RELATED APPLICATION

[0001] This Application is related to and claims priority benefit of U.S. Provisional Application No. 63/020,412, filed May 5, 2020, which is incorporated herein by reference for all purposes.

FIELD

[0002] The present embodiments relate to methods of aquaculture and aquafeed comprising plant-sourced Omega 3 fatty acids. Fish, such as salmonids, fed this aquafeed exhibited better health and mortality, and produced superior fillets.

BACKGROUND

[0003] Currently, feeds manufactured for use in farmed fish, such as salmonids, contain marine ingredients. Although many inputs of marine origin are renewable resources, there are limits to the quantities of these products that the world's oceans can supply. In the long term, the growing demand for such inputs results in sustained price increases and stress on over-fished oceans. Thus, the shortage of supply of marine raw materials is a problem for aquaculture. Moreover, fish farmed at industrial scale experience a variety of environmental stresses. Adequate supply of long chain polyunsaturated fatty acids (LC PUFA) is needed to maintain or improve the health of farm-raised fish, and LC PUFA are typically sourced by marine raw materials. There remains a need for aquafeed that includes sustainable, e.g., land-based, materials that provides healthy fish and quality products.

SUMMARY

[0004] The present embodiments provide methods of farming fish to improve the health of farm-raised fish by providing a plant-sourced Omega 3 nutritional component that provides Omega 3 LC PUFA in aquafeed. In at least one embodiment, the method improves the health, mortality, or yield of the farmed fish. In one embodiment, the farmed fish are salmonids. At least one embodiment provides aquafeed with significant levels of ALA, DHA, and other LC PUFA, with a low ratio of Omega 6 to Omega 3 fatty acids obtained from canola oil. In one embodiment, the canola oil is Aquaterra® Omega 3 oil. In one aspect of the present embodiments, fish receiving the Aquaterra® Omega 3 oil as a nutritional component in aquafeed in commercial scale sea water farming exhibited an increased survivability by an average of about 2% compared with control (fish oil) diets. [0005] An aspect of the present embodiments provides a method for providing a farm- raised fish comprising providing said fish with aquafeed comprising a plant-sourced Omega 3 nutritional component, wherein at harvest said fish is healthier than a farm-raised fish that has not been provided the plant-sourced Omega 3 nutritional component. Another aspect provides a method for providing farm-raised fish comprising providing said fish with aquafeed comprising a plant-sourced Omega 3 nutritional component, wherein at harvest said fish have a lower mortality rate than farm-raised fish that have not been provided the plant-sourced Omega 3 nutritional component. In at least one embodiment, the plant-sourced Omega 3 nutritional component comprises about 8% to about 10% DHA, inclusive, and about 18% to about 22% ALA, inclusive (% total fatty acid content). In at least one embodiment, the plant-sourced Omega 3 nutritional component comprises about 9.2% DHA and about 20.2% ALA (% total fatty acid content). In at least one embodiment, the plant-sourced Omega 3 oil nutritional component has a ratio of Omega 6 to Omega 3 fatty acids of about 0.15 to about 0.35, such as about 0.23. In at least one embodiment, the plant-sourced Omega 3 nutritional component has the fatty acid profile as shown for the Aquaterra® Omega 3 oil in Table 1. In at least one embodiment, the plant-sourced Omega 3 nutritional component is Aquaterra® Omega 3 oil.

[0006] In at least one embodiment, the methods provided herein improve FIFO and FDDR compared with fish farming using conventional aquafeed that lacks the Omega 3 nutritional component.

[0007] In at least one embodiment, the farmed fish have improved color in fillets compared with a method that does not comprise inclusion of the Omega 3 nutritional component.

[0008] In at least one embodiment, the Omega 3 nutritional component provides a lower fish mortality of about 1.5 to about 2% compared with a method that does not comprise inclusion of the Omega 3 nutritional component. In another embodiment, the healthier fish or lower fish mortality rate improves the economic feed conversion factor (FCRe).

[0009] In at least one embodiment, providing aquafeed with the Omega 3 nutritional component provides the fish with higher resistance or more robust responses to environmental stresses compared with fish lacking the Omega 3 nutritional component.

[0010] In another aspect of the present embodiments provides an aquafeed comprising an Omega 3 nutritional component comprising the fatty acid profile as shown for the Aquaterra® Omega 3 oil in Table 1. In at least one embodiment, the incorporation of the Omega 3 nutritional component is from about 3% to about 7%, inclusive, of total components in aquafeed. In at least one embodiment, the Omega 3 nutritional component replaces about 30% to about 60%, inclusive of fish oil included in typical aquafeed. [0011] Another aspect of the present embodiments provides a fish fillet obtained from the farm-raised fish of provided an Omega 3 nutritional component as in any one of the preceding claims. In at least one embodiment, the fillet is a Norwegian quality cut having the fatty acid content as shown for Aquaterra in Table 3.

DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a bar graph showing the mortality rate (%) for each trial (control and experimental diet comprising a plant-sourced Omega 3 nutritional component (Aquaterra Omega 3 oil)), consolidated in the processing plant.

[0013] FIG. 2 presents a graphical representation of a cage-to-cage analysis of the evolution of the monthly and cumulative mortality rate (Trial 2). A color version of this graphic and other figures is presented in Silva et al., Applied Research on the Use of Aquaterra®, a New Source of Omega-3 for Use in Salmon Feed - Successful results of industrial-scale trials (2020) available at the Aquaterra® Omega 3 oil website (aquaterraomega3) and incorporated herein by reference.

[0014] FIG. 3 is a bar graph depicting the fatty acid content comparison of control and experimental diets according to saturated, monounsaturated, and polyunsaturated fatty acid categories. Black: saturated fatty acids; gray: monosaturated fatty acids; light gray: polyunsaturated fatty acids.

[0015] FIG. 4 is a bar graph showing the comparison of fatty acid content for fatty acids of interest, shown as g/lOOg in fillets (Norwegian quality cut) (NQC), between control and test diets in each trial.

[0016] FIG. 5 is a bar graph reflecting the comparison (g/lOOg NQC) according to fatty acid categories Omega 6 fatty acids (co6) and Omega 3 fatty acids (co3). Light gray: total Omega 6 fatty acids; dark gray: total Omega 3 fatty acids.

[0017] FIG. 6 is a bar graph showing the ratio comparisons of Omega 6 fatty acids (co6) to Omega 3 fatty acids (co3) for all three trials described herein.

[0018] FIG. 7 is a bar graph showing EPA and DHA content comparisons as g/lOOg of fillets (NQC) of wild or farmed salmon. For each year or trial indicated, left bar: EPA; middle bar: DHA; right bar: EPA+DHA.

[0019] FIG. 8 is a bar graph showing the astaxanthin content in NQC in ppm. For each indicated trial, left bar: astaxanthin in control diets; right bar: astaxanthin in diets including a plant-sourced Omega 3 nutritional component: Aquaterra® Omega 3 oil. [0020] FIG. 9 is a bar graph reflecting the color expression in fillet, according to Salmofan™ scale. For each indicated scale value (y axis), top bar: Aquaterra® Omega 3 oil diet; bottom bar: control diet.

[0021] FIG. 10 shows the sensory profile of salmon samples. More specifically, this figure is an intensity graph by attribute. Intensity 0 = the attribute is not perceived;

9 = maximum intensity of the attribute.

[0022] FIG. 11 is a bar graph of the sustainability index showing the comparison of indexes related to use of marine ingredients in feed using FIFO (BAP) and FFDRO (ASC) sustainability indicators. Light gray (left bar): control oil diet; dark gray (right bar): Aquaterra® Omega 3 oil diet.

DETAILED DESCRIPTION

[0023] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. The embodiments described herein are not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary.

[0024] All patents and other publications identified are incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present embodiments, but are not to provide definitions of terms inconsistent with those presented herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0025] As used herein and in the claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, “comprise,” “comprises,” and “comprising” are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers. The term “or” is inclusive unless modified, for example, by “either.” Thus, unless context indicates otherwise, the word “or” means any one member of a particular list and also includes any combination of members of that list.

[0026] All values are approximate as there is some fluctuation in fatty acid composition due to environmental conditions. Values are typically expressed as area percent, which approximates percent by weight, of total fatty acid or percent weight of the total oil.

Accordingly, other than in the operating examples, or where otherwise indicated, all numbers expressing quantities or reaction conditions used herein should be understood as modified in all instances by the term “about,” that means ± 1% to ± 10% depending on the context as understood by one skilled in the art.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. In order that the present disclosure can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0028] Diet is a major factor in the health, growth, and quality of farmed fish. In particular, the long chain polyunsaturated fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are critical nutrients in fish feed. These nutrients are historically sourced from ocean-caught fish; however, the ocean may not provide enough of these nutrients to sustainably supply the rapidly growing aquaculture industry. As a result, the aquaculture industry needs alternative sources of Omega 3 nutrients. In at least one embodiment provides an Omega 3 nutritional component from a plant-sourced Omega 3 oil as a source of DHA and EPA, delivered from canola oil at fish oil equivalency (9%-l 1% DHA + EPA), as well as a source of alpha linolenic acid (ALA) important for achieving a desirable Omega 6/Omega 3 (co6/co3) ratio. In one embodiment, the oil with this fatty acid profile is Aquaterra® Omega 3 oil (commercially available from Nuseed, West Sacramento, California, U.S.A.). A least one embodiment provides a method of feeding fish with an aquafeed comprising, as a component, Aquaterra® Omega-3 oil as a partial replacement for fish oil in aquaculture feed.

[0029] The present embodiments provide a method of commercial scale fish farming comprising providing as a component in aquafeed a plant-based source of Omega 3 fatty acids for a superior aquafeed that provides healthier commercial scale farmed fish (e.g., healthier salmonids), compared with traditional fish oil-based sources of Omega 3 fatty acids. The plant- based source of the Omega 3 nutritional component also provides a superior fish product, such as fillets. In at least one embodiment, the farmed fish is a salmonid, such as Atlantic salmon {Salmo salar ) or Rainbow trout ( Oncorhynchus mykiss).

[0030] The present embodiments provide a method for farming fish at commercial scale comprising providing the fish with aquafeed comprising a plant-based Omega 3 (co3) nutritional component, wherein at harvest said fish is healthier (e.g., lower mortality) than farm-raised fish that have not been provided the plant-based co3 nutritional component. The co3 nutritional component is an oil that may comprise about 8% to about 10% DHA, inclusive, such as about 9.2% DHA, and about 19% to about 22% ALA, inclusive, such as about 20% ALA (% total fatty acid content of oil), and a ratio of co6/co3fatty acids of about 0.15 to about 3.5, inclusive, such as about 0.2. Accordingly, at least one embodiment provides an aquafeed comprising a canola oil having the fatty acid profile shown in Table 1 or similar to that profile. In another embodiment, the co3 nutritional component is Aquaterra® Omega 3 oil.

[0031] In at least one embodiment, the plant-based source of Omega 3 fatty acids is Aquaterra® Omega 3 oil or an oil with the fatty acid content (e.g., high DHA, high ALA, low co6/co3 ratio) as described herein. In at least one embodiment, the plant-sourced Omega 3 fatty acid is obtained from seed of transgenic Brassica, such as Brassica napus event NS-B50027-4 or progeny thereof. Accordingly, in one embodiment, the fatty acid content of the plant-sourced Omega 3 nutritional component is an oil comprising at least 8.0% DHA, such as about 8.10% to about 10% DHA, inclusive, or more DHA, as wt.% of the total fatty acids of the oil; at least 18% ALA, about 18.1% to about 22% ALA inclusive, or more ALA, as wt.% of the total fatty acids of the oil; at least 10% LC-PUFA, about 10.25% LC-PUFA to about 12% LC PUFA, inclusive, or more LC PUFA, as the sum of EPA, DP A, and DHA, as wt.% of the total fatty acids of the oil; and an co6/co3 ratio of about 0.15 to about 0.3. See U.S. Patent No. 10,570,405, Elite event canola NS-B50027-4. In a further embodiment, the fatty acid content of the oil of the Omega 3 nutritional component comprises about 4% palmitic acid (C16:0n-7); about 2% stearic acid (Cl 8:0); about 38% to about 46% oleic acid (C18:ln-9c); about 7% to about 8% linoleic acid (C18:2n-6c); about 2% stearidonic acid (SDA) (C18:4n-3); about 0.4% eicosapentaenoic acid (EPA) (C20:5n-3); and about 0.8% to about 0.9% docosapentaenoic acid (DP A) (C22:5n-3) as wt.% of the total fatty acids of the oil. See U.S. Patent No. 10,570,405.

[0032] In understanding the importance of the present embodiments, by 2050 the earth’s population may reach over 9 billion people, and a substantial improvement in the conservation and use of resources will be necessary to achieve the expected 50% increase in food demand by that date. It is expected that aquaculture can take on a good part of that challenge, primarily because it is an industry that continues to grow at interesting rates compared to other animal production systems intended for human consumption. Rabobank, Global Animal Protein Outlook 2020: Seeking Opportunities in an Uncertain World ’(2020).

[0033] Wild-catch fisheries have maintained production levels since the late 1980s, while aquaculture has delivered an impressive increase in the supply of fish for human consumption. Though aquaculture production is expanding rapidly, foraged fish harvests are increasing at a much slower rate. FAO, El estado mundial de la pesca y la acuicultura 2018,

Fao. Roma.: Licencia: CC BY-NC-SA 3.0 IGO., p. 250 (2018). [0034] Currently, all feeds manufactured for use in fish such as salmonids contain marine ingredients. Although many inputs of marine origin are renewable resources, there are limits to the quantities of these products that the world's oceans can supply. In the long term, the growing demand for such inputs results in sustained price increases. Thus, the shortage of supply of marine raw materials is a problem for aquaculture and for the economic and environmental sustainability of fisheries.

[0035] Regarding the impact on salmon feed formulation, according to SalmonChile {Informe de sustentabilidad Gestion 2018 (2020)), 84% of salmonids produced in Chile in 2018 meet at least one of these certification standards: Best Aquaculture Practices, Global-Gap, and largely Aquaculture Stewardship Council. These standards specify criteria on including marine origin inputs in the feed. Global Salmon Initiative member companies represent the world’s major salmon producers in Chile, Norway, North America, and other regions. Member companies have achieved ASC certification for 273 farms with 82 in process; 60% of the production is certified and these companies have a commitment to reach 100%. GSI 2020, Section Find a Farm, available at the asc-aqua website (last access April 23, 2020). These standards include requirements on the incorporation of inputs of marine origin into feed, which results in indicators such as Fish ImFish Out (FIFO) and Forage Fish Dependency Ratio (FFDR).

[0036] These factors have drastically influenced the formulation of fish feed, leading to high incorporation of plant-derived and animal by-products, replacing inputs of marine origin. Skretting, Reporte de -Sustentabilidad 2014, at 34 (2014); Marine Harvest, SALMON FARMING INDUSTRY HANDBOOK 2016 at 94. When the availability of oils of marine origin declines, they must be replaced by other lipid sources. According to Ytrestoyl et al., Utilisation of feed resources in production of Atlantic salmon (Salmo salar) in Norway, 448 Aquaculture 365-74 (2015)), the replacement of marine oils with plant-based sources has been increasing in Norway, with marine oil content declining from 24% in 1990 to 10.9% in 2013. According to Mowi’s annual report for 2018, the use of fish oil in salmon diets was 10%. Mowi, SALMON FARMING INDUSTRY HANDBOOK 2019. It is estimated that in 2017 the level of incorporation of marine oils was similar in Chile and Norway. Marine Harvest, SALMON FARMING INDUSTRY HANDBOOK at 113 (2018).

[0037] The formulation of fish feed must consider the importance of Omega 3 fatty acids, especially LC PUFA Omega 3 fatty acids, for both human and fish nutrition. Increasingly, consumers take an interest in food functionality. One of the most cited examples of interest is in salmonids as the source of LC PUFA, due to their health benefits. Thus EPA (eicosapentaenoic acid, 20:5n-3) and DHA (docosahexaenoic acid, 22:6n-3) are considered fatty acids of particular interest in animal and human nutrition.

[0038] A decline in EPA and DHA content in salmon produced in Norway (Nofima/Nifes, Oppdatering av utredningen (2016)) and Scotland (Sprague et al., Impact of sustainable feeds on omega-3 long-chain fatty acid levels in farmed Atlantic salmon, 2006-2015, 6 Sci. Reports 1-9 (2016)) has been reported, and the same trend is indicated for Chile. Nevertheless, farmed salmon still offers more EPA+DHA for human consumption than most other fish species and more than any land animals (Sprague et al., 2016).

[0039] Both EPA and DHA are considered to have beneficial effects in neurodegenerative diseases (Martinez et al., 2018), cardiovascular (Balk & Lichtenstein, Omega-3 fatty acids & cardiovascular disease: Summary of the 2016 agency of healthcare research & quality evidence review, 9(8) Nutrients (2017)), hypertension (Colussi et al., Omega- 3 Polyunsaturated Fatty Acids in Blood Pressure Control & Essential Hypertension, Update on Essential Hypertension, doi: 10.5772/63501 (2016)), inflammation (Laye et eά , Anti inflammatory effects of omega-3 fatty acids in the brain: Physiological mechanisms & relevance to pharmacology, 70(1) Pharmacol. Rev. 12-38 (2018)) and cancer (D’Eliseo & Velotti, Omega-3 Fatty Acids and Cancer Cell Cytotoxicity: Implications for Multi-Targeted Cancer Therapy, 5(2) J. Clin. Medicine, 15 (2016)). DHA has a positive influence on fluidity and permeability of cells membrane. Stillwell & Wassail, Docosahexaenoic acid: Membrane properties of a unique fatty acid, 126(1) Chemistry & Physics of Lipids 1-27 (2003). DHA is a predominant fatty acid in the central nervous system and retina and plays an essential role in brain development. Singh, Essential fatty acids, DHA human brain, 72(3) Indian J. Pediatrics 239-42 (2005). EPA acts as a precursor to eicosanoids that include prostaglandins and leukotrienes. Eicosanoids are also formed from arachidonic acid (ARA, 20:4n-6). Long chain Omega-3 fatty acids are crucial for the synthesis of eicosanoids which play an essential role in vascular physiology. Punia et al., Omega 3-metabolism, absorption, bioavailability and health benefits-A review, 10 PharmaNutrition 100162 (2019).

[0040] The composition of dietary fatty acids can affect many aspects of fish growth, development and health. According to Roberts (FISH PATHOLOGY, 4th Ed, Wiley-Blackwell, Oxford, 2012), nutritionally compromised diets often increase a species’ susceptibility to infectious diseases. In this regard, there is now growing concern about the effect that low availability of Omega 3 fatty acids, currently sourced from marine sources (e.g., fish meal and fish oil), could have on the ability of fish to address disease or environmental challenges.

[0041] Earlier studies have estimated that the minimum LC Omega 3 fatty acid requirement in Atlantic salmon {Salmo salar ) and rainbow trout ( Oncorhynchus mykiss ) is 1%. Hardy, Rainbow Trout, Oncorhynchus mykiss, Chapter 14, in NUTRIENT REQUIREMENTS & FEEDING OF FINFISH FOR AQUACULTURE (Eds: Webster & Lim, Cabi Publishing, 2001); Storebakken, Atlantic salmon, Salmo salar, Chapter 6, in NUTRIENT REQUIREMENTS & FEEDING OF FINFISH FOR AQUACULTURE (Eds: Webster & Lim, Cabi Publishing, 2001). Some long-term experiments in tanks have agreed with this requirement in Atlantic salmon. Rosenlund et ak, Atlantic salmon require long-chain n-3 fatty acids for optimal growth throughout the seawater period , 5 J. Nutr. Sci. el9 (2016).

[0042] A long-term experiment in Atlantic salmon grown in sea cages, however, showed that 1% of EPA and DHA in the feed was insufficient under adverse environmental conditions. More specifically, salmon fed 1% EPA + DHA or less had significantly higher mortality rates than salmon fed 1.7% EPA + DHA when the fish were exposed to stress, and the 1% EPA + DHA fish had increased accumulation of lipids in the liver and visceral fat, compressed vertebrae, and changes in the epithelial tissue of the mid intestine. Ruyter et ak, Langtidseffekter av lave omega-3-nivaer i for pa flskens helse - fhf.no, 02.06.2016, Nofima at 5 (2016).

[0043] Another seawater trial in Atlantic salmon from 150 g to 5,000 g tested two fish stocks with feed containing 1.6% or 2.6% EPA+DHA. In this case the population fed with the low-Omega-3 diet had a higher frequency of melanin spots in fillets (28.2% vs 21.5%), possibly because of the role that fatty acid balance plays in the inflammatory process. The authors concluded that EPA+DHA levels of 1.6% and higher in fish feed appear to be sufficient under certain conditions, although there were some indications that higher levels may be positive in some disease situations. Sissener et ak, Long-term feeding of Atlantic salmon in seawater with low dietary long-chain n-3 fatty acids affects tissue status of the brain, retina & erythrocytes, 115(11) Br. J. Nutr. 1919-29 (2016).

[0044] A more recent study in Atlantic salmon suggests that low dietary EPA and DHA may interrupt the barrier function of fish skin, due to changes in the phospholipid profile. Cheng et ak, Reduced dietary levels of EPA & DHA have a major impact on the composition of skin membrane lipids in Atlantic salmon (Salmo salar L.), 66 J. Agricultural & Food Chem. (2018). On the other hand, dietary DHA supplementation related with the capacity to maintain normal structure and ameliorate the deficiency symptoms in the intestinal epithelium such as swollen enterocytes and vacuolization. Bou et ak, Requirements of n-3 Very Long-chain PIJFA in Atlantic Salmon (Salmo Salar L): Effects of Different Dietary Levels of EPA & DHA on Fish Performance & Tissue Composition & Integrity, 117 Br. J. Nutr. 30-47 (2017). A study conducted with Atlantic salmon in freshwater (around 50-190 g) concluded that EPA appeared likely dispensable and even detrimental when provided in excess. The majority of EPA from the diet is catabolized or converted into DHA, and little-to-no DHA is catabolized so the DHA from the diet is efficiently deposited in tissues. Emery et al., Uncoupling EPA & DHA in Fish Nutrition: Dietary Demand is Limited in Atlantic Salmon & Effectively Met by DHA Alone, 51 Lipids 399-412 (2015). According to Bou et al. (2017) a trial in Atlantic salmon reared in seawater (around 50g - 400g) suggest that dietary EPA was converted to DHA in group diets lacking DHA and a very limited retroconversion of DHA to EPA. These studies suggest that a low EPA:DHA ratio in the fish diet could be beneficial.

[0045] The aquaculture industry is faced with a need for new sources of oils for fish nutrition. These sources must provide the critical Omega 3 fatty acids contributes to meeting the nutritional requirements of the fish and allow for good growth and enable the fish to meet the challenges of the fish farm (stressful conditions). These fatty acids should also be delivered at a certain level to maintain the appropriate composition in the fish (e.g., salmon) fillet that confers its reputation as a source of healthy lipids.

[0046] Faced with this need, Nuseed - in collaboration with the Commonwealth Scientific and Industrial Research Organization (CSIRO) and the Grains Research and Development Corporation (GRDC) - applied advanced plant genetics to develop a land-based Omega 3 oil (brand name Aquaterra®) that delivers the nutritional benefits of microalgae via canola to benefit the aquaculture industry. See, e.g., U.S. Patents No. 8,816,111, No. 10,570,405, No. 10,563,218; Petrie et al., Development of a Brassica napus (Canola) Crop Containing Fish Oil-Like Levels of DHA in the Seed Oil, 11 Frontiers Plant Sci. 727 (2020). Aquaterra®

Omega 3 oil contains significant levels of DHA and other LC PUFA. The oil also contains higher levels of alpha linolenic acid (ALA, 18:3n-3) and has a much lower Omega 6 to Omega 3 (co6/co3) ratio in comparison with conventional canola oil (see Table 1).

[0047] The unique combination of high DHA, high ALA, and low co6/co3 ratio are attributes of the Omega 3 nutritional component provided herein and embodied in Aquaterra® Omega 3 oil. Indeed, this unique profile distinguishes Aquaterra® Omega 3 oil from other plant- based sources of oil, such as Camelina, flax, or other transgenic Brassica oils. Accordingly, at least one embodiment provides a component for aquafeed consisting of an oil having a fatty acid content as described in Table 1 as for Aquaterra® Omega 3 oil. At least one embodiment provides a component for aquafeed comprising an Omega 3 nutritional component as an oil with a ratio of Omega 6 to Omega 3 fatty acid of about 0.15 to about 0.35, such as about 0.23; or about 18% to about 22% ALA, such as about 20% ALA, and about 8% to about 10% DHA, such as about 9% DHA. Inclusion of this oil component in aquafeed is shown herein to decrease mortality of fish farmed at commercial scale compared with aquafeed that does not include this oil component.

[0048] A previous study indicated that Aquaterra® Omega 3 oil was safe and effective in young fish. More specifically, a 2016 collaboration with Nuseed the Norwegian Institute of Food, Fisheries and Aquaculture (NOFIMA) validated the safety and effectiveness of Aquaterra® Omega 3 oil as an ingredient in salmon feed. The objectives of the NOFIMA study were: (1) to evaluate the partial replacement of fish oil with Aquaterra® Omega 3 oil in the diets of 1 g to 20 g fry, in freshwater; (2) to evaluate the effect on the replacement of conventional canola oil by Aquaterra® Omega 3 oil in diets of salmon from 450 g to 1,500 g, in seawater; and (3) to determine feed safety in the use of Aquaterra® Omega 3 oil by analyzing the oil for pollutants and unwanted substances.

[0049] The study showed that growth and survival were excellent in all diets, in both freshwater and seawater, and no difference in mortality was observed. The replacement of fish oil by Aquaterra® Omega 3 oil in the freshwater trial had no adverse effect on salmon growth and survival. In the seawater phase, in which Aquaterra® Omega 3 oil replaced conventional canola oil, growth and survival were also excellent, and again no difference in mortality was observed. Additionally, in that trial the content of ALA and DHA in the fillets increased as the inclusion rate of Aquaterra® Omega 3 oil increased. The EPA content of Aquaterra® Omega 3 oil is relatively low (0.5%) compared to fish oil. Nevertheless, the EPA content in the fillet also increased, suggesting that the fish had the physiological capacity to generate EPA from ALA. This was confirmed by a separate experiment that showed that some of the ALA in the diet was converted to EPA, thus generating a compensatory effect in favor of Aquaterra® Omega 3 oil. Concerning fish health, the hepatosomatic and cardio-somatic index did not differ, nor were there any differences in biological markers (expression of genes and enzymes) associated with health status. The study also showed there were no significant levels of pesticides or organic pollutants in the oil. More recent independent analyses of Aquaterra® Omega 3 oil have confirmed these results.

[0050] As important as these previous studies have been in understanding the requirements for and metabolism of Omega 3 fatty acids in the aquaculture diet, these studies have been done in controlled environments lacking natural stressors, and have not been attempted at commercial scale. Subsequently, Nuseed initiated a collaboration with the Chilean aquaculture industry in order to study the feasibility of using Aquaterra® Omega 3 oil — already validated previously under small-scale experimental conditions — according to each company’s criteria, to evaluate the performance of Aquaterra® Omega 3 oil in the challenging large-scale commercial production conditions in Chile.

[0051] More specifically, the present embodiments are supported by aquafeed trials that brought together Nuseed’ s expertise, feed producers, fish producers, processing plants, analytical laboratories (from Chile and the United States), technical departments of the participating companies and scientific advisors. The trials involved five companies among Chilean salmon and feed producers, with a total population of 2,650,000 salmon in trials spread over three fish farms, two in the region 10 and one in the region 11. The embodiments described herein are the first Chilean aquaculture industry experiments using plant oil with high DHA Omega 3 content for use under large-scale field production conditions. This trial also included processing plant quality reports for production batches from the experimental ocean cages.

[0052] Three feeding tests were carried out on populations of Atlantic salmon ( Salmo salar ), considering an equivalent number of cages randomized at the same site. Trial 1 consisted of sixteen ocean cages with approximately 40,000 fish in each cage, with an initial fish weight of approximately 1.6 kg. Trial 2 included twenty-four ocean cages with about 45,000 fish in each cage and an initial weight of approximately 1.2 kg. Trial 3 included eighteen ocean cages with approximately 50,000 fish and an initial weight of approximately 150 g.

[0053] The standard formulation used by each company was considered the control diet. Other than replacement of control oil with Aquaterra® Omega 3 oil, manufacture of aquafeed was carried out with commercial components and methods well-known in the art. Incorporation of Aquaterra® Omega 3 oil varied between 3% and 7%, inclusive, of the feed, with a replacement of fish oil with Aquaterra® Omega 3 oil between 30% and 60%, inclusive. The EPA+DHA content (predominantly DHA) in the feed ranged from 1.7 to 2.0%. [0054] Feed Conversion Factor (FCR), Specific Growth Rate (SGR) and Survival Rate were recorded as the most relevant productive aspects to evaluate growth. The composition of fatty acids, astaxanthin content, and color expression were evaluated on the Norwegian quality cut (NQC) of the fish at harvest by SGS Chile Ltda. and Tracelab, both accredited and experts in their respective fields. Analysis of the fatty acid profile (via gas chromatography) and lipid content (via Soxhlet extractor) were performed by SGS Chile, while the portion of the counterpart side was used by Tracelab for analysis of astaxanthin content (via HPLC) and color expression (SalmoFan™ color reference guide; DSM Nutritional Products).

[0055] In Trial 1, measurements were made at the beginning and at time of harvest, while in Trials 2 and 3 initial, intermediate, and harvest sampling was carried out, generating a large amount of data on fatty acid deposition and lipid compositions. In this disclosure, final sampling results are presented for all three trials.

[0056] In addition, in Trial 2 a sensory panel, composed of eight people selected and trained according to strict criteria established in ISO 8586 (“Sensory analysis - General guidelines for the selection, training and monitoring of selected assessors and expert sensory assessors), carried out an organoleptic evaluation in Dictuc’s aroma and flavor centre (Santiago, Chile). This trial also included processing plant quality reports associated with production batches from the experimental cages.

[0057] As sustainability indicators, the Fish in to Fish out ratio (FIFO ratio) used in the Best Aquaculture Practices Certification Standard (BAP) and the Forage Fish Dependency Ratio for Oil (FFDRo) used in the Aquaculture Stewardship Council standard were used.

[0058] The data were evaluated using Statgraphics™ software. In the case of comparisons presented herein, the assumptions of normality and homoscedasticity were assessed. If such assumptions were met, Student’s T test was used; otherwise, the Wilcoxon test was used. Statistical comparisons covered only the contrast of the two dietary treatments in each trial, but differences between trials were not compared because different companies used different nutritional criteria and the study periods. For multiple comparisons of sensory analysis, normality and homoscedasticity were verified. ANOVA and Tukey’s test were applied to establish differences.

[0059] Regarding productive performance, as shown in Table 2, there were no significant differences in productive performance between the control and Aquaterra® Omega 3 oil-containing diets. There was a difference, however, in the level of mortality, which influenced the economic feed conversion factor (FCRe).

[0060] As shown in Table 2, in Trials 1, 2 and 3 there was 1.49%, 1.90%, and 1.87% less mortality, respectively, in the total consolidated cages fed with Aquaterra® Omega 3 oil compared to the control diets. In terms of biomass, this is equivalent to 44.6 tons, 77.8 tons, and 19 tons, respectively, at the three sites with the Aquaterra® Omega 3 oil compared with the control.

[0061] Importantly, Trial 2 included data on several environmental stresses, including low oxygen concentration, an amebiasis outbreak and SRS (salmon rickettsial septicaemia), resulting in a high mortality situation. Mortality increased with both diets, but was lower in the case of the fish on the Aquaterra® Omega 3 oil diet (FIG. 1). To establish whether this result was attributable to a single cage, mortality in each individual cage was examined (FIG. 2). It is clear from these data that the fish in ocean cages in which aquafeed comprised Aquaterra® Omega 3 oil had higher resistance to environmental stresses, expressed as lower mortality. This is the first ocean-based commercial scale trial to report lower mortality in fish fed aquafeed comprising a plant-sourced oil with the fatty acid content (high DHA, high ALA, low co6:co3 ratio) as provided herein. Accordingly, the present embodiments provide a method of farming fish wherein inclusion of the Omega 3 nutritional component in aquafeed provides fish a higher resistance or more robust response to environmental stresses. The increased survivability of fish fed the Omega 3 nutritional component further evidences the improved health of these fish.

[0062] Additionally, the composition of lipid and fatty acids of content (ALA, EPA, DHA and oleic acid), and the proportion of saturated, monounsaturated and polyunsaturated fatty acids in the NQC of harvested fish were determined for each trial. The data, shown in Table 3, are expressed as g/100 g of NQC. In addition, the sum of EPA+DHA and ratios of interest, such as the proportion of co3 and co6 fatty acids, are included in Table 3.

[0063] Additionally, FIG. 3 shows the proportion of saturated, monounsaturated and polyunsaturated fatty acids in the fish at harvest in the three trials. There were no differences in the content of saturated, monounsaturated and polyunsaturated fatty acids in the fillets of fish at harvest in the three trials, apart from an increase in polyunsaturated fatty acids in fish on the Aquaterra® Omega-3 oil diet in Trial 1 (Table 3 and FIG. 3). The only statistically significant difference in the total lipid content was in Trial 1, in which the PUFA content was higher in of the case of the Aquaterra® Omega 3 oil.

[0064] Regarding the composition of fatty acids, the level of DHA was higher in fish fed the Aquaterra® Omega-3 oil diets in all trials, with statistically significant differences. The EPA level was slightly lower in the fish fed the Aquaterra® Omega-3 oil diets in Trials 1 and 2. However, in the full sea cycle trial (Trial 3), there was no significant difference in the EPA level between diets. Furthermore, there were no differences in the sum of EPA+DHA between the two diets in any trial (FIG. 4). This may be a consequence of the ALA to EPA conversion previously observed in the 2016 NOFIMA study. Additionally, there was a significant increase in the ALA fillet content in fish fed the Aquaterra® Omega-3 oil diet in all three trials (FIG. 4).

[0065] In the three trials the level of total Omega 3 fatty acids was higher in fish fed the Aquaterra® Omega 3 oil diet, with statistically significant differences in all trials (FIG. 5). This is reflected in a significant decrease in the Omega 6 to Omega 3 fatty acids ratio (co-6/co-3) in all trials (FIG. 6).

[0066] In addition, a comparison of the EPA and DHA content of fillets (measured in the NQC) from the three trials was made with levels found in wild Norwegian salmon and farmed salmon. It is interesting to note that the diets used in the three trials represent low, intermediate and high levels of EPA+DHA when compared with the historical Norwegian diets (FIG. 7), with the diet in Trial 3 resulting in the highest levels of EPA+DHA in the fillets.

[0067] These studies show that the fillets produced in these trials constitute an important source of EPA and DHA for human consumption. According to the recommendation of the European Food Safety Authority {Labelling reference intake values for n-3 & n-6 polyunsaturated fatty acids, 1176 EFSA J. 1-11 (2009)), a 250 mg/d intake of Omega 3 long chain fatty acids (EPA plus DHA) is recommended, which is in accordance with the available evidence on the relationship between intake of these fatty acids and cardiovascular health in healthy populations. In this regard, a 150 g portion of fish from Trials 1, 2, and 3 would cover the requirement for 3.5, 6.2, and 8.1 days, respectively.

[0068] At least one embodiment described herein provides a finished product of suitable quality. Regarding yield in the processing plant, in Trial 2, it was possible to track fish to the processing plant to evaluate performance in head-on eviscerated product and Trim D fillet. The processing plant had access to the performance sampling data that is usually developed as a control method, isolating the fish corresponding to diet-fed cages (control and Aquaterra® Omega 3 oil). There was no difference in the weight of the viscera in percentage terms, with a median of 10.5% in both cases (n = 130). For the trim D fillet yield, expressed in percentage terms, the median was 60.7% in both dietary treatments.

[0069] Regarding astaxanthin content and fillet color, there were no significant differences in astaxanthin content between the control diet and the Aquaterra® Omega 3 oil diet, in any of the trials, with all values falling between 6.1 and 7.2 ppm (FIG. 8).

[0070] Regarding the expression of color, Trial 3 was considered to be the only one that included the entire cycle from sea entry to fillet production. Rather than show an average value for color (the usual practice in the industry), a frequency chart including the frequencies of the color readings on the loin and NQC was used. As shown in FIG. 9, the color results were satisfactory in both diets, although with more readings in categories 25 and 26 in fish fed the Aquaterra® Omega 3 oil diet.

[0071] Regarding organoleptic analysis, there was no difference in the organoleptic aroma, taste, and texture analysis of samples of raw salmon between those fed the Aquaterra® Omega 3 oil diet and the control diet. This is evident in the almost perfect overlap of the radial graph for fish fed the two diets (FIG. 10).

[0072] Regarding sustainability, as shown in FIG. 11 the sustainability indicators improved markedly with the use of Aquaterra® Omega 3 oil as a component in aquafeed. In the case of Trial 2 the difference is accentuated by the effect of the difference in the FCR and in large part by the effect of the higher survival rate of fish fed the Aquaterra® Omega 3 oil diet ( see Table 2).

[0073] The disclosures herein show that under commercial scale Chilean conditions the use of Aquaterra® Omega 3 oil consistently reduced total mortality in all three trials, resulting in an improvement in FCRe. Weight gain was excellent in all three trials, with no differences between the two diets.

[0074] Although literature generated in controlled small scale trials support the notion that differences in fatty acid content of the feed may influence the survival rate of fish growing under stress conditions in commercial production (Sissener et al., 2016), this is the first series of commercial scale trials to show the consistent lower mortality rate in all the populations fed with Aquaterra® Omega 3 oil diets in the trials. Without being bound by theory, the consistent lower mortality rate in all the fish populations fed with Aquaterra® Omega-3 oil diets may be due to the unique fatty acid profile (e.g., high DHA, high ALA, and low co6/co3 ratio) of this nutritional component.

[0075] The major difference in fatty acid content of fillets from fish was an increase in DHA and ALA content in fish on the Aquaterra® Omega 3 oil diet compared to the control, reflecting the high DHA and ALA content of Aquaterra® Omega 3 oil. The Aquaterra®

Omega 3 oil diet led to a significant increase in the total level Omega3 fatty acids and, in this way, resulted in a more favorable co-6/co-3 ratio. Interestingly in Trial 1, although there was more EPA in fillets of fish on the control diet, the difference was minimal, only 19 mg/100 g of fillet. In the full-cycle Trial 3, the EPA level in the fillets was equivalent, with no significant statistical difference. This indicates that despite the low EPA level of Aquaterra® Omega 3 oil it is possible to obtain a fillet EPA profile equal to that in a typical diet. That suggests that some of the ALA in the diet may be converted to EPA, in agreement with results obtained by others. A comparison of the EPA+DHA content in fish from these trials with wild and farmed salmon originating in Norway shows that Aquaterra® Omega 3 oil contributes to the achievement of a healthy nutritional composition of the fillet. With an appropriate nutritional strategy, it is feasible to achieve EPA+DHA levels comparable to those obtained in farmed and wild salmon.

[0076] The composition of saturated, monounsaturated, and polyunsaturated fatty acids in harvested fillets was consistent across all three trials. The total lipid content in the feed varied across the three trials, and this was reflected in the lipid content of the harvested fish (Table 3). Fish fed the Aquaterra® Omega 3 oil diet showed an increase in ALA and DHA content, which reflects the high content of these fatty acids in Aquaterra® Omega 3 oil.

[0077] The inclusion of Aquaterra® Omega-3 oil as a component in fish feed did not impact the deposition of astaxanthin in the fillets. There was a higher number of fillets scoring Salmofan categories 25 and 26 from fish fed the Aquaterra® Omega-3 oil diet in Trial 3, with no differences in Trials 1 and 2. As for sensory analysis, no adverse effects were detected on the taste, aroma, or texture (to the palate) of fish raised on a diet including Aquaterra® Omega-3 oil.

[0078] Importantly, the level of incorporation of Aquaterra® Omega 3 oil in aquafeed contributed to an improvement in FIFO and FDDRo as shown herein. A surprising difference between current feeding methods and the methods provided herein was demonstrated in Trial 2, in which higher survival was obtained using Aquaterra® oil as a component in the diet in the face of the stress situation suffered by the fish at that site. Thus, inclusion of a plant-sourced oil with the Omega 3 profile described herein as a component in aquafeed reduces the dependence on marine ingredients while addressing the sustainability goals of the industry.

[0079] As shown herein, industry trials confirm that Aquaterra® Omega 3 oil has been is an excellent Omega 3-rich oil and a safe and effective partial replacement of fish oil that contributes to the sustainable development of the industry and satisfies the nutritional requirements of commercially raised fish such as salmon. Aquaterra® Omega 3 oil, or an oil with a similar Omega 3 profile (e.g., high DHA, high ALA, low co6/co3 ratio) provides a unique fatty acid profile that leads to improved nutritional status for the fish and maintains the characteristic composition of fatty acids in the fillet that confers the identity of a healthy product typical of salmon and that is valued by the consumer market.