Effects of dietary oxidized fish oil with vitamin E supplementation on growth performance and reduction of lipid peroxidation in tissues and blood of red sea bream Pagrus major

Aquaculture 356-357 (2012) 73–79

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Effects of dietary oxidized fish oil with vitamin E supplementation on growth performance and reduction of lipid peroxidation in tissues and blood of red sea bream Pagrus major Jian Gao a,⁎, Shunsuke Koshio a, Manabu Ishikawa a, Saichiro Yokoyama a, Roger Edward P. Mamauag a, Yuzhe Han b a b

Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Kagoshima 890‐0056, Japan Applied Science of Marine Resources, The United Graduate School of Agricultural Science, Kagoshima University, Kagoshima 890‐8580, Japan

a r t i c l e

i n f o

Article history: Received 21 February 2012 Received in revised form 19 May 2012 Accepted 24 May 2012 Available online 4 June 2012 Keywords: Oxidized lipid Red sea bream Vitamin E TBARS Oxidative stress

a b s t r a c t A study was conducted to determine the effects of dietary oxidized fish oil (OFO) and vitamin E (VE) supplementation on growth and reduction of oxidative stress in juvenile red sea bream. An 8-week feeding trial on juveniles (average weight of 1.8 g) was conducted in triplicate groups with test diets containing two degrees of OFO (83.8 and 159.0 meq/kg) supplement with three levels of VE (0, 100 and 200 ppm), respectively. Fresh fish oil (13.5 meq/kg) with supplementation of 100 ppm VE was employed as control group. No significant differences were found on growth performance between fish fed with 100 or 200 ppm VE supplemented groups and control group. However, fish fed without VE supplement diets indicated significantly lower growth than control group. Increased dietary VE levels led to reduce liver thiobarbituric acid reactive substances value, while the highest value was observed in fish fed high OFO without VE supplemented diets. Liver vitamin C concentrations increased with increasing dietary VE levels. In conclusion, the results demonstrated that dietary OFO increased the oxidative stress condition of fish, but supplement of more than 100 mg/kg VE may prevent tissues from lipid oxidation, and improve growth and health of juvenile red sea bream. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Red sea bream Pagrus major, is one of the most economically cultured marine fish species in Japanese aquaculture, due to its economic feasibility and traditional food habits (Koshio, 2002). This fish has a dietary requirement for n− 3 highly unsaturated fatty acids (n− 3 HUFA) such as eicosapentaenoic acid (EPA, 20:5n−3) and docosahenaenoic acid (DHA, 22:6n− 3) (Furuita et al., 1996; Takeuchi et al., 1990). The n− 3 HUFA are essential for a variety of physiological functions including healthy growth, mortality and skeletal development for marine fish species (Sargent et al., 1999; Takeuchi et al., 1992; Tocher and Ghioni, 1999). However, large amount of n− 3 HUFA is susceptible to lipid peroxidation. Ingestion of oxidized lipids can influence oxidative stress because they are capable of being absorbed through the digestive tract (Kanazawa, 1991). The undesirable effects on fish by the consumption of dietary oxidized lipid have been reported in many studies (Gao et al., 2012a, 2012b; Koshio et al., 1994; Kosutarak et al., 1995; Tocher et al., 2003; Zhong et al., 2007,

⁎ Corresponding author. Tel.: + 81 99 286 4181; fax: + 81 99 286 4184. E-mail address: [email protected] (J. Gao). 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.05.034

2008). For successful aquaculture it is important to reduce oxidative stress. Vitamin E (α-tocopherol, α-Toc) has long been known as an essential nutritional factor for animal, which plays an important role in prevention of lipid peroxidation to protect the integrity of tissues in fish including turbot (Stephan et al., 1995; Tocher et al., 2002), gilthead sea bream (Tocher et al., 2002), Atlantic halibut (Lewis-McCrea and Lall, 2007) and black sea bream (Peng et al., 2009). Lipid oxidation in tissues can be prevented by increasing filet α-Toc concentrations through dietary supplementation of vitamin E (Bai and Gatlin, 1993; Gatlin et al., 1992). On the other hand, the requirement of vitamin E in fish is affected by dietary levels of lipid oxidation (Baker and Davies, 1996a, 1997a; Lewis-McCrea and Lall, 2007; Mourente et al., 2002; Zhong et al., 2008). Other than vitamin E, vitamin C or ascorbic acid (AsA) is another major antioxidant in body. Although some sparing and/or regenerating effects between these vitamins have been shown in a number of studies (Lee and Dabrowski, 2003; Lim et al., 2010; Shiau and Hsu, 2002), the protective role of α-Toc on AsA in fish remains unclear. Hamre et al. (1997) reported that liver AsA concentration of Atlantic salmon increased by increasing supplementation of vitamin E. On the contrary, a significant decrease in liver AsA concentration was observed in turbot fed with higher concentration of vitamin E supplementation (Ruff et al., 2003). The difference was

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J. Gao et al. / Aquaculture 356-357 (2012) 73–79

probably due to a different level of oxidative stress status of fish. Thus, there is a need for further research in effects of α-Toc on AsA of fish under different levels of lipid oxidation. It is generally accepted that α-Toc requirements of fish are increased by consumption of dietary oxidized lipid. However, there is no information regarding the effects of vitamin E supplementation with different degrees of lipid oxidation on reduction of oxidative stress in red sea bream. Therefore, this study was conducted to evaluate the effects of feeding different levels of dietary OFO with vitamin E supplementation on growth performance, lipid peroxidation, tissues α-Toc and AsA concentrations, fatty acid composition and blood parameters of juvenile red sea bream.

2. Materials and methods 2.1. Lipid oxidation and test diets The oxidized fish oil was prepared by the following procedure: cod liver oil without any antioxidant was obtained from Nippon Suisan Kaisha Ltd. (Tokyo, Japan) and was oxidized by heating at 70 °C with vigorous aeration. Peroxide value (POV) was monitored every 8 h intervals until the values of 83.8 meq/kg as low oxidation, and 159.0 meq/kg as high oxidation, respectively, were reached. The basal diet was formulated to contain about 47% protein, 19% lipid and 11% ash (Table 1). Seven semi-purified diets were formulated (Table 1) to contain about 47.6% protein, 15.0% lipid and 11.4% ash. Three levels of α-Toc (0, 100 and 200 mg/kg diet) combined with two degrees of oxidation of fish oil (83.1 meq/kg and 159.0 meq/ kg), respectively. 100 mg/kg of α-Toc with fresh fish oil (13.5 meq/ kg) was employed as a control. POV, α-Toc concentration, thiobarbituric acid reactive substances (TBARS) and fatty acid composition of experimental diets are presented in Table 2. All experimental diets were stored at −80 °C until the time of feeding.

Table 1 Ingredient and proximate composition of basal diets. Ingredients

%

Brown fish meala Activated gluten Starch Dextrin Fish oilb,c Mineral mixtured Vitamin mixture (E free)e AMP-Na/Caf α-cellulose + α-Toc

67.0 8.0 4.0 4.0 10.0 3.0 2.97 0.03 1.0

Proximate composition Moisture (%) Lipid (% dry matter) Ash (% dry matter) Protein (% dry matter)

9.0 15.0 11.4 47.6

a

Nippon Suisan Co. Ltd., Japan. Cod liver oil without antioxidants, Nippon Suisan, Tokyo, Japan. c Peroxide values of lowly and highly oxidized fish oil were 83.8 and 159.0 meq/kg, respectively. d Mineral mixture (mg/kg diet): MgSO4 5070; Na2HPO4 3230; K2HPO4 8870; Fe Citrate 1100; Ca Lactate12090; Al (OH)3 10; ZnSO4 130; CuSO4 4; MnSO4 30; Ca (IO3)2 10; CoSO4 40. e Vitamin mixture (mg/kg diet): ß-carotene 95.3; Vitamin D3 9.58; Menadione NaHSO3.3H2O (K3) 45.37; Thiamine-Nitrate (B1) 57.17; Riboflavin (B2) 190.45; Pyridoxine-HCl (B6) 45.37; Cyanocobalamin (B12) 0.07; d-Biotin 5.72; Inositol 3180.7; Niacin (Nicotinic acid) 762.03; Ca Pantothenate 266.8; Folic acid 14.26; Choline chloride 7790.61; ρ-Aminobenzoic acid 379.42. f L-Ascorbyl-2-Monophosphate (AMP-Na/Ca, DSM Nutrition Japan K.K.). b

Table 2 Peroxide value (POV, meq/kg), analyzed α-tocopherol content (mg/kg) thiobarbituric acid reactive substances (TBARS, μmol MDAa/mg dry mass) and fatty acid composition of test diets. Experiment dietb FFO/ Low Low 100E OFO/ OFO/ 0E 100E Analyzed α-Toc POV TBARS Fatty acid composition (% of total fatty acid) ∑ Saturates ∑ Monoenes ∑ n−6 ∑ n−3

Low OFO/ 200E

High OFO/ 0E

High OFO/ 100E

High OFO/ 200E

93.5 8.7 35.0

Ndc 27.5 58.1

87.8 26.4 60.9

165.7 26.9 62.7

Nd 39.5 101.9

71.0 36.8 108.9

143.8 36.5 98.1

27.2 41.3 3.3 24.1

29.1 41.6 3.2 21.1

28.0 41.8 3.2 21.8

27.3 41.3 3.2 22.3

29.1 43.9 3.2 18.0

29.3 43.5 3.2 18.3

29.8 43.9 3.1 17.9

∑ Saturates includes 14:0, 16:0 and 18:0. ∑ Monoenes includes 16:1n − 7, 18:1n − 9, 18:1n − 7, 20:1n − 9, 22:1n − 11 and 22:1n − 9. ∑ n− 6 includes 18:2n − 6, 20:2n − 6 and 20:4n − 6. ∑ n− 3 includes 18:3n − 3, 18:4n − 3, 20:4n − 3, 20:5n − 3, 22:5n − 3 and 22:6n − 3. a MDA, malondialdehyde. b FFO, fresh fish oil; OFO, oxidized fish oil. c Nd, not detected.

2.2. Fish and feeding trial Juveniles were obtained from a local hatchery, in Miyazaki prefecture, Japan, and transported alive to the Kamoike Marine Production Laboratory, Faculty of Fisheries, Kagoshima University, Kagoshima, Japan. Prior to the feeding trial, all fish were acclimated to the indoor rearing conditions for 2 weeks and fed a commercial pelleted fish feed (Higashimaru Co., Ltd. Kagoshima, Japan). At the start of the feeding experiment, fish (initial body weight 1.8 ± 0.1 g) were stocked in twenty-one 100-l tanks with 12 fish per tank in triplicate per treatment. Fish were fed the test diets to satiation for 8 weeks. The daily ration was divided into two, and fed to the fish at 08:00 and 16:00 h. The fish were weighed every 2 weeks and their ration adjusted accordingly. Uneaten diet was collected and dried to determine feed intake (FI). The water flow to the tanks was at 1.8 l/min with artificial aeration and natural light/dark regime throughout the duration of the experiment. The pH and salinity of the tank water during the experiment were 8.0 ± 0.1 and 33.5 ± 0.3 ppt, respectively. The water temperature ranged from 24.6 ± 2.5 °C (mean ± SD) throughout the trial. 2.3. Sample collection At the end of feeding trial, all fish were individually weighed and measured for fork length. Three fish from each tank were randomly collected and stored at − 20 °C for proximate analysis. Blood was collected using heparinized (1800 IU m/l), needle (25G × 1 in.) and syringes (1 ml) from 3 fish per tank and pooled into 1 tube as a blood sample. And from the other fish, liver was dissected out and weighed individually to calculate hepatosomatic index (HSI). Liver and muscle (without skin) samples were collected, pooled and stored at −80 °C until analysis for α-Toc, AsA and TBARS contents. 2.4. Analysis Proximate composition of diets and whole body was analyzed in triplicates for protein, ash and moisture (AOAC, 1990). Total lipid was measured following the method of Bligh and Dyer (1959). POVs of fish oil and diets (extracted by acetone) were determined by I.U.P.A.C. (1987). The measurement of TBARS was carried out using a method adapted from Yagi (1987). The AsA content of liver was

J. Gao et al. / Aquaculture 356-357 (2012) 73–79

quantified by the method of Sakakura et al. (1998). Conditions of high-performance liquid chromatography (HPLC) analysis for AsA content of liver were previously described by Gao et al. (2012b). The α-Toc contents in fish tissues and diet were determined by HPLC with fluorescence detector (FLD) according to Weber (1987). Fatty acid composition of diet and liver samples were analyzed according to Gao et al. (2012a). Total-cholesterol (T-Cho) and triglyceride (TG) were determined by a SPOTCHEM (SPOTCHEMtm EZ model SP-4430, Arkray, Inc. Kyoto, Japan). Biological antioxidant potential (BAP) and reactive oxygen metabolites (d-ROMs) were also measured spectrophotometrically from blood plasma with an automated analyzer FRAS, Diacron International s.r.l., Grosseto, Italy by following Kader et al. (2010). 2.5. Statistical analysis Statistical analysis was performed with analysis of variance (ANOVA) using a program (package super-ANOVA, Abacus 19 Concepts, Berkeley, California, USA). The Data from each group were compared to control using Turkey Kramer test (one-way ANOVA). Differences between treatments were considered significant when P b 0.05. Two-way ANOVA was also used to test the effects of dietary vitamin E level and degree of dietary oxidized lipid, and their interactions excluding the control diet. 3. Results Dietary OFO level did not affect body weight gain of fish fed with 100 or 200 mg/kg vitamin E supplemented diets after 8 weeks (Table 3). However, body weight gain of fish fed with no vitamin E supplemented diets was significantly lower than control. Within OFO group, supplementation of vitamin E was a major factor affecting on body weight gain. Survival rate and FI were similar in all treatments.

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Whole-body fat contents from fish fed with no vitamin E supplemented diets were significantly higher than control (Table 4). However, the whole-body fat contents decreased with incremental level of dietary vitamin E. Supplementation of vitamin E also reduced HSI value. Whole-body protein contents were significantly decreased by dietary OFO, while no differences were found for moisture and crude ash among treatments. The α-Toc concentrations and TBARS values of liver and muscle are presented in Table 5. In comparison with control, fish fed high OFO without vitamin E supplemented diets showed significantly lower α-Toc concentrations in liver and muscle. Increasing dietary vitamin E concentration significantly increased liver α-Toc concentrations, but did not affect muscle α-Toc concentrations. The α-Toc concentrations in liver and muscle were decreased by feeding of dietary OFO, especially the high OFO. On the other hand, tissue lipid oxidations were determined from TBARS value. Fish fed OFO without vitamin E supplementation diets had significantly higher levels of TBARS as compared to control. At a level of vitamin E of 100 or 200 mg/kg supplemented diets, liver and muscle TBARS had no significant differences compared to control. Within the groups that fish fed OFO diets, the results indicated that increasing dietary vitamin E concentrations significantly decreased liver and muscle TBARS values. Liver AsA concentrations are shown in Fig. 1. Fish fed with low or high OFO diets had lower liver AsA concentrations as compared to those of fish fed with fresh fish oil containing the same levels of vitamin E supplementation. Increasing the level of dietary vitamin E increased liver AsA concentrations. When supplementation of dietary vitamin E was increased to 200 mg/kg, the liver AsA concentration of fish in low OFO group was higher than control, while fish from high OFO groups had lower AsA concentrations than control. However, fish fed with high OFO without vitamin E supplemented diets had significantly lower AsA concentrations than control. In general, Fish fed with high OFO diets had lower liver AsA concentrations than those of fish fed low OFO diets with equivalent vitamin E concentration. Fatty acid composition of liver is also presented in Table 6. The degree of dietary OFO significantly increased in percentages of 14:0,

Table 3 Growth performances of red sea bream fed test diets for 8 weeksa. Experiment dietb

Final wt BWGc (g) (%)

SGRd (%)

FCRe

SRf (%)

FIg (g/fish/56 days)

FFO/100E

23.3 ± 0.9 19.1 ± 0.5* 20.6 ± 0.5 22.0 ± 1.5 19.1 ± 1.3* 20.8 ± 0.7 22.7 ± 2.0

4.5 ± 0.1 4.2 ± 0.1* 4.3 ± 0.1 4.4 ± 0.1 4.2 ± 0.1* 4.3 ± 0.1 4.5 ± 0.1

0.9 ± 0.0 1.0 ± 0.0* 0.9 ± 0.1 1.0 ± 0.2 1.1 ± 0.1* 0.9 ± 0.1 0.8 ± 0.1

88.9 ± 4.8 83.3 ± 8.4 86.1 ± 4.8 88.9 ± 12.7 91.7 ± 8.4 91.7 ± 8.4 88.9 ± 12.7

21.3 ± 0.6

Low OFO/0E Low OFO/ 100E Low OFO/ 200E High OFO/0E

High OFO/ 100E High OFO/ 200E Two way ANOVA Vitamin E 0.002 level Oxidation Ns degree Interaction Ns a

1164.1 ± 53.4 936.3 ± 31.9* 1026.2 ± 40.2 1104.2 ± 77.1 941.8 ± 69.5* 1030.6 ± 43.5 1129.5 ± 102.5

Table 4 Whole body proximate analysis and somatic parameters in red sea bream fed test diets for 8 weeksa.

19.0 ± 2.2

Experiment dietb

19.0 ± 1.6

FFO/100E

22.1 ± 3.8 20.3 ± 2.2 18.9 ± 2.7 18.9 ± 1.2

0.002

0.002

0.002

Ns

Ns

Ns

Ns

Ns

Ns

Ns

Ns

Ns

Ns

Ns

Ns

Values are expressed as mean ± SE from triplicate groups. Data with an asterisk in a column are significantly different from those of FFO/100E (P b 0.05). FFO/100E is not included in two-way ANOVA. b FFO, fresh fish oil; OFO, oxidized fish oil. c BWG: body weight gain = 100 ⁎ (final weight-initial weight) / (initial weight). d SGR: specific growth rate = 100 ⁎ (ln final weight − ln initial weight) / (duration). e FCR: feed conversion ratio = dry feed intake (g)/wet weight gain (g). f SR: survival rate = 100 ⁎ (initial fish number − dead fish number) / (initial fish number). g FI: feed intake = total feed intake (g) / number of fishes in 56 days feeding period.

Crude Fat Moisture Crude ash Crude protein CF (%) (%) (%) (%)

27.8 ± 2.0 Low OFO/0E 35.6 ± 1.2* Low OFO/100E 33.0 ± 0.8 Low OFO/200E 32.4 ± 0.9 High OFO/0E 35.2 ± 1.4* High OFO/ 31.9 100E ± 1.8 High OFO/ 32.1 200E ± 1.1 Two way ANOVA Vitamin E 0.021 level Oxidation 0.006 degree Interaction Ns

a

70.2 ± 0.5 69.1 ± 0.7 69.3 ± 0.6 69.4 ± 0.6 69.7 ± 1.6 70.7 ± 0.9 70.2 ± 1.1

17.9 ± 0.3

57.1 ± 2.3

16.1 ± 0.9

53.9 ± 2.1

16.4 ± 0.5

56.4 ± 3.5

16.3 ± 0.3

52.1 ± 0.3

16.2 ± 11.3 16.6 ± 0.4

50.1 ± 3.5

17.2 ± 0.1

47.1 ± 0.4

Ns

Ns

Ns Ns

HSIc (%)

2.5 ± 0.2 2.4 ± 0.1 2.3 ± 0.2 2.5 ± 0.2 2.2 ± 0.1 2.4 ± 0.1 2.6 ± 0.2

1.4 ± 0.1 1.6 ± 0.2 1.4 ± 0.1 1.4 ± 0.1 1.7 ± 0.1 1.5 ± 0.3 1.4 ± 0.2

Ns

0.041

0.042

Ns

b0.001

Ns

Ns

Ns

Ns

Ns

Ns

48.2 ± 0.6

Values are expressed as mean ± SE from triplicate groups. Data with an asterisk in a column are significantly different from those of FFO/100E (P b 0.05). FFO/100E is not included in two-way ANOVA. Crude protein, crude lipid and ash are expressed on a dry weight basis. b FFO, fresh fish oil; OFO, oxidized fish oil. c HSI: hepatosomatic index = 100 ⁎ (liver weight / body weight).

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J. Gao et al. / Aquaculture 356-357 (2012) 73–79

Table 5 TBARS (μmol MDA/mg wet) and α-tocopherol contents (mg/kg wet) in liver and muscle of juvenile red sea bream fed test diets for 8 weeksa. Experiment dietb

FFO/100E Low OFO/0E Low OFO/100E Low OFO/200E High OFO/0E High OFO/100E High OFO/200E Two way ANOVA Vitamin E level Oxidation degree Interaction

Liver

Muscle

α-Tocopherol

TBARS

α-Tocopherol

TBARS

126.1 ± 12.7 30.2 ± 9.5* 109.9 ± 13.5 136.2 ± 17.6 27.1 ± 7.4* 85.5 ± 12.4 112.0 ± 24.4

52.1 ± 14.7 116.2 ± 16.9* 81.4 ± 22.2 69.4 ± 11.0 124.5 ± 17.3* 94.2 ± 19.6 82.0 ± 18.2

14.2 ± 3.4 9.9 ± 0.7 10.6 ± 1.1 11.6 ± 3.1 8.0 ± 0.5* 9.9 ± 0.6 10.8 ± 0.5

5.3 ± 1.9 9.0 ± 3.6 8.1 ± 3.0 7.6 ± 1.7 15.5 ± 3.1* 8.0 ± 1.2 7.7 ± 1.1

0.001 0.004 Ns

0.006 Ns Ns

Ns 0.021 Ns

0.012 Ns Ns

a Values are expressed as mean ± SE from triplicate groups. Data with an asterisk in a column are significantly different from those of FFO/100E (P b 0.05). FFO/100E is not included in two-way ANOVA. b FFO, fresh fish oil; OFO, oxidized fish oil.

18:0 and decreased in total n− 3 HUFA, like EPA, 22:5n−3 and DHA. The proportions of EPA, DHA, 22:5n− 3 and total n −3 HUFA of fish fed OFO without vitamin E supplementation had lower values than control. EPA, DHA and total n− 3 HUFA were increased by supplementation of dietary vitamin E, while 14:0, 16:0 and 18:0 were decreased. Plasma T-Cho, TG and oxidative status (d-Roms and BAP) of fish fed with test diets for 8 weeks are shown in Table 7. Increasing levels of dietary vitamin E decreased TG and d-Roms values, and increased BAP in both low and high OFO groups. Fig. 2 shows the pattern of combined effects of d-Roms and BAP. Diet with low OFO/100E, low OFO/200E, high OFO/200E and FFO/100E was located in zone A, while high OFO/100E in zone C, low OFO/0E and high OFO/0E in zone D, respectively. 4. Discussion Generally, the results of the present study were in agreement with previous studies in which feeding of OFO without vitamin E supplemented diets reduced growth of fish including juvenile hybrid tilapia (Huang and Huang, 2004), African catfish (Baker and Davies, 1997b), turbot and halibut (Tocher et al., 2003) and rainbow trout (Cowey et al., 1984). Reduced weight gain of animal fed dietary oxidized lipid was caused by a combined effect of oxidation products and a-Toc deficiency (Sheehy et al., 1994), since vitamin E is an essential nutrient that has a function in protection of organisms against lipid oxidation (Hamre, 2011). In the present study, supplement of adequate vitamin E (100 or 200 mg/kg diet, about 5–10 fold higher

AsA content (µg/g wet)

50 40 30 *

20

*

10 0

FFO/100E

Low Low Low High High High OFO/0E OFO/100E OFO/200E OFO/0E OFO/100E OFO/200E

Treatment Fig. 1. Ascorbic acid concentration in liver of fish fed test diets for 8 weeks. Values are expressed as mean± SE from triplicate groups. Data with an asterisk in a column are significantly different from those of FFO/100E (P b 0.05). FFO/100E is not included in two-way ANOVA where vitamin E = 0.022, oxidation degree= b0.001, interaction = no significant.

than the requirement of most marine specie) improved growth of red sea bream fed OFO diets was detected. This finding was in agreement with Tocher et al. (2003) who reported vitamin E supplementation improved growth in gilthead sea bream fed oxidized oil diet. Moreover, Peng et al. (2009) showed that supplementation of a dose of more than 150 mg a-Toc/kg diet could improve black sea bream's growth performance when oxidized oils existed in diet. Therefore, these results demonstrated that vitamin E supplementation was necessary to maintain the concentrations of tissue α-Toc in protecting against lipid oxidation for normal growth of fish fed OFO. On the other hand, FI was the other major factor affecting on growth of fish fed OFO. Dietary lipid oxidation altered the odor and consequently the taste of the diet reduced FI and led to retarded growth of fish. However, FI in the present study has no significant difference among treatments, confirming our previous study (Gao et al., 2012b). Similarly, another study (Kosutarak et al., 1995) from our laboratory worked on the same species reported that FI was not affected by feeding of higher level of OFO (256 meq/kg). On the contrary, Gao et al. (2012a) found that FI in Japanese sea bass was significantly decreased by dietary oxidized lipid in similar levels of dietary lipid oxidation (26 meq/kg) as this study. Furthermore, Tocher et al. (2003) reported that the extent of peroxidative stress and deleterious effects appeared inversely proportional to responses of the hepatic antioxidant defense enzyme activities. In their work, feeding of OFO diets, a gradient of deleterious effects was observed from very few in sea bream to considerable stress in halibut with turbot intermediate. Although we did not measure antioxidant defense enzyme activities in this study, it may be possible that red sea bream might tolerate strong dietary rancid odor and have strong antioxidant defense system. In the present study, fish fed OFO without vitamin E diets had higher whole body fat content. Supplementation of vitamin E significantly reduced whole body fat content and HSI value. HSI value is correlated with the amount of fat deposition, fish fed diet without vitamin E supplement, lipid radicals cannot to be digestive, probably result in fatty infiltration into liver cells and to enlarge the live size (Baker and Davies, 1996b). The value tended to be lower in fish fed with vitamin E supplemented diets was detected in this study. Similar results have also been reported in gilthead bream (Mourente et al., 2002) and black sea bream (Peng et al., 2009). In contrast with that, a significant decrease in HSI value has been found in halibut (Tocher et al., 2003). Moreover, Lewis-McCrea and Lall (2007) reported that HSI was not affected by dietary vitamin E or oxidized lipid. On the other hand, whole body protein contents decreased in fish fed OFO diets. A similar study was also found in Gao et al. (2012b). It is possibly because basic amino acids were blocked by reaction with oxidized lipids but the bonds formed became resistant to acid hydrolysis only in hydroperoxide maximum (Pokorny et al., 1990). Although lipid hydroperoxides were considered to be poorly absorbed into the body (Artman, 1969), in this study, feeding with high OFO diets significantly reduced more α-Toc concentrations in liver and muscle than those fed with low OFO diets, suggesting that lipid oxidation products were absorbed by red sea bream and were taken up into the liver and muscle. The reduced levels of α-Toc could be due to the destruction of a-Toc in the gastrointestinal tract or intestine by free radicals present in OFO (Sheehy et al., 1994). Since oxidation products were taken up into the body, it should be a response to an increased consumption of α-Toc in tissues (Hamre et al., 2001). The similar results were observed in yellowtail (Sakai et al., 1992), African catfish (Baker and Davies, 1997a), gilthead sea bream (Mourente et al., 2002), Atlantic Cod (Zhong et al., 2007) and black sea bream (Peng et al., 2009). On the contrary, increasing dietary oxidation did not significantly affect liver and muscle α-Toc concentration in Atlantic halibut fed low OFO diets (POV of 7.5 and 15 meq/kg) (Lewis-McCrea and Lall, 2007). The responses to increasing levels of dietary vitamin E were similar to those observe in previous studies in which dietary vitamin E supplemented levels elevated

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Table 6 Liver fatty acid composition (% total fatty acids) of fish fed different experimental diets for 8 weeksa. Fatty acid

14:0 16:0 18:0 ∑ Saturated 16:1n−7 18:1n−9 18:1n−7 20:1n−9 22:1n−11 ∑ Monoenes 18:2n−6 20:4n−6 ∑ n−6 fatty acids 18:3n−3 18:4n−3 20:4n−3 20:5n−3 22:5n−3 22:6n−3 ∑ n−3 fatty acids

Experiment dietb

Two way ANOVA

FFO/100E

Low OFO/0E

Low OFO/100E

Low OFO/200E

High OFO/0E

High OFO/100E

High OFO/200E

VE

OF

VE × OF

3.6 ± 0.4 20.8 ± 0.7 3.9 ± 0.1 28.4 ± 0.7 6.7 ± 0.1 15.0 ± 0.6 4.8 ± 0.1 6.9 ± 0.1 4.3 ± 0.0 37.8 ± 0.5 2.2 ± 0.0 1.0 ± 0.1 2.5 ± 0.5 0.8 ± 0.0 1.2 ± 0.1 0.3 ± 0.5 10.9 ± 0.1 2.4 ± 0.0 15.3 ± 0.3 30.9 ± 0.4

4.6 ± 0.1* 21.7 ± 1.7 4.4 ± 0.3 30.8 ± 1.7 7.4 ± 0.5 14.3 ± 1.0 5.1 ± 0.2 6.9 ± 0.3 4.9 ± 0.1 38.8 ± 0.6 2.2 ± 0.1 0.7 ± 0.2 2.4 ± 0.3 0.6 ± 0.1 1.1 ± 0.2 0.4 ± 0.4 9.1 ± 0.6* 2.0 ± 0.0* 12.8 ± 0.3** 25.8 ± 0.6*

4.5 ± 0.1* 22.0 ± 0.6 4.3 ± 0.5 30.8 ± 0.7 7.0 ± 0.5 15.0 ± 0.0 5.5 ± 0.7 7.0 ± 0.3 4.3 ± 0.1 38.9 ± 1.1 2.3 ± 0.1 0.9 ± 0.3 2.5 ± 0.3 0.6 ± 0.1 1.0 ± 0.0 0.4 ± 0.4 9.8 ± 0.5 2.2 ± 0.1** 13.7 ± 0.4 27.5 ± 0.1*

4.1 ± 0.0 21.6 ± 0.1 4.1 ± 0.1 29.8 ± 0.1 6.8 ± 0.2 15.0 ± 0.1 5.3 ± 0.1 7.0 ± 0.0 4.2 ± 0.2 38.3 ± 0.5 2.4 ± 0.1 0.8 ± 0.2 2.7 ± 0.6 0.6 ± 0.1 1.0 ± 0.1 0.2 ± 0.4 10.0 ± 0.7 2.3 ± 0.1** 14.4 ± 0.4 28.1 ± 0.2

4.8 ± 0.2* 21.3 ± 0.8 5.1 ± 0.1* 31.2 ± 0.9 7.6 ± 0.4 16.1 ± 0.4 5.3 ± 0.8 6.4 ± 0.6 4.6 ± 0.3 40.4 ± 0.6* 1.6 ± 0.6 0.6 ± 0.1 2.2 ± 0.6 0.7 ± 0.0 1.1 ± 0.1 0.3 ± 0.2 8.2 ± 0.9* 2.0 ± 0.0* 11.2 ± 0.2* 23.1 ± 0.2*

4.5 ± 0.3* 20.5 ± 2.8 5.0 ± 0.1* 31.0 ± 1.8 7.8 ± 0.2* 15.7 ± 0.7 5.0 ± 0.6 7.2 ± 0.2 5.2 ± 0. 41.0 ± 0.6* 1.8 ± 0.6 0.7 ± 0.1 2.6 ± 0.6 0.7 ± 0.0 1.2 ± 0.1 0.3 ± 0.2 8.5 ± 0.1* 2.0 ± 0.0* 11.4 ± 0.5* 24.1 ± 0.6*

4.6 ± 0.1* 20.3 ± 0.5 4.7 ± 0.5 30.5 ± 0.3 7.9 ± 0.2* 15.5 ± 0.3 5.0 ± 0.5 7.1 ± 0.1 5.3 ± 0.1 40.9 ± 0.6* 2.2 ± 0.7 0.8 ± 0.2 3.0 ± 0.6 0.7 ± 0.0 1.2 ± 0.1 0.4 ± 0.3 9.0 ± 0.5* 2.0 ± 0.1* 11.6 ± 0.2* 25.0 ± 0.8*

0.007 Ns Ns Ns Ns Ns Ns 0.035 Ns 0.001 Ns Ns Ns Ns Ns Ns Ns 0.001 0.001 0.001

0.020 Ns 0.001 Ns 0.002 0.002 Ns Ns 0.001 Ns 0.05 Ns Ns Ns Ns Ns 0.005 0.001 0.001 0.001

Ns Ns Ns Ns Ns Ns Ns Ns 0.001 Ns Ns Ns Ns Ns Ns Ns Ns 0.018 0.025 Ns

a Values are expressed as mean ± SE from triplicate groups. Data with an asterisk in a row are significantly different from those of FFO/100E (P b 0.05). FFO/100E is not included in two-way ANOVA. VE; vitamin E, OF; oxidation degree; VE × OF; interaction. b FFO, fresh fish oil; OFO, oxidized fish oil.

tissue α-Toc concentrations (Bai and Gatlin, 1993; Bai and Lee, 1998; Gatlin et al., 1992; Tocher et al., 2003). This effect was more pronounced in the liver than in the muscle. These results were in accordance with a previous study by Stephan et al. (1995), who reported that the liver α-Toc concentration was higher than the muscle in sea bass Dicentrarchus labrax. It was also reported by Liu and Huang (1996), who found that the increased a-Toc concentration was more pronounced in tissues rich in a-Toc of rats fed oxidized soybean oil diets through a depletion–repletion period, possibly because of the relatively fast turnover and/or catabolism of the α-Toc in these tissues. In this present study, liver and muscle TBARS contents negatively correlated with liver and muscle α-Toc concentrations. Fish fed OFO without vitamin E supplemented diets had significantly higher liver TBARS as compared to those of fish fed control diets. It is well known that enhancement of α-Toc concentrations in tissues through dietary vitamin E supplementation may protect against lipid oxidation of tissues (Baker and Davies, 1996b; Frigg et al., 1990; Peng et al., 2009; Stephan et al., 1995; Tocher et al., 2002). In this study, increasing dietary vitamin E concentrations significantly reduced liver

and muscle TBARS contents, and this had also been reported by Murata and Yamauchi (1989), who found that α-Toc concentration in tissues from same fish species was positively correlated with those of their diets and negatively correlated with respective tissue TBARS value. In contrast, Zhong et al. (2007) found a significant increased in liver TBARS content in Atlantic cod fed OF with 300 mg α-Toc/kg of vitamin E supplementation as compared to those of fish fed OF without vitamin E supplementation. The interactive effects of vitamins C and E on preventing lipid peroxidation have been reported for several fish species (Chen et al., 2004; Lewis-McCrea and Lall, 2010; Lim et al., 2000; Montero et al., 1999; Shiau and Hsu, 2002). In the present study, increasing dietary vitamin E levels increased liver AsA concentration, suggesting that α-Toc in tissues may regenerate and/or spare AsA. This was consistent with the finding of Hamre et al. (1997) who reported that liver AsA in juvenile Atlantic salmon was significantly increased by increasing levels of dietary vitamin E. Moreover, a slight increase in liver AsA concentration in turbot fed diets containing vitamin E raised from 20 to 320 mg/kg (Stephan et al., 1995). On the contrary, a significant reduction in liver AsA concentration was observed in market33.5

Experiment dietb

T-Cho (%)

TG (g/dl)

d-ROM (U. Carr)

BAP (μmol/l)

FFO/100E Low OFO/0E Low OFO/100E Low OFO/200E High OFO/0E High OFO/100E High OFO/200E Two way ANOVA Vitamin E level Oxidation degree Interaction

260.3 ± 23.0 308.0 ± 65.2 297.3 ± 14.6 284.0 ± 38.0 295.7 ± 38.2 289.3 ± 45.8 236.3 ± 23.4

122.7 ± 27.0 185.3 ± 54.8 156.3 ± 42.0 150.7 ± 19.7 221.0 ± 50.6* 128.0 ± 17.6 115.0 ± 17.8

89.0 ± 11.1 100.7 ± 18.6 86.7 ± 7.2 77.3 ± 9.8 124.0 ± 17.6 87.0 ± 7.0 76.7 ± 2.1

3532.0 ± 141.5 2760.7 ± 415.4 3306.7 ± 145.0 3321.3 ± 173.6 2641.3 ± 381.4 3388.0 ± 428.5 3288.3 ± 212.5

Ns Ns Ns

0.013 Ns Ns

b 0.001 Ns Ns

0.014 Ns Ns

a Values are expressed as mean ± SE from triplicate groups. Data with an asterisk in a row are significantly different from those of FFO/100E (P b 0.05). FFO/100E is not included in two-way ANOVA. b FFO, fresh fish oil; OFO, oxidized fish oil.

A

BAP (µmoli/L) ,*100

Table 7 Total cholesterol (T-Cho), triglyceride (TG), reactive oxygen metabolites (d-Rom) and biological antioxidant potential (BAP) in red sea bream fed test diets for 8 weeksa.

B

32.5

FFO/100E Low OFO/200E

31.5

High OFO/200E Low OFO/100E

30.5 High OFO/100E

29.5

Low OFO/0E

28.5

High OFO/0E

C

27.5

0

25

50

75

100

125

D

150

d-ROMs (U CARR) Fig. 2. Oxidative stress parameter in red sea bream fed test diets for 8 weeks (Values are expressed as mean ± SE from triplicate groups. Central axis based on combined value of d-Roms and BAP from each treatment, respectively.) Zone A: High antioxidant potential and low reactive oxygen metabolites (highly preferred). Zone B: High antioxidant potential and high reactive oxygen metabolites (acceptable). Zone C: Low antioxidant potential and low reactive oxygen metabolites (acceptable). Zone D: Low antioxidant potential and high reactive oxygen metabolites (not acceptable).

78

J. Gao et al. / Aquaculture 356-357 (2012) 73–79

size turbot fed higher level of vitamin E supplementation in diet (Ruff et al., 2003). Results of the present showed that liver AsA concentrations in fish fed with low OFO/200E diet were higher, while in fish fed with high OFO/200E diet was lower than those of fish fed FFO/100E diet, respectively. These findings may be due to excess α-Toc in tissues that may regenerate and/or spare AsA, the AsA was stored under low level of oxidative stress condition, but the amount of AsA would be consumed under high oxidative stress condition. Because of the free radical scavenging effects of α-Toc, producing a high amount of α-Toc radicals requires a large amount of AsA to recycle these α-Toc radicals. The present study indicated that the percentages of EPA, DHA and 22:5n − 3 were increased with incremental levels of dietary vitamin E. Similar result was also found by Baker and Davies (1996b) who reported that the production of DHA of tissues in catfish fed oxidized oils diets increased with incremental levels of dietary vitamin E. On the contrary, Zhong et al. (2007) found that PUFA content in muscle phospholipids of fish fed oxidized oil was decreased by α-Toc supplementation. α-Toc may affect the desaturation reaction even more indirectly through affecting general cellular peroxide tone/antioxidant status, due to an increase in peroxide tone. Moreover, whether achieved by restricted dietary intake of one or more antioxidants and/or by oxidized lipid appeared to result in the activation of fatty acyl desaturation and elongation (Mourente et al., 2007). Although the role of α-Toc as an antioxidant is well investigated, the effects of α-Toc on fatty acid metabolism remain unclear. Thus, further investigation should be made to determine these effects of αToc on fatty acid metabolism, especial fatty acid desaturation and elongation. The blood parameters of fish species are indicators of the state of health of the organism (Kader et al., 2010). In this study, fish fed OFO without vitamin E supplemented diets had higher value of blood TG, whereas TG value was reduced with incremental levels of dietary vitamin E. This may be related to chylomicron pathway of α-Toc, where α-Toc reduced T-Cho and TG in chylomicrons (Gao et al., 2012c). Oxidative stress was measured using the free radical analytical system (FRAS 4) to assess the derivatives of d-ROMs and BAP. Recently, in our laboratory, d-Rom and BAP have been used for evaluation of the oxidative stress condition of fish (Kader et al., 2011; Gao et al., 2012b, 2012c). As indicated in Fig. 2, it is interesting to note that FFO/100E, Low OFO/100E, Low OFO/200E and High OFO/200E diet groups were located in zone A. Vitamin E free diet (Low OFO/0E and High OFO/0E) groups were located in zone D. This evidence clearly indicated that more than 100 mg/kg of vitamin E in diet fed fish could suppress lipid peroxidation in plasma, thus protecting the cells from oxidative stress and similar health condition with those fed fresh fish oil. In summary, results showed that red sea bream fed OFO diet without vitamin E reduced growth and health condition. However, an increase of α-Toc concentration through dietary vitamin E supplementation could improve growth performance and reduce tissue lipid peroxidation and blood oxidative stress status. Results also showed that an excess α-Toc in tissues may regenerate and/ or spare AsA under low level of lipid oxidation condition, and amount of AsA would be consumed under high oxidative stress condition. In conclusion, dietary OFO increased the oxidative stress condition of fish, but supplementation of more than 100 mg/kg VE may prevent tissue lipid oxidation, and improve growth and health of juvenile red sea bream.

Acknowledgments The authors wish to acknowledgment the Ministry of Education, Culture, Sports, Science and Technology of Japan (MONBUKAGAKUSHO) for supporting this research work. The research was partially funded by the

Management Expenses Grants of the United Graduate School of Agricultural Sciences, Kagoshima University provided to S. Koshio.

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