The effects of sustained exercise and hypoxia upon oxygen tensions in the red muscle of rainbow trout

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1 The Journl of Experimentl Biology 27, Pulished y The Compny of Biologists 24 doi:1.1242/je The effects of sustined exercise nd hypoxi upon oxygen tensions in the red muscle of rinow trout D. J. McKenzie 1, *, S. Wong 2, D. J. Rndll 3, S. Egginton 4, E. W. Tylor 1 nd A. P. Frrell 2 1 School of Biosciences, University of Birminghm, Birminghm B15 2TT, UK, 2 Deprtment of Biologicl Sciences, Simon Frser University, 8888 University Drive, Burny, BC, V5A 1S6, Cnd, 3 Deprtment of Biology nd Chemistry, City University of Hong Kong, Tt Chee Avenue, Kowloon, Hong Kong, Chin nd 4 Deprtment of Physiology, University of Birminghm, Birminghm B15 2TT, UK *Author for correspondence t present ddress: CNRS/IFREMER, CREMA L Houmeu, BP 5, L Houmeu, Frnce (e-mil: Dvid.Mckenzie@ifremer.fr) Accepted 2 July 24 Teleost fish possess discrete locks of oxidtive red muscle (RM) nd glycolytic white muscle, wheres tetrpod skeletl muscles re mixed oxidtive/glycolytic. It hs een suggested tht the ntomy of RM in teleost fish could led to higher intrmusculr O 2 prtil pressures (P O ) thn in mmmlin skeletl muscles. This study provides the first direct experimentl support for this suggestion y using novel opticl fire sensors to discover men (± S.E.M., N=6) normoxic stedy-stte red muscle P O (PRMO ) of 61±1 mmhg (1 mmhg=133.3 P) in freeswimming rinow trout Oncorhynchus mykiss. This is significntly higher thn literture reports for mmmlin muscles, where the P O never exceeds 4 mmhg. Aeroic RM powers sustined swimming in rinow trout. During grded incrementl exercise, PRMO declined from 62±5 mmhg t the lowest swim speed down to 45±3 mmhg t mximum rtes of eroic work, ut then rose gin to 51±5 mmhg t exhustion. These mesurements of PRMO during exercise indicted, Summry therefore, tht O 2 supply to the RM ws not mjor limiting fctor t exhustion in trout. The current study found no evidence tht teleost hemogloins with Root effect cuse extremely elevted O 2 tensions in eroic tissues. Under normoxic conditions, PRMO ws significntly lower thn rteril P O (119±5 mmhg), nd remined lower when the rteril to tissue P O grdient ws reduced y exposure to mild hypoxi. When two sequentil levels of mild hypoxi (3 min t wter P O of 1 mmhg then 3 min t 75 mmhg) cused P O to fll to 84±2 mmhg then 61±3 mmhg, respectively, this elicited simultneous reductions in PRMO, to 51±6 mmhg then 41±5 mmhg, respectively. Although these hypoxic reductions in PRMO were significntly smller thn those in PO 2, the effect could e ttriuted to the sigmoid shpe of the trout hemogloin O 2 dissocition curve. Key words: O 2-sensitive optode, Root effect, O 2 prtil pressure, rteril lood O 2 content, O 2 consumption, swimming. Introduction The ntomy of the skeletl musculture in teleost fish differs significntly from tht of the tetrpod vertertes. Teleosts possess distinct locks of highly vsculrised oxidtive slow-twitch fires ( red muscle, RM), rrnged longside locks of less vsculrised glycolytic fst-twitch fires ( white muscle, WM), wheres tetrpod muscles ll comprise mixture of oxidtive slow-twitch nd glycolytic fst-twitch fires (Bone, 1978; Young, 1981). The different ntomicl rrngement of the oxidtive RM of teleost fish hs led to the suggestion tht the prtil pressures of oxygen (P O ) in their red muscle fires (PRMO ) my e significntly higher thn typiclly found in skeletl muscles of mmmls (Egginton, 22). Recent mesurements of the P O in vrious skeletl muscles of mmmls never seem to exceed pproximtely 4 mmhg under resting conditions in normoxi (Hutter et l., 1999; Jung et l., 1999; Behnke et l., 21; Suttner et l., 22). Although rteril nd venous lood PRMO vlues re known for teleost fishes such s the rinow trout Onchorhynchus mykiss (Holeton nd Rndll, 1967; Stevens nd Rndll, 1967; Kiceniuk nd Jones, 1977; Thoms nd Hughes, 1982; Thoms et l., 1987; Frrell nd Clutterhm, 23), we re unwre of ny PRMO mesurements tht hve tested this prediction. In fct, mesurements of the P O of muscle pper to e limited to those of Jnkowsky (1966), who reported very low vlue of <5 mmhg in the glycolytic WM of eels (Anguill sp.). Mesurements of O 2 tensions in the skeletl musculture of teleost fish would e prticulrly informtive for two other resons. One of these is to investigte the extent to which convective O 2 supply might e limiting fctor in the

2 363 D. J. McKenzie nd others performnce of sustined eroic exercise. During sustined exercise in tetrpods, incresed muscle O 2 demnd reltive to rtes of supply cuses reduction in muscle P O (Jung et l., 1999; Behnke et l., 21), nd ftigue is ssocited with severe decline in intrmusculr O 2 tension (Molé et l., 1999; Howlett nd Hogn, 21). Fish support sustined swimming ctivity with their RM, while WM powers the fster, unstedy sprint nd urst swimming ctivities (Bone, 1978). Therefore, mesurements of PRMO during swimming would provide insight into whether RM O 2 supply is fctor limiting the performnce of sustined swimming, tht is, whether exhustion is ssocited with profound decline in PRMO. Another reson why PRMO of teleosts might e prticulrly interesting reltes to unique chrcteristic of some teleost hemogloins, the Root effect (Root, 1931). When lood ph drops, hemogloins with Root effect exhiit mrkedly reduced cpcity to ind O 2, nd hence will relese ound O 2 (Root, 1931; Rndll, 1998; Pelster nd Rndll, 1998). A well estlished physiologicl role for the Root effect is found in specilised vsculr eds (retes), where high rtes of lctic cid nd CO 2 production y specilised cells generte low ph, resulting in loclised P O vlues tht re considerly higher thn in rteril lood leving the gills, due to unloding of O 2 from hemogloin. In prticulr, the choroid rete ensures tht photoreceptors in the retin re well oxygented, while the rete mirilis provides O 2 to inflte the swimldder nd mintin uoyncy s fish descend in the wter column (Jensen et l., 1998; Pelster nd Rndll, 1998). Theoreticlly, the Root effect my lso promote the relese of O 2 from hemogloin t other respiring tissues. This is ecuse in vitro evidence shows tht diffusion of respirtory CO 2 into the teleost erythrocyte, nd its cronic nhydrsectlysed hydrtion to HCO 3 nd H +, occurs more rpidly thn diffusionl relese of O 2 from hemogloin in response to P O grdient (Bruner nd Rndll, 1998; Pelster nd Rndll, 1998). Consequently, trnsient drop in erythrocyte ph following the ctlysed hydrtion of CO 2 could elicit Root effect nd generte high P O vlues in well-vsculrised eroic tissues. The presence nd extent of this effect in tissues other thn retes, such s RM, hs not een studied. If mesurements of PRMO reveled tht it ws higher thn the P O of rteril lood leving the gills (P O ), then this would e drmtic evidence tht the Root effect influences O 2 tensions in eroic tissues. In the current study, novel O 2 -sensitive opticl fire sensors ( micro-optodes ) were used to mesure the PRMO of conscious free-swimming rinow trout, species with pronounced Root effect (Binotti et l., 1971). Mesurements were mde under three regimes: during normoxi, to compre with dt reported for mmmlin skeletl muscles (Hutter et l., 1999; Jung et l., 1999; Suttner et l., 22; Behnke et l., 21) nd to investigte whether the Root effect contriutes to elevted PRMO ; during grded sustined exercise, to gin insights into RM O 2 supply during n increse in demnd, nd lso during mild hypoxi, to investigte whether reducing the rteril-to-tissue P O grdient would expose n impct of the Root effect upon PRMO. Mterils nd methods Experimentl nimls Rinow trout Oncorhynchus mykiss Wlum with men (± S.D.) mss of 697±152 g nd fork length of 36±2 cm, were trnsported from Sun Vlley Trout Frm (Mission, BC, Cnd) to Simon Frser University, where they were held outside in 1 l circulr fireglss tnks provided with flow of fresh wter t sesonl tempertures of C (men temperture 13.8±.4 C). Fish were cclimted to these conditions for t lest 2 weeks, nd fed dily. Individul trout were strved for 24 h prior to surgery. Surgicl preprtion nd mesurement of red muscle P O Fish were nesthetised in.1 mg l 1 MS-222 uffered with.1 mg l 1 NHCO 3, nd then trnsferred to n operting tle where their gills were irrigted with erted wter contining diluted nesthetic (.5 mg l 1 MS-222 nd NHCO 3 ). A smll incision ws mde in the skin just dorsl to the lterl line to revel the underlying RM sheet. A lunted surgicl needle (15G Terumo, Leuven, Belgium) ws then dvnced under the skin for pproximtely 1 cm, with the foremost end of the lunted needle evel ginst the underside of the skin. Gret cre ws tken to void penetrting the underlying musculture. An oxygen-sensitive opticl chemicl fire sensor (PreSens; Precision Sensing GmH, Regensurg, Germny), with tpered Teflon-coted tip (dimeter <1 µm), ws inserted into the ore of the needle nd dvnced until the tip reched the end of the needle. The needle ws then ngled t pproximtely 45 to the skin such tht the evelled end rested flt ginst the musculture nd the tip of the optode dvnced gently, t the previling ngle of 45, for pproximtely 3 mm into the underlying sheet of RM. The needle ws then withdrwn long the optode led, nd the optode secured in position with sutures to the skin. Trout were then cnnulted in the dorsl ort (DA) using the technique descried y Soivio et l. (1975). While the trout were still under nesthesi, the optode ws connected to Microx 1 oxygen meter (PreSens), connected in turn vi seril port to PC with dedicted softwre, which displyed PRMO t the optode tip every 1 s nd sved mesure of PRMO every 1 min in n ASCII file. Prior to surgery, ech optode ws clirted in oxygen-free nd irsturted wter, nd the tip soked for 1 min in 1 i.u. ml 1 heprin (Frrell nd Clutterhm, 23). The position of the proe in the RM ws confirmed post-mortem y creful dissection under inoculr microscope. Dt re reported only for those experiments where the proe could e reclirted, post-mortem, to correct for ny drift, ccording to the mnufcturer s instructions. In one cse where lood clotting nd tissue dmge were visile round the tip of the proe, the results were disregrded. Fish were recovered for pproximtely 42 h in normoxic

3 Oxygen tension in trout red muscle 3631 wter while swimming gently t speed equivlent to.5 ody lengths s 1 (BL s 1 ) in the Brett-type swimming respirometer descried in Gllugher et l. (1995), nd PRMO ws mesured every 1 min throughout. The DA cnnul ws flushed every 24 h with heprinised (1 i.u. ml 1 ) teleost sline. Mesurements of control normoxic PRMO vlues were mde while the nimls were swimming gently so s to estlish constnt level of musculr work nd consequent O 2 demnd nd to reduce spontneous chnges in ctivity level, thus minimising vriility in PRMO (see Fig. 1). Sustined exercise 7 Exercise performnce ws mesured y B exposing the fish to.5 BL s 1 increments in swimming speed every 3 min until ftigue. 6 Mximum sustinle swimming speed (U crit ) ws clculted s descried y Brett (1964). The PRMO ws mesured every 5 1 min throughout, while P O, rteril lood totl O 2 content (C O ) nd rteril lood ph (ph) were mesured once t ech 4 swimming speed, t ftigue, nd t 1 h nd 2 h post-ftigue. The P O ws mesured y gently withdrwing lood long the DA 3 ctheter nd into glss cuvette (D616, Rdiometer, Copenhgen, Denmrk) contining n oxygen electrode (Rdiometer E546), thermosttted to the experimentl temperture, with the signl displyed on Rdiometer PHM72 cid se nlyser. A susmple of this rteril lood ws withdrwn (3 µl) nd C O mesured s descried y Tucker (1967) using Rdiometer O 2 electrode thermosttted to 37 C, nd ph mesured using Rdiometer BMS2 cpillry ph electrode thermosttted to the sme wter temperture s the fish, with the signls displyed on Rdiometer PHM73 cid se nlyser. The remining lood, plus 3 µl of sline, ws returned to the niml. Wter P O (Pw O ) ws monitored continully using n oxygen-sensitive glvnic cell nd ssocited meter (HO1G, Oxygurd, Birkerød, Denmrk) with the signl displyed on chrt-recorder. The Pw O recording ws used to mesure oxygen consumption y the fish (M O, in mg kg 1 h 1 ) in the seled respirometer over 2 min t ech swimming speed, then for 3 min period centred round 1 h nd 2 h recovery, using the techniques descried in Gllugher et l. (1995). For the nlysis of the effects of exercise, men vlues were derived for the mesured vriles under control conditions (i.e. exercising gently t.5 BL s 1 ); for fish swimming t common degree of sustined exercise (1 BL s 1 ); for the mximum speed which the fish were le to PRMO2 (mmhg) A Time (h) Time (min) Fig. 1. (A) Representtive trce of red muscle P O (PRMO ) in rinow trout s mesured every 1 min during pproximtely 42 h recovery from implnttion of the micro-optode proe under nesthesi. (B) Expnded view of the dotted ox in A, with rrows indicting where the fish ws oserved to struggle violently in the respirometer. sustin for complete 3 min mesurement intervl (this rnged from 1 to 1.5 BL s 1 ); immeditely t exhustion; nd then t 1 h nd 2 h of recovery. An indiction of chnges in lood O 2 supply during eroic exercise ws otined y resolving the Fick eqution: M O = D O (P O PRMO ), (1) where D O is n index of rtes of lood O 2 delivery. This index ws resolved with the vlues of M O, P O nd PRMO mesured t the lowest swim speed (.5 BL s 1 ) nd then compred with those mesured t mximum rtes of oxygen uptke. Exposure to hypoxi While swimming gently t speed of.5 BL s 1, the trout were exposed to two levels of mild hypoxi, comprising 3 min t 1 mmhg, followed y 3 min t 75 mmhg, followed y 1 h recovery to normoxi (14 mmhg). The PRMO ws monitored every 1 min throughout the exposure

4 3632 D. J. McKenzie nd others protocol. Wter P O ws monitored continully, nd wter entering the respirometer mde hypoxic y pssing it countercurrent to flow of compressed 1% N 2 in gs-exchnge column. The Pw O recording ws used to mesure M O in the seled respirometer for 3 min in normoxi, for 3 min t oth levels of hypoxi, nd for 3 min centred upon 1 h recovery to normoxi. A mesurement of P O ws mde every 5 min y gently withdrwing lood long the DA ctheter nd into the O 2 electrode cuvette. Smples of rteril lood (3 µl, replced immeditely with n equl volume of sline) were collected from the DA cnnul in normoxi, t 3 min exposure to ech level of hypoxi, nd following 1 h recovery to normoxi, to mesure C O nd ph. The H O 2 dissocition curve derived for rinow trout t 14 C y Frrell nd Clutterhm (23) ws used to identify the percentge hemogloin sturtions tht would previl in lood t the P O mesured in the dorsl ort nd in the RM, in normoxi nd t ech level of hypoxi. The totl O 2 content of lood in the RM (CRMO, in mmol ml 1 ) ws then estimted s follows: CRMO = [H st RM / H st DA] C O, (2) where H st RM nd H st DA re the percentge sturtions of hemogloin in the red muscle nd dorsl ort, respectively. C O CRMO is then n estimte of the mount of O 2 relesed etween the DA nd RM. It ws ssumed tht, if the Root effect ws cusing PRMO to e high, then these estimtes of pprent O 2 unloding would decline drsticlly s P O fell in hypoxi, in mnner tht could not e ccounted for y the simultneous mesurements of whole-niml M O. Dt nlysis nd sttistics One-wy nlysis of vrince (ANOVA) for repeted mesures ws used to revel effects of exercise or hypoxi on ny single vrile. A two-wy repeted-mesures ANOVA ws used to ssess the effects of progressive hypoxi on P O versus PRMO. Where chnges in P O were expressed s percentge of the normoxic vlue, dt were rc-sine trnsformed prior to nlysis y ANOVA. In ll cses, Bonferroni post-hoc tests were used to identify where significnt differences ly. A proility of less thn 5% (P<.5) ws tken s the fiducil level for sttisticl significnce. Results Chrcteristics of red muscle P O in normoxi In ll fish, PRMO ws close to zero under nesthesi, ut grdully rose over few hours during recovery (Fig. 1) nd, t full recovery, ws pproximtely 6 mmhg (Tle 1). Under stedy stte normoxi, men PRMO ws significntly lower thn oth men Pw O nd P O (Tle 1). There ws vriility in normoxic PRMO mong fish, rnging from low of 39 mmhg to high of 11 mmhg, ut no fish exhiited higher P O in their muscle thn in either their inspired wter or rteril lood. Therefore, there ws no drmtic evidence Tle 1. Prtil pressures of O 2 in the red muscle nd rteril lood of rinow trout under control normoxic conditions, nd the effects of reducing wter P O in mild hypoxi Pw O (mmhg) 14 (normoxi) 1 75 PRMO (mmhg) 61±1 51±6 41±5 c P O (mmhg) 119±5 d 84±2 e 61±3, P O PRMO (mmhg) 58±1 33±6 2±4 c Chnge in PRMO from normoxi (%) 13±7 29±8 Chnge in P O from normoxi (%) 29±3 48±3 c PRMO, prtil pressure of O 2 in the red muscle; P O, prtil pressure of O 2 in rteril lood; Pw O, wter P O. All vlues re mens ± S.E.M., N=6. The PRMO vlue for ech fish, used to derive the men vlue shown here, ws clculted s the men of 3 mesurements mde every 1 min during either normoxi or either level of hypoxi, wheres the P O for ech fish ws clculted s the verge of two mesurements, mde t the eginning nd end of the 3 min period (see text for further detils). The P O PRMO is the prtil pressure grdient etween rteril lood nd the RM. For the P O nd PRMO dt, common superscript indictes no significnt difference y two-wy repeted-mesures ANOVA with Bonferroni post-hoc comprisons mongst mens. The sme ANOVA ws performed on the % chnge dt, following their rc-sine trnsformtion. For the P O PRMO dt, common superscript indictes no significnt difference y one-wy repeted-mesures ANOVA with Bonferroni post-hoc comprisons mongst mens. In ll cses, significnce ws ttriuted t P<.5. tht the Root effect influenced PRMO. In contrst to the stle PRMO oserved while fish were swimming stedily during normoxi, shrp reductions in PRMO occurred if the niml struggled in the respirometer, followed y grdul return to the previous P O (Fig. 1), ut PRMO never decresed elow ~4 mmhg. Effects of sustined exercise As expected, exercise cused n exponentil increse in O 2 uptke, nd the mximum rte of M O ws oserved t the mximum speed which the fish were le to sustin for complete 3 min intervl (Fig. 2). Exercise lso cused decline in ph, prticulrly t U crit (Fig. 2), which is evidence of switch to glycolytic metolism prior to exhustion. Nevertheless, trout in the current study did not exercise exceptionlly well, reching U crit of 1.38±.16 BL s 1 (N=5), which is lower thn the U crit of pproximtely 2. BL s 1 reported erlier for chroniclly instrumented rinow trout t the sme temperture (e.g. Shingles et l., 21; Frrell nd Clutterhm, 23). Arteril lood P O lso showed significnt reduction during sustined exercise nd t exhustion, ut recovered rpidly, unlike oth M O nd ph, which slowly returned towrds control vlues during 2 h of recovery. Despite the rteril cidosis, C O ws unchnged throughout exercise nd t exhustion (Fig. 2).

5 Oxygen tension in trout red muscle 3633 Fig. 2. Effects of sustined exercise, ftigue, nd susequent recovery, on rtes of O 2 uptke (M O ; men ± S.E.M.); rteril lood ph (ph), P O (P O ) nd totl O 2 content (C O ); red muscle P O (PRMO ), nd the rteril to red muscle P O grdient (P O PRMO ). Dt re provided for rinow trout swimming t two sustined speeds in ody lengths s 1 (BL s 1 ); t their mximum sustined swimming speed (Mx); immeditely upon ftigue (note tht M O dt re not ville for this instnce), nd t 1 h nd 2 h of recovery (Rec). NA, dt not ville. N=5 in ll cses, for those vriles where exercise elicited sttisticlly significnt effects y one-wy ANOVA for repeted vriles, common superscript indictes no significnt difference etween mens y Bonferroni post-hoc test (P<.5). During sustined exercise, PRMO showed significnt drop nd lthough the men vlue remined ove 4 mmhg, PRMO never exceeded P O. Moreover, PRMO rose significntly t the moment of exhustion to level tht ws not sttisticlly different from the control, nd remined thus for the ensuing 2 h recovery period. The prtil pressure grdient etween rteril lood nd the RM dropped s exercise intensity incresed, to low t ftigue, ut then returned rpidly to control vlues during recovery (Fig. 2). Given tht the prtil pressure grdient dropped s M O incresed during exercise, resolution of the Fick eqution reveled tht O 2 delivery, hence lood flow, to the red muscle would hve to increse y fctor of 4.6±1.1 times (men ± S.E.M., N=5) etween swimming speeds of.5 BL s 1 nd 1.38 BL s 1. Effects of exposure to hypoxi Mild hypoxi hd no significnt effects on M O nd C O (Tle 2), so it could e ssumed tht rtes of tissue O 2 demnd nd lood O 2 trnsport cpcity did not chnge significntly. Furthermore, the sence of ny chnges in ph indictes tht there ws no mjor increse in the relese of lctic cid nd CO 2 from the tissues (Tle 2). Thus, the most significnt effect of mild hypoxi ws decrese in the P O of rteril lood s it left the gills (Tle 1). Correspondingly, the PRMO showed close temporl sensitivity to chnges in Pw O nd P O during exposure to hypoxi nd the return to normoxi (Fig. 3). Red muscle P O ws significntly reduced from normoxic vlues t oth levels of hypoxi (Pw O =1 mmhg nd 75 mmhg), ut chnges in PRMO were significntly less thn those in P O, nd so the rteril to RM P O grdient declined s hypoxi deepened (Fig. 3, Tle 1). Proportionl (%) chnges in P O, reltive to normoxic vlues, were much more pronounced thn the net MO2 (mmol kg 1 h 1 ) PO2 (mmhg) PRMO2 (mmhg) BL s 1, 1 BL s 1 c Mx NA c Ftigue Rec 1 h Rec 2 h ph CO2 (mmol ml 1 ) PO2 PRMO2 (mmhg) chnges in the RM ut, nonetheless, were significntly smller in the RM thn in the rteril lood (Fig. 3, Tle 1). At no time during either hypoxi or recovery did ny fish exhiit higher PRMO thn their P O. In fct, the estimtes of pprent O 2 unloding did not decrese s hypoxi deepened ut, rther, incresed slightly (Tle 2). Discussion Chrcteristics of red muscle P O in normoxi Frrell nd Clutterhm (23) used the sme micro-optodes to mesure mixed venous P O (Pv O ) in the ductus Cuvier of rinow trout t similr temperture. They found tht Pv O declined to 2 mmhg immeditely fter surgery, nd recovered to stedy-stte vlue of pproximtely 35 mmhg.5 BL s 1 1 BL s 1 Mx c c Ftigue d Rec 1 h, Rec 2 h

6 3634 D. J. McKenzie nd others Tle 2. Trout oxygen uptke, rteril lood O 2 content, rteril ph nd pprent rtes of lood O 2 unloding etween the dorsl ort nd the red muscle, s function of wter P O Pw O (mmhg) 14 (normoxi) 1 75 M O (mmol kg 1 h 1 ) 4.76± ± ±.64 C O (mmol ml 1 ) 4.76± ± ±.35 ph 7.82± ± ±.3 Apprent O 2 unloding 1.46± ± ±.37 (mmol ml 1 ) M O, rte of oxygen uptke; C O, rteril lood O 2 content; ph, rteril ph; PwO 2, wter P O. All vlues re men ± S.E.M., N=6. Apprent O 2 unloding refers to the decline in totl O 2 content etween lood in the dorsl ort nd in the RM, clculted s descried in the text. There ws no significnt effect of hypoxi upon ny vrile. within 3 min. These mesurements of Pv O re very different from our mesurements for RM, where PRMO declined lmost to zero during surgery nd, lthough it then rose rpidly during the first few hours of recovery, it did not chieve stedy-stte vlue for pproximtely 2 h. This result clerly shows tht the RM muscle cn ecome severely hypoxic during deep nesthesi nd the slow recovery of PRMO my reflect reduced distriution of lood to the RM, s consequence of post-surgicl crdic depression nd decresed totl peripherl resistnce, nd/or incresed muscle O 2 demnd, perhps to metolise the nesthetic or repy n O 2 det. Frrell nd Clutterhm (23) lso found tht Pv O dropped precipitously to round 2 mmhg whenever the fish struggled, nd ttriuted this to sudden increses in muscle O 2 extrction. The shrp reductions in PRMO tht were oserved when fish struggled could e due either to incresed O 2 demnd nd extrction, or to decrese in locl lood flow ssocited with struggling ehviours. The ltter my e the min contriuting fctor, s struggling ehviours re ssocited with rdycrdi nd reduced crdic output (Stevens et l., 1972; Frrell, 1982; Frrell nd Jones, 1992), nd would lso result in hypoperfusion if incresed intrmusculr pressure compresses the supplying segmentl rteries. Even so, the fct tht men PRMO did not decrese elow ~4 mmhg is novel finding indicting tht RM remined well supplied with oxygen during spontneous struggling ehviours in rinow trout. These oservtions suggest tht the previously oserved precipitous decrese in Pv O (Frrell nd Clutterhm, 23) during struggling is driven y tissues in ddition to RM, the most likely cndidte eing the WM. Another novel finding of the present study is tht the PRMO of pproximtely 6 mmhg mesured in the freeswimming normoxic trout is pprecily higher thn the P O of 2 4 mmhg mesured with microelectrodes in the WM of Chnge in PO2 from normoxi (%) PO2 (mmhg) A B Time (min) Fig. 3. (A) Temporl chnges (men ± S.E.M.) in red muscle P O (PRMO, red dimonds) nd rteril lood P O (P O, lue dimonds) in rinow trout during exposure to mild hypoxi nd recovery to normoxi (wter P O, Pw O, shown s the simple line). (B) Percentge chnges (men ± S.E.M.) in PRMO (lue dimonds), P O (red dimonds) nd Pw O (simple line), from their respective normoxic vlues, over the sme period. N=6 in ll cses. eels (Jnkowsky, 1966). Unfortuntely, this erlier study does not detil exctly how the proes were implnted nd whether the eels were conscious during susequent mesurements (Jnkowsky, 1966). Therefore, given our oservtion of low PRMO during nesthesi, further studies with WM re wrrnted to confirm this difference etween RM nd WM. In contrst, it is very cler tht the normoxic PRMO in rinow trout is significntly higher thn in the skeletl muscle of mmmls, where P O vlues mesured with implnted microelectrodes rnge from 25 mmhg to 35 mmhg in conscious humns Homo spiens (Jung et l., 1999; Suttner et l., 22) nd dogs Cnis cnis (Hutter et l., 1999). Similr vlues were otined in nesthetised rts Rttus norvegicus, using phosphorescence quenching techniques (Behnke et l., 21). In view of this difference, we provide the first direct evidence to support the erlier suggestions y Egginton (22) tht the ntomy nd

7 Oxygen tension in trout red muscle 3635 physiology of RM in teleost fish could led to n elevted P O compred with mmmls. If the P O in respiring tissues is determined y the rte t which O 2 is supplied in the lood, the distnce nd speed it diffuses, nd the rte t which the tissue consumes it (Egginton, 22), then comprison of these vriles etween teleost RM nd skeletl muscles of mmmls might provide insight into why PRMO is so high. Mss-specific lood flow rtes to trout RM my e up to twice the level reported for mmmlin skeletl muscles t rest, lthough they re similr during exercise (Egginton, 1987, 22; Tylor et l., 1996). Rinow trout hemogloin hs similr ffinity for O 2 to tht of, for exmple, humns nd rts (Wilmer et l., 2). Egginton (22) clculted nd compred the men geometric supply re (domin of influence) s well s the men Krogh s diffusion distnce for cpillries in the tiilis nterior (TA) of oth rts nd Syrin hmsters Mesocritus urtus versus those in the RM of oth rinow trout nd striped ss Morone sxtilis. In these two teleosts, domins nd diffusion distnces were pproximtely 2% smller thn in the hmster, wheres rt domins were pproximtely eightfold lrger nd diffusion distnces pproximtely twofold lrger thn in the other three species (Egginton, 22). Thus, the higher lood flow nd smller cpillry domins clerly fvour higher P O in the red muscle of the rinow trout reltive to the skeletl muscles of the rt (Behnke et l., 21). The lower ody temperture of the fish, however, will led to significnt reduction in O 2 diffusivity, n effect tht is only prtilly offset y concurrent reduction in tissue O 2 consumption (Tylor et l., 1997; Egginton, 22). Insights into red muscle O 2 supply during grded exercise Our mesurements of PRMO in fish during grded exercise, t exhustion nd during recovery re lso novel. Furthermore, it is evident tht they contrst with results in exercising mmmls. In oth conscious humns nd the nesthetised rt, sustined exercise reduces intrmusculr P O from round 3 mmhg to elow 2 mmhg, chnge tht is ttriuted to incresed rtes of O 2 extrction y the working muscle (Jung et l., 1999; Behnke et l., 21). The significnt decrese in PRMO during sustined exercise in the current study presumly occurred for the sme reson. However, PRMO declined to only 45 mmhg t the mximum rtes of exercise performnce nd M O, which is considerly higher thn the P O vlues oserved in mmmls (Jung et l., 1999; Behnke et l., 21). Egginton et l. (2), using morphologicl dt nd nlysis of the resulting physico-chemicl conditions for O 2 diffusion, estimted tht the P O grdient etween cpillries nd the centre of red muscle fire my e less thn 4 mmhg in trout t mximum sustined exercise. Thus, the high PRMO suggests tht rinow trout RM my not ecome hypoxic t high levels of sustined exercise, suggestion tht is supported y two other lines of evidence. First, resolution of the Fick eqution reveled tht the reduction in the rteril to RM P O grdient tht occurred etween swimming speed of.5 BL s 1 nd mximl exercise would hve required n pproximtely fivefold increse in lood supply to meet the mesured increse in M O. This increse compres fvourly with the eightfold increse in lood supply to RM mesured with microspheres during mximum sustined exercise in rinow trout (Tylor et l., 1996). Second, t exhustion PRMO incresed rther thn decresed. This contrsts with tetrpod skeletl muscles, where ftigue is ssocited with profound decline in P O to elow 5% of resting vlues (Molé et l., 1999; Howlett nd Hogn, 21). This suggests tht when WM is recruited to power swimming speeds ove 7% of U crit (Burgetz et l., 1998; Lee et l., 23) susequent exhustion is not linked to mjor reductions in RM O 2 supply. Consequently, convective O 2 supply to the RM seems not to e limiting fctor for mximum eroic performnce in rinow trout. Prolonged exercise t 9% of U crit leds to depletion of oxidtive sustrtes in trout RM (Richrds et l., 22), so this my e the cuse of ftigue. Alterntively, RM my simply reduce its ctivity when WM is recruited during incrementl exercise, git trnsition representing n orderly nd necessry trnsition to muscle tht cn generte the required increse in tilet frequency nd musculr power output (see Jones nd Rndll, 1978). One consequence of this git trnsition is tht PRMO remins high, higher thn Pv O t ftigue (Frrell nd Clutterhm, 23). Further reserch into this re is clerly required, not lest to determine the vlidity of n incrementl grded exercise protocol in investigting fctors limiting mximum rtes of eroic metolism nd performnce in fish. Insights into the impct of the Root effect upon red muscle O 2 tensions There ws no evidence tht the Root effect influenced tissue O 2 tension enough to rise PRMO ove P O in the rinow trout. In fct, the opposite ws lwys true, oth in normoxi nd in mild hypoxi, when the P O to PRMO grdient ws reduced. Indeed, PRMO ws sensitive to chnges in P O nd lthough the proportionl chnges in PRMO during hypoxi were significntly less thn the chnges in P O, this could e ttriuted to the sigmoid shpe of the trout H O 2 dissocition curve. Thus, s P O declined, the rteril to RM P O difference shifted left towrds the steep portion of the dissocition curve, such tht smller drop in P O ws required to elicit the sme degree of O 2 unloding. In ddition to PRMO eing elevted compred with mesurements in mmmlin muscles, it is interesting tht PRMO ws lso consistently higher thn pulished vlues for mixed Pv O in the trout, oth in normoxi nd t comprle degrees of hypoxi (Holeton nd Rndll, 1967; Frrell nd Clutterhm, 23). Indeed, the mesured vlues for PRMO lie lmost exctly midwy etween pulished vlues for P O nd Pv O t the pproprite wter P O (Holeton nd Rndll, 1967). In contrst, the reported rnge for mmmlin intrmusculr P O (Hutter et l., 1999; Jung et l., 1999; Behnke et l., 21; Suttner et l., 22) is consistently lower thn tht of mixed Pv O, which is typiclly round 4 mmhg (Hutter et l., 1999; Wilmer et l., 2). The higher PRMO reltive to Pv O in the trout cn e interpreted in one of two wys. One possiility is

8 3636 D. J. McKenzie nd others tht venous return from RM is reltively smll contriution to mixed venous lood. The other possiility is tht the high PRMO of rinow trout reltive to mmmls could e, t lest in prt, consequence of Root effect in lood perfusing the RM. The Root effect would e engendered y trnsient chnges in erythrocyte ph cused y the fster rtes of CO 2 diffusion thn O 2 diffusion, nd the strong coupling of O 2 nd CO 2 movements tht re known to exist in trout lood (Bruner nd Rndll, 1998; Bruner et l., 2). Tht is, when rteril lood enters the RM of trout, rpid diffusion of metolic CO 2 into the erythrocyte would cuse trnsient drop in ph nd cuse Root off-shift, driving O 2 off the hemogloin nd rising P O. The deoxygented hemogloin would, however, then ind protons (the Hldne effect) nd cuse lood ph to rise gin, eliciting Root on-shift tht inds O 2 ck onto the hemogloin nd lowers P O in the venous lood leving the tissue. While such role of the Root effect is conjecture t this time, we cn eliminte the possiility tht we mesured n rtefct of mixed rteril, tissue nd venous P O vlues rther thn intrmusculr P O. If this hd een the cse, PRMO should hve vried directly with P O during hypoxi nd exercise, which it did not. Furthermore, similr concerns would, presumly, exist for mmmlin studies of intrmusculr P O tht involved the implnttion of microelectrodes (Hutter et l., 1999; Jung et l., 1999; Suttner et l., 22). Thus, in ddition to the ntomicl resons for elevted PRMO tht hve een rised y Egginton (22), the current study hs not eliminted the possiility tht O 2 tensions re lso influenced y the ction of the Root effect within the muscle vsculture. Future investigtions should perhps e imed t experimentl mnipultion of the Root effect to investigte how this chnge influences PRMO reltive to P O nd mixed Pv O. Conclusions The results show tht the P O previling in the RM of rinow trout is higher thn tht reported for skeletl muscles of rts nd humns. While there ws significnt decrese in PRMO during sustined exercise, it did not decline elow 4 mmhg nd incresed slightly t exhustion. These oservtions re tken s strong indiction tht O 2 supply to the RM does not ecome limiting either t the moment of recruitment of WM or t exhustion. We found no drmtic evidence tht the Root effect rises O 2 tensions in red muscle ecuse PRMO remined lmost exctly midwy etween previously pulished vlues of P O nd Pv O for rinow trout nd ws sensitive to reductions in P O during mild hypoxi. Further work is needed to explin the higher P O in the RM reltive to mixed venous lood ecuse, while this my reflect limited contriution from RM to mixed venous return, the phenomenon might lso e consequence of trnsient Root effect in the RM vsculture. This study ws supported y NSERC grnts to A.P.F. nd D.J.R. 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