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Final Report Summary - OMEGA3MAX (Maximizing marine omega-3 retention in farmed fish: sustainable production of healthy food.)

The Omega3max-IAPP network aims at increasing the competitiveness of the EU aquaculture industry by outlining ways to improve production costs and the nutritional value and quality of aquaculture products. This network also intends to strengthen the research and educational potential on aquaculture of both sectors industry and academia. The network consist of four partners, including two public non-commercial organisations, namely the Universidad Politécnica de Madrid (UPM-Spain) and the Christian-Albrechts University Kiel (CAU-Germany) as well as two commercial enterprises, namely Skretting Aquaculture Research Centre (Skretting ARC-Norway) and Lucta (Spain).

Reducing the fish oil inclusion level in aquaculture feeds while maintaining high levels of EPA and DHA in fish products to meet consumer expectations is a major problem in modern aquaculture. In order to solve this problem it is necessary to examine in depth nutritional strategies aimed at maximising EPA and DHA retention in fish tissues. To this end, the current project explores the potential of using vegetable-derived oil blends together with novel antioxidant additives in salmon feeding during the growth phase. A major goal of Omega3max is to determine optimal antioxidant sources and concentrations to limit in vivo oxidative stress in fish tissues helping to preserve EPA and DHA. Moreover, not only the quantity of EPA and DHA, but also their position in triacyglycerols (TAG) and phospholipids, seem to affect function and bioavailability and is an increasingly important aspect for human consumers. Also, the accumulation of antioxidants in the fish muscle is desirable from a consumer point of view as it might increase the functional value of the food. In addition to contributing to basic knowledge on the physiology of fish, our research could have practical applications in the aquaculture industry, since optimal husbandry conditions, good health and adequate feed, well-suited to the physiological needs of the farmed aquatic animals are essential for optimal growth and production.

The overall objectives of Omega3max were:
-to explore the potential of novel antioxidant additives in salmon feeding during the growth phase, and
-to transfer knowledge and expertise between academia and the aquaculture feed industry.

These objectives were reached through the participation of 18 fellows. Three of them were recruited (2 female and 1 male), 7 were seconded from academia to industry partners and 8 were seconded from industry partners to academia. The Omega3max project was organised in 15 work packages (WP), including three in vivo feeding trials with Atlantic salmon, research, transfer of knowledge and dissemination activities. Throughout the whole joint research and knowledge transfer activities attention to the following aspects was paid:
-Chemical composition of ingredients and antioxidants used for feed formulations.
-Productive performance and health records in all the in vivo trials with Atlantic salmon including transfer of knowledge on fish management.
- Antioxidant enzyme activities, lipid peroxidation and gene expression analysis of redox regulated transcription factor measurements.
- Lipid profiles, total fatty acid analysis and their regiospecific studies in TAG and phospholipids in salmon muscle and livers.
- The analysis of expression of genes involved in liver lipid metabolism and genes encoding proteins involved in the eicosanoid biosynthesis pathways.
- Vitamin E (alpha- and gamma-tocopherol) concentration in muscle and livers.

The scientific work plan was divided in two major phases: (i) Finding an adequate antioxidant or a mixture of antioxidants that limit in vivo lipid peroxidation in fish tissues and help to preserve EPA and DHA; and (ii) to optimize antioxidant functioning. The work plan was completed with WPs 1, 2, 3, 4, 7, 9, 12 and 13.

Work performed and summary of results:
-Twelve substances with antioxidant capacity including tocopherols, polyphenols (hydroxytyrosol, resveratrol, quercetin, soy isoflavones), phenolic compounds (chlorogenic acid), organic acids (fumaric acid) and cofactors (Q10, Lipoic acid and Vitamin C) were tested in in vivo trials performed with Atlantic salmon post-smolts. This represents the first report of Atlantic salmon feeding trials with some of the above mentioned antioxidants.
- Of these, two substances quercetin and soy isoflavones were found to enhance EPA and DHA concentrations respectively in the liver and fillets compared to an identically composed control diet without the supplemented substance. Also, it was of relevance from a consumer point of view the observed enrichment of salmon fillets with gamma tocopherol in salmon fed a diet supplemented with gamma tocopherol.
- To confirm the results obtained with soy isoflavones and quercetin and to optimize both substances, the second phase of the project included a dose-response study and to test synergies between the quercetin and soy isoflavones at the optimal concentration and the use of lecithin to increase the availability of both substances at low (6ºC) and high (12ºC) temperatures. After both studies we were able to confirm only the positive effects of soy isoflavones on DHA retention in the salmon tissues. However, at the desired concentration of isoflavones to maximize DHA retention a significant reduction in fish performance (final body weight, feed intake, specific growth rate and feed conversion ratio) was observed. This negative effect on performance was more pronounced at low temperatures. Quercetin does not improve or even worsen the negative effects of isoflavones on productive traits. Although, these results dissuade the use of soy isoflavones in Atlantic salmon feeds to increase tissue DHA concentrations the consistent effect of this polyphenol on DHA retention merits a more in depth research on the mechanism behind such effect.
- To better characterize fish nutritional studies including polyphenols we developed a method to trace quercetin and genistein in the feed.
- The nutritional and functional characteristics of dietary fat for the consumer are related to the fatty acid (FA) composition and its positional distribution in the triacylglycerol fraction. Therefore, to better assess the impact of fish diets on the nutritional quality of the fillet we implemented a method to study the regiospecificity of FA in the fish TAG. Our results indicate that feeding salmon with vegetable and fish oil providing different EPA + DHA concentrations does not affect their positional distribution in the fillet TAG.

Training, education and dissemination were performed in WPs 5, 8, 10 and 14 hosted by the two academic partners Christian-Albrechts University Kiel (CAU-Germany) and Universidad Politécnica de Madrid (UPM-Spain). This activity consisted mainly on seminaries on fish nutrition and feed additives conducted by seconded researchers from the industrial partners to graduate, master, PhD students and researchers of the host academic partners. Other dissemination activities included the presentation of the project results in scientific conferences and workshops, and in events open to the general public such as the Researchers night in 2014 and the Science Week in Madrid. At the moment 3 peer reviewed articles have been published and 3 Bachelor and 2 Master-Thesis were completed within the Omega3max project.

In conclusion, although much remains to be done for the full implementation of new antioxidant sources in Atlantic salmon feeds the knowledge generated in the frame of this project provide a first approach towards the potential use of some natural antioxidants including the polyphenols. Omega3max produced novel scientific knowledge on the function of antioxidants in salmon physiology including their nutritional-regulated gene expression and also significant amounts of scientific and technical know-how regarding polyphenol usage in salmon diets. From a consumer point of view we suggest that Atlantic salmon may provide a marine functional source capable of enriching gamma tocopherol for human consumption. Also, it is of interest for the functional characteristics of dietary fat for the consumer knowing that feeding Atlantic salmon with vegetable and fish oil providing different EPA + DHA concentrations does not affect their positional distribution in the fillet TAG. Thus, in addition to the scientific contributions of this work, the aquaculture industry and the consumers are also expected to benefit from the results of this project.

The network have provided its members exposure to the integration of academia and industry with the aim of developing novel cost-effective feeding strategies that will support the sustainable development of the European aquaculture industry. Finally, the knowledge gained coupled with the direct involvement of the academic partners in the entire process going from research to innovation have reinforced their educational capabilities thereby improving institutional teaching in aquaculture. In turn, this will contribute to maintain the entry of well-qualified technical and managerial staff to the aquaculture sector in the future.

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