Aquaculture 518 (2020) 734870
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Re-evaluating the dietary requirement of EPA and DHA for Atlantic salmon in freshwater C. Qian, B. Hart, S.M. Colombo
T
⁎
Department of Animal Science and Aquaculture, Faculty of Agriculture, Dalhousie University, Bible Hill, Nova Scotia B2N 5E3, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Atlantic salmon Essential fatty acid Growth performance Requirement
The requirement for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is recommended at 0.50–1.0% of the dry diet for juvenile Atlantic salmon (Salmo salar). Current commercial aquafeed formulations for Atlantic salmon tend to contain a physiological excess of these fatty acids. The aim of this study was to reevaluate the EPA and DHA requirement by Atlantic salmon parr in freshwater since it was determined 20 years ago, and feed formulations and salmon production have changed. This study evaluated the effect of different dietary levels of EPA and DHA (0.25 to 2.0%) on growth performance, overall health, and whole-body EPA and DHA content. Salmon (initial weight 22.6 g ± 1.2) were fed four levels (0.25, 0.5, 0.75 and 2%) of EPA + DHA diets for 6 weeks in freshwater at 12 °C. Diets were fed to triplicate tanks, with 20 fish per tank. There were no mortalities throughout the trial and fish nearly doubled their weight after 6 weeks. Categorically, there were no discrete significant differences in growth performance among treatments after 6 weeks. However, weight gain was exponentially related to diet EPA + DHA levels (p < .0001; r2 = 28.3%), and the feed conversion ratio was inversely related. In both cases, these relationships suggested that the asymptote is reached between 0.25% and 0.5% dietary EPA + DHA. Whole body DHA and EPA showed positive linear relationships with diet DHA (p < .001; r2 = 88.7%) and EPA (p < .001; r2 = 92.9%). Based on growth performance and whole-body fatty acid content, salmon fed a diet containing 0.50% EPA + DHA diet exhibited similar growth performance and haematocrit with control diet (2%). Therefore, 0.5% is considered the lowest required EPA + DHA level for Atlantic salmon parr in freshwater in this study.
1. Introduction
implementation, which protects fish catch quotas and limits FM and FO harvest (FAO, 2018). The aquaculture feed industry has used alternative ingredients (plant-sourced protein and oils, animal-sourced protein and fats) to partially replace FM and FO in feed largely over the last two decades (Turchini et al., 2019; Tacon and Metian, 2015; Hixson, 2014). However, plant oils and animal fats do not supply the omega-3 (n-3) long chain polyunsaturated fatty acids (LC-PUFA) that are required in fish diets. The n-3 LC-PUFA, namely eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), provide several critical functions through molecular, cellular and physiological actions in vertebrates, for both fish and humans (Tocher, 2015). Even with such a significant decrease in the use of FM and FO in commercial feeds, a minimum level is still required since they are currently the only commercial source of these nutrients, yet there is a gap between supply and demand (Tocher et al., 2019). The NRC (2011) requirement for n-3 PUFA for Atlantic salmon is
Atlantic salmon (Salmo salar) is a prevalent cold-water farmed species in the global fish market, with a combined production of over 2 million metric tonnes, mainly by Norway, Chile, Scotland, and Canada (Ytrestøyl et al., 2015). Salmon production relies on feeds containing marine-sourced ingredients, specifically wild pelagic fish (e.g., anchovy, sardine) to produce fish meal (FM) and fish oil (FO), which is the main source of protein and fat in aquafeeds for carnivorous fish like salmon. However, with the increasing global demand for seafood, and aquaculture industry production needs, the use of wild fish as a raw material resource must be significantly reduced as they face fluctuations in price and availability and as a result, limit the needed development of aquaculture industry. Global fisheries have been at maximum levels of exploitation for the past three decades, while the demand for seafood is increasing with the growing human population. This has attracted attention in policy-making and management
⁎ Corresponding author at: Department of Animal Science and Aquaculture, Faculty of Agriculture, Dalhousie University, 58 Sipu Awti, Bible Hill, Nova Scotia, B2N 5E3, Canada. E-mail address:
[email protected] (S.M. Colombo).
https://doi.org/10.1016/j.aquaculture.2019.734870 Received 21 September 2019; Received in revised form 15 December 2019; Accepted 15 December 2019 Available online 16 December 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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0.50–1.0% EPA + DHA (based on Ruyter et al., 2000a; Ruyter et al., 2000b). However, the total n-3 PUFA dietary requirement of salmonids, including α-linolenic acid (ALA; 18:3n-3), EPA, and DHA, has been reported to range from 1.0 to 2.5% of the diet. Bou et al. (2017) found that essential fatty acid requirements for normal growth are met by approximately 0.5% of EPA + DHA in the diet, which is in agreement with other studies showing similar requirements in Atlantic salmon (Ruyter et al., 2000a; Ruyter et al., 2000b; Emery et al., 2016). Early studies in Atlantic salmon fry (4 g/fish) determined that n-3 PUFA levels ranging from 0.5 to 1.0% in the feed were needed to attain acceptable growth (Ruyter et al., 2000a). However, this requirement was determined for salmon that were fed a low lipid diet with only 8% total fat content and with lower growth rates compared with commercial standards today. Salmon farming conditions have evolved over the past two decades, and today high-lipid diets are now commonly used to support fast growth, so this stated required level may be different 20 years later in a commercial setting with salmon that have been selectively bred for growth. Furthermore, with greater emphasis on nutrient-based approaches to formulating aquafeeds, rather than raw materials (Turchini et al., 2019), accurate requirements should be defined for assessing novel sources of n-3 LC-PUFA in the future. Current commercial aquafeed formulations for Atlantic salmon tend to contain a physiological excess of n-3 LC PUFA to ensure a high level of deposition of these fatty acids into the fillet tissue. Atlantic salmon have a demonstrated the ability to bioconvert ALA to DHA (Mock et al., 2019; Torstensen et al., 2004; Tocher et al., 1997). This has been estimated at a rate of ~25% bioconversion in Atlantic salmon (Colombo et al., 2018; Hixson et al., 2014), but also depends on environmental conditions (salinity, temperature), age/life stage, and dietary fatty acid content, particularly dietary ALA as a substrate, the n-3/n-6 ratio, and concentrations of dietary EPA and DHA. Given that bioconversion is possible, commercial aquafeed formulations may contain what is considered a physiological excess of n-3 LC PUFA to ensure a high level of performance and deposition of these fatty acids into the fillet tissue. While maximizing the deposition efficiency of dietary EPA and DHA into the final edible product is important for human consumers, accurately defining the lowest level of EPA and DHA needed to produce healthy, quality salmon is key to economical, sustainable diets. The aim of this study was to re-evaluate the EPA and DHA requirement by Atlantic salmon parr in freshwater, since it was determined 20 years ago, and given that feed formulations and salmon production have changed. This study evaluated the effect of different dietary levels of EPA and DHA (0.25 to 2.0%) on growth performance, overall health, and whole body EPA and DHA content.
Table 1 Formulation and proximate composition (as-fed) of experimental diets containing increasing levels of fish oil (FO) in the diet1. Ingredient (%)
0.25
0.50
0.75
2.0
Fish meal Fish oil (Herring) Ground wheat Empyreal™752 Canola oil Poultry byproduct meal Blood meal Vitamin/Mineral Mix Dicalcium phosphate Special mix Lysine HCL Choline chloride
15 0.4 13.1 25 17.5 17 8 0.2 2 0.25 0.5 1.05
15 2.25 13.1 25 15.65 17 8 0.2 2 0.25 0.5 1.05
15 4.15 13.1 25 13.75 17 8 0.2 2 0.25 0.5 1.05
15 13.5 13.1 25 4.4 17 8 0.2 2 0.25 0.5 1.05
97.55 7.67 52.42 22.55
97.68 7.83 52.67 22.57
97.51 7.90 53.24 22.60
Proximate composition (Analyzed values) Dry matter (%) 97.80 Ash (%) 7.79 Crude protein (%) 52.90 Crude fat (%) 22.27 1 2
Ingredients provided by Northeast Nutrition, Truro, Nova Scotia, Canada. Corn protein concentrate.
inclusion levels (Table 2). Saturated fatty acids (SFA) were lowest in the 0.25 diet (11.2%) and highest in the 2.0 diet (25.1%). Monounsaturated fatty acids (MUFA) were highest in the 0.25 diet (59.9%) and lowest in the 2.0 diet (38.7%). Polyunsaturated fatty acids (PUFA) were lowest in the 0.25 diet (28.7%) and highest in the 2.0 diet (35.7%). Both EPA and DHA (and their sum) were lowest in diets with lower FO inclusion, and highest in the 2.0 diet. The 18‑carbon fatty acids (namely 18:1n-9, ALA, LNA) generally decreased with greater FO inclusion, except 18:4n-3. The 16‑carbon fatty acids (namely 16:0 and 16:1n-7) generally increased with greater FO inclusion. 20:4n-6 was highest in the control diet and lower in diets with low FO. Total n-6 PUFA were highest in the 0.25 diet, and lowest in the 2.0 diet; total n-3 PUFA showed the opposite trend. The n-3/n-6 ratio was highest in the 2.0 diet, and lowest in the 0.25 diet. Diet fatty acid data presented as mg/g is available in the Supplementary Information (Table S1). 2.2. Experimental fish and feeding trial Atlantic salmon parr in freshwater were received from Cape D'Or Seafoods Inc., Wentworth, NS, Canada. The feeding trial was conducted at the Aquaculture Centre, Faculty of Agriculture, Dalhousie University and followed procedures outlined in the approved animal care protocol (#2018–041). A total of 240 fish with an average initial weight of 22.6 g ± 1.2 g were distributed randomly into 12 recirculating rearing tanks (50 L, 3 tanks per diet, 20 fish per tank), supplied with 2.7 L/min freshwater. The temperature varied between 10.6 °C and 13.0 °C (mean 12 °C). The O2 saturation level varied between 92% and 120% (mean 102%). The pH varied between 8 and 8.2 (mean 8.15). Temperature and oxygen measurements were recorded daily throughout the experiment. Fish were hand fed 2 mm pellets to satiation twice per day for six weeks. Feed consumption (per tank) and water quality assessment (pH, alkalinity, total ammonia nitrogen, nitrite, nitrate) were recorded weekly. There were no mortalities to report throughout the duration of the trial.
2. Methods 2.1. Experimental diets Diets were formulated as isonitrogenous (52–53%), isolipidic (22–23%) and isoenergetic, and were produced at the Faculty of Agriculture, Dalhousie University (Truro, Nova Scotia, Canada). Experimental diets were created by formulating different proportions of FO and canola oil to achieve increasing dietary levels of EPA + DHA that was sourced from FO. Diets were formulated to contain 0.25, 0.50, 0.75, and 2.0% EPA + DHA. The diet with 2.0% EPA + DHA resembles a commercial diet and is referred to as control diet. Other ingredients, except the lipid source, were the same in all four diets (Table 1). The experimental diets are referred in the text according to their percentage supplementation in the feed from 0 to 2.0%. All diets were steam pelleted using a laboratory size pellet mill (California Pellet Mill, San Francisco, USA) to produce 2 mm pellets and stored at −20 °C. Diets were analyzed for proximate composition by the Nova Scotia Department of Agriculture (Truro, Nova Scotia, Canada; Table 1). The fatty acid content of the diets (% of total FAME) is in Table 2. Range in FA content (% FAME) was dependent on FO and canola oil
2.3. Sampling Five fish per tank were randomly sampled at Week 0, measured for length, weight, and blood samples, and whole body frozen for nutritional composition. Fish were fasted for 24 h prior to sampling to empty gut contents. Fish were euthanized humanely with overdose of tricaine methane sulfonate (MS222), then length and weight were measured. Subsequently, the caudal fin was cut to obtain blood samples in 2
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Condition factor = 100 × [Biomass/(length)3].
Table 2 Fatty acid content (% of total FAME) of experimental diets (dry weight) fed to Atlantic salmon parr1. Fatty acid
2
14:0 14:13 15:0 16:0 16:1n-9 16:1n-7 16:1n-5 16:4n-1 17:0 17:1 18:0 18:1n-9 18:1n-7 18:2n-6 (LNA) 18:3n-6 18:3n-3 (ALA) 18:4n-3 20:0 20:1n-11 20:1n-9 20:1n-7 20:2n-6 20:4n-6 20:5n-3 (EPA) 22:0 22:1n-11 22:1n-9 22:1n-7 22:5n-6 22:5n-3 22:6n-3 (DHA) 24:1 EPA + DHA SFA4 MUFA5 PUFA6 n-3 n-6 n-3/n-6
0.25
0.5
0.75
2.0
0.44 0.02 0.06 7.38 0.12 1.22 0.04 0.07 0.08 0.06 2.39 51.1 2.81 19.5 0.01 6.42 0.10 0.51 0.22 1.90 0.11 0.07 0.08 0.41 0.29 1.56 0.22 0.07 0.04 0.09 0.94 0.21 1.35 11.2 59.9 28.7 8.22 19.8 0.41
0.89 0.02 0.09 8.39 0.13 1.74 0.05 0.19 0.11 0.07 2.55 48.0 2.78 18.4 0.03 6.04 0.28 0.47 0.24 1.94 0.13 0.07 0.13 1.67 0.28 1.62 0.23 0.07 0.04 0.21 1.66 0.23 3.33 12.8 57.5 29.5 10.3 18.8 0.53
1.38 0.04 0.13 9.55 0.14 2.31 0.06 0.34 0.14 0.07 2.72 44.2 2.76 17.1 0.05 5.53 0.49 0.45 0.19 1.90 0.14 0.08 0.19 2.88 0.27 1.70 0.25 0.08 0.05 0.36 2.60 0.25 5.48 14.7 54.3 30.8 12.2 17.6 0.69
3.99 0.11 0.33 16.1 0.23 4.99 0.11 0.98 0.31 0.05 3.78 24.5 2.68 9.82 0.13 2.51 1.50 0.35 0.27 1.98 0.25 0.11 0.45 8.56 0.22 2.08 0.30 0.11 0.17 1.07 7.05 0.44 15.6 25.1 38.7 35.7 21.7 10.8 2.0
Blood was drawn from the caudal vein with 3 mL capillary tubes, which were centrifuged at 3000 ×g for 10 min to obtain the fractions of plasma and red blood cells (RBC), and haematocrit was evaluated by measuring the proportion of RBC in the total length of the blood sample in the capillary tube, and calculated according to the following formula: Haematocrit = 100× (Length of RBC mm)/(Length of whole blood mm). 2.5. Proximate analysis Whole body composition was analyzed according to the following procedure. Frozen fish samples were taken from −80 °C freezer and thawed at ambient temperature for 1 h. The caudal, pectoral, and dorsal fins were removed, and the whole fish was grinded with 50 mL of water until tissues were liquified. The homogenate was frozen, then freezedried for 48 h to remove moisture. The samples were weighed before and after freeze drying to determine the moisture content of the whole fish. The freeze-dried samples were then ground in a small coffee grinder to obtain a homogenized powder prior to analyzing protein and lipid. Total lipid content was analyzed using ANKOM (TX15 Extractor, Macedon NY). Nitrogen content was analyzed using a Leco FP-528 (Leco Corporation, St. Joseph, MI, USA) analyzer. Nitrogen values were converted to protein values using the conversion factor 6.25. 2.6. Fatty acid analysis Whole body freeze-dried samples were then subsampled and individually ground to a fine powder in liquid nitrogen using a mortar and pestle, and the resulting powder was weighed to the nearest microgram. Total lipid was extracted using a modified Folch method (Folch et al., 1957), as in Hixson et al. (2017). In brief, each sample was extracted three times, using 2 mL of chloroform/methanol (2:1; v/v) and then pooled (total 6 mL). Polar impurities were removed by adding 1.6 mL KCl solution (0.9% w/v). The organic layer was removed using a lipid-cleaned glass pipette and pooled. The resulting lipid-containing solvent was concentrated to 2 mL by evaporating with nitrogen gas. The lipid extract was then prepared for gas chromatography by derivatizing into fatty acid methyl esters (FAME) using the Hildich reagent (sulfuric acid) as the catalyst (Christie, 2003). Fatty acid methyl esters were extracted twice using hexane: diethyl ether (1:1; v/v), then dried under a gentle stream of nitrogen. The dry FAME extract was re-dissolved in hexane and individual FAME were separated using a gas chromatograph (GC). All solvents used in the extraction and FAME derivatization procedures were of high purity HPLC grade (> 99%). A known concentration of 5 alpha-cholestane (C8003, Sigma-Aldrich, St. Louis, Missouri) was added to each sample prior to extraction to act as the internal standard to estimate extraction and instrument recovery efficiency. FAMES were analyzed by the Marine Lipids Lab at Dalhousie University (Halifax, NS, Canada).
1
The dietary groups are named according to their percentage in the feed as 0, 0.5, 0.75, and 2.0% of the diet. 2 Reported fatty acids are over 0.05% of total FAME. Analytical replicates n = 3. 3 14:1 is the sum of 14:1n-5, 14:1n-7, 14:1n-9. 4 SFA = sum of saturated fatty acids. 5 MUFA = sum of monounsaturated fatty acids. 6 PUFA = sum of polyunsaturated fatty acids.
capillary tubes. Finally, whole fish were put into plastic bags and sealed, frozen at −80 °C and stored for subsequent analysis. The same procedure was implemented at Week 6 with the remaining 15 fish per tank. 2.4. Growth performance
2.7. Statistical analysis
Weight and length were measured at Week 0 and Week 6. Weight gain (final weight – initial weight) and specific growth rate (SGR) were calculated as:
Data are reported as mean values ± standard deviation for each treatment. For analysis of growth data, a two-level nested analysis of variance (ANOVA) was performed using the general linear model (Minitab 16 Statistical Software, State College, PA, USA) on data collected from individuals (weight, length, condition factor, haematocrit, fatty acids). The model was designed to test the effect of diet (fixed factor) on the growth performance (response variable) and nested fish individuals (random factor) within tanks to negate variability among tanks and individuals, and also tested for tank effects. For analysis of growth data that depend on comparison to an initial measurement and thus must be pooled per tank (i.e. weight gain, SGR, apparent feed intake, FCR), a one-way ANOVA was performed to test the effect of diet,
SGR (%BW/d) = 100 × [ln (final BW)–ln (initial BW)]/days. Recorded feed intake (weekly, dry matter) and weight gain in each tank were used to calculate the feed conversion ratio (FCR), according to the following formula:
FCR (g/fish) = (Feed intake g/fish)/(weight gain g/fish). Individual final weight and fork lengths were used to calculate the condition factor (CF), according to the following formula: 3
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Table 3 Growth performance of individual Atlantic salmon after fed experimental diets containing different dietary EPA + DHA levels for 6 weeks1. Growth parameter
0.25
Initial weight (g) Final weight (g) Weight gain (g) Initial length (cm) Final length (cm) Initial haematocrit Final haematocrit Condition factor Specific growth rate Apparent feed intake2 Feed conversion ratio2
22.1 37.8 15.6 12.6 14.5 36.3 43.9 1.22 1.27 13.3 0.86
1 2
0.50 ± ± ± ± ± ± ± ± ± ± ±
1.2 1.7b 2.5 0.4 0.2 0.7 4.5 0.04 0.2 1.3 0.06
23.4 43.5 20.1 12.8 15.3 37.7 44.6 1.22 1.48 13.1 0.76
0.75 ± ± ± ± ± ± ± ± ± ± ±
1.4 1.3a 2.5 0.2 0.4 1.2 3.4 0.09 0.2 1.5 0.12
22.0 40.0 18.0 12.6 14.9 37.2 45.5 1.20 1.42 13.6 0.76
2.0 ± ± ± ± ± ± ± ± ± ± ±
1.2 1.2ab 1.6 0.2 0.2 2.0 4.2 0.05 0.1 0.5 0.06
22.7 41.7 18.9 12.7 15.4 36.9 42.4 1.14 1.44 14.0 0.75
± ± ± ± ± ± ± ± ± ± ±
1.3 1.3ab 3.3 0.3 0.6 0.8 3.3 0.05 0.2 1.0 0.08
F-value
p-value
0.72 4.17 1.62 0.31 3.22 0.35 0.65 1.21 0.68 0.35 2.52
0.576 0.047 0.259 0.821 0.083 0.787 0.604 0.368 0.591 0.780 0.132
Means are ± standard deviation (n = 3). Different superscripts indicate significant differences among means. Apparent feed intake and feed conversion ratio are reported on a dry matter basis.
with tank as the experimental unit (n = 3). In all cases, where significant differences occurred (p < .05), treatment means were differentiated using the Tukey HSD multiple comparison. For fatty acid analysis, FA raw data were converted to percentages, and Bonferroni post hoc confidence intervals with appropriate correction of the α level for multiple comparisons were used to determine differences among treatments. The relationships among growth performance (i.e., weight gain, FCR) and diet EPA + DHA, and diet vs. tissue EPA and DHA, were determined using the Curve Fit function in Sigma Plot version 14.0 (Systat Software Inc., Chicago, IL, USA).
3. Results 3.1. Growth performance and haematocrit Growth performance data were measured at initial (week 0) and final (week 6) sampling of the fish (Table 3). The fish doubled their weight to a mean final weight of 40.7 g ± 2.8 over the six-week study. Salmon fed the 0.25% diet had significantly lower final weight than salmon fed the 0.50% treatment. However, weight gain, condition factor, apparent feed intake, FCR, and haematocrit were not significantly different among treatments. Numerically, fish fed the lowest EPA + DHA treatment group (0.25) showed the highest FCR and lowest SGR compared to other groups. The haematocrit of all diet groups increased from the beginning to the end of trial (p < .001 in two sample t-test). Nonlinear regression models were used to relate weight gain and FCR with diet EPA + DHA. A single 2-parameter exponential rise to maximum showed the best fit for the relationship between weight gain and diet EPA + DHA (p < .0001; r2 = 28.3%; WG = 19.0243*(1-exp (−1.3226*diet EPA + DHA); Fig. 1). A 2-parameter inverse polynomial showed the best fit for the relationship between FCR and diet EPA + DHA (p < .0001; r2 = 48.3%; FCR = 0.7486 + (201.2/ (EPA + DHA)) + (5.584/(EPA + DHA)^2); Fig. 2).
Fig. 1. Relationship between dietary EPA + DHA content (mg/g), corresponding to 0.25, 0.50, 0.75 and 2.0% of the diet) and weight gain (WG) of Atlantic salmon, fed diets containing increasing EPA + DHA levels over 16 weeks. The model fit is a single 2-parameter exponential rise to maximum.
3.2. Whole body chemical composition After six weeks of feeding, the whole body content of protein decreased (p = .018 in two sample t-test) but there was no significant difference among treatments (Table 4). The whole body content of lipid increased from Week 0 to Week 6 (p < .001 in a two sample t-test). However, after six weeks of feeding experimental diets, there was no significant difference in whole body lipid content among groups fed varying levels of EPA + DHA (Table 4). Initial and final dry matter content was consistent between 28 and 29% and did not vary among treatments.
Fig. 2. Relationship between dietary EPA + DHA content (mg/g), corresponding to 0.25, 0.50, 0.75 and 2.0% of the diet) and the feed conversion ratio (FCR) of Atlantic salmon, fed diets containing increasing EPA + DHA levels over 16 weeks. The model fit is an inverse second-order polynomial.
4
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acid, a stepwise pattern was observed in salmon that were fed the 0.25 diet to the control diet, with the highest level of EPA + DHA, which was reflective of the diet FA content. SFA were higher in salmon fed the control than salmon fed any other treatment with reduced FO (p < .001). MUFA was higher in salmon that were fed the 0.25 and 0.50 diets than the 0.75 diets and control diets (p < .001). PUFA was highest in salmon fed the control, followed by salmon fed the 0.75 and 0.50 diets (which did not differ from each other), and the 0.25 diet (which did not differ from salmon fed the 0.50 diet; p < .001). The sum of EPA + DHA was highest in salmon fed the control, while salmon fed the 0.25 diet showed lower EPA + DHA levels than salmon fed the 0.75 diet (p < .001). The same pattern was observed for both EPA (p < .001) and DHA (p < .001). 16‑carbon fatty acids such as 16:0 and 16:1n-7 were highest in salmon fed the control and were not different among salmon fed lower FO diets (p < .001). 18:2n-6 and 18:3n-3 were the same in salmon fed reduced FO treatments, but lower in salmon fed the control (p < .001). 20:4n-6 was highest in salmon fed the low FO and control treatments, but lower in salmon fed the high FO treatment (p < .001). Total n-6 PUFA were lower in salmon fed the control diet but did not differ among any other treatment (p < .001). Total n-3 PUFA were highest in salmon fed the control diet, followed by the 0.75 diet, then 0.25 diet (p < .001). The n-3/n-6 ratio was highest
Table 4 Mean ( ± standard deviation, n = 3) initial & final protein,fat, and dry matter percentage of whole body in Atlantic salmon after fed four different dietary DHA + EPA level diets for 6 weeks.
Initial protein % Final protein % Initial lipid % Final lipid % Initial dry matter % Final dry matter %
0.25
0.50
0.75
2.0
p-value
54.2 ± 1.4
54.3 ± 3.3
54.8 ± 0.1
54.6 ± 1.4
0.989
50.4 10.0 13.0 29.1
52.8 10.2 13.7 28.6
2.1 0.5 1.0 0.7
52.8 ± 1.3 9.6 ± 1.2 13.0 ± 1.5 28.9 ± 0.4
54.0 ± 0.7 9.6 ± 2.2 12.3 ± 0.6 29.0 ± 0.3
0.071 0.969 0.418 0.519
29.7 ± 0.5
29.8 ± 0.7
29.9 ± 0.4
0.632
± ± ± ±
1.2 2.4 0.6 0.5
29.5 ± 0.3
± ± ± ±
3.3. Whole body fatty acid content Overall, fatty acid content (% FAME) in salmon differed based on dietary treatment (Table 5). After six weeks, salmon fed the control diet showed a fatty acid profile that appeared to be most similar to the pooled initial sample, from salmon that were fed a commercial diet prior to beginning the experiment. In general, depending on the fatty
Table 5 Fatty acid content (% FAME)1 of Atlantic salmon parr (whole body, dry weight) at initial (pooled) and after six weeks of feeding experimental diets. Fatty acid 14:0 14:12 15:0 16:0 16:1n-9 16:1n-7 16:1n-5 16:4n-1 17:0 17:1 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-6 18:3n-3 18:4n-3 20:0 20:1n-11 20:1n-9 20:1n-7 20:2n-6 20:4n-6 20:5n-3 22:0 22:1n-11 22:1n-9 22:1n-7 22:5n-6 22:5n-3 22:6n-3 24:1 EPA + DHA SFA3 MUFA4 PUFA5 n-3 n-6 n-3/n-6
Initial 3.74 0.10 0.26 14.7 0.33 6.38 0.13 – 0.27 – 3.95 22.8 3.57 8.72 0.23 1.73 1.49 0.18 1.14 1.81 0.23 0.51 0.78 4.76 0.06 1.44 0.23 0.06 0.05 2.05 11.6 0.45 16.0 23.2 39.5 37.0 23.1 11.3 2.07
± ± ± ± ± ± ±
0.25 0.12 0.01 0.08 0.37 0.02 0.14 0.01
± 0.01 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.21 0.6 0.01 0.21 0.01 0.25 0.06 0.02 0.30 1.21 0.02 0.06 0.44 0.24 0.02 0.14 0.00 0.00 0.02 0.12 0.6 0.03 1.75 0.52 0.94 0.71 0.66 1.1 0.16
2.13 0.05 0.14 11.1 0.30 3.80 0.08 0.25 0.15 0.12 3.26 37.8 2.92 12.2 1.16 2.67 1.69 0.26 0.78 1.77 0.14 0.52 0.68 2.43 0.14 1.27 0.25 0.05 0.22 1.06 6.58 0.40 9.00 17.3 50.5 32.0 14.6 15.5 0.95
0.50 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
bc
0.25 0.01b 0.07c 0.75b 0.01a 0.41b 0.00b 0.03b 0.06c 0.03b 0.19b 1.61a 1.03 0.50a 0.23a 0.11a 0.15a 0.02a 0.47 0.61 0.05b 0.03bc 0.06a 0.28c 0.00a 0.05b 0.01 0.00c 0.03a 0.13b 0.59c 0.03b 0.85c 1.27b 1.17a 1.14c 1.20c 1.81a 0.12c
2.11 0.07 0.17 11.1 0.28 3.74 0.08 0.27 0.18 0.12 3.21 37.1 3.20 12.5 0.80 2.90 1.47 0.25 0.54 2.02 0.17 0.58 0.57 2.52 0.13 1.26 0.26 0.05 0.18 1.08 7.04 0.42 9.57 17.2 49.7 32.9 15.9 15.2 1.05
0.75 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
c
0.27 0.00b 0.02bc 0.58b 0.01ab 0.50b 0.00b 0.04b 0.01bc 0.03b 0.22b 2.25ab 0.07 0.52a 0.14b 0.18a 0.10bc 0.02a 0.07 0.17 0.01b 0.06ab 0.05ab 0.41bc 0.01a 0.03b 0.03 0.00bc 0.01b 0.19b 0.89bc 0.03ab 1.2bc 1.03b 1.84a 1.39bc 1.46bc 0.68a 0.13bc
2.37 0.08 0.19 11.8 0.28 4.00 0.09 0.31 0.19 0.10 3.35 35.3 3.18 12.4 0.58 2.94 1.35 0.26 0.51 2.00 0.18 0.62 0.49 2.84 0.13 1.26 0.27 0.05 0.18 1.18 7.82 0.42 10.7 18.4 47.8 33.7 17.0 15.4 1.11
2.0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
b
0.15 0.02b 0.01b 0.54b 0.02b 0.23b 0.00b 0.02b 0.01b 0.04b 0.12b 1.00b 0.10 0.4a 0.08c 0.05a 0.05c 0.02a 0.10 0.07 0.01ab 0.05a 0.19b 0.17b 0.01a 0.08b 0.04 0.00b 0.02b 0.07b 0.37b 0.03b 0.51b 0.87b 0.82b 0.61b 0.50b 1.56a 0.12b
3.60 0.10 0.28 14.2 0.29 5.83 0.12 0.49 0.26 0.16 3.65 24.9 3.27 9.43 0.26 1.85 1.51 0.17 0.66 1.78 0.22 0.37 0.63 5.41 0.09 1.44 0.26 0.06 0.22 1.96 11.0 0.45 16.4 22.3 40.1 37.3 23.1 11.1 2.08
Reported fatty acids are over 0.05% of total FAME. 1 Means are ± standard deviation (n = 3). Different superscripts indicate significant differences among means. 2 14:1 is the sum of 14:1n-5, 14:1n-7, 14:1n-9. 3 SFA = sum of saturated fatty acids. 4 MUFA = sum of monounsaturated fatty acids. 5 PUFA = sum of polyunsaturated fatty acids. 5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
a
0.10 0.01a 0.00a 0.29a 0.02ab 0.29a 0.00a 0.17a 0.00a 0.00a 0.16a 0.37c 0.06 0.16b 0.04d 0.71b 0.05b 0.06b 0.14 0.05 0.01a 0.01c 0.04a 0.30a 0.01b 0.09a 0.02 0.00a 0.00a 0.06a 0.33a 0.01a 0.61a 0.48a 0.49c 0.79a 0.72a 0.12b 0.09a
F-stat
p-value
141.3 14.69 46.52 75.62 3.19 75.83 90.74 12.73 29.18 2.84 9.65 907.57 1.23 132.31 100.45 26.24 21.84 17.20 1.78 2.15 17.58 5.50 6.65 289.16 27.31 40.02 0.58 23.16 23.76 180.96 194.75 6.89 248.03 64.30 238.75 44.64 117.97 21.91 143.96
< 0.001 0.001 < 0.001 < 0.001 0.084 < 0.001 < 0.001 < 0.002 < 0.001 0.106 0.005 < 0.001 0.361 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.229 0.173 0.001 0.023 0.015 < 0.001 < 0.001 < 0.001 0.645 < 0.001 < 0.001 < 0.001 < 0.001 0.013 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001
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aquaculture. Relatively limited knowledge exists about the EPA and DHA requirement in Atlantic salmon, and the NRC requirement (NRC, 2011) is based on just two studies from nearly two decades ago. While the present study was not a true “requirement” study in terms of determining dietary deficiencies below a given threshold, we tested practical levels of FO, with consistently low FM across all treatments to reflect commercial-type diets. Though we were logistically limited to four graded levels (0.25%, 0.5%, 0.75%, and 2.0%), we tested practical and feasible dietary EPA + DHA levels which can be easily replicated in practical diets. After six weeks of feeding, and nearly doubling their initial weight, there were no discrete differences in growth performance. However, weight gain was exponentially-related to diet EPA + DHA levels, and the FCR was inversely-related. In both cases, the visual representation of these relationships (see Figs. 1 and 2) suggested that the asymptote appeared to be reached between 0.25% and 0.5% dietary EPA + DHA, which aligns with current recommendations for Atlantic salmon (NRC, 2011; Bou et al., 2017). Fig. 3. Relationship between dietary DHA content (% fatty acid methyl ester, FAME) and whole body DHA content of Atlantic salmon, fed diets containing increasing DHA levels over 6 weeks.
4.1. Growth performance Based on growth performance, salmon fed the diet with 0.5% EPA + DHA (supplied at 0.4% of the diet as-fed, and equated to 3.33% EPA + DHA as a % of total FA) suggests that it is the lowest required level to achieve the best growth in this study. The NRC requirement (2011) is based on the results from Ruyter et al. (2000a), which showed that up to 1% of EPA + DHA resulted in faster growth, in Atlantic salmon fry (4 g) and that < 0.5% resulted in slower growth. This is the cited study in the NRC (2011) requirement for Atlantic salmon, which states 0.5–1.0% n-3 LC-PUFA is required, or 1.0% ALA. The n-3 LCPUFA can satisfy the essential fatty acid requirements at lower levels than ALA and increase growth over that obtained with ALA alone (Ruyter et al., 2000a). Bou et al. (2017) stated that the requirement was 0.5%, however, salmon were larger post-smolts (50 g initial weight to 400 g final weight). The 0.5% refers to the amount supplied in the diet, which corresponds to a measured value of 1.39% EPA + DHA (as a % of total fatty acids) in the Bou et al. (2017). This corresponds to our value of 1.35% of the diet. This is also in agreement with other studies showing similar requirements in Atlantic salmon (Ruyter et al., 2000a, 2000b; Emery et al., 2016). Clearly size and/or life stage (freshwater vs. seawater) is an important determinant in the n-3 requirement. Smaller fish may have a physiological need for higher EPA + DHA contents for development. It is also important to note that the total n-3 content of the diets in our study was over 8%, meaning that the ALA supplied in the diet also provided available substrate for EPA and DHA synthesis, which is stated as part of NRC's n-3 requirement for salmon. EPA and DHA dietary requirements cannot be based exclusively on growth; tissue integrity and fish health also need to be considered (Bou et al., 2017). Overall health was evaluated by haematocrit analysis. The normal haematocrit range of healthy Atlantic salmon is ~44–49% (Waagbø et al., 1988). Haematocrit values increased after six weeks of growth, suggesting normal improvement in the capacity of blood oxygen transportation with growth and development (Thomas and Perry, 1992). Diseases like infectious salmon anaemia can result in haematocrit values below 25% (Evensen et al., 1991), and even lower than 15% (Dannevig et al., 1993). However, the haematocrit values were ~37% in initial sampling which was normal in that stage, suggesting that fish were in good health at the start of the trial, in addition to normal appearance and feeding behaviour. There were no significant differences in hameatocrit in salmon that were fed decreasing levels of EPA + DHA.
Fig. 4. Relationship between dietary EPA content (% fatty acid methyl ester, FAME) and whole body EPA content of Atlantic salmon, fed diets containing increasing EPA levels over 6 weeks.
in salmon fed the control diet and followed the same pattern as n-3 PUFA (p < .001). Salmon whole body fatty acid data presented as mg/ g is available in the Supplementary Information (Table S2). The linear relationship between diet and whole body fatty acid content (% FAME) was significant for both DHA (p < .0001; r2 = 88.7%; DHA = 5.809 + 0.7332*Diet DHA; Fig. 3) and EPA (p < .0001; r2 = 92.9%; EPA = 1.9898 + 0.3882 * Diet EPA; Fig. 4). Comparatively, the linear relationship between diet and whole body fatty acid content as total amounts (mg/g) was also significant for both DHA (p < .0001; r2 = %; DHA = 7.8389 + 0.2508*Diet DHA; Supplementary Information Fig. S1) and EPA (p < .0001; r2 = 54.5%; EPA = 2.5654 + 0.1847*Diet EPA); Supplementary Information Fig. S2), although were not as explanatory as comparing fatty acid data as a % FAME, as individual variability in mg/g data was higher. 4. Discussion
4.2. Whole body composition
Currently, commercial aquafeed formulations for Atlantic salmon contain a physiological excess of n-3 LC PUFA to ensure a high level of deposition of these fatty acids into the fillet tissue (Mock et al., 2019). Understanding the accurate dietary requirement of EPA and DHA for salmon may be the most practical method to reduce FO/FM use in
Protein content in salmon decreased after six weeks; however, this could be due to the slightly lower protein content in the feed compared to the commercial diet. As for whole body fat, there was an increase 6
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from the initial sampling to final sampling due to a higher fat supply in the experimental feeds and subsequent growth of the fish. However, there were no differences in whole body fat or protein content as a function of differences in dietary EPA + DHA level. Generally, whole body FA content linearly corresponded to diet FA content. Whole body EPA and DHA both showed linear relationships with diet EPA and DHA; however, the slope (or rate of retention) was different for each. The slope of DHA (0.73) suggests that a majority (73%) of diet DHA was retained. This is slightly lower than previous reports that the relationship between diet DHA and tissue DHA is at least 1:1 or higher (Colombo et al., 2018; Hixson et al., 2017). This obviously can depend on tissue. The slope of EPA (0.39) was also slightly lower than previous reports but was expected to be < 50%, according to a previous study (Colombo et al., 2018). This is in agreement with the previously reported selective deposition of DHA over EPA (Trushenski et al., 2012; Bou et al., 2017; Colombo et al., 2018) and suggests different biological roles, and consequently, needs for these FA. Dietary EPA is the n-3 precursor for eicosanoid synthesis, and some of the EPA may be also be modified toward DHA production. EPA is known to be more dispensable than DHA (Trushenski et al., 2012; Glencross et al., 2015; Emery et al., 2016), and is preferentially catabolized in salmonids, if dietary requirements have been met (Murray et al., 2014). The lower retention values for EPA and DHA (especially DHA) could be because sufficient dietary values did not require full retention, since the requirement appears to have been met for all levels except at 0.25%, and synthesis of DHA was not required (which would have resulted in a slope over 1). It has been shown previously that salmonids, Atlantic salmon and rainbow trout (Oncorhynchus mykiss), can be net producers of n-3 LC-PUFA by feeding diets with only low levels of fish oil (Crampton et al., 2010; Sanden et al., 2011; Turchini et al., 2011; Colombo et al., 2018). This suggests that it may be possible that salmon could perform well when fed 0.5 EPA + DHA diets, or less. However, given that the retention efficiency of DHA in this study was estimated at 73%, it's likely that sufficient DHA was supplied. It is also interesting to note that despite provision of ALA (6.25% of total FA, 2% of the diet), salmon fed a diet containing 0.25% EPA + DHA still showed lower performance. It is quite well known when provided a diet containing relatively high levels ALA and low levels of EPA and DHA, Atlantic salmon possesses a capacity for the endogenous synthesis of n-3 LC PUFA via in vivo fatty acid bioconversion (Mock et al., 2019; Hixson et al., 2017; Glencross et al., 2015; Thomassen et al., 2012). This is why the NRC requirement states that 1.0% ALA is required, or 0.5–1.0% DHA. Although the growth performance data suggests that n-3 LC-PUFA synthesis was not sufficient in salmon fed the 0.25% treatment, we can't conclude that synthesis had not occurred. It is also important to note that growth rate is linked to metabolic activity, including intermediary metabolism such as LC-PUFA biosynthesis, therefore, differences in growth rate could affect production rates and the time required from hatch to harvest. However, growth rate is less likely to affect production harvest yield (per g), but rather the time required to achieve slaughter weight is longer (Sprague et al., 2019). While our study did not look at phospholipid content, supplementing dietary choline chloride is intended to provide the ‘building blocks’ necessary for phosphatidylcholine (PC), which often stores EPA and DHA. The role of phospholipids appears to be independent of fatty acid requirements, although the presence of an unsaturated fatty acid at the sn-2 position may be important (Tocher et al., 2008). However, not all lipid classes are equally effective in delivering essential fatty acids to fish. This is possibly linked to specific requirements for certain lipid classes, particularly phospholipids, and the requirement for phospholipid bases like choline (Sargent et al., 2002). Further, the EFA requirements of young fish and early developing larvae are mostly met by the phospholipid content. While our study may have determined that FM and FO can be lowered in the diet to meet the minimum EPA and DHA requirement, replacing FO with plant-based oils may be
problematic because they contain very little, if any, phospholipid in comparison to FO (Sargent et al., 2002). So, while the EPA and DHA requirement may be met with very low FO in the diet, possible phospholipid requirements may be a significant issue when using plantbased diets (Tocher et al., 2008). 5. Conclusion It is probably not optimal to estimate requirements based primarily on the growth performance and survival rate alone (Bou et al., 2017). Several additional aspects must be considered when evaluating nutritional requirements to foresee possible long-term health impacts related to EFA deficiencies. Based on growth performance and whole body fatty acid content, salmon fed a diet containing 0.50% EPA + DHA diet exhibited almost the same growth performance and health conditions with control diet (2%). Therefore, 0.5% is considered the lowest required EPA + DHA level for Atlantic salmon parr in freshwater in this study. The diet with 0.25% EPA + DHA showed lower weight gain and higher feed conversion compared with supplying over 0.50% EPA + DHA. The current inclusion of EPA and DHA via FO in commercial feed is about at least 2% of the diet, which means it could be reduced four-fold according to our study. This can reduce feed costs overall, whether it is FO or future commercial sources of EPA + DHA. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors would like to thank Dr. Suzanne Budge for use of the gas chromatography system; Margaret Hartling and Paul MacIsaac for technical assistance; Zeyu Zhang, Minmin Wei, and Angelisa Osmond for assistance in fish sampling; and Northeast Nutrition for supplying the feed ingredients used in the present study. This study was funded by S. Colombo's NSERC Discovery Grant (2018-05400). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.aquaculture.2019.734870. References Bou, M., Berge, G.M., Baeverfjord, G., Sigholt, T., Østbye, T.K., Romarheim, O.H., Ruyter, B., 2017. Requirements of n-3 very long-chain PUFA in Atlantic salmon (Salmo salar L): effects of different dietary levels of EPA and DHA on fish performance and tissue composition and integrity. Br. J. Nutr. 117 (1), 30–47. Christie, W.W., 2003. Preparation of derivates of fatty acids. In: Christie, W.W. (Ed.), Lipid Analysis: Isolation, Separation and Structural Analysis of Lipids, 3rd ed. J. Barnes and Associates, pp. 205–225. Colombo, S.M., Parrish, C.C., Wijekoon, M.P.A., 2018. Optimizing long chain-polyunsaturated fatty acid synthesis in salmonids by balancing dietary inputs. PLoS One 13 (10), e0205347. Crampton, V.O., Nanton, D.A., Ruohonen, K., Skjervold, P.O., El-Mowafi, A., 2010. Demonstration of salmon farming as a net producer of fish protein and oil. Aquac. Nutr. 16, 437–446. Dannevig, B.H., Falk, K., Krogsrud, J., 1993. Leucocytes from Atlantic salmon, Salmo salar L., experimentally infected with infectious salmon anaemia (ISA) exhibit an impaired response to mitogens. J. Fish Dis. 16 (4), 351–359. Emery, J.A., Norambuena, F., Trushenski, J., Turchini, G., 2016. Uncoupling EPA and DHA in fish nutrition: dietary demand is limited in Atlantic salmon and effectively met by DHA alone. Lipids. 51, 399–412. Evensen, O., Thorud, K.E., Olsen, Y.A., 1991. A morphological study of the gross and light microscopic lesions of infectious anaemia in Atlantic salmon (Salmo salar). Res. Vet. Sci. 51 (2), 215–222. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Food and Agriculture Organization (FAO), 2018. The State of the World Fisheries and
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