Growth, survival and stress resistance of tiger barb (Puntius tetrazona) larvae fed on linseed oil-enriched Artemia franciscana nauplii

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1 Irnin Journl of Fisheries Sciences 13(3) Growth, survivl nd stress resistnce of tiger r (Puntius tetrzon) lrve fed on linseed oil-enriched Artemi frnciscn nuplii Downloded from jifro.ir t 17: on Sundy Ferury 18th 2018 Aolhsni M.H. 1 ; Hosseini S.A. 1 ; Ghorni R. 1 ; Soudgr M. 1 ; Hoseini S.M. 1 * Received: Octoer 2012 Accepted: Ferury 2014 Astrct Effect of feeding on linseed oil (LO)-enriched Artemi ws investigted on growth, survivl nd stress resistnce of tiger r (Puntius tetrzon) lrve. Lrve were fed y LO-enriched (2.5, 5 nd 7.5 %) s well s non-enriched Artemi nuplii for 14 d, followed y 14 d feeding on non-enriched Artemi. Fish fed on enriched nuplii showed significnt increse in growth performnce t oth 14 th nd 28 th d. Also, fish fed on enriched Artemi, especilly those fed on 7.5 % LO, showed greter resistnce in response to osmotic nd hypoxi stress t 28 th d. The results suggest tht LO enriched Artemi nuplii re cple to promote growth nd stress response in tiger r lrve. Keywords: Ctfish, Ftty cid, Liver, Ftty tissue 1-Fculty of Fisheries nd Environment, Deprtment of Fisheries, Gorgn University of Agriculturl Sciences nd Nturl Resources, Gorgn, Irn *Corresponding uthor emil: seyyedmortez.hoseini@gmil.com

2 577 Aolhsni et l., Growth, survivl nd stress resistnce of Tiger Br (Puntius tetrzon) lrve fed on Downloded from jifro.ir t 17: on Sundy Ferury 18th 2018 Introduction Lipids re n importnt source of energy nd essentil ftty cids (EFA) in quculture. The importnce of long-chin highly unsturted ftty cids (HUFA), such s docoshexenoic cid (DHA, 22:6n-3), eicospentenoic cid (EPA, 20:5n- 3), nd rchidonic cid (ARA, 20:4n-6) in fish nutrition is well estlished (Srgent et l., 2002). These ftty cids re required for fish optiml growth, development nd reproduction (Srgent et l., 1999). Despite efforts to develop rtificil lterntives (Leger et l., 1986; Jones et l., 1993) rine shrimp (Artemi sp.) nuplii remin n importnt live food for fish lrve. Artemi sp. nuplii consists of 50-60% protein (ll min mino cids in enough content nd of 20-50% long chin ftty cids in much content) tht is importnt in nutrition of qutic cretures (Sorgeloos et l., 1986). Due to compulsory nutrition, there is the ility of trnsferring different mterils in different mounts to fish y Artemi sp. nuplius. Artemi sp. is importnt in fish culture due to high nutrient vlue nd ese of ccess. Its vrious forms re pplied t different stges of growth s primry food. Its usility s crrier of vitmins, pigments, chemicls, vccines nd hormones hs mde it crucil mens of enriched food source in quculture (Sorgeloos et l., 1986). Biochemicl composition is different in vrious Artemi types nd this difference could e decresed y vrious nutrient enrichment (Leger et l., 1986). Artemi sp. is deficient in the cse of some EFA, especilly DHA (Figueiredo et l., 2009). Considering the importnce of EFA in erly life stges of fish, it is necessry to enrich Artemi nuplii with EFA. Linseed oil is n idel source of linolenic cid (18: 3n-3) (Mtthews et l., 2000; Rhimi et l., 2009). Although it does not contin high mounts of ARA, it contins reltively high mount of linolenic cid (18: 2n-6 Cis) (Mtthews et l., 2000) which could e converted to ARA (Srgent et l., 2002). Therefore Artemi sp. nuplii enrichment with LO is eneficil for optimizing EFA levels of nuplii. Tiger r, (Puntius tetrzon) is nturlly found in Sumtr, Borneo, Thilnd nd Mlysi (Welcomme, 1988). However, it ws introduced to the other prts of the world minly s populr ornmentl fish species. On the other hnd it is rtificilly red nd rered for its nturl popultion conservtion, sle to fish hoyists, s well s reintroducing to hitts in which they hve een eliminted (Ng nd Tn, 1997). Like mny other species, erly life of tiger r is dependent on live food, especilly Artemi sp. nuplii. Thus the im of the present study ws to determine the effect of LO-enriched Artemi frnciscn nuplii on growth performnce, survivl nd resistnce to stress in tiger r lrve. Mterils nd methods A. nuplii nd enrichment protocol

3 Irnin Journl of Fisheries Sciences13(3) Downloded from jifro.ir t 17: on Sundy Ferury 18th 2018 Htching technique ws sed on Dhont nd Vn Stppen (2003). Briefly, cysts of A. frnciscn were rtificilly htched y dding 1 g cysts to 1 liter wter (33 g/l). Temperture nd light intensity ws mintined t 28ºC nd 2000 lux, respectively. Continuous ertion ws provided to ensure dissolved oxygen up to 5 mg/l s well s for suitle turulence for cysts. Htching rte ws determined y counting nd verging the nuplii of three smples (0.1 ml) under stereoscopic loupe (Olympus, SZX7, Jpn). LO emulsion ws prepred y mixing of 0.5 g lecithin with 100 ml distilled wter, y n electric lender for 5 min. To this mixture, 2.5, 5 nd 7.5 g LO ws dded nd mixed for 1.5 min (Clwson nd Lovell 1992). Three tches of nuplii were dded to three conicl-ottom cylinders (1 L wter). Two milliliters of LO emulsions (2.5, 5 nd 7.5%) were inoculted to the cylinders. After 6 hours, inocultion ws repeted s the first one. Six hours fter the second inocultion nuplii were dded to the corresponding fish quriums. Lrve nd Growth tril Tiger r lrve were otined from rtificil propgtion. A totl of 1440 lrve were used for the growth tril. The lrve were distriuted into 12 glss quriums. They were fed on Artemi nuplii for 2 dys s cclimtion period. Then, the quriums were ssigned into 4 groups; one control nd three enriched groups. The control group ws fed on nonenriched Artemi, while the enriched groups were fed (until stition, four times dy) on one of the 2.5, 5 nd 7.5 % LOenriched Artemi for 14 dys. After 14 dys, ll groups were fed (until stition) on non-enriched Artemi, for nother 14- dys period. The lrve weight nd length were mesured t the eginning of the experiment s well s 14 nd 28 dys therefter. Wter temperture, ph nd dissolved oxygen were mintined t 28 ± 0.7 ºC, 7.2 ± 0.12 nd 6.4 ± 0.34 mg/l. Stress resistnce After 28 dys, ll groups were exposed to osmotic nd hypoxi stress. For osmotic stress, the fish were exposed to 6, 10 nd 12 g/l slinity, in 24-hours-intervl sis. Three tnks were ssigned for ech tretment, ech contining 20 fish. Survivl ws recorded every 24 hours, efore exposure to the next slinity. For hypoxi chllenge, ertion ws cesed for dissolved oxygen to e dropped down to 4 mg/l. Fish remined under this condition for 4 dys, when survivl ws recorded. Three tnks were ssigned for ech tretment, ech contining 20 fish. Sttisticl nlyses Normlity of dt ws confirmed y Shpiro-Wilk test. Dt were sujected to one-wy ANOVA nd Duncn test to detect significnt difference. All nlyses were performed using the sttisticl softwre SPSS version 16. P < 0.05 ws considered significntly different. Dt re presented s tretment men ± SD. Results Growth performnce The fish fed on enriched Artemi showed significnt increse in weight, length, weight gin (WG) nd specific growth rte (SGR), t oth 14 th nd 28 th dy (Tle 1;

4 SGR (%/d) 579 Aolhsni et l., Growth, survivl nd stress resistnce of Tiger Br (Puntius tetrzon) lrve fed on Fig. 1 nd 2). There ws no significnt chnge in totl mortlity mong the tretments, during 14 nd 28 dys rering periods (Tle 2). Downloded from jifro.ir t 17: on Sundy Ferury 18th 2018 Tle 1: Growth performnce of the tiger r fed on enriched nd non-enriched Artemi over 14 nd 28 dys. Different letters in ech row show significnt difference (Duncn test). n = 3. Control 2.5 LO 5 LO 7.5 LO Initil Weight (mg) 2.3 ± ± ± ± 0.1 Totl length (mm) 6.15 ± ± ± ± dys Weight (mg) 17.8 ± ± ± 0.4 c 23.6 ± 0.7 Totl length (mm) ± ± ± 0.3 c ± 0.2 Cumultive mortlity (%) 9.16 ± ± ± ± dys Weight (mg) 110 ± ± 5.3 c 130 ± ± 9.8 Totl length (mm) 14.2 ± ± ± ± 0.4 c Cumultive mortlity (%) 15.8 ± ± ± ± c d 28 d 0 C 2.5 LO 5 LO 7.5 LO Tretments Figure 1: SGR of the tiger r fed on enriched (2.5 LO, 5 LO nd 7.5 LO) nd non-enriched (C) Artemi over 14 nd 28 dys. Different letters ove the rs show significnt difference t 14 th or 28 th dy, seprtely (Duncn test).

5 Weight gin (%) Irnin Journl of Fisheries Sciences13(3) c Downloded from jifro.ir t 17: on Sundy Ferury 18th Figure 2: Weight gin of the tiger r fed on enriched (2.5 LO, 5 LO nd 7.5 LO) nd non-enriched (C) Artemi over 14 nd 28 dys. Different letters ove the rs show significnt difference t 14 th or 28 th dys, seprtely (Duncn test). Stress resistnce The control group showed significntly impired survivl compred with the enriched groups, following oth osmotic nd hypoxi stresses (Fig. 3 nd 4). There were no significnt differences in stress resistnce mong the enriched groups. Survivl of the control group exposed to 10 nd 13 g/l sltwter reched zero, while C 2.5 LO 5 LO 7.5 LO Tretments c 14 d 28 d the enriched groups showed ner 80 nd 50 % survivl, respectively (Fig. 3). The fish fed on 7.5 LO-enriched nuplii showed significntly higher survivl rte compred with the other groups, following hypoxi (Fig. 4). The control group showed the lowest survivl rte compred with the other groups following hypoxi (Fig. 4).

6 Survivl (%) Survivl (%) 581 Aolhsni et l., Growth, survivl nd stress resistnce of Tiger Br (Puntius tetrzon) lrve fed on ppt ppt 13 ppt 60 Downloded from jifro.ir t 17: on Sundy Ferury 18th C 2.5 LO 5 LO 7.5 LO Tretments Figure 3: Osmotic stress resistnce of the tiger r following 28 dys feeding on enriched (2.5 LO, 5 LO nd 7.5 LO) nd non-enriched (C) Artemi. Different letters ove the rs show significnt difference for ech slinity, seprtely (Duncn test) c 0 C 2.5 LO 5 LO 7.5 LO Tretments Figure 4: Hypoxi stress resistnce of the tiger r following 28 dys feeding on enriched (2.5 LO, 5 LO nd 7.5 LO) nd non-enriched (C) Artemi. Different letters ove the rs show significnt difference (Duncn test). Discussion Artemi is often n inferior food source for lrve compred with wild zooplnkton. However, the cpility of Artemi to produce ny mount of iomss over short time, in contrst to zooplnkton, nd the possiility of enrichment with essentil nutrients ensures its widespred use in lrviculture (Støttrup nd McEvoy, 2008). Tiger r showed etter performnce

7 Irnin Journl of Fisheries Sciences13(3) Downloded from jifro.ir t 17: on Sundy Ferury 18th 2018 when fed y LO-enriched Artemi compred with non-enriched Artemi. Previous studies on mrine fish lrve such s turot (Scophthlmus mximus) (Gtesoupe nd Le Milinire, 1985), Jpnese flounder (Prlichthys olivceus), red se rem (Pgrus mjor) (Izquierdo et l., 1989), gilthed se rem (Sprus urt) (Koven et l., 1993; Rinuzzo et l., 1997), Limnd ferrugine (Copemn et l., 2002) nd freshwter fish lrve such s Belug (Huso huso) (Jlli et l., 2008) showed the dvntge of n-3 HUFA on growth performnce. Linseed oil contins out 50 nd 15 %, of linoleic nd linolenic cid, respectively (Rhimi et l., 2009). Linolenic cid is precursor of EPA nd DHA nd linolenic cid is precursor of ARA (Srgent et l., 2002). Thus higher growth performnce of the lrve fed on LO-enriched nuplii might e due to the endogenous production of EPA, DHA nd ARA. In this study, the positive effects of feeding LO-enriched Artemi for 14 dys were kept y the fish over the next 14 dys (during which they fed on non-enriched Artemi). It showed tht the fst growth ility of tiger r lrve, when fed on enriched live food cn e mintined even if feeding continues with non-enriched live food. Jlli et l. (2008) reported similr oservtion in Belug lrve fed on fish oil-enriched Artemi followed y nonenriched Dphni sp. Tiger r fed on enriched Artemi lso demonstrted etter survivl rte thn those fed on non-enriched Artemi during osmotic nd hypoxi chllenges. It is elieved tht eicosnoids' production vi their precursor, linolenic cid, ws responsile for such stress resistnce in the fish fed on LO-enriched nuplii. Eicosnoid hs n importnt role in stressful conditions (Srgent et l., 2002). Eicosnoid production is norml physiologicl process, with excess eicosnoid production often occurring in pthologicl conditions (Srgent et l., 2002). On the other hnd, it is possile tht higher stress resistnce in the tiger r fed on enriched Artemi ws relted to endogenous production of DHA (from linolenic cid) compred to those fed on non-enriched Artemi. In fct, DHA is importnt in rpid development of visul nd neurl tissues in lrvl stges s reported for Coryphen hippuus (Srgent et l., 2002). Moreover, lrve fed on higher DHA gined igger ody size, which might e relted to their etter stress resistnce, s reported in C. hippuus (Krul et l., 1993). Likewise, Jlli et l. (2008) reported higher survivl rte, nd lrger ody size in Belug (H. huso) lrve fed on HUFA enriched Artemi compred to those fed on non-enriched Artemi. It is concluded tht tiger r lrve fed on LO-enriched Artemi, over 14 dys, showed etter growth performnce, which lsted until 28 dys. They lso demonstrted etter stress resistnce compred to those fed on non-enriched Artemi, which could e ttriuted to the higher EPA intke or the higher ody size, s result of higher DHA intke.

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9 Irnin Journl of Fisheries Sciences13(3) Downloded from jifro.ir t 17: on Sundy Ferury 18th 2018 Ng, P.K.L. nd Tn H. H., Freshwter fishes of Southest Asi: potentil for the qurium fish trde nd conservtion issues. Aqurium Sciences nd Conservtion, 1(2), Rhimi, M.M., Mohmdi, G.N., Aeinehnd, A., Afshr, I. nd Mofpourin, G., Effects of sowing time nd different nitrogen levels on quntittive nd qulittive chrecteristics of oil flx (Linum usittissimum L.). Seed nd Plnt Production Journl, 25, Pe79-Pe91. Rinuzzo, J. R., Reitn, K. I. nd Olsen Y., The significnce of lipids t erly stges of mrine fish: review. Aquculture. 155(1), Srgent, J. R, Bell, G., McEvoy, L., Tocher, D. nd Estevez, A., Recent developments in the essentil ftty cid nutrition of fish. Aquculture, 177(1), Srgent, J.R., Tocher, D.R. nd Bell, J.G., The Lipids. In Fish Nutrition, (ed. J.E., Hlver nd R.W. Hrdy), Acdemic Press. pp Sorgeloos, P., Lvens, P., Leger, P., Tckert, W. nd Versichele, D., Mnul for the culture nd use of Brine Shrimp Artemi in quculture. Stte University of Gent, Belgium, 319P. Støttrup, J.G. nd McEvoy, L.,2008. Live feeds in mrine quculture. Wiley-Blckwell. 313P. Welcomme, R.L., Interntionl introductions of inlnd qutic species. FAO Fish. Tech. Pp P.