# Effect of Supplemental Dietaryα-linolenic Acid (18:3n-3) on the Growth Performance, Body Composition, and Fatty Acid Profile of Coho Salmon (Oncorhynchus kisutch) Alevins Cultured in Freshwater

Authors: Hairui Yu; Lingyao Li; Leyong Yu; Ling Zhang; Fanghui Li; Mengjie Guo; Jiayi Zhang; Jiyun Hou; Yijing Zhang
Journal: Aquaculture Research
 (2023)

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## Abstract

The present study evaluated the effects of supplemental dietaryα-linolenic acid (ALA, 18:3n-3) on growth performance, body composition, hepatic fat metabolizing enzymes, and fatty acid profiles of coho salmon (Oncorhynchus kisutch) alevins cultured in freshwater. Six isonitrogenous and isolipidic experimental diets were formulated to attain different levels of ALA (0.09, 0.41, 0.76, 1.03, 1.32, and 1.68% dry weight) by adding linseed oil in a basal diet, respectively. Each diet was fed to triplicate groups of alevins (50 fish in each tank) with initial mean body weight of 0.352 ± 0.002 g. Fish were reared in a freshwater flow-through rearing system and fed to apparent satiation 4 times daily for 12 weeks. The specific growth rate (SGR) increased with increasing dietary ALA level, which reached the peak at 1.03% ALA group (p<0.05). The final body weight (FBW) and SGR showed linear, quadratic, and cubic responses with the dietary ALA level. The whole-body crude protein increased with increasing dietary ALA level while lipid content was the opposite trend (p<0.05). However, the content of crude lipid in the whole-body of fish showed quadratic and cubic responses with the dietary ALA level. In addition, except for fatty acid synthase (FAS), the hepatic enzymes activities of lipoprotein lipase (LPL), hepatic lipase (HL), and malate dehydrogenase (MDH) showed linear, quadratic, and cubic response with the dietary ALA level (p<0.05) The activities of hepatic LPL and HL in 1.32% ALA group were significantly higher than other groups (p<0.05). The activities of hepatic MDH and FAS in 1.32% ALA group were the lowest, which were significantly lower than those in ALA groups (form 0.09% to1.03%) (p<0.05). Except for the content of MUFA, the contents of other fatty acids showed linear, quadratic, and cubic response with the dietary ALA level. The ALA and ∑n-3 PUFA content significantly increased with increasing dietary ALA levels (p<0.05). These results indicated that dietary ALA actively impacted fish growth performance, hepatic lipid metabolizing enzymes, and muscle fatty acid profile. The cubic regression analysis based on SGR as evaluation indices indicated that the optimal dietary ALA content was 1.33% dry weight of diet for alevins.


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## Body

## 1. Introduction

As the main component of fat, fatty acids (FA) are composed of a terminal carboxyl group and a long hydrocarbon chain. The FA can be divided into saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) [1, 2]. For fish, the essential fatty acids (EFAs) are recognized to comprise PUFA with carbon chain lengths of 18 and highly unsaturated fatty acids (HUFA) with carbon chain lengths of 20 and 22, both the n-3 and n-6 series [1]. The EFA cannot be synthesized by fish themselves and need to be obtained from feed to maintain growth and normal physiological functions. The n-3 and n-6 series of PUFA are important EFA for fish [3]. There are certain differences in the EFA requirements of fish due to differences in fish species and growth environments. Tocher and Dick [4] found that salinity had a great impact on the EFA requirements of fish and there was a big difference between freshwater fish and marine fish. The main reason is that different living environment, diet, and access to preformed dietary DHA leads to different synthesis of long-chain PUFA. Since freshwater fish could effectively convert α-linolenic acid (ALA, 18:3n-3) to docosahexaenoic acid (DHA, 22:6n-3), marine fish lack this ability to extend. ALA is a major EFA in freshwater fish [5, 6].PUFA plays important roles in maintaining the integrity of fish body cell structure and function, which regulates the activity of protein kinase C and the immune response, promotes the biological properties of growth factors, and also can reduce cholesterol and triglycerides [7–9]. ALA is the parent of the omega-3 series of FAs, which have 18 carbon atoms and three methylene discontinuous double bonds, starting at number 3 carbon at the omega end of the molecule (as opposed to the carboxyl group) [10]. It is important to promote animal growth, enhance immunity, improve meat quality, decrease blood lipids, and so on. Because n-3 fatty acids include ALA and DHA, which can be used to maintain the fluidity and permeability of biofilm at low temperature, it is generally believed that the requirement of n-3 fatty acids for cold-water fish is greater than that of n-6 fatty acids [11–14]. In addition, bioconversion of ALA is reported to account for 25% of total net intake in the diets of Atlantic salmon (Salmo Salar) that have completely replaced fish oil with camelina oil (ALA content of camelina oil is 30% total fatty acids) [13, 15].Coho salmon (Oncorhynchus kisutch) is an important aquaculture Pacific salmon species [16], which belongs to cold-water fish and higher economic and nutritional value. In recent years, farming of salmon has started to be promoted in China. Previous studies have shown that the EFA demand greatly varies depending on the fish species, growth stage, and environment of the fish, but most studies focus on juvenile and adult fish stages, and studies on EFAs in the early stage have not attracted enough attention [4, 17–19]. Currently, linoleic acid (18 : 2n-6, LA) and eicosapentaenoic acid (20:5n-3, EPA) are characterized as EFAs [20, 21], there is a lack of information about the ALA requirements for this species. Therefore, this study investigated the effects of dietary ALA levels on the growth, body composition, and fatty acid profile of coho salmon alevins. To determine the optimal requirement of ALA is also helpful to improve the database of nutrition parameters of this fish species.


## 2. Materials and Methods

### 2.1. Ethics Statement

The study was performed in strict accordance with the commendations of Care and Use of Laboratory Animals in China, Animal Ethical and Welfare Committee of China Experimental Animal Society. The experimental protocol and procedures were approved by the Institutional Animal Care and Use Committee of Weifang University (approval number 202104132) (Weifang, China).


### 2.2. Experimental Diets

Six experimental diets were prepared by supplementing with graded levels (0.00, 0.60, 1.20, 1.80, 2.40, and 3.00%) of linseed oil (Fatty acid profile: C16:0, 5.83%; C18:0, 2.91%; C18:1n-9, 21.12%; C18:2n-6, 13.18%; and C18:3n-3, 54.32%). The final concentration of ALA in diets was 0.09, 0.41, 0.76, 1.03, 1.32, and 1.68% (Tables1 and 2), respectively. The diets were made by using a double-screw extruder with a 2.0-mm-diameter pellet. No steam was used and the pellet temperature at diets was ranged from 90 to 100°C. And dried in a ventilated oven for 12 h at 50°C, ground and sieved into particles of 0.6–0.8 mm size, and stored in plastic bags at −20°C until used.Table 1
Formulation and proximate composition of the experimental diets forOncorhynchus kisutch alevins (% in dry matter).
IngredientsDietary ALA level (%)0.000.601.201.802.403.00Degreasing fish meal140.0040.0040.0040.0040.0040.00Soybean protein concentrate15.005.005.005.005.005.00Soybean meal120.0020.0020.0020.0020.0020.00Peanut meal19.809.809.809.809.809.80α-Starch113.8013.8013.8013.8013.8013.80Sodium alginate12.002.002.002.002.002.00Fish oil12.002.002.002.002.002.00Lard oil6.005.404.804.203.603.00Linseed oil20.000.601.201.802.403.00Mineral premix30.500.500.500.500.500.50Vitamin premix40.500.500.500.500.500.50Vitamin C phosphate0.050.500.500.500.500.50Choline chloride0.300.300.300.300.300.30Ethoxyquin0.050.050.050.050.050.05Proximate compositionMoisture (%)7.737.717.737.757.217.98Crude protein (%)46.9146.8046.8846.8346.9546.97Crude lipid (%)12.3012.8912.8712.7912.8212.45Ash (%)9.319.349.519.329.319.30Linolenic acid (%)0.090.410.761.031.321.681Provided by Shandong Conqueren Marine Technology Co., Ltd., Weifang, China. 2Linseed oil: ALA content, 50%. 3Composition (mg/kg mineral premix): AlK (SO4)2·12H2O, 123.7; CaCl2, 17879.8; CuSO4·5H2O, 31.7; CoCl2·6H2O, 48.9; FeSO4·7H2O, 707.4; MgSO4·7H2O, 4316.8; MnSO4·4H2O, 31.1; ZnSO4·7H2O, 176.7, KCl, 1191.9; KI, 5.3; NaCl, 4934.5; Na2SeO3·H2O, 3.4; Ca (H2PO4)2·H2O, 12457.0; KH2PO4, 9930.2. 4Composition (IU or g/kg vitamin premix): retinal palmitate, 10,000 IU; cholecalciferol, 4,000 IU; α-tocopherol, 75.0 IU; menadione, 22.0 g/kg; thiamine-HCl, 40.0 g/kg; riboflavin, 30.0 g/kg; D-calcium pantothenate, 150.0 g/kg; pyridoxine-HCl, 20.0 g/kg; meso-inositol, 500.0 g/kg; D-biotin, 1.0 g/kg; folic acid, 15.0 g/kg; ascorbic acid, 200.0 g/kg; niacin, 300.0 g/kg; cyanocobalamin, 0.3 g/kg.Table 2
Fatty acids composition of the experimental diets (% total fatty acids).
Fatty acidsDietary ALA level (%)0.090.410.761.031.321.68C14:00.360.340.320.300.270.26C16:011.1011.0511.0010.9210.7010.69C18:016.8112.779.736.633.480.43∑SFA28.2724.1621.0517.8514.4511.38C16:1n-70.340.360.400.420.410.44C18:1n-90.381.743.235.225.607.13∑MUFA0.722.13.635.646.017.57C18:3n-30.743.386.2610.1310.8713.83C20:5n-30.420.490.490.460.480.46C22:6n-30.450.510.540.560.580.57∑n-3 PUFA1.614.387.2911.1511.9314.56


### 2.3. Experimental Procedures

Feed-trained coho salmon alevins were obtained from one base of Shandong Collaborative Innovation Center of Coho Salmon Health Culture Engineering Technology, Linyi, China. A total of 900 coho salmon alevins (initial average weight 0.352 ± 0.002 g) were randomly assigned to 18 experimental fiberglass tanks (80 cm × 60 cm × 60 cm, water volume 240-L) connected to freshwater in a re-circulating aquaculture system. Each diet treatment contained triplicate tanks (50 fish/tank). The feeding management was followed by the previous report [22]. The alevins were fed four times a day at 7 : 30, 11 : 00, 14 : 30, and 18 : 00 for 12 weeks.


### 2.4. Sampling Procedures

At the end of the feeding trial, fish were fasted 24 hrs, and then anaesthetized (MS 222, 20 mg·L−1). Fish in each tank were netted out before being bulk weighed and counted. Nine fish were randomly from each tank stored in −20°C for chemical composition analyses. Six anesthetic fish were rapidly dissected, removed liver and muscle, and stored at −80°C until analysis of hepatic biochemical activities and muscle FA composition. Three additional anaesthetized fish were used to record fish body lengths and weights, and then dissected to excise liver and intestine for morphological indices. The liver samples were then frozen in liquid nitrogen and stored at −80°C for determining hepatic lipoprotein lipase (LPL), hepatic lipase (HL), malate dehydrogenase (MDH), and fatty acid synthase (FAS) activity.


### 2.5. Growth Performance

The calculation formulae for the indexes mentioned above are as follows:(1)SurvivalrateSR,%=finalnumberoffishinitialnumberoffish×100,SpecificgrowthrateSGR,%day=lnfinalbodyweight−lninitialbodyweightdays×100,ConditionfactorCF,gcm3=100×bodyweightbodylength3,HepatosomaticindexHSI,%=100×liverweightbodyweight,IntestinesomaticindexISI,%=100×intestineweightbodyweight.


### 2.6. Assays for Chemical Composition and Fatty Acid Profile

The proximate compositions of diets and tissue samples were measured according to AOAC [23]. The whole-body sample was freeze-dried for 48 h and ground in a mortar. Then, FAs were detected with a GC-MS chromatograph (Agilent Technologies 7890-5977A, USA) according to a previous method [24].


### 2.7. Assays for Testing Lipid Metabolism-Related Enzymes Activities

Liver samples were homogenized in 0.1 M pH 7.4 Tris-HCl buffers at 4°C. The supernatants were used for enzyme analysis. The activities of lipase, including LPL and HL activity, were also determined using a commercial kit by the procedures described by Dong et al. [25]. LPL and HL can decompose triglycerides (TG) and hydrolyze them into glycerol and free fatty acids (FFAs). LPL and HL activity can be determined by the amount of FFA produced with a copper reagent. HL belonging glycoprotein is not needed to be activated by apolipoprotein CII and some ions and is not inhibited by high-concentration salt and protamine. 1 μmol of free fatty acid produced by per mg of tissue protein per hour at 37.0°C in the reaction system, namely, is regarded as one unit of enzyme activity (FFA μmol/mg prot. hour). The reaction mixture was used for estimation of malate dehydrogenase (MDH) with different substrate as 1 mg oxaloacetate/mL of chilled triple distilled water [26]. The activities of FA synthetase (FAS) were assayed with the commercial kits purchased from Zhuocai biology Co., Ltd. (Shanghai, China) [27].


### 2.8. Statistical Analysis

Results are presented as mean ± SD. The normality and homogeneity of variances among groups were tested and results were subjected to one-way analysis of variance (ANOVA), Duncan multiple comparison was used for the significantly different groups. With ALA level as independent variable, linear, quadratic, and cubic regression analyses were performed on the responses data. Difference was considered significant atp<0.05. All the result data was performed using SPSS version 20.0 software (SPSS Inc., USA).


## 2.1. Ethics Statement

The study was performed in strict accordance with the commendations of Care and Use of Laboratory Animals in China, Animal Ethical and Welfare Committee of China Experimental Animal Society. The experimental protocol and procedures were approved by the Institutional Animal Care and Use Committee of Weifang University (approval number 202104132) (Weifang, China).


## 2.2. Experimental Diets

Six experimental diets were prepared by supplementing with graded levels (0.00, 0.60, 1.20, 1.80, 2.40, and 3.00%) of linseed oil (Fatty acid profile: C16:0, 5.83%; C18:0, 2.91%; C18:1n-9, 21.12%; C18:2n-6, 13.18%; and C18:3n-3, 54.32%). The final concentration of ALA in diets was 0.09, 0.41, 0.76, 1.03, 1.32, and 1.68% (Tables1 and 2), respectively. The diets were made by using a double-screw extruder with a 2.0-mm-diameter pellet. No steam was used and the pellet temperature at diets was ranged from 90 to 100°C. And dried in a ventilated oven for 12 h at 50°C, ground and sieved into particles of 0.6–0.8 mm size, and stored in plastic bags at −20°C until used.Table 1
Formulation and proximate composition of the experimental diets forOncorhynchus kisutch alevins (% in dry matter).
IngredientsDietary ALA level (%)0.000.601.201.802.403.00Degreasing fish meal140.0040.0040.0040.0040.0040.00Soybean protein concentrate15.005.005.005.005.005.00Soybean meal120.0020.0020.0020.0020.0020.00Peanut meal19.809.809.809.809.809.80α-Starch113.8013.8013.8013.8013.8013.80Sodium alginate12.002.002.002.002.002.00Fish oil12.002.002.002.002.002.00Lard oil6.005.404.804.203.603.00Linseed oil20.000.601.201.802.403.00Mineral premix30.500.500.500.500.500.50Vitamin premix40.500.500.500.500.500.50Vitamin C phosphate0.050.500.500.500.500.50Choline chloride0.300.300.300.300.300.30Ethoxyquin0.050.050.050.050.050.05Proximate compositionMoisture (%)7.737.717.737.757.217.98Crude protein (%)46.9146.8046.8846.8346.9546.97Crude lipid (%)12.3012.8912.8712.7912.8212.45Ash (%)9.319.349.519.329.319.30Linolenic acid (%)0.090.410.761.031.321.681Provided by Shandong Conqueren Marine Technology Co., Ltd., Weifang, China. 2Linseed oil: ALA content, 50%. 3Composition (mg/kg mineral premix): AlK (SO4)2·12H2O, 123.7; CaCl2, 17879.8; CuSO4·5H2O, 31.7; CoCl2·6H2O, 48.9; FeSO4·7H2O, 707.4; MgSO4·7H2O, 4316.8; MnSO4·4H2O, 31.1; ZnSO4·7H2O, 176.7, KCl, 1191.9; KI, 5.3; NaCl, 4934.5; Na2SeO3·H2O, 3.4; Ca (H2PO4)2·H2O, 12457.0; KH2PO4, 9930.2. 4Composition (IU or g/kg vitamin premix): retinal palmitate, 10,000 IU; cholecalciferol, 4,000 IU; α-tocopherol, 75.0 IU; menadione, 22.0 g/kg; thiamine-HCl, 40.0 g/kg; riboflavin, 30.0 g/kg; D-calcium pantothenate, 150.0 g/kg; pyridoxine-HCl, 20.0 g/kg; meso-inositol, 500.0 g/kg; D-biotin, 1.0 g/kg; folic acid, 15.0 g/kg; ascorbic acid, 200.0 g/kg; niacin, 300.0 g/kg; cyanocobalamin, 0.3 g/kg.Table 2
Fatty acids composition of the experimental diets (% total fatty acids).
Fatty acidsDietary ALA level (%)0.090.410.761.031.321.68C14:00.360.340.320.300.270.26C16:011.1011.0511.0010.9210.7010.69C18:016.8112.779.736.633.480.43∑SFA28.2724.1621.0517.8514.4511.38C16:1n-70.340.360.400.420.410.44C18:1n-90.381.743.235.225.607.13∑MUFA0.722.13.635.646.017.57C18:3n-30.743.386.2610.1310.8713.83C20:5n-30.420.490.490.460.480.46C22:6n-30.450.510.540.560.580.57∑n-3 PUFA1.614.387.2911.1511.9314.56


## 2.3. Experimental Procedures

Feed-trained coho salmon alevins were obtained from one base of Shandong Collaborative Innovation Center of Coho Salmon Health Culture Engineering Technology, Linyi, China. A total of 900 coho salmon alevins (initial average weight 0.352 ± 0.002 g) were randomly assigned to 18 experimental fiberglass tanks (80 cm × 60 cm × 60 cm, water volume 240-L) connected to freshwater in a re-circulating aquaculture system. Each diet treatment contained triplicate tanks (50 fish/tank). The feeding management was followed by the previous report [22]. The alevins were fed four times a day at 7 : 30, 11 : 00, 14 : 30, and 18 : 00 for 12 weeks.


## 2.4. Sampling Procedures

At the end of the feeding trial, fish were fasted 24 hrs, and then anaesthetized (MS 222, 20 mg·L−1). Fish in each tank were netted out before being bulk weighed and counted. Nine fish were randomly from each tank stored in −20°C for chemical composition analyses. Six anesthetic fish were rapidly dissected, removed liver and muscle, and stored at −80°C until analysis of hepatic biochemical activities and muscle FA composition. Three additional anaesthetized fish were used to record fish body lengths and weights, and then dissected to excise liver and intestine for morphological indices. The liver samples were then frozen in liquid nitrogen and stored at −80°C for determining hepatic lipoprotein lipase (LPL), hepatic lipase (HL), malate dehydrogenase (MDH), and fatty acid synthase (FAS) activity.


## 2.5. Growth Performance

The calculation formulae for the indexes mentioned above are as follows:(1)SurvivalrateSR,%=finalnumberoffishinitialnumberoffish×100,SpecificgrowthrateSGR,%day=lnfinalbodyweight−lninitialbodyweightdays×100,ConditionfactorCF,gcm3=100×bodyweightbodylength3,HepatosomaticindexHSI,%=100×liverweightbodyweight,IntestinesomaticindexISI,%=100×intestineweightbodyweight.


## 2.6. Assays for Chemical Composition and Fatty Acid Profile

The proximate compositions of diets and tissue samples were measured according to AOAC [23]. The whole-body sample was freeze-dried for 48 h and ground in a mortar. Then, FAs were detected with a GC-MS chromatograph (Agilent Technologies 7890-5977A, USA) according to a previous method [24].


## 2.7. Assays for Testing Lipid Metabolism-Related Enzymes Activities

Liver samples were homogenized in 0.1 M pH 7.4 Tris-HCl buffers at 4°C. The supernatants were used for enzyme analysis. The activities of lipase, including LPL and HL activity, were also determined using a commercial kit by the procedures described by Dong et al. [25]. LPL and HL can decompose triglycerides (TG) and hydrolyze them into glycerol and free fatty acids (FFAs). LPL and HL activity can be determined by the amount of FFA produced with a copper reagent. HL belonging glycoprotein is not needed to be activated by apolipoprotein CII and some ions and is not inhibited by high-concentration salt and protamine. 1 μmol of free fatty acid produced by per mg of tissue protein per hour at 37.0°C in the reaction system, namely, is regarded as one unit of enzyme activity (FFA μmol/mg prot. hour). The reaction mixture was used for estimation of malate dehydrogenase (MDH) with different substrate as 1 mg oxaloacetate/mL of chilled triple distilled water [26]. The activities of FA synthetase (FAS) were assayed with the commercial kits purchased from Zhuocai biology Co., Ltd. (Shanghai, China) [27].


## 2.8. Statistical Analysis

Results are presented as mean ± SD. The normality and homogeneity of variances among groups were tested and results were subjected to one-way analysis of variance (ANOVA), Duncan multiple comparison was used for the significantly different groups. With ALA level as independent variable, linear, quadratic, and cubic regression analyses were performed on the responses data. Difference was considered significant atp<0.05. All the result data was performed using SPSS version 20.0 software (SPSS Inc., USA).


## 3. Results

### 3.1. Survival and Growth Performance

No significant differences were observed for SR among the dietary groups based on the linear analysis (p>0.05), but which showed quadratic and cubic responses with the dietary ALA level (Table 3). The CF, HSI, and ISI of fish were not significantly affected by the dietary ALA level (p>0.05). The final body weight (FBW) and SGR showed linear, quadratic, and cubic responses with the dietary ALA level. With the increase of the dietary ALA content, SGR increased significantly, the fish fed diet with 1.32% ALA level had the highest SGR, which was significantly higher than that of the 0.09% ALA group (p<0.05), but did not differ significantly from 0.41% to 1.68% ALA groups (p>0.05). Based on the cubic regression analysis, the optimal dietary ALA level for maximum SGR was 1.33% (Y = −0.048X3–0.016X2 + 0.2962 + 2.8939, R2 = 1.0000) (Figure 1).Table 3
Survival and growth performance and feed utilization ofOncorhynchus kisutch alevins fed the experimental diets with different dietary ALA level for 12 weeks (means ± SD, n = 3).
Dietary ALA level (%)SEMRegression analysis (P,R2)0.090.410.761.031.321.68LinearQuadraticCubicSR98.4398.5798.7898.8998.8098.500.0360.252, 0.0590.000, 0.7980.000, 0.930IBW (g)0.3510.3520.3510.3530.3520.3510.0000.303, 0.0480.559, 0.0540.762, 0.055FBW (g)4.08a4.41ab4.70ab4.89b4.95b4.81b0.0890.001, 0.3790.001, 0.4910.003, 0.496SGR (%/day)2.92a3.01ab3.09ab3.13b3.15b3.12b0.0230.001, 0.4090.000, 0.5360.001, 0.538CF (g/cm3)1.181.141.091.161.091.100.0360.536, 0.0390.814, 0.0450.934, 0.050HSI (%)1.141.041.421.391.241.030.0580.971, 0.0000.117, 0.3790.156, 0.461ISI (%)1.611.221.601.501.471.660.0570.442, 0.0600.445, 0.1640.567, 0.213Means in the same raw with different superscript letters are significantly different (p<0.05).Figure 1
Relationship between specific growth rate (SGR) and dietaryα-linolenic acid (ALA) levels based on the cubic regression analysis showed the predicted dietary ALA requirement was 1.33% for Oncorhynchus kisutch alevins.


### 3.2. Proximate Composition and Fatty Acid Profile

The whole-body moisture and ash contents did not differ significantly among dietary groups (p>0.05) (Table 4). The whole-body crude protein significantly increased with increasing levels of dietary ALA from 0.09% to 1.68% (p<0.05). The content of crude lipid in the whole-body of fish showed quadratic and cubic responses with the dietary ALA level. Compared to the dietary 0.09% ALA level, the ALA levels of 0.41, 0.76, 1.03, 1.32, and 1.68% ALA levels decreased the content of crude lipid in the whole-body of fish; however, there was no significant difference between 0.09% and 1.32% ALA level groups (p>0.05).Table 4
Whole-body composition ofOncorhynchus kisutch alevins fed the experimental diets with different dietary ALA level for 12 weeks (means ± SD, n = 3).
Dietary ALA level (%)SEMRegression analysis (P,R2)0.090.410.761.031.321.68LinearQuadraticCubicMoisture (%)77.8577.3677.3777.3977.3977.310.0770.095, 0.2530.129, 0.3660.126, 0.493Crude protein (%)12.23a12.63a12.70a13.79b13.99b14.11b0.2320.264, 0.1230.419, 0.1760.510, 0.239Crude lipid (%)6.55b6.48b6.38ab5.18ab5.07ab4.86a0.2510.451, 0.0580.015, 0.6090.016, 0.708Ash (%)3.273.233.243.253.223.270.0090.053, 0.3240.170, 0.3250.180, 0.439Means in the same column with different superscript letters are significantly different (p<0.05).Except for the content of MUFA, the contents of other fatty acids showed linear, quadratic, and cubic response with the dietary ALA level. The content of C14:0, C16:0, and C18:0 fatty acids were significantly decreased, but that of C18:3n-3 was significantly increased with increasing dietary ALA levels. Compared to the fish fed 0.09% ALA level, the fish fed ALA diets supplemented resulted in a decrease of SFA and MUFA in whole-body fish (p<0.05, Table 5). Fish fed diet with 1.32% ALA level had the lowest SFA and MUFA contents. The C18:3n-3 and n-3 PUFA in the whole-body were higher in fish fed ALA diets than those in fish fed 0.09% ALA level (p<0.05). The contents of EPA and DHA in the whole body were significantly decreased with increasing dietary ALA, and got the lowest with increasing dietary ALA level to 1.32%.Table 5
Effects of different dietary ALA levels on fatty acid composition ofOncorhynchus kisutch alevins (Mean ± SD).
Dietary ALA level (%)SEMRegression analysis (P,R2)0.090.410.761.031.321.68LinearQuadraticCubicC14:01.97c1.85b1.77b1.58a1.51a1.57a0.0500.000, 0.8630.000, 0.9280.000, 0.976C16:018.49c17.81bc17.55b15.99a15.62a15.63a0.3440.000, 0.8980.000, 0.9120.000, 0.955C18:05.06d4.87c4.41b3.14a3.10a3.13a0.2550.000, 0.8480.000, 0.8680.000, 0.954∑SFA25.52c24.53b23.73b20.71a20.23a20.33a0.6490.000, 0.8840.000, 0.9030.000, 0.961C16:1n-76.16d5.18c5.54c4.55b4.18a4.34ab0.2110.000, 0.7870.000, 0.8230.002, 0.829C18:1n-913.30d13.20d12.86c10.97b9.77a10.84b0.4050.000, 0.7380.002, 0.7450.000, 0.947∑MUFA19.46e18.38d18.4c15.52b13.95a15.18b1.2220.035, 0.3730.106, 0.3920.062, 0.581C18:3n-30.40a3.46b5.60c9.34d9.47d9.60d1.0560.000, 0.8870.000, 0.9670.000, 0.979C20:5n-3 (EPA)1.14d0.98c0.90bc0.90bc0.75a0.81ab0.0360.000, 0.7970.000, 0.8850.000, 0.886C22:6n-3 (DHA)2.80d2.46c2.45c2.25b1.92a2.22b0.0820.001, 0.6930.000, 0.8220.001, 0.859∑n-3 PUFA4.34a6.90b8.95c12.14d12.49e12.63e0.9470.000, 0.8830.000, 0.9600.000, 0.970Means in the same column with different superscript letters are significantly different (p<0.05).


### 3.3. Lipid Metabolism-Related Enzymes Activities

The hepatic enzymes activities of LPL and HL showed linear, quadratic, and cubic response with the dietary ALA level (p<0.05), which significantly increased as dietary ALA level increased from 0.09% to 1.32%, and then showed a significant decrease trend when ALA level further increased to 1.68% (p<0.05) (Table 6, Figures 2(a) and 2(b)). However, compared to the 0.09% ALA group, the hepatic LPL and HL activities of the 1.68% ALA group were significantly increased (p<0.05), the highest value were found in 1.32% group. Similarly, the hepatic enzymes activity of MDH showed linear, quadratic, and cubic response (p<0.05), however, which decreased significantly with the increased of ALA levels (p<0.05) (Table 6, Figure 2(c)). The hepatic enzymes activity of FAS decreased with the increase of dietary ALA level, and the lowest value was found in 1.68% group (p<0.05) (Table 6, Figure 2(d)).Table 6
Effects of different dietary ALA levels on the activity of LPL, HL, MDH, and FAS in liver ofOncorhynchus kisutch alevins (Mean ± SD).
Dietary ALA level (%)SEMRegression analysis (P, R2)0.090.410.761.031.321.68LinearQuadraticCubicLPL (U/mgprot.)0.95a1.01a1.14b1.41c1.69d1.16b0.0540.000, 0.4320.002, 0.4560.004, 0.476HL (U/mgprot.)0.96a1.01a1.59b1.86c2.73d1.50b0.1240.000, 0.5790.000, 0.7160.000, 0.884MDH (U/mgprot.)1.27c1.26c1.13b1.10b0.73a0.76a0.0460.000, 0.7230.000, 0.7710.000, 0.792FAS (U/mgprot.)2.893b2.892b2.892b2.891b2.865a2.850a0.0050.867, 0.0030.060, 0.4640.147, 0.470Means in the same column with different superscript letters are significantly different (p<0.05).Figure 2
The activity of LPL, HL, MDH, and FAS in liver ofOncorhynchus kisutch alevins fed the experimental diets with graded dietary ALA levels after 12 weeks. (a) LPL; (b) HL; (c) MDH; (d) FAS.
(a)(b)(c)(d)


## 3.1. Survival and Growth Performance

No significant differences were observed for SR among the dietary groups based on the linear analysis (p>0.05), but which showed quadratic and cubic responses with the dietary ALA level (Table 3). The CF, HSI, and ISI of fish were not significantly affected by the dietary ALA level (p>0.05). The final body weight (FBW) and SGR showed linear, quadratic, and cubic responses with the dietary ALA level. With the increase of the dietary ALA content, SGR increased significantly, the fish fed diet with 1.32% ALA level had the highest SGR, which was significantly higher than that of the 0.09% ALA group (p<0.05), but did not differ significantly from 0.41% to 1.68% ALA groups (p>0.05). Based on the cubic regression analysis, the optimal dietary ALA level for maximum SGR was 1.33% (Y = −0.048X3–0.016X2 + 0.2962 + 2.8939, R2 = 1.0000) (Figure 1).Table 3
Survival and growth performance and feed utilization ofOncorhynchus kisutch alevins fed the experimental diets with different dietary ALA level for 12 weeks (means ± SD, n = 3).
Dietary ALA level (%)SEMRegression analysis (P,R2)0.090.410.761.031.321.68LinearQuadraticCubicSR98.4398.5798.7898.8998.8098.500.0360.252, 0.0590.000, 0.7980.000, 0.930IBW (g)0.3510.3520.3510.3530.3520.3510.0000.303, 0.0480.559, 0.0540.762, 0.055FBW (g)4.08a4.41ab4.70ab4.89b4.95b4.81b0.0890.001, 0.3790.001, 0.4910.003, 0.496SGR (%/day)2.92a3.01ab3.09ab3.13b3.15b3.12b0.0230.001, 0.4090.000, 0.5360.001, 0.538CF (g/cm3)1.181.141.091.161.091.100.0360.536, 0.0390.814, 0.0450.934, 0.050HSI (%)1.141.041.421.391.241.030.0580.971, 0.0000.117, 0.3790.156, 0.461ISI (%)1.611.221.601.501.471.660.0570.442, 0.0600.445, 0.1640.567, 0.213Means in the same raw with different superscript letters are significantly different (p<0.05).Figure 1
Relationship between specific growth rate (SGR) and dietaryα-linolenic acid (ALA) levels based on the cubic regression analysis showed the predicted dietary ALA requirement was 1.33% for Oncorhynchus kisutch alevins.


## 3.2. Proximate Composition and Fatty Acid Profile

The whole-body moisture and ash contents did not differ significantly among dietary groups (p>0.05) (Table 4). The whole-body crude protein significantly increased with increasing levels of dietary ALA from 0.09% to 1.68% (p<0.05). The content of crude lipid in the whole-body of fish showed quadratic and cubic responses with the dietary ALA level. Compared to the dietary 0.09% ALA level, the ALA levels of 0.41, 0.76, 1.03, 1.32, and 1.68% ALA levels decreased the content of crude lipid in the whole-body of fish; however, there was no significant difference between 0.09% and 1.32% ALA level groups (p>0.05).Table 4
Whole-body composition ofOncorhynchus kisutch alevins fed the experimental diets with different dietary ALA level for 12 weeks (means ± SD, n = 3).
Dietary ALA level (%)SEMRegression analysis (P,R2)0.090.410.761.031.321.68LinearQuadraticCubicMoisture (%)77.8577.3677.3777.3977.3977.310.0770.095, 0.2530.129, 0.3660.126, 0.493Crude protein (%)12.23a12.63a12.70a13.79b13.99b14.11b0.2320.264, 0.1230.419, 0.1760.510, 0.239Crude lipid (%)6.55b6.48b6.38ab5.18ab5.07ab4.86a0.2510.451, 0.0580.015, 0.6090.016, 0.708Ash (%)3.273.233.243.253.223.270.0090.053, 0.3240.170, 0.3250.180, 0.439Means in the same column with different superscript letters are significantly different (p<0.05).Except for the content of MUFA, the contents of other fatty acids showed linear, quadratic, and cubic response with the dietary ALA level. The content of C14:0, C16:0, and C18:0 fatty acids were significantly decreased, but that of C18:3n-3 was significantly increased with increasing dietary ALA levels. Compared to the fish fed 0.09% ALA level, the fish fed ALA diets supplemented resulted in a decrease of SFA and MUFA in whole-body fish (p<0.05, Table 5). Fish fed diet with 1.32% ALA level had the lowest SFA and MUFA contents. The C18:3n-3 and n-3 PUFA in the whole-body were higher in fish fed ALA diets than those in fish fed 0.09% ALA level (p<0.05). The contents of EPA and DHA in the whole body were significantly decreased with increasing dietary ALA, and got the lowest with increasing dietary ALA level to 1.32%.Table 5
Effects of different dietary ALA levels on fatty acid composition ofOncorhynchus kisutch alevins (Mean ± SD).
Dietary ALA level (%)SEMRegression analysis (P,R2)0.090.410.761.031.321.68LinearQuadraticCubicC14:01.97c1.85b1.77b1.58a1.51a1.57a0.0500.000, 0.8630.000, 0.9280.000, 0.976C16:018.49c17.81bc17.55b15.99a15.62a15.63a0.3440.000, 0.8980.000, 0.9120.000, 0.955C18:05.06d4.87c4.41b3.14a3.10a3.13a0.2550.000, 0.8480.000, 0.8680.000, 0.954∑SFA25.52c24.53b23.73b20.71a20.23a20.33a0.6490.000, 0.8840.000, 0.9030.000, 0.961C16:1n-76.16d5.18c5.54c4.55b4.18a4.34ab0.2110.000, 0.7870.000, 0.8230.002, 0.829C18:1n-913.30d13.20d12.86c10.97b9.77a10.84b0.4050.000, 0.7380.002, 0.7450.000, 0.947∑MUFA19.46e18.38d18.4c15.52b13.95a15.18b1.2220.035, 0.3730.106, 0.3920.062, 0.581C18:3n-30.40a3.46b5.60c9.34d9.47d9.60d1.0560.000, 0.8870.000, 0.9670.000, 0.979C20:5n-3 (EPA)1.14d0.98c0.90bc0.90bc0.75a0.81ab0.0360.000, 0.7970.000, 0.8850.000, 0.886C22:6n-3 (DHA)2.80d2.46c2.45c2.25b1.92a2.22b0.0820.001, 0.6930.000, 0.8220.001, 0.859∑n-3 PUFA4.34a6.90b8.95c12.14d12.49e12.63e0.9470.000, 0.8830.000, 0.9600.000, 0.970Means in the same column with different superscript letters are significantly different (p<0.05).


## 3.3. Lipid Metabolism-Related Enzymes Activities

The hepatic enzymes activities of LPL and HL showed linear, quadratic, and cubic response with the dietary ALA level (p<0.05), which significantly increased as dietary ALA level increased from 0.09% to 1.32%, and then showed a significant decrease trend when ALA level further increased to 1.68% (p<0.05) (Table 6, Figures 2(a) and 2(b)). However, compared to the 0.09% ALA group, the hepatic LPL and HL activities of the 1.68% ALA group were significantly increased (p<0.05), the highest value were found in 1.32% group. Similarly, the hepatic enzymes activity of MDH showed linear, quadratic, and cubic response (p<0.05), however, which decreased significantly with the increased of ALA levels (p<0.05) (Table 6, Figure 2(c)). The hepatic enzymes activity of FAS decreased with the increase of dietary ALA level, and the lowest value was found in 1.68% group (p<0.05) (Table 6, Figure 2(d)).Table 6
Effects of different dietary ALA levels on the activity of LPL, HL, MDH, and FAS in liver ofOncorhynchus kisutch alevins (Mean ± SD).
Dietary ALA level (%)SEMRegression analysis (P, R2)0.090.410.761.031.321.68LinearQuadraticCubicLPL (U/mgprot.)0.95a1.01a1.14b1.41c1.69d1.16b0.0540.000, 0.4320.002, 0.4560.004, 0.476HL (U/mgprot.)0.96a1.01a1.59b1.86c2.73d1.50b0.1240.000, 0.5790.000, 0.7160.000, 0.884MDH (U/mgprot.)1.27c1.26c1.13b1.10b0.73a0.76a0.0460.000, 0.7230.000, 0.7710.000, 0.792FAS (U/mgprot.)2.893b2.892b2.892b2.891b2.865a2.850a0.0050.867, 0.0030.060, 0.4640.147, 0.470Means in the same column with different superscript letters are significantly different (p<0.05).Figure 2
The activity of LPL, HL, MDH, and FAS in liver ofOncorhynchus kisutch alevins fed the experimental diets with graded dietary ALA levels after 12 weeks. (a) LPL; (b) HL; (c) MDH; (d) FAS.
(a)(b)(c)(d)


## 4. Discussion

ALA has an important physiological function, since it is an EFA for freshwater fish. As one of the best vegetable oil sources, linseed oil contains a large amount of ALA [28, 29]. The decomposition of ALA provides energy for the growth of organisms. The result showed that growth performance of alevins was greatly affected by the dietary levels of ALA and could be enhanced remarkably by the diets rich in ALA, which was in agreement with previous studies in European sea bass [30], Japanese seabass (Lateolabrax japonicus) [31], rainbow trout [32], common carp [33], and Atlantic salmon [34, 35]. In contrast, Kanazawa et al. [36] reported that no growth improvement was found in tilapia fed a diet rich in ALA. Lacking or excess ALA in diet would impact the normal growth and development in fish. With the lacking of dietary ALA content, the feed utilization rate and SGR of yellow catfish was significantly reduced [37]. Gordon Bell et al. [38] found that 19% linseed oil supplementation in feed inhibited the growth of the juvenile turbot. It can be inferred from that adding appropriate ALA could promote the growth of fish. In addition, the differences in fish species, specifications, and breeding environment make different fish have different requirements for ALA. Fish feeding with nutritionally unbalanced diets often have abdominal swelling and liver hypertrophy, and the edible part is relatively reduced. In this experiment, the level of ALA had no significant effect on CF, HSI, and ISI. The results showed that adding ALA did not cause hepatic hypertrophy and change the form of the fish.The studies have shown that the composition of feed has a significant effect on the chemical composition of fish [39, 40]. Different dietary ALA levels affected the whole-body protein and lipid contents of Atlantic salmon [41] and Japanese seabass [31]. In the present study, with the increase of dietary ALA content, the whole-body crude protein contents increased while the crude lipid contents decreased (quadratic and cubic responses), which indicated that the increase of dietary ALA content might decrease the whole-body lipid deposition of alevins. It is speculated that different ALA levels have effects on the expression of fatty acid synthesis and oxidation-related genes, thereby having an effect on the whole-body chemical composition. The present study showed that different ALA levels did not affect moisture and ash contents, whose result is similar to that of reported by Chen et al. [42] in Nile tilapia (Oreochromis niloticus).It has also been reported that the activities of lipid metabolism-related enzymes including LPL, HL, and FAS are affected by HUFAs [43–46]. Current research indicated that low dietary ALA level significantly reduced whole-body lipid contents, which might be due to promoting the lipolysis in fish with increasing LPL and HL activities (linear, quadratic, and cubic response with the dietary ALA level). The LPL and HL activities were the highest with 1.32% ALA group, indicating that lipid utilization reached its peak at the point. However, the reduction in LPL and HL activities with dietary ALA level (>1.32% or <1.32%) might be associated with a form of liver protection, limiting lipid uptake from plasma lipoproteins. Nevertheless, Arantza-mendi [47] found that complete substitution of dietary linseed oil for fish oil did not have an effect on the LPL activity in perivisceral adipose tissue, muscle, or liver in rainbow trout. From the above, dietary ALA deficiency can affect lipid metabolism and related enzyme activities of fish. Similarly, MDH is the key enzyme for lipogenesis in the liver [48]. However, MDH activity (linear, quadratic, and cubic response) showed an opposite pattern of change compared with LPL activity in this study. Evidence suggests that MDH activity of juvenile grass carp (Ctenopharyngodon idellus) decreased with the increasing dietary n-3 HUFAs levels [49]. FAS enzyme is the key rate-limiting enzyme for fatty acid synthesis. This activity is of great significance for fat synthesis and fat deposition in animal body [50]. The brain is the main site for FAS transcription, followed by the kidney and liver. Studies have shown that the content of PUFA increases would inhibit FAS activity [51, 52]. In this experiment, it showed that the hepatic FAS enzyme activity decreased with the ALA content increased, which was the same as reported by Huang et al. [53] in spotted seabass (Lateolabrax maculatus).The composition of dietary fatty acid directly affected the fatty acid composition of fish, which has been proved by many studies [31, 54, 55]. With the increase of dietary ALA level, muscle ALA content increased while the content of SFA and MUFA decreased. The content of ∑n-3PUFA increased in the muscle with the increased of the dietary ALA level. Similar results was also found in Nile tilapia, whereas the fish fed with flax seed oil had higher ∑n-3 fatty acid content than those fed with alone sunflower seed oil in Nile tilapia [56]. The muscle DHA and EPA showed downward with the increase of dietary ALA content, and the highest content obtained in 0.09% ALA content. The reason may be inferred that higher dietary ALA contents caused the increase of oxidative decomposition DHA and EPA in the muscle.


## 5. Conclusions

In conclusion, supplement of ALA could increase growth performance and the hepatic HL and LPL activities, and decreased the FAS and MDH activities in coho salmon alevins. Based on the results analysis of SGR, the optimal dietary ALA level for coho salmon alevins was estimated to be 1.33%.



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*Source: 4869006-2023-02-07.xml*