Indian Journal of BiotechnologyVol. 2, April 2003, pp. 195-202

Improved Post-larval Production in Giant Prawn, Macrobrachium rosenbergii,through Modulation of Antioxidant Defence System by Dietary Vitamin-E

Jagneshwar Dandapat', Gagan B N Chainy'" and K Janardhana Rao2

'Centre for Biotechnology and Biochemistry Unit, Department of Zoology, Utkal University, Bhubaneswar 751 004. India2Central Institute of Freshwater Aquaculture, Kau alyaganga, Bhubaneswar 751 002, India

Received 25 June 2002; accepted 6 September 2002

Giant freshwater prawn, Macrobrachium rosenbergii (deMan) (Crustacea-Decapoda) is an economicallyimportant species widely cultured in both freshwater and low saline water (brackish water) aquaculture. Theexperiments were conducted to study the effects of dietary supplementation of vitamin-E on the post-larvalproduction (seed production), lipid peroxidation (LPX) and antioxidant defence system in the post larvae of M.rosenbergii. Objective of the study is to refine the existing seed production technology through the modulation ofantioxidant defence system during larval progression. Results of the first feeding trial clearly exhibit the increasedpost-larval production in response to different levels of supplementary vitamin-E (50, 100, 200 or 400 mglkg feed).Results of the second feeding experiment with a selective dose of vitamin-E (200 mglkg feed) were also consistent withthat and it was further observed that vitamin-E supplemented diet (200 mglkg feed) can reduce the level of LPX inthe post larvae. The activity of superoxide dismutase (SOD) and catalase (CAT) was reduced significantly but that ofglutathione peroxidase (GPX) was elevated in the post larvae receiving vitam.in-E supplementation. Though ascorbicacid content of the post larvae was elevated in response to vitamin-E supplemented diet, glutathione (GSH) contentremained unaltered. The present findings indicate that modulation of antioxidant defence system in response tovitamin-E supplementation in the diet might have a positive role in improving post-larval production.

Keywords: Prawn seed production, vitamin-E, lipid peroxidation, antioxidant defence system

IntroductionVitamin-E (a-tocopherol) is an essential nutrient

for normal life processes like growth, developmentand reproduction. It is also a membrane bound, lipidsoluble antioxidant and prevents peroxidation ofpolyunsaturated fatty acids (PUFA) from theoxidative assault caused by reactive oxygen species(ROS) (National Research Council, 1983).Superoxide radical (02'-), hydroxyl radical (OH),H202, etc., are well known ROS producedcontinuously during cellular metabolism. ROS beinghighly reactive, can damage biomoleculesnonspecifically (Halliwell & Gutteridge, 2001).However, among the biomolecules, lipids andparticularly PUFA are highly susceptible to ROSattack (Tappel, 1975; Gardner, 1989). Oxidativeattack to lipids is referred to as lipid peroxidation(LPX), a well acknowledged index of oxidative stress(imbalance between the generation and neutralisationof ROS in favour of the former).

*Author for correspondence:Tel: 0674-2581503, 2587389; Fax: 0674 - 2509037E-mail: chainyg@sify.com

Oxidative stress in the aquatic animals is generallyelevated during hypoxia, increased temperature,nutrient deficiency and even due to increasedincorporation of PUFA in the diet (Kolkovski et at,2000; Hwang & Lin, 2002; Mourente et at, 1999).Augmented oxidative stress has been linked to anumber of pathological conditions in the aquaticanimals and may lead to apoptosis (Faverney et at,2001). Antioxidant enzymes like superoxidedismutase (SOD), catalase (CAT), glutathioneperoxidase (GPX) and low molecular weightsubstances such as reduced glutathione (GSH),vitamins E and C are important antioxidants that limitthe oxidative assault to various biomolecules. It is anestablished fact that PUFA of both n-3 and n-6 typesare extremely important for the synthesis ofbiomembranes during cellular differentiation andorganogenesis. Further, the requirement of PUFA forthe larval development and growth of the aquaticanimals is also well documented (Watanabe, 1993;Glencross et at, 2002). Hence, PUFA should be wellprotected from the oxidative damage during larvaldevelopment and metamorphosis. Although a definiterole of ROS and antioxidants is established in various

196 INDIAN J BIOTECHNOL, APRIL 2003

cellular processes such as development,differentiation and regeneration (Allen & Venkatraj,1992; Mahapatra et al, 2001, 2002), informationdepicting the involvement of ROS duringdevelopment of aquatic animals in general andcrustaceans in particular is scanty.

The seed resources of giant freshwater prawn,Macrobrachuim rosenbergii (deMan), an importantcrustacean aquatic crop having potentiality for largescale aquaculture both in freshwater as well as lowsaline water (~7 parts per thousand-PPT), is thelimiting factor for the expansion of its culture. Hence,an alternative is the optimum production of seedsthrough a suitable hatchery technology(Subramoniam, 1999). Management of suitablehusbandry condition by avoiding stress is a key factorfor optimum seed production in the hatchery. It isevident that research to-date has focussed primarilyon the role of proteins, fatty acids and probiotics forthe enhancement of aquaculture production (Hebb etal, 1997; Glencross et al, 2002; Yasuda & Kitao,1980), but limited attention has been given towardsmicronutrients and antioxidants. Though a few reportssuggest the involvement of vitamin-E in the improvedproduction of aqua products (He & Lawrence, 1993;Gatta et al, 2000), none of the findings clearly unveilsthe mechanism of vitamin-E mediated effects.Recently, the authors reported the modulation ofantioxidant defence system by vitamin-E in thefreshwater prawn, which also suggests that thisantioxidant can attenuate oxidative stress through themodulation of antioxidant defence system (Dandapatet al, 2000). In the present study, the authorsdemonstrated the effect of supplementation of (J.-

tocopherol acetate in a formulated larval diet toapprise whether supplementation of vitamin-E canimprove post-larval production in crustaceans, and ifso, whether the improved post-larval production isdue to the modulation of antioxidant defence system.The specific objective of the study is to upgrade andrefine the existing seed production technology of M.rosenbergii, to improve post-larval output through themodulation of antioxidant defence system.

Materials and MethodsChemicals

Thiobarbituric acid (TBA), bovine serum albumin(BSA), glutathione reductase (GR), cumenehydroperoxide, sephadex G-25 and 5,5-Dithio-bis (2-nitrobenzoic acid)-DTNB, of analar grade werepurchased from Sigma Chemical Co USA. NADPH,

GSH, GSSG and ascorbic acid also of analar gradewere obtained from SISCO Research Laboratory,India.

Lipid Peroxidation (LPX) AssayFor the study of LPX, tissue homogenates (10%,

w/v homogenate in l.15% KC1) were centrifuged at1000 x g for 10 min at 4°C. The resulting postnuclearsupernatant was used for the estimation of LPX bymonitoring the formation of thiobarbituric acidreactive substances (TBARS) by the method ofOhkawa et al (1979) and was expressed as nmolTBARS formedimg protein. Sensitivity to in vitroLPX was estimated by incubating identical sampleswith 1000 /lM FeS04 at 37°C for 30 min in a shakingwater bath prior to LPX assay. Protein content of thesamples was measured according to the method ofLowry et al (1951) using BSA as standard.

Assay of Antioxidant EnzymesA 10% (w/v) homogenate was prepared in ice-cold

50 mM phosphate buffer (PH 7.4) with the help of amotor driven glass teflon homogeniser andcentrifuged at 10,000 x g for 20 min at 4°C. Thesupernatant was used directly for assay of catalase(CAT-EC 1.1l.1.6) activity following the decrease inabsorbency of H202 at 240 nm (Aebi, 1974), and wasexpressed as pkatlmg protein (1 katal = 1 mol see").One ml of the supernatant containing around 8 mg ofprotein was passed through a 5 ml column ofSephadex G-25 and the elute was used for estimationof superoxide dismutase (SOD-EC 1.15.1.1). TheSOD activity was estimated according to the methodof Das et al (2000), which involves generation ofsuperoxide radical by photoreduction of riboflavinand its detection by nitrite formation fromhydroxylamine hydrochloride at 543 nm using Greissreagent. To discriminate between CN-sensitive andCN-resistant SOD activities, identical samples wereincubated with 5 mM KCN (final concentration) for40 min at 37°C prior to SOD assay. Glutathioneperoxidase (GPX-EC 1.11.1.9) and glutathionereductase (GR-EC 1.6.4.2) activities were measuredaccording to the method of Paglia & Valentine (1967)and Massey & Williams (1965), respectively. Enzymeactivities were expressed as nmol NADPHoxidised/min/mg protein. Total GPX activity wasmeasured taking cumine-hydroperoxide as thesubstrate, whereas the Se-dependent enzyme activitywas assayed taking tert-butyl-hydroperoxide assubstrate.

DANDAPAT et al.: ROLE OF ANTIOXIDANT IN PRAWN SEED PRODUCTION

Assay of Antioxidant Small MoleculesA 10% (w/v) homogenate of the tissue in 5% (w/v)

metaphosphoric acid was centrifuged at 1000 x g for30 min at room temperature, and the deproteinizedsupernatant was used for assay of GSH using DTNB(Ellman, 1959), and ascorbic acid following thestoichiometric reduction of phosphomolybdate byascorbic acid (Mitusi & Ohata, 1961).

Diet Preparation and its Proximate BiochemicalAnalyses

Diet for different stages of the larvae was preparedfrom locally available ingredients. Percentagecomposition of various ingredients of the prepareddiet and its proximal biochemical analyses are givenin Tables 1 and 2, respectively. The ingredients forthe larval diet were mixed thoroughly in a mixer andwas cooked in steam. The prepared feed was keptunder refrigeration and was prepared fresh every fivedays. For proximate biochemical analyses and tomeasure the energy value, the oven-dried feed wasroutinely analysed following the methods of AOAC(1990). The energy content of the feed was measuredby Parr bomb calorimeter.

Table I -Percentage composition of various ingredients of thelarval diet

Ingredients % Composition

E<H'bb

Skimmed milk powderFish fleshWheat flour(Supplevite-M)* Vitamin andmineral premixCod liver oilCarboxy methyl cellulose (binder)

502020081.0

0.80.2

*Supplevite-M contains following amounts of vitamins andmineraisllOOg of mixture and it was purchased from SarabhaiChemicals, India.Vitamin A = 20, 000 International Unit (LU.), D3 = 40, 000LU, B2 = 0.08 g, E = 30 LU, K = 0.04 g, Cal. Pantothenate =O.l g, Nicotinamide = 0.4 g, B 12 = 0.24 g, Choline Chloride =0.24 g, Calcium = 30 g, Manganese = 1.I g, Iodine = 0.04 g,Iron = 0.3 g, Zinc = 0.6 g, Copper = 0.08 g, Cobalt = 0.018 g

Table 2-Proximate biochemical analyses and energy value of thelarval diet

Proximate analyses % Composition

Crude proteinEther extractCrude fiberAshNitrogen free extractives (NFE)Gross energy

48.2011.831.236.5632.18

4.56 kcal/g

197

Statistical AnalysisResults are presented as mean ± standard error of

mean (SEM). Difference between the control andtreatment means were compared by t-test, whereasdifference among the control and various treatmentmeans were analysed by one way analysis of variance(ANOV A), followed by Duncan's new multiple rangetest. Differences were considered statisticallysignificant when P < 0.05.

Experimental ProtocolTo study the effect of supplementation of vitamin-E

on post-larval production and their antioxidant defencesystem, two feeding experiments were conducted withhealthy zoea larvae of stages V & VI (Z- V and VI) of M.rosenbergii. As early larvae can not survive on artificialdiets (Deru, 1990), the experiments were performed withlater stages of the larvae (Z- V and VI) and continued till'metamorphosis. Healthy and active larvae (Z- V & VI)from individual mother prawn (collected from thebroodstock pond of Central Institute of FreshwaterAquaculture, Kausalyaganga, Bhubaneswar) werestocked (35 Nos/I) in 12 ppt saline water. To theingredient mixture of the basal diet, vitamin-E (0.-tocopherol acetate) was added to prepare four differenttest diets with graded levels of vitarnin-E (50, 100, 200and 400 mglkg feed, respectively referred to as E-50, E-100, E-200 and E-400). Before feeding the diet wascrumbled to suitable particle sizes and fed ad libitumfour times a day (at 08.00, 10.00, 14.00 and 17.00hours). Artemia nauplii were given as a night feed. Thetanks were cleaned everyday by siphoning before theArtemia feeding. 40% of the water was exchanged inevery alternate day. Experiments were done underconstant aeration and natural photoperiod (12 hrs light:12 hrs dark). Important water quality parameters weremaintained at the optimum level. After completion oflarval cycle (final metamorphosis) post larvae (PL) fromthe respective tanks were counted to assess the rate ofsurvival.

On the basis of the results of the above feedingexperiment a separate feeding trial was conductedwith a selective dose of vitamin-E (200 mg/kg feed).At the end of the experiment post-larval productionwere quantified as described above. Average finalweight and average final length of the PL wererecorded. In addition, various parameters ofantioxidant defence system and LPX were studied inthe control and vitamin-E supplemented post larvae.

198 INDIAN J BIOTECHNOL, APRIL 2003

ResultsPost-larval Production

Post-larval production increased in response todifferent doses of supplementary vitamin-E (Fig. 1).However, no significant difference was observed inthe post-larval production when the larvae were fedeither with E-200 or E-400. When the second feedingexperiment was conducted with a selective dose ofsupplementary vitamin-E (200 mg/kg feed), inaddition to the increased post-larval production, theaverage length and weight of the post larvae (PL)were significantly higher than the respective controls(Table 3).

Lipid Peroxidation and Antioxidant DefencesIt is clearly observed that vitamin-E (200 mg/kg

feed) significantly reduced both endogenous (by 33%)and FeS04-induced LPX (by 30%) in the PL incomparison to control value (Fig. 2).

Superoxide dismutase (both total and CN--sensitive) and catalase (CAT) activities were reducedsignificantly in the post-larval tissues in response tovitamin-E supplemented diet (Fig. 3). Both total aswell as Se-dependent GPX activities were elevatedwithout any alteration in Se-independent GPX activityin response to vitarnin-E supplemented diet (Fig. 4a).GR activity was also elevated in the post larvaereceiving vitamin-E supplemented diet (Fig. 4b).Although ascorbic acid content of the PL waselevated in response to the vitamin-E supplementeddiet, glutathione (GSH) level remained unaltered(Fig. 5).

DiscussionIn the present study, marked elevation in post-

larval production was recorded in vitamin-Esupplemented groups compared to the control. This

Table 3--Effect of supplementary vitamin-E (200 mg/kg feed-E-200) on average length and weight of post-larvae of M.rosenbergii. Data are expressed as mean ± S.E.M (ne l O)

Category No. of Average length Average weightlarvae of post-larvae of post-larvae

stocked inmm in g

35/1 10.70 0.0089± 0.82 ± 0.0017

35/l 13.40* 0.015*±0.69 ± 0.001

Control

E-200

*Denotes significant difference from respective controls(p<0.05)

80 PL-Productionc::::

60c b b ....~ -x::J :.:.~ 40c -x•...0- ....;#! 20 ........:.:...

0 ..Control E-50 E-100 E-200 E-400

Fig. I-Effect of supplementary vitamin-E (50, 100, 200 and 400mg/kg feed) on post-larval (PL) production. Data are expressed aspercentage mean ± S.E.M. (n=S). Bars having different,superscripts are significantly different (p<0.05).

~c LPX2 8

ea.

E6....(/)

OCI

~ 4f-

'0cC 2

Xa,..J

! 0 Endogenous

o 30 m In In cuoanon• F~SO. s tuu utat e d

CONTROL VtT·E(200 mg/kg feed)

Fig. 2-Effect of supplementary vitamin-E (200 mg/kg feed) onlipid peroxidation (LPX: nmol TBA-RS/mg protein) in the postlarvae of M. rosenbergii. Data are expressed as mean ± S.E.M.(n=5). *Denotes significant difference from the respectivecontrols (p<0.05).

indicates a significant role of vitamin-E in survival,growth and general metabolism of the larvae. Resultsof the present investigation corroborate the earlierreports in fishes (NRC-1983, Halver, 1985; Sinha &Sinha, 1994; Kokabas & Gatlin III, 1999) and inshrimps (Kanazawa, 1985; He et al, 1992). In thesecond feeding experiment it was also observed thatsupplemented vitamin-E (200 mg/kg feed) increasedthe mean body weight and mean body length of thepost larvae in comparison to the control. Duringexperimentation the larvae were fed with natural livefood organisms (Artemia) as well as the preparedlarval diet with and without vitamin-Esupplementation. Artemia nauplii are used as naturalfeed for the larvae of prawns in general and they areconsidered to be a rich source of lipids and PUFA(Leger et al, 1986; Sorgeloos & Leger, 1992). PUF Aare highly essential for normal metabolism and

DANDAPAT et al.: R'OLE OF ANTIOXIDANT IN PRAWN SEED PRODUCTION

12(a) SOD

10

c2 8

ec.6

Cll.§~ 4c:>

2

0CONTROL

60 (b) CAT

e'Ci) 40'0C. ..CI.E~ 20c.

oTolal SOD

&;I eN 4·Sensilive

_eN -·Resistant

VIT-E(200 mglkg feed)

o

o CONTROLfillV1T-E (200 mgl1<g feed)

Fig. 3-Effect of supplementary vitamin-E on activities ofsuperoxide dismutase (SOD: units/mg protein) (a) and catalaseactivity (CAT: pkatlmg protein) (b) in the post larvae of M.rosenbergii. Data are expressed as mean ± S.E.M. (n=S).*Denotes significant difference from the respective controls(p<O.05).

growth in fishes as well as in crustacea (Devresse etal, 1990; Sorgeloos & Leger, 1992; New, 1995;Murthy, 1998; Kolkovski et al, 2000). However, theelevated levels of PUFA in the tissues of animalsmake them more susceptible to oxidative stress(enhanced LPX) (Mourente et ai, 1999). Theincreased growth rate and survival in the larvae of M.rosenbergii resulting in enhanced post-larvalproduction by vitarnin-E supplemented diet may bedue to the attenuation of oxidative stress bypreventing oxidative damage of PUFA as evidentfrom the reduced LPX level in the experimental groupin comparison to the control. He et ai (1992) observedthat shrimps reared in vitamin-E deficient dietexhibited reduced growth and significantly poorsurvival. The fact is supported by the results ofStephan et al (1995) who reported that in vivo and invitro oxidation of lipids in turbot larvae was reducedwhen a-tocopherol was supplemented in the diet.Protective role of vitamin-E in reducing LPX value inRBC membrane of channel catfish was also observed

199

(a) GPX oTolal GPXm Se-Dependent• Non-Se-Dependent

120

80

40

CONTROL VIT·E (200 m g/kg feed)

(b) GR o CONTROLIli'IVlT-E (200

mg!kg feell)

20

10

o ~------~~~--

Fig. 4--Effect of supplementary vitamin-E on the activities ofglutathione peroxidase (GPX) (a) and glutathione reductase (GR)(nmol NADPH oxidised/min/mg protein) (b) in the postlarvae ofM. rosenbergii. Data are expressed as mean ± S.E.M. (n=S).*Denotes significant difference from respective controls (p<O.05).

by Wise et al (1993). Similarly, diets having morethan 100 mg of vitamin-E/kg feed are reported to beeffective in suppressing ascorbic acid-stimulatedmitochondrial and microsomal LPX in muscles andhepatopancreas of shrimp, Paeneus vanamei (He &Lawrence, 1993).

The activities of both total and CN--sensitive SODhave been reduced significantly in response tovitarnin-E supplementation. Decreased production/availability of the substrate (02-) in the tissues inresponse to vitamin-E supplementation may be areason for the observed attenuation in SOD activities.Vitamin-E is well known to scavenge O2- inbiological system (Cay & King, 1980). It has beenobserved that vitamin-E can also regulate O2-

generation in human neutrophils (Ando et al, 1996),monocytes (Cachia et al, 1998) and in rat livermitochondria (Chow et al, 1999). Palace et al (1993)noticed an increased SOD activity in the tissues ofRainbow trout fed with vitamin-E deficient diet which

200 INDIAN J BIOTECHNOL, APRIL 2003

D CONTROL(a) GSH III VIT-E (200 mg/kg feed)

OJ-.oE3,C 2(])

->oJCooIC/)CD o

D CONTROL(b) ASA ~ VIT-E(200 mg/kg feed)

J

...-.. 60 -(])

::JCfJCfJ

Cl 40 '""-...rn3,

<l::C/) 20 -<l::

Fig. 5--Effect of supplementary vitamin-E on glutathione (GSH:urnol/g tissue) (a) and ascorbic acid (ASA: Ilg/g tissue) (b)content in the post-larvae of M. rosenbergii. Data are expressed asmean ± S.E.M. (n=5). *Denotes significant difference fromrespective controls (p<0.05).

indirectly suggests that less dietary tocopherolaugments the SOD activity to balance the antioxidantstatus. Supplementation of vitamin-E in the presentstudy might have elevated tissue vitamin-E levelwhich, in turn compensated the function of SOD.Although vitamin-E content of the post-larvae has notbeen measured in the present experiment, earlierstudies confirm that dietary vitamin-E fairly improvesthe level of tissue vitamin-E in fishes (Cowey et at,1981; Stephan et at, 1995; Bai & Lee, 1998; Gatta etat, 2000). Decreased catalase activity in the PLreceiving vitamin-E supplemented diet can beattributed to the low H202 content of the tissue, whichmay be resulted due to low SOD activity. Level of

H202 may further decrease due to diffusion of H202

through the gills to the surrounding medium. Filho etat (1994) demonstrated that elimination of H202

through gill diffusion is an important physiologicaladaptation in fishes. Activity of the other counterpartof peroxide scavenging enzyme, the GPX, has beenfound to be elevated in the vitamin-E supplementedgroup. The induction of GPX system may be acompensatory adapti ve response to reduce the H202

level of the tissue when CAT activity is reduced. Itwill not be out of context to mention here that whencellular H202 level is less, GPX is more functionalthan CAT in the removal of H202 and GPX isreported to protect cells against low level oxidantstress (Yan & Harding, 1997). Further, this increasedGPX activity is mainly attributed to its Se-containingiso-enzymes, i.e. Se-dependent GPX, which can alsobe correlated with the increased availability of Se inthe tissue from the surrounding medium (Se contentsof the sea water is high). The synergistic function ofselenium with vitamin-E to increase the GPX activityhas been reported in the liver of fishes (Gatlin III etat, 1986; Wise et at, 1993), lymphocytes of horses(Avellini et al, 1999) and also in cultured cells(BALB/e MK2) of mouse keratinocytes (Stewart et al,1999).

Glutathione reductase activity was found to beelevated in the experimental animals. The elevatedGR can be correlated to maintain the thiol status ofthe cell by regenerating GSH from GSSG. Ascorbicacid level of the post-larval tissue was found to beelevated in response to vitamin-E supplementationwithout any alteration in the GSH level. Since GSH isknown to reduce dehydroascorbic acid (DHAA) backto ascorbic acid in animal cells and tissues (Winkler etat, 1994; Vethanayagam et at, 1999), the increasedconversion of DHAA to ASA may be a reason for theelevated ASA content of the post larvae. As ascorbicacid also acts synergistically with vitamin-E (Niki etat, 1982), the observed elevation in ASA level in thepost larvae may also be attributed to the sparingaction of vitamin-E, resulting in decreased utilisationof ASA in the tissue.

On the basis of findings of the present investigationit can be concluded that the larval diet supplementedwith a-tocopherol acetate has a positive influence onthe growth, survival and post-larval production of M.rosenbergii. The attenuation of LPX and the changesin antioxidant defence system in the post larvae alsosuggest the active involvement of vitamin-E inmodulating the antioxidant defence status. This

DANDAPAT et al.: ROLE OF ANTIOXIDANT IN PRAWN SEED PRODUCTION

further strengthens the fact that vitamin-E modulatedimproved post-larval production may be mediatedthrough optimum cellular performance duringdevelopment and metamorphosis influenced bydietary antioxidant and reduced oxidative stress.

AcknowledgementThe financial assistance from the Department of

Biotechnology, Govt. of India and University GrantsCommission, New Delhi is gratefully acknowledged.Authors wish to thank the Director, Central Instituteof Freshwater Aquaculture, Kausalyaganga,Bhubaneswar and the Head, Department of Zoology,Utkal University, Bhubaneswar for providingnecessary facilities.

ReferencesAebi H, 1974. Catalase. in Methods in Enzymatic Analysis, Vol

II, edited by H U Bergmayer. Academic Press, New York. Pp673-680.

Allen R G & Venkatraj V S, 1992. Oxidants and antioxidants indevelopment and differentiation. J Nutr, 122,631-635.

Ando M et ai, 1996. Regulation of neutrophil superoxidegeneration by a-tocopherol in human peripheral andumbilical cord blood. J Obstet Gynaecol Res, 22, 507-516.

AOAC, 1990. Official Methods of Analysis, 15th edn.,Association of Official Analytical Chemists. Inc, Arlington.Pp 1298.

Avellini L et ai, 1999. Effect of exercise training, Selenium andvitarnin-E on some free radical scavengers in horses (Equuscaballusi. Comp Biochem Physiol, 123B, 147-154.

Bai S C & Lee K J, 1998. Different levels of dietary DL-a-tocopheryl acetate affect the vitarnin-E status of juvenileKorean rockfish, Sebastes schlegeli. Aquaculture, 161, 405-414.

Cachia 0 et ai, 1998. a-tocopherol inhibits the respiratory burst inhuman monocytes. Attenuation of p47Phox membranetranslocation and phosphorylation. J Bioi Chem, 273, 32801-32805.

Cay P B & King M M, 1980. Vitamin-E. Its role as a biologicalfree radical scavenger and its relationship to the microsomalmixed function oxidase system. ill Vitamin-E. AComprehensive Treatise: Basic and Clinical Nutrition I,edited by L J Machlin. Marcel Dekker, New York. Pp. 289-317.

Chow C K et ai, 1999. Vitarnin-E regulates mitochondrialhydrogen peroxide generation. Free Rad Bioi Med, 27, 580-587.

Cowey C B et ai, 1981. Tissue distribution, uptake and requirementfor a-tocopherol of rainbow trout iSalmo gairdnerii fed dietswith a minimal content of unsaturated fatty acids. J Nutr, 111,1556-1567.

Dandapat J et ai, 2000. Dietary vitarnin-E modulates antioxidantdefence system in giant freshwater prawn, Macrobrachiumrosenbergii. Comp Biochem Physiol, 127C, IOI-lI5.

Das K et ai, 2000. A modified spectrophotometric assay ofsuperoxide dismutase using nitrite formation by superoxideradicals. Indian J Biochem Biophys, 37,201-204.

201

Deru J, 1990. Studies on the development and nutnuon of thecaridean prawn, Macrobrachium rosenbergii (deMan)(Crustacea: Decapoda). Ph D Thesis, University of Wales,Bangor.

Devresse B et ai, 1990. Improved larvi culture outputs in the giantfreshwater prawn Macrobrachium rosenbergii fed diet ofAnemia enriched with (00-3) HUFA and phospholipid. WorldAquacult, 21, 123-125.

Ellman G L, 1959. Tissue sulfhydryl groups. Arch BiochemBiophys, 82, 72-77.

Faverney C R et ai, 2001. Cadmium induces apoptosis andgenotoxicity in rainbow trout hepatocytes through generationof reactive oxygen species. Aquat Toxicol, 53,65.

Filho D W et ai, 1994. Gill diffusion as a physiologicalmechanism for hydrogen peroxide elimination by fish. Bra: JMed Bioi Res, 27, 2879-2882.

Gardner H W, 1989. Oxygen radical chemistry of polyunsaturatedfatty acids. Free Rad Biol Med, 7, 65-86.

Gatlin III D M et al, 1986. Effects of singular and combineddietary deficiencies of selenium and vitamin-E on fingerlingchannel catfish (lctalurus punctatusi. J Nutr, 116, 1061-1067.

Gatta P P et ai, 2000. The influence of different levels of dietaryvitarnin-E on sea bass, Dicentrarchus labrax flesh quality.Aquacult Nutr, 6,47-52.

Glencross B D et al, 2002. Optimising the essential fatty acids inthe diet for weight gain of the prawn, Penaeus monodon.Aquaculture, 204,85-99.

Halliwell B & Gutteridge J M C, 2001. Free Radicals in Biology andMedicine, 3rd edn, Clerendon Press, Oxford.

Halver J E, 1985. Recent advances in vitamin nutrition andmetabolism in fish. in Nutrition and Feeding in Fish, editedby C B Cowey, A M Mackie & J G Bell. Academic Press,London. Pp 415-429.

He H & Lawrence A L, 1993. Vitarnin-E requirements of Penaeusvannamei. Aquaculture, 118,245-255.

He H et ai, 1992. Evaluation of dietary essentiality of fat solublevitamins, A, D, E & K for penaeid shrimp (Penaeusvannameii. Aquaculture, 103,177-185.

Hebb C D et al, 1997. Nutritional studies on growth and proteinutilisation during the juvenile stage of winter flounderiPleuronecies americanusi. Bull Aquacult Assoc Canada, 2,45-47.

Hwang D F & Lin T S, 2002. Effects of temperature on dietaryvitamin C requirement and lipid in common carp. CompBiochem Physiol, BIB, 1-7.

Kanazawa A, 1985. Nutrition of penaeid prawn and shrimp. inProc First Int Conf Culture Penaeid prawn/shrimps, edited byY Taki, L H Primavera & J A Lobrera. Aquacult Depr,Southeast Asian Fish Dev Center, Iloilo, Philippines. Pp 123-130.

Kokabas A M & Gatlin III D M, 1999. Dietary vitarnin-Erequirement of hybrid striped baas (Morone chrysops femalex M. saxatilis male). Aquacult Nutr, 5,3-7.

Kolkovski S et ai, 2000. The effect of vitamin-C and E in (n-3)highly unsaturated fatty acids-enriched Anemia nauplii ongrowth, survival and stress resistance of freshwater walleye,Stizostedion vitreum larvae. Aquacult Nutr, 6, 199-206.

Leger P et al, 1986. The use and nutritional value of Artemia as afood source. Oceanogr Mar Bioi Annu Rev, 24,521-623.

Lowry 0 H et al, 1951. Protein measurement with the Folin phenolreagent. J BioI Chem, 193,265-275.

202 INDIAN J BlOTECHNOL, APRIL 2003

Mahapatra P K et aL,200 l. Changes in oxidative stress parametersand acid phosphatase activity in the pre-regressing andregressing tail of Indian jumping frog Polypedates macuLatus(Anura, Rhacophoridae). Comp Biochem PhysioL, 130C, 28 1-288.

Mahapatra P K et al, 2002. Oxidative stress during vitamin A-induced abnormal tails regeneration in the tadoples ofpoLypedates maculatus. Comp Biochem Physiol, 131B, 403-410.

Massey V & Williams C H, 1965. On the reaction mechanism ofyeast glutathione reductase. J Bioi Chem, 240,4470-448 I.

Mitusi A & Ohata T, 1961. Photooxidative consumption andphotoreductive formation of ascorbic acid in green leaves.PLant Cell Physiol, 2, 31-44.

Mourente G et al, 1999. Relationships between antioxidants,antioxidant enzyme activities and lipid peroxidation productsduring early development in Dentex dentex eggs and larvae.Aquaculture, 179,309-324.

Murthy H S, 1998. Effect of enriched Artemia on metamorphosisand survival of larvae of giant freshwater prawn,Macrobrachium rosenbergii. JAqua Trop, 13,215-222.

National Research Council-NRC, 1983. Nutrient requirements ofdomestic animals, nutrient requirements of warm waterfishes and shellfishes. National Academy Press, Washington,DC. Pp 102.

New M B, 1995. Status of freshwater prawn farming: A review.Aquacult Res, 26, 1-54.

Niki E et aL, 1982. Regeneration of vitamin-E from a-chromanoxyl radical by glutathione and vitamin C. ChemLeu. 789-792.

Ohkawa H et al, 1979. Assay for lipid peroxides in animal tissuesby thiobarbituric acid reaction. AnaL Biochem, 95, 351-358.

Paglia D E & Valentine W N, 1967. Studies on quantitative andqualitative characterization of erythrocyte glutathioneperoxidase. J Lab Clin Med, 70, 158-169.

Palace V Pet al, 1993. Interactions among antioxidant defences inliver of rainbow trout (Oncorhynchus mykiss) exposed tocadmium. Can J Fish Aquat Sci, S0, 156-162.

Sinha A & Sinha Y K P, 1994. Role of vitamin-E in growth of anIndian major carp, Catla (Catla catla). Proc Nat SympAquacrops, 24, 91-96.

Sorgeloos P & Leger P, 1992. Improved larviculture outputs ofmarine fish, shrimp and prawn. J World Aquacult Sac, 23,251-264.

Stephan G et al, 1995. Lipid peroxidation in turbot iScophthalmusmaximusi tissue:Effect of dietary vitamin-E and dietary n-6or n-3 polyunsaturated fatty acids. Aquaculture. 130, 251-268.

Stewart M S et al, 1999. Selenium compounds have disparateabilities to impose oxidative stress and induce apoptosis.Free Rod BioLMed, 26,42-48.

Subramoniam T, 1999. Endocrine regulation of egg production ineconomically important crustaceans. Curr Sci, 76, 350-360.

Tappel A L, 1975. Lipid peroxidation and fluorescent moleculardamage to membranes. in Pathology of Cell Membranes, Vol I,edited by B F Trump & A U Arstila, Academic Press, NewYork. Pp 145-173.

Vethanayagam J G G et al, 1999. Glutathione-dependentascorbate recycling activity of rat serum albumin. Free RodBioi Med, 26,1591-1598.

Watanabe T, 1993. Importance of docosahexaenoic acid in marinelarval fish. J World Aquacult Sac, 24, 152-161.

Winkler B S et aL, 1994. The redox couple between glutathioneand ascorbic acid: A chemical and physiological perspective.Free Rod Bioi Med, 17, 333-349.

Wise D J et al, 1993. Effects of dietary selenium and vitarnin-E onred blood cell peroxidation, glutathione peroxidase activity,and macrophase superoxide anion production in channel catfish. J Aquat Anim Health,S, 177-182.

Yan H & Harding J J, 1997. Glycation-induced inactivation andloss of antigenicity of catalase and superoxide dismutase.Biochem J, 328,599-605.

Yasuda K & Kitao T, 1980. Bacterial flora in the digestive tract ofprawn, Penaeus japonicus, Bate. Aquaculture. 19,229-234.