iologicl indictors of stress in pcu (Pirctus mesopotmicus) fter cpture breu, JS., Tkhshi, LS. d, Hoshib, M. c nd Urbinti, EC. b,d * Deprtmento de Zootecni e Extensão Rurl, Fculdde de gronomi e Medicin Veterinári FMEV, Universidde Federl do Mto Grosso UFMT, v. Fernndo Corrê d Cost, s/n, Coxipó, CEP 786-9, Cuibá, MT, rzil b Centro de qüicultur C, Universidde Estdul Pulist UNESP Vi de cesso Prof. Pulo Donto Cstelne, CEP 14884-9, Jboticbl, SP, rzil c Instituto de Ciêncis Sociis e plicds e Zootecni do ixo mzons, Universidde Federl do mzons UFM, Ru Príb, 2186, Cmpus Universitário do ixo mzons, CEP 6912-1, Printins, M, rzil d Fculdde de Ciêncis gráris e Veterináris FCV, Universidde Estdul Pulist UNESP, Vi de cesso Prof. Pulo Donto Cstelne, CEP 14884-9, Jboticbl, SP, rzil *e-mil: bethurb@cunesp.unesp.br Received ugust 17, 27 ccepted Jnury, 28 Distributed My 1, 29 (With figures) bstrct The effects of cpture (chsing, netting nd ir exposure) on cortisol, glucose, chloride, sodium, potssium nd clcium concentrtions, osmollity, hemtocrit, hemoglobin concentrtion, red blood cells count (RC) nd men corpusculr volume (MCV) were investigted in pcu (Pirctus mesopotmicus). totl of 12 fish (49.7 ± 11.7 g) were subjected to cpture nd minutes ir exposure nd cpture nd ir exposure. Nine fish t ech tretment were smpled t, 1,, 6 minutes nd fter the procedure. Nine undisturbed fish were scrificed before the hndling nd used s controls. Cpture resulted in rise in blood cortisol nd glucose nd, respectively, fter both ir exposures. oth indictors returned to resting levels fter cpture. In both fish groups, plsm chloride decresed 6 minutes fter cpture, not recovering the resting levels within fter, nd serum sodium rose t 1 nd minutes nd recovered the resting levels lter. There were no significnt chnges neither in potssium, clcium nd osmollity nor in hemtocrit, hemoglobin, RC nd MCV s consequence of cpture. The sequentil stressors imposed to pcu during cpture ctivted the brin-pituitry-interrenl xis (cortisol nd glucose responses) but the ctivtion of the brin-sympthetic-chromffin cell xis ws pprently moderte (ionic nd hemtologicl responses). Keywords: cpture, ir exposure, stress, Pirctus mesopotmicus. Indicdores biológicos de estresse em pcu (Pirctus mesopotmicus) pós cptur Resumo Os efeitos d cptur (perseguição, contenção em puçá e exposição ére) no perfil snguíneo do cortisol, glicose, cloreto, sódio, potássio, cálcio e n osmolridde, hemtócrito, hemoglobin, número de céluls vermelhs (CV) e volume corpusculr médio (VCM) form investigdos no pcu (Pirctus mesopotmicus). Um totl de 12 peixes (49,7 ± 11,7 g) foi submetido à cptur com ou minutos de exposição ére. Nove peixes de cd trtmento form mostrdos, 1,, 6 minutos e 24 hors depois e outros nove peixes form mostrdos ntes d cptur e considerdos controle. cptur resultou em umento do cortisol e glicose no sngue e minutos depois d cptur, respectivmente, independente do tempo de exposição ére. mbos os indicdores recuperrm os vlores controle em 24 hors. Nos dois grupos de peixes, o cloreto plsmático diminuiu 6 minutos pós cptur e não recuperou os vlores controle, enqunto o sódio sérico umentou entre 1 e minutos recuperndo condição controle em 24 hors. Não houve lterção significtiv nos vlores de potássio, cálcio, osmolridde ou no hemtócrito, hemoglobin, CV e VCM como consequênci d cptur. Os estressores sequenciis plicdos no pcu durnte cptur tivrm o eixo cérebro-pituitári-interrenl (resposts do cortisol e glicose), ms tivção do eixo cérebro-sistem simpático-céluls cromfins foi prentemente moderd (resposts iônics e hemtológics). Plvrs-chve: cptur, exposição ére, estresse, Pirctus mesopotmicus. rz. J. iol., 69(2): 41-421, 29 41
breu, JS. et l. 1. Introduction There re mny potentil pplictions of the stress response. Experimentl biologists need to know the bseline from which to ssess whtever response they re studying. It is lso importnt to know whether fish under intensive quculture re in or out of stressed stte. quculture development depends on the estblishment of pproprite mngement prctices. Thus the fetures of the physiologicl stress responses cn serve this purpose. Vrious stressors, including fish cpture (Mugnier et l., 1998; rends et l., 1999; rcellos et l., 21; Roch et l., 24; Morles et l., 2), re necessry components of modern intensive quculture (Wendelr ong, 1997). The fish response to such stressors involves series of physiologicl chnges in n ttempt to compenste for the chllenge imposed upon it nd, thereby cope with the stress. Such chnges hve been brodly ctegorized into primry, secondry nd tertiry responses including hormonl, metbolic, osmoionic nd hemtologicl disturbnces nd hve been used to chrcterize the degree of stress fish experienced (McDonld nd Millign, 1997; Wendelr ong, 1997; Wojtszek et l., 22). The mjority of interntionl reserch effort hs gone into the stress responses of slmonids (McCormick et l., 1998; ckermn et l., 2; rton, 2), but other species such s crp (Cyprinus crpio L.) (Vinen et l., 21), tilpi (Oreochromis niloticus L.) (Vn der Slm et l., 2), turbot (Scophthlmus mximus L.) (Vn Hm et l., 2) nd se brem (Sprus urt L.) (rends et l., 1999) hve gined ttention due to their potentil for quculture in mny countries. However, little reserch hs been done on ntive South mericn fishes. mong the rzilin frmed fish, reserch on stress hs been crried out on mtrinxã (rycon cephlus) (Günther, 1869) (Crneiro nd Urbinti, 21; Ide et l., 2; Roch et l., 24; Urbinti et l., 24), tmbqui (Colossom mcropomum) (Cuvier, 1818) (Gomes et l., 2), pirrucu (rpims gigs) (Schinz, 1822) (Gomes et l., 2b) nd jundiá (Rndi quelen) (Quoy nd Gimrd, 1824) (rcellos et l., 21). Despite pcu (Pirctus mesopotmicus) (Holmberg, 1887) being considered one of the most importnt ntive species (Queiroz et l., 2) nd studies on its reproduction (Romgos et l., 199), lrviculture (Jomori et l., 2), feeding nd nutrition (Souz et l., 2; echr et l., 2; Tkhshi et l., 26; bimord et l., 27) re vilble, there is sprse knowledge on the stress response of the species (Krieger et l., 1989; Mrtins et l., 2; Tkhshi et l., 26). In the present study, the physiologicl responses (hormonl, metbolic, ionic nd hemtologicl) to cpture (chsing, netting nd ir exposure) were investigted in the pcu. 2. Mteril nd Methods 2.1. Fish nd experimentl procedure Juveniles of pcu (12 fish, 49.7 ± 11.7 g) were rndomly distributed in thirty three 1 L boxes (4 fish per box), with constnt wter nd ir flow, where they were kept for 1 dys to cclimte to the experimentl conditions. Feeding ws stopped before the cpture tht consisted in chsing, netting nd exposed the fish to the ir. Fish were submitted to the conditions: T1: undisturbed fish (control) ( boxes), T2: chsing nd minutes of ir exposure (1 boxes), T: chsing nd of ir exposure (1 boxes). Nine fish of T2 nd T ( fish/ boxes) were smpled t ech smpling time (, 1,, 6 minutes nd fter hndling). The control fish (n = 9) were smpled before hndling. Fish were nesthetized (benzocine, 66 mg.l 1 ) nd bled by cudl vessels puncture nd serum nd plsm were seprted. The four fish of ech box were simultneously nesthetized but only were smpled. 2.2. nlyses Glucose (King nd Grner, 1947), hemtocrit, red blood cell count (RC), men corpusculr volume (MCV) nd hemoglobin were determined in totl blood (Celm D-), chloride (kit Lbtest) nd osmollity (Osmometer Wescor Mod ) in plsm nd cortisol (RI, kit Dignostics Products Corportion), sodium, potssium nd clcium (ion selector Iselb Drke) in serum. Wter temperture (29.1 ±. C) nd dissolved oxygen (4.4 ±.4 mg.l 1 ) (Yellow Springs Instruments, Yellow Springs, OH, US, YSI ), ph (1.2 ±.4) (YSI 6) nd totl mmoni (.12 ±.4 mg.l 1, Nessler Rective) were monitored throughout the experiment. 2.. Sttisticl nlysis completely rndomized design (CRD) ws employed nd results were nlyzed by two-wy nlysis of vrince (NOV), with 2 tretments (cpture with or ir exposure) nd smpling times (, 1, nd 6 minutes nd fter cpture) s the fctors plus the resting condition (control fish). Dt were expressed in mens ± stndrd devition of the men. Mens were compred by Tukey test (p <.).. Results No mortlity ws observed in ny group during the experiment. There were hormonl nd metbolic chnges fter the hndling imposed on the fish. Serum cortisol concentrtions in ir-exposed fish either for minutes (T2) or (T) rose within minutes (p =.19) nd then dropped until lter. The rise ws higher in fish of tretment (p =.74) regrdless of the smpling (Figure 1). lood glucose in both ir-exposed fish (T2 nd T) incresed within (p =.1) compred to control (undisturbed fish) nd recovered the resting vlues within (Figure 1). Cpture elicited mild chnges in ionic blnce of pcu. Plsm chloride levels dropped in fish of tretments 2 nd within 6 minutes, not recovering the resting levels within (p =.291) (Figure 2). Serum sodium incresed (p =.26) t 416 rz. J. iol., 69(2): 41-421, 29
iologicl indictors of stress in pcu Serum cortisol (ng.ml 1 ) 2 18 16 14 12 1 8 6 4 2 b 1 minutes 6 minutes b ir exposure lood glucose (mg.dl 1 ) 12 1 8 6 4 2 C 1 minutes 6 minutes C ir exposure Smpling times Smpling times Figure 1. lood cortisol nd glucose of pcu undisturbed nd ir exposed for nd. Different cpitl letters indicte differences mong smpling times (ll ir exposure times re represented in the sme br) nd smll letters between tretments (ll smplings re represented in the sme br). rs represent mens of tretments or smplings. Verticl brs represent SEM. Plsm chloride (meq.l 1 ) 14 1 12 11 1 9 8 7 6 4 2 1 1 C minutes CD 6 minutes D ir exposure Serum sodium (meq.l 1 ) 16 14 12 1 8 6 4 2 1 minutes 6 minutes ir exposure Smpling times Smpling times Figure 2. Plsm chloride nd serum sodium of pcu undisturbed nd ir exposed for nd. Different cpitl letters indicte differences mong smpling times (ll ir exposure times re represented in the sme br) nd smll letters between tretments (ll smplings re represented in the sme br). rs represent mens of tretments or smplings. Verticl brs represent SEM. 1 nd minutes in both ir-exposed fish (T2 nd T) nd returned to resting levels lter (Figure 2). Potssium concentrtion (p =.291) nd osmollity (p =.2) did not chnge fter ir exposure (Tble 1). In fish of tretments 2 nd, clcium levels did not differ significntly until 6 minutes fter hndling nd decresed fter (p =.1) compred to the other smplings but not to the control condition (Tble 1). Excepting for MCV, which rose minutes fter cpture nd returned to resting levels lter (p =.44), the hemtocrit (Tble 1), red blood cells count (RC) nd hemoglobin (Figure ) were not ffected by cpture (p =.882; p =.298 nd p =.46, respectively). 4. Discussion Stress is biologicl response of dpttion to dverse conditions nd fish respond by ctivting responses such s incresed circulting cortisol nd glucose (Wendelr ong, 1997), chnges in blood ionic blnce (McDonld nd Millign, 1997) nd hemtologicl profile (Wojtszek et l., 22). lthough lbortory studies cn be criticized for lck of relism, they llow for systemtic determintion of generl behviorl nd physiologicl principles of stress response tht is not possible in the field. Interctions of sequentil stressors, s those to which the pcu ws exposed in the present study, often cuse incresed stress in fish. Cpture, for instnce, includes rz. J. iol., 69(2): 41-421, 29 417
breu, JS. et l. Tble 1. Hemtocrit, serum potssium nd clcium nd osmollity of pcu undisturbed nd ir exposed for nd. Different cpitl letters indicte differences mong smplings nd smll letters between tretments. Smpling times ir exposure 1 minutes 6 minutes Mens 4. ±.21 Hemtocrit (%) Serum potssium (meq.l 1 ) Serum clcium (meq.l 1 ) Osmollity (mosmol.l 1 ) minutes 1.4 ± 2.22,2 ± 2.22 4.7 ± 1.. ± 1.1 7.1 ±.77 4. ± 2.4 4.9 ± 1.68 4.2 ± 2.42 2.8 ± 1.76.8 ±.9 6.6 ± 1. 4. ± 2.1 Mens.2 ± 2.8.7 ± 2.16.7 ± 1.76. ± 2.27 6.9 ±.99 2.4 ±.49 minutes. ±.74 2. ±.2 2.61 ±.48 2. ±.42 2.9 ±.1 2.66 ±.49 2.4 ±. 2.4 ±.44 2.2 ±. 2.64 ±.4 2.77 ±.1 2.48 ±.4 Mens 2.7 ±.61 2.4 ±.4 2.4 ±. 2. ±.4 2.86 ±. 1. ±.6 minutes 1.48 ±.22 1.4 ±.16 1.88 ±.24 1.74 ±.47 1.1 ±.19 1. ±.9 1.4 ±. 1.1 ±.8 1.6 ±.22 1.79 ±.4 1. ±.8 1.48 ±.1 Mens 1.46 ±.14 1.±.11 1.74 ±.26 1.77 ±.9 1.2 ±.1 6. ±.8 minutes 1.9 ± 6.2 12. ±.9 7. ± 1.8 9.7 ± 7.1 298.1 ± 16. 7.6 ± 8.9 14. ± 4. 14.8 ± 1.7 14.8 ± 7.8 6. ± 8.4 17. ± 4.6 1. ± 6. Mens 12.7 ±.2 1.4 ±.1 11.2 ± 6.4 8. ± 7.2 7. ± 14.9 Erythrocyte number (cell.ul 1 ) x 1 6. 2. 2. 1. 1... MCV (u ) 18 16 14 12 1 8 6 4 2 16 14 Hemoglobin (g.dl 1 ) 12 1 8 6 4 2 1 minutes 6 minutes ir exposure Smpling times Figure. Number of erythrocytes, men corpusculr volume (MCV) nd hemoglobin concentrtion of pcu undisturbed nd ir exposed for nd. Different cpitl letters indicte differences mong smpling times (ll ir exposure times re represented in the sme br) nd smll letters between tretments (ll smplings re represented in the sme br). rs represent mens of tretments or smplings. Verticl brs represent SEM. 418 rz. J. iol., 69(2): 41-421, 29
iologicl indictors of stress in pcu the chsing nd swimming of fish, physicl injury provoked by the contct mong fish nd with the net nd noxi by ir exposure (Mugnier et l., 1998; rends et l., 1999; Ross nd Ross, 1999; rcellos et l., 21; Morles et l., 2). Confirming the literture regrding fish stress, elevted circulting cortisol nd glucose of pcu were short-term responses in both ir-exposed fish fter cpture nd the recovery occurred within. However, the mgnitude of glucose responses ws low (from 6 to 94 mg.dl 1 ) compred to vlues found previously in pcu in stressed stte (Krieger et l., 1989; Mrtins et l., 2). ir exposure of Sprus urt for minutes resulted, within minutes, in n increse in plsm concentrtions of cortisol nd glucose. fter 2 hours, plsm cortisol nd fter 12 hours plsm glucose hd returned to control concentrtions (rends et l., 1999). On the other hnd, netting nd exposure of juvenile turbot to ir for 1-4 minutes hd no immedite effect on plsm cortisol concentrtions (Mugnier et l., 1998). The stress response of pcu involved ctivtion of the brin-pituitry-interrenl xis, s indicted by the cortisol nd glucose elevtions nd it ws probbly elicited by the combined stimuli of fish swimming when chsed, netting nd the hypoxic condition provoked by ir exposure. Mny stressors ffect the ionic blnce in fish (Wendelr ong, 1997) fcilitted by cthecolmineinduced increse of gill permebility responsible for chloride nd sodium exchnge with the environment (McDonld nd Millign, 1997). Decrese in plsm concentrtion of chloride nd sodium were found in other stressed freshwter fish s in rycon cephlus fter trnsport (Crneiro nd Urbinti, 21; Urbinti et l., 24). In our experiment, prtil nd moderte disturbnce of the ionic blnce ws found in both ir exposed fish. In stressed pcu, chloride decresed 6 minutes fter cpture wheres sodium concentrtion incresed trnsiently between 1 nd 6 minutes fter cpture. dditionlly, clcium nd potssium levels nd osmollity were not ffected by the sequentil stressors during cpture. pprently only moderte nd rther specific loss of permebility control occurred s consequence of cpture. The low mgnitude of glucose responses, mild chloride nd sodium disturbnces, nd the lcking potssium, clcium nd osmollity responses indicted low ctivtion of the brin-sympthetic-chromffin cell xis, nd hence low relese of ctecholmines, which seemed though to occur to higher extent t severe stressors (Mzeud nd Mzeud, 1981). Severl studies re known to initite ctecholmine secretion in fish including physicl disturbnce (Ristori nd Lurent, 198) nd hypoxi (Ristori nd Lurent, 1989). However, the degree of hypoxi required to initite the responses is highly vrible. Studies hve shown tht there exist hypoxi-tolernt species such s crp (Vinen et l., 21) in contrst to hypoxi-intolernt species such s rinbow trout (outilier et l., 1988). recent study tht provided the first dt on plsm ctecholmines level in tropicl fish (Perry et l., 24) hs shown tht plsm ctecholmines levels remined constnt in pcu exposed to cute hypoxi, suggesting n inopertive or bsent humorl drenergic stress response in this species. The results of the hemtologicl ssessement of stressed pcu confirmed the low degree of ctivtion of the brin-sympthetic-chromffin cell xis. Periphericl blood nlysis hs been used s dignosis to ssess helthy stte in fish nd the effect of severl stressors on them (Wojtszek et l., 22). The most significnt effect of ctecholmines relese during stress is to enhnce blood O 2 trnsport by incresing the crrying cpcity (Wells nd Weber, 199) nd by enhncing Hb-O 2 binding ffinity (Cossins nd Richrdson, 198). No significnt chnges were found in the hemtologicl prmeters tested in pcu fter the sequence of stressors during the cpture. Concluding, the results of this work indicte tht the submission of sequentil stressors on pcu during cpture ctivted the brin-pituitry-interrenl xis (cortisol nd glucose responses) but the ctivtion of the brin-sympthetic-chromffin cell xis ws pprently moderte (ionic nd hemtologicl responses). cknowledgements We would like to thnk Mrs. Dmres Perecim Roviero for the technicl support, to Piscicultur Águs Clrs (Mococ, SP) for the fish dontion nd to the quculture Centre of So Pulo Stte University for the fcilities. References IMORD, EG., CRNEIRO, DJ. nd URINTI, EC., 27. Growth nd metbolism of pcu (Pirctus mesopotmicus) juveniles fed diets contining different protein, lipid, nd crbohydrte levels. quculture Reserch, vol. 8, no. 1, p. 6-44. CKERMN, P., FORSYTH, R., MZUR, CF. nd IWM, GK., 2. Stress hormones nd the cellulr stress response in slmonids. Fish Physiology nd iochemistry, vol. 2, no. 4, p. 27-6. RENDS, RJ., MNCER, JM., MUNOZ, JL., WENDELR ONG, SE. nd FLIK, G., 1999. The stress response of the gilthed se brem (Sprus urt L.) to ir exposure nd confinement. Journl of Endocrinology, vol. 16, no. 1, p. 149 17. RCELLOS, LJG., WOEHL, VM., WSSERMNN, GF., QUEVEDO, RM., ITTZÉS, I. nd KRIEGER, MH., 21. Plsm levels of cortisol nd glucose in response to cpture nd tnk trnsference in Rhmdi quelen (Quoy nd Gimrd), South mericn ctfish. quculture Reserch, vol. 2, no. 2, p. 121-12. RTON,., 2. Slmonid fishes differ in their cortisol nd glucose responses to hndling nd trnsport stress. North mericn Journl of quculture, vol. 62, no. 1, p. 12-18. ECHR, J., ROUX, JP., DIS, FJR., QUINTN, CIF. nd MEE, CL., 2. The effect of dietry protein level on pond wter qulity nd feed utiliztion efficiency of pcu Pirctus mesopotmicus (Holmberg, 1887). quculture Reserch, vol. 6, no. 6, p. 46-. rz. J. iol., 69(2): 41-421, 29 419
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