The Journl of Experimentl Biology 26, 3569-3579 23 The Compny of Biologists Ltd doi:1.1242/je.588 3569 Influene of sesonl temperture on the repet swimming performne of rinow trout Onorhynhus mykiss Kmini E. Jin nd Anthony P. Frrell* Biologil Sienes Deprtment, Simon Frser University, Burny, BC, V5A 1S6, Cnd *Author for orrespondene (e-mil: frrell@sfu.) Aepted 7 July 23 While the temperture dependene of exerise performne in fishes is resonly well doumented, informtion on the temperture dependene of metoli reovery nd reperformne is snt. This study exmined the reovery of swimming performne fter exhustive exerise in rinow trout Onorhynhus mykiss t sesonl tempertures rnging from 5 to 17 C nd explored the reltionship etween performne nd preeding metoli stte. The primry ojetive of the study ws to test the hypothesis tht inresed temperture inreses the pility of rinow trout to repet ritil swimming speed (U rit ), s ssessed y two onseutive ritil swimming speed tests seprted y 4 min rest intervl. An dditionl expettion ws tht ertin plsm ioni, metoli nd humorl prmeters would e orrelted with how well fish reperformed nd so plsm levels of ltte, potssium, mmoni, osmollity, sodium nd ortisol, s well s hemtorit, were monitored efore, during nd fter the swim hllenges vi n indwelling nnul in the dorsl ort. As expeted, performne in the first U rit test (U rit1 ) ws positively relted to temperture. However, the reltionship etween U rit1 nd reperformne (U rit2 ) ws not dependent on limtion temperture in simple mnner. Contrry to our expettions, U rit2 ws less thn U rit1 for wrmlimted fish (14.9±1. C), wheres U rit2 equled U rit1 for old-limted fish (8.4±.9 C). Cold-limted fish lso exhiited lower U rit1 nd less metoli disruption Summry ompred with wrm-limted fish. Thus, while wrm limtion onferred fster U rit1, similr swimming speed ould not e ttined on susequent swim fter 4 min reovery period. This finding does not support the hypothesis tht the ility of rinow trout to reperform on U rit test is improved with temperture. Both plsm ltte nd plsm potssium levels were strongly orrelted with U rit1 performne. Therefore, the higher U rit1 of wrm-limted fish my hve een due in prt to greter neroi swimming effort ompred with old-limted fish. In ft, signifint orreltion existed etween the plsm ltte onentrtion prior to the strt of the seond test nd the susequent U rit2 performne, suh tht U rit2 deresed when threshold plsm ltte level of round 12.2 mmol l 1 ws surpssed for the initil swim. No other mesured plsm vrile showed signifint reltionship with the U rit2 performne. We onlude tht wrm-limted fish, y pprently swimming hrder nd possily more neroilly ompred with old-limted fish, were unle to reovery suffiiently well during the fixed reovery period to repet this initil level of performne, nd this poorer repet performne ws orrelted with elevtions in plsm ltte levels. Key words: fish, rinow trout, Onorhynhus mykiss, ritil swimming speed, temperture limtion, repet swimming, plsm, ltte threshold, mmonium. Introdution An extensive literture exists on the reovery of metolites nd ions following exhustive exerise in fish (see reviews y Driedzi nd Hohhk, 1978; Millign, 1996; Kieffer, 2). Considerly fewer studies hve mesured how quikly or how well swimming performne reovers following exhustive exerise (e.g. Stevens nd Blk, 1966; Rndll et l., 1987; Bruner et l., 1994; Jin et l., 1998; Frrell et l., 1998, 21, 23). Given tht metoli reovery in skeletl musle (musle ltte, ATP nd glyogen, ut not PCr) ours more rpidly t wrm thn old tempertures in exhusted rinow trout Onorhynhus mykiss nd Atlnti slmon Slmo slr (Kieffer et l., 1994; Wilkie et l., 1997; Kieffer, 2), the expettion is tht swimming performne is restored fster t higher temperture. This expettion would e onsistent with the known inrese in oth mximum oxygen uptke nd mximum rdi output with temperture (e.g. Butler et l., 1992; Frrell, 1997; Tylor et l., 1997) euse n improved oxygen delivery system ould support more rpid reovery of the metoli det inurred with exhustive exerise. However, when Atlnti slmon were ngled rther thn hsed to exhustion, musle glyogen, intrellulr ph nd ltte were restored more rpidly under old onditions thn wrm onditions (Wilkie et l.,
357 K. E. Jin nd A. P. Frrell 1996). Therefore, given this unertinty nd the sene of ny study tht hs diretly mesured how limtion temperture ffets the reovery of swimming performne, the primry ojetive of the present study ws to test the hypothesis tht the ility of rinow trout to repet ritil swimming speed (U rit ) test is improved with temperture. A seond ojetive of the present study ws to serh for orreltions etween the ility to reperform fter n exhustive U rit swim nd the ltertion in plsm levels of ions, metolites nd hormones during exerise. In prtiulr, possile linkges were sought etween the reovery of swimming performne nd the post-exhustion levels of plsm potssium, ltte nd totl mmoni onentrtions (T mm ), ll of whih hve een linked with musulr exhustion in oth mmmls nd fish. For exmple, high intensity exerise in mmmls produes potssium loss from the musle (Sjøgrd et l., 1985; Vøllestd et l., 1994; Hllén, 1996), whih ould derese the musle memrne exitility nd ompromise tension development (reviewed y Sjøgrd, 1991). Plsm potssium levels inrese in rinow trout just prior to U rit nd, moreover, exerise trining inresed U rit while lunting nd delying the inreses in plsm potssium nd ltte just prior to exhustion (Holk nd Lykkeoe, 1998). Plsm ltte onentrtion hs long een onsidered useful inditor of eroi limittions nd neroi pilities in exerise studies. Indeed, rinow trout refused to perform repetitive outs of urst exerise when plsm ltte onentrtion exeeded 13 mmol l 1 (Stevens nd Blk, 1966) nd poorer repet U rit ws found for sokeye slmon Onorhynhus nerk when plsm ltte onentrtion ws >1 mmol l 1 (Frrell et l., 1998). In mmmls, elevted plsm T mm hs een implited in exerise ftigue (reviewed y Muth nd Bnister, 1983) due to inhiitory influenes on neroi metolism (Zleski nd Bryl, 1977; Su nd Storey, 1994), eroi metolism (MKhnn nd Tower, 1961; Avillo et l., 1981) nd neuromusulr oordintion (Binstok nd Ler, 1969; O Neill nd O Donovn, 1979). Plsm T mm lso inreses in rinow trout during exerise (Turner et l., 1983; Wng et l., 1994; ut see Beumont et l., 1995,). Furthermore, when routine T mm ws elevted in rown trout Slmo trutt, s result of exposure to idi, opper-ontining wter, the susequent U rit performne ws inversely relted to pre-exerise plsm T mm onentrtion (Beumont et l., 1995). Thus, euse plsm levels of potssium, ltte nd T mm re good inditors of exhustion in fish, we ntiipted tht they re potentilly strong inditors of repet swimming pility in rinow trout. If this is the se, the expettion is tht individul vrition in these plsm vriles prior to seond swim would e orrelted with individul vrition in the performne of seond U rit test ompred to the initil performne. Mterils nd methods Fish Rinow trout Onorhynhus mykiss Wlum [mss=871.49±43.34 g (men ± stndrd error of the men, S.E.M.); fork length (FL)=4.95±.77 m, N=15] were otined from lol hthery (Sun Vlley Trout Frm, Mission, British Columi, Cnd). They were held outdoors in 2 liter round fierglss qurium provided with erted nd dehlorinted Vnouver muniipl wter, ph 6.7, hrdness 5.2 6. mg l 1 CCO 3, nd mient temperture 5 17 C. Experiments were performed etween Novemer 1997 nd April 1998, nd Septemer Otoer 1998. All experimentl work onformed to the guidelines set out y the Cndin Counil on Animl Cre, s pproved y the Simon Frser University Animl Cre Committee. Swim tunnel Fish were swum in modified Brett-type swim tunnel, similr to tht desried y Gehrke et l. (199). The swim hmer ws 21 m dimeter nd 97 m length, with metl grid t eh end. The rer grid ws equipped with n eletril pulse genertor (4 V) tht, when ontted y the fish, provided mild stimultion to enourge the fish to swim forwrd. Wter speed ws uniform ross the swim tunnel throughout the speed rnge used in these experiments. The wter urrent in the tunnel ws produed y 3-phse indution motor nd entrifugl pump tthed to thometer whose redings (Hz) were lirted with known wter veloities, s mesured with Vleport urrent meter (Vleport Mrine Sientifi Ltd., Drtmouth, UK). Protool for rteril nnultion The dorsl ort ws nnulted to permit smpling of lood prior to nd during the swimming tests, nd during the reovery periods. Arteril nnultion ws performed under nesthesi (.1 g l 1 uffered MS-222; Syndel Lortories, Vnouver, BC, Cnd), using the method of Smith nd Bell (1964). Fish mss, fork length, mximum width nd mximum depth were lso mesured t this time. Cnnulted fish were either pled in the swim tunnel to reover or returned to the outdoor tnk, where they reovered for up to 3 dys efore eing pled in the swim tunnel. During susequent trnsfer from the outdoor tnk to the tunnel, fish were lightly nd riefly nesthetized (.5 g l 1 uffered MS-222). There ws no signifint reltionship etween post-nnultion reovery time nd mesured swimming performne (dt not presented). Hitution to the swim tunnel nd high wter veloities Fish reovered from nesthesi in the tunnel t wter speed of 1 m s 1 for t lest 45 min. After this time, fish performed 2 min prtie swim, s suggested in Jin et l. (1997), during whih wter speed ws inresed in 9 1 m s 1 inrements every 2 min to speed of ~41 m s 1. Wter speed ws then returned to 1 m s 1 for 2 min nd gin inresed in the sme fshion to speed of either 55 or 59 m s 1, depending on the fish s swimming pility. The prtie swim, whih did not exhust the fish, prevented the trining effet often oserved with nive fish on seond U rit (Frlinger nd Bemish, 1977; Jin et l., 1997). Fish then reovered overnight (14 16 h) t wter speed of 1 m s 1.
Effet of temperture on repet swimming performne 3571 Swimming protool All experiments were strted etween 8: h nd 1: h. Fish performed rmp-u rit test (Jin et l., 1997). The first U rit test ws followed y 4 min reovery period t wter speed of 1 m s 1 nd then seond rmp-u rit test followed y nother reovery period. Eh rmp-u rit test involved inresing wter speed to ~5% of U rit over 5 min period, fter whih wter speed ws inresed in 1 m s 1 inrements (~15% of U rit ) every 2 min until exhustion. Exhustion ws tken s the point t whih the fish filed to swim wy from the eletrified rer grid fter 2 s of ontt. The rmp-u rit protool produes similr vlues for U rit to the more stndrd U rit testing protool in whih the longer time intervls re used from the onset of the test (Jin et l., 1997). U rit vlues were lulted for the first (U rit1 ) nd seond (U rit2 ) swims, s desried y Brett (1964): U rit = u i + (t i /t ii u ii ), (1) where u i is the highest speed t whih the fish swm for the full time period (m s 1 ); u ii is the inrementl speed inrese (m s 1 ); t i is the time the fish swm t the finl speed (min), nd t ii is the presried period of swimming per speed (2 min). As the ross-setionl re of eh fish ws <2% ut sometimes >1% of tht of the swimming hmer, the lirted wter speed ws orreted for the solid loking effet ording to the lultions desried y Bell nd Terhune (197): orreted U rit = U rit {1 + [.4FL /.5(w+d)] (.25πdw/A t ) 1.5 }, (2) where FL is fork length (m), w is mximum fish width (m), d is mximum fish depth (m) nd A t is tunnel ross-setionl re. Wter temperture did not flutute y more thn.5 C from mient temperture during the period tht the fish spent in the tunnel. Blood smpling Blood smples (.9 ml) were tken through the dorsl ort nnul to mesure plsm ion nd metolite levels. Normlly, smples were tken immeditely prior to the swimming protool (routine smples), t exhustion for oth swim tests (U rit exhustion smples), nd fter 4 min reovery for oth tests (reovery smples; the reovery smple for the first U rit swim lso served s the smple tken immeditely efore the seond U rit swim). In 14 of the 16 fish, lood smple ws tken during eroi swimming, i.e. fter 15 min t 45 m s 1 (pprox. 69% U rit ). (These dt re not reported s they simply provided intermedite vlues etween the routine nd U rit vlues.) An equl volume of physiologil sline solution ws used to reple ll lood smples (Gllugher et l., 1992). Routine hemtorit ws never less thn 23% nd remined elevted throughout the swim tests (see Fig. 2D). Anlytil tehniques Hemtorit ws mesured in miropillry tues fter entrifugtion t 2 g for 3 min. The reminder of the lood ws entrifuged t 1 g for 5 min to otin plsm, whih ws stored t 8 C. Within 1 week of testing, plsm ltte nd gluose onentrtions were mesured on 25 µl smples using YSI 23 ltte/gluose nlyzer (Yellow Springs, OH, USA) tht lirted utomtilly every five smples. Plsm potssium nd sodium onentrtions were mesured using model 51 Turner flme photometer (Plo Alto, CA, USA). Plsm (5 µl) ws diluted 1:2 with prepred 15 meq l 1 lithium diluent for nlysis. The mhine ws lirted prior to use nd heked ginst stndrd pproximtely every six smples. The mesurement ws repeted if there ws disgreement etween duplites eyond 2% of solute vlue. Osmollity ws mesured on duplite 1 µl smples using lirted Wesor Vpour Pressure Osmometer, Model 55 (Wesor, Logn, UT, USA). The mesurement ws repeted if there ws disgreement etween duplites eyond 3% of solute vlue. The thermoouple heds were lened periodilly in order to mintin onsisteny. Plsm ortisol onentrtion ws mesured using ommeril rdiommunossy kit (ICN Biomedils, In., Cost Mes, CA, USA), with detetion limit of 1.5 ng ml 1. Plsm mmoni onentrtion (T mm ) ws mesured spetrophotometrilly on.1 ml plsm smples (Sigm Dignostis kit no. 171, St Louis, MI, USA) with lirtion every seven smples. Dt nlysis All plsm metolites nd ions were mesured in duplite nd verged for individul dt. Fish were sudivided into two temperture limtion groups sed on their swimming performne (see Results) nd vlues (men ± S.E.M.) re presented for old- nd wrm-limted fish. One wrmlimted femle fish tht ws overtly grvid ws not inluded in the sttistil nlysis to eliminte ny onfounding effet, euse reprodutive mturity is known to negtively ffet U rit performne in slmon (Willims et l., 1986). Sttistil omprisons within temperture groups were mde with onewy repeted mesures nlysis of vrine (ANOVA) followed y post ho Tukey test. With this test, the vlues ssoited with eh fish were ompred to other levels t other smpling times for the sme fish to determine whether either swimming speed or metolite levels hnged throughout testing. Comprisons of swimming performne nd metolite levels etween temperture groups were mde using t-tests. U rit1 ws ompred to U rit2 using Blnd Altmn plot. Blnd nd Altmn (1986, 1995) introdued this method of grphil nlysis to ssess the equivleny of two testing pprohes (here U rit1 nd U rit2 ), while removing the is tht omes from ssuming tht one method represents the true vlue (independent vrile). The Blnd Altmn plot uses the men of oth methods s the independent vrile nd the differene etween the two testing methods s the dependent vrile. If the liner regression of the points is non-signifint, then the two testing proedures (i.e. U rit1 nd U rit2 here) n e onsidered to e
3572 K. E. Jin nd A. P. Frrell equivlent testing proedures. Su-groups n e identified within dt set in Blnd Altmn plot y demonstrting different signifint liner regressions from eh other. In the present study, different regressions would identify su-groups with different reltionships etween U rit1 nd U rit2. Reltionships etween U rit vlues nd plsm vriles were fitted with the est-fitting regressions using the options provided in Sigm-Plot (SPSS In.; Chigo, IL, USA). P<.5 ws used to estlish sttistil signifine. Results Swimming performne As wter speed inresed, fish progressed from stedy swimming mode to one tht inluded periods of urst-ndglide swimming. In onjuntion with higher speeds, fish rmventilted their gills, exept during urst-nd-glide swimming when tive ventiltion ws oserved. Visully, swimming ehvior did not pper to e different for the first nd seond U rit tests. A Blnd Altmn plot reveled tht U rit1 nd U rit2 were equivlent testing proedures (P=.98), ut visul inspetion of the plot reveled tht overll the fish ould e divided into two su-groups eh with different nd signifint liner reltionship (Fig. 1A). Eh of the two su-groups orresponded to different limtion tempertures nd herefter re termed wrm- nd old-limted fish (14.9±1. C nd 8.4±.9 C, respetively; see Tle 1). U rit1 performne ws temperture dependent (Fig. 1B; r 2 =.74, P<.5). U rit1 (78.9±1. m s 1 ) for wrmlimted fish ws signifintly greter (P<.5) thn tht for old-limted fish (59.1±2.5 m s 1 ; Tle 1). However, U rit2 did not show ny temperture dependeny. Unexpetedly, U rit2 performne (65.8±2.7 m s 1 ) for wrm-limted fish ws signifintly lower thn U rit1, wheres U rit2 for old-limted fish (58.±4.2 m s 1 ) ws U rit1 U rit2 (m s 1 ) 3 2 1 1 A Cold-limted fish: r 2 =.99 y=3.41.5x P<.1 4 5 6 7 8 Averge U rit (m s 1 ) Wrm-limted fish: r 2 =.57 y=139. 1.74x P=.8 U rit1 (m s 1 ) 8 B 7 6 5 8 12 16 Temperture ( C) U rit2 (m s 1 ) 8 7 6 5 4 C Cold-limted fish Wrm-limted fish r 2 =.81 U rit2 =U rit1 Reovery rtio 1.2 1..8.6 D r 2 =.62 5 6 7 8 U rit1 (m s 1 ) 8 12 16 Temperture ( C) Fig. 1. (A) Blnd Altmn plot ompring onseutive U rit tests (U rit1 nd U rit2) performed y rinow trout, seprted y 4 min reovery period. Regression lines indite the existene of two su-groups, old-limted (filled symols) nd wrm-limted (open symols) fish, sed on the visile groupings in this grph. (B) U rit1 versus mient wter temperture for rinow trout. Fish re divided into two sugroups, old-limted (filled symols) nd wrm-limted (open symols) fish. Regression: y=4.44+2.42x, r 2 =.74; P<.1. (C) U rit2 versus U rit1 for individul rinow trout performing two U rit tests seprted y 4 min reovery period. Fish re divided into oldlimted nd wrm-limted groups. The thin line is the line of identity where x=y, i.e. the predited line if U rit1=u rit2 independent of temperture, nd this ws not the se. Regression (thik line): y= 24.3+7.57x.5x 2, r 2 =.81, P<.1. (D) Reovery rtios for individul rinow trout s funtion of limtion temperture (filled symols, old-limted group; open symols, wrm-limted group). The regression line for these dt illustrtes tht wrm-limted fish ould not ttin the sme U rit fter 4 min reovery.
Effet of temperture on repet swimming performne 3573 Tle 1. Critil swimming speed of the old- nd wrmlimted groups of rinow trout for the first nd seond swim tests Cold-limted Wrm-limted fish (N=9) fish (N=6) U rit1 (m s 1 ) 59.1±2.5 78.9±1. U rit2 (m s 1 ) 58.9±4.2 65.9±2.7 U rit1 (FL s 1 ) 1.51±.1 1.84±.4 U rit1 (FL s 1 ) 1.48±.14 1.54±.8 Cold-limtion temperture = 8.4±.9 C; wrm-limtion temperture = 14.9±1. C. U rit1, first swim test; U rit2, seond swim test; FL, fork length. Vlues re mens ± S.E.M. Sttistilly signifint differene (P<.5) ompred with old su-group; sttistilly signifint differene (P<.5) etween omprle U rit1 nd U rit2 vlues. the sme s their U rit1 vlues (Tle 1). As result, the overll reltionship etween U rit1 nd U rit2 ws est desried y polynomil eqution (y= 24.3+7.57x.5x 2 ; P<.1; Fig. 1C), with old-limted fish lying lose to the line of identity nd wrm-limted fish lying elow the line of identity. Thus, while wrm limtion onferred fster U rit1, similr swimming speed ould not e ttined fter 4 min reovery period, s shown y reovery rtios tht re less thn unity for wrm-limted fish (Fig. 1D). Plsm sttus efore, during nd fter U rit tests There were no signifint differenes etween the old- nd wrm-limted groups of fish in terms of routine vlues for plsm levels of ltte, potssium, T mm, sodium, gluose, ortisol nd osmollity nd hemtorit. When old-limted fish were exhusted t U rit1, plsm levels of ltte, potssium nd T mm, s well s hemtorit, ll inresed signifintly (Fig. 2A D). Plsm ortisol (Fig. 2E) nd sodium (Fig. 2F) levels were unhnged t exhustion for U rit1. After 4 min reovery from U rit1, plsm ltte inresed signifintly eyond the level oserved t exhustion, plsm T mm deresed to the routine level, nd plsm potssium nd hemtorit remined elevted t the sme level. As result, plsm ltte nd potssium levels, nd hemtorit were ll signifintly elevted t the outset of the U rit2 test. For old-limted fish exhusted t U rit2, plsm levels of ltte, potssium, sodium nd T mm, nd hemtorit, were gin signifintly elevted ompred with the routine vlues, ut no more so thn for U rit1. In ft, ompred with the reovery vlues for U rit1, plsm ltte levels hd deresed signifintly (Fig. 2A) t exhustion for U rit2, while T mm hd inresed signifintly (Fig. 2C). Similr to U rit1, plsm ltte inresed during the 4 min reovery from U rit2 to level signifintly higher thn tht oserved t exhustion, plsm T mm deresed to the routine level, nd plsm potssium nd hemtorit remined elevted t the sme level. As result, none of the reovery vlues for U rit2 in oldlimted fish were signifintly different to those for U rit1. Plsm levels of ortisol, gluose nd osmollity remined unhnged throughout oth swimming protools (dt not shown). Therefore, the seond swim for old-limted fish hd no dditive effets on ny of the mesured plsm vriles. When wrm-limted fish were exhusted t U rit1, plsm T mm nd hemtorit inresed y the sme mount s for oldlimted fish (Fig. 2C,D). In ontrst, the fster U rit1 of the wrm-limted fish ws ssoited with signifintly lrger inreses in plsm levels of ltte nd potssium (Fig. 2A,B) ompred with old-limted fish. Furthermore, wrmlimted fish signifintly inresed plsm sodium nd ortisol levels t exhustion for U rit1 (Fig. 2E,F), unlike oldlimted fish. After 4 min reovery from U rit1, the levels of plsm ltte, potssium, T mm, sodium nd ortisol, s well s hemtorit ll remined signifintly elevted in wrmlimted fish, wheres only plsm levels of ltte, potssium nd hemtorit remined elevted in oldlimted fish (Fig. 2). In ddition, plsm ltte, potssium, sodium nd ortisol remined elevted in wrm-limted fish t levels tht were signifintly greter thn those oserved in old-limted fish during reovery. In ft, the plsm ltte level ws out threefold higher nd plsm potssium lmost twofold higher. These results suggest tht the higher U rit1 of wrm-limted fish my hve een prtly due to greter neroi swimming effort ompred with old-limted fish, nd (or) ltte nd potssium were relesed from musle to plsm to greter extent. Compred with old-limted fish, wrm-limted fish lerly egn the seond U rit test with greter plsm ioni nd metoli disruption nd, s result in these fish, U rit2 ws signifintly lower thn U rit1. In ddition, while U rit2 for wrm-limted nd old-limted fish ws the sme, wrm-limted fish displyed signifint, further inrese in plsm potssium levels (Fig. 2B) t exhustion nd signifint, further inrese in plsm ltte levels (Fig. 2A) during the reovery from U rit2. However, plsm T mm did not reover to routine level, s it did in the old-limted fish (Fig. 2C). Therefore, the seond U rit swim of wrmlimted fish produed signifint dditive effets on some of the plsm vriles, unlike in old-limted fish where there were none. Correltionl nlysis The initil swimming performne of individul fish ws relted to the pperne of ltte in the plsm. Plsm ltte onentrtions mesured t U rit1 nd fter 4 min reovery were oth linerly relted to U rit1 (Fig. 3; r 2 =.73, P<.5 nd r 2 =.79, P<.5, respetively). As might e expeted from Fig. 3, plsm ltte onentrtions were highly orrelted with eh other (2 nd exhustion with 1 st exhustion: r 2 =.95, P<.5; 1 st exhustion with 1 st reovery: r 2 =.92, P<.5; 2 nd exhustion with 1 st reovery: r 2 =.94, P<.5; 2 nd reovery with 2 nd exhustion: r 2 =.94, P<.5).
3574 K. E. Jin nd A. P. Frrell The differene in swimming performne etween U rit1 nd U rit2 ws signifintly relted to the plsm ltte onentrtion prior to the seond U rit test (Fig. 4). This reltionship ws desried y either polynomil (r 2 =.74), or 2-prmeter power (r 2 =.65) regression. Both types of nlysis suggest tht the redution in U rit2 reltive to U rit1 ourred when fish rehed plsm ltte of 12.2 mmol l 1 (95% onfidene intervls of 7.9 nd 16.5 mmol l 1 ) 4 min fter eing exhusted y n initil U rit swim test. Only wrm-limted fish rehed this threshold plsm ltte level. Swimming effort in the initil swim ws lso relted to the pperne of potssium in the lood. Plsm potssium onentrtion mesured t U rit1 ws linerly relted to U rit1 (r 2 =.6, P<.5). However, there ws no signifint orreltion etween plsm potssium levels nd performne on the seond swim. Plsm T mm t exhustion ws not signifintly relted to U rit1, ut T mm vlues for the 1 st [Ltte] (mmol l 1 ) 25 A 2 15 1 5 [Potssium] (mequiv l 1 ) 8 7 6 5 4 3 2 1 B Cold-limted fish Wrm-limted fish Routine E1 R1 E2 R2 Routine E1 R1 E2 R2 [Cortisol] (ng ml 1 ) [Ammoni] (µmol l 1 ) 12 1 8 6 4 2 4 35 3 25 2 15 1 5 C E Routine E1 R1 E2 R2 Hemtorit (%) [Sodium] (mequiv l 1 ) Routine E1 R1 E2 R2 Routine E1 R1 E2 R2 35 3 25 2 15 1 5 16 14 12 1 8 6 4 2 D F Routine E1 R1 E2 Fig. 2. Blood prmeters in old-limted (N=8 9) nd wrm-limted (N=5 6) fish efore testing (Routine), t filure in the first U rit test (E1), fter 4 min reovery (R1; this ws lso immeditely efore the strt of the seond U rit test), t filure in seond U rit test (E2), nd fter nother 4 min reovery period (R2). Level different from the routine vlue; level different from the previous smpling time; vlue for wrm-limted fish different from the orresponding vlue for old-limted fish. (A) Plsm ltte onentrtion. (B) Plsm potssium onentrtion. (C) Plsm mmoni onentrtion. (D) Hemtorit. (E) Plsm ortisol onentrtion. (F) Plsm sodium onentrtion.
Effet of temperture on repet swimming performne 3575 reovery were relted to U rit1 (Fig. 5; r 2 =.34, P<.5). There were no other signifint orreltions for plsm T mm. The influene of limtion temperture on the plsm ioni nd metoli responses to exerise is illustrted y the signifint liner orreltions tht existed etween plsm Plsm [ltte] (mmol l 1 ) 2 15 1 5 1 st reovery: r 2 =.79 5 6 7 8 9 U rit1 (m s 1 ) 1 st exhustion: r 2 =.73 Fig. 3. Plsm ltte onentrtion t 1 st exhustion (irles) nd 1 st reovery (tringles) smpling times versus U rit1 for rinow trout. Fish re divided into two su-groups, old-limted (filled symols) nd wrm-limted (open symols) fish. Regression for 1 st exhustion plsm [ltte] (solid line): y= 1.48+.22x, r 2 =.73; P<.1; for 1 st reovery plsm [ltte] (roken line): y= 24.52+.51x, r 2 =.79; P<.1. Urit2 Urit1 1 1 2 Wrm-limted fish Cold-limted fish r 2 =.65 r 2 =.74 3 5 1 15 2 1 st reovery plsm [ltte] (mmol l 1 ) Fig. 4. U rit2 U rit1 versus the plsm ltte onentrtion prior to the seond U rit test (reovery 1) for individul rinow trout. Fish were divided into two su-groups: old-limted (filled symols) fish nd wrm-limted (open symols) fish. The dt ould e desried y either polynomil (roken line; r 2 =.74) or 2- prmeter power (solid line; r 2 =.65) reltionship. ltte, ortisol nd potssium levels nd temperture (Tle 2). There were no signifint orreltions with temperture nd the other prmeters mesured (T mm, [sodium] nd hemtorit). One overtly grvid, wrm-limted femle fish ws treted s n outlier, sed on its slow swimming performne, nd ws not used for ny orreltion nlysis. However, it is importnt to note tht ll the plsm hnges oserved in this fish were onsistent with the slower swimming performne of the old-limted fish. Disussion This study tested the hypothesis tht wrm-limted rinow trout would perform etter in repeted U rit swimming tests thn old-limted fish. The present findings, however, do not support this hypothesis euse U rit2 ws signifintly lower thn U rit1 in wrm-limted fish thn in oldlimted fish. At U rit1, the wrm-limted fish showed greter metoli disturne in the plsm ompred with old-limted fish nd lso showed dditive effets for the seond U rit, unlike the old-limted fish. Therefore, lthough wrm-limted fish swm etter thn oldlimted fish for U rit1, s expeted, the onsequene of this fster U rit1 ws redued performne on the seond U rit test. If nything, it ppered tht wrm-limted fish, y pprently swimming hrder nd possily more neroilly, were unle to reover suffiiently well during the fixed reovery period to repet this initil level of performne. For old-limted fish, however, the 4 min reovery period ws suffiient for dequte reovery nd llowed swimming performne to e repeted. Therefore, we re left with the 1st reovery plsm [mmoni] (µmol l 1 ) 175 15 125 1 75 5 25 Cold-limted fish Wrm-limted fish 5 6 7 8 9 U rit1 (m s 1 ) Fig. 5. Plsm mmoni onentrtion t first reovery versus U rit1 for rinow trout. Fish re divided into two su-groups, oldlimted (filled symols) nd wrm-limted (open symols) fish. Regression: y= 56.51+2.2x, r 2 =.3, P<.5.
3576 K. E. Jin nd A. P. Frrell Tle 2. Signifint liner regressions etween mient wter temperture nd individul plsm vriles during repetitive swim tests in rinow trout Plsm vrile P-vlue r 2 [Ltte] 1 st exhustion <.1.72 1 st reovery <.1.74 2 nd exhustion <.1.68 2 nd reovery <.1.69 [Potssium] 1 st exhustion <.1.65 1 st reovery <.1.69 2 nd exhustion <.1.74 2 nd reovery <.1.78 [Cortisol] 1 st exhustion <.1.41 1 st reovery <.5.27 2 nd exhustion <.5.33 onlusion tht overll reovery, s it pertins to repet swimming pilities nd time llowed for reovery, ws superior for the old-limted ompred with the wrmlimted group of rinow trout. Our originl hypothesis, whih we now rejet, ws sed on the estlished temperture dependene of post-exerise metoli nd ioni reovery when slmonids re hsed to exhustion to produe similr levels of intrellulr idosis, ltte umultion nd glyogen depletion in white musle regrdless of temperture (Kieffer et l., 1994; Wilkie et l., 1997). However, when Atlnti slmon were ngled to exhustion t wrmer temperture, essentilly the opposite effet of temperture on post-exerise musle reovery ws otined; they displyed greter depletion of musle glyogen, greter intrellulr idosis nd slower reovery of musle metolites t the wrmer temperture ompred with older tempertures (Booth et l., 1995; Wilkie et l., 1996). The present findings for U rit swim tests re more in line with dt otined when fish re ngled rther thn hsed to exhustion euse the metoli disturnes were higher nd performne reovery slower t wrmer tempertures. We suggest tht the disprity mong studies ould simply reflet differenes in the degree of exhustion nd the methods used to exhust the fish, with fish eoming more exhusted euse they pereive the hsing protool s more of thret or provotion thn either ngling or U rit testing. Given this possiility, old-limted fish ould opt to stop swimming sooner thn wrm-limted fish to preserve glyogen reserves. A U rit vlue, like time-to-exhustion t presried wter speed (e.g. Fey nd Grossmn, 199; Mitton nd MDonld, 1993), llows quntifition of the swimming effort, something tht is not esily done when fish re hsed or ngled to exhustion. U rit tests lso enompss spetrum of swimming speeds, with the eroi demnds of swimming up to mximum oxygen uptke eing met y rdiorespirtory djustments, while white musle reruitment nd neroi metolism inresingly supports the higher musulr power output ner U rit (Burgetz et l., 1998), ulminting in exhustion (Brett, 1964; Bemish, 1978). The simplest explntion for the higher U rit1 vlues otined for wrm-limted ompred with oldlimted fish is greter involvement of neroi swimming, given the signifintly lrger ltertions in plsm metolites oserved for wrm-limted rinow trout. Certinly, the wrm-limted fish were more stressed thn the oldlimted fish, s judged y the greter elevtion in plsm ortisol levels. However, sine musle metolites were not mesured here, we nnot exlude other possiilities. The higher levels of plsm potssium, ltte nd T mm, s well s the dditive effets of the seond swim, ould simply reflet greter relese of ltte nd potssium into the plsm euse the relese of ltte nd hydrogen ions from white musle to the lood is known to e temperture dependent (see Kieffer, 2). Nevertheless, it is unlikely tht different musle glyogen levels were ftor sine these re unffeted y limtion temperture (Kieffer, 2). Rome et l. (1985) showed tht utely exposing wrmlimted rp Cyprinus rpio to old wter resulted in white musle fires eing reruited t lower swimming speed, nd this ompression of reruitment order led to erlier ftigue nd redued sustined swimming speed. However, when the rp were old-limted, they reruited white musle t higher swimming speed thn wrm-limted fish, presumly euse old temperture limtion hd improved the mehnil performne of the red musle. The present findings re onsistent with this erlier work with rp in tht the old-limted rinow trout ppered to rely less on neroi white musle thn wrm-limted fish, ut the two studies differ in tht old-limted rinow trout hd lower U rit thn wrm-limted rinow trout wheres oldlimted rp swm to the sme mximum speed s wrmlimted fish (Rome et l., 1985). Rome et l. (1985) suggested three possile physiologil differenes in oldlimted fish ompred with wrm-limted fish: (1) higher mehnil power output from eroi musle, (2) limittions on the neurl ontrol of loomotory musle nd (3) limittions of the respirtory nd irultory systems in supplying oxygen. The present findings suggest fourth possiility: fish my opt to swim to different sttes of exhustion depending on either the temperture or resulting physiologil ondition. One enefit of limiting the level of exhustion under old onditions ppers to e more resonle reovery rte, whih llows for repeted performne. At wrm tempertures, fish enefit from higher initil level of performne ut, y exhusting themselves to reltively greter degree, hve the disdvntge of more prolonged reovery period. An dditionl disdvntge, ut for unknown resons, is tht exhustive exerise t wrm, ut not t old tempertures, n result in ppreile levels of postexerise mortlity (see Kieffer, 2). The present onlusions re lso in line with the results of MKenzie et l. (1996) working with Nile tilpi Oreohromis
Effet of temperture on repet swimming performne 3577 niloti. They found tht wrm-limted fish hd greter ost of reovery ( higher nd more prolonged post-exerise oxygen onsumption) fter eing hsed to exhustion thn old-limted fish. Interestingly, white musle ltte umultion ws similr for 16 C-limted nd 23 Climted tilpi, suggesting tht musle ltte my not lwys e relile mesure of post-exerise reovery. However, 23 C-limted tilpi exreted over twie the mount of mmoni post-exerise thn 16 C-limted fish. Kieffer et l. (1998) similrly found tht mmoni exretion t 75% U rit ws lmost threefold higher for 15 C-limted thn 5 C-limted rinow trout, while protein utiliztion t 75% U rit ws 3% t 15 C versus 15% t 5 C. Likewise, in the present study, we oserved signifintly higher plsm T mm in wrm-limted rinow trout. As disussed y MKenzie et l. (1996), the elevted mmoni prodution ould e result of either inresed protein metolism to fuel loomotion or inresed protein degrdtion from tissue dmge. Sine elevted T mm is thought to hve inhiitory tions on neurl nd musle tivity in fish (Bemount et l., 1995), the lrger elevtion in plsm T mm in wrmlimted fish is perhps ritil to survivl post-exhustion. On the other hnd, tissue dmge might negtively ffet U rit2. U rit vlues were omprle to those reported erlier y Jin et l. (1997) for rinow trout of the sme size in the sme swim tunnel [1.64 1.66 ody lengths (BL) s 1 ] nd higher thn those reported for 822 1118 g rinow trout (.94 BL s 1 nd.53 BL s 1 t 11 C nd 18 C, respetively; Tylor et l., 1996). Comprisons lso n e mde with studies on smller rinow trout, whih re expeted to ttin slightly higher U rit vlues (Brett, 1964) thn the 879 g fish used here. U rit vlues of 1.8 to 2. BL s 1 re reported for 53 73 g rinow trout t 18 19 C (Gllugher et l., 1992) nd 2.13 BL s 1 for 431 483 g rinow trout t 7 11 C (Burgetz et l., 1998). For 32 52 g rown trout, U rit vlues were 2.2 BL s 1 t 15 C nd 1.85 BL s 1 t 5 C (Butler nd Dy, 1993; Butler et l., 1992). As ntiipted, 4 min reovery period llowed full reovery of swimming performne for old-limted fish. Originlly it ws suggested tht slmonids e given 4 h etween U rit tests (Brett, 1964) to ensure return to routine O 2 onsumption ut not neessrily to routine glyogen levels. Susequently, reovery times of 2 h (Bruner et l., 1994), 1 h (Rndll et l., 1987), 45 min (Frrell et l., 1998, 23) nd 4 min (Jin et l., 1998) hve ll een shown to e suffiient for slmonids to repet U rit tests without ny signifint deline in performne. Here fish were provided with low speed wter urrent during reovery nd this my hve ided their reovery, sine reent studies with rinow trout (Millign et l., 2) nd oho slmon Onorhynhus kisuth (Frrell et l., 21) hve shown tht low to moderte swimming post-exhustion gretly ids metoli reovery through wrm-down effet. In ontrst, reovery time without wrm-down is >2 h for optiml performne on time-to-exhustion test (Mitton nd MDonld, 1994). Wng et l. (1994) reported tht musle phosphoretine nd ATP levels were restored within 3 min of rinow trout eing hsed to exhustion, while the post-exerise deline of oxygen onsumption lsted 3 3.5 h (Srello et l., 1991). However, routine oxygen onsumption does not hve to e restored efore dult sokeye slmon n repet seond U rit test (Frrell et l., 1998, 23). There ws generlly good greement etween the routine plsm vriles reported here nd those reported in previous studies (Butler nd Dy, 1993; Eros nd Millign, 1996; Pgnott et l., 1994; Thorrensen et l., 1994; Wng et l., 1994). However, the plsm ltte onentrtions t U rit in this study, espeilly those for the wrm-limted fish (7.3 mmol l 1 ), were t the high end of literture vlues for U rit swimming (1.5 5.5 mmol l 1 ) (Butler nd Dy, 1993; Gllugher et l., 1992; Thorrensen et l., 1993; Holk nd Lykkeoe, 1998; Frrell et l., 1998). Millign (1996) ites rnge for plsm ltte levels of 2 13 mmol l 1 immeditely fter hsing, inresing to pek vlues of 12 2 mmol l 1 t 2 h post-exerise. The vlues reported here for old-limted fish of 4.3 mmol l 1 t U rit nd 8.9 mmol l 1 4 min lter re t the low end of this rnge, wheres those for the wrmlimted fish (7.3 mmol l 1 t U rit nd 16.6 mmol l 1 4 min lter) re t the upper end of the rnge nd pprohed the level rehed (17.8 mmol l 1 ) pproximtely 9 min fter hypoxi U rit test (Frrell et l., 1998). The seond ojetive of the present study ws to determine whether ny of the mesured metolites displyed threshold levels tht, if surpssed in the first swim hllenge, were inditive of metoli ondition tht negtively ffeted susequent swimming performne. Plsm ltte level ws the only ndidte: the plsm ltte level efore U rit2 ws signifintly orrelted to the susequent swimming performne (U rit2 ). The threshold plsm ltte level of pproximtely 12.2 mmol l 1 (95% CI 7.9 16.4) grees with tht of 13 mmol l 1 reported y Stevens nd Blk (1966) for urst exerise with rinow trout nd 1 mmol l 1 for sokeye slmon (Frrell et l., 1998). In the erlier studies, fish refused to swim if the ltte threshold ws surpssed. However, no fish refused to swim outright in the present study nd insted U rit performne ws redued y 8 31%. Thus, euse neroi metolism is inresingly required to support swimming speeds greter thn 7% U rit (Burgetz et l., 1998), elevted levels of ltte ove the ltte threshold is proly inditive of filure to fully reruit neroi metolism in white musle (e.g. through deresed musle ph nd glyogen stores). This ide needs further study, however, euse plsm ltte dynmis re omplex, refleting rtes of prodution in the musle, rtes of relese from the musle nd rtes of lerne from the lood. While the present study suggests tht prodution my e greter t wrmer tempertures, relese rte is dependent on temperture (Keiffer et l., 1994) nd lerne rte is inversely relted to temperture (Kieffer nd Tufts, 1996). Beumont et l. (1995,) reported tht opper-exposed rown trout in wter of low ph hd poor U rit vlues nd
3578 K. E. Jin nd A. P. Frrell suggested tht the elevted plsm T mm inhiited white musle tivity either diretly or through CNS inhiitory mehnisms, euse elevted plsm T mm levels were orrelted with the redued U rit vlues. In the present study, we found no signifint orreltions etween swimming performne nd plsm T mm. However, our dt re not neessrily t odds with the suggestion of Beumont et l., (1995,) euse the plsm T mm levels reported in the present work were hlf those mesured in opper-exposed rown trout nd, in the erlier studies, U rit ws not redued ppreily until plsm T mm rehed levels >2 µmol l 1. A plsm T mm level >6 µmol l 1 resulted in fish refusing to swim. In the present study, T mm rehed only 1 µmol l 1 nd ws restored etween U rit tests for old-limted fish, lthough not for wrm-limted fish (Fig. 2). Severl studies report temperture optimum for U rit. For sokeye slmon, 15 C ws the optimum temperture for U rit, metoli sope (Brett, 1964) nd rdi performne (Brett, 1971; Dvis, 1968). The preferred temperture for sokeye slmon, however, ppers to e slightly ooler (1 12 C; Birtwell et l., 1994; Spohn et l., 1996). Grside nd Tit (1958) suggested preferred temperture rnge for rinow trout of 11 16 C, whih oinides with the optimum temperture rnge suggested for rdi performne (Frrell et l., 1996; Tylor et l., 1997; Frrell, 22). The present experiments show tht the shift in responses to repeted swimming for old- nd wrm-limted fish ourred t round 12 C. Therefore, the fish s preferred temperture my reflet su-mximl rtes for ertin tivities euse of negtive onsequenes in terms of rtes of reovery. In summry, we provide evidene tht wrm-limted rinow trout hve higher U rit thn old-limted fish, ut ssoited with this higher U rit is greter metoli nd ioni disturne. A onsequene of this elevted disturne is tht wrm-limted fish do not reover well enough fter 4 min rest to perform seond test t the sme level s the first one, wheres old-limted fish do. 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