Effect of aerial O₂ partial pressure on bimodal gas exchange and air-breathing behaviour in Trichogaster leeri

Ventilation in bimodal fish (i.e. those that respire simultaneously in air and water) is characterised by variable branchial ventilation patterns and intermittent and arrhythmic air breathing(Shelton et al., 1986). It has been suggested therefore that air breathing in these animals is an on-demand phenomenon that is stimulated by afferent feedback from one or more peripheral receptors (Shelton et al.,1986). Studies on the air-breathing lungfish Protopterusand gar Lepisosteus show that O₂ chemoreceptors are located diffusely throughout the gills, as is the case for most fish, and evidence points to the existence of both internally and externally oriented O₂ chemoreceptors that respond to changes in blood O₂partial pressure (P_O2) and aquatic P_O2, respectively(Johansen and Lenfant, 1968; Lahiri et al., 1970; Smatresk, 1986).

Since there is evidence to suggest that chemoreceptors are located at the site of aquatic respiration in air-breathing fish, it is reasonable to contemplate the existence of chemoreceptors at the site of aerial respiration,i.e. within the air-breathing organ (ABO). A common method used to identify sites of chemoreceptors in conscious air-breathing fish is to observe behavioural responses to independent changes in aquatic and aerial gas contents (e.g. Burggren, 1979; Graham et al., 1995; Hedrick and Jones, 1993; Hughes and Singh, 1970; Johansen and Lenfant, 1968; Sanchez et al., 2001). Studies involving manipulations of aquatic O₂ content by far exceed studies involving manipulations of aerial O₂ content. However, experimental manipulations of aerial O₂ content have the potential to offer insight into the presence and role of ABO-O₂ chemoreceptors. Air-breathing fish generally respond to aerial hypoxia by reducing apnoea(i.e. breath-hold) duration, whereas hyperoxia often lengthens it(Shelton et al., 1986). However, conclusions arising from experiments involving aerial O₂manipulations are limited in that the activity of chemoreceptors within the ABO cannot be discriminated from those located remotely, in the efferent vasculature (Graham, 1997). Simulated breathing by injection of either hypoxic or hyperoxic gas into the ABO produces the same results as changes in aerial gas composition (e.g. Johansen and Lenfant, 1968). However, gas injection also increases the volume of the ABO, and such experiments may therefore be confounded by activation of ABO mechanoreceptors that transmit information about the rate or extent of organ wall deformation(Milsom, 1990; Pack et al., 1990).

Evidence for ABO-O₂ chemoreceptors was found for the swamp eel, Monopterus albus, by Graham et al.(Graham et al., 1995). M. albus was observed to expel severely hypoxic or anoxic breaths within a few seconds of inspiration. The rapidity of the gas-voiding reflex suggested the presence of an ABO chemoreceptor, because it occurred about two to four times faster than would be expected if O₂ levels in the ABO had to be conveyed by blood flow to remote vascular receptors located somewhere in the systemic circulation. By contrast, the response of the blue gourami, Trichogaster trichopterus, to changes in aerial P_O2 was not immediate, suggesting that chemoreceptors were more centrally located(Burggren, 1979).

The present study investigates the effect of aerial O₂ partial pressure (P_O2,air) on bimodal gas exchange and air-breathing behaviour in an air-breathing fish, the pearl gourami, Trichogaster leeri Bleeker 1852 (sub-order Anabantoidei, family Belontiidae). T. leeri is a freshwater pelagic fish that has a pair of suprabranchial chambers serving as its ABO(Peters, 1978). Changes in air-breathing frequency (f_ab), aerial and aquatic O₂ consumption rate(V̇_O2) and mean O₂ uptake per breath (V_O2/breath)are analysed. Using a novel approach, apnoeic V̇_O2(V̇_O2,ap=rate of O₂ uptake from the air within the ABO while the fish is submerged)is derived by a breath-by-breath assessment of O₂ uptake and apnoea duration. V̇_O2,apis used to address two questions: (1) is the response of T. leeri to changes in P_O2,air regulated by ABO-O₂ chemoreceptors and (2) is a change in mean V_O2/breath with changing P_O2,air facilitated by a change in the air–blood P_O2 gradient as well as a change in apnoea duration?

To address the first question, V̇_O2,ap and estimated measurements of ABO volume (V_ABO) are used to calculate the P_O2 in the ABO at the end of apnoea. It is hypothesised that if the P_O2 in the ABO is regulated by ABO-O₂ chemoreceptors, then the P_O2 in the ABO at the end of apnoea should not be significantly different across treatments of varying P_O2,air. This would suggest the existence of an ABO-P_O2 threshold, which, once reached,triggers fish to renew the gas in their ABO.

The second question arises because O₂ uptake during apnoea may change simply as a result of changes in apnoea duration, i.e. V_O2/breath may increase during aerial hyperoxia because T. leeri holds its breath for longer. However, a change in the air–blood P_O2 gradient may also facilitate O₂ uptake during apnoea. Thus, it is hypothesised that if a change in apnoea duration is solely responsible for a change in mean V_O2/breath then P_O2,air will not have an effect on mean V̇_O2,ap. This is because mean V̇_O2,ap accounts for variations in apnoea duration between treatments, and thus if mean V̇_O2,ap changes with changing P_O2,air then a change in the air–blood P_O2 gradient must also be responsible. To our knowledge, this is the first study to examine how changes in the air–blood P_O2 gradient associated with aerial hypoxia and hyperoxia influence O₂ consumption in an air-breathing fish.

Experiments were performed on seven adult pearl gourami Trichogaster leeri Bleeker 1852 (wet mass 5.15±0.97 g; length 59.1±1.2 mm; means ± s.d.) that were obtained from a local aquarium supplier. Fish were maintained in aquaria filled with dechlorinated Adelaide tap water at 25°C. All fish were maintained under these conditions for at least one year prior to experimentation.

Prior to experimentation, food was withheld for 24 h and fish were individually placed in 1 litre bottles (Schott Duran, Germany) that were covered in black plastic to minimise stress. Bottles containing fish were held in a constant-temperature water bath maintained at 25±1°C by a heater (Thermomix 1419; B. Braun, Melsungen, Germany). Fish were allowed 12 h to acclimate to the chambers and to recover from handling prior to each experiment. An air stone gently aerated the water during the acclimation period.

Respiratory studies were conducted with an open-flow respirometer system. O₂ and N₂ were supplied from compressed cylinders (BOC Gases, Adelaide, Australia) and delivered to the respirometry chamber at a desired O₂ content (Fi_O2)and flow rate (V̇i; 100, 150 or 200 ml min^-1). The flow rate for each trial was selected to minimise the washout time while providing reliably detectable changes in the excurrent O₂ content(Fe_O2). O₂ content and flow rate were controlled by mass flow controllers (model GFC171; Aalborg Instruments and Controls, New York, NY, USA; 0–1 l min^-1,rated accuracy ±15 ml min^-1) using a PC running digital–analogue control software and hardware (PowerDAQ™ PD2-AO and ProfessorDAQ™; United Electronic Industries, Canton, MA, USA). Flow controllers were calibrated to each gas with a 3.5 litre calibrator (model 1057A Vol-U-Meter Calibrator; Brooks Instruments, Hatfield, PA, USA; accuracy of calibrated controllers was better than 5% of reading, usually 1–2% of reading). To ensure uniform mixing of O₂ and N₂, these gases were passed through a 1 litre, rigid mixing chamber before entering the respirometer. Nevertheless, the O₂ level was not as constant as expected in normal open-flow respirometry with atmospheric air, so the variability had to be accounted for in the analysis (see below).

Upon exiting the respirometry chamber, gases passed through a U-tube containing Drierite™ (Hammond Drierite Co. Ltd, Xenia, OH, USA) to remove water vapour and into a differential O₂ analyser (FC-2 Oxzilla I; Sable Systems International, Las Vegas, NV, USA). The analyser was calibrated using dried (Drierite™), CO₂-free(Ascarite™; Arthur H. Thomas Company, Swedesboro, NJ, USA) atmospheric air (0.2095 O₂). The analyser measured Fe_O2 approximately every 1.25 s and used a running five-sample average for each data point. The data output from the O₂ analyser was received simultaneously in an analogue and digital format. The analogue output was recorded at 2 s intervals with a digital multimeter (model TX3 True RMS and WaveStar™ Version 2.2;Tektronix, Beaverton, OR, USA) interfaced with a PC via the RS232 port. Depending on the analogue output scaling chosen, these voltage values were converted to Fe_O2 using relationships provided by Sable Systems International. The Oxzilla digital output was received by a DOS terminal program (SERIN; Sable Systems International) and recorded at intervals of approximately 1.1 s on a second PC. For analyses, WaveStar data were preferred due to SERIN's unstable timing interval, but SERIN data were used in two cases where high-frequency noise in the analogue data signal prevented reliable detection of breaths.

Aerial P_O2(P_O2,air) in the respirometry chamber was manipulated to nominal levels of 5, 10, 21 (control), 40 or 60 kPa (actual P_O2,air range: 5.4–5.5, 9.6–10.1,19.9–20.7, 38.4–39.0 and 57.5–57.9 kPa, respectively). Treatments were executed in random order and an arbitrary exposure time of 1 h was set so as to reduce the effect of declining aquatic P_O2. However, due to the instability of Fe_O2, some fish were exposed for longer than 1 h (maximum exposure time was 3 h), in order to accumulate 1 h worth of interpretable data. During trials, aquatic P_O2 dropped 2.3, 1.8, 1.5, 1.2 and 0.6 kPa h^-1 in the 5, 10, 21, 40 and 60 kPa treatments, respectively. To determine whether declining aquatic P_O2 had an effect on aerial respiration, f_ab, aerial V̇_O2 and V̇_O2/breath in the first and final 15 min of each trial were compared. In each case, there was no significant difference between the first and final 15 min (P=0.22,0.94 and 0.56, respectively). Following exposure to each treatment, fish were given at least 24 h rest before the next treatment. During this time, either the water in the chamber was aerated with an air stone or the fish were returned to their aquarium.

Each breath was integrated individually to arrive at a breath-by-breath estimate of O₂ uptake from the ABO during the apnoeic period(V_O2/breath; ml). This was summed across the trial and divided by trial duration to calculate aerial V̇_O2. Additionally, this approach measured f_ab (breaths h^-1) by tallying the number of breaths during a trial and dividing by trial duration.

To calculate oxygen uptake from the ABO during apnoea(V̇_O2,ap;ml h^-1), V_O2/breath (ml) was plotted against the apnoea duration (h) for each breath of each fish under each treatment (e.g. Fig. 2). A linear regression was fitted to the data, with the derived slope of the regression representing V̇_O2,ap (ml h^-1) (e.g. Fig. 2).

End-apnoea ABO-P_O2 assuming both a constant and totally compliant ABO volume was determined for each fish and compared between treatments.

To ascertain whether a change in apnoea duration is solely responsible for a change in mean V_O2/breath with changing P_O2,air, or whether a change in the air–blood P_O2 gradient is also a contributing factor, mean V̇_O2,ap was calculated for each treatment and plotted against P_O2,air.

Where appropriate, mass-specific values were used to account for the variation attributed to mass differences between fish(McNab, 1999). Although this procedure does not completely remove mass effects(Packard and Boardman, 1999)because V̇_O2scales allometrically in fish (White et al., 2006), the body size range was too small to determine the intraspecific allometric exponent for T. leeri accurately and arrive at mass-independent values.

A repeated-measures analysis of variance (ANOVA) was performed in JMP Version 5.1 (SAS Institute, Cory, NC, USA) to determine the effect of P_O2,air on all variables. To fulfil assumptions of normality and homogeneity of variance, P_O2,air was log transformed for the analysis of f_ab, which was not transformed; aerial V̇_O2, aquatic V̇_O2, V̇_O2/breath,end-apnoea ABO-P_O2 (assuming constant ABO volume) and end-apnoea ABO-P_O2 (assuming a totally compliant ABO) were log transformed together with P_O2,air; and V̇_O2,ap and total V̇_O2 were log transformed without P_O2,air being transformed. A Tukey's HSD test was used in post-hoc analyses where repeated-measures ANOVA revealed significant treatment effects. Statistical significance for all tests was determined with α=0.05.

There was a significant negative effect of P_O2,air on f_ab(F_1,23=120.4, P<0.0001), with f_ab decreasing from 46.3 breaths h^-1 at 5 kPa to 14.3 breaths h^-1 at 60 kPa(Fig. 3A). There was a significant positive effect of P_O2,air on aerial V̇_O2(F_1,23=37.2, P<0.0001), with aerial V̇_O2 increasing from 20.1 ml kg^-1 h^-1 at 5 kPa to 60.8 ml kg^-1 h^-1 at 60 kPa(Fig. 3B). Complementary to this, there was a significant positive effect of P_O2,air on V_O2/breath (F_1,23=221.5, P<0.0001), with V_O2/breath increasing from 0.5 ml kg^-1 at 5 kPa to 5.5 ml kg^-1 at 60 kPa (Fig. 3C).

There was a significant negative effect of P_O2,air on aquatic V̇_O2(F_1,27=17.8, P=0.0003), with a significant increase in aquatic V̇_O2 under the 5 kPa treatment (Fig. 3B). There was no significant effect of P_O2,air on total V̇_O2(F_1,23=2.49, P=0.13)(Fig. 3B).

Regardless of whether it was assumed that the ABO volume was constant or that the ABO was totally compliant, there was a significant positive effect of P_O2,air on end-apnoea ABO-P_O2 (F_1,21=544, P<0.0001, and F_1,21=1091, P<0.0001, respectively) (Fig. 4). When ABO volume was assumed to be constant, end-apnoea ABO-P_O2 increased from 4.3 kPa at P_O2,air=5 kPa to 40.6 kPa at P_O2,air=60 kPa, and when ABO volume was assumed to be totally compliant, end-apnoea ABO-P_O2increased from 4.4 to 47.3 kPa with the same change in P_O2,air.

A repeated-measures ANOVA revealed a significant positive correlation between mean V̇_O2,ap and P_O2,air (F_1,21=34.7, P<0.0001), with mean V̇_O2,ap increasing from 13.2 ml kg^-1 h^-1 at 5 kPa to 59.8 ml kg^-1 h^-1 at 60 kPa. The relationship between mean V̇_O2,ap and P_O2,air was described by the logarithmic curve: V̇_O2,ap=45.8log(P_O2,air)–19.2(r² =0.63) (Fig. 5).

Under normoxic conditions at 25°C, the mass-specific total (air +water) V̇_O2 of T. leeri was 108.2 ml kg^-1 h^-1. This value is lower than that found for the closely related T. trichopterus (156.2 ml kg^-1 h^-1)(Burggren and Haswell, 1979)and T. pectoralis (126.9 ml kg^-1 h^-1)(Natarajan and Rajulu, 1982),all normalised to a common body temperature of 25°C with a Q₁₀of 1.65 (White et al., 2006). When viewed in comparison to an allometric relationship between V̇_O2 and body mass, the data obtained here for T. leeri are well within the 95%prediction intervals (31.8–290 ml kg^-1 h^-1) for new data (White et al., 2006),indicating that the data obtained in this study are reliable.

The breathing frequency (f_ab) of T. leeriunder normoxic conditions was 21.8±5.9 breaths h^-1 (mean± s.d.), which is greater than that observed for T. trichopterus (12.8± 4.1 breaths h^-1)(Burggren, 1979). However, like T. trichopterus, f_ab of T. leeri increased as the gas phase became increasingly hypoxic (Fig. 3A). At a P_O2,air of 5 kPa, f_ab was twofold higher than control (normoxic) levels,whereas T. trichopterus increased f_ab almost threefold. However, f_ab calculated for T. leeriat 5 kPa may be an underestimate, because only three fish yielded results where some individual breaths could be isolated. For the remaining fish, no or few breathing events were apparent. This suggests that the air–blood P_O2 gradient was insufficient to promote O₂ uptake. The air–blood P_O2gradient may have in fact been reversed, resulting in fish losing O₂ gained from the water to the air. This futile behaviour would be expected, however, because severe aerial hypoxia is not encountered in the natural environment, and fish are unlikely to have evolved an appropriate response to such conditions.

An increase in f_ab in response to aerial hypoxia has been observed in species that have a reduced gill surface area and are therefore heavily reliant on aerial respiration. For example, M. albus showed a significant reduction in apnoea duration when inspiring gas mixtures containing 16 kPa O₂ or less, and breaths containing 1.5 kPa O₂ were exhaled almost immediately(Graham et al., 1995). Monopterus cuchia increased tidal volume as well as f_ab during hypoxic breathing(Lomholt and Johansen, 1974),but no such change in ABO tidal volume occurred in T. trichopterus(Burggren, 1979). Protopterus aethiopicus increased f_ab when N₂ gas was injected into the ABO, and, interestingly, air breathing was stimulated by N₂ injection even when arterial P_O2 was higher than that towards the end of a normal apnoea period (Johansen and Lenfant, 1968). Amia calva, a fish less reliant on aerial respiration, increased f_ab twofold when air containing only 8 kPa O₂ was inspired(Hedrick and Jones, 1993). In contrast to Amia, other fish with efficient aquatic gas exchange do not show a change in f_ab when N₂ gas is injected into the lung (Neoceratodus forsteri)(Johansen et al., 1967) or air bladder (Lepisosteus oculatus)(Smatresk and Cameron, 1982b). The first and second gill arches of T. leeri are large and fully developed (Munshi, 1968), and T. leeri is sensitive to aquatic hypoxia(Miller, 2003), unlike Protopterus (Johansen and Lenfant, 1968) and Monopterus(Lomholt and Johansen, 1974). It therefore seems likely that, although T. leeri is not as reliant on aerial respiration as these species, aquatic respiration may not be sufficient to meet its metabolic demands, making it an obligate air breather(Graham, 1997). T. trichopterus is considered an obligate air breather at temperatures above 20–25°C, as it shows signs of distress if denied access to air(Burggren, 1979).

Despite an increase in f_ab during aerial hypoxic exposure, T. leeri was unable to sustain aerial V̇_O2 equal to that under normoxic conditions (Fig. 3B). Complementary to the observed decrease in aerial V̇_O2 with decreasing P_O2,air, V_O2/breath also showed a decline(Fig. 3C).

The contribution of aerial V̇_O2 to total V̇_O2 decreased from 33% in normoxia to 25% when P_O2,air was 10 kPa, and to 16% at 5 kPa. Similarly, under normoxic conditions at 27°C,the ABO of T. trichopterus accounted for 42% of the total V̇_O2 and less than 15% when P_O2,air was reduced to 7.2 kPa(Burggren, 1979). Both species showed an increase in aquatic V̇_O2 to compensate for their reduced ability to extract O₂ from the air(Fig. 3B). It is reasonable to assume that this increase in aquatic V̇_O2 arises almost entirely from gas exchange via the gills, because cutaneous gas exchange in air-exposed T. trichopterus accounts for only ∼10% of the total gas exchange (Burggren and Haswell, 1979). Aquatic V̇_O2 may be increased via an increase in branchial ventilation frequency,branchial tidal volume or both. Branchial ventilation is known to increase initially in most air-breathing fish as aquatic P_O2 falls(Hughes and Singh, 1971; Johansen et al., 1970; Pettit and Beitinger, 1985; Smatresk and Cameron, 1982a),but the effect of aerial hypoxia on branchial ventilation appears not to have been investigated.

At a P_O2,air of 60 kPa, f_ab significantly decreased in T. leeri(Fig. 3A). Similarly, T. trichopterus decreased f_ab when P_O2,air was increased to 80 kPa(Burggren, 1979). A decrease in f_ab has been observed in almost all species exposed to aerial hyperoxia: P. ethiopicus(Lahiri et al., 1970), L. oculatus (Smatresk and Cameron,1982b), Electrophorus electricus(Johansen et al., 1968b) and M. albus (Graham et al.,1995).

Despite a decrease in f_ab in hyperoxic air, V̇_O2,ap increased by almost twofold when P_O2,air was 40 kPa;however, no significant increase occurred when P_O2,air was increased to 60 kPa(Fig. 3B).

Although aerial V̇_O2 increased significantly in aerial hyperoxia compared with normoxia, there was no corresponding decrease in aquatic V̇_O2, and total V̇_O2 was unchanged(Fig. 3B). However, because aerial and aquatic respiration were measured separately, inherent variability may have obscured the expected correlations.

The responsiveness of T. leeri to changes in aerial O₂content lends insight to the question of the existence of ABO-O₂chemoreceptors. Graham et al. give support for the existence of ABO-O₂ chemoreceptors in some air-breathing species(Graham et al., 1995). They found that the rapidity of the gas-voiding reflex of M. albus to changed aerial O₂ content was indicative of ABO-O₂chemoreceptors, not chemoreceptors in the systemic circulation. They suggested that an ABO-O₂ chemoreceptor would be advantageous in the regulation of cardiac responses to an air-breathing event. A common pattern of cardiac arrhythmia found in fishes during an air-breathing event is inspiration-induced tachycardia followed by the gradual onset of bradycardia as the ABO-O₂ content falls, leading to pronounced bradycardia with exhalation (Farrell, 1978; Johansen et al., 1968a; Singh and Hughes, 1973; Smatresk, 1988; Smatresk, 1990). Therefore, an ABO-O₂ chemoreceptor may be important in the modulation of mechanoreceptor and other stimuli affecting air-breathing tachycardia, in attenuating tachycardia as ABO-O₂ declines and in terminating the breath when ABO-P_O2 becomes too low to promote O₂ uptake (Graham and Baird,1984). This would result in the effective matching of ventilation and perfusion (Johansen, 1966; Johansen, 1970).

However, the findings of Graham et al.(Graham et al., 1995) contrast with Burggren's indication of more centrally located chemoreceptors (i.e. in the brain) in T. trichopterus, which was based on the lag in ventilation response time (several seconds) to stepwise changes in aerial O₂ content (Burggren,1979). Burggren argued that because aerial hypoxia is rarely, if ever, encountered in the natural environment, selection pressures should not be strong for the evolution of a peripheral chemoreceptor control system able to differentiate between reduced systemic blood O₂ resulting from gill ventilation with hypoxic water and that resulting from ABO ventilation with hypoxic gas.

More substantial evidence for ABO-O₂ chemoreceptors in T. leeri would have been an end-apnoea ABO-P_O2 that was independent of P_O2,air. This would have suggested the existence of an ABO-P_O2 threshold that, once reached, triggered T. leeri to renew the gas in its ABO. However,this was not the case in this study; end-apnoea P_O2 increased with increasing P_O2,air(Fig. 4). This lack of correlation between ABO-P_O2 and renewal of ABO gas has also been recognised in lungfish(Johansen and Lenfant, 1968)and in Pacific tarpon that uses the swimbladder as an ABO(Seymour et al., 2007). It has also been shown that apnoea termination occurs at high ABO-P_O2 levels when the rate of decline in ABO-P_O2 is rapid(Shelton et al., 1986). This corresponds with the findings in this study; mean apnoeic V̇_O2(V̇_O2,ap)increased under hyperoxic conditions and end-apnoea ABO-P_O2 was higher than that under normoxic conditions. These findings indicate that if ABO-O₂ chemoreceptors are present, then their regulation of bimodal control in air-breathing fish is partial, and that respiration is mainly affected by chemoreceptors elsewhere in the central and peripheral nervous system and possibly by mechanoreceptors in the ABO.

Mean V̇_O2,apincreased with increasing P_O2,air(Fig. 5), supporting the hypothesis that the observed increase in V_O2/breath with increasing P_O2,air(Fig. 3C) was facilitated not only by an increase in apnoea duration but also by an increase in the air–blood P_O2 gradient. The logarithmic function between V̇_O2,ap and P_O2,air suggests that when there is no O₂ uptake occurring in the ABO (i.e. V̇_O2,ap is equal to zero), P_O2,air is equal to 2.62 kPa. Therefore, the air and blood are in equilibrium and the efferent blood from the gills can be assumed to have a P_O2approximating this value. The relationship also indicates that in hyperoxia V̇_O2,ap reaches a maximum where it becomes independent of P_O2,air(Fig. 5). Because the haemoglobin would be expected to be completely saturated in the hyperoxic ABO(Herbert and Wells, 2001),aerial V̇_O2plateaus because the blood reaches a point where it can take up no more than can be dissolved in the plasma.

List of symbols and abbreviations

 
• ABO

  air-breathing organ

 
• βO2

  oxygen capacitance

 
• f_ab

  air-breathing frequency

 
• Fe_O2

  excurrent oxygen content

 
• Fi_O2

  incurrent oxygen content

 
• P_O2

  oxygen partial pressure

 
• P_O2,air

  aerial oxygen partial pressure

 
• t_ap

  apnoea duration

 
• V_ABO

  volume of the air-breathing organ

 
• V_ABO(t₀)

  ABO volume at the beginning of the apnoea period

 
• V̇i

  oxygen flow rate

 
• V̇_O2

  oxygen consumption rate

 
• V̇_O2,ap

  apnoeic V̇_O2

 
• V_O2,end

  volume of oxygen in the ABO at the end of apnoea

 
• V_O2/breath

  oxygen uptake per breath

This work was supported by the Australian Research Council. It was carried out under permit from the Animal Ethics Committee of the University of Adelaide. We would like to thank Philip Matthews for his technical assistance.