Act Scientirum http://www.uem.br/ct ISSN printed: 1806-2636 ISSN on-line: 1807-8672 Doi: 10.4025/ctscinimsci.v34i1.11898 Ammoni excretion t different life stges of silver ctfish Lucino de Oliveir Grci 1, Neiv Brun 2, Alexssndro Geferson Becker 1, Vni Lúci Loro 3 nd Bernrdo Bldisserotto 1* 1 Lbortório de Fisiologi de Peixes, Deprtmento de Fisiologi e Frmcologi, Universidde Federl de Snt Mri, Av. Rorim, 1000, 97105-900, Snt Mri, Rio Grnde do Sul, Brzil. 2 Deprtmento de Aquicultur, Universidde Federl de Snt Ctrin, Florinópolis, Snt Ctrin, Brzil. 3 Lbortório de Bioquímic Adpttiv, Deprtmento de Químic, Universidde Federl de Snt Mri, Snt Mri, Rio Grnde do Sul, Brzil. *Author for correspondence. E-mil: bbldisserotto@hotmil.com ABSTRACT. This study exmined mmoni excretion t different life stges (eggs, lrve nd juveniles) in silver ctfish (Rhmdi quelen) nd determined the influence of fsting time on mmoni excretion. Eggs nd lrve were collected from incubtors t different times fter fecundtion nd plced in chmbers. Juveniles were seprted into two weight clsses (2-50 g nd 150-320 g) nd plced in individul chmbers fter feeding. Wter ws collected from ech chmber to determine mmoni excretion. Ammoni excretion by the eggs ws low, but when htching begn pproximtely 28h fter fecundtion, excretion incresed until 48h fter fecundtion. In fsting silver ctfish juveniles, there ws significnt negtive correltion between mmoni excretion nd weight. Moreover, mmoni excretion decresed significntly fter 12 nd 48h of fsting (compred to 6h fsting) in the smllest nd lrgest specimens, respectively. Consequently, during incubtion of silver ctfish eggs, wter renovtion must be incresed t htching time to void build-up in the concentrtion of mmoni. In ddition, s mmoni excretion in this species increses fter feeding, feed must be discontinued when mmoni levels in the tnks re high to void further increse of this metbolite nd consequent mortlity of silver ctfish. Keywords: eggs, fsting time, nitrogenous compound, lrve. Excreção de môni em diferentes estágios de vid do jundiá RESUMO. Verificou-se excreção de môni em diferentes estágios de vid (ovos, lrvs e juvenis) e determinou-se influênci do tempo de jejum n excreção de môni no jundiá, Rhmdi quelen. Ovos e lrvs form coletdos ds incubdors em diferentes tempos pós fecundção e colocdos em recipientes. Juvenis form seprdos em dus clsses de peso (2-50 e 150-320 g) e pós limentção form colocdos em recipientes individuis. As águs form coletds de cd recipiente pr determinção d excreção de môni. A excreção de môni pelos ovos foi bix, ms inicid eclosão 28h pós fecundção, el umentou té s 48h. Nos juvenis em jejum, houve significtiv correlção negtiv entre excreção de môni e o peso. Além disso, excreção de môni diminuiu significtivmente pós 12 e 48h de jejum nos espécimes menores e miores, respectivmente. Portnto, renovção d águ deve ser umentd no momento d eclosão dos ovos de jundiá pr evitr umento d concentrção de môni. Como ness espécie excreção de môni ument pós limentção, qundo os níveis nos tnques forem elevdos limentção deve ser suspens pr evitr umento dicionl deste metbólito e mortlidde. Plvrs-chve: ovos, tempo de jejum, composto nitrogendo, lrv. Introduction Ammoni is the dominnt end product of nitrogen metbolism in most teleosts, nd is toxic t low concentrtions, prticulrly in NH 3 (unionized mmoni) form (CHEW et l., 2006; FELIPO; BUTTERWORTH, 2002; IP et l., 2004; WICKS; RANDALL, 2002). The min internl source of mmoni in fish is through the ctbolism of proteins, nd most of this wste product is produced in the liver of fish during the trnsmintion of mino cids followed by the demintion of glutmte (WICKS; RANDALL, 2002). Ammoni is minly excreted through the gills (WILKIE, 2002), nd teleosts usully increse the excretion of mmoni fter feeding (ALTINOK; GRIZZLE, 2004). Compred to the number of studies conducted on dult stges of teleosts, only smll number of studies hve exmined nitrogen metbolism during the erly development of teleosts. Ammoni ppers to be the dominnt end product during the embryonic nd yolk sc stge of freshwter teleosts (DABROWSKI et l., 1984; OLIVA-TELES; KAUSHIK, 1990; WRIGHT et l., 1995). Ammoni excretion in Cregonus lvretus incresed during the pre-htching period, from 52.1 to
16 Grci et l. 163.2 μg h -1 10 3 eggs -1 (DABROWSKI et l., 1984), nd high rtes of mmoni excretion were observed (> 100 nd > 50 μg N h -1 10 3 eggs -1 ) in common crp, Cyprinus crpio, t htching nd t the onset of freeswimming stges, respectively (KAUSHIK et l., 1982). Altinok nd Grizzle (2004) reported tht dietry protein intke is the most importnt fctor ffecting mmoni excretion. In fish fed until stition, mmoni production my be 10 times higher thn in strved fish (WOOD, 1993). The silver ctfish, Rhmdi quelen (Quoy nd Gimrd, 1824; Heptpteride), is found from southern Mexico to centrl Argentin; the species is bred more intensively in Brzil, Uruguy nd Argentin. Moreover, the species shows two spwning peks yer -1 (GOMES et l., 2000); embryologicl development is fst, nd lrvl development occurs in 3-5 dys (AMORIM et l., 2009; PEREIRA et l., 2006). Therefore, the im of this study ws to exmine mmoni excretion t different life stges (eggs, lrve nd juveniles) nd determining the influence of fsting time on mmoni excretion in silver ctfish. Mteril nd methods Silver ctfish eggs (men weight = 5.0 mg) were obtined fter induced spwning. The brood fish received one dose of crp pituitry extrct (femle = 5 mg kg -1 ; mle = 3 mg kg -1, ccording to LEGENDRE et l., 1996) nd were then extruded fter nine hours. The oocyte mss ws plced in plstic continer, nd milt ws then dded to provide fertiliztion. Hlf liter of wter t the sme temperture s the wter in which the brood fish were mintined ws dded to the continer to llow egg hydrtion (10 min.). Twelve minutes lter, the ded eggs were mnully seprted on Petri dishes, nd the vible fertilized eggs (determined by the observtion of the initil cell division on microscope) were plced in incubtors (4 L polyethylene bottles 600 eggs per bottle) t temperture of 24 ± 1.0ºC nd ph of 7.2 ± 0.2 units. Ech bottle received continuous ertion with 20 W ir pump (3.2 L min. -1 ir flow) tht lso promoted wter movement. The incubtors were clened dily by suction, nd, consequently, t lest 30% of the wter in the bottles ws replced with wter previously djusted to the sme conditions of temperture nd ph. Eggs nd lrve were collected from the incubtors t 0, 12, 24, 36, nd 192h fter fecundtion nd plced in continuously erted 25 ml chmbers (ten replictions in ech time, n=10 for ech repliction) for 12h. Wter smples (5 ml) were tken t the beginning nd end of ech 12h period nd the mmoni concentrtion ws immeditely mesured. Afterwrds, ll eggs nd lrve from ech repliction were weighed in scle (precision ± 0.00001 g). Lrve received commercil diet (32% crude protein) fter htching (pproximtely 28h fter fecundtion), in constnt intervls (every two hours). Silver ctfish juveniles (2-50 g nd 150-320 g) were bought from commercil fish frm ner the city of Snt Mri, Rio Grnde do Sul Stte, Brzil, nd trnsported to the Fish Physiology lbortory t the UFSM, where they were mintined for four dys in continuously erted 250 L tnks (erted using two 20 W ir pumps; ph 6.7 ± 0.5 units; temperture 23 ± 1.0ºC; nd hrdness 32.0 ± 1.0 mg CCO 3 L -1 ). After this cclimtion period, the silver ctfish were fed nd plced in individul chmbers (ten fish for ech size rnge) with pproximtely 20 times their volume in wter. Wter ws collected from ech chmber t 0, 6, 12, 24, 36, nd 48h fter the trnsfer nd the mmoni concentrtion ws immeditely mesured. Fish were weighed t the end of wter collection nd were not fed when inside the chmbers. Dissolved oxygen (6.17 ± 0.8 mg L -1 ) nd temperture (21.5 ± 1.5 o C) were monitored with n oxygen meter (model Y5512). The ph levels (7.2 ± 0.2 units) were verified with DMPH-2 ph meter, while nitrite vlues (0.3 ± 0.1 mg L -1 ) were ssessed using colorimetric method (commercil kit), nd wter lklinity (40 ± 1.3 mg CCO 3 L -1 ) nd hrdness vlues (36 ± 1.5 mg CCO 3 L -1 ) were determined ccording to Boyd nd Tucker (1992). Ammoni concentrtions in the wter smples were mesured ccording to Verdouw et l. (1978), nd mmoni excretion (efflux) ws clculted ccording to Gonzlez et l. (1998). These prmeters were observed in ll experiments (eggs, lrve nd juveniles groups). Dt re reported s mens ± S.E.M. Homogeneity of vrinces between groups ws tested by Levene s test. Comprisons of mmoni excretion t different times of embryonic development nd lrviculture nd fter fsting in juveniles were conducted using onewy ANOVA nd Tukey s test (Sttistic softwre v.5.1). The reltionship between weight nd mmoni excretion in silver ctfish juveniles ws ssessed using Sigm Plot 11.0 softwre. Results nd discussion All physicochemicl prmeters of the wter were kept within the rnge recommended for this species
Ontogenetic nitrogen excretion in Rhmdi quelen 17 throughout the experiment (BALDISSEROTTO, 2004). Ammoni excretion by eggs ws low, but when htching strted pproximtely 28h fter fecundtion, excretion incresed until 48h fter fecundtion (Figure 1). Lrger lrve (192h fter fecundtion) presented lower vlues of mmoni excretion. Therefore, the increse observed during the embryonic stge probbly reflects the smll but growing mount of respiring tissues nd lso the elimintion of metbolites. However, lthough the egg cpsule or chorion is permeble to mmoni (RAHAMAN-NORONHA et l., 1996), elimintion of mmoni is slow in the bsence of respirtory convection (ROMBOUGH; MOROZ, 1990, 1997) nd direct contct with bulk wter. The present results regrding silver ctfish re in greement with studies of mmoni excretion of embryos nd lrve of other freshwter teleost species (BUCKING; WOOD, 2008; DABROWSKI et l., 1984; KAUSHIK et l., 1982; OLIVA-TELES; KAUSHIK, 1987, 1990; TERJESEN et l., 1997; WRIGHT et l., 1995). Ammoni excretion incresed mrkedly for up to 48h fter htching, but the trend did not continue t the next smpling point. Interestingly, similr pttern with n pprent lg phse ws observed in studies of other freshwter teleost lrve (DABROWSKI et l., 1984; KAUSHIK et l., 1982; OLIVA-TELES; KAUSHIK, 1987; TERJESEN et l., 1997), nd it could result from high mino cid ctbolism ssocited with the process of htching. 2.4 1.6 0.8 00 0 24 48 72 96 120 140 168 192 Time fter fecundtion (h) Figure 1. Ammoni excretion in silver ctfish eggs nd lrve t different times fter fecundtion. In fsting silver ctfish, there ws significnt negtive reltionship between mmoni excretion nd weight fter 48h (Figure 2). This is in greement with higher mmoni urinry excretion ssocited with lower body mss observed in silver ctfish (BOLNER; BALDISSEROTTO, 2007), s well s in wlleye, Snder vitreus (YAGER; SUMMERFELT, 1993) nd tmbqui, Colossom mcropomum (ISMIÑO-ORBE et l., 2003). This cn be ttributed t lest prtilly to ontogenetic chnges in the metbolic intensities of the different tissues, which tend to decline s fish increse in size (WOOD, 1993). 1.2 1.0 0.8 0.6 0.4 0.2 00 0 100 200 300 Weight (g) Figure 2. Ammoni excretion in silver ctfish s function of weight. This reltionship is described by the following eqution: ŷ = -0.976 + 2.163 / (1 + (X / 461.57) 0.42 ), r 2 = 0.960; where ŷ = mmoni excretion (μg h -1 g weight -1 ) nd x = fish weight (g). Ammoni excretion decresed significntly fter 12 nd 48h of fsting (compred to 6h of fsting) in the smllest nd lrgest specimens, respectively (Figure 3). Ammoni excretion is ssocited with feeding chnges during the postprndil period. Severl studies (Dosdt et l. (1996), with five teleost fish species divided into two weight clsses, 10 nd 100 g; Gelineu et l. (1998), with 70 g rinbow trout; Leung et l. (1999), with 83 g Epinephelus reoltus; Bucking nd Wood (2008), with 300-400 g rinbow trout) hve documented tht elevted mmoni excretion rtes occurred between 2 nd 12h fter feeding. As reported bove, mmoni excretion increses during the postprndil period in fish, suggesting tht internl mmoni levels hve risen fter feeding (WICKS; RANDALL, 2002). Interestingly, mmoni excretion incresed gin in smll silver ctfish fter 24-48h of fsting. As explined erlier, smll fish hve higher metbolic rte thn lrger fish, nd it is possible tht fter 24h fsting they use protein rther thn lipid nd crbohydrte resources to generte energy. This protein ctbolism would led to n increse in mmoni excretion. In greement with this hypothesis, rinbow trout fsted for one week presented mmoni excretion levels s high s those observed in trout 6-12h fter feeding (BUCKING; WOOD, 2008). Postprndil nitrogenous excretion is known to be influenced by the type of feed (ENGIN;
18 Grci et l. CARTER, 2001; LAM et l., 2008; WEBB JR.; GATLIN III, 2003) nd fish size (JOBLING, 1981; LEUNG et l., 1999), nd thus it is essentil tht future studies exmine the intricte reltionships between feed types qulities -1 nd the induction of N retention mechnisms in silver ctfish t different stges of development. 1.00 0.80 0.60 0.40 0.20 0.00 b Weight clsses (g) 2-50 150-320 b b 0 12 24 36 48 Time fter feeding (h) Figure 3. Ammoni excretion in silver ctfish of two different weight clsses s function of fsting time. Different letters indicte significnt differences between the times fter feeding within the sme weight clss (p < 0.05). Conclusion In conclusion, during the incubtion of silver ctfish eggs, wter renovtion must be incresed t htching time to void build-up of mmoni. Additionlly, since mmoni excretion in this species increses fter feeding, feeding must be discontinued when mmoni levels in the tnks re high, to void further increse of this metbolite nd consequent fish mortlity. Acknowledgements N. Brun received CAPES (Coordenção de Aperfeiçomento de Pessol de Nível Superior, Brzil) fellowships, nd A. G. Becker, V. L. Loro nd B. Bldisserotto received CNPq (Conselho Ncionl de Desenvolvimento Científico e Tecnológico) fellowships. This study ws supported by CNPq (process number 475017/03-0). References ALTINOK, I.; GRIZZLE, J. M. Excretion of mmoni nd ure by phylogeneticlly diverse fish species in low slinities. Aquculture, v. 238, n. 1-4, p. 499-507, 2004. AMORIM, M. P.; GOMES, B. V. C.; MARTINS, Y. S.; SATO, Y.; RIZZO, E.; BAZZOLI, N. Erly development of the silver ctfish Rhmdi quelen (Quoy & Gimrd, 1824) (Pisces: Heptpteride) from the São Frncisco River bsin, b b b Brzil. Aquculture Reserch, v. 40, n. 2, p. 172-180, 2009. BALDISSEROTTO, B. Silver ctfish culture. World Aquculture, v. 35, n. 4, p. 65-67, 2004. BOLNER, K. C. S.; BALDISSEROTTO, B. Wter ph nd urinry excretion in silver ctfish Rhmdi quelen. Journl of Fish Biology, v. 70, n. 1, p. 50-64, 2007. BOYD, C. E.; TUCKER, C. S. Wter qulity nd pond soil nlysis for quculture. Albm Agriculturl Experiment Sttion. Albm: Auburn University, 1992. BUCKING, C.; WOOD, C. M. The lkline tide nd mmoni excretion fter voluntry feeding in freshwter rinbow trout. The Journl of Experimentl Biology, v. 211, n. 15, p. 2533-2541, 2008. CHEW, S. F.; WILSON, J. M.; IP, Y. K.; RANDALL, D. J. Nitrogenous excretion nd defense ginst mmoni toxicity. In: VAL, A.; ALMEIDA-VAL, V.; RANDALL, D. J. (Ed.). Fish physiology. The physiology of tropicl fishes. New York: Acdemic Press, 2006. p. 307-395. DABROWSKI, K.; KAUSHIK, S. J.; LUQUET, P. Metbolic utiliztion of body stores during the erly life of whitefish Coregonus lvretus L. Journl of Fish Biology, v. 24, n. 6, p. 721-729, 1984. DOSDAT, A.; SERVAIS, F.; METAILLER, R.; HUELVAN, C.; DESBRUYERES, E. Comprison of nitrogenous losses in five teleost fish species. Aquculture, v. 141, n. 1-2, p. 107-127, 1996. ENGIN, K.; CARTER, C. G. Ammoni nd ure excretion rtes of juvenile Austrlin short-finned eel (Anguill ustrlis ustrlis) s influence by dietry protein level. Aquculture, v. 194, n. 1-2, p. 123-136, 2001. FELIPO, V.; BUTTERWORTH, R. F. Neurobiology of mmoni. Progress in Neurobiology, v. 67, n. 4, p. 259-279, 2002. GELINEAU, A.; MEDALE, F.; BOUJARD, T. Effect of feeding time on postprndil nitrogen excretion nd energy expediture in rinbow trout. Journl of Fish Biology, v. 52, n. 4, p. 655-664, 1998. GOMES, L. C.; GOLOMBIESKI, J. I.; CHIPPARI- GOMES, A. R.; BALDISSEROTTO, B. Biologi do jundiá (Rhmdi quelen; Teleostei, Pimelodide). Ciênci Rurl, v. 30, n. 1, p. 179-185, 2000. GONZALEZ, R. J.; WOOD, C. M.; WILSON, R. W.; PATRICK, M. L.; BERGMAN, H. L.; NARAHARA, A.; VAL, A. L. Effects of wter ph nd clcium concentrtion on ion blnce in fish of the Rio Negro, Amzon. Physiologicl Zoology, v. 71, n. 1, p. 15-22, 1998. IP, Y. K.; CHEW, S. F.; WILSON, J. M.; RANDALL, D. J. Defenses ginst mmoni toxicity in tropicl irbrething fishes exposed to high concentrtions of environmentl mmoni: review. Journl of Comprtive Physiology - Prt A, v. 174, n. 7, p. 565-575, 2004. ISMIÑO-ORBE, R. A.; ARAUJO-LIMA, C. A. R. M.; GOMES, L. C. Excreção de môni por tmbqui (Colossom mcropomum) de cordo com vrições n tempertur d águ e mss do peixe. Pesquis Agropecuári Brsileir, v. 38, n. 10, p. 1243-1247, 2003.
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