Update on GM canola crops as novel sources of omega-3 fish oils

There is considerable interest in new sources of omega‐3 long‐chain (here defined as fatty acids ≥C20) polyunsaturated fatty acids (LC‐PUFA), specifically eicosapentaenoic acid (EPA; 20:5n‐3) and docosahexaenoic acid (DHA; 22:6n‐3), commonly known as omega‐3 fish oils, to supplement the limited supplies of oceanic fish oil (Tocher, 2015). These alternative sources include extraction of other diverse marine organisms (e.g. krill, plankton etc.), algal fermentation, and the genetic engineering of microbes such as yeasts. Another approach is the synthesis of omega‐3 fish oils in transgenic plants (reviewed in Napier et al., 2015), and this short article will discuss the recent results obtained by two major industry collaborations

Biochemical composition of copepods for evaluation of feed quality in production of juvenile marine fish

To increase current knowledge on the nutritional value of natural prey organisms, the biochemical components of mainly three copepods (Acartia grani, Centropages hamatus, and Eurytemora affinis) from a marine pond system were analysed once a week from spring until late fall, over two years. The analysed components were total lipid, lipid class composition, total lipid fatty acid composition, free amino acids, total protein, protein-bound amino acids, pigment (astaxanthin and ß-carotene), and vitamins (A, thiamine, riboflavin, C, D3, and E). Copepod dry weight (DW), dry matter (% of wet weight), and ash content (% of DW) were also determined. The data are unique due to the homogenous content of copepods in the samples and the long time span of sampling. The copepods were characterised by moderate levels of lipids (6.9-22.5% of DW), with polar lipids accounting for 37.9 to70.2% of the total lipid. The most abundant fatty acids in total lipid (as % of total lipid) were 16:0 (palmitic acid, 10.8-17.1%), 20:5n-3 (EPA, 8.3-24.6%), and 22:6n-3 (DHA, 13.9-42.3%). The amount of 20:4n-6 (ARA) was generally low (0-2.6%), giving an EPA/ARA range between 7.5and 49.5. The DHA/EPA ratio was between 1.0 and 4.9. Free amino acids (FAA) constituted between 4.3 and 8.9% of copepod DW, and varied with salinity. Glycine, taurine, and arginine dominated FAA, and the fraction of indispensable amino acids varied between 15.5 and 26.8%. Protein, as back-calculated from the protein-bound amino acids (PAA), amounted to 32.7-53.6% of copepod DW, and contained a stable fraction of indispensable amino acids (37.3-43.2% of PAA). Glutamine/glutamic acid, asparagine/aspartic acid, leucine, alanine, and glycine were the most abundant PAA. Astaxanthin was abundant in the copepods (413-1422 μg/g DW), while ß-carotene was not found. High but variable concentrations of vitamin C (38-1232 μg/g DW) and vitamin E (23-209 μg/g DW) were found, while vitamin A and D3 occurred in trace amounts or were not detected. Detectable levels were found for both thiamine (3.5-46.0 μg/g DW) and riboflavin (23.2-35.7 μg/g DW). The data may generate an important base for improvement of live feed enrichment emulsions or formulated feeds used during larval and early juvenile stages in marine fish culture

Algal oil gives control of long-chain omega-3 levels in full-cycle production of Atlantic salmon, without detriment to zootechnical performance and sensory characteristics

The levels of eicosapentaenoic (EPA) and docosahexaenoic (DHA) in salmon fillets have decreased because of the progressive replacement of fish oil (FO). This study contributes to enabling the sustainable growth of aquaculture by confirming the effects of partially or fully replacing FO with microalgal oil (AO) on growth, muscle fatty acid profiles, and muscle quality of farmed Atlantic salmon. Crucially, this is now done throughout the entire post-smolt production cycle and up to a harvest weight of 3 kg. Three experiments were performed using fish ranging from 145 g to 3 kg and testing different diets, replacing FO up to 100%. Zootechnical performance was similar among treatments in all experiments. Changing the lipid source in the diet resulted in EPA and DHA digestibility of greater than 96%. Sensory characteristics of raw fish fillets were similar among treatments, supporting a similar sensorial experience with the replacement of FO with no impact on consumers. Overall, results confirm that the AO tested here enables the sustainable growth of Atlantic salmon aquaculture by helping to maintain a level of EPA and DHA in the fish fillets, without detriment to zootechnical performance and sensory characteristics, while simultaneously contributing to a reduced marine footprint for aquafeeds.publishedVersio

CRISPR/Cas9-mediated editing of Δ5 and Δ6 desaturases impairs Δ8-desaturation and docosahexaenoic acid synthesis in Atlantic salmon (Salmo salar L.)

The in vivo functions of Atlantic salmon fatty acyl desaturases (fads2), Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads2 in long chain polyunsaturated fatty acid (LC-PUFA) synthesis in salmon and fish in general remains to be elucidated. Here, we investigate in vivo functions and in vivo functional redundancy of salmon fads2 using two CRISPR-mediated partial knockout salmon, Δ6abc/5Mt with mutations in Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads2, and Δ6bcMt with mutations in Δ6fads2-b and Δ6fads2-c. F0 fish displaying high degree of gene editing (50–100%) were fed low LC-PUFA and high LC-PUFA diets, the former containing reduced levels of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids but higher content of linoleic (18:2n-6) and alpha-linolenic (18:3n-3) acids, and the latter containing high levels of 20:5n-3 and 22:6n-3 but reduced compositions of 18:2n-6 and 18:3n-3. The Δ6abc/5Mt showed reduced 22:6n-3 levels and accumulated Δ6-desaturation substrates (18:2n-6, 18:3n-3) and Δ5-desaturation substrate (20:4n-3), demonstrating impaired 22:6n-3 synthesis compared to wildtypes (WT). Δ6bcMt showed no effect on Δ6-desaturation compared to WT, suggesting Δ6 Fads2-a as having the predominant Δ6-desaturation activity in salmon, at least in the tissues analyzed. Both Δ6abc/5Mt and Δ6bcMt demonstrated significant accumulation of Δ8-desaturation substrates (20:2n-6, 20:3n-3) when fed low LC-PUFA diet. Additionally, Δ6abc/5Mt demonstrated significant upregulation of the lipogenic transcription regulator, sterol regulatory element binding protein-1 (srebp-1) in liver and pyloric caeca under reduced dietary LC-PUFA. Our data suggest a combined effect of endogenous LC-PUFA synthesis and dietary LC-PUFA levels on srebp-1 expression which ultimately affects LC-PUFA synthesis in salmon. Our data also suggest Δ8-desaturation activities for salmon Δ6 Fads2 enzymes.publishedVersio

Molecular Regulation of Biosynthesis of Long Chain Polyunsaturated Fatty Acids in Atlantic Salmon

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Triploid Atlantic salmon × brown trout hybrids have similar seawater growth and welfare issues as triploid Atlantic salmon, but both were heavier at harvest than their diploid counterparts

Interspecific hybridisation may improve the farm performance of sterile triploid salmonids via heterosis (i.e. hybrid vigour). We assessed growth over the final 293 days in seawater, and harvest quality, in diploid and triploid Atlantic salmon (Salmo salar) × brown trout (Salmo trutta) hybrids compared to diploid and triploid Atlantic salmon. We measured vertebral deformities, cataracts, flesh colour, gut mass, and body shape at harvest. In triploids, hybridisation had no effect on harvest size, vertebral deformities, cataracts, or body shape, but did improve fillet colouration (Mean digital SalmoFan™ score [95% CI]: 24.6 [24.4–24.9] and 26.0 [25.7–26.2] for triploid salmon and triploid hybrids, respectively) and lower relative gut size (34% lower). Compared to diploid salmon, triploid salmon were significantly heavier at harvest, triploid hybrids tended to be heavier (Post-hoc, least square means, p = 0.08), whereas diploid hybrids were 83% lighter (Mean mass [g] at harvest [95% CI]: 2676 [2470–2898], 3395 [3134–3679], 462 [401–534], and 3086 [2832–3363] for diploid salmon, triploid salmon, diploid hybrids, and triploid hybrids, respectively). However, both triploid groups had a significantly higher incidence of fish with one or more deformed vertebra (Mean % [95% CI]: 23 [14–35], 60 [47–71], 38 [20–60], and 44 [31–57] % in diploid salmon, triploid salmon, diploid hybrids, and triploid hybrids, respectively), more severe cataracts (Mean cataract score [95% CI]: 3.0 [2.7–3.3], 3.5 [3.2–3.8], 2.2 [1.7–2.6], 3.6 [3.3–4.0] for diploid salmon, triploid salmon, diploid hybrids, and triploid hybrids, respectively), and a smaller relative gut size (21% smaller) compared to diploid counterparts. In conclusion, triploid hybrids have no growth advantage over triploid salmon and suffer from similar welfare issues while only benefiting from increased fillet colour.publishedVersio

CRISPR/Cas9-mediated ablation of elovl2 in Atlantic salmon (Salmo salar L.) inhibits elongation of polyunsaturated fatty acids and induces Srebp-1 and target genes

Atlantic salmon can synthesize polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (20:5n-3), arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) via activities of very long chain fatty acyl elongases (Elovls) and fatty acyl desaturases (Fads), albeit to a limited degree. Understanding molecular mechanisms of PUFA biosynthesis and regulation is a pre-requisite for sustainable use of vegetable oils in aquafeeds as current sources of fish oils are unable to meet increasing demands for omega-3 PUFAs. By generating CRISPR-mediated elovl2 partial knockout (KO), we have shown that elovl2 is crucial for multi-tissue synthesis of 22:6n-3 in vivo and that endogenously synthesized PUFAs are important for transcriptional regulation of lipogenic genes in Atlantic salmon. The elovl2-KOs showed reduced levels of 22:6n-3 and accumulation of 20:5n-3 and docosapentaenoic acid (22:5n-3) in the liver, brain and white muscle, suggesting inhibition of elongation. Additionally, elovl2-KO salmon showed accumulation of 20:4n-6 in brain and white muscle. The impaired synthesis of 22:6n-3 induced hepatic expression of sterol regulatory element binding protein-1 (srebp-1), fatty acid synthase-b, Δ6fad-a, Δ5fad and elovl5. Our study demonstrates key roles of elovl2 at two penultimate steps of PUFA synthesis in vivo and suggests Srebp-1 as a main regulator of endogenous PUFA synthesis in Atlantic salmon.publishedVersio

Molecular Mechanism Involved in Carotenoid Metabolism in Post-Smolt Atlantic Salmon: Astaxanthin Metabolism During Flesh Pigmentation and Its Antioxidant Properties

A better understanding of carotenoid dynamics (transport, absorption, metabolism, and deposition) is essential to develop a better strategy to improve astaxanthin (Ax) retention in muscle of Atlantic salmon. To achieve that, a comparison of post-smolt salmon with (+ Ax) or without (− Ax) dietary Ax supplementation was established based on a transcriptomic approach targeting pyloric, hepatic, and muscular tissues. Results in post-smolts showed that the pyloric caeca transcriptome is more sensitive to dietary Ax supplementation compared to the other tissues. Key genes sensitive to Ax supplementation could be identified, such as cd36 in pylorus, agr2 in liver, or fbp1 in muscle. The most modulated genes in pylorus were related to absorption but also metabolism of Ax. Additionally, genes linked to upstream regulation of the ferroptosis pathway were significantly modulated in liver, evoking the involvement of Ax as an antioxidant in this process. Finally, the muscle seemed to be less impacted by dietary Ax supplementation, except for genes related to actin remodelling and glucose homeostasis. In conclusion, the transcriptome data generated from this study showed that Ax dynamics in Atlantic salmon is characterized by a high metabolism during absorption at pyloric caeca level. In liver, a link with a potential of ferroptosis process appears likely via cellular lipid peroxidation. Our data provide insights into a better understanding of molecular mechanisms involved in dietary Ax supplementation, as well as its beneficial effects in preventing oxidative stress and related inflammation in muscle.publishedVersio