1 TRANSPORT OF PIRARUCU Arpim gigs JUVENILES IN PLASTIC BAG Levy de Crvlho GOMES 1, Rodrigo ROUBACH 2, Bruno Adn Sgrtzki CAVERO 2, Mnoel PEREIRA-FILHO 2, Elisbeth Criscuolo URBINATI 3 ABSTRACT - This study exmined the stress response of pirrucu juveniles in closed system trnsport. Pirrucu (Arpim gigs) is ntive Amzonin fish species from the Osteoglosside fmily nd n obligted ir brether. A short durtion trnsport tril (6h) ws undertken compring closed polyethylene bgs filled with tmospheric ir (Air group) nd bgs filled with pure oxygen (Oxi group). Dissolved oxygen ws the only wter prmeter tht presented difference between fish groups, nd ws sturted in the oxi group s expected. There ws no mortlity in either group fter trnsport. Fish feeding ws observed 36 h fter trnsport for ll fish, nd norml feeding consumption ws observed t 72 h. In both groups physiologicl responses were similr. Cortisol did not show ny significnt ltertion during the smpled period. Unlike most fish species, cortisol vlues were unltered in both groups during smpling, while glucose presented significnt chnge up to 12 h fter trnsport. The results showed tht pirrucu trnsport in plstic bgs could be mde with either tmospheric ir or pure oxygen, since physiologicl response to stress, wter qulity nd feeding behvior fter 36 h were similr in two groups. Key-words: quculture, Arpim gigs, stress, cortisol, glucose Trnsporte de juvenis de pirrucu Arpim gigs em scos plásticos RESUMO - Este estudo exminou s resposts de estresse de juvenis de pirrucu trnsportdos em sistem fechdo. Pirrucu (Arpim gigs) é um peixe ntivo d bci Amzônic, d fmíli Osteoglosside que possui respirção ére obrigtóri. Foi relizdo um trnsporte de curt durção (6 h) em scos de polietileno infldos com r tmosférico (grupo r) ou com oxigênio puro (grupo oxi). O oxigênio dissolvido foi o único prâmetro de qulidde d águ que presentou diferenç esttístic entre os grupos, e como esperdo, o oxigênio estv supersturdo pr o grupo oxi. Não houve mortlidde pós o trnsporte em mbos os grupos. Os peixes se limentrm 36 h pós o trnsporte e presentrm um consumo de rção hbitul pós 72 h. As resposts fisiológics form semelhntes nos dois grupos. O cortisol não presentou mudnç significtiv durnte o período de mostrgem. Ao contrário d miori ds espécies, os vlores de cortisol se presentrm inlterdos nos dois grupos durnte mostrgem, enqunto glicose teve um umento significtivo té 12 h pós o trnsporte. Os resultdos mostrm que o trnsporte de pirrucu em scos de polietileno pode ser relizdo com r tmosférico ou oxigênio puro, um vez que s resposts de estresse, qulidde d águ e o comportmento limentr pós 36 h form similr entre os grupos. Plvrs-chve: qüicultur, Arpim gigs, estresse, cortisol, glicose INTRODUCTION Pirrucu (Arpim gigs) is n exclusively ir brether nd crnivorous fish species. It is highly prized ntive species from the Amzon region nd hs experienced huge increse in its demnd due to its growth rte in intensive nd semi-intensive systems reching up to 10 kg in twelve-month cycle (Sint-Pul, 1986; Imbirib, 2001; Cvero, 1 Embrp Amzôni Ocidentl, CP 319, , Mnus, AM. 2 Instituto Ncionl de Pesquiss d Amzôni - INPA, CP 478, , Mnus, AM 3 UNESP - Centro de Aqüicultur/CAUNESP, , Jboticbl, SP Trnsport ACTA AMAZONICA of pirrucu... 33(3): 1
2 2002). Broodstock recruitment nd reproduction for seed mss production re still the min issue for further reserch efforts. There is still lck of informtion vilble towrds stndrd nd n efficient trnsporttion method for pirrucu, minly to 1 to 2 kg fish. Since most fish trnsporttion uses the closed system within polyethylene bgs filled with oxygen (Piper, 1989; Gomes et l., 1999; Ross & Ross, 1999), this method should be strting point with pirrucu. With the incresing demnd for pirrucu of vrious sizes for the grow-out opertions nd broodstock formtion, the determintion of n pproprite, simple, relible, nd inexpensive method for efficient trnsporttion without stress is required. Pirrucu is n obligted ir brething fish, therefore the use of closed system with polyethylene bgs without the use of pure oxygen cn be fesible option for its trnsporttion. For this trnsporttion tril with short durtion (6 h), we compred the closed system (polyethylene bgs) filled with tmospheric ir versus pure oxygen. The intensity nd durtion of fish stress through physiologicl responses (cortisol nd glucose) were mesured long with the wter qulity fter the opertion, fish survivl nd feeding response within 96 h. MATERIALS AND METHODS Cptive rered pirrucu juveniles were obtined from commercil fish frm from Cori (Amzons, Brzil) trnsported to the Aquculture Deprtment t Instituto Ncionl de Pesquiss d Amzoni/INPA, where it were rered for 6 months in 75-m 2 pond. Ten fish (weight 1,123 ± g nd totl length 51.3 ± 3.5 cm; men ± SEM) were trnsferred to 1-m 3 fish cge in the sme pond nd held off feeding for 24 h to gstrointestinl emptying (Grottum et l., 1997). Fish were individully plced in ten 30- L polyethylene bgs with 10 L of wter, in two different groups (5 bgs per group): one group with bgs inflted with pure oxygen (commercil grde oxygen, White Mrtins S.A., Mnus, Brzil) (Oxi group) nd the other group with bgs inflted with tmospheric ir (Air group). Bgs were tied with rubber strings nd pcked in styrofom boxes, ccording to Gomes et l. (2002). Simulted trnsport took 6 h nd during this period, fish were periodiclly disturbed by shking the boxes. After trnsporttion, ech group of fish ws trnsferred to two 1-m 3 cges for subsequent monitoring. Commercil extruded feed ws offered to the fish 36 h fter trnsporttion to observe fish feeding behvior. Wter prmeters were mesured before trnsporttion, nd t the opening of the bgs (fter 6 h). Wter temperture nd DO were mesured using YSI 55 oxygen meter (Yellow Springs Instruments, Yellow Springs, Ohio, USA), nd the wter ph using digitl ph meter (Digimed model DMPH-2, São Pulo, SP, Brzil). Determintion of totl mmoni ws mde ccording to Verdow et l. (1978). Concentrtion of un-ionized mmoni (NH 3 ) ws clculted ccording to Boyd (1982). Levels of CO 2 concentrtion were determined by titrtion (APHA, 1992). Fish stress responses were evluted in fish from the cge (control; before trnsporttion); immeditely fter trnsporttion (AT) nd t 6 h AT, 12 h AT nd 24 h AT intervls, respectively. All fish were smpled in ech smpling time, similr proceedings s used by Brnett & Pnkhurst (1998). Blood ws withdrwn from the fish cudl vein with heprinized syringes. Blood glucose concentrtion ws mesured using the Advntge blood glucose system (Boehringer Mnnheim Gmbh, Mnnheim D-68298, Germny). Blood plsm ws seprted by centrifugtion (3,000 g, 10 min) nd stored t 20 C for further nlysis of cortisol using rdiommunossy technique (Cot-A-Countâ kit, DPCâ, CA, USA). Wter qulity fter trnsporttion nd stress prmeters of control group were compred by t-test (P < 0.05). Stress prmeters of both groups t different smplings times were compred ginst the control group by ANOVA nd Dunnett s test (P < 0.05). RESULTS There ws no mortlity fter trnsporttion in both groups. Fish feeding ws 2 Gomes et l.
3 observed 36 h fter trnsporttion for ll fish nd, the norml feeding consumption ws observed t 72 h. In both groups fish physiologicl responses were similr. Comprison of the control groups between both tretments with t test did not show ny difference for plsm cortisol (df = 8, P=0.292) or for glucose (df = 7, P=0.528). Cortisol did not show ny significnt ltertion through the smpled period (Fig. 1A). Immeditely fter trnsport there ws significnt increse on plsm glucose concentrtions, which were stble up to 12 h AT. At 24 h AT glucose level recovered the control group vlues (Fig. 1B). Wter qulity prmeters fter trnsport re presented on Tble 1. As expected, DO concentrtion ws significntly higher for the Oxi group. All other wter prmeters did not chnge significntly in both groups. Cortisol (ng/dl) A Control AT 6h AT 12h AT 24h AT Smpling Times Air group Oxi group Glucose (mg/dl) B * * * * * * Air group Oxi group 20 0 Control AT 6h AT 12h AT 24h AT Smpling Times Figure 1 - Cortisol (A) nd glucose (B) concentrtions during pirrucu trnsport procedures. Dt re mens ± SEM of 5 fish for ech group. control - before trnsport; AT - immeditely fter trnsport; 6h AT, 12h AT nd 24h AT - 6, 12 nd 24 h fter trnsport. Columns mrked with * re significntly different from the control (Dunnett s test; P < 0.05). Trnsport of pirrucu... 3
4 Tble 1 - Wter qulity from the control nd immeditely fter 6 h of trnsporting two groups pirrucu in closed system: (1) in plstic bgs inflted with ir (ir group) nd (2) in plstic bgs inflted with oxygen (oxi group). Dt re mens ± SEM of five replictes for ech group. Mens within row followed by different letters re significntly different (P < 0.05), s determined by t-test. Prmeters Control Air Groups Oxy o Temperture ( C) ± ± DO (mg/l) ± > b ph (units) ± ± CO 2 ( mg/l) ± ± NH 3 ( µmol/l) ± ± DISCUSSION Monitoring physiologicl prmeters during stressing opertions, like fish trnsporttion, cn provide us importnt dt for the estblishment of dequte mngement prctices, even for situtions where we re not ble to observe ny fish mortlity. For successful fish hndling nd trnsporting stronger effort towrds niml well being is more desirble thn surveying for fish mortlity. Usully severe stress condition could compromise fish dptive cpcity to new environment nd even result in temporry growth interruption, long with higher susceptibility to prsite infections due to lower feeding ctivity (Wedemeyer, 1996). Plsm glucose is useful stress indictor, besides being esy to monitor (Morgn & Iwm, 1997; Wells & Pnkhurst, 1999; Gomes et l., 2001). Cortisol is lso good indictor, nd one of the most studied hormones for stress monitoring (Mommsen et l., 1999; Brton, 1997). The present results re importnt for future studies towrds the stress meliortion in pirrucu husbndry nd mngement s it does give some bseline vlues for plsm cortisol nd glucose, in comfort zone (control) nd in stressful situtions (trnsporttion). Pirrucu vlues for plsm cortisol nd glucose in the control group (80-97 ng/ml nd mg/dl, respectively) were similr to vlues found for other Amzonin species, s for tmbqui (Colossom mcropomum) (80 ng/ ml nd mg/dl for plsm cortisol nd glucose, respectively) (Wood et l., 1998; Gomes et l., 2001) nd for mtrinxã (Brycon cephlus) (100 ng/ml nd 40 mg/dl for plsm cortisol nd glucose, respectively) (Crneiro & Urbinti, 2001). For most fish species, cortisol is the min hormone tht ctivtes glucose, which provides n increse in the energy supply, so fish cn withstnd the stress sitution (Mommsen et l., 1999). This pttern ws not observed with pirrucu, where cortisol levels were unltered t both groups during smpling, while glucose presented significnt increse up to 12 h fter trnsporttion. For further studies with pirrucu this is n importnt spect to pproch, since there re other hormones tht could be the precursors of glycemic stte in stressful situtions, s the cthecholmines (Mommsen et l., 1999). Another importnt result ws not significnt chnge on pirrucu plsm cortisol levels even when fish were submitted to continuous smpling. Observtions with the se rven (Hemitripterus mericnus), fish with sedentry nd sluggish life-style nd low metbolic ctivity, presented higher threshold for the endocrine response to stress (cortisol nd ctecholmines) nd delyed relese of cortisol, which ws ttributed to n dpttion to prevent the excessive mobiliztion of energy stores to suit its life-style (Vijyn & Moon, 1994). On the other hnd, Brnett & Pnkhurst (1998) observed tht continuous smpling in greenbck flounder (Rhombosole tpirin) 4 Gomes et l.
5 cused progressive increse on fish plsm cortisol vlues. This shows tht, like in the se rven, pirrucu hs gret cpcity to keep cortisol t low levels even when successive stress occurs within short time intervl (24 h). Glucose results cn be relted to the continuous smplings, mostly during smplings t AT, 6 nd 12 h AT. The glycemic rise fter trnsport could be relted to the stress during trnsporttion procedures. In ddition, the short time intervls of AT, 6 nd 12 h AT of fish smpling could hve hd n effect on the mintennce of high glucose vlues. Between 12 h AT nd 24 h AT smpling procedures there ws bigger time intervl, llowing fish to return to norml glycemic levels. In such cses, pirrucu did not present n ccumultive stress effect, becuse even with successive smplings, blood glucose levels kept similr vlues from AT until 12 h AT smpling. Brton et l. (2000) observed tht juvenile pllid sturgeon (Scphirhynchus lbuns) nd the hybrid of pllid x shovelnose (S. lbuns x S. pltorynchus) presented low physiologicl response to trnsporttion, cute hndling nd severe confinement, nd suggested tht it cn be relted either to the species evolutionry history, neuroendocrine mechnisms involved in the corticosteroid responses, or with the ntomy structure of the intr-renl tissues. Pirrucu is lso fish with long evolutionry history, nd n obligte ir brether. Therefore, the low responses observed in pirrucu could lso be relted to its evolutionry history or different physiologic mechnisms due to its respirtory mode. Besides tht, pirrucu is sluggish type of fish, nd s suggested by Vijyn & Moon (1994), it could lso explin the delyed relese of cortisol s n dpttion to prevent the excessive mobiliztion of energy stores to suit its life-style. Wter qulity is n importnt stress precursor under poor conditions, like in fish trnsporttion (Berk, 1986; Erikson et l., 1997). In the present study, wter qulity fter trnsporttion, ws t cceptble levels in both groups ccording to Berk (1986). Therefore it ws probbly not the triggering cuse for the plsm glucose increse. DO ws the only wter prmeter tht presented difference between fish groups. As expected in the Oxi group, DO in the wter ws sturted. Deleterious effects due to oxygen sturted wter during trnsporttion hve lredy been described by Wedemeyer (1996), lthough this probbly did not hppen with pirrucu, since cortisol nd glucose vlues were similr in both groups. The fct tht oxygen sturtion does not hve n dverse effect on pirrucu could be n dvntge for long distnce trnsporttion. Even though oxygen consumption ws not mesured, it ws probbly low, since fish frequency to breth t the surfce followed its norml pttern observed in fish in n erth pond. The results showed tht pirrucu trnsporttion using closed system with plstic bgs cn be relized with tmospheric ir or with pure oxygen, since fish physiology response to stress, wter qulity nd feeding behvior fter 36 h were similr between groups. More studies re needed with pirrucu to evlute longer durtion fish trnsport nd the possibility of utilizing sedtive products to help reduce fish glycolytic stress. ACKNOWLEDGMENTS The uthors thnk Richrd Brinn from CAUNESP for his ssistnce during cortisol nlysis. This reserch ws prtilly supported by Project Pirrucu/CNPq. Urbinti nd Pereir-Filho re reserch fellowship recipients of CNPq/Brzil. LITERATURE CITED APHA (Americn Public Helth Assocition, Americn Wter Works Assocition, Wter Environment Federtion) Stndrd methods for the exmintion of wter nd wstewter, 18th edition. Americn Public Helth Assocition, New York, 1220 p. Brnett, C.W.; Pnkhurst, N.W The effects of common lbortory nd husbndry prctices on the stress response of greenbck flounder Rhombosole tpirin (Günther, 1862). Aquculture. 162: Trnsport of pirrucu... 5
6 Brton, B.A Stress in finfish: pst, present nd future historicl perspective. In: Iwm, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B. (Eds.). Fish stress nd helth in quculture. Society for Experimentl Biology Seminr Series 62. Cmbridge University Press, New York, NY. p Brton, B.A.; Bolling, H.; Huskins, B.; Jnsen, C.R Juvenile pllid (Scphirhynchus lbuns) nd hybrid pllid x shovelnose (S. lbuns x S. pltorynchus) sturgeons exhibit low physiologicl responses to cute hndling nd severe confinement. Comprtive Biochemistry Physiology. 126A: Berk, R The trnsport of live fish. A review. EIFAC Technicl Ppers 48, FAO, Rome. 52 p. Boyd, C.E Wter qulity mngement for pond fish culture. Elsevier Science Publishers, Amsterdm. 318 p. Crneiro, P.C.F.; Urbinti, E.C Slt s stress response mitigtor of mtrinxã, Brycon cephlus (Günther), during trnsport. Aquculture Reserch. 32: Cvero, B. A. S Densidde de estocgem de juvenis de pirrucu, Arpim gigs (Cuvier, 1829), em tnques-rede de pequeno volume. Dissertção de mestrdo, Instituto Ncionl de Pesquiss d Amzôni/Fundção Universidde do Amzons. Mnus, Amzons. 51 p. Erikson, U.; Sigholt, T.; Selnd, A Hndling stress nd wter qulity during live trnsporttion nd slughter of Atlntic slmon (Slmo slr). Aquculture. 149: Gomes, L.C.; Chippri-Gomes, A.R.; Lopes, N.P.; Roubch, R.; Arujo-Lim, C.A.R.M Efficcy of benzocine s nesthetic for tmbqui juveniles (Colossom mcropomum). Journl of the World Aquculture Society. 31: Gomes, L.C.; Golombieski, J.I.; Chippri- Gomes, A.R.; Bldisserotto, B Effect of slt in the wter of trnsport on survivl nd N + nd K + body levels in fingerlings of silver ctfish Rhmdi quelen (Pimelodide). Journl of Applied Aquculture. 9:1-9. Gomes, L.C.; Roubch, R.; Arujo-Lim, C.A.R.M Trnsporttion of tmbqui juveniles (Colossom mcropomum) in Amzon: min problems. World Aquculture Mgzine. 33: Grottum, J.A.; Sturnes, M.; Sigholt, T Effect of oxygention, ertion nd ph control on wter qulity nd survivl of turbot, Scophthlmus mximus (L.), kept t high densities during trnsport. Aquculture Reserch. 28: Imbirib, E.P Potencil d crição de pirrucu, Arpim gigs, em ctiveiro. Act Amzônic. 31: Mommsen, T.P.; Vijyn, M.M.; Moon, T.W Cortisol in teleosts: dynmics, mechnisms of ction, nd metbolic regultion. Reviews in Fish Biology nd Fisheries. 9: Morgn, J.D.; Iwm, G.K Mesurements of stressed sttes in the field. In: Iwm, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B. (Eds.). Fish stress nd helth in quculture. Society for Experimentl Biology Seminr Series 62. Cmbridge University Press, New York, NY. p Piper, G.R.; McElwin, I.B.; Orme, L.E.; McCrren, J.P.; Fowler, L.G.; Leonrd, J.R Fish htchery mngement. United Sttes Deprtment of the Interior, Fish nd Wildlife Service, Wshington, D.C. 517 p. Ross, L.G.; Ross, B Anesthethic nd sedtive techniques for qutic nimls. Blckwell Science, Oxford. 159 p. Sint-Pul, U Potentil for quculture of South Americn freshwter fishes: review. Aquculture. 54: Verdow, H.; Vnechted, C.J.A.; Dekkers, E.M.J Ammoni determintion bsed on indophenol with sodium slicylte. Wter Reserch. 12: Gomes et l.
7 Vijyn; M.M.; Moon, T.W The stress response nd plsm disppernce of corticosteroid nd glucose in mrine teleost, se rven. Cndin Journl of Zoology. 72: Wedemeyer, G.A Physiology of fish in intensive culture systems. Chpmn nd Hll, New York. 232 p. Wells, R.M.G.; Pnkhurst, N.W Evlution of simple instruments for the mesurement of blood glucose nd lctte, nd plsm protein stress indictor in fish. Journl of the World Aquculture Society. 30: Wood, C.M.; Wilson, R.W.; Gonzlez, R.J.; Ptrick, M.L.; Bergmn, H.L.; Nrhr, A.; Vl, A.L Responses of n Amzonin teleost, the tmbqui (Colossom mcropomum), to low ph in extremely soft wter. Physiologicl Zoology. 71: Recebido: 14/10/2002 Aceito: 04/09/2003 Trnsport of pirrucu... 7
8 8 Gomes et l.