TUNA KELESTEMUR. Fisheries Faculty, Firat University, 23119, Elazig, Turkey Received : Accepted : ABSTRACT

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1 Indin J. Anim. Res., 46 (1) : 22-27, 2012 AGRICULTURAL RESEARCH COMMUNICATION CENTRE ccjournls.com / indinjournls.com nls.com EFFECTS OF β-carotene AND VITAMIN - C ON BLOOD ELECTROLYTE LEVELS OF JUVENILE RAINBOW TROUT ( (ONCORHYNCHUS MYKISS W ) UNDER HYPOXIC STRESS Gülüzr TUNA KELESTEMUR Fisheries Fculty, Firt University, 23119, Elzig, Turkey Received : Accepted : ABSTRACT In this study,, the effect of dietry supplementtion of β-crotene nd vitmin C on lood electrolytes (N +, K +, C +2,Mg +2, Cl - ) nd ph of juvenile rinow trouts under hypoxic stress (4.5 mg/l O2) ws investigted. Three diets were prepred differing only in the type of supplementtion s follows: Bc dietry group with 70 mg/kg β-crotene, A dietry y group with 250 mg/kg Vitmin C nd control (C) dietry y group with no β-crotene nd Vitmin C. Bsed on sttisticl evlutions, it ws oserved tht serum N + level of A group ws significntly higher thn Bc nd C groups (p<0.05) nd the lest serum N + level ws found in C group. Serum K + nd Cl - levels of A nd Bc groups were determined to e significntly higher thn C group (p<0.05). Serum C + 2 level of C group ws determined to e significntly higher thn the other groups (p<0.05) wheres serum Mg +2 level of C group ws found to e significntly lower thn other groups (p<0.05). Serum ph level ws not sttisticlly different mong the groups (p>0.05). Key words: Rinow trout, Vitmin C, β-crotene, Blood electrolytes, Stress, Hypoxi. INTRODUCTION Hypoxi in qutic ecosystems refers to wters where there is reduction of dissolved oxygen concentrtion due to nturl mens (eutrophiction, wter movements nd sesonl iologic, chemicl or physicl chnges etc.) or humn-sed sources (chemicl nd iologic wstes). The reduction in oxygen levels limits the vitl functions such s growth, development, reproduction nd nutrition of qutic nimls, especilly fish nd cuses deth in chroniclly ongoing conditions (Krmer nd McClure, 1982; Chpmn et l., 2010). When the stress fctors strt to hve n effect on metolic ctivities, the regultory mechnisms of the orgnism ttempts to minimize the dmge s result of n immune response cused y tissue dmge (Conte, 2004; Johnnessen nd Dhl, 1996). The regultory mechnisms of the orgnism must mintin optimum cellulr ph nd osmolrity. Osmoregultion is mintined with homeostsis of intrcellulr nd extrcellulr ions (Ntochin et. l., 1985; Johnston nd Cheverie, 1985). In prllel with physiologicl chnges for the mintennce of *Author s E mil: gkelestemur.firt.edu.tr homeostsis in metolism, chnges in some of the prmeters of lood tissue provide importnt dt for the evlution of the stress levels. Those prmeters tht exist in certin concentrtions in lood plsm re lood electrolytes (Hndy et l., 1999). Potssium (K + ) is the mjor ction of intrcellulr fluid wheres sodium (N + ) nd chloride (Cl - ) re the mjor ions of extrcellulr fluid. N +, K + nd Cl re importnt ions which provide the sustinility of the osmotic pressure of ody fluids s well s cid-se lnce. Mgnesium ion (Mg +2 ) is the second most undnt ction tht exist in intrcellulr fluid. It cts s co-fctor in function of mny enzymes. It lso plys n importnt role in neurochemicl impulse trnsmission nd muscle excitility. Mg is essentil in sorption of clcium ion from intestines (Krnky, 1998; Rilo et l., 1985). As in ll living eings, the min two fctors which ffecting cid-se lnce in fish re the nutrition nd environment. In intensive cultivtion, ny interventions mde to mintin this lnce must improve the immune system (Moeller nd

2 Vol. 46, No. 1, Roert, 2001; Mores, 2004). Mny studies showed Diets tht supplementtion of vitmin A, C, E nd β- In this study, experimentl diets were crotene into the rtions of cultured rinow trouts prepred s follows: Bc diet with 70 mg/kg β- for the purpose of eliminting the stress fctors ws crotene, A diet with 250 mg/kg Vitmin C proven to e eneficil for the development of fish (Synthetic Rovimix β-crotene, 10 % nd Synthetic (Tun Kelestemur nd Ozdemir, 2011). Dietry levels L-scoric cid 99 %) nd C 0 (control) diet with no of these vitmins hve een reported to influence β-crotene nd vitmin C. Rovimix β-crotene nd ntioxidnt defenses of fish (Zhng nd Omley, 2001; vitmin C (L-scoric cid) were supplied y Turkey Redge et l., 1997; Choi et l., 2004; Bry, 2000). DSM nutritionl productions firm. The mounts of This study ims to determine the lood supplemented β-crotene nd Vitmin C in the electrolytes such s N +, K +, C +2, Mg +2, Cl - nd rtions were determined y tking the recommended lood ph of juvenile rinow trout fed the â- mounts for fish exposed to stress into considertion crotene nd vitmin C supplemented diets under (Hilton et l., 1977). Composition of the sl diet hypoxic stress t the level of 4.5 mg/l dissolved (NRC, 1993) is shown in Tle 1. oxygen. Dry mtter (105 C, overnight), sh (650 C, MATERIALS AND METHODS 6 h), crude protein (nitrogen 6.25), ether extrct, Experimentl fish crude fire of diets ws nlyzed y methods of Rinow trout (Oncorhynchus mykiss) AOAC (1990). The experimentl fish were fed two were produced t the Çircir Htchery (Elzig, times dy t nd hours for 4 weeks. Turkey) were trnsferred Ken Dm Lke Dily feed llownce ws 3% ody weight per dy. Generl Directorte of Stte Hydrulic Works Blood Smples Lortory. After the cclimtion, fish were At the end of the experiment, lood ws selected nd rndomly stocked. Fish (initil otined from the cudl vsculture of 10 fish per verge weight, 42.27±2.13 g; initil length, tnk. Blood ws otined from the cudl vein of 15.39±1.01 cm) were distriuted into nine individul fish fter nesthesi with Qinldine (15 fierglss rectngulr tnks ( cm) in mg/l Quinldin). The lood smples were centrifuged triplicte groups t density of 10 fish/tnk. (3500 rpm for 7 min) to otin serum nd serum Experimentl design smples were stored -20 C until nlysis (Shn et Experimentl fish were rndomly distriuted l., 2003). The electrolyte vlues of the lood in ech tnk s (cpcity of tnk is 360 L with smples were determined using Autonlyzer dimension of 200 cm long, 40 cm height nd 40 cm (Roche Hitchi Cos 6000) device in in vitro wide) t groups of 10 fish. Experiments were lortory. conducted in tnk, supplied with well wter flow Sttisticl nlysis rte t 0.9 l min -1. At wter flow rte 0.9 l/min, 0 2 Biochemicl dt were nlyzed with SPSS concentrtions were s seen 4.5±0.3 mg/l. The flow 11.5 for Windows using one-wy nlyses of rtes were stilized through the use of the wter vrince (ANOVA) nd significnt mens were inflow control vlves (Tun Keleºtemur nd Özdemir, sujected to multiple comprison test (Duncn) t 2010). Wter temperture rnged from 9.3±0.5 0C P<0.05. (SPSS 11.0, SPSS Ltd. Working, Surrey, nd wter ph rnged from 8.4±0.5. The ph of the UK). wter in the tnks where the fish were situted ws RESULTS TS AND DISCUSSION determined y portle Checker rnd ph meter Serum N +, K +, C + 2, Mg +2, Cl - levels, nd nd the dissolved oxygen nd the temperture vlues ph vlues of fish fed with the vrious experimentl were recorded through the use of portle YSI 55 diets re presented in Figure 1. The results of onewy ANOVA test showed tht lood ion levels of Model 51/12 oxygen proe throughout the durtion of the reserch. The study ws initited fter 1 weekdpttion period of fish to the hypoxic condition supplementtion of β-crotene nd vitmin C. Serum fish were positively ffected y dietry t the level of 4.5 mg/l dissolved oxygen. N + level ws significntly ffected y 250 mg/kg vitmin C supplemented diet group (A), followed

3 24 INDIAN JOURNAL OF ANIMAL RESEARCH y the 70 mg/kg β-crotene supplemented diet group (Bc) (p<0.05). The highest serum K +, Cl - nd Mg +2 levels were otined vitmin C supplemented group nd â-crotene supplemented group (p<0.05), while the lowest K +, Cl - nd Mg +2 levels were otined in the C group (p<0.05). Serum C +2 level ws higher in the C group (p<0.05) thn other diet groups. ph vlue of rinow trout under hypoxic stress ws not sttisticlly different mong ll dietry tretments. Diminished oxygen vilility to cells nd tissues (hypoxi) nd the complete sence of oxygen (noxi) re physiologicl stressors tht cn cuse significnt nd rpid reduction in the production nd supply of ATP (Rndll nd Hungy, 2004; Tuvikene et l., 2001). There follows consequent loss of cellulr homeostsis nd memrne integrity, which susequently leds to cellulr degrdtion nd deth (Chpmn et l., 2010). Severl reserchers hve reported tht the concentrtion of the dissolved oxygen in wter should not e llowed to fll elow 5 mg/l nd tht the optiml concentrtion of dissolved oxygen vries etween 7 nd 9 mg/l in trout stock frming (Clrk, 2003). Petersen (1987) hs determined tht the nturl oxygen requirement for the trout is 8 mg/l tht t concentrtions of 4 mg/l difficulties in rething ppered nd tht t concentrtions of 3 mg/l slow deths occurred. In this study, effect of β- crotene nd vitmin C supplementtion into the rtions of juvenile rinow trout on lood electrolytes (N +, K +, C +2, Mg +2, Cl - ) ws investigted under hypoxic conditions. It ws reported tht N + plys n importnt role in mintennce of cid-se lnce of lood, function of nerve nd muscle, ph of lood. Serum N + levels re found to e etween nd mmol/l. It ws reported tht serum K + levels of rinow trout otined from nturl environment ws out mmol/l. Serum Cl - levels of rinow trout found in nturl environments were determined s mmol/l. Reserchers found tht serum C +2 nd Mg +2 levels of rinow trout were mmol/l nd in verge, respectively (Çelik, 2006). Vrious stressors effect lood ion concentrtions of fish. Lyytikinen et l. (2002) were determined tht there were no sttisticl TABLE 1: Composition nd proximte nlysis of the experimentl diets Ingredient Percent of dry weigth Fish (nchovy) mel 50 Soyen mel 23.1 Whet flour 19.8 Sunflower oil 6 Antioxidnt 0,10 Vitmin premix 0.90 Minerl premixc 0.10 Proximte composition Dry mtter Crude protein 44,48 Crude ft 8,02 Crude fire 4,12 Crude sh 13,10 Antioxidnt (mg/kg dry diet): Butilen Hydroxytoluene (BHT); mg/ kg Vitmin premix (IU or mg/kg dry diet): Mendion 3.000, Rioflvin 6.000, Pridoksin 5.000, kolmin 15, Nisin , Biotin 40, Folik sit 1.000, Kolin klorid 300, Klsiyum D-pntothent 8.000, Klsiferol , Vitmin A 1000, Vitmin E IU. c Minerl premix (mg/kg dry diet): Mn , Fe , Zn , Cu 5.000, I 2.000, Co 400, Se 150. differences in lood clcium, sodium nd chloride concentrtions of Arctic chrr (Slvelinus lpinus) etween fluctuting nd constnt therml conditions (p>0.05), the influence of stress on plsm clcium, sodium nd potssium concentrtions ws similr under fluctuting nd constnt conditions (p>0.05), the influence of stress on plsm potssium concentrtions ws sttisticlly significnt in these conditions (p<0.05). Xugng et l. (2009) were determined tht the lood N +, C +2 nd C1 - levels of Chinese sturgeon (Acipenser sinensis) incresed significntly fter direct trnsfer from fresh wter to rckish wter. In this study, serum N + levels of C, Bc nd A dietry groups were determined s 141±2.23 mmol/l, 149±3.45 nd 157±4.77 mmol/l, respectively. Serum N + levels of A group fed the 250 mg/kg vitmin C ws determined to e higher thn the other groups. In this study, serum K + levels of juvenile rinow trout fed the β-crotene nd vitmin C were determined to e significntly higher thn the C group (p<0.05). Serum K + levels of Bc nd A groups were determined s 3.88±0.34 mmol/ l nd 3.25±0.67, respectively wheres serum K+ levels of C group ws determined s 1.53±0.45 mmol/l. Serum Cl - levels of juvenile rinow trout

4 Vol. 46, No. 1, () Serum N levels (mmol/l) () Serum K levels (mmol/l) c 4,5 4 3,5 3 2,5 2 1,5 1 0,5 125 C Bc A 0 C Bc A (c) Serum C levels (mmol/l) (d) Serum Mg levels (mmol/l) C Bc A 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 C Bc A (e) Serum Cl levels (mmol/l) (f) Serum ph levels ,35 7, ,25 7, , ,1 105 C Bc A 7,05 C Bc A FIGURE 1: Serum N (), K (), C (c), Mg (d), Cl (e), ph (f) vlues of the rinow trouts fed with C, Bc nd A diets under hypoxic stress during four weeks.,,c Indictes significnt differences with respect to controls (P<0.05). (Hypoxi= 4.5 mgo2/l). All dt points re the verge of n =12 with ±SD.

5 26 INDIAN JOURNAL OF ANIMAL RESEARCH fed the Bc nd A rtions were determined to e s 127±3.27mmol/l nd 133±3.15 mmol/l, respectively nd re significntly higher (p<0.05) thn C group (119±4.12 mmol/l). Highest C +2 level of fish under hypoxic stress ws oserved in C group (5.27±0.12 mmol/l) wheres serum C +2 serum levels of Bc nd A groups were determined s 2.35±0.07 mmol/l nd 2.33±0.04 mmol/l, respectively (p<0.05) thus sttisticlly lower thn C group. Serum ph level did not sttisticlly differ mong the groups (p>0.05). However, the lowest ph level ws oserved in C group (Fig.1). In conclusion, this study determined tht feeding juvenile rinow trout with 250 mg/kg Vitmin C nd 70 mg/kg β-crotene diets promotes preservtion of lood electrolytes such s N +, K +, Mg +2 nd Cl - in fish under hypoxic stress t the level of 4.5 mg/kg oxygen. It ws found tht 250 mg/kg vitmin C supplementtion ws more effective thn 70 mg/kg et crotene in preservtion of serum N + level. Also serum C 2+ level of C group ws oserved to e significntly higher thn the other groups. The reson for serum C 2+ level of C group eing higher thn other groups is postulted s result of decrese of Mg +2 levels in lood cusing C 2+ trnsfer from tissues to lood. Artificil wter products, limited wter res used in their cultivtion systems, (pool, qurium, ot vrieties etc.) stock density, feed nd metolic wstes re stress fctors inevitly led the formtion of hypoxi in fish due to reduction in levels of oxygen in wter. It ws determined tht supplementtion of vitmin C nd β-crotene which re ntioxidnt vitmins into the rtions of juvenile rinow trout hs positive effects in preservtion of lood electrolyte levels of fish which provides cidse lnce during homeostsis period. REFERENCES AOAC, (1990). Officil Methods of Anlysis Assocition of Agriculturl Acedemy Press. Wshington, 1158 pp. Bry, T.M., (2000). Dietry ntioxidnts nd ssessment of oxidtive stress. Nutrition, 16: Chpmn, C.A., Hrhush B.K., Renshw GMC., (2010). The physiologycl tolernce of grey crpet shrk (Chiloscyllium puncttum) nd the epulette shrk (Hemiscyllium ocelltum) to noxic exposure t three sesonl tempertures. Fish. Phy. nd Biochem. doi: /s y. Clrk M.L., (2003). Comprýson of wter qulýty, rýnow trout productýon nd economýcs ýn oxygented nd erted rcewys oxygented nd erted rcewys Thesis Virgini Polytechnic Institute nd Stte University, 66 pp. Conte, F.S., (2004). Stress nd The Welfre of Cultured Fish, Applied Ani. Behviour Sci., 86: Choi, W., Benzic F., Collins A., Hnnigon M. nd Strin J., (2004). Vitmin C nd E: cut interctive effects on iomrkers of ntioxidnt defnce nd oxidtive stress. Muttion Res., 551: Çelik, E.ª., (2006). Stndrdistion of lood electrolytes for some fish species. Erciyes Üniversitesi J. Sci. Enstituet, 36: Hndy, R.D., Sims D.W., Giles A., Cmpell H.A. nd Musond M.M., (1999). Metolic trde-off etween locomotion nd detoxifiction for mintennce of lood chemistry nd growth prmeters y rinow trout (Oncorhynchus mykiss) during chronic dietry exposure to copper. Aqutic Toxicology, 47: Hilton, J.W., C.Y. Cho nd S.J. Slinger, (1977). Fctors ffecting the stility of supplementl scoric cid in prcticl trout diets. J. Fisheries Res. Bord of Cnd, 34: Johnnessen, T. nd Dhl E., (1996). Declines in oxygen concentrtions long the Norwegin Skgerrk cost, (1993): signl of ecosystem chnges due to eutrophiction? Limnology nd Ocenogrphy, 41: Johnston, C.E., Cheverie J.C., (1985). Comprtive nlysis of ionoregultion in rinow trout (Slmo girdneri) of different sizes following rpid nd slow slinity dpttion. Cn. J. Fish Aqut. Sci. 42: Krnky, K. J., (1998). Osmotic nd ionic regultion. In: Evns DH. The physiology of fishes, 2nd edn. CRC Press, Boc Rton, Fl., pp. Krmer, D.L., nd McClure M., (1982). Aqutic surfce respirtion, widespred dpttion to hypoxi in tropicl freshwter fishes. Environ. Biology Fishes, 7: Lyytikinen,T., Pylkko P, Ritolc O. nd Lindström-Seppd P., P., (2002). The effect of cute stress nd temperture on plsm cortisol nd ion concentrtions nd growth of Lke Inri Arctic chrr, Slvelinus lpinus. Env. Biol. Fishes., 64: Moeller, J. nd Roert B., (2001). Diseses of Fish. Cliforni Animl Helt nd Food Sfety, Cliforni Press, 34 pp. Mores, G., (2004). Metolicl responses in dpttion to stress in fish. Ýnterntionl Congress on the Biology of Fish, Brzil. Ntochin, Y.V., Lukinenko V.I.,, Kirsnov V.I., Lvrov E.A., Mctolloy G.F. nd, Shkhmotov H.J., (1985). Fetures of osmotic nd ionic regultions in Russin sturgeon (Acipenser guldenstdtii Brndt). Comp. Biochem. Physiol., 80:

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