SUMMARY

Teleost fish possess discrete blocks of oxidative red muscle (RM) and glycolytic white muscle, whereas tetrapod skeletal muscles are mixed oxidative/glycolytic. It has been suggested that the anatomy of RM in teleost fish could lead to higher intramuscular O2 partial pressures(PO2) than in mammalian skeletal muscles. This study provides the first direct experimental support for this suggestion by using novel optical fibre sensors to discover a mean (± s.e.m., N=6) normoxic steady-state red muscle PO2(PrmO2) of 61±10 mmHg (1 mmHg=133.3 Pa) in free-swimming rainbow trout Oncorhynchus mykiss. This is significantly higher than literature reports for mammalian muscles,where the PO2 never exceeds 40 mmHg. Aerobic RM powers sustained swimming in rainbow trout. During graded incremental exercise, PrmO2 declined from 62±5 mmHg at the lowest swim speed down to 45±3 mmHg at maximum rates of aerobic work, but then rose again to 51±5 mmHg at exhaustion. These measurements of PrmO2 during exercise indicated, therefore, that O2 supply to the RM was not a major limiting factor at exhaustion in trout. The current study found no evidence that teleost haemoglobins with a Root effect cause extremely elevated O2 tensions in aerobic tissues. Under normoxic conditions, PrmO2 was significantly lower than arterial PO2 (119±5 mmHg), and remained lower when the arterial to tissue PO2 gradient was reduced by exposure to mild hypoxia. When two sequential levels of mild hypoxia (30 min at a water PO2 of 100 mmHg then 30 min at 75 mmHg) caused PaO2 to fall to 84±2 mmHg then 61±3 mmHg, respectively, this elicited simultaneous reductions in PrmO2, to 51±6 mmHg then 41±5 mmHg, respectively. Although these hypoxic reductions in PrmO2 were significantly smaller than those in PaO2, the effect could be attributed to the sigmoid shape of the trout haemoglobin–O2 dissociation curve.

Introduction

The anatomy of the skeletal musculature in teleost fish differs significantly from that of the tetrapod vertebrates. Teleosts possess distinct blocks of highly vascularised oxidative slow-twitch fibres (`red' muscle, RM),arranged alongside blocks of less vascularised glycolytic fast-twitch fibres(`white' muscle, WM), whereas tetrapod muscles all comprise a mixture of oxidative slow-twitch and glycolytic fast-twitch fibres(Bone, 1978; Young, 1981). The different anatomical arrangement of the oxidative RM of teleost fish has led to the suggestion that the partial pressures of oxygen(PO2) in their red muscle fibres(PrmO2) may be significantly higher than typically found in skeletal muscles of mammals(Egginton, 2002). Recent measurements of the PO2 in various skeletal muscles of mammals never seem to exceed approximately 40 mmHg under resting conditions in normoxia (Hutter et al.,1999; Jung et al.,1999; Behnke et al.,2001; Suttner et al.,2002). Although arterial and venous blood PrmO2 values are known for teleost fishes such as the rainbow trout Onchorhynchus mykiss(Holeton and Randall, 1967; Stevens and Randall, 1967; Kiceniuk and Jones, 1977; Thomas and Hughes, 1982; Thomas et al., 1987; Farrell and Clutterham, 2003),we are unaware of any PrmO2measurements that have tested this prediction. In fact, measurements of the PO2 of muscle appear to be limited to those of Jankowsky (1966), who reported a very low value of <5 mmHg in the glycolytic WM of eels (Anguillasp.).

Measurements of O2 tensions in the skeletal musculature of teleost fish would be particularly informative for two other reasons. One of these is to investigate the extent to which convective O2 supply might be a limiting factor in the performance of sustained aerobic exercise. During sustained exercise in tetrapods, increased muscle O2 demand relative to rates of supply causes a reduction in muscle PO2 (Jung et al., 1999; Behnke et al.,2001), and fatigue is associated with a severe decline in intramuscular O2 tension(Molé et al., 1999; Howlett and Hogan, 2001). Fish support sustained swimming activity with their RM, while WM powers the faster,unsteady sprint and burst swimming activities(Bone, 1978). Therefore,measurements of PrmO2 during swimming would provide insight into whether RM O2 supply is a factor limiting the performance of sustained swimming, that is, whether exhaustion is associated with a profound decline in PrmO2.

Another reason why PrmO2 of teleosts might be particularly interesting relates to a unique characteristic of some teleost haemoglobins, the Root effect(Root, 1931). When blood pH drops, haemoglobins with a Root effect exhibit a markedly reduced capacity to bind O2, and hence will release bound O2(Root, 1931; Randall, 1998; Pelster and Randall, 1998). A well established physiological role for the Root effect is found in specialised vascular beds (retes), where high rates of lactic acid and CO2 production by specialised cells generate low pH, resulting in localised PO2 values that are considerably higher than in arterial blood leaving the gills, due to unloading of O2 from haemoglobin. In particular, the choroid rete ensures that photoreceptors in the retina are well oxygenated, while the rete mirabilis provides O2 to inflate the swimbladder and maintain buoyancy as fish descend in the water column (Jensen et al., 1998; Pelster and Randall, 1998).

Theoretically, the Root effect may also promote the release of O2 from haemoglobin at other respiring tissues. This is because in vitro evidence shows that diffusion of respiratory CO2into the teleost erythrocyte, and its carbonic anhydrase-catalysed hydration to HCO3 and H+, occurs more rapidly than diffusional release of O2 from haemoglobin in response to a PO2 gradient(Brauner and Randall, 1998; Pelster and Randall, 1998). Consequently, a transient drop in erythrocyte pH following the catalysed hydration of CO2 could elicit a Root effect and generate high PO2 values in well-vascularised aerobic tissues. The presence and extent of this effect in tissues other than retes,such as RM, has not been studied. If measurements of PrmO2 revealed that it was higher than the PO2 of arterial blood leaving the gills (PaO2), then this would be dramatic evidence that the Root effect influences O2 tensions in aerobic tissues.

In the current study, novel O2-sensitive optical fibre sensors(`micro-optodes') were used to measure the PrmO2 of conscious free-swimming rainbow trout, a species with a pronounced Root effect(Binotti et al., 1971). Measurements were made under three regimes: during normoxia, to compare with data reported for mammalian skeletal muscles(Hutter et al., 1999; Jung et al., 1999; Suttner et al., 2002; Behnke et al., 2001) and to investigate whether the Root effect contributes to elevated PrmO2; during graded sustained exercise, to gain insights into RM O2 supply during an increase in demand, and also during mild hypoxia, to investigate whether reducing the arterial-to-tissue PO2 gradient would expose an impact of the Root effect upon PrmO2.

Materials and methods

Experimental animals

Rainbow trout Oncorhynchus mykiss Walbaum with a mean (± s.d.) mass of 697±152 g and fork length of 36±2 cm, were transported from Sun Valley Trout Farm (Mission, BC,Canada) to Simon Fraser University, where they were held outside in 1000 l circular fibreglass tanks provided with a flow of fresh water at seasonal temperatures of 13–15°C (mean temperature 13.8±0.4°C). Fish were acclimated to these conditions for at least 2 weeks, and fed daily. Individual trout were starved for 24 h prior to surgery.

Surgical preparation and measurement of red musclePO2

Fish were anaesthetised in 0.1 mg l–1 MS-222 buffered with 0.1 mg l–1 NaHCO3, and then transferred to an operating table where their gills were irrigated with aerated water containing diluted anaesthetic (0.05 mg l–1 MS-222 and NaHCO3). A small incision was made in the skin just dorsal to the lateral line to reveal the underlying RM sheet. A blunted surgical needle (15G Terumo, Leuven, Belgium) was then advanced under the skin for approximately 1 cm, with the foremost end of the blunted needle bevel against the underside of the skin. Great care was taken to avoid penetrating the underlying musculature. An oxygen-sensitive optical chemical fibre sensor (PreSens;Precision Sensing GmbH, Regensburg, Germany), with a tapered Teflon-coated tip(diameter <10 μm), was inserted into the bore of the needle and advanced until the tip reached the end of the needle. The needle was then angled at approximately 45° to the skin such that the bevelled end rested flat against the musculature and the tip of the optode advanced gently, at the prevailing angle of 45°, for approximately 3 mm into the underlying sheet of RM. The needle was then withdrawn along the optode lead, and the optode secured in position with sutures to the skin. Trout were then cannulated in the dorsal aorta (DA) using the technique described by Soivio et al.(1975).

While the trout were still under anaesthesia, the optode was connected to a Microx 1 oxygen meter (PreSens), connected in turn via a serial port to a PC with dedicated software, which displayed PrmO2 at the optode tip every 1 s and saved a measure of PrmO2 every 1 min in an ASCII file. Prior to surgery, each optode was calibrated in oxygen-free and air-saturated water, and the tip soaked for 10 min in 100 i.u. ml–1 heparin (Farrell and Clutterham, 2003). The position of the probe in the RM was confirmed post-mortem by careful dissection under a binocular microscope. Data are reported only for those experiments where the probe could be recalibrated, post-mortem, to correct for any drift, according to the manufacturer's instructions. In one case where blood clotting and tissue damage were visible around the tip of the probe, the results were disregarded.

Fish were recovered for approximately 42 h in normoxic water while swimming gently at a speed equivalent to 0.5 body lengths s–1(BL s–1) in the Brett-type swimming respirometer described in Gallaugher et al.(1995), and PrmO2 was measured every 1 min throughout. The DA cannula was flushed every 24 h with heparinised (10 i.u. ml–1) teleost saline. Measurements of control normoxic PrmO2 values were made while the animals were swimming gently so as to establish a constant level of muscular work and consequent O2 demand and to reduce spontaneous changes in activity level, thus minimising variability in PrmO2 (see Fig. 1).

Fig. 1.

(A) Representative trace of red muscle PO2(PrmO2) in a rainbow trout as measured every 1 min during approximately 42 h recovery from implantation of the micro-optode probe under anaesthesia. (B) Expanded view of the dotted box in A, with arrows indicating where the fish was observed to struggle violently in the respirometer.

Fig. 1.

(A) Representative trace of red muscle PO2(PrmO2) in a rainbow trout as measured every 1 min during approximately 42 h recovery from implantation of the micro-optode probe under anaesthesia. (B) Expanded view of the dotted box in A, with arrows indicating where the fish was observed to struggle violently in the respirometer.

Sustained exercise

Exercise performance was measured by exposing the fish to 0.5 BLs–1 increments in swimming speed every 30 min until fatigue. Maximum sustainable swimming speed (Ucrit) was calculated as described by Brett (1964). The PrmO2 was measured every 1 min throughout, while PaO2, arterial blood total O2 content (CaO2) and arterial blood pH (pHa) were measured once at each swimming speed, at fatigue, and at 1 h and 2 h post-fatigue. The PaO2 was measured by gently withdrawing blood along the DA catheter and into a glass cuvette (D616,Radiometer, Copenhagen, Denmark) containing an oxygen electrode (Radiometer E5046), thermostatted to the experimental temperature, with the signal displayed on a Radiometer PHM72 acid–base analyser. A subsample of this arterial blood was withdrawn (300 μl) and CaO2 measured as described by Tucker(1967) using a Radiometer O2 electrode thermostatted to 37°C, and pHa measured using a Radiometer BMS2 capillary pH electrode thermostatted to the same water temperature as the fish, with the signals displayed on a Radiometer PHM73 acid–base analyser. The remaining blood, plus 300 μl of saline, was returned to the animal. Water PO2(PwO2) was monitored continually using an oxygen-sensitive galvanic cell and associated meter (HO1G, Oxyguard,Birkerød, Denmark) with the signal displayed on a chart-recorder. The PwO2 recording was used to measure oxygen consumption by the fish(O2, in mg kg–1 h–1) in the sealed respirometer over 20 min at each swimming speed, then for a 30 min period centred around 1 h and 2 h recovery, using the techniques described in Gallaugher et al.(1995). For the analysis of the effects of exercise, mean values were derived for the measured variables under control conditions (i.e. exercising gently at 0.5 BLs–1); for fish swimming at a common degree of sustained exercise (1 BL s–1); for the maximum speed which the fish were able to sustain for a complete 30 min measurement interval (this ranged from 1 to 1.5 BL s–1); immediately at exhaustion; and then at 1 h and 2 h of recovery. An indication of changes in blood O2 supply during aerobic exercise was obtained by resolving the Fick equation:
\[\ \mathit{{\dot{M}}}_{\mathrm{O}_{2}}=\mathit{D}_{\mathrm{O}_{2}}{\times}(\mathit{P}\mathrm{a}_{\mathrm{O}_{2}}-P\mathrm{\mbox{\textsc{rm}}}_{\mathrm{O}_{2}}),\]
(1)
where DO2 is an index of rates of blood O2 delivery. This index was resolved with the values of O2, PaO2 and PrmO2 measured at the lowest swim speed (0.5 BL s–1) and then compared with those measured at maximum rates of oxygen uptake.

Exposure to hypoxia

While swimming gently at a speed of 0.5 BL s–1,the trout were exposed to two levels of mild hypoxia, comprising 30 min at 100 mmHg, followed by 30 min at 75 mmHg, followed by 1 h recovery to normoxia (140 mmHg). The PrmO2 was monitored every 1 min throughout the exposure protocol. Water PO2 was monitored continually, and water entering the respirometer made hypoxic by passing it counter-current to a flow of compressed 100% N2 in a gas-exchange column. The PwO2 recording was used to measure O2 in the sealed respirometer for 30 min in normoxia, for 30 min at both levels of hypoxia, and for 30 min centred upon 1 h recovery to normoxia. A measurement of PaO2 was made every 5 min by gently withdrawing blood along the DA catheter and into the O2 electrode cuvette. Samples of arterial blood (300 μl, replaced immediately with an equal volume of saline) were collected from the DA cannula in normoxia, at 30 minexposure to each level of hypoxia, and following 1 h recovery to normoxia,to measure CaO2 and pHa.

The Hb–O2 dissociation curve derived for rainbow trout at 14°C by Farrell and Clutterham(2003) was used to identify the percentage haemoglobin saturations that would prevail in blood at the PO2 measured in the dorsal aorta and in the RM,in normoxia and at each level of hypoxia. The total O2 content of blood in the RM (CrmO2, in mmol ml–1) was then estimated as follows:
\[\ \mathit{C}\mathrm{\mbox{\textsc{rm}}}_{\mathrm{O}_{2}}=[\mathrm{Hb}{\ }\mathrm{sat}{\ }\mathrm{RM}{/}\mathrm{Hb}{\ }\mathrm{sat}{\ }\mathrm{DA}]{\times}\mathit{C}\mathrm{a}_{\mathrm{O}_{2}},\]
(2)
where `Hb sat RM' and `Hb sat DA' are the percentage saturations of haemoglobin in the red muscle and dorsal aorta, respectively. CaO2CrmO2is then an estimate of the amount of O2 released between the DA and RM. It was assumed that, if the Root effect was causing PrmO2 to be high, then these estimates of apparent `O2 unloading' would decline drastically as PaO2 fell in hypoxia, in a manner that could not be accounted for by the simultaneous measurements of whole-animal O2.

Data analysis and statistics

One-way analysis of variance (ANOVA) for repeated measures was used to reveal effects of exercise or hypoxia on any single variable. A two-way repeated-measures ANOVA was used to assess the effects of progressive hypoxia on PaO2versus PrmO2. Where changes in PO2 were expressed as a percentage of the normoxic value, data were arc-sine transformed prior to analysis by ANOVA. In all cases, Bonferroni post-hoc tests were used to identify where significant differences lay. A probability of less than 5%(P<0.05) was taken as the fiducial level for statistical significance.

Results

Characteristics of red musclePO2in normoxia

In all fish, PrmO2 was close to zero under anaesthesia, but gradually rose over a few hours during recovery(Fig. 1) and, at full recovery,was approximately 60 mmHg (Table 1).Under steady state normoxia, mean PrmO2 was significantly lower than both mean PwO2 and PaO2 (Table 1). There was variability in normoxic PrmO2 among fish, ranging from a low of 39 mmHg to a high of 101 mmHg, but no fish exhibited a higher PO2 in their muscle than in either their inspired water or arterial blood. Therefore, there was no dramatic evidence that the Root effect influenced PrmO2. In contrast to the stable PrmO2 observed while fish were swimming steadily during normoxia, sharp reductions in PrmO2 occurred if the animal struggled in the respirometer, followed by a gradual return to the previous PO2 (Fig. 1), but PrmO2 never decreased below ∼40 mmHg.

Table 1.

Partial pressures of O2 in the red muscle and arterial blood of rainbow trout under control normoxic conditions, and the effects of reducing water PO2in mild hypoxia

PwO2 (mmHg)
140 (normoxia)10075
PrmO2 (mmHg) 61±10a 51±6b 41±5c 
PaO2 (mmHg) 119±5d 84±2e 61±3a,b 
PaO2PrmO2(mmHg) 58±10a 33±6b 20±4c 
Change in PrmO2 from normoxia (%) -13±7a -29±8b 
Change in PaO2 from normoxia (%) -29±3b -48±3c 
PwO2 (mmHg)
140 (normoxia)10075
PrmO2 (mmHg) 61±10a 51±6b 41±5c 
PaO2 (mmHg) 119±5d 84±2e 61±3a,b 
PaO2PrmO2(mmHg) 58±10a 33±6b 20±4c 
Change in PrmO2 from normoxia (%) -13±7a -29±8b 
Change in PaO2 from normoxia (%) -29±3b -48±3c 

PrmO2, partial pressure of O2 in the red muscle; PaO2, partial pressure of O2 in arterial blood; PwO2, water PO2.

All values are means ± s.e.m., N=6.

The PrmO2 value for each fish,used to derive the mean value shown here, was calculated as the mean of 30 measurements made every 1 min during either normoxia or either level of hypoxia, whereas the PaO2 for each fish was calculated as the average of two measurements, made at the beginning and end of the 30 min period (see text for further details).

The PaO2PrmO2is the partial pressure gradient between arterial blood and the RM.

For the PaO2 and PrmO2 data, a common superscript indicates no significant difference by two-way repeated-measures ANOVA with Bonferroni post-hoc comparisons amongst means. The same ANOVA was performed on the % change data, following their arc-sine transformation.

For the PaO2PrmO2data, a common superscript indicates no significant difference by one-way repeated-measures ANOVA with Bonferroni post-hoc comparisons amongst means.

In all cases, significance was attributed at P<0.05.

Effects of sustained exercise

As expected, exercise caused an exponential increase in O2uptake, and the maximum rate of O2 was observed at the maximum speed which the fish were able to sustain for a complete 30 min interval (Fig. 2). Exercise also caused a decline in pHa, particularly at Ucrit(Fig. 2), which is evidence of a switch to glycolytic metabolism prior to exhaustion. Nevertheless, trout in the current study did not exercise exceptionally well, reaching a Ucrit of 1.38±0.16 BL s–1(N=5), which is lower than the Ucrit of approximately 2.0 BL s–1 reported earlier for chronically instrumented rainbow trout at the same temperature (e.g. Shingles et al., 2001; Farrell and Clutterham, 2003). Arterial blood PO2 also showed a significant reduction during sustained exercise and at exhaustion, but recovered rapidly,unlike both O2and pHa, which slowly returned towards control values during 2 h of recovery. Despite the arterial acidosis, CaO2 was unchanged throughout exercise and at exhaustion(Fig. 2).

Fig. 2.

Effects of sustained exercise, fatigue, and subsequent recovery, on rates of O2 uptake(O2; mean± s.e.m.); arterial blood pH (pHa), PO2 (PaO2) and total O2 content (CaO2); red muscle PO2(PrmO2), and the arterial to red muscle PO2 gradient(PaO2PrmO2). Data are provided for rainbow trout swimming at two sustained speeds in body lengths s–1 (BL s–1); at their maximum sustained swimming speed (Max); immediately upon fatigue (note that O2 data are not available for this instance), and at 1 h and 2 h of recovery (Rec). NA, data not available. N=5 in all cases, for those variables where exercise elicited statistically significant effects by one-way ANOVA for repeated variables, a common superscript indicates no significant difference between means by Bonferroni post-hoc test (P<0.05).

Fig. 2.

Effects of sustained exercise, fatigue, and subsequent recovery, on rates of O2 uptake(O2; mean± s.e.m.); arterial blood pH (pHa), PO2 (PaO2) and total O2 content (CaO2); red muscle PO2(PrmO2), and the arterial to red muscle PO2 gradient(PaO2PrmO2). Data are provided for rainbow trout swimming at two sustained speeds in body lengths s–1 (BL s–1); at their maximum sustained swimming speed (Max); immediately upon fatigue (note that O2 data are not available for this instance), and at 1 h and 2 h of recovery (Rec). NA, data not available. N=5 in all cases, for those variables where exercise elicited statistically significant effects by one-way ANOVA for repeated variables, a common superscript indicates no significant difference between means by Bonferroni post-hoc test (P<0.05).

During sustained exercise, PrmO2showed a significant drop and although the mean value remained above 40 mmHg, PrmO2 never exceeded PaO2. Moreover, PrmO2 rose significantly at the moment of exhaustion to a level that was not statistically different from the control, and remained thus for the ensuing 2 h recovery period. The partial pressure gradient between arterial blood and the RM dropped as exercise intensity increased, to a low at fatigue, but then returned rapidly to control values during recovery (Fig. 2). Given that the partial pressure gradient dropped as O2 increased during exercise, resolution of the Fick equation revealed that O2delivery, hence blood flow, to the red muscle would have to increase by a factor of 4.6±1.1 times (mean ± s.e.m., N=5) between swimming speeds of 0.5 BL s–1 and 1.38 BLs–1.

Effects of exposure to hypoxia

Mild hypoxia had no significant effects on O2 and CaO2 (Table 2), so it could be assumed that rates of tissue O2demand and blood O2 transport capacity did not change significantly. Furthermore, the absence of any changes in pHa indicates that there was no major increase in the release of lactic acid and CO2from the tissues (Table 2). Thus, the most significant effect of mild hypoxia was a decrease in the PO2 of arterial blood as it left the gills(Table 1). Correspondingly, the PrmO2 showed close temporal sensitivity to changes in PwO2 and PaO2 during exposure to hypoxia and the return to normoxia (Fig. 3). Red muscle PO2 was significantly reduced from normoxic values at both levels of hypoxia(PwO2=100 mmHg and 75 mmHg), but changes in PrmO2 were significantly less than those in PaO2, and so the arterial to RM PO2 gradient declined as hypoxia deepened(Fig. 3, Table 1). Proportional (%)changes in PO2, relative to normoxic values,were much more pronounced than the net changes in the RM but, nonetheless,were significantly smaller in the RM than in the arterial blood(Fig. 3, Table 1). At no time during either hypoxia or recovery did any fish exhibit a higher PrmO2 than their PaO2. In fact, the estimates of apparent O2 unloading did not decrease as hypoxia deepened but, rather,increased slightly (Table 2).

Table 2.

Trout oxygen uptake, arterial blood O2 content, arterial pH and apparent rates of blood O2 unloading between the dorsal aorta and the red muscle, as a function of waterPO2

PwO2 (mmHg)
140 (normoxia)10075
O2 (mmol kg-1 h-14.76±0.79 5.11±0.90 4.63±0.64 
CaO2 (mmol ml-14.76±0.45 4.41±0.36 4.20±0.35 
pHa 7.82±0.02 7.81±0.03 7.84±0.03 
Apparent O2 unloading (mmol ml-11.46±0.37 1.80±0.29 2.26±0.37 
PwO2 (mmHg)
140 (normoxia)10075
O2 (mmol kg-1 h-14.76±0.79 5.11±0.90 4.63±0.64 
CaO2 (mmol ml-14.76±0.45 4.41±0.36 4.20±0.35 
pHa 7.82±0.02 7.81±0.03 7.84±0.03 
Apparent O2 unloading (mmol ml-11.46±0.37 1.80±0.29 2.26±0.37 

O2, rate of oxygen uptake; CaO2, arterial blood O2 content; pHa, arterial pH; PwO2, water PO2.

All values are mean ± s.e.m., N=6.

Apparent O2 unloading refers to the decline in total O2 content between blood in the dorsal aorta and in the RM,calculated as described in the text. There was no significant effect of hypoxia upon any variable.

Fig. 3.

(A) Temporal changes (mean ± s.e.m.) in red muscle PO2(PrmO2, red diamonds) and arterial blood PO2 (PaO2,blue diamonds) in rainbow trout during exposure to mild hypoxia and recovery to normoxia (water PO2, PwO2, shown as the simple line). (B) Percentage changes (mean ± s.e.m.) in PrmO2 (blue diamonds), PaO2 (red diamonds) and PwO2 (simple line), from their respective normoxic values, over the same period. N=6 in all cases.

Fig. 3.

(A) Temporal changes (mean ± s.e.m.) in red muscle PO2(PrmO2, red diamonds) and arterial blood PO2 (PaO2,blue diamonds) in rainbow trout during exposure to mild hypoxia and recovery to normoxia (water PO2, PwO2, shown as the simple line). (B) Percentage changes (mean ± s.e.m.) in PrmO2 (blue diamonds), PaO2 (red diamonds) and PwO2 (simple line), from their respective normoxic values, over the same period. N=6 in all cases.

Discussion

Characteristics of red musclePO2in normoxia

Farrell and Clutterham(2003) used the same micro-optodes to measure mixed venous PO2(PvO2) in the ductus Cuvier of rainbow trout at a similar temperature. They found that PvO2declined to 20 mmHg immediately after surgery, and recovered to a steady-state value of approximately 35 mmHg within 30 min. These measurements of PvO2 are very different from our measurements for RM, where PrmO2 declined almost to zero during surgery and, although it then rose rapidly during the first few hours of recovery, it did not achieve a steady-state value for approximately 20 h. This result clearly shows that the RM muscle can become severely hypoxic during deep anaesthesia and the slow recovery of PrmO2 may reflect reduced distribution of blood to the RM, as a consequence of post-surgical cardiac depression and decreased total peripheral resistance, and/or increased muscle O2 demand, perhaps to metabolise the anaesthetic or repay an O2 debt.

Farrell and Clutterham(2003) also found that PvO2 dropped precipitously to around 20 mmHg whenever the fish struggled, and attributed this to sudden increases in muscle O2 extraction. The sharp reductions in PrmO2 that were observed when fish struggled could be due either to increased O2 demand and extraction, or to a decrease in local blood flow associated with struggling behaviours. The latter may be the main contributing factor, as struggling behaviours are associated with bradycardia and reduced cardiac output(Stevens et al., 1972; Farrell, 1982; Farrell and Jones, 1992), and would also result in hypoperfusion if increased intramuscular pressure compresses the supplying segmental arteries. Even so, the fact that mean PrmO2 did not decrease below ∼40 mmHg is a novel finding indicating that RM remained well supplied with oxygen during spontaneous struggling behaviours in rainbow trout. These observations suggest that the previously observed precipitous decrease in PvO2(Farrell and Clutterham, 2003)during struggling is driven by tissues in addition to RM, the most likely candidate being the WM.

Another novel finding of the present study is that the PrmO2 of approximately 60 mmHg measured in the free-swimming normoxic trout is appreciably higher than the PO2 of 2–4 mmHg measured with microelectrodes in the WM of eels(Jankowsky, 1966). Unfortunately, this earlier study does not detail exactly how the probes were implanted and whether the eels were conscious during subsequent measurements(Jankowsky, 1966). Therefore,given our observation of a low PrmO2during anaesthesia, further studies with WM are warranted to confirm this difference between RM and WM. In contrast, it is very clear that the normoxic PrmO2 in rainbow trout is significantly higher than in the skeletal muscle of mammals, where PO2 values measured with implanted microelectrodes range from 25 mmHg to 35 mmHg in conscious humans Homo sapiens (Jung et al.,1999; Suttner et al.,2002) and dogs Canis canis(Hutter et al., 1999). Similar values were obtained in anaesthetised rats Rattus norvegicus, using phosphorescence quenching techniques(Behnke et al., 2001). In view of this difference, we provide the first direct evidence to support the earlier suggestions by Egginton(2002) that the anatomy and physiology of RM in teleost fish could lead to an elevated PO2 compared with mammals.

If the PO2 in respiring tissues is determined by the rate at which O2 is supplied in the blood, the distance and speed it diffuses, and the rate at which the tissue consumes it(Egginton, 2002), then a comparison of these variables between teleost RM and skeletal muscles of mammals might provide insight into why PrmO2 is so high. Mass-specific blood flow rates to trout RM may be up to twice the level reported for mammalian skeletal muscles at rest, although they are similar during exercise(Egginton, 1987, 2002; Taylor et al., 1996). Rainbow trout haemoglobin has a similar affinity for O2 to that of, for example, humans and rats (Wilmer et al.,2000). Egginton(2002) calculated and compared the mean geometric supply area (domain of influence) as well as the mean Krogh's diffusion distance for capillaries in the tibialis anterior (TA) of both rats and Syrian hamsters Mesocritus auratus versus those in the RM of both rainbow trout and striped bass Morone saxatilis. In these two teleosts, domains and diffusion distances were approximately 20% smaller than in the hamster, whereas rat domains were approximately eightfold larger and diffusion distances approximately twofold larger than in the other three species (Egginton, 2002). Thus,the higher blood flow and smaller capillary domains clearly favour a higher PO2 in the red muscle of the rainbow trout relative to the skeletal muscles of the rat(Behnke et al., 2001). The lower body temperature of the fish, however, will lead to a significant reduction in O2 diffusivity, an effect that is only partially offset by a concurrent reduction in tissue O2 consumption(Taylor et al., 1997; Egginton, 2002).

Insights into red muscle O2 supply during graded exercise

Our measurements of PrmO2 in fish during graded exercise, at exhaustion and during recovery are also novel. Furthermore, it is evident that they contrast with results in exercising mammals. In both conscious humans and the anaesthetised rat, sustained exercise reduces intramuscular PO2 from around 30 mmHg to below 20 mmHg, a change that is attributed to increased rates of O2 extraction by the working muscle(Jung et al., 1999; Behnke et al., 2001). The significant decrease in PrmO2 during sustained exercise in the current study presumably occurred for the same reason. However, PrmO2 declined to only 45 mmHg at the maximum rates of exercise performance and O2, which is considerably higher than the PO2 values observed in mammals (Jung et al.,1999; Behnke et al.,2001). Egginton et al.(2000), using morphological data and analysis of the resulting physico-chemical conditions for O2 diffusion, estimated that the PO2gradient between capillaries and the centre of a red muscle fibre may be less than 4 mmHg in trout at maximum sustained exercise. Thus, the high PrmO2 suggests that rainbow trout RM may not become hypoxic at high levels of sustained exercise, a suggestion that is supported by two other lines of evidence. First, resolution of the Fick equation revealed that the reduction in the arterial to RM PO2 gradient that occurred between a swimming speed of 0.5 BL s–1 and maximal exercise would have required an approximately fivefold increase in blood supply to meet the measured increase in O2. This increase compares favourably with the eightfold increase in blood supply to RM measured with microspheres during maximum sustained exercise in rainbow trout(Taylor et al., 1996). Second,at exhaustion PrmO2 increased rather than decreased. This contrasts with tetrapod skeletal muscles, where fatigue is associated with a profound decline in PO2 to below 50% of resting values (Moléet al., 1999; Howlett and Hogan, 2001). This suggests that when WM is recruited to power swimming speeds above 70% of Ucrit(Burgetz et al., 1998; Lee et al., 2003) subsequent exhaustion is not linked to major reductions in RM O2 supply. Consequently, convective O2 supply to the RM seems not to be a limiting factor for maximum aerobic performance in rainbow trout. Prolonged exercise at 90% of Ucrit leads to depletion of oxidative substrates in trout RM (Richards et al.,2002), so this may be the cause of fatigue. Alternatively, RM may simply reduce its activity when WM is recruited during incremental exercise, a gait transition representing an orderly and necessary transition to a muscle that can generate the required increase in tailbeat frequency and muscular power output (see Jones and Randall,1978). One consequence of this gait transition is that PrmO2 remains high, higher than PvO2 at fatigue(Farrell and Clutterham,2003). Further research into this area is clearly required, not least to determine the validity of an incremental graded exercise protocol in investigating factors limiting maximum rates of aerobic metabolism and performance in fish.

Insights into the impact of the Root effect upon red muscle O2 tensions

There was no evidence that the Root effect influenced tissue O2tension enough to raise PrmO2 above PaO2 in the rainbow trout. In fact, the opposite was always true, both in normoxia and in mild hypoxia, when the PaO2 to PrmO2 gradient was reduced. Indeed, PrmO2 was sensitive to changes in PaO2 and although the proportional changes in PrmO2 during hypoxia were significantly less than the changes in PaO2,this could be attributed to the sigmoid shape of the trout Hb–O2 dissociation curve. Thus, as PaO2 declined, the arterial to RM PO2 difference shifted left towards the steep portion of the dissociation curve, such that a smaller drop in PO2 was required to elicit the same degree of O2 unloading.

In addition to PrmO2 being elevated compared with measurements in mammalian muscles, it is interesting that PrmO2 was also consistently higher than published values for mixed PvO2 in the trout, both in normoxia and at comparable degrees of hypoxia(Holeton and Randall, 1967; Farrell and Clutterham, 2003). Indeed, the measured values for PrmO2 lie almost exactly midway between published values for PaO2 and PvO2 at the appropriate water PO2(Holeton and Randall, 1967). In contrast, the reported range for mammalian intramuscular PO2 (Hutter et al., 1999; Jung et al.,1999; Behnke et al.,2001; Suttner et al.,2002) is consistently lower than that of mixed PvO2, which is typically around 40 mmHg(Hutter et al., 1999; Wilmer et al., 2000). The higher PrmO2 relative to PvO2 in the trout can be interpreted in one of two ways. One possibility is that venous return from RM is a relatively small contribution to mixed venous blood. The other possibility is that the high PrmO2 of rainbow trout relative to mammals could be, at least in part, a consequence of a Root effect in blood perfusing the RM. The Root effect would be engendered by transient changes in erythrocyte pH caused by the faster rates of CO2 diffusion than O2 diffusion, and the strong coupling of O2 and CO2 movements that are known to exist in trout blood(Brauner and Randall, 1998; Brauner et al., 2000). That is,when arterial blood enters the RM of trout, rapid diffusion of metabolic CO2 into the erythrocyte would cause a transient drop in pH and cause a Root `off-shift', driving O2 off the haemoglobin and raising PO2. The deoxygenated haemoglobin would, however, then bind protons (the Haldane effect) and cause blood pH to rise again, eliciting a Root `on-shift' that binds O2 back onto the haemoglobin and lowers PO2 in the venous blood leaving the tissue.

While such a role of the Root effect is conjecture at this time, we can eliminate the possibility that we measured an artefact of mixed arterial,tissue and venous PO2 values rather than intramuscular PO2 If this had been the case, PrmO2 should have varied directly with PaO2 during hypoxia and exercise, which it did not. Furthermore, similar concerns would, presumably, exist for mammalian studies of intramuscular PO2 that involved the implantation of microelectrodes (Hutter et al., 1999; Jung et al.,1999; Suttner et al.,2002). Thus, in addition to the anatomical reasons for elevated PrmO2 that have been raised by Egginton (2002), the current study has not eliminated the possibility that O2 tensions are also influenced by the action of the Root effect within the muscle vasculature. Future investigations should perhaps be aimed at experimental manipulation of the Root effect to investigate how this change influences PrmO2 relative to PaO2 and mixed PvO2.

Conclusions

The results show that the PO2 prevailing in the RM of rainbow trout is higher than that reported for skeletal muscles of rats and humans. While there was a significant decrease in PrmO2 during sustained exercise, it did not decline below 40 mmHg and increased slightly at exhaustion. These observations are taken as a strong indication that O2 supply to the RM does not become limiting either at the moment of recruitment of WM or at exhaustion. We found no dramatic evidence that the Root effect raises O2 tensions in red muscle because PrmO2 remained almost exactly midway between previously published values of PaO2 and PvO2 for rainbow trout and was sensitive to reductions in PaO2 during mild hypoxia. Further work is needed to explain the higher PO2 in the RM relative to mixed venous blood because, while this may reflect a limited contribution from RM to mixed venous return, the phenomenon might also be a consequence of a transient Root effect in the RM vasculature.

Acknowledgements

This study was supported by NSERC grants to A.P.F. and D.J.R. D.J.M. was employed by a research project within the 5th Framework of the CEC.

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