Effects of replacing fish oil with wheat germ oil on growth, fat deposition, serum biochemical indices and lipid metabolic enzyme of juvenile hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂)

Aquaculture 505 (2019) 54–62

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Effects of replacing fish oil with wheat germ oil on growth, fat deposition, serum biochemical indices and lipid metabolic enzyme of juvenile hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂)

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Li Baoshan, Wang Jiying , Huang Yu, Hao Tiantian, Wang Shixin, Huang BingShan, Sun Yongzhi Key Laboratory of Marine Ecological Restoration, Shandong Marine Resources and Environment Research Institute, Yantai 264006, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Hybrid grouper Fish oil Wheat germ oil Replacement Growth Lipid metabolism

Fish oil was one of most important feed ingredients for carnivorous marine fish species, but the production might not cover all the necessary quantity for aquaculture. Meanwhile wheat germ oil could be a potential alternative for fish oil in aquatic feed. Hence, a 70 day feeding trial was conducted to investigate the effects of dietary fish oil replaced by wheat germ oil (WGO) on the growth, lipid and fatty acids deposition, and lipid metabolic enzymes activities of juvenile hybrid grouper (initial weight 55.31 ± 0.68 g), E. fuscoguttatus♀ × E. lanceolatus♂. Six isonitrogenous and isoenergetic experimental diets were formulated with 0%, 20%, 40%, 60%, 80% and 100% replacement of fish oil by WGO. Survival rate, weight gain rate, specific growth rate, and feed conversion ratio were similar among fish fed with different diets. Viserosomatic index and mesenteric fat ratio were significantly increased by dietary WGO. No significant differences were observed in the compositions of whole body, muscle and liver. C18: 3n-3 and C18:2n-6 contents in tissues were significantly increased, but EPA and DHA were significantly decreased by dietary WGO. No significant differences were found on the activities of hepatopancreas lipoprotein lipase, hepatic lipase and fatty acid synthase, while the activity of intestinal lipase was decreased. Total cholesterol, high density lipoprotein cholesterol, and low density lipoprotein cholesterol in plasma were not affected, but triglyceride was increased by dietary WGO. It can be concluded from the results that about 71.8% dietary fish oil can be replaced by WGO in diets with 50% crude protein and 12% crude lipid for subadult hybrid grouper, and fatty acids deposition and biosynthesis of this species was quite different from that of other fish species.

1. Introduction Fish oil is one of most important feed ingredients for aquatic animals, particularly for marine carnivorous fish. With the fast development of fish culture industry, fish oil production might not cover all the necessary quantity for aquaculture (Kaushik, 2004; Tacon, 2005). Fish oil substitution has come to a hot topic in the field of aquatic animal nutrition. In recent years, vegetable oils became one of the best alternatives to fish oil because of its steadily increasing production, high availability and better economic value. A lot of researches have been reported that fish oil can be partial or total replaced by soybean oil (Yu et al., 2017; Peng et al., 2008), linseed oil (Bell et al., 2004; Liu et al., 2016), palm oil (Huang et al., 2016), algae oil (Hixson et al., 2013), and vegetable oil blend (Aeo et al., 2007) in different fish species (Nasopoulou and Zabetakis, 2012). Wheat germ oil (WGO) is one of byproducts of wheat procession. It is rich in unsaturated fatty acids

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(oleic acid, linoleic acid and linolenic acid) and physiological active components, such as vitamin E and octacosanol. WGO has been attributed to reducing plasma and liver cholesterol, improving physical endurance, and delaying aging (Kahlon, 1989). It has been reported that WGO can be used as a fertility agent, an antioxidant, and an additive in natural food and health and cosmetic products (Wang and Lawrence, 2001). The world wheat production reached 749 million tons in 2018 (FAO), and theoretically producing 749 thousand tons of wheat germ oil (He et al., 2006). It may be a potential alternative for fish oil in aquatic feed. Huang et al. (2015) has reported that WGO can replace up to 60% dietary fish oil of Cynoglossus semilaevis Günter. Hybrid grouper (E.fuscoguttatus♀ × E. lanceolatus♂) is one of newly cultured species in China and south-east Asia. It owns the advantages of growing fast from giant grouper (E. lanceolatus) and diseases resistance from brown-marbled grouper (E. fuscoguttatus) (Yashiro, 2008; Sugama et al., 2008). It has been reported that the optimal dietary lipid level for

Corresponding author. E-mail address: [email protected] (W. Jiying).

https://doi.org/10.1016/j.aquaculture.2019.02.037 Received 15 October 2018; Received in revised form 12 February 2019; Accepted 16 February 2019 Available online 18 February 2019 0044-8486/ © 2019 Published by Elsevier B.V.

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Table 1 Composition and nutrient levels of experimental diets (g kg−1).

Table 2 Fatty acids composition of fish oil, wheat germ oil and diets (% total fatty acid).

D0

D20

D40

D60

D80

D100

Fatty acids

Fish oil

Wheat germ oil

D0

D20

D40

D60

D80

D100

Ingredients Fishmeala Soy protein concentrateda Wheat gluten Alpha-starch Methionine Phospholipids Fish oil Wheat germ oilb Choline chloride Ca(H2PO4)2 Vitamin mixturec Mineral mixtured Betaine Ethoxyquin

400 300 20 141.5 5 10 75 0 5 10 20 10 3 0.5

400 300 20 141.5 5 10 60 15 5 10 20 10 3 0.5

400 300 20 141.5 5 10 45 30 5 10 20 10 3 0.5

400 300 20 141.5 5 10 30 45 5 10 20 10 3 0.5

400 300 20 141.5 5 10 15 60 5 10 20 10 3 0.5

400 300 20 141.5 5 10 0 75 5 10 20 10 3 0.5

Nutrient levels(DM basis)/% Crude protein Crude lipid Crude ash Gross energy/(KJ/g)

508.5 123 120.3 18.81

510 123 120.8 18.59

507.5 120.5 120.6 18.63

511.2 121.1 119.5 18.60

512.4 119.7 120.2 18.49

513.5 119.5 120 18.69

C14:0 C15:0 C16:0 C18:0 C20:0 ∑SFA C16:1n-7 C18:1n-9 C18:1n-7 C20:1n-7 C22:1n-9 ∑MUFA C20:2n-9 C18:2n-6 C18:3n-3 C20:4n-6 C20:5n-3 C22:6n-3 ∑PUFA ∑n-3PUFA ∑n-3HUFA ∑n-6PUFA n-3/n-6 EPA/DHA

8.48 1.02 24.5 4.04 0.83 39.63 7.42 12.42 3.46 3.25 4.39 30.94 2.04 2.21 1.43 0.64 5.85 8.25 20.99 16.09 14.10 2.85 5.64 0.71

0.07 0.03 16.11 0.79 0.11 17.14 0.14 13.83 1.41 1.12 – 16.5 – 60.7 4.19 – – – 64.9 4.19 – 60.7 0.07 –

6.6 0.68 21.92 4.23 0.69 34.68 6.5 8.96 3.31 1.9 2.47 23.13 2 6.14 1.56 0.81 11.36 11.17 34.29 25.34 22.53 6.95 3.65 1.02

5.63 0.56 21.13 4.02 0.56 32.35 5.76 9.41 2.94 1.71 2.03 21.85 1.63 13.5 1.95 0.7 9.97 9.33 38.21 22.38 19.30 14.2 1.58 1.07

4.71 0.47 20.31 3.43 0.48 29.79 4.86 9.89 2.95 1.5 1.53 20.74 1.4 20.86 2.33 0.63 8.80 7.72 42.78 19.89 16.52 21.49 0.93 1.14

3.82 0.37 19.68 3.21 0.34 27.73 4.08 10.37 2.58 1.33 0.97 19.34 1.11 28.74 2.75 0.54 7.54 5.90 47.57 17.18 13.44 29.28 0.59 1.28

2.95 0.24 18.78 2.71 0.21 25.11 3.28 10.86 2.49 1.08 0.45 18.17 0.78 35.79 3.13 0.4 6.60 4.56 52.11 15.13 11.16 36.19 0.42 1.45

2.16 0.14 17.97 2.50 0.11 23.07 2.61 10.82 2.37 0.88 – 16.68 0.33 41.99 3.53 0.33 5.62 3.72 56.36 13.72 9.34 42.32 0.32 1.51

a Raw materials were supplied by Shengsuo Feed Company (Shandong, China), white fishmeal (crude protein,716.9 g kg−1, crude lipid, 73.6 g kg−1), soybean meal concentrated (crude protein, 643.8 g kg−1, crude lipid, 7.6 g kg−1). b Provided by Shandong Fushikang Industry and trade Co., LTD. c One kilogram of vitamin premix contained the following: retinol acetate 38.0 mg, alpha-tocopherol 210.0 mg, cholecalciferol 13.2 mg, thiamin 115.0 mg, riboflavin 380.0 mg, pyridoxine HCl 88.0 mg, pantothenic acid 368.0 mg, niacin acid 1030.0 mg, biotin 10.0 mg, folic acid 20.0 mg, vitamin B12 1.3 mg，inositol 4000.0 mg. d One kilogram of mineral premix contained the following: MgSO4·7H2O 3568.0 mg, KCl 3020.5 mg, KAl(SO4)2 8.3 mg, CoCl2 28.0 mg, ZnSO4·7H2O 353.0 mg, CuSO4·5H2O 9.0 mg, KI 7.0 mg, MnSO4·4H2O 63.1 mg, Na2SeO3 1.5 mg, C6H5O7Fe·5H2O 1533.0 mg, NaCl 100.0 mg, NaF 4.0 mg, NaH2PO4·2H2O 25,568.0 mg, Ca-lactate 15,968.0 mg.

Note: SFA: saturated fatty acids; MUFA: monounsaturated fatty acid; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; ARA: arachidonic acid; PUFA: polyunsaturated fatty acids; “–” represents the fatty acid content < 0.1%.

2.2. Fish, rearing condition and feeding regime Grouper juveniles were obtained from a commercial fish farm in Laizhou, China. Prior to the beginning of the experiment, a total of 1000 juveniles were reared in a recirculating aquatic system for 2 weeks to acclimate to the experimental conditions. Fish were fed twice daily with a commercial feed to satiation during this period (CP 500 mg kg−1, CL 120 mg kg−1, supported by Shandong Shengsuo Feed Co., LTD). At the beginning of the trial, all fish were starved for 24 h, and then 360 juveniles with initial body weight 55.35 g were selected and randomly distributed into 18 green cylindrical fiberglass tanks (300 L) and each tank was stocked with 20 juveniles. Each diet was randomly assigned to triplicate tanks. Fish were hand-fed to satiation twice a day (8.30 and 16.00). Thirty minutes after feeding, the uneaten feed was collected from the drainage tube of the aquarium and the pellets counted. The feeding trial lasted for 70 days. During this period, the temperature ranged from 26.9–27.4 °C, the salinity was held at 30, dissolved oxygen was approximately 6 mg L–1, and both the total ammonia nitrogen were maintained at < 0.1 mg L–1.

this species is 14% at 50% protein level (Rahimnejad et al., 2015), which is higher than E. malabaricus (Lin and Shiau, 2003). Jiang et al. (2015) has reported that dietary lipid levels from 7% to 13% had no significantly influences on growth performance. It seemed that dietary protein not lipid limited the growth of hybrid grouper, and dietary fish oil can be large scale replaced by other kinds of lipids. Hence, the aim of this study was to determine the effects of replacing dietary fish oil by WGO on growth performance, lipid and fatty acids deposition, plasma biochemical indices and lipid metabolic enzyme of juvenile hybrid grouper.

2. Materials and method 2.1. Experimental diets

2.3. Sampling and analysis Using fishmeal and soy protein concentrated as protein sources and fish oil as lipid source, six isonitrogenous, isolipidic and isoenergetic diets were formulated to contain 510 g kg−1 protein, 120 g kg−1 lipids and 18.6 kJ g-1 in a dry-weight basis. Fish oil was replaced by WGO at 20% gradient until totally replaced (D0, D20, D40, D60, D80, D100). Ingredients and nutrient levels were given in Table 1, and fatty acids compositions were shown in Table 2. All solid ingredients were weighted individually before mixed thoroughly in a feed mixer for 30 min. Oil was added and mixed again for 15 min, then dissolved water was added and mixed to form dough. The mixture was transformed into pellets using a single-screw extruder (Fishery Machinery and Instrument Research Institute, China Academy of Fishery Science, Shanghai, China). All diets were stored at −20 °C for the duration of the trial.

At the end of the trial, the fish were starved for 24 h before harvesting. The total number, body weight and length of each fish were measured after anesthetizing them with MS-222 (100 mg kg−1, Jiangxi Jian Reagent, China). The values obtained were used to calculate survival rate (SR), weight gain rate (WGR), daily feed intake (DFI), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), and condition factor (CF). Three fish per tank were randomly selected for whole fish samples. The remaining fish were weighed individually. Blood samples were taken from the caudal vessels using 2ml sterile injection syringe, transferred into and mixed in 5-ml anticoagulant tube and plasma harvested after centrifugation at 4000 ×g for 10 min at 4 °C using a high-speed refrigerated centrifuge (Hitachi Crg Series, Japan). Dorsal muscle, viscera, liver, intestine and mesenteric fat 55

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3.2. Proximate compositions and fatty acids of tissues

were dissected out and measured for analysis of proximate composition or calculation of organ indices. All samples were stored at −20 °C for further analyses. Proximate composition analyses of feed ingredients, experimental diets, and tissues samples were performed using established standard methods (AOAC, 1990). Moisture was determined by drying samples in an oven at 105 °C overnight. Crude protein was determined by measuring nitrogen (N × 6.25) using the Kjeldahl method. Crude lipid was determined by ether extraction using Soxhlet, and energy was determined using an adiabatic bomb calorimeter (PARR 6100, USA). Fatty acids composition was determined on the total lipid extract. Total lipids were extracted and measured gravimetrically according to Folch et al. (1957) using dichloromethane. Fatty acid methyl esters were prepared by acid-catalyzed transmethylation of total lipids using boron trifluoride-methanol according to Metcalfe et al. (1966) and were analyzed in a high performance gas chromatograph (GC-2010, Hitachi, Japan). The chromatograph was equipped with a 100 m × 0.25 mm × 0.20 μm chromatographic column (SP-2560，Supelco，Bellefonte，PA，USA) and the precolumn pressure was 357.6 KPa. High pure nitrogen was used as carrier gas (1.8 mL min−1). The thermal gradient was 140–240 °C at 4 °C min–1 and a constant temperature of 240 °C for 10 min. One microliter sample was split injected and split ratio was 90:1. Both injector and flame ionization detector temperatures were 260 °C. Fatty acid methyl esters were identified by comparison with known artificial fatty acid standard mixtures (supelco，Bellefonte, PA, USA). The relative content of fatty acid was calculated by area normalization method. The liver and intestine samples were homogenized on ice in 10 volumes (w/v) of ice-cold physiological saline and centrifuged at 3000 rpm at 4 °C for 10 min. The supernatant was used for measuring activities of fatty acid synthetase (FAS) and lipase (LPS). Plasma bio-chemical indices, such as triglyceride (TG), total cholesterol (T-CHO), high density lipoprotein cholesterol (HDL–C), and low density lipoprotein cholesterol (LDL-C) were analyzed using an automatic analyzer (7020, Hitachi, Japan) with commercial kits supplied by Leaderman Co. LTD (Beijing). Total lipase (TL), hepatic lipase (HL), hepatopancreas lipoprotein lipase (LPL) in plasma, FAS in liver, and LPS in intestine were analyzed using commercial kits (Shanghai Enzyme-linked Industrial Co., Ltd., Shanghai, China).

Effects of replacing fish oil by WGO on proximate composition of hybrid grouper were presented in Table 4. All of contents of moisture, crude protein, crude lipid, and crude ash of whole fish, dorsal muscle and liver were not affected by dietary WGO (P > .05). Effects of replacing fish oil by WGO on fatty acids compositions of dorsal muscle, liver and mesenteric fat were listed in Tables 5, 6 and 7. The percentages of C14:0 and C15:0 in dorsal muscle and mesenteric fat were decreased by dietary WGO, but was not affected in liver. Palmitic acid (C16:0) and stearic acid (C18:0) in dorsal muscle were significantly decreased by dietary WGO (P < .05), meanwhile these two kinds of fatty acids in liver and mesenteric fat were not significantly affected (P > .05). The percentages of palmitic acid (C16:1n-7), paullinic acid (C20:1n-7), ARA(C20:4n-6), EPA(C20:5n-3), and DHA (C22:6n-3) were decreased, however linoleic acid (C18:2n-6) and linolenic acid (C18:3n3) were increased with the increase of dietary WGO content in all three tissues (P < .05). Oleic acid (C18:1n-9) was not affected by dietary WGO (P > .05). Eicosadienoic acid (C20:2n-9) in dorsal muscle and mesenteric fat was decreased by dietary WGO, but it was not been detected in liver. The ratio of EPA to DHA (EPA/DHA) in dorsal muscle and mesenteric fat was not affected, but the ratio in liver was significantly increased by dietary WGO. C18:1n-9/n-3 HUFA was increased by dietary WGO in three tissues. Correlation coefficients, k and p values of dietary fatty acid concentrations vs. fatty acid concentrations in fillets were shown in Table 8. Most of fatty acids in fillet were positively correlated with the fatty acids in diet. k values of C16:0, C18:1n-7, and C18:2n-6 were higher than 0.6. Correlation coefficients (R2) of dietary fatty acid concentrations vs. fatty acid concentrations in tissues were showed in Fig. 1. All of C18:2n6, C18:3n-3, ARA, EPA, DHA and C20:2n-9 in tissues had a positive correlation with the fatty acid in diet. The deposition of C18:2n-6 in muscle was quicker than mesenteric fat and liver. The depositions of C18:3n-3 and EPA in muscle and mesenteric fat were quicker than liver. The depositions of ARA and DHA in all tissues were more consistent. 3.3. Lipid metabolism enzymes activities All of total lipase (TL), hepatic lipase (HL), hepatopancreas lipoprotein lipase (LPL), and fatty acid synthetase (FAS) were not affected, but the lipase (LPS) was decreased significantly by dietary WGO (Table 9).

2.4. Statistic analysis Data from the trial was analyzed using one-way analysis of variance (ANOVA) and the SPSS program Version for windows (SPSS Inc., IL, USA). When significant differences were identified by ANOVA, multiple comparisons among means were made with a Duncan's multiple-range test with the significance cut-off set at P < .05. The results are presented as the means ± SD (standard deviation). Fatty acid in tissues was further analyzed using the regression model of analysis (Microsoft excel 2010) for assessing the linear (k) and quadratic (R) trends taking the same dietary fatty acid level as a factor.

3.4. Plasma parameters Effects of replacing fish oil by WGO on plasma parameters were listed in Table 10. Triglyceride (TG) was increased by dietary WGO, but there were no difference on the contents of total cholesterol (T-CHO), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) among all groups.

3. Results

4. Discussion

3.1. Growth, feed utilization and figure indices

4.1. Effects of replacing fish oil by WGO on growth performances of hybrid grouper

Effects of replacement of fish oil by WGO on growth, feed utilization and figure indices of hybrid grouper were listed in Table 3. Survival rates of all experimental groups were above 98%, and not affected by dietary WGO. There were no differences on WGR, DFI, SGR, FCR, and PER among all groups (P > .05). Viscerosomatic index (VSI) and mesenteric fat ratio (MFR) were increased with the increasing of dietary WGO significantly (P < .05). Hepatosomatic index (HSI), intestinal somatic index (ISI), spleen somatic index (SSI), and condition factor (CF) were not affected by dietary WGO (P > .05).

Results of this trial demonstrate that in the diet with 40% fishmeal, WGO could totally replace fish oil without any adverse effect on growth performance of this hybrid grouper. A lot of researches had been reported that fish oil can partly or totally substituted by vegetable oil in marine fish diet (Izquierdo et al., 2003; Fountoulaki et al., 2009; Shapawi et al., 2008; Yıldız et al., 2018) on the basis of meeting the requirements of the essential fatty acids, especially for EPA and DHA (Lee et al., 2003; Sourabié et al., 2018). There was no report on the dietary n-3HUFA requirement for this hybrid species, but Zhu et al. 56

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Table 3 Effects of replacement of fish oil by WGO on growth, feed utilization and figure indices of hybrid grouper. Indices

D0

D20

D40

D60

D80

D100

IBW FBW WGRa DFIa SGRa FCRa PERa SRa VSIb HSIb MFRb ISIb SSIb CFb

55.13 ± 0.23 146.43 ± 3.17 158.79 ± 12.49 1.09 ± 0.02 1.40 ± 0.03 0.88 ± 0.05 2.44 ± 0.11 98.33 ± 2.36 9.92 ± 0.09a 3.05 ± 0.11 2.39 ± 0.05a 0.89 ± 0.05 0.16 ± 0.01 3.02 ± 0.13

55.23 ± 0.29 142.03 ± 5.52 156.04 ± 7.15 1.07 ± 0.04 1.35 ± 0.05 0.86 ± 0.05 2.49 ± 0.12 100.00 ± 0.00 10.05 ± 0.06ab 2.9 ± 0.11 2.50 ± 0.06ab 0.93 ± 0.01 0.16 ± 0.03 3.04 ± 0.06

55.27 ± 0.40 148.37 ± 4.33 150.70 ± 24.43 1.07 ± 0.08 1.41 ± 0.05 0.93 ± 0.06 2.33 ± 0.13 98.33 ± 2.36 10.14 ± 0.05bc 3.38 ± 0.07 2.55 ± 0.07b 0.93 ± 0.08 0.17 ± 0.02 3.00 ± 0.06

55.37 ± 0.15 145.63 ± 5.60 160.02 ± 10.97 1.13 ± 0.04 1.38 ± 0.06 0.90 ± 0.01 2.36 ± 0.01 98.33 ± 2.36 10.21 ± 0.04cd 3.26 ± 0.47 2.63 ± 0.06bc 0.89 ± 0.06 0.14 ± 0.01 2.99 ± 0.03

55.57 ± 0.12 146.87 ± 14.09 162.66 ± 28.91 1.12 ± 0.12 1.38 ± 0.14 0.89 ± 0.01 2.37 ± 0.02 98.33 ± 2.36 10.26 ± 0.05cd 2.78 ± 0.08 2.73 ± 0.06cd 0.94 ± 0.08 0.14 ± 0.02 3.03 ± 0.09

55.33 ± 0.25 144.93 ± 12.77 155.62 ± 31.73 1.09 ± 0.08 1.37 ± 0.12 0.90 ± 0.07 2.34 ± 0.15 98.33 ± 2.36 10.35 ± 0.08d 3.18 ± 0.23 2.81 ± 0.07d 0.94 ± 0.05 0.16 ± 0.04 3.08 ± 0.05

IBW (g·fish−1), initial body weight; FBW (g·fish−1), final body weight; WGR (%) = 100 × (FBW-IBW)/IBW; SGR (%·d−1) = 100 × [(lnFBW-lnIBW)/70]; DFI = 100 × feed consumption (g)/ [(FBW + IBW)/2 × 70]; FCR = feed intake (g)/(FBW-IBW); PER (%) = 100 × weight gain/protein intake; SR (%) = 100 × final fish number/initial fish number. Organ indices [VSI, HSI, MFR ISI, and SSI] (%) = organ weight (viscera, liver, intestine, mesenteric fat, spleen, g)/ Body weight (g) × 100; CF (g cm–3) = weight of fish/length of fish3 × 100. a Values (mean ± SD.) (n = 3) with the different letters in the same line are significantly different at P < .05. b Values (mean ± SD.) (n = 17) with the different letters in the same line are significantly different at P < .05. Table 4 Effects of replacing of fish oil by WGO on proximate composition of hybrid grouper (% wet weight). Whole fish Moisture Crude protein Crude lipid Crude ash Muscle Moisture Crude protein Crude lipid Crude ash Liver Moisture Crude protein Crude lipid Crude ash

69.99 ± 0.33 17.68 ± 0.18 8.32 ± 0.23 3.49 ± 0.12

69.43 ± 0.67 17.81 ± 0.19 8.21 ± 0.35 3.83 ± 0.21

69.44 ± 0.33 17.59 ± 0.34 8.02 ± 0.21 3.75 ± 0.26

69.88 ± 0.22 17.82 ± 0.19 8.29 ± 0.21 3.64 ± 0.14

70.32 ± 0.48 17.98 ± 0.13 8.33 ± 0.17 3.87 ± 0.21

69.59 ± 0.34 17.9 ± 0.24 8.26 ± 0.33 3.71 ± 0.20

76.25 ± 0.20 21.03 ± 0.09 1.77 ± 0.07 1.31 ± 0.07

76.07 ± 0.10 20.76 ± 0.16 1.82 ± 0.08 1.38 ± 0.06

75.92 ± 0.31 21.02 ± 0.04 1.75 ± 0.14 1.39 ± 0.07

76.19 ± 0.20 20.87 ± 0.09 1.73 ± 0.09 1.41 ± 0.08

76.00 ± 0.31 20.82 ± 0.11 1.73 ± 0.06 1.33 ± 0.05

76.34 ± 0.19 20.91 ± 0.15 1.82 ± 0.05 1.35 ± 0.05

64.86 ± 0.51 24.06 ± 0.41 5.37 ± 0.15 1.31 ± 0.04

64.81 ± 0.67 23.87 ± 0.37 5.58 ± 0.19 1.33 ± 0.02

64.73 ± 0.58 23.73 ± 0.16 5.51 ± 0.31 1.34 ± 0.04

64.54 ± 0.74 23.87 ± 0.31 5.5 ± 0.16 1.36 ± 0.03

64.35 ± 0.46 24.33 ± 0.22 5.46 ± 0.16 1.32 ± 0.02

64.4 ± 0.34 24.16 ± 0.31 5.62 ± 0.24 1.35 ± 0.02

Values (mean ± SD.) (n = 3) with the different letters in the same line are significantly different at P < .05.

still inhibited the growth performances. And many other reports illustrated that the essential fatty acids requirements varied among species, the total dietary lipid level and test time (Carvalho et al., 2018; Rombenso et al., 2016; Trushenski et al., 2012). It may be the reason for the difference reported on its lipid requirement (Rahimnejad et al., 2015; Jiang et al., 2015; Zhou et al., 2014). It can be concluded from the reports that lipid requirement should be reevaluated on the basis of satisfying the requirement of n-3 HUFA for these species. In another words, fish oil can be totally substituted with vegetable oil on the basis of satisfying the requirement of n-3 HUFA in hybrid grouper diet (Liu et al., 2016).

(2012) had reported that dietary n-3 HUFA requirement for E. coioides was 1.27–1.42%. Chu and Sheen (2016) reported that the optimal dietary lipid requirements of E. cocoides and E. lanceolatus were 95 and 75 g lipid kg–1 diet respectively by using the same semi-purified diets, but the weight gain percentage of two grouper species fed diets included 5% to 20% lipid showed no significant difference. It seemed that diet D100 could provide adequate levels of EFA to support normal growth of juvenile hybrid grouper. There was 9.34% n-3 HUFA of total fatty acids in completely substitution group, which may satisfied the requirement, and resulted into no difference in growth. The same results had been reported by Niu et al. (2007) who completely replaced fish oil with maize oil in the diet of E. coioides. Torstensen et al. (2005) reported that up to 100% of the fish oil can be replaced by vegetable oil blend without compromising growth or flesh quality if vegetable oil blend provided balanced levels of dietary fatty acids. However, quantitative requirements of essential fatty acids in this hybrid grouper cannot be determined based on the current results. Drew et al. (2007) had reported that fish oil can be totally replaced by vegetable oil when dietary fishmeal was 40%, but fish growth decreased when dietary fishmeal was reduced to 10 or 20%. Caballero et al. (2003) and Regost et al. (2003) had reported that in spite of the content of EPA and DHA in diets was higher than the requirement of gilthead seabream (Sparus aurata) and turbot (Psetta maxima), the high proportion substitution of fish oil with vegetable oil

4.2. Effects of replacing fish oil by WGO on body compositions of hybrid grouper Substitution of fish oil with WGO had no effects on body composition of juvenile hybrid grouper, which was in agreement with many other reporters with minor differences. Peng et al. (2008) and Niu et al. (2007) had reported that only dorsal muscle lipid was affected by the substitution of fish oil, and Huang et al. (2016) and Yu et al. (2017) had reported that only liver lipid was affected by the substitution of fish oil. Huang et al. (2015) reported that lipid contents in both whole body and liver of Cynoglossus semilaevis were affected by dietary WGO. It can be concluded that changes in dietary lipid sources can lead to changes in 57

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Table 5 Effects of replacing of fish oil by WGO on fatty acid composition in the muscle of hybrid grouper (% total fatty acids). Fatty acid C14:0 C15:0 C16:0 C18:0 C20:0 ∑SFA C16:1n-7 C18:1n-9 C18:1n-7 C20:1n-7 C22:1n-9 ∑MUFA C20:2n-9 C18:2n-6 C18:3n-3 C20:3n-3 C20:4n-6 ARA C20:5n-3 EPA C22:6n-3 DHA ∑PUFA ∑n-3PUFA ∑n-6PUFA n-3/n-6 EPA/DHA 18:1n-9/n-3HUFA

D0

D20 a

5.03 ± 0.24 0.54 ± 0.04a 25.57 ± 0.45a 6.13 ± 0.05a 0.50 ± 0.03a 37.77 ± 0.73a 6.09 ± 0.03a 13.79 ± 0.06 3.36 ± 0.05a 2.08 ± 0.06a 1.80 ± 0.06a 27.12 ± 0.18a 0.75 ± 0.02a 5.74 ± 0.18a 1.20 ± 0.03a 0.15 ± 0.01 0.74 ± 0.04a 5.46 ± 0.13a 8.45 ± 0.23a 24.05 ± 0.33a 16.81 ± 0.16a 6.48 ± 0.19a 2.60 ± 0.05a 0.65 ± 0.03 0.88 ± 0.01a

D40 ab

4.74 ± 0.35 0.49 ± 0.02b 24.78 ± 0.38b 5.88 ± 0.10b 0.44 ± 0.02b 36.32 ± 0.65b 5.61 ± 0.09b 13.88 ± 0.12 3.13 ± 0.07b 1.97 ± 0.01b 1.52 ± 0.07b 26.12 ± 0.06b 0.67 ± 0.02b 9.70 ± 0.36b 1.45 ± 0.02b 0.16 ± 0.01 0.66 ± 0.02b 5.17 ± 0.16b 8.04 ± 0.17b 27.37 ± 0.44b 16.33 ± 0.22b 10.36 ± 0.34b 1.58 ± 0.05b 0.64 ± 0.03 0.93 ± 0.02b

D60 b

4.36 ± 0.16 0.43 ± 0.02c 24.11 ± 0.22b 5.63 ± 0.11c 0.40 ± 0.01b 34.94 ± 0.26c 5.26 ± 0.08c 13.77 ± 0.17 3.02 ± 0.04c 1.85 ± 0.06c 1.23 ± 0.04c 25.13 ± 0.24c 0.63 ± 0.04b 13.72 ± 0.40c 1.61 ± 0.03c 0.15 ± 0.01 0.62 ± 0.02bc 4.86 ± 0.04c 7.65 ± 0.20b 30.77 ± 0.56c 15.80 ± 0.22c 14.34 ± 0.40c 1.10 ± 0.02c 0.63 ± 0.02 0.97 ± 0.03c

D80 c

3.64 ± 0.14 0.35 ± 0.01d 23.19 ± 0.18c 5.56 ± 0.07c 0.34 ± 0.02c 33.07 ± 0.14d 4.63 ± 0.08d 13.94 ± 0.17 2.89 ± 0.03d 1.72 ± 0.06d 0.94 ± 0.04d 24.12 ± 0.38d 0.54 ± 0.04c 17.92 ± 0.47d 1.82 ± 0.03d 0.14 ± 0.01 0.58 ± 0.04c 4.53 ± 0.13d 6.93 ± 0.18c 33.95 ± 0.50d 14.92 ± 0.21d 18.49 ± 0.48d 0.81 ± 0.03d 0.65 ± 0.03 1.06 ± 0.03d

D100 cd

3.32 ± 0.15 0.30 ± 0.02de 22.47 ± 0.32d 5.30 ± 0.12d 0.29 ± 0.03d 31.69 ± 0.53e 4.05 ± 0.11e 13.94 ± 0.17 2.73 ± 0.03e 1.60 ± 0.02e 0.75 ± 0.05e 23.06 ± 0.23e 0.48 ± 0.01d 23.49 ± 0.37e 2.05 ± 0.02e 0.15 ± 0.01 0.50 ± 0.01d 4.09 ± 0.08e 6.19 ± 0.14d 38.42 ± 0.55e 13.96 ± 0.18e 23.98 ± 0.38e 0.58 ± 0.02e 0.66 ± 0.02 1.17 ± 0.03de

3.04 ± 0.23d 0.26 ± 0.02e 21.90 ± 0.19e 5.22 ± 0.16d 0.26 ± 0.02d 30.68 ± 0.01f 3.43 ± 0.06f 13.72 ± 0.12 2.62 ± 0.06f 1.54 ± 0.04e 0.58 ± 0.01f 21.88 ± 0.14f 0.32 ± 0.01e 27.77 ± 0.25f 2.26 ± 0.02f 0.14 ± 0.01 0.46 ± 0.05d 3.49 ± 0.16f 5.70 ± 0.22e 41.56 ± 0.19f 13.00 ± 0.15f 28.24 ± 0.27f 0.46 ± 0.03f 0.61 ± 0.05 1.28 ± 0.03e

Values (mean ± SD.) (n = 3) with the different letters in the same line are significantly different at P < .05. Table 6 Effects of replacing of fish oil by WGO on fatty acid composition in liver of hybrid grouper (% total fatty acids). Fatty acid

D0

D20

D40

D60

D80

D100

C14:0 C15:0 C16:0 C18:0 C20:0 ∑SFA C16:1n-7 C18:1n-9 C18:1n-7 C20:1n-7 C22:1n-9 ∑MUFA C18:2n-6 C18:3n-3 C20:3n-3 C20:4n-6 ARA C20:5n-3 EPA C22:6n-3 DHA ∑PUFA ∑n-3PUFA ∑n-6PUFA n-3/n-6 EPA/DHA 18:1n-9/n-3HUFA

4.42 ± 0.48 0.26 ± 0.02 30.91 ± 0.53 6.71 ± 0.25 0.27 ± 0.01a 42.57 ± 0.81 8.39 ± 0.14a 23.44 ± 0.38 3.50 ± 0.10a 2.78 ± 0.14a 0.75 ± 0.03a 38.86 ± 0.56a 2.30 ± 0.09a 0.49 ± 0.01a 0.14 ± 0.02 0.38 ± 0.02a 1.41 ± 0.10a 4.12 ± 0.10a 10.40 ± 0.28a 7.72 ± 0.11a 2.68 ± 0.07a 2.88 ± 0.07a 0.34 ± 0.02a 3.25 ± 0.15a

4.43 ± 0.44 0.26 ± 0.01 30.88 ± 0.57 6.35 ± 0.24 0.25 ± 0.03a 42.18 ± 0.77 8.12 ± 0.18ab 23.55 ± 0.14 3.44 ± 0.11ab 2.61 ± 0.13ab 0.64 ± 0.04b 38.36 ± 0.13ab 3.78 ± 0.24b 0.50 ± 0.01a 0.14 ± 0.01 0.34 ± 0.02b 1.34 ± 0.06ab 3.63 ± 0.10b 11.05 ± 0.16b 6.93 ± 0.09b 4.12 ± 0.23b 1.69 ± 0.04b 0.37 ± 0.01a 3.66 ± 0.07b

4.41 ± 0.24 0.26 ± 0.03 30.61 ± 0.33 6.86 ± 0.17 0.22 ± 0.01b 42.35 ± 0.27 7.78 ± 0.13b 23.58 ± 0.15 3.41 ± 0.08ab 2.51 ± 0.15abc 0.57 ± 0.04b 37.85 ± 0.34b 5.90 ± 0.14c 0.50 ± 0.01a 0.13 ± 0.01 0.27 ± 0.02c 1.22 ± 0.02bc 2.88 ± 0.06c 12.05 ± 0.12c 5.88 ± 0.04c 6.17 ± 0.12c 0.95 ± 0.02c 0.42 ± 0.01b 4.38 ± 0.02c

4.56 ± 0.31 0.25 ± 0.03 30.56 ± 0.49 6.62 ± 0.21 0.20 ± 0.01bc 42.18 ± 0.77 7.28 ± 0.17c 23.57 ± 0.19 3.24 ± 0.07b 2.37 ± 0.09bcd 0.43 ± 0.05c 36.89 ± 0.35c 7.77 ± 0.24d 0.57 ± 0.02b 0.13 ± 0.01 0.21 ± 0.01d 1.01 ± 0.07cd 2.31 ± 0.05d 13.13 ± 0.11d 5.14 ± 0.16d 7.99 ± 0.25d 0.65 ± 0.04d 0.47 ± 0.02c 5.16 ± 0.23d

4.31 ± 0.27 0.24 ± 0.01 30.86 ± 0.08 6.44 ± 0.37 0.18 ± 0.01cd 42.04 ± 0.09 6.94 ± 0.38c 23.54 ± 0.22 2.97 ± 0.13c 2.24 ± 0.14cd 0.30 ± 0.01d 36.00 ± 0.56cd 10.05 ± 0.12e 0.63 ± 0.02c 0.14 ± 0.01 0.18 ± 0.01de 0.97 ± 0.05d 1.82 ± 0.05e 14.69 ± 0.20e 4.47 ± 0.09e 10.22 ± 0.11e 0.44 ± 0.01e 0.53 ± 0.01d 6.14 ± 0.15e

4.39 ± 0.23 0.24 ± 0.02 30.77 ± 0.09 6.49 ± 0.31 0.16 ± 0.01d 42.05 ± 0.4 6.43 ± 0.22d 23.86 ± 0.14 2.71 ± 0.09d 2.16 ± 0.12d 0.18 ± 0.02e 35.34 ± 0.37d 11.92 ± 0.28f 0.69 ± 0.03d 0.13 ± 0.02 0.14 ± 0.02e 0.75 ± 0.04e 1.21 ± 0.05f 15.53 ± 0.30f 3.47 ± 0.12f 12.06 ± 0.28f 0.29 ± 0.01f 0.62 ± 0.01e 8.59 ± 0.27f

Values (mean ± SD.) (n = 3) with the different letters in the same line are significantly different at P < .05.

Japanese flounder (Paralichthys olivaceus) (Xue et al., 2004), and Cynoglossus semilaevis Günter (Huang et al., 2015), C18:1n-9/n-3HUFA was used as an indicator of the deficiency of the essential fatty acids, but it may be not suitable for this grouper which the percentages of C18:1n-9 were not changed in all tissues, and it be may resulted by the difference of lipid metabolism. The variation of k values represents the difference of fatty acid deposition or synthesis rate (Table 8 & Fig. 1), although fatty acid profiles of fillet, liver and mesenteric fat reflected profiles of the corresponding dietary treatment, which implied the accumulating differences among tissues (Annette et al., 2018; Wu and Chen, 2012). The deposition rate of C18:2n-6 and C18:3n-3 in liver was much slower than

lipid deposition and metabolism in tissues, and had minor effects on other nutrients, which in accordance with the changes of the activities of lipid metabolism enzymes (Kim and Lee, 2004). It had been reported that most of fatty acids compositions in tissues were positively correlated with the dietary fatty acids compositions (Montero et al., 2003; Bell et al., 2004; Xu et al., 2012; Seno-O et al., 2010). In the present study, the percentages of C16:0, C18:0 and C18:1n-9 in all 3 tissues were minor affected by dietary WGO, and it was in agreement with other grouper, such as E. akaara (Wang et al., 2016), Cromileptes altivelis (Shapawi et al., 2008), and E. coioides (Li et al., 2016), but there was variations among vegetable oils (Shapawi et al., 2008; Lee et al., 2018). In many other marine species, such as 58

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Table 7 Effects of replacing of fish oil by WGO on fatty acids composition in mesenteric fat of hybrid grouper (% total fatty acids). Fatty acid

D0

C14:0 C15:0 C16:0 C18:0 C20:0 ∑SFA C16:1n-7 C18:1n-9 C18:1n-7 C20:1n-7 C22:1n-9 ∑MUFA C20:2n-9 C18:2n-6 C18:3n-3 C20:3n-3 C20:4n-6 ARA C20:5n-3 EPA C22:6n-3 DHA ∑PUFA ∑n-3PUFA ∑n-6PUFA n-3/n-6 EPA/DHA 18:1n-9/n-3HUFA

D20 a

D40 b

5.74 ± 0.11 0.60 ± 0.02b 22.45 ± 0.47 4.19 ± 0.11 0.51 ± 0.02b 33.97 ± 0.47ab 6.21 ± 0.26ab 13.97 ± 0.17 3.15 ± 0.05a 2.15 ± 0.08ab 2.14 ± 0.07b 27.62 ± 0.44b 1.25 ± 0.05b 10.53 ± 0.24b 1.90 ± 0.04b 0.20 ± 0.02 0.58 ± 0.02b 6.92 ± 0.15b 8.76 ± 0.14b 31.96 ± 0.31b 19.60 ± 0.31b 11.11 ± 0.23b 1.76 ± 0.06b 0.79 ± 0.01 0.79 ± 0.01b

6.11 ± 0.04 0.63 ± 0.02a 22.52 ± 0.61 4.13 ± 0.16 0.56 ± 0.01a 34.46 ± 0.78a 6.61 ± 0.27a 13.92 ± 0.18 3.14 ± 0.08a 2.26 ± 0.03a 2.37 ± 0.08a 28.29 ± 0.24a 1.63 ± 0.04a 8.26 ± 0.16a 1.74 ± 0.04a 0.20 ± 0.01 0.64 ± 0.02a 7.21 ± 0.12a 9.18 ± 0.11a 30.73 ± 0.12a 20.20 ± 0.21a 8.90 ± 0.16a 2.27 ± 0.06a 0.79 ± 0.01 0.75 ± 0.01a

D60 c

5.23 ± 0.13 0.55 ± 0.02c 22.26 ± 0.36 4.07 ± 0.08 0.46 ± 0.01c 33.02 ± 0.23bc 5.81 ± 0.10bc 13.86 ± 0.10 3.06 ± 0.09ab 2.02 ± 0.09bc 1.93 ± 0.02c 26.68 ± 0.19c 1.02 ± 0.09c 13.66 ± 0.16c 2.06 ± 0.04c 0.21 ± 0.01 0.55 ± 0.01bc 6.47 ± 0.04c 8.28 ± 0.05c 34.00 ± 0.13c 18.77 ± 0.06c 14.21 ± 0.16c 1.32 ± 0.01c 0.78 ± 0.01 0.83 ± 0.01c

D80 d

4.89 ± 0.03 0.47 ± 0.02d 22.2 ± 0.32 4.16 ± 0.13 0.38 ± 0.02d 32.5 ± 0.31cd 5.48 ± 0.22c 13.69 ± 0.07 3.04 ± 0.05ab 1.95 ± 0.07c 1.70 ± 0.01d 25.87 ± 0.12d 0.94 ± 0.09c 18.16 ± 0.24d 2.21 ± 0.04d 0.20 ± 0.01 0.51 ± 0.01c 5.91 ± 0.17d 7.50 ± 0.19d 37.06 ± 0.34d 17.45 ± 0.40d 18.67 ± 0.24d 0.94 ± 0.03d 0.79 ± 0.01 0.90 ± 0.03d

D100 e

4.45 ± 0.07 0.42 ± 0.01e 22.43 ± 0.76 4.13 ± 0.12 0.32 ± 0.02e 32.09 ± 0.95cd 5.02 ± 0.18d 13.97 ± 0.16 2.94 ± 0.10b 1.65 ± 0.08d 1.32 ± 0.04e 24.9 ± 0.08e 0.80 ± 0.06d 21.91 ± 0.34e 2.41 ± 0.02e 0.20 ± 0.01 0.44 ± 0.03d 5.20 ± 0.11e 6.68 ± 0.09e 39.20 ± 0.49e 16.05 ± 0.23e 22.35 ± 0.32e 0.72 ± 0.01e 0.78 ± 0.01 1.02 ± 0.02e

4.02 ± 0.08f 0.38 ± 0.01f 22.28 ± 0.23 3.99 ± 0.10 0.28 ± 0.01f 31.25 ± 0.34d 4.50 ± 0.15e 14.00 ± 0.18 2.74 ± 0.06c 1.47 ± 0.03e 1.04 ± 0.02f 23.74 ± 0.18f 0.66 ± 0.03e 24.46 ± 0.22f 2.65 ± 0.04f 0.21 ± 0.02 0.39 ± 0.02e 4.55 ± 0.10f 5.78 ± 0.09f 40.16 ± 0.45f 14.66 ± 0.20f 24.85 ± 0.24f 0.59 ± 0.01f 0.79 ± 0.01 1.18 ± 0.02f

Values (mean ± SD.) (n = 3) with the different letters in the same line are significantly different at P < .05.

percentages of EPA and DHA in 3 tissues (mesenteric fat > muscle > liver). It seemed that essential fatty acids can be stored in the form of mesenteric fat by this grouper, and the requirement of n-3 HUFA of liver was smaller than other 2 tissues. The ratio of EPA to DHA (EPA/ DHA) in muscle and mesenteric fat was not affected by the change of dietary lipid source, and it meant DHA was preferential deposited in these 2 tissues or EPA and DHA was deposited in a certain proportion. EPA/DHA was increased with the increasing of dietary WGO levels, which was in agreement with the change of diet. More interesting, the maximum of EPA/DHA of liver was close to the value of muscle, and the ratios of all of 3 tissues were smaller than that of diet, which indicated that DHA was more important than EPA for this species. More attention was paid on the fatty acids variations in the research of fish oil substitution (Aeo et al., 2007; Fountoulaki et al., 2009; Izquierdo et al., 2003), but it was also very important of the reactions on lipid digestion, absorption and synthesis (Li et al., 2016; MongeOrtiz et al., 2017;Yıldırım et al., 2013). Lipid metabolism enzyme system was constituted by LPS, TL and FAS. Blood lipid can reflect lipid metabolism in the body. Lipid bioavailability was not been affected in the present study, while the digestive ability was decreased by dietary WGO, which in accord with the report on greater amberjack (Seriola dumerili) (Monge-Ortiz et al., 2017). In many other reports, lipid bioavailability was significantly changed by substitution of dietary fish oil, such as TG was decreased and T-CHO was increased by the replacement of fish oil by soybean oil or linseed oil in European sea bass (Dicentrarchus labrax) and rainbow trout (Oncorhynchus mykiss) (Figueiredo-Silva et al., 2015) or hybrid sturgeon (Liu et al., 2016), and FAS was decreased by substitution of fish oil in Pacific white shrimp (Litopenaeus vannamei) (Xu et al., 2012) and spotted grouper (E. coioides) (Li et al., 2016). But in this study, there was no difference on TL, FAS, TCHO, HDL-C and LDL-C among groups, and TG was significantly increased by dietary WGO. Fatty acids biosynthesis activities were not affected by the alternation of lipid resource in the present study. It may be associated with the accumulation of mesenteric fat (Gao et al., 2006).

Table 8 Correlation coefficients (R2), k and p values of dietary fatty acid concentrations vs. fatty acid concentrations in fillets. Fatty acids

Liner regression

R2

k

P

C14:0 C16:0 C18:0 C20:0 C16:1n-7 C18:1n-7 C20:1n-7 C20:2n-9 C18:2n-6 C18:3n-3 C20:4n-6 C20:5n-3 C22:6n-3

Yfillet = 0.479Xdiet + 1.954 Yfillet = 0.952Xdiet + 4.659 Yfillet = 0.494Xdiet + 3.964 Yfillet = 0.417Xdiet + 0.205 Yfillet = 0.67Xdiet + 1.82 Yfillet = 0.748Xdiet + 0.883 Yfillet = 0.548Xdiet + 1.025 Yfillet = 0.253Xdiet + 0.259 Yfillet = 0.611Xdiet + 1.398 Yfillet = 0.529Xdiet + 0.386 Yfillet = 0.566Xdiet + 0.271 Yfillet = 0.33Xdiet + 1.855 Yfillet = 0.368Xdiet + 4.556

0.98 0.991 0.979 0.997 0.985 0.96 0.987 0.986 0.993 0.997 0.995 0.955 0.964

0.479 0.952 0.494 0.417 0.67 0.748 0.548 0.253 0.611 0.529 0.566 0.33 0.368

< .05 < .05 < .05 < .05 < .05 < .05 < .05 < .05 < .05 < .05 < .05 < .05 < .05

The greater the k value, the faster the accumulation of fatty acids.

dorsal muscle and mesenteric fat (k value was smaller), which represented the difference of utilization or the preference of fatty acids among tissues. Gao (2009) had reported that tissue involved in lipid metabolism was greatly influenced by the dietary fatty acid profile, but there was still differences on the abilities of biosynthesis among fatty acid. For example, accumulations of linoleic acid in muscle were much quicker than other tissues (k value was bigger than others). Most of marine fish were unable to produce EPA and ARA from linolenic acid and linoleic acid. Most of n-3 HUFAs in tissues were from diet, and the accumulations were almost similar in 3 tissues (k values were close). Kalogeropoulos et al. (1992) had reported that accumulation of C20:2n9 was resulted by the deficiency of HUFA in gilthead bream (Sparus aurata). But in our research, the content of C20:2n-9 was decreased with the decreasing of dietary C20:2n-9. Besides, the deposition rate of C20:2n-9 in mesenteric fat was much quicker than muscle (The bigger of k value, the faster of decreasing), and it was not detected in liver. That illustrated the differences on the utilization among tissues. Most of C20:2n-9 was deposited in mesenteric fat, which meant that it was not the essential fatty acid for grouper. All of EPA and DHA in tissues were from diet. It was varied of the 59

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Fig. 1. Correlation coefficients (R2) of dietary fatty acid concentrations vs. fatty acid concentrations in tissues. m is abbreviation for muscle, l is for liver, mf is for mesenteric fat, and d is for diet. In the model of “Y = kx + b”, the greater the k value, the faster the accumulation of fatty acids.

5. Conclusions

Data availability statement

About 71.8% dietary fish oil can be replaced by WGO in diets with 50% crude protein and 12% crude lipid for subadult hybrid grouper, however, quantitative requirements of essential fatty acids in this hybrid grouper cannot be determined based on the current results. Furthermore results different tissues owns different mechanisms of lipid metabolism, meanwhile fatty acids deposition and biosynthesis of this species was quite different from that of other fish species.

All data generated or analyzed during this study are included in this published article and its supplementary information files. Acknowledgements This work was financially supported by Key Research and Development Plan of Shandong Province (2016GSF115005) and

Table 9 Effects of replacement of fish oil by WGO on lipid metabolism enzymes activities of hybrid grouper. Indices −1

LPL (U mgprot ) HL (U mgprot−1) TL (U mgprot−1) FAS (nmol L−1) LPS (U gprot−1)

D0

D20

D40

D60

D80

D100

1.08 ± 0.10 2.04 ± 0.21 3.12 ± 0.12 0.84 ± 0.01 134.36 ± 3.48a

1.07 ± 0.10 2.05 ± 0.29 3.12 ± 0.35 0.84 ± 0.01 123.08 ± 3.76b

1.05 ± 0.08 2.08 ± 0.34 3.13 ± 0.26 0.82 ± 0.02 109.31 ± 4.63c

1.01 ± 0.10 2.00 ± 0.13 3.01 ± 0.11 0.83 ± 0.01 98.79 ± 3.94d

1.08 ± 0.14 2.09 ± 0.24 3.17 ± 0.24 0.82 ± 0.01 89.52 ± 5.03e

1.06 ± 0.14 1.96 ± 0.21 3.01 ± 0.35 0.83 ± 0.02 81.01 ± 3.29e

Values (mean ± SD.) (n = 9) with the different letters in the same line are significantly different at P < .05. 60

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Table 10 Effects of replacement of fish oil by WGO on plasma parameters of hybrid grouper. Indices

D0 −1

TG(mmol L ) TCHO(mmol L−1) HDL-C(mmol L−1) LDL-C(mmol L−1)

0.33 1.77 0.09 0.92

D20 ± ± ± ±

0.05 0.03 0.01 0.03

a

0.37 1.76 0.10 0.91

D40 ± ± ± ±

ab

0.02 0.02 0.02 0.02

0.40 1.74 0.10 0.91

D60 ± ± ± ±

0.03 0.03 0.01 0.02

abc

0.42 1.74 0.09 0.91

D80 ± ± ± ±

abc

0.05 0.03 0.01 0.04

0.45 1.71 0.09 0.90

D100 ± ± ± ±

bc

0.04 0.04 0.02 0.02

0.50 1.70 0.09 0.89

± ± ± ±

0.06c 0.03 0.01 0.02

Values (mean ± SD.) (n = 9) with the different letters in the same line are significantly different at P < .05.

Innovation Projects on Agricultural Technology of Shandong Province, as well as Yantai Science and Technology Promoting Plan (2016ZH068, 2018ZHGY066).

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