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4 National Centre for Marine Conservation and Resource Sustainability, University of Tasmania, Launceston, Tasmania 7250, Australia and 5 Commonwealth Scientific and Industrial Research Organisation (CSIRO) Food Futures Flagship and Division of Marine and Atmospheric Research, Hobart, Tasmania 7001, Australia
* To whom correspondence should be addressed. E-mail: millerm{at}crop.cri.nz.
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ABSTRACT |
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6 desaturase (FAD6) step in the (n-3) LC-PUFA biosynthetic pathway. EO was fed to seawater Atlantic salmon for 12 wk and compared with fish fed a diet containing canola oil (CO), a source of 
-linolenic acid [ALA; 18:3(n-3)] or fish oil (FO) that provides (n-3) LC-PUFA. Fatty acid (FA) composition of liver, white muscle, and whole fish was measured to show whether dietary precursors were endogenously biosynthesized to LC-PUFA. Gene expression of liver FA elongase and FAD5 was upregulated in EO fish compared with FO fish. Furthermore, dietary precursors affected the FA concentrations of direct biosynthetic products in all tissues. The increased gene expression in the EO fish was reflected by an increased FA concentration of eicosapentaenoic acid [20:5(n-3)] in the liver compared with the CO fish. However, the high concentrations of (n-3) LC-PUFA found in seawater Atlantic salmon fed diets rich in FO were not attained via biosynthesis from precursors (ALA or SDA) in diets.
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Introduction |
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C20) PUFA [(n-3) LC-PUFA],7 largely supplied from marine fishery products. Alternative oil sources are needed to ensure the sustainability of intensive aquaculture of carnivorous fish, particularly Atlantic salmon, which dominates marine finfish production (3). Atlantic salmon can grow when they are fed diets with 75% replacement of fish oil by vegetable oils without compromising growth, performance, fish health, or welfare (4–6). Vegetable oils are generally high in monounsaturated fatty acids (MUFA) and (n-6) PUFA and can contain large amounts of SFA (e.g. palm oil) but do not contain any (n-3) LC-PUFA (7). Atlantic salmon fed vegetable oils have reduced (n-3) LC-PUFA content (5,6,8–14). However, beneficial effects of (n-3) LC-PUFA are being increasingly recognized in human nutrition (15–17). Atlantic salmon offer a good source of (n-3) LC-PUFA for human consumption, but reduced (n-3) LC-PUFA may compromise their nutritional benefit to consumers (18). Oils that utilize the endogenous biosynthetic capacity of fish would provide sustainable (n-3) LC-PUFA sources for future aquafeeds, although it is unclear how fish respond to such sources (19–21).
The biosynthesis of eicosapentaenoic acid [EPA; 20:5(n-3)] and docosahexaenoic acid [DHA; 22:6(n-3)] from -linolenic acid [ALA; 18:3(n-3)] is inefficient in marine fish, an evolutionary consequence of a natural diet rich in (n-3) LC-PUFA (22,23). Conversion is better in freshwater fish, possibly due to higher concentrations of ALA and limited DHA in their natural diet (22). Consequently, changes in the fatty acid (FA) metabolism of Atlantic salmon, which migrate from fresh to sea water, are of interest (24–26). The biosynthetic pathways for PUFA are well known, but the mechanism underpinning (n-3) LC-PUFA biosynthesis from precursors is not (22,23). The 6 desaturation step, which has a multi-functional role in the conversion of ALA to DHA, is the rate limiting step (27). Diets rich in stearidonic acid [SDA; 18:4(n-3)], the biosynthetic precursor of (n-3) LC-PUFA, provide a relatively high concentration of FA that bypasses the initial conversion requiring 6 desaturase (FAD6). Theoretically, SDA can be con-verted more efficiently than ALA to EPA and DHA (27). Hence, it is of interest to determine whether dietary SDA affects elongase and desaturase gene expression.
Oil from the plant Echium plantagineum L., Boraginaceae, contains 
14% SDA. Previous dietary replacement studies with SDA in Atlantic cod (Gadus morhua L.) and Arctic charr (Salvelinus alpinus L.) have shown SDA conversion to eicosatetraenoic acid [20:4(n-3)] but not to EPA and DHA (20,21). We have recently shown that freshwater Atlantic salmon parr can maintain (n-3) LC-PUFA, in particular EPA and DHA, concentrations in muscle tissue over 6 wk when fed a diet rich in SDA and containing only trace levels of (n-3) LC-PUFA (19). This result indicated that SDA-rich aquafeeds may be an alternative to (n-3) LC-PUFA sources, such as fish oil in freshwater aquaculture, and therefore warranted further investigation in seawater Atlantic salmon. Most of the Atlantic salmon production cycle occurs in seawater where fish are grown from 100 g to >3 kg (3). The effect of dietary SDA on (n-3) LC-PUFA biosynthesis in seawater Atlantic salmon has yet to be determined.
We aimed to determine whether dietary SDA and ALA affected elongase and desaturase gene expression in seawater Atlantic salmon and the resultant effect on the accumulation or biosynthesis of (n-3) LC-PUFA in whole fish, white muscle, and the liver. Consequently, any differences in gene expression and FA composition were due directly to differences in dietary oil source and the effects of differences in weight or in growth rate were discounted (28,29). An SDA-rich oil diet from Echium oil (EO) was compared with a canola oil (CO) diet, which had no (n-3) LC-PUFA and high concentrations of ALA, the precursor to SDA. A fish oil diet (FO) was used as a traditional aquafeed rich in EPA and DHA. We used real-time quantitative RT-PCR (qRT-PCR) to measure the expression of the genes involved in (n-3) LC-PUFA biosynthesis. To our knowledge, this study is the first examining the in vivo gene expression of Atlantic salmon fed an SDA-rich diet.
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Materials and Methods |
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87.9 g) were obtained from Springfield Fisheries hatchery, acclimated for 14 d in 300-L tanks at the School of Aquaculture, University of Tasmania (Launceston, Tasmania, Australia), and fed a commercial feed (Skretting). Prior to the experiment, the fish were slowly adapted to seawater over a 21-d period. The tanks were held at a constant 12.0°C under a natural photoperiod. Water was treated through physical, UV, and biofilters. Dissolved oxygen, pH, ammonia, nitrate, nitrite, and salinity were monitored daily to ensure water quality remained within the limits recommended for Atlantic salmon (31). The experiment followed University of Tasmania Animal Ethics guidelines (investigation A0008392). At the start of the experiment, fish were anesthetized (50 mg/L benzocaine), their weight and length measured, and 4 fish killed to measure initial lipid content and composition [initial fish (INT)]. Twenty-five fish were randomly reallocated into each of 12 300-L tanks. Fish were marked on the ventral surface by a Panjet (28,32) to assess individual performance. The 3 diets were fed in quadruplicate by hand at a ration of 1.1% body weight/d. Every 3 wk, all fish in each tank were anesthetized (50 mg/L benzocaine) and batch-weighed. Fish were starved on the day prior to weighing. Total feed consumption (kg dry matter) was estimated daily from the amount of feed uneaten.
At the end of the experiment, fish were starved on the day before being anesthetized (50 mg/L benzocaine) and their weight and length measured. Fish with similar minimum performance were selected and the first 3 fish that had at least doubled their known initial weight were sampled from each tank. This prevented any small fish with low feeding hierarchy rank and growth from being sampled (28). Fish were killed by a blow to the head after immersion in anesthetic. Samples of red (mean 0.5 ± 0.0 g) and white muscle (0.8 ± 0.1 g), dissected from below the dorsal fin, and liver (2.6 ± 0.1 g) were frozen at –80°C until analysis (33). Two other fish per tank, which had doubled their initial weight, were killed and frozen for total carcass analysis. Specific growth rate (SGR) was calculated as SGR (%/d) = 100 x [ln (Wf/Wi)]/d, where Wf and Wi are the final and initial weights (g) and d is the number of days of the experiment.
Lipid extraction and analyses. Samples were freeze-dried and extracted overnight using a modified Bligh and Dyer protocol (34). This involved a single-phase overnight extraction, CHCl3:MeOH:H2O (1:1:0.9, v:v:v), followed by phase separation to yield a total lipid extract (35). Lipid classes were analyzed by an Iatroscan MK V TLC-flame ionization detector analyzer (Iatron Laboratories) (35,36). An aliquot of the total lipid extract was transmethylated in methanol:chloroform:hydrochloric acid (10:1:1, v:v:v) for 1 h at 100°C to obtain FAME. We performed GC with an Agilent Technologies 6890N GC with an Equity-1 fused silica capillary column (15-m x 0.1-mm i.d., 0.1-µm film thickness), an flame ionization detector, a split/splitless injector, and an Agilent Technologies 7683 Series autosampler and injector. Helium was the carrier gas (19). Molecular weights used for mmol calculations were 903.4 for triacylglycerols (TAG), 806.7 for polar lipids (PL), 282.4 for FFA, 386.7 for sterols (ST), and 270.4 for hydrocarbons.
Chemical analysis. Standard methods were used to determine dry matter (freeze-dry to constant weight), crude fat (34), nitrogen (Kjeldahl using a selenium catalyst; crude protein was calculated as N x 6.25), and energy (bomb calorimeter, Gallenkamp Autobomb, calibrated with benzoic acid).
RNA isolation and preparation. Total RNA was extracted from white muscle and liver tissue stored in an RNA preservation reagent (25 mmol/L sodium citrate, 10 mmol/L EDTA, 10 mol/L ammonium sulfate, pH 5.2) and purified using TRI Reagent (Molecular Research Center), including DNAse treatment (DNA-free, Ambion). RNA yield (A260) and purity (A260/230 and A260/280) were determined spectrophotometrically and the integrity of the RNA was estimated from gel electrophoresis on a 1% agarose gel.
RT. First-strand cDNA was synthesized from total RNA (1 µg) using a SensiMix kit (Quantace) with oligo(dT)18 priming according to the manufacturer's instructions. The reactions were incubated at 65°C for 10 min, and then 42°C for 50 min before the RT enzyme was inactivated at 70°C for 15 min. First-strand cDNA reactions (20 µL) were diluted to 80 µL using nuclease-free water (Sigma-Aldrich) and stored at –20°C before quantitative PCR (qPCR).
qPCR. Real-time PCR primers (Supplemental Table 1) were designed using gene sequences available on GenBank and a 1147-bp expressed sequence tag contig (SGP.Contig7470) identified as polyubiquitin (PolyUb) (94% nucleotide identity to O. mykiss PolyUb; accession no. AF361365) by searching the salmon genome project database. The RNA polymerase II primers were designed previously (37) from a 556-bp salmon sequence (accession no. CA049789). qPCR used SYBR Green chemistry on a MyiQ Real-Time PCR Detection system (Bio-Rad). Each reaction (25 µL) contained primers (200 nmol/L each), 1x SensiMixPlus SYBR and Fluorescein PCR master mix (Quantace), and 2 µL cDNA. All samples were assayed for each gene in duplicate with no-template controls and a 5-step, 2-fold cDNA dilution series for PCR efficiency calculation on the same plate. The reaction was incubated at 95°C for 10 min to activate the heat-activated Taq DNA polymerase followed by 40 cycles of 95°C for 15 s, 55°C for 30 s and 72°C for 25 s. At the end of the 40 cycles, a melt curve analysis was performed to test the specificity of reaction.
Relative expression.
mRNA expression levels were normalized using the geometric mean of 4 stably expressed reference genes (eukaryotic elongation factor 1 , β-actin, RNA polymerase II, and PolyUb as determined by the geNorm software) (38). Automated analysis of qPCR data used qBase software (39), with a modified -Ct relative quantification model with PCR efficiency correction and multiple reference gene normalization.
Statistical analysis.
Values are reported as means ± SEM. Normality and homogeneity of variance were confirmed and percentage data were arcsin-transformed prior to analysis. Comparison between treatments of FA concentration means was by 1-way ANOVA followed by multiple comparison using Tukey-Kramer honestly significant difference at P 
0.05. Correlations were determined by a 1-tailed Spearman bivariate correlation (n = 18). We used SPSS for Windows version 11 for statistical analysis. The normalized relative quantities generated from the qBase software were exported to SigmaStat 3.5 (SPSS) and the nonparametric Mann-Whitney U-test (n = 6) was used to evaluate the significant difference in mean normalized relative quantities of the treatments compared with the control.
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Results |
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Lipid class composition. Lipid class composition of whole carcass and white muscle did not differ among fish fed the 3 diets (Tables 2 and 3). However, the livers of CO fish had more FFA and ST and less PL than in those of EO and FO fish (P < 0.01; Table 4). The dominant lipid class was TAG in both whole carcass and white muscle and was PL in liver.
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(n-3) Biosynthetic pathway. The concentration of SDA was greater in EO fish compared with the FO and CO fish (P < 0.01) (Tables 2–4). Concentrations of 20:4(n-3) were greater in all tissues of EO fish compared with FO and CO fish (P < 0.01). An important difference was that concentrations of EPA were greater in liver of EO fish (2.3 mg/g) than in CO fish (P < 0.01), although both were lower than concentrations in FO fish (P < 0.01). Concentrations of DHA were higher in all tissues in FO fish than in fish fed the other diets (P < 0.01). There was more total (n-3) in the INT, FO, and EO fish than in CO fish (P < 0.01).
(n-6) Biosynthetic pathway. Concentrations of 18:2(n-6) were higher in CO and EO fish than in FO fish (P < 0.01) (Tables 2–4). EO fish had more 18:3(n-6) in white muscle and whole carcass than FO and CO fish (P < 0.01), more 18:3(n-6) in the liver than FO fish (P < 0.01), and more 20:3(n-6) in the whole carcass than FO and CO fish (P < 0.01). However, there was a lower concentration of arachidonic acid [ARA; 20:4(n-6)] in liver in the EO fish (P < 0.01).
Gene expression. Measurements of mRNA abundance by qRT-PCR showed gene expression in liver was affected by dietary oil. In liver of EO fish, there was significantly higher FA elongase (FAE) and FAD5 gene expression compared with FO fish (Fig. 1). There was also significantly higher FAE and both FAD5 and FAD6 gene expression in liver of CO fish than in FO fish.
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) = 0.66; P < 0.01], FAD6 ( = 0.62; P < 0.01), and FAD6 ( = 0.55; P < 0.01) genes. Dietary ALA concentrations was positively correlated with the expression of both FAE ( = 0.53; P < 0.01) and FAD5 ( = 0.47; P < 0.02) genes, whereas dietary SDA was negatively correlated with FAD6 ( = –0.45; P < 0.03) gene expression. There was modest negative correlations between dietary (n-3) LC-PUFA concentrations and hepatic gene expression of FAE (EPA, = –0.68, P < 0.01; DHA, = –0.66, P < 0.01), FAD5 (EPA, = –0.59, P < 0.01; DHA, = –0.55, P < 0.01), and FAD6 (EPA, = –0.45, P < 0.03; DHA, = –0.62, P < 0.01). |
Discussion |
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FA profiles.
The concentrations of individual FA in salmon indicate how fish store dietary FA in tissues and they may also show an endogenous biosynthetic capacity. Defatted fish meal removes virtually all LC-PUFA from the diet, so differences in LC-PUFA concentrations in tissues between the CO and EO fish are presumably due to biosynthesis rather than accumulation. EPA concentration in liver of EO fish significantly increased compared with CO fish, indicating that where the FAD6 was bypassed with dietary SDA, increased biosynthesis occured through to EPA. In all sampled tissues, fish fed the EO diet showed increased 20:4(n-3) and 20:3(n-6), the immediate biosynthetic elongation products of SDA and 
-linolenic acid [GLA; 18:3(n-6)] provided by the EO diet. The FA profiles provide information about the extent to which Atlantic salmon can endogenously biosynthesize (n-3) LC-PUFA from biosynthetic precursors. FA profiles of muscle and carcass of EO fish suggest C18 PUFA were readily biosynthesized to their direct elongase product in both the (n-3) and (n-6) LC-PUFA pathways. Overall, FA profiles of sampled tissues reflected the FA profile of their diet. Comparison to FO fish, there was a reduction of (n-3) LC-PUFA in the tissues for fish fed EO and CO diets, increased oleic acid [18:1(n-9)] in fish fed CO diet, and increased SDA and GLA in fish fed EO diet. In many oil replacement trials, the FA profile of Atlantic salmon reflects their diet (6,9–11,13,30,35). In this study, compared with the FO fish, EO fish did not attain whole carcass, muscle, or liver concentrations of (n-3) or (n-6) LC-PUFA that were achieved with salmon parr (19).
SDA is converted to EPA demonstrated with in the plasma and liver of other animals (41) and the plasma of humans (42). In a salmon cell line, SDA was converted in vitro to 20:4(n-3) and then further desaturated and elongated to EPA (43). Furthermore, radiolabeled [U-14C] SDA was transferred in vitro to DHA in cell culture (44), indicating that SDA can be converted along the (n-3) pathway to DHA. Liver FA profiles suggest that salmon have an increased ability to convert SDA through to EPA compared with ALA. However, tissue FA profiles indicated that further biosynthesis in the (n-3) pathway was negligible.
The FA results indicate that FAE activity is greater for the (n-6) pathway than the (n-3) pathway. As CO and EO diets provide virtually no LC-PUFA, conversion can be estimated from calculations of biosynthesis from C18 precursors. Conversion from GLA to 20:3(n-6) was greater than for SDA to 20:4(n-3) in both CO and EO fish in all tissues. This increased conversion resulted in significantly higher concentrations of ARA in white muscle and liver of CO fish compared with EO fish, even though EO fish had significantly higher concentrations of the precursor product 20:3(n-6). FA results are consistent with an increased biosynthesis in the (n-6) pathway in CO fish to ARA, which is important in the production of fish leukotrienes (45). The increased (n-6) LC-PUFA in CO fish and not in EO fish is likely associated with the markedly higher (n-6):(n-3) FA ratio in the CO diet and increased competition between (n-6) and (n-3) PUFA for desaturase enzymes.
Gene expression. There are many potential mechanisms by which FA, in particular (n-3) LC-PUFA, can affect or regulate gene expression (46). These include changes in membrane composition and signaling, eicosanoid production, oxidant stress, nuclear receptor activation, and /or covalent modification of specific transcription factors (47,48). Gene expression could also be affected indirectly through specific enzyme-mediated pathways and can affect structural, metabolic, and regulatory components of cells (48).
The mechanisms involved in the increased elongase and desaturase gene expression that we showed in liver of both EO and CO fish are not yet known. Dietary FA, environmental factors, and life stage (hormonal) can affect elongase and desaturase activity (46,49–51). Each step along the (n-3) LC-PUFA pathway depends on the amount of substrate, and therefore the activity of the preceding enzymatic step if it is not supplied by the diet, and also the removal of subsequent products (51). Therefore, increased elongase and desaturase gene expression could be influenced by either increased dietary concentrations of substrate (ALA in CO; ALA and SDA in EO) or removal or absence of the product [EO and CO diets having no (n-3) LC-PUFA].
In this study, dietary SDA influenced the molecular mechanisms involved in the biosynthesis of (n-3) LC-PUFA. SDA affected expression in the liver of FAE and FAD5 genes compared with FO fish, as did ALA in the CO diet. These results could also be interpreted as the absence of dietary (n-3) LC-PUFA in the CO and EO diets, resulting in increased expression of FAE, FAD5, and FAD6 genes. The altered expression of the genes involved with the (n-3) pathway may indicate what is driving the compositional changes in the tissue FA profiles. The FAE, FAD5, and FAD6 genes also act upon the (n-6) pathway. Increased expression of FAE, FAD5, and FAD6 genes measured could result from conversion of LA and GLA to AA. FA profiles and conversion ratios indicate that there is a preference for the (n-6) pathway, in particular for CO fish. We demonstrated that dietary concentrations of substrate (ALA, SDA, LA, and GLA) had a positive correlation with expression of the FAE and FAD5 genes, which is similar to previous trials for Atlantic salmon (50,51). There was a negative correlation with dietary (n-3) LC-PUFA, in particular EPA, DPA, and DHA and FAE, FAD5, and FAD6 gene expression. However, there are more factors than just substrate/product presence/absence in the diet that may alter enzymatic activity.
Conflicting results have been reported on FAE gene expression in salmon liver for the replacement of dietary fish oil with vegetable oil (50–52). FAE gene expression increased in liver with a graded replacement of linseed oil (51). However, in a more recent trial, liver FAE gene expression was not increased by a vegetable oil blend (52). Our study confirmed the earlier result (51) with liver FAE gene expression increasing for both alternative oil diets (EO and CO) compared with FO.
Elsewhere, we demonstrated that Atlantic salmon parr fed a SDA-rich oil had similar concentrations of (n-3) LC-PUFA in all tissues to that of FO fish (19). The 6-wk trial indicated that parr fed the SDA-rich diet maintained concentrations of (n-3) LC-PUFA similar to a FO diet (19). However, freshwater fish and salmon parr approaching smolting have an enhanced ability to biosynthesize (n-3) LC-PUFA (22,50). As most salmon production is marine, it is fundamental to ascertain how SDA is further biosynthesized and whether (n-3) LC-PUFA concentrations can be maintained in seawater fish. In the present study SDA-fed smolt increased endogenous metabolism due to elevated FAD5 and FAE gene expression but did not biosynthesize (n-3) LC-PUFA to levels similar to FO fish. Salmon in this trial were previously fed a commercial fish oil-based diet throughout their life, including transfer to seawater and smolting. This history of (n-3) LC-PUFA in their diet may have influenced the expression of FAE and desaturase genes.
In summary, we showed that dietary (n-3) LC-PUFA precursors can directly affect the FA profile of tissues in seawater Atlantic salmon. FA profiles indicated that Atlantic salmon endogenously convert dietary SDA, as demonstrated by increased concentrations of EPA, 20:4(n-3), and GLA in liver of Atlantic salmon smolt and increased concentrations of 20:4(n-3) and GLA in white muscle and whole carcass of EO fish compared with CO fish. However, EO fish did not attain (n-3) LC-PUFA concentrations similar to those of FO fish as demonstrated in an Atlantic salmon parr trial. The high concentrations of (n-3) LC-PUFA found in FO fish will not be provided by increased metabolism of fish fed diets rich in ALA or SDA.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosures: A. R. Bridle, P. D. Nichols, and C. G. Carter, no conflicts of interest.
3 Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org.
6 Present address: Crop and Food Research, Food and Bioresources Innovation, PO Box 5114, Nelson, New Zealand 7000.
7 Abbreviations used: ALA, -linolenic acid; ARA, arachidonic acid; CO, canola oil; DHA, docosahexaenoic acid; EO, Echium oil; EPA, eicosapentaenoic acid; ETA, eicosatetraenoic acid; FA, fatty acid; FAD5, fatty acid 5 desaturase; FAD6, fatty acid 6 desaturase; FAE, fatty acid elongase; FO, fish oil diet; GLA, -linolenic acid; INT, initial fish; (n-3) LC-PUFA, (n-3) long-chain (C20) PUFA; MUFA, monounsaturated fatty acid; PL, polar lipid; PolyUb, polyubiquitin; qPCR, quantitative PCR; qRT-PCR, real-time quantitative RT-PCR; RPL2, RNA polymerase II; SDA, stearidonic acid; SGR, specific growth rate; ST, sterol; TAG, triacylglycerol.
Manuscript received 18 April 2008. Initial review completed 14 May 2008. Revision accepted 14 August 2008.
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LITERATURE CITED |
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