Aquaculture 430 (2014) 21–27

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Effects of vitamin E on growth performance and antioxidant status injuvenile grass carp Ctenopharyngodon idellus

Jie Li, Xu-Fang Liang ⁎, Qingsong Tan, Xiaochen Yuan, Liwei Liu, Yi Zhou, Bin LiCollege of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University Freshwater Aquaculture Collaborative Innovation Center of Hubei Province,Wuhan, Hubei 430070, PR China

⁎ Corresponding author at: College of Fisheries, HuaNo. 1, Shizishan Street, Hongshan District, Wuhan, HubTel.: +86 27 8728 8255; fax: +86 27 8728 2114.

E-mail address: xufang_liang@hotmail.com (X.-F. Lian

http://dx.doi.org/10.1016/j.aquaculture.2014.03.0190044-8486/© 2014 Published by Elsevier B.V.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 November 2013Received in revised form 14 March 2014Accepted 14 March 2014Available online 22 March 2014

Keywords:All-rac-α-tocopherol acetateCtenopharyngodon idellusGrowthAntioxidantSerum biochemistry

An 8-week feeding trial was conducted to investigate the effects of dietary vitamin E on growth performanceand antioxidant status of juvenile grass carp. Semi-purified diets containing six levels of all-rac-α-tocopherylacetate (0, 25, 50, 100, 200, 400 mg kg−1) were assayed in triplicate. Growth performance, body composition,antioxidant activity and serum biochemistry parameter were determined. The maximum weight gain (WG),specific growth ratio (SGR) and feed intake (FI) were achieved in fish fed on a diet supplemented with100 mg kg−1 vitamin E. Vitamin E supplementation appeared to improve hepatic glutathione peroxidase(GPx) activity significantly, and higher activity of hepatic superoxide dismutase (SOD) was displayed in dietswith excess vitamin E. However, serum SOD and catalase (CAT) activity were enhanced in grass carp fed50 mg vitamin E kg−1 diet, and then decreased significantly as the content of vitamin E in the diet increased.A consistent decline in the hepatic and serum malondialdehyde (MDA) content was observed in fish fed dietswith the increased supplementation of vitamin E. In addition, with the increasing level of vitamin E, serumalanine aminotransferase (ALT) was reduced. Serum alkaline phosphatase (AKP) activity was enhanced in fishfed 50 and 100 mg vitamin E kg−1 diet, whereas it decreased as the content of vitamin E in the diet increased.Based on the broken-line regression analysis of WG, 100.36 mg kg−1 vitamin E was the optimal requirementof juvenile grass carp. The benefits of excess vitamin E levels need to be weighed carefully: on the one handmight enhance the antioxidant capacity, on the other hand might decrease growth.

© 2014 Published by Elsevier B.V.

1. Introduction

Vitamin E is the generic term for a group of lipid-soluble molecules,of which, α-tocopherol has the highest vitamin E activity (NationalResearch Council, 1993). It is an indispensable micro-nutrient whichplays an important role in various biochemical and physiologicalprocesses like protecting polyunsaturated lipids against oxidation(Hamre, 2011; National Research Council, 2011). What's more, vitaminE has also been proved to increase growth in fish and may be involvedindirectly in modulating growth by contributing to control of metaboliccosts and tissue damage during stress (Agradi et al., 1993; Tocher et al.,2002). However, vitamin E cannot be synthesized by fish based on itsdietary intake (Peng and Gatlin, 2009). Vitamin E deficiency maylead to increased lipid oxidation, anemia (Kocabas and Gatlin, 1999),liver degeneration (Hamre et al., 1994), muscular degeneration(Frischknecht et al., 1994) and even death. Nevertheless, excess dosesof α-tocopherol might also exerted a pro-oxidant effect on lipid tissues,

zhong Agricultural University,ei Province, 430070, PR China.

g).

and this has been demonstrated in rainbow trout Oncorhynchus mykiss(Tokuda and Takeuchi, 1995, 1999) and yellowtail Seriola quinqueradiata(Ito et al., 1999). Dietary vitamin E requirement has been demonstratedin a number of fish, such as 131.91 mg kg−1 diet for rohu Labeo rohitafry (Sau et al., 2004), 61–115 mg kg−1 for grouper Epinephelusmalabaricus (Lin and Shiau, 2005), 31 mg kg−1 for red drum Sciaenopsocellatus (Peng and Gatlin, 2009) and 78–111 mg kg−1 for juvenilecobia Rachycentron canadum (Zhou et al., 2013). Considering the interac-tions with other nutrients, different body levels and organ distributionsof vitamin E in different animal species (Hamre and Lie, 1995), it is notsurprising that the vitamin E requirement varies depending on speciesand experimental conditions.

Lipid peroxidation is a process of oxidative degradation of lipidswhere free radicals gain electrons from lipids, especially from polyun-saturated fatty acids (PUFA) in cellmembranes, resulting in cell damage(Kanazawa, 1993). As one of themost important free radical scavengers,vitamin E plays very important roles in the maintenance of redoxpotential and prevention of lipid peroxidation (Mourente et al., 2007).It competeswith PUFA in donating a hydrogen atom to the lipid peroxylradical (Buettner, 1993), thereby breaking the chain of reactionsinvolved in lipid peroxidation. In order to protect against free radicals,organisms have also developed a variety of other antioxidant defenses,

Table 1Formulation and proximate composition of the basal diet (dry matter).

Ingredient Content (g/kg)

Fish meal (vitamin E free)a 240.00Caseina 200.00Corn starcha 200.00Fish oil 20.00Soybean oil 20.00Vitamin premix (vitamin E free)b 10.00Mineral premixc 20.00Cellulose 253.50Monocalcium phosphate (MCP) 20.00Choline chloride (50%) 6.00Ethoxyquin (30%) 0.50Carboxymethylcellulose sodium 10.00Proximate composition (%)Crude protein 31.81Crude fat 5.28Carbohydrate 22.98Gross energy (MJ kg−1) 16.34

a Fulong Dietary company, Wuhan, Hubei, China.b Vitamin premix (per kg of diet): vitamin A, 2000 IU; vitamin B1 (thiamin), 5 mg;

vitamin B2 (riboflavin), 5 mg; vitamin B6, 5 mg; vitamin B12, 0.025 mg; vitamin D3,1200 IU; vitamin K3, 2.5 mg; folic acid, 1.3 mg; biotin, 0.05 mg; pantothenic acidcalcium, 20 mg; inositol, 60 mg; ascorbic acid (35%), 110 mg; niacinamide, 25 mg.

c Mineral premix (per kg of diet):MnSO4, 10 mg;MgSO4, 10 mg; KCl, 95 mg;NaCl,165 mg; ZnSO4, 20 mg; KI, 1 mg; CuSO4, 12.5 mg; FeSO4, 105 mg; Na2SeO3, 0.1 mg;Co, 1.5 mg.

22 J. Li et al. / Aquaculture 430 (2014) 21–27

such as catalase (CAT), superoxide dismutase (SOD) and glutathioneperoxidase (GPx). CAT and SOD are scavengers of active oxygen species,acting on hydrogen peroxide (H2O2) and superoxide (O2−), respectively(Miller et al., 1993). GPx, a cytosolic and mitochondrial enzyme, canreduce lipid peroxides (ROOH) to their corresponding alcohols (ROH),an important reaction for quenching lipid-peroxidizing chain reactions(Reed, 1990). However, after PUFA having been peroxidized, the prod-ucts of endoperoxide reactions include ketones and various toxic alde-hydes (Sargent et al., 2002). Among these aldehydes, malondialdehyde(MDA), measured as thiobarbituric acid reactive substances (TBARS),is an important indicator of the oxidative changes of lipid (Shahidi andHong, 1991), and it is the direct evidence of membrane injury causedby free radicals (Traystman et al., 1991).

Alanine aminotransferase (ALT) and aspartate aminotransferase(AST) belong to theplasmanon-functional enzymeswhich are normallylocalized within the cells of liver, kidneys, heart, and other organs (Hadiet al., 2009). These enzymes are considered to be important in assessingthe state of liver (Coz-Rakovac et al., 2005). Alkaline phosphatase (AKP)is a hydrolase enzyme responsible for removing phosphate groups frommany types of molecules, including nucleotides, proteins, and alkaloids;it is an indicator of osteoblast function and indirectly reflects physicalcondition (Zhou et al., 2012). Among the blood proteins, albumin(ALB) is themajor protein, which plays a significant role in the immuneresponse. And it has been used as an indicator of liver impairment(Silverman et al., 1986).

Grass carp has been regarded as the largest freshwater culturingspecies by artificial diet in China. There are many researches about itsnutritional requirements, but not much information was publishedconcerning the vitamin E requirement and nutritional function. Further-more, there is no information about antioxidant effect and serumbiochemistry parameters of vitamin E on grass carp, which just areimportant factors affecting fish health. Therefore, the objective of thisstudywas to estimate the dietary vitamin E requirement and to investi-gate the effects of dietary vitamin E on growth performance, bodycomposition, antioxidant capacity and serum biochemistry parameterin juvenile grass carp.

2. Materials and methods

2.1. Experimental diets

The formulation and proximate composition of the basal dietare shown in Table 1. Fish meal (vitamin E free) and casein were usedas major dietary protein sources. Fish oil and soybean oil were utilizedas the lipid sources. The ingredients were purchased from Shentianyuand Fulong Dietary Company (Wuhan, China). The basal diets weresupplemented with six all-rac-α-tocopherol acetate (Sigma ChemicalCo., Steinheim, Germany) levels (0, 25, 50, 100, 200 and 400 mg kg−1

diet) at the expense of small amounts of cellulose. The basal dietwithout addition of all-rac-α-tocopherol acetate was referred to as thecontrol diet. The dietary vitamin E concentrations were analyzed byhigh-performance liquid chromatography (HPLC) to be 3.38, 27.25,51.77, 103.16, 207.44, 404.52 mg kg−1 diets, respectively. The actualquantities of vitamin E differed slightly from expected content, mostprobably because of the manufacturing technique.

Each mixture was then pelleted (2 mm diameter) by a laboratorypellet machine (Fishery Machinery and Instrument Research Instituteof Chinese Academy of Fishery Sciences, Shanghai, China) within30 min and the outlet temperature was 72 ± 2 °C. Then the pelletswere air-dried and stored in a freezer at−20 °C until used.

2.2. Fish and experimental conditions

Grass carp was obtained from the Xiantao Hatchery, Hubei, China.Prior to feeding experiment, the juveniles were acclimatized tolaboratory conditions for 4 weeks in two 1000-L plastic tanks. And

they were fed to apparent satiation with a commercial diet (40.0%protein; 9.0% fat; 6.9% moisture; 7.6% ash) twice a day at 08:00 and16:00. After the 4-week acclimation, fish were starved for 24 h tomeasure the body initial weight. Then, 360 fish with mean weightof 11.2 ± 0.03 g (mean ± S.E.M.) were stocked in 18 aquariums(20 fish in each aquarium with near uniform biomass). Each experi-ment diet was fed to three randomly assigned aquaria. The fish werefed to apparent satiation twice per day (08:00 and 16:00) during thewhole 8-week feeding trial. Each aquarium was part of a closedrecirculating system. The filtered flow-through tap water was keptat a flow-rate of 3 L min−1. Dissolved oxygen (DO) value wasapproximately 7 mg L−1, the water temperature ranged from 23 to26 °C, the ammonia content was about 0.27 ± 0.02 mg L−1 and pHranged from 7.1 to 7.6 during the experimental period.

2.3. Sample collection

At the end of the feeding experiment, animals were fasted for 24 hand then final body weight was carried out by every aquarium asone unit. The fish were deeply anesthetized with 3-aminobenzoicacid ethyl ester methanesulfonate (MS-222, 200 mg L−1 water). Fiveindividuals from each aquarium were sampled randomly for bloodcollection from the caudal vein by a non-heparinized syringe. Theblood sample was centrifuged at 3500 ×g for 10 min and resultingserum samples were stored at −80 °C for subsequent analyses.Viscerosomatic index (VSI), hepatosomatic index (HSI) and intraperito-neal fat (IPF) ratio values were determined from three individual fishper aquarium by obtaining viscera, liver and IPF samples and expressingas a percent of body weight. The liver samples were dissected andstored at −20 °C until analyzed. Three fish were randomly capturedfrom each aquarium for whole body proximate composition.

2.4. Composition analyses

The proximate composition of feed andwhole bodywas determinedby standard methods (AOAC, 1995). Crude protein (N ∗ 6.25) wasdetermined following the Kjeldahl method after an acid digestionusing a Kjeltec system (Kjeltec 2300 Analyzer, Foss Tecator, Sweden).Crude lipid was evaluated by the ether-extraction method using Soxtec

23J. Li et al. / Aquaculture 430 (2014) 21–27

System HT (Soxtec System HT6, Tecator, Sweden). Ash was measuredusing a muffle furnace at 550 °C for 12 h. Moisture was determinedby oven drying at 105 °C for 6 h. Carbohydrate content of the dietswas analyzed by the 3, 5-dinitro salicylic acid method (Yu et al.,1997). Energy content of the diets was measured by bomb calorime-try using a Parr 6200 calorimeter equipped with a Parr 1108 OxygenBomb and a Parr 6510 water handling system (Parr InstrumentCompany, Moline, IL, USA).

2.5. Tissue treatment and enzyme activity assay

The liver samples were thawed, rinsed with 0.65% physiologicalsaline, and thenhomogenized infive volumes v/wof ice-cold physiolog-ical saline on ice (Han et al., 2011). The homogenate was then centri-fuged at 12,000 ×g for 15 min at 4 °C, being the upper lipid layerdiscarded. The supernatant was collected and kept at−20 °C for enzy-matic determinations. The activity of GPx, SOD, and CAT and the contentof MDA were determined in liver extracts and serum using commercialkits (Jiancheng Bioengineering Institute, Nanjing, China).

SOD activity was determined according to McCord and Fridovich(1969). It wasmeasured by its ability to inhibit superoxide anion gener-ated by the xanthine and xanthine oxidase reaction system, and oneactivity unit was defined as the amount of enzyme necessary to producea 50% inhibition of color formationmeasured at 450 nm. The SOD activ-ity was expressed as unit per milliliter serum and unit per milligramhepatic protein. CAT activity was analyzed according to the method ofAebi (1984). It was measured following hydrogen peroxide reductionat 405 nm, and one activity unit was defined as per milliliter serumdecomposition the amount of 1 μmol hydrogen peroxide per second,and expressed as unit per milliliter serum. Activity of hepatic GPx wasmeasured according to the method of Noguchi et al. (1973). One unitof GPx activity was defined as the amount of enzyme that reduced theglutathione (GSH) concentration in the reaction system at 1 μmol L−1

per minute. MDA was measured as TBARS according to Beuge andAust (1978) and expressed as nanomole per milligram protein andnanomole per milliliter serum.

Serum ALT, AST, and AKP activity and ALB content were determinedusing an automatic biochemical analyzer [Abbott Aeroset Analyzer(Abbott Laboratories, Abbott Park, IL, USA)] in the Zhongnan Hospitalof Wuhan University. The protein content of the hepatic supernatantwas determined using a commercial kit (Jiancheng BioengineeringInstitute, Nanjing, China), with bovine serum albumin as a standard.

2.6. Statistical analysis

All data were presented as mean ± S.E.M. (standard error of themean). The normality of data was assessed by using SPSS softwarewith the Shapiro–Wilk test. All data were subjected to one-way analysisof variance (one-way ANOVA) using SPSS 18.0 software. Differencesbetween the means were tested by Tukey's test after homogeneityof variances was checked. The level of significance was chosen atP b 0.05. Dietary vitamin E requirement of grass carp was estimatedby the broken-line regression.

Table 2Proximate whole-body composition (% in dry weight) of juvenile grass carp fed different dieta

0 25 50

Crude protein (%) 47.94 ± 1.11 49.82 ± 2.00 49.58 ±Crude lipid (%) 34.55 ± 2.64 35.29 ± 0.94 36.43 ±Ash (%) 10.62 ± 0.67 11.20 ± 0.25 10.44 ±Moisture (%) 71.09 ± 1.56 72.57 ± 0.47 71.44 ±

Values represent means ± S.E.M. of three replicates, each of three fish.

3. Results

As shown in Table 2, the content of crude protein, crude lipid, ashand moisture was not significantly influenced by the dietary vitamin Elevels (P N 0.05).

The growth performance and feed utilization of fish fed withdifferent vitamin E diets after the 8-week feeding trial are presentedin Table 3. Fish fed diet with 100 mg kg−1 vitamin E had signifi-cantly the highest weight gain (WG) and specific growth ratio(SGR) (P b 0.05), followed by fish fed diets with 50 mg kg−1 and200 mg kg−1 vitamin E, and lowest in fish fed diets with 0 mg kg−1

and 400 mg kg−1 vitamin E. Significantly higher feed intake (FI) wasfound in fish fed 100 mg vitamin E kg−1 diet (P b 0.05) and the lowestin the control group. Therewas no significant difference in feed efficien-cy (FE), protein efficiency ratio (PER) and survival ratio (SR) among thegroups. The dietary vitamin E levels had no significant effect onviscerosomatic index (VSI), hepatosomatic index (HSI) and intraperito-neal fat ratio (IPF) (P N 0.05). However, HSI was lower in fish fed dietwith 50 and 100 mg kg−1 vitamin E than that of the other groups.

The hepatic GPx activity increased significantly with the increaseof dietary vitamin E (P b 0.05, Table 4). Higher activity of hepatic SODwas displayed in diets with increased vitamin E levels. Inversely,there was a significant decrease of the hepatic MDA content under thesupplementation of vitamin E (P b 0.05), and the lowest MDA value inthe liver was observed in fish fed with 400 mg kg−1 vitamin E diet.At the same time, the activity of serumSOD and CATwas affected signif-icantly by the dietary vitamin E (P b 0.05), displaying SOD and CAT thehigher activity in fish fed diet with 50 mg kg−1 vitamin E. Serum MDAconcentration showed a significant negative correlation with theincreased supplementation of dietary vitamin E (P b 0.05).

Fish fed the control diet had significantly higher serum ALT activitythan those fed other diets (P b 0.05, Table 5), and fish fed diet with400 mg kg−1 vitamin E had the lowest ALT activity. However, vitaminE did not notably affect the serum AST activity and ALB content. Fishfed 100 mg kg−1 dietary vitamin E had significantly higher AKP activitythan that of control group (P b 0.05).

4. Discussion

Among natural antioxidants, vitamin E has been found to offer aprotective role against the adverse influence of reactive oxygen andother free radicals, triggering the oxidation of polyunsaturated mem-brane phospholipids, critical proteins, or both (National ResearchCouncil, 1993). Vitamin E requirements for commonly cultured fishspecies have been demonstrated to be ranging from 6.26 to200 mg kg−1 α-tocopherol (Hamre, 2011; National ResearchCouncil, 2011). Many factors such as dietary lipid content andsource, or other antioxidants presented in diet as well as the fishbody condition may affect the vitamin E requirement of fish. Dietaryvitamin E requirement for common carp Cyprinus carpio has been de-termined to be 100 mg kg−1 (Watanabe et al., 1970). Later, it hasbeen reported that elevated dietary linolenic fatty acid (18:2n6)levels increased the α-tocopherol requirement of common carp tobe 200 to 300 mg kg−1 (Watanabe et al., 1977). Further, Watanabeet al. (1981) determined that in common carp, an increase in

ry vitamin E levels for 8 weeks.

100 200 400

0.93 50.57 ± 1.49 48.07 ± 0.17 47.79 ± 0.701.92 36.96 ± 0.89 38.37 ± 0.57 37.93 ± 1.430.56 11.39 ± 0.41 10.16 ± 0.15 11.10 ± 0.480.67 72.10 ± 0.67 71.56 ± 0.35 71.95 ± 0.66

Table 3Growth performance and nutritional and biometric indices of juvenile grass carp fed different dietary vitamin E levels for 8 weeks.

0 25 50 100 200 400

WG (%) 103.28 ± 8.22a 121.66 ± 7.47b 155.26 ± 3.72c 185.90 ± 4.54d 155.35 ± 4.52c 102.61 ± 3.51a

SGR (%) 1.26 ± 0.01a 1.42 ± 0.01b 1.66 ± 0.04c 1.88 ± 0.03d 1.67 ± 0.02c 1.26 ± 0.03a

FI (g) 0.33 ± 0.03a 0.37 ± 0.01ab 0.58 ± 0.12bc 0.67 ± 0.05c 0.59 ± 0.08bc 0.46 ± 0.04abc

FE 0.54 ± 0.03 0.58 ± 0.06 0.56 ± 0.05 0.55 ± 0.01 0.54 ± 0.01 0.45 ± 0.06PER 1.68 ± 0.10 1.84 ± 0.18 1.77 ± 0.16 1.74 ± 0.04 1.68 ± 0.17 1.41 ± 0.17VSI (%) 11.78 ± 0.45a 11.83 ± 0.43a 11.61 ± 0.45a 12.42 ± 0.47ab 13.43 ± 0.38b 12.52 ± 0.49ab

HSI (%) 2.49 ± 0.20ab 2.63 ± 0.20ab 2.09 ± 0.27a 2.20 ± 0.25a 2.59 ± 0.14ab 2.94 ± 0.22b

IPF (%) 2.64 ± 0.10a 2.74 ± 0.14a 2.91 ± 0.08ab 2.88 ± 0.10ab 3.11 ± 0.04b 2.91 ± 0.09ab

SR (%) 83.334 ± 6.00 88.33 ± 6.67 86.67 ± 6.00 88.33 ± 3.33 90.00 ± 2.89 85.00 ± 5.77

Values represent means ± S.E.M. of three replicates, each of three fish. Values within the same row with different letters are significantly different (P b 0.05).WG (weight gain, %) = 100 × (W2 − W1) / W1, W1 and W2 indicated the initial weight and final mean weight (g), respectively; SGR (specific growth ratio, %) = 100 ×(lnW2 − lnW1) / time (days); FI (feed intake, g per fish per day) = feed consumed (g, dry weight) / fish amount / time (days); FE (feed efficiency) = (W2 − W1) / feedconsumed (g, dry weight); PER (protein efficiency ratio, %) = (W2 − W1) / total protein fed (g); VSI (viscerosomatic index, %) = 100 × [viscera weight (g) / whole body weight (g)];HSI (hepatosomatic index, %) = 100 × [liver weight (g) / whole body weight (g)]; IPF (intraperitoneal fat ratio, %) = 100 × [intraperitoneal fat weight (g) / whole body weight (g)];SR (survival ratio, %) = 100 × final amount of fish / initial amount of fish.

24 J. Li et al. / Aquaculture 430 (2014) 21–27

unsaturation in the dietary lipid caused an increase in α-tocopherol re-quirement. Based on the broken-line regression analysis of WG data, itwas determined that 100.36 mg kg−1 vitamin E was the optimalrequirement of grass carp in the present study (Fig. 1). This result waslower than the reported 200 mg kg−1 α-tocopherol, below whichlordosis was observed in grass carp (Takeuchi et al., 1992).

Diets containing different levels of vitamin E did not significantlyaffect whole body proximate composition. Similar results have beenreported by Gatta et al. (2000) and Zhou et al. (2013) in sea bassDicentrarchus labrax and juvenile cobia, respectively. However, Huanget al. (2003) found that the body protein content of hybrid tilapiaOreochromis niloticus × Oreochromis aureus fed the lowest level ofvitamin E was significantly lower than fish fed other diets. Sau et al.(2004) also found significant difference in crude protein of rohu fryamong the treatments. Watanabe and Takashima (1977) indicatedthat reduced whole-body crude protein and lipid, and high moisturecontent in the vitamin E-deficient fish were associated with severityof muscular atrophy.

In the present study, WG and FI were enhanced by the vitamin E,but it tends to be decreased in diet with higher vitamin E levels(N100 mg kg−1). The finding is in agreement with the study of finger-ling Channa punctatus which accepted dietary vitamin E at a high level(N140 mg kg−1) and resulted in a significant reduction in WG (Abdel-Hameid et al., 2012). Kiron et al. (2004) reported poor growth andfeed utilization by incorporating 1000 mg kg−1 vitamin E in the dietof rainbow diet. The growth depression at super-optimum levels ofdietary vitamin E may be because of the imbalance and accumulationof vitamin E radicals, which may act as pro-oxidants (Hamre et al.,1997). Similarly, Takeuchi et al. (1992) also reported a decreasedtendency in the WG of grass carp after the vitamin E supplementationexceeded 94 mg kg−1, while no significant effect was found in thedaily feed consumption and FE. However, in the study of red drum,

Table 4Antioxidant enzymes activities and MDA values in the liver and serum of juvenile grass carp fe

Parameter 0 25 50

LiverGPx (U mg−1 protein) 2377.71 ± 16.49a 2878.74 ± 38.81b 2916.2SOD (U mg−1 protein) 294.57 ± 5.91a 294.12 ± 11.10a 294.5MDA (nmol mg−1 protein) 15.06 ± 0.60d 8.56 ± 0.25c 7.2

SerumSOD (U mL−1) 57.97 ± 2.64bc 59.55 ± 5.00bc 63.8CAT (U mL−1) 3.84 ± 0.27c 4.51 ± 0.23c 6.6MDA (nmol mL−1) 13.77 ± 0.60d 9.97 ± 0.51c 9.8

Values representmeans ± S.E.M. of three replicates, each of three fish (liver) and five fish (seruGPx = glutathione peroxidase; GSH = reduced glutathione; SOD = superoxide dismutase; M

both WG and FE were significantly altered by the dietary vitamin Elevels without dose-dependent responses (Peng and Gatlin, 2009).Furthermore, vitamin E deficient diets did not have significant effecton growth in gilthead sea bream Sparus aurata L. (Montero et al.,2001), juvenile turbot Scophthalmus maximus L., halibut Hippoglossushippoglossus L. (Tocher et al., 2002), white sturgeon Acipensertransmontanus (Moreau and Dabrowski, 2003) and juvenile goldenshiner Notemigonus crysoleucas (Chen et al., 2004). The differencemight be due tofish species, individual size, development stage, cultiva-tion environment and other interacting nutrients in the experimentaldiets, such as vitamin C (Yildirim-Aksoy et al., 2008) and selenium(Bell et al., 1985). In our study, different vitamin E levels had no signif-icant influence on the FE and PER, but FIwas changed obviously. This in-dicated that vitamin E had a significant effect in increasing the weights,at least in part, by improving the feed intake. Previous reports of vitaminE did not mention FI of fish. There might be a lipid peroxidation that oc-curred in the vitamin E deficiency diets. Dietary lipid oxidation alteredthe odor and consequently the taste of the diet reduced (Gao et al.,2012). And vitamin E may improve the palatability of the feed bypreventing lipid oxidation to promote ingestion.

Biometric indices of particular organs or tissues relative to total bodymass (such as condition factor, VSI and HSI) were used as indices ofchange in nutritional and energy status (Goede and Barton, 1990).In our study, HSI was lower in fish fed diets with 50 mg kg−1 and100 mg kg−1 vitamin E than that of other groups. The increase of HSIin fish fed diets free of vitamin E and at sub-optimum levels(b50 mg kg−1) may be due to the accumulation of lipid peroxidates(Baker and Davies, 1996). And this is consistent with the result of highhepatic MDA value in fish fed vitamin E deficiency diets. In addition,liver is the main retention place for α-tocopherol (Hamre, 2011). Theincreased HSI in diets with higher vitamin E levels (N100 mg kg−1)might be due to the deposition of vitamin E along with liver fat

d different dietary vitamin E levels for 8 weeks.

100 200 400

3 ± 18.60b 2862.26 ± 47.68b 3150.38 ± 51.34c 3306.29 ± 35.93d

5 ± 12.66a 298.90 ± 4.19ab 299.14 ± 9.05ab 322.75 ± 3.29b

4 ± 0.19b 7.75 ± 0.55bc 0.72 ± 0.06a 0.66 ± 0.01a

4 ± 3.12c 55.26 ± 2.52abc 49.77 ± 1.98ab 46.32 ± 2.21a

7 ± 0.15d 4.21 ± 0.16c 3.16 ± 0.24b 1.20 ± 0.12a

6 ± 0.43c 8.48 ± 0.28b 9.20 ± 0.19bc 4.93 ± 0.24a

m). Valueswithin the same rowwith different letters are significantly different (P b 0.05).DA = malondialdehyde; CAT = catalase.

Table 5Serum biochemistry parameters of juvenile grass carp fed different dietary vitamin E levels for 8 weeks.

Enzyme 0 25 50 100 200 400

ALT (mU mL−1) 11.25 ± 0.65c 9.85 ± 0.05b 9.07 ± 0.30ab 9.13 ± 0.27ab 9.30 ± 0.53ab 8.30 ± 0.06a

AST (mU mL−1) 56.15 ± 2.95 56.35 ± 3.95 49.03 ± 5.34 47.97 ± 1.52 49.53 ± 5.81 47.80 ± 0.56AKP (mU mL−1) 151.45 ± 3.35a 167.97 ± 8.02ab 253.20 ± 5.00c 273.20 ± 5.03d 169.27 ± 5.26ab 178.50 ± 5.83b

ALB (mg mL−1) 10.10 ± 0.90 9.95 ± 0.05 12.00 ± 1.80 12.00 ± 0.67 10.83 ± 0.47 11.30 ± 0.06

Values represent means ± S.E.M. of three replicates, each of five fish. Values within the same row with different letters are significantly different (P b 0.05).ALT = alanine transaminase; AST = aspartate transaminase; AKP = alkaline phosphatase; ALB = albumin.

25J. Li et al. / Aquaculture 430 (2014) 21–27

(Amlashi et al., 2011). The same tendency has been observed in finger-ling C. punctatus (Abdel-Hameid et al., 2012) and gilthead sea bream(Mourente et al., 2002).

Dietary vitamin E also showed some relationships with the activitiesof antioxidant defense system. Mourente et al. (2000) and Tocher et al.(2002) showed that the level of dietary tocopherol had significanteffects on the activities of the liver antioxidant enzymes in juvenilemarine fish. It is well established that GPx is particularly important inpreventing free radical initiation in membranes since it is a very effec-tive scavenger of H2O2 (Cay and King, 1980). And the synergistic func-tion of selenium and vitamin E to increase GPx activity has alreadybeen well established in the case of the liver of fishes (Wise et al.,1993). However, excess supplementation ofα-tocopherol to the biolog-ical system leads to acceleration of lipid peroxidation (Kaewsrithonget al., 2001). Thus, the induction of GPx may be a compensatory physi-ological adaptive response to reduce the oxidative stress (Dandapatet al., 2000). What's more, our results of the significantly enhancedGPx activity and the increased SOD activity in the liver in response tovitamin E supplementation also corroborate with the decreased hepaticMDA content. These results might support the hypothesis that the in-creased activities of both enzymes in the liver might be able to counter-act the pro-oxidant effect induced by the excess doses of α-tocopherol.However, no significant interaction was observed between dietary vita-min E and hepatic GPx activity in Atlantic salmon Salmo salar L. (Lygrenet al., 2000). Jaramillo et al. (2009) reported that the lack of dietaryvitamin E supplementation increased whole-body glutathione per-oxidase expression and increased selenium digestion, absorptionand deposition in the whole-body of hybrid striped bass Moronechrysops × Morone saxatilis. As pointed out by Gatellier et al.(2004), the effect of vitamin E on GPx activity was strongly controversial,which merited further investigations.

However, a decrease in SOD and CAT activities was found in theabsence of dietary vitamin E supplements (b50mg kg−1). The activitiesof these two enzymeswould be expected to parallel each other based onthe known mechanisms of the enzymes and the fact that superoxideanions are known to be efficiently scavenged by vitamin E in biologicalsystems (Cay and King, 1980). The decrease of antioxidant enzymesactivities might be responsible for scavenging superoxide anion

y = 0.8386x + 100.49R² = 0.9964

0

20

40

60

80

100

120

140

160

180

200

0 50 100 150 200 250 300 350 400 450

Wei

ght g

ain

(%)

Dietary vitamin E concentration (mg kg-1)

100.36 mg kg-1

Ymax = 184.65

Fig. 1. The effect of dietary vitamin E on weight gain (%) of juvenile grass carp. Each pointrepresents the mean of three aquaria of fish with 20 fish per aquarium (n = 3). Dietaryvitamin E requirement derived from the broken-line regression model is 100.36mg kg−1.

(Wang and Chen, 2005). On the other hand, the decreased activitiesof SOD and CAT as observed in our study may be responsible for theelevated lipid peroxidation (Chaudiere and Ferrari-Illiou, 1999). Atthe same time, we also discovered a decreasing trend of serum SODactivity when vitamin E supplementation is beyond 50 mg kg−1.The decreasing serum SOD activity may be indicative of a decreasingneed to detoxify the superoxide radical (Tocher et al., 2002). SerumCAT activity was enhanced in grass carp fed 50 mg vitamin E kg−1

diet, whereas it also decreased significantly as the content of vitaminE in the diet increased. It has been suggested that CAT can eliminateH2O2 effectively and rapidly due to the direct stimulatory action ofvitamin E (Liu et al., 2007). Thus, the low concentration of H2O2

may be responsible for the decrease CAT activity in the serum asCAT is an ineffective scavenger of H2O2 at low concentration(Dandapat et al., 2000). These changes of the serum SOD and CAT ac-tivities indicated that an adequate protection against oxidative stresscould be achieved by a moderate dose of vitamin E, and that a highsupplementation may be unnecessary, similar to results in rainbowtrout (Puangkaew et al., 2004) and sea cucumber Apostichopusjaponicus (Wang et al., 2013).

In the present study, the MDA values in both hepatic and serumwere inversely related to the dietary vitamin E levels. The resultsuggested that increased vitamin E could reduce the level of peroxida-tion in tissues. Tocher et al. (2002) reported that inadequacy of dietaryvitamin E could enhance the level of fatty acid peroxidation, mainlybecause of the formation of considerable amounts of oxidative radicalsin the tissues. The same tendency was also found in the study on hybridtilapia (Huang and Huang, 2004), grouper (Lin and Shiau, 2005), reddrum (Peng and Gatlin, 2009), and juvenile cobia (Zhou et al., 2013),that tocopherol improved the protection against peroxidation andresulted in decreased levels of TBARS.

Wells et al. (1986) reported that the increase of plasma AST and ALTactivities may give information on liver injury or dysfunction. In thepresent study, serumALT activitywas decreasedwith the supplementa-tion of vitamin E, indicating that a better trend occurred in the liverunder increasing dietary vitamin E levels. And this might be associatedwith the antioxidant function of vitamin E which stabilizes biologicalmembranes (Wang and Quinn, 2000; Zimmer et al., 1993) and avoidscell damage (Kanazawa, 1993). AKP has been reported to be involvedin immune defense mechanisms and is correlated with the immunecompetence (Yan et al., 2014). Decrease in AKP activity may be takenas an index of disturbance of membrane transport system (Öner et al.,2008). The increase of serum AKP activity in our study indicated thatvitamin E might have stimulated the innate immune responses ingrass carp; however, a high dose of vitamin E inhibited the activity ofserum AKP. Amlashi et al. (2011) reported that vitamin E might be in-volved indirectly in modulating growth by its effect on immunologicalparameters. Thus, reduced AKP activity might be associated with thegrowth depression at super-optimum levels of dietary vitamin E inthis study. Similarly, Jiang et al. (2009) showed that 7.0 μgmL−1 vitaminE gave lowered AKP of fish enterocytes. Nevertheless, Zhou et al. (2013)found no aspect of vitamin E on plasma AKP activities of juvenile cobia.To the best of our knowledge, the increase in serumALB level is thoughtto be associated with a stronger innate immune response of fish(Wiegertjes et al., 1996). However, we observed no difference inserum ALB between all treatments. Sahoo and Mukherjee (2002) also

26 J. Li et al. / Aquaculture 430 (2014) 21–27

found that high α-tocopherol (1000 mg kg−1 feed) had no significantinfluence on the serum ALB of Indianmajor carp L. rohita in comparisonwith the control group (100 mg kg−1 feed). There was not much in-formation about the effect of vitamin E on the serum ALB; therefore,a further study is needed.

In conclusion, the results indicated that appropriate vitamin Eenhanced growth performance and antioxidant status in juvenile grasscarp. The supplementation of vitamin E at 100.36 mg kg−1 dietarycould be considered as a suggested dose in balancing the growthperformance and antioxidant status. The benefits of excess vitaminE levels need to be weighed carefully: on the one hand might en-hance the antioxidant capacity, on the other hand might decreasethe growth.

Acknowledgments

This work was financially supported by the National Natural ScienceFoundation of China (31272641 and 31172420), the National BasicResearch Program of China (2014CB138601), the Special Fund forAgro-Scientific Research in the Public Interest of China (201003020)and the Fundamental Research Funds for the Central Universities(2011PY030).

References

Abdel-Hameid, N.A.H., Abidi, S.F., Khan, M.A., 2012. Dietary vitamin E requirementfor maximizing the growth, conversion efficiency, biochemical composition andhaematological status of fingerling Channa punctatus. Aquac. Res. 43, 226–238.

Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126.Agradi, E., Abrami, G., Serrini, G., Mckenzie, D., Bolis, C., Bronzi, P., 1993. The role of dietary

n−3 fatty acid and vitamin E supplements in growth of sturgeon (Acipenser naccarii).Comp. Biochem. Physiol. A 105, 187–195.

Amlashi, A.S., Falahatkar, B., Sattari, M., Gilani, M.H.T., 2011. Effect of dietary vitamin Eon growth, muscle composition, hematological and immunological parameters ofsub-yearling beluga Huso huso L. Fish Shellfish Immunol. 30, 807–814.

AOAC (Association of Official Analytical Chemists), 1995. Official Methods of Analysis ofOfficial Analytical Chemists International, 16th edn. AOAC, Arlington, Virginia, USA.

Baker, R.T.M., Davies, S.J., 1996. Oxidative nutritional stress associated with feeding rancidoils to African catfish, Clarias gariepinus (Burchell) and the protective role ofα-tocopherol. Aquac. Res. 27, 795–803.

Bell, J.G., Cowey, C.B., Adron, J.W., Shanks, A.M., 1985. Some effects of vitamin E andselenium deprivation on tissue enzyme levels and indices of tissue peroxidation inrainbow trout (Salmo gairdneri). Br. J. Nutr. 53, 149–157.

Beuge, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52,302–310.

Buettner, G.R., 1993. The pecking order of free radicals and antioxidants: lipid peroxida-tion, α-tocopherol, and ascorbate. Arch. Biochem. Biophys. 300, 535–543.

Cay, P.B., King, M.M., 1980. Vitamin E: its role as a biological free radical scavenger and itsrelationship to the microsomal mixed function oxidase system. In: Machlin, L.J. (Ed.),Vitamin E, A Comprehensive Treatise: Basic and Clinical Nutrition, vol. 1. MarcelDekker, New York, pp. 289–317.

Chaudiere, J., Ferrari-Illiou, R., 1999. Intracellular antioxidant from chemical to biochemicalmechanism. Food Chem. Toxicol. 37, 949–962.

Chen, R.G., Lochmanna, R., Goodwina, A., Praveenb, K., Dabrowskic, K., Lee, K.J., 2004.Effects of dietary vitamins C and E on alternative complement activity, hematology,tissue composition, vitamin concentrations and response to heat stress in juvenilegolden shiner (Notemigonus crysoleucas). Aquaculture 242, 553–569.

Coz-Rakovac, R., Strunjak-Perovic, I., Hacmanjek,M., Topic Popovic, N., Lipej, Z., Sostaric, B.,2005. Blood chemistry and histological properties of wild and cultured sea bass(Dicentrarchus labrax) in the North Adriatic Sea. Vet. Res. Commun. 29, 677–687.

Dandapat, J., Chainy, G.B.N., Rao, K.J., 2000. Dietary vitamin-E modulates antioxidantdefence system in giant freshwater prawn, Macrobrachium rosenbergii. Comp.Biochem. Physiol. C 127, 101–115.

Frischknecht, R., Wahli, T., Meier, W., 1994. Comparison of pathological changes due todeficiency of vitamin C, vitamin E and combinations of vitamins C and E in rainbowtrout, Oncorhynchus mykiss (Walbaum). J. Fish Dis. 17, 31–45.

Gao, J., Koshio, S., Ishikawa, M., Yokoyama, S., Mamauag, R.E.P., Han, Y., 2012. Effects ofdietary oxidized fish oil with vitamin E supplementation on growth performanceand reduction of lipid peroxidation in tissues and blood of red sea bream Pagrusmajor. Aquaculture 356, 73–79.

Gatellier, P., Mercier, Y., Renerre, M., 2004. Effect of diet finishing mode (pasture or mixeddiet) on antioxidant status of Charolais bovine meat. Meat Sci. 67, 385–394.

Gatta, P.P., Pirini, M., Testi, S., Vignola, G., Monetti, P.G., 2000. The influence of differentlevels of dietary vitamin E on sea bass Dicentrarchus labrax flesh quality. Aquacult.Nutr. 6, 47–52.

Goede, R.W., Barton, B.A., 1990. Organismic indices and an autopsy based assessment asindicators of health and condition of fish. Am. Fish. Soc. Symp. 8, 93–108.

Hadi, A., Shokr, A., Alwan, S., 2009. Effects of aluminum on the biochemical parameters offresh waterfish Tilapia zillii. J. Sci. Appl. 3, 33–41.

Hamre, K., 2011. Metabolism, interaction, requirements and functions of vitamin E in fish.Aquacult. Nutr. 17, 98–115.

Hamre, K., Lie, Ø., 1995. α-Tocopherol levels in different organs of Atlantic salmon(Salmo salar L.) — effect of smoltification, dietary levels of polyunsaturated fattyacids and vitamin E. Comp. Biochem. Physiol. A 111, 547–554.

Hamre, K., Hjeltnes, B., Kryvi, H., Sandberg, S., Lorentzen, M., Lie, Ø., 1994. Decreasedconcentration of hemoglobin, accumulation of lipid oxidation products andunchanged skeletal muscle in Atlantic salmon (Salmo salar) fed low dietaryvitamin E. Fish Physiol. Biochem. 12, 421–429.

Hamre, K., Waagbø, R., Berge, R.K., Lie, Ø., 1997. Vitamins C and E interact in juvenileAtlantic salmon (Salmo salar L.). Free Radical Biol. Med. 22, 137–149.

Han, D., Xie, S., Liu, M., Xiao, X., Liu, H., Zhu, X., Yang, Y., 2011. The effects of dietaryselenium on growth performances, oxidative stress and tissue selenium concen-tration of gible carp (Carassius auratus gibelio). Aquacult. Nutr. 17, 741–749.

Huang, C.H., Huang, S.L., 2004. Effect of dietary vitamin E on growth, tissue lipidperoxidation, and liver glutathione level of juvenile hybrid tilapia, Oreochromisniloticus × O. aureus, fed oxidized oil. Aquaculture 237, 381–389.

Huang, C.H., Chang, R.J., Huang, S.L., Chen, W.L., 2003. Dietary vitamin E supplementationaffects tissue lipid peroxidation of hybrid tilapia, Oreochromis niloticus × O. aureus.Comp. Biochem. Physiol. B 134, 265–270.

Ito, T., Murata, H., Tsuda, T., Yamada, T., Yanauchi, K., Ukawa,M., Yamaguchi, T., Yoshida, T.,Sakai, T., 1999. Effects of α-tocopherol levels in extrusion pellets on in vivo lipidperoxidation levels and antioxidant activities in cultured yellowtail Seriolaquinqueradiata injected with the causative bacteria of fish jaundice. Fish. Sci. 65,679–683.

Jaramillo, J.R.F., Peng, L.I., Gatlin III, D.M., 2009. Selenium nutrition of hybrid striped bass(Morone chrysops × M. saxatilis) bioavailability, toxicity and interaction with vitaminE. Aquacult. Nutr. 15, 160–165.

Jiang, J., Zheng, T., Zhou, X.Q., Liu, Y., Feng, L., 2009. Influence of glutamine and vitamin Eon growth and antioxidant capacity of fish enterocytes. Aquacult. Nutr. 15, 409–414.

Kaewsrithong, J., Ohshima, T., Ushio, H., Nagasaka, R., Maita, M., Sawada, M., 2001. Effectsof an excess dose of dietary α-tocopherol on hydroperoxide accumulation anderythrocyte osmotic fragility of sweet smelt Plecoglossus altivelis (Temmincket Schlegel). Aquac. Res. 32, 191–198.

Kanazawa, K., 1993. Tissue injury induced by dietary products of lipid peroxidation. In:Corongiu, F. (Ed.), Free Radicals and Antioxidants in Nutrition. Richelieu Press,London, pp. 383–399.

Kiron, V., Puangkaew, J., Ishizka, K., Satoh, S., Watanabe, T., 2004. Antioxidant status andnonspecific immune responses in rainbow trout (Oncorhynchus mykiss) fed twolevels of vitamin E along with three lipid sources. Aquaculture 234, 361–379.

Kocabas, A.M., Gatlin III, D.M., 1999. Dietary vitamin E requirement of hybrid striped bass(Morone chrysops female × M. saxatilis male). Aquacult. Nutr. 5, 3–7.

Lin, Y.H., Shiau, S.Y., 2005. Dietary vitamin E requirement for grouper, Epinephelusmalabaricus, at two lipid levels, and their effects on immune responses. Aquaculture248, 235–244.

Liu, Y., Wang, W.N., Wang, A.L., Wang, J.M., Sun, R.Y., 2007. Effects of dietary vitamin Esupplementation on antioxidant enzyme activities in Litopenaeus vannamei (Boone,1931) exposed to acute salinity changes. Aquaculture 265, 351–358.

Lygren, B., Hamre, K., Waagbø, R., 2000. Effect of induced hyperoxia on the antioxidantstatus of Atlantic salmon Salmo salar L. fed three different levels of dietary vitaminE. Aquac. Res. 31, 401–407.

McCord, J.M., Fridovich, I., 1969. Superoxide dismutase: an enzymatic function forerythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049–6055.

Miller, J.K., Brzezinska-Slebodzinska, E., Madsen, F.C., 1993. Oxidative stress, antioxidants,and animal function. J. Dairy Sci. 76, 2812–2823.

Montero, D., Tort, L., Robaina, L., Vergara, J.M., Izquierdo, M.S., 2001. Low vitamin E indiet reduces stress resistance of gilthead sea bream (Sparus aurata) juveniles. FishShellfish Immunol. 11, 473–490.

Moreau, R., Dabrowski, K., 2003. α-Tocopherol downregulates gulonolactone oxidaseactivity in sturgeon. Free Radical Biol. Med. 34, 1326–1332.

Mourente, G., Díaz-Salvago, E., Tocher, D.R., Bell, J.G., 2000. Effects of dietarypolyunsaturated fatty acid/vitamin E (PUFA/tocopherol) ratio on antioxidantdefence mechanisms of juvenile gilthead sea bream (Sparus aurata L., Osteichthyes,Sparidae). Fish Physiol. Biochem. 23, 337–351.

Mourente, G., Díaz-Salvago, E., Bell, J.G., Tocher, D.R., 2002. Increased activities of hepaticantioxidant defence enzymes in juvenile gilthead sea bream (Sparus aurata L.) feddietary oxidised oil: attenuation by dietary vitamin E. Aquaculture 214, 343–361.

Mourente, G., Bell, G.J., Tocher, D.R., 2007. Does dietary tocopherol level affect fatty acidmetabolism in fish? Fish Physiol. Biochem. 33, 269–280.

National Research Council, 1993. Nutrient Requirement of Fish. National Academy Press,Washington, DC.

National Research Council, 2011. Nutrient Requirements of Fish and Shrimp. NationalAcademy Press, Washington, DC.

Noguchi, T., Cantor, A.H., Scott, M.L., 1973. Mode of action of selenium and vitamin E inprevention of exudative diathesis in chicks. J. Nutr. 103, 1502–1511.

Öner, M., Atli, G., Canli, M., 2008. Changes in serum biochemical parameters of freshwaterfish Oreochromis niloticus following prolonged metal (Ag, Cd, Cr, Cu, Zn) exposures.Environ. Toxicol. Chem. 27, 360–366.

Peng, L.I., Gatlin III, D.M., 2009. Dietary vitamin E requirement of the red drum Sciaenopsocellatus. Aquacult. Nutr. 15, 313–319.

Puangkaew, J., Kiron, V., Somamoto, T., Okamoto, N., Satoh, S., Takeuchi, T., Watanabe,T., 2004. Nonspecific immune response of rainbow trout (Oncorhynchus mykissWalbaum) in relation to different status of vitamin E and highly unsaturatedfatty acids. Fish Shellfish Immunol. 16, 25–39.

27J. Li et al. / Aquaculture 430 (2014) 21–27

Reed, D.J., 1990. Glutathione: toxicological implications. Annu. Rev. Pharmacol. Toxicol.30, 603–631.

Sahoo, P.K., Mukherjee, S.C., 2002. Influence of high dietary α-tocopherol intakes onspecific immune response, nonspecific resistance factors and disease resistance ofhealthy and aflatoxin B1-induced immunocompromised Indian major carp, Labeorohita (Hamilton). Aquacult. Nutr. 8, 159–167.

Sargent, J.R., Tocher, D.R., Bell, J.G., 2002. The lipids, In: Halver, J.E., Hardy, R.W. (Eds.), FishNutrition, 3rd edn. Academic Press, San Diego, pp. 182–246.

Sau, S.K., Paul, B.N., Mohanta, K.N., Mohanty, S.N., 2004. Dietary vitamin E requirement,fish performance and carcass composition of rohu (Labeo rohita) fry. Aquaculture240, 359–368.

Shahidi, F., Hong, C., 1991. Evaluation ofmalonaldehyde as a marker of oxidative rancidityin meat products. J. Food Biochem. 15, 97–105.

Silverman, L.M., Christenson, R.H., Grant, G.H., 1986. Amino acids and proteins. In:Norbert, W.T. (Ed.), Textbook of Clinical Chemistry. W.B. Saunders Publishers,Philadelphia, PA, pp. 579–585.

Takeuchi, T., Watanabe, K., Satoh, S., Watanabe, T., 1992. Requirement of grass carpfingerlings for α-tocopherol. Nippon Suisan Gakkaishi 58, 1743–1749.

Tocher, D.R.,Mourente, G., Van, D.E.A., Evjemo, J.O., Diaz, E., Bell, J.G., Geurden, I., Lavens, P.,Olsen, Y., 2002. Effects of dietary vitamin E on antioxidant defence mechanisms ofjuvenile turbot (Scophthalmus maximus L.), halibut (Hippoglossus hippoglossus L.)and sea bream (Sparus aurata L.). Aquacult. Nutr. 8, 195–207.

Tokuda, M., Takeuchi, M., 1995. Effects of excess doses of α-tocopherol on the lipids andfunction of rainbow trout liver. J. Nutr. Sci. Vitaminol. 41, 25–32.

Tokuda, M., Takeuchi, M., 1999. Effects of excess doses of α-tocopherol on the lipid inserum and muscle of rainbow trout. Fish. Sci. 65, 496–497.

Traystman, R.J., Kirsch, J.R., Koehler, R.C., 1991. Oxygen radical mechanisms of brain injuryfollowing ischemia and reperfusion. J. Appl. Physiol. 71, 1185–1195.

Wang, L.U., Chen, J.C., 2005. The immune response of white shrimp Litopenaeus vannameiand its susceptibility to Vibrio alginolyticus at different salinity levels. Fish ShellfishImmunol. 18, 269–278.

Wang, X.Y., Quinn, P.J., 2000. The location and function of vitamin E in membranes(review). Mol. Membr. Biol. 17, 143–156.

Wang, J., Xu, Y.P., Li, X.Y., Li, J.G., Bao, P.Y., Che, J., Li, S.Y., Jin, L.J., 2013. Vitamin Erequirement of sea cucumber (Apostichopus japonicus) and its effects on nonspecificimmune responses. Aquac. Res. 1–10.

Watanabe, T., Takashima, F., 1977. Effect ofα-tocopherol deficiency on carp-VI. Deficiencysymptoms and changes of fatty acid and triglyceride distribution in adult carp. Bull.Jpn. Soc. Sci. Fish. 43, 819–830.

Watanabe, T., Takashima, F., Ogino, C., Hibiya, T., 1970. Requirements of young carpfor α-tocopherol. Bull. Jpn. Soc. Sci. Fish. 36, 972–976.

Watanabe, T., Takeuchi, T., Matsui, M., Ogino, C., Kawabata, T., 1977. Effect ofα-tocopheroldeficiency on carp. VII: the relationship between dietary levels of linoleate andα-tocopherol requirement. Bull. Jpn. Soc. Sci. Fish. 43, 935–946.

Watanabe, T., Takeuchi, T., Wada, M., 1981. Dietary lipid levels and α-tocopherolrequirement of carp. Bull. Jpn. Soc. Sci. Fish. 47, 935–946.

Wells, R., McIntyre, R., Morgan, A., Davie, P., 1986. Physiological stress responses in biggamefish after exposure: observation on plasma chemistry and blood factors.Comp. Biochem. Physiol. A 64, 565–571.

Wiegertjes, G.F., Stet, R.J.M., Parmentier, H.K., Muiswinkel, W.B.V., 1996. Immunogeneticsof disease resistance in fish: a comparative approach. Dev. Comp. Immunol. 20,365–381.

Wise, D.J., Tomasso, J.R., Gatlin III, D.M., Bai, S.C., Blazer, V.S., 1993. Effects of dietary sele-nium and vitamin E on red blood cell peroxidation, glutathione peroxidase activity,and macrophage superoxide anion production in channel catfish. J. Aquat. Anim.Health. 5, 177–182.

Yan, F.J., Tian, X.L., Dong, S.L., Fang, Z.H., Yang, G., 2014. Growth performance, immuneresponse, and disease resistance against Vibrio splendidus infection in juvenile seacucumber Apostichopus japonicus fed a supplementary diet of the potential probioticParacoccus marcusii DB 11. Aquaculture 420, 105–111.

Yildirim-Aksoy, M., Lim, C., Li, M.H., Klesius, P.H., 2008. Interaction between dietary levelsof vitamins C and E on growth and immune responses in channel catfish, Ictaluruspunctatus (Rafinesque). Aquac. Res. 39, 1198–1209.

Yu, S.K., Olsen, C.E., Marcussen, J., 1997. Methods for the assay of 1, 5-anhydro-D-fructoseand α-1, 4-glucanlyase. Carbohydr. Res. 305, 73–82.

Zhou, Q.C., Wang, L.G., Wang, H.L., Xie, F.J., Wang, T., 2012. Effect of dietary vitamin C onthe growth performance and innate immunity of juvenile cobia (Rachycentroncanadum). Fish Shellfish Immunol. 30, 969–975.

Zhou, Q.C., Wang, L.G., Wang, H.L., Wang, T., Elmada, C.Z., Xie, F.J., 2013. Dietaryvitamin E could improve growth performance, lipid peroxidation and non-specificimmune responses for juvenile cobia (Rachycentron canadum). Aquacult. Nutr. 19,421–429.

Zimmer, G., Thurich, T., Scheer, B., 1993. Membrane fluidity and vitamin E. In: Packer, L.,Fuchs, J. (Eds.), Vitamin E in Health and Disease. Marcel Dekker Inc., New York, Basel,Hong Kong, pp. 207–222.