in the freshwater facilities of the IoA in August 2021 and was completed in sea water in May 2022. In parallel, a small trial was run in fresh water in the UoA facilities. During the trial a PAMPS and a sea lice trial were carried out. Preliminary results indicated that there were not differences in growth during the fresh water phase and fish for all treatments successfully smoltified. Results of the sea lice challenge showed that fish fed diets containing microalgal oil seemed to have a better protection towards this parasitic infestation. At present we have analysed approximately 80% of the samples generated.
In addition, a method based on mass spectrometry has been developed to analyse fatty acids in tissues of fish fed on GM derived ingredients (Broughton et al., 2020).
Exploitation Route The outcomes of the present project are highly applicable to the industry as most of the products that are being tested are commercially available or on the way to their approval as feed ingredients. The information generated will contribute to this. This technique will be very useful in this project and in other working on these novel ingredients.
Sectors Agriculture, Food and Drink
 
Description COVID has severely affected this project and, consequently, there are no economic and societal impacts to report thus far. The following text describes precisely the challenges that we are facing to achieve the anticipated impacts despite the COVID pandemic. Impact of COVID lockdown on NOSIFISH Project This research project, which was scheduled to run from 1 July 2019 to 30 June 2022, aims to determine the impact of novel dietary sources of "omega-3 fatty acids, EPA and DHA, including oils from GM crops and microalgal oil on salmon health including resistance to pathogens and immune responses, and to elucidate the underpinning mechanisms focussing on lipid metabolism and fatty acid inflammatory mediators and pathways. The project is a collaboration between the Universities of Stirling (UoS), Aberdeen (UoA) and Glasgow (UoG), and Rothamsted Research (joint project BB/S006400/1) with the industrial partner BioMar (fish feed company). The project involves a large feeding trial in Atlantic salmon to be performed at the UoS. The fish trial was due to be initiated in April 2020 and to run for 12 months until May/June 2021. Two of the university PDRAs were already in post, at UoS this was a 36-month post, and at UoG this was an 18-month post. The UoA PDRA was also an 18-month post. As per the Scottish Government's Coronavirus lockdown instructions, the UoS shut down completely on 20 March 2020, and all new fish trials that had not already started at UoS fish trial facilities were postponed or cancelled completely. Therefore, as the long-term (12-month) fish trial in NOSIFISH had not started, it was postponed. The fish feeding trial was/is absolutely key and central to the entire research project, delivering all the samples and research material for the research partners. The feeding trial involves feeding the fish for a 12-month period, six months prior to and six months post seawater transfer. Without this trial there could be no project. While Phase 2 of the Scottish Government's plans for easing of lockdown 1 included the possible reopening of research laboratories access, it was not possible until August at the earliest. Unfortunately, the fish required for the feeding trial, which had been raised from eggs in the UoS facilities specifically for this trial, had grown and were too large to now fit the trial schedule including the key seawater transfer window. No other suitably-sized fish were available. Therefore, the salmon feeding trial had to be postponed for a full year to enable us to source more eggs in December 2020 that could be raised to supply fish of the correct specification in May/June 2021 when the feeding trial will be initiated with a new end date of May-June 2022 (just one month before the original end date of the project. Project Extension Obviously, this would leave essentially no time for the extensive laboratory analyses required and, therefore, we requested that the NOSIFISH research grant be considered for a twelve-month extension with a new termination date of 30th June 2023. On 7 July 2020, we received confirmation from Mr Harry Pollock (Operations Administrator, UK Research & Innovation Corporate Funding Services) that the 12 month no-cost extension of NOPSIFISH (grant reference BB/S005919/1) had been approved. The revised end date is now 30/06/2023, with the Final Statement of Expenditure now due on 29/09/2023 (enquiry reference number 1- 579794350). Financial impacts At the time of applying for the project extension, it was already anticipated that there would be some negative consequences of the 12-month delay. Thus, while the UK Government Coronavirus Job Retention Scheme (JRS) was utilised to furlough the UoS PDRA (11 weeks, 11 May - 31 July), this only preserved around 3-months of salary, which still left a very significant shortfall in funding of up to 9-months salary. Similarly, the UoG PDRA was in post but unable to be furloughed and, as such, 3-months of laboratory time was lost due to the first lockdown. The UoA PDRA was not planned to be employed until May 2021. Therefore, while we are confident that the approved twelve-month extension will be sufficient time to enable all the post fish trial laboratory analyses to be completed, this will only possible with all the PDRAs in post during the extension. Therefore, when the full impact of the Coronavirus lockdowns are established, additional funding will likely be required to enable this project to be completed successfully. UKRI COVID19 Grant Extension Allocation (CoA) Further analyses of the financial impacts were performed when the UKRI COVID19 Grant Extension Allocation (CoA) was announced. The trial had to be postponed for a full year and while BBSRC granted a 12 month no-cost extension, this had two financial impacts. 1. Fish trial. We required to source more fish (eggs) in Dec 2020 and raise them in UoS facilities to have the fish available at the appropriate size later in 2021. The additional costs for fish (£1000) and the tanks/husbandry at UoS (£4000) came to a total of £5,000. We believe that with careful budgeting we can vire funds (consumables/travel) to cover these additional costs. 2. PDRAs. The PDRAs are absolutely essential for this project to be completed successfully. In UoS, we require an additional 12-months salary. The PDRA started on 1 September 2019 (2-months after the start of the project), plus the PDRA was furloughed for 11 weeks and so only up to 5-months are available in the PDRA salary code. This leaves a significant shortfall of 7-months salary. The UHI/UoG PDRA post also lost 3-months of salary (March-June 2020). Unfortunately, the UoS determined that the conditions of the UKRI COVID19 Grant Extension Allocation (CoA) scheme meant that NOSIFISH was ineligible and so could not receive any of this allocation. Activities March 2020 - March 2021 Two project meetings have been held. Meeting 1, the Project Initiation (Start-Up) Meeting, was held on 3 September 2019 in UoS. Meeting 2 was held in the run-up to the initiation of the salmon feeding trial and went ahead on 4 March 2020 at the University of Aberdeen. All aspects of the feeding trial (dietary oils, feeds and fish), samplings (schedule and protocols), and analyses (scope and methodologies) were discussed in some detail. However, approximately 2 weeks later the COVID lockdown was announced and all our Universities were shut down on 20 March 2020 and the feeding trial cancelled/postponed for 12 months (until April/May 2021) for the reasons described in detail above. Between July 2019 and March 2020, considerable laboratory work was performed in UoS as part of Tasks 1.2 (Lipid, fatty acid and molecular species analyses of dietary oils) and 1.3 (Development and application of methodology for molecular species analyses of fish membrane phospholipids). This has led to 2-3 outputs including a Conference Abstract (Poster) [Broughton, R., Betancor, M.B. and Tocher, D.R. (2019) Development of a C18 SFC-MS/MS methodology for lipidomics and its application to salmon flesh lipidome profiling; trialling fish oil substitutes derived from genetically modified oilseeds. Abstract for Scottish Metabolomics Network Symposium, 14-15 November 2019, Glasgow, Scotland] and a published paper [Broughton, R., Tocher, D.R. and Betancor, M.B. (2020) Development of a C18 SFC-MS/MS methodology for the analysis of VLCPUFA lipid matrices and its application to fish oil substitutes derived from genetically modified oilseeds in the aquaculture sector. ACS Omega 5, 22289-22298. https://dx.doi.org/10.1021/acsomega.0c02631 (published 24 August 2020)]. A second paper is in preparation with submission projected for summer 2021 [Broughton, R., Tocher, D.R. and Betancor, M.B. Lipid molecular species in fish feeds and impacts on lipidomic profiles of tissues of Atlantic salmon (Salmo salar). In preparation] Salmon Feeding Trial 2021 This is now planned to start in June/July 2021. All 8 dietary oils to be tested in the trail have been sourced (Task 1.1) and are in place at the BioMar Tech Centre, Brande, Denmark, and feeds have been designed and formulated (Task 2.1) with manufacture scheduled for the last week in March. Most dietary oils were already sourced for the trial in Feb/March 2020 but, where appropriate and/or necessary, new batches of most oils have been obtained for the rescheduled trial. Salmon eggs were obtained in December 2020, and were hatched and fry produced. The fish will be vaccinated as routine prior to the initiation of the feeding trial when the fish will be 40-50g (Task 2.2). Feed pellets of 2mm and 3mm will be produced for each diet, sufficient to take the fish through the freshwater phase to seawater transfer at 80-100g in November. Dates are approximate based on water temperatures and growth performance.
First Year Of Impact 2020
 
Description BioMar 
Organisation BioMar
Country United Kingdom 
Sector Private 
PI Contribution Consultancy and advice. Project management. Laboratory analyses.
Collaborator Contribution BioMar are an international feed company and have formulated and manufactured all feeds used in nutritional trials (in-kind contribution). Funded PhD studentships and collaborative research
Impact Joint publications. Papers (4 published, one submitted as of Feb 2017. doi:10.1038/srep08104 http://dx.doi.org/10.1016/j.aquaculture.2015.03.020 doi:10.1007/s11745-016-4191-4 http://dx.doi.org/10.1371/journal.pone.0159934 Betancor, M.B., Li K; Sprague M; Sayanova O; Usher S; Måsøval K; Torrissen O; Napier JA; Tocher DR; Olsen RE; (2017) An oil containing EPA and DHA from transgenic Camelina sativa to replace marine fish oil in feeds for Atlantic salmon (Salmo salar L.): Effects on intestinal transcriptome, histology, tissue fatty acid profiles and plasma biochemistry. PLoS ONE, submitted. Conference abstracts (9) Betancor, M.B. Napier, J.A., Bell, J.G., Sayanova, O., Campbell, P.J. and Tocher, D.R. (2014) Replacement of marine fish oil with high-EPA oil from transgenic Camelina sativa in feeds for Atlantic salmon (Salmo salar). Oral presentation, p.5. Proceedings XVI International Symposium on Fish Nutrition & Feeding (ISFNF) 25-30 May 2014, Cairns, Australia. Tocher, D.R., Betancor, M.B., Campbell, P.J. and Napier, J.A. (2014) Oil from a transgenic oilseed as a source of n-3 LC-PUFA in feeds for Atlantic salmon (Salmo salar). Abstract Oral O26, p.41. Proceedings 55th International Conference on the Bioscience of Lipids (ICBL), 23 - 27 June 2014, Aberdeen, Scotland. Robert Gordon University, Aberdeen. Betancor, M.B. Napier, J.A., Bell, J.G., Sayanova, O., Campbell, P.J. and Tocher, D.R. (2014) Use of high-EPA oil from transgenic Camelina sativa in feeds for aquaculture. Abstract Oral 01.10.1615.003, p.33-34. 11th Congress of the International Society for the Study of fatty Acids and Lipids (ISSFAL) 28 June - 2 July 2014, Stockholm, Sweden. Betancor, M.B., Sprague, M., Campbell, P.J., Napier, J.A. and Tocher, D.R. (2014) High-EPA oil from transgenic Camelina sativa as a replacement for marine fish oil in Atlantic salmon (Salmo salar) feeds. Marine Alliance for Science and Technology for Scotland (MASTS) Annual Science Meeting, 3 - 5 September, Heriot-Watt University, Edinburgh. Betancor, M.B. Sprague, M., Sayanova, O., Usher, S., Campbell, P.J., Napier, J.A. and Tocher, D.R. (2014) High-EPA oil from transgenic Camelina sativa as a replacement for marine fish oil in feeds for Atlantic salmon (Salmo salar). Abstract (oral) for Aquaculture Europe 2014 "Adding Value", 14-17 October 2014, Donostia-San Sebastian, Spain, European Aquaculture Society, p. 1329-1330. Betancor, M.B., Sprague, M., Sayanova, O., Usher, S., Campbell, P.J., Napier, J.A. and Tocher, D.R. (2015) Replacement of marine fish oil with oils from transgenic Camelina sativa in feeds for gilthead sea bream (Sparus aurata). Proceedings of the 13th Euro Fed Lipid Congress: Fats, Oils and Lipids: New Challenges in Technology, Quality Control. p.XX. Florence, Italy, 27-30 September, 2015. European Federation for the Science and Technology of Lipids. Tocher, D.R., Betancor, M.B. Sprague, M., Sayanova, O., Usher, S., Campbell, P.J. and Napier, J.A. (2015) Transgenic Camelina sativa as a source of oils to replace marine fish oil in aquaculture feeds. Abstract (oral) for Aquaculture Europe 2015 "Aquaculture, Nature and Society", 20-23 October 2015, Rotterdam, Netherlands, European Aquaculture Society, p. 794-795. Betancor. M.B., Sprague, M., Sayanova, O., Campbell, P.J., Izquierdo, M., Napier, J.A. and Tocher, D.R. (2016) Oils from a transgenic oilseed crop, Camelina sativa, to replace marine fish oil in aquafeeds. Abstract presentation, XVII International Symposium on Fish Nutrition & Feeding (ISFNF) 5-10 June 2016, Sun Valley, Idaho, USA. Betancor, M.B., Li, K., Bardal, T., Sprague, M., Sayanova, O., Usher, S., Måsøval, K., Torrissen, O., Napier, J.A., Tocher, D.R. and Olsen, R.E. (2016) An oil containing EPA and DHA from transgenic Camelina sativa to replace marine fish oil in feeds for Atlantic salmon (salmo salar): effects on intestinal transcriptome. Abstract (oral) for Aquaculture Europe 2016 "Food for Thought", 20-23 September 2016, Edinburgh, Scotland, European Aquaculture Society, p.106-107.
Start Year 2012
 
Description Glasgow University Lipidomics Group 
Organisation University of Glasgow
Department Polyomics Facility
Country United Kingdom 
Sector Academic/University 
PI Contribution The University of Stirling is leading then project and running the salmon feeding trial that all the experimental samples will be derived from. Briefly, the feeding trial will be performed in the facilities of UoS, initially in the Neil Bromage Freshwater Research Unit (NBFRU, Buckieburn) before transfer to seawater at the Marine Environmental Laboratory (MERL, Machrihanish). The trial will run for 12 months and include 6-months in freshwater prior to seawater transfer, the fish will then undergo parr-smolt transformation prior to sea water transfer using natural photoperiod, with a further 6-months post seawater transfer. Approximately 1800 Atlantic salmon parr (initial weight, ~10g) will be randomly distributed among 24 (triplicate) tanks at NBFRU and, at seawater transfer, 80 fish (~100g) per diet will be transferred to 16 (40 fish/duplicate tank) seawater tanks at MERL and grown on to approximately 500g at termination. These fish will be the basis for lipid metabolic assays and also selected fish will be used for the different immune function and challenge experiments outlined in WP3. Individual fish (30/tank) will be anaesthetised (MS-222) and standard measures taken to monitor growth (initial & final weights, specific growth rate, thermal growth coefficient) and feed efficiency (as determined by food conversion ratio). Fish (6 per tank) will be sampled after lethal anaesthesia (MS-222) for biometry (hepato-somatic index, viscero-somatic index and condition factor K), lipid analysis and gene expression at the initiation of the trial, pre- and post seawater transfer, and immediately prior to pathogen challenges.
Collaborator Contribution Initially, the Lipidomics Group at the UHI were responsible for the quantitative analysis of pro-inflammatory and pro-resolving lipid mediators derived from n-6 and n-3 LC-PUFA. However, Prof Phil Whitfield, Head of the UHI Lipidomic Group took up a new appointment as the Head of Metabolomics at the University of Glasgow, Polyomics Facility in March 2020, with his group moving to join him later in 2020. Thus, from mid-2020, the lipidomic work in the NOSIFISH project will be carried out largely at the University of Glasgow. Until then, preliminary work has been carried out at UHI and will continue until full transfer. Briefly, samples of head kidney, liver, skeletal muscle, brain, plasma and primary macrophage cultures will be analysed using validated methods for the measurement of these lipids. The lipid mediators will be extracted in methanol and isolated by solid-phase extraction chromatography. Appropriate stable isotope labelled internal standards that are representative of eicosanoids and related lipid mediators will be added to the experimental system. The lipids will be identified and quantified by LC-MS/MS on a Sciex QTRAP 6500 platform. The analyses will be performed in negative ion mode with multiple reaction monitoring (MRM). The LC-MS/MS raw data will be processed and the concentrations of individual lipid mediators determined by comparison to an appropriate calibration curve. Alterations in the lipid mediator profiles will be correlated with disease resistance (samples from Tasks 3.1 and 3.3) and immune function in salmon to provide insights into the mechanisms of action of n-3 LC-PUFA that mediate the physiological benefits.
Impact None yet.
Start Year 2020
 
Description Rothamsted Research 
Organisation Rothamsted Research
Department Biological Chemistry and Crop Protection
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution UoS and RRES collaborate to produce n-3 LC-PUFA in transgenic crops for use as feed ingredients for aquaculture (fish farming). In UoS, we test the oils from GM crops produced by RRES as ingredients in fish diets.
Collaborator Contribution RRES are the crop scientists producing the transgenic oilseed crops and thus the novel omega-3-rich oils.
Impact Joint publications. Papers (10 published, as of March 2020). doi:10.1038/srep08104 http://dx.doi.org/10.1016/j.aquaculture.2015.03.020 doi:10.1007/s11745-016-4191-4 http://dx.doi.org/10.1371/journal.pone.0159934 ISSN: 0968-297X https://doi.org/10.1371/journal.pone.0175415 DOI: https://doi.org/10.1017/S0007114518001125 doi:10.3390/nu11010089 doi: 10.1111/pbi.13045 Project presented at Cheltenham Science Festival May 2014, Royal Highland Show June 2014 and Great British Bioscience Festival, London November 2014 (BBSRC 20th Anniversary events) Project reported in oral presentations at 2 national (MASTS 2012 and MASTS 2013) and 10 international conferences (ISFNF 2014, Cairns Australia May 2014; ISSFAL, Stockholm June 2014; ICBL 2014, Aberdeen June 2014; Aquaculture Europe, San Sebastian, October 2014; Aquaculture Europe, Rotterdam, October 2015; ISFNF 2016, Idaho, USA; Aquaculture Europe, Edinburgh, September 2016; ISFNF 2018, Las Palmas, Spain May 2018; Marine Biotechnology Conference, Shizuoka, Japan, September 2019; Aquaculture Europe, Berlin, October 2019). Abstracts below. Betancor, M.B. Napier, J.A., Bell, J.G., Sayanova, O., Campbell, P.J. and Tocher, D.R. (2014) Replacement of marine fish oil with high-EPA oil from transgenic Camelina sativa in feeds for Atlantic salmon (Salmo salar). Oral presentation, p.5. Proceedings XVI International Symposium on Fish Nutrition & Feeding (ISFNF) 25-30 May 2014, Cairns, Australia. Tocher, D.R., Betancor, M.B., Campbell, P.J. and Napier, J.A. (2014) Oil from a transgenic oilseed as a source of n-3 LC-PUFA in feeds for Atlantic salmon (Salmo salar). Abstract Oral O26, p.41. Proceedings 55th International Conference on the Bioscience of Lipids (ICBL), 23 - 27 June 2014, Aberdeen, Scotland. Robert Gordon University, Aberdeen. Betancor, M.B. Napier, J.A., Bell, J.G., Sayanova, O., Campbell, P.J. and Tocher, D.R. (2014) Use of high-EPA oil from transgenic Camelina sativa in feeds for aquaculture. Abstract Oral 01.10.1615.003, p.33-34. 11th Congress of the International Society for the Study of fatty Acids and Lipids (ISSFAL) 28 June - 2 July 2014, Stockholm, Sweden. Betancor, M.B., Sprague, M., Campbell, P.J., Napier, J.A. and Tocher, D.R. (2014) High-EPA oil from transgenic Camelina sativa as a replacement for marine fish oil in Atlantic salmon (Salmo salar) feeds. Marine Alliance for Science and Technology for Scotland (MASTS) Annual Science Meeting, 3 - 5 September, Heriot-Watt University, Edinburgh. Betancor, M.B. Sprague, M., Sayanova, O., Usher, S., Campbell, P.J., Napier, J.A. and Tocher, D.R. (2014) High-EPA oil from transgenic Camelina sativa as a replacement for marine fish oil in feeds for Atlantic salmon (Salmo salar). Abstract (oral) for Aquaculture Europe 2014 "Adding Value", 14-17 October 2014, Donostia-San Sebastian, Spain, European Aquaculture Society, p. 1329-1330. Betancor, M.B., Sprague, M., Sayanova, O., Usher, S., Campbell, P.J., Napier, J.A. and Tocher, D.R. (2015) Replacement of marine fish oil with oils from transgenic Camelina sativa in feeds for gilthead sea bream (Sparus aurata). Proceedings of the 13th Euro Fed Lipid Congress: Fats, Oils and Lipids: New Challenges in Technology, Quality Control. p.XX. Florence, Italy, 27-30 September, 2015. European Federation for the Science and Technology of Lipids. Tocher, D.R., Betancor, M.B. Sprague, M., Sayanova, O., Usher, S., Campbell, P.J. and Napier, J.A. (2015) Transgenic Camelina sativa as a source of oils to replace marine fish oil in aquaculture feeds. Abstract (oral) for Aquaculture Europe 2015 "Aquaculture, Nature and Society", 20-23 October 2015, Rotterdam, Netherlands, European Aquaculture Society, p. 794-795. Betancor. M.B., Sprague, M., Sayanova, O., Campbell, P.J., Izquierdo, M., Napier, J.A. and Tocher, D.R. (2016) Oils from a transgenic oilseed crop, Camelina sativa, to replace marine fish oil in aquafeeds. Abstract presentation, XVII International Symposium on Fish Nutrition & Feeding (ISFNF) 5-10 June 2016, Sun Valley, Idaho, USA. Betancor, M.B., Li, K., Bardal, T., Sprague, M., Sayanova, O., Usher, S., Måsøval, K., Torrissen, O., Napier, J.A., Tocher, D.R. and Olsen, R.E. (2016) An oil containing EPA and DHA from transgenic Camelina sativa to replace marine fish oil in feeds for Atlantic salmon (salmo salar): effects on intestinal transcriptome. Abstract (oral) for Aquaculture Europe 2016 "Food for Thought", 20-23 September 2016, Edinburgh, Scotland, European Aquaculture Society, p.106-107. Napier, J., Haslam, R., Han, L., Sayanova, O., Betancor, M.B. and Tocher, D.R. (2018) Heterologous synthesis of omega-3 long chain polyunsaturated fatty acids in transgenic plants: a terrestrial source of fish oils. Abstract 3.03, 18th International Symposium on Fish Nutrition & Feeding (ISFNF) 3-7 June 2018, Las Palmas de Gran Canaria, Spain. Tocher, D.R., Betancor, M.B., Sprague, M. and Napier, J.A. (2019) Engineering Camelina sativa as a novel de novo source of marine omega-3 long-chain polyunsaturated fatty acids. Keynote lecture O14-4, p.92. Joint Marine Biotechnology Conference, 12th International Marine Biotechnology Conference (IMBC2019)/12th Asia Pacific Marine Biotechnology Conference, Shizuoka, Japan, 9 - 13 September 2019. Betancor, M.B., MacEwan, A., M. Sprague, M., Montero, D., Norambuena, F., Sayanova, O., Han, L., Napier, J.A., Izquierdo, M. and TOCHER, D.R. (2019) Feasibility of an oil derived from a GM-oilseed crop as a substitute for fish oil in feeds for European sea bass (Dicentrarchus labrax). Abstract for Aquaculture Europe 2019 "Our Future Growing from Water", 7- 10 October 2019, Berlin, Germany, European Aquaculture Society, p.xxx-xxx.
Start Year 2012
 
Description UHI, University of the Highlands and Islands 
Organisation University of the Highlands and Islands
Country United Kingdom 
Sector Academic/University 
PI Contribution The University of Stirling is leading then project and running the salmon feeding trial that all the experimental samples will be derived from. Briefly, the feeding trial will be performed in the facilities of UoS, initially in the Neil Bromage Freshwater Research Unit (NBFRU, Buckieburn) before transfer to seawater at the Marine Environmental Laboratory (MERL, Machrihanish). The trial will run for 12 months and include 6-months in freshwater prior to seawater transfer, the fish will then undergo parr-smolt transformation prior to sea water transfer using natural photoperiod, with a further 6-months post seawater transfer. Approximately 1800 Atlantic salmon parr (initial weight, ~10g) will be randomly distributed among 24 (triplicate) tanks at NBFRU and, at seawater transfer, 80 fish (~100g) per diet will be transferred to 16 (40 fish/duplicate tank) seawater tanks at MERL and grown on to approximately 500g at termination. These fish will be the basis for lipid metabolic assays and also selected fish will be used for the different immune function and challenge experiments outlined in WP3. Individual fish (30/tank) will be anaesthetised (MS-222) and standard measures taken to monitor growth (initial & final weights, specific growth rate, thermal growth coefficient) and feed efficiency (as determined by food conversion ratio). Fish (6 per tank) will be sampled after lethal anaesthesia (MS-222) for biometry (hepato-somatic index, viscero-somatic index and condition factor K), lipid analysis and gene expression at the initiation of the trial, pre- and post seawater transfer, and immediately prior to pathogen challenges.
Collaborator Contribution Initially, the Lipidomics Group at the UHI were responsible for the quantitative analysis of pro-inflammatory and pro-resolving lipid mediators derived from n-6 and n-3 LC-PUFA. Samples of head kidney, liver, skeletal muscle, brain, plasma and primary macrophage cultures will be analysed using validated methods for the measurement of these lipids. The lipid mediators will be extracted in methanol and isolated by solid-phase extraction chromatography. Appropriate stable isotope labelled internal standards that are representative of eicosanoids and related lipid mediators will be added to the experimental system. The lipids will be identified and quantified by LC-MS/MS on a Sciex QTRAP 6500 platform. The analyses will be performed in negative ion mode with multiple reaction monitoring (MRM). The LC-MS/MS raw data will be processed and the concentrations of individual lipid mediators determined by comparison to an appropriate calibration curve. Alterations in the lipid mediator profiles will be correlated with disease resistance (samples from Tasks 3.1 and 3.3) and immune function in salmon to provide insights into the mechanisms of action of n-3 LC-PUFA that mediate the physiological benefits.
Impact None yet
Start Year 2019
 
Description University of Aberdeen 
Organisation University of Aberdeen
Country United Kingdom 
Sector Academic/University 
PI Contribution The University of Stirling (UoS) are the lead partner in this project. The project PI (Prof Douglas Tocher) and the UoS team contribute expertise in fish nutrition, salmon biology, and and fish biochemistry and physiology especially in relation to lipid and fatty acid metabolism in salmon. As such the UoS has all the facilities (laboratories) and equipment (HPLC. GC, GCMS, LC-MS PCR, qPCR and Seq) required for biochemical and molecular analyses. The UoS team also brings the background IP in the application of the "nutritional programming" concept in fish having successfully carried out preliminary research into this as a principle consortium member in a recently completed EU FP7 project "ARRAINA" project. This initial study resulted in two publications in the peer-reviewed scientific literature. The UoS will also provide the on-campus and external fish culture facilities required to run the salmon nutritional trials from first feeding through the entire freshwater phase (in temperate aquarium, UoS campus, and at the UoS Buckieburn FW Unit), and after seawater transfer at the UoS Marine Environmental Research laboratory (MERL), Machrihanish, Scotland).
Collaborator Contribution The University of Aberdeen (UoA) are key partners in this project having a global reputation in fish immunology and health, recently investing in forming the 'International Centre for Aquaculture Research and Development', which complements the established world-leading 'Scottish Fish Immunology Research Centre'. The UoA Co-I (Prof SAM Martin) and his team contributing expertise in fish gut microbiome and intestinal immune function and responses particularly in relation to Atlantic salmon. As such the UoA team has all the facilities (laboratories and aquaria) and equipment required for analysing microbiomes and carrying out research into biological, physiological (e.g. FACS at the UoA Iain Fraser Cytometry Centre) and molecular aspects of immune function in salmon including 16S rRNA gene analysis and gene expression analysis (NGS/Illumina MiSeq RNAseq platforms). Sequencing in the project will be carried out at UoA Centre for Genome Enabled Biology.
Impact Too early as project only started on 1 January 2019.
Start Year 2019
 
Description University of Aberdeen 
Organisation University of Aberdeen
Country United Kingdom 
Sector Academic/University 
PI Contribution The University of Stirling (UoS) are the lead partner in this project. The project PI (Prof Douglas Tocher) and the UoS team contribute expertise in fish nutrition, salmon biology, and and fish biochemistry and physiology especially in relation to lipid and fatty acid metabolism in salmon. As such the UoS has all the facilities (laboratories) and equipment (HPLC. GC, GCMS, LC-MS PCR, qPCR and Seq) required for biochemical and molecular analyses. The UoS team also brings the background IP in the application of the "nutritional programming" concept in fish having successfully carried out preliminary research into this as a principle consortium member in a recently completed EU FP7 project "ARRAINA" project. This initial study resulted in two publications in the peer-reviewed scientific literature. The UoS will also provide the on-campus and external fish culture facilities required to run the salmon nutritional trials from first feeding through the entire freshwater phase (in temperate aquarium, UoS campus, and at the UoS Buckieburn FW Unit), and after seawater transfer at the UoS Marine Environmental Research laboratory (MERL), Machrihanish, Scotland).
Collaborator Contribution The University of Aberdeen (UoA) are key partners in this project having a global reputation in fish immunology and health, recently investing in forming the 'International Centre for Aquaculture Research and Development', which complements the established world-leading 'Scottish Fish Immunology Research Centre'. The UoA Co-I (Prof SAM Martin) and his team contributing expertise in fish gut microbiome and intestinal immune function and responses particularly in relation to Atlantic salmon. As such the UoA team has all the facilities (laboratories and aquaria) and equipment required for analysing microbiomes and carrying out research into biological, physiological (e.g. FACS at the UoA Iain Fraser Cytometry Centre) and molecular aspects of immune function in salmon including 16S rRNA gene analysis and gene expression analysis (NGS/Illumina MiSeq RNAseq platforms). Sequencing in the project will be carried out at UoA Centre for Genome Enabled Biology.
Impact Too early as project only started on 1 January 2019.
Start Year 2019