SUMMARY
The emperor penguin (Aptenodytes forsteri) thrives in the Antarctic underwater environment, diving to depths greater than 500 m and for durations longer than 23 min. To examine mechanisms underlying the exceptional diving ability of this species and further describe blood oxygen(O2) transport and depletion while diving, we characterized the O2–hemoglobin (Hb) dissociation curve of the emperor penguin in whole blood. This allowed us to (1) investigate the biochemical adaptation of Hb in this species, and (2) address blood O2 depletion during diving, by applying the dissociation curve to previously collected partial pressure of O2 (PO2) profiles to estimate in vivo Hb saturation (SO2)changes during dives. This investigation revealed enhanced Hb–O2 affinity (P50=28 mmHg, pH 7.5) in the emperor penguin, similar to high-altitude birds and other penguin species. This allows for increased O2 at low blood PO2 levels during diving and more complete depletion of the respiratory O2 store. SO2 profiles during diving demonstrated that arterial SO2 levels are maintained near 100%throughout much of the dive, not decreasing significantly until the final ascent phase. End-of-dive venous SO2 values were widely distributed and optimization of the venous blood O2store resulted from arterialization and near complete depletion of venous blood O2 during longer dives. The estimated contribution of the blood O2 store to diving metabolic rate was low and highly variable. This pattern is due, in part, to the influx of O2 from the lungs into the blood during diving, and variable rates of tissue O2 uptake.
INTRODUCTION
Extreme environments, particularly those with limited oxygen(O2) availability, are especially challenging to most life forms,yet still host diverse and abundant life. Species that flourish in such environments are ideal models in which to examine the physiological, cellular and biochemical mechanisms underlying tolerance to hypoxia and O2depletion. The emperor penguin (Aptenodytes forsteri), the consummate avian diver, thrives in the extreme Antarctic underwater environment, diving to depths greater than 500 m (Kooyman and Kooyman, 1995; Wienecke et al., 2007) and for durations longer than 23 min(Ponganis et al., 2007). Recent measurements of air sac and blood partial pressure of O2(PO2) in diving emperor penguins have revealed exceptional tolerance to low O2 in this species(Ponganis et al., 2007; Stockard et al., 2005). It has been hypothesized that this underlying tolerance, well below the limits of many birds and mammals, may necessitate biochemical and molecular adaptations including a shift in the emperor penguin O2–hemoglobin (Hb)dissociation curve, relative to other birds.
The O2–Hb dissociation curve of whole blood of emperor penguins has not been determined. In general, the hemoglobin of birds has lower O2 affinity than that of mammals. This may reflect a shift toward favoring O2 unloading to the tissues, as the avian respiratory system is inherently more efficient at O2 uptake(Piiper and Scheid, 1975; Powell, 2000; Powell et al., 1989). Consequently, the P50 (PO2at which Hb is 50% saturated) values of most birds (44–52 mmHg) are much higher than those of mammals (25–31 mmHg)(Christensen and Dill, 1935; Hirsowitz et al., 1977; Lenfant et al., 1970; Lutz, 1980; Wastl and Leiner, 1931). However, Adélie (Pygoscelis adeliae), chinstrap (P. antarctica), and gentoo penguins (P. papua), and the bar-headed goose (Anser indicus), a bird adapted to life at high altitudes, have P50 values in the mammalian range(Black and Tenney, 1980; Lenfant et al., 1969b; Milsom et al., 1973; Petschow et al., 1977),favoring O2 uptake from the lungs when PO2 is low. The P50 for isolated emperor penguin Hb (36 mmHg) has been measured(Tamburrini et al., 1994). Determination of the P50 and dissociation curve in whole blood for this species remains necessary, however, as the P50 of isolated Hb of this species was highly sensitive to the presence of various cofactors(Tamburrini et al., 1994).
We characterized the O2–Hb dissociation curve of the emperor penguin in whole blood in order to (1) investigate the biochemical adaptation of Hb in emperor penguins, and (2) address blood O2depletion during diving, by applying the dissociation curve to previously collected PO2 profiles(Ponganis et al., 2009; Ponganis et al., 2007) to estimate in vivo Hb saturation (SO2)changes during dives. It was hypothesized that the P50 of the emperor penguin would be similar to that of the bar-headed goose and other penguin species, an adaptation which could contribute to tolerance to low O2 in the emperor penguin (Black and Tenney, 1980; Lenfant et al., 1969b; Milsom et al.,1973; Ponganis et al.,2007). Hemoglobin with O2 affinity in this range would also be consistent with blood PO2 and O2 contents of emperor penguins at rest(Ponganis et al., 2007). The magnitude of the Bohr effect was expected to be similar to those in other birds and penguins (–0.4 to –0.6)(Lenfant et al., 1969b). A similar range of Bohr effects, which should favor O2 unloading to the tissues, has also been observed in a variety of marine mammals(Lenfant et al., 1970; Qvist et al., 1981; Willford et al., 1990). An accurate determination of the Bohr effect in emperor penguin whole blood also allows refinement of the estimation of SO2 from PO2 profiles since PCO2 and pH data are available at various points in dives of this species(Ponganis et al., 2009; Ponganis et al., 2007).
The extent and rate of respiratory O2 store depletion have been previously determined for the emperor penguin(Stockard et al., 2005). The addition of blood O2 store depletion data provides the next component of the investigation of total O2 consumption in this exceptional diver. It was hypothesized that SO2profiles would reflect the wide range of PO2values at the end of dives (Ponganis et al., 2009; Ponganis et al.,2007) and that the rate of decrease of blood O2 would vary inversely with dive duration since heart rate in this species progressively decreases during longer dives(Meir et al., 2008). An inverse relationship to dive duration has been demonstrated in the respiratory O2 store depletion rate of the diving emperor penguin(Stockard et al., 2005) and in blood O2 store depletion rates of seals(Elsner et al., 1964; Stockard et al., 2007).
MATERIALS AND METHODS
As in past studies (Kooyman et al.,1992; Ponganis et al.,2001), non-breeding emperor penguins (Aptenodytes forsteri Gray; ∼15 birds/season, 20–30 kg) were captured near the McMurdo Sound ice edge or at Terra Nova Bay in October 2003–2005,2007 and 2008 and were maintained for 6 weeks at an isolated dive hole enclosed within a corral at the Penguin Ranch on the McMurdo Sound sea ice(77°41′, 165°59′). All procedures were approved under a UCSD Animal Subjects Committee protocol and US Antarctic Treaty Permit. All birds were returned to the ice edge and released upon completion of the study.
PO2 electrode deployments
PO2 electrodes (Licox C1.1 Revoxode; Integra LifeSciences, Plainsboro, NJ, USA) and thermistors (model 554, Yellow Springs Instruments, Yellow Springs, OH, USA) were inserted percutaneously into the aorta or vena cava of emperor penguins under general isoflurane anesthesia as described previously (Ponganis et al.,2007; Ponganis et al.,2001; Ponganis et al.,2004; Ponganis et al.,2003). An additional arterial data set was obtained in 2008 from a bird equipped with only an arterial PO2electrode as described in the previous study(Ponganis et al., 2009). The electrodes were connected to a custom-built PO2/temperature recorder (UFI, Morro Bay, CA,USA) and an Mk9 time–depth recorder (TDR) was also attached (Wildlife Computers, Redmond, WA, USA) as previously described(Stockard et al., 2005). After overnight recovery from anesthesia, birds were allowed to dive at the isolated dive hole. The instrumented birds dived for 1–2 days, after which catheters, probes and recorders were removed under general anesthesia. All PO2 values were corrected to 38°C for construction of the PO2 profiles, as previously described (Ponganis et al.,2007).
O2-Hb dissociation curve characterization
O2–Hb dissociation curves on fresh whole blood were determined with the mixing technique of tonometered blood(Scheid and Meyer, 1978). Blood samples were obtained from the Penguin Ranch emperor penguins during anesthesia, placed on ice, and processed immediately. All dissociation curve analyses were completed within 6 h of blood collection in order to prevent depletion of labile organic phosphates such as inositol pentaphosphate (IPP)which would influence the dissociation curve, and to avoid prolonged metabolism of these nucleated avian red blood cells. The mixing technique consisted of the volumetric mixing of 0% O2-saturated blood and 100% O2-saturated blood to achieve the desired SO2 at various points (i.e. 90, 70, 50, 40, 20,10, 5% SO2) along the curve with subsequent measurement of the PO2 of the resulting mixture using an i-STAT blood gas analyzer (37°C; Abbott Point of Care, Princeton,NJ, USA) (Black and Tenney,1980; Johansen et al.,1987; Nörgaard-Pedersen et al., 1972; Qvist et al.,1981). Use of the i-STAT analyzer also allowed verification of pH and PCO2, and Tucker chamber analyses(Tucker, 1967) provided verification of blood O2 content. The CO2 Bohr effect was determined by changing the CO2 concentration of the gas in the tonometer in order to adjust pH. Dissociation curves were determined at pH values of 7.5, 7.4, 7.3 and 7.2. The log[SO2/(100–SO2)]vs log(PO2) was plotted and linear regression analysis performed in order to generate the equation for the O2–Hb dissociation curve at each pH(Nicol, 1991). As in similar studies (Nicol, 1991), all SO2 points from all birds were combined in order to generate a general O2–Hb dissociation curve for this species. This is justified as, with the exception of disease, the structure of hemoglobin varies among species, not individuals of a species. The Bohr coefficient was derived from linear regression of the log P50 on pH (each point averaged from all data of all birds for pH 7.5, 7.4, 7.3 and 7.2) (Nicol,1991; Willford et al.,1990). In addition, the fixed acid Bohr effect was determined by titrating with HCl to pH 7.4, 7.3 and 7.2 while PCO2 was maintained at the level required for the standard pH 7.5 curve in that penguin(Willford et al., 1990). The effect of lactic acid on the dissociation curve was also evaluated in whole blood by determining additional dissociation curve points after adding lactic acid (L6661, Sigma, St Louis, MO, USA) to the blood to a final concentration of 5 and 10 mmol l–1(Nicol, 1991).
In order to validate the specific equipment and methods used in this study,dissociation curves were also determined with blood from an avian (chicken, Gallus gallus domesticus) and pinniped (Leptonychotes weddelli,Phoca vitulina, Zalophus californianus) species with previously published O2–Hb binding data(Bartels et al., 1966; Hirsowitz et al., 1977; Lenfant, 1969; Lenfant et al., 1969a). Mixing technique tests were also conducted to verify that no hemolysis occurred during mixing.
% Hb saturation (SO2)calculations
Percent hemoglobin O2 saturation(SO2) values were obtained by applying data from PO2 profiles [from previous studies(Ponganis et al., 2009; Ponganis et al., 2007) and current studies] to the linear regression equation generated by the log[SO2/(100–SO2)]vs log(PO2) plot and solving for SO2 (at the appropriate pH, see below).
Blood O2 store depletion calculations
Blood samples taken for the O2–Hb dissociation curve were also used for hemoglobin analyses [cyanomethemoglobin technique(Ponganis et al., 1993)] in order to obtain Hb concentration. Oxygen content for initial and final(end-of-dive) time points for each dive were calculated from the corresponding SO2 values, using a Hb concentration of 18.3 g dl–1 ([this study, agrees well with previous findings(Kooyman and Ponganis, 1998; Ponganis et al., 1997a)] with the formula: O2 content (ml O2 dl–1blood)= (1.34 ml O2 g–1 Hb)×[Hb](g dl–1)×SO2+(0.003×PO2). Initial SO2 (SO2at the start of the dive) was estimated with the pH 7.5 dissociation curve,and the final SO2 value (from the final PO2 value, measured within the last 5 or 15 s of the dive depending on the specific recorder) was estimated with the pH 7.4 dissociation curve. The effect of pH is critical in estimation of final SO2. For example, from data of Weddell seals(Qvist et al., 1981), a PO2 of 10 mmHg corresponds to 8%SO2 at pH 7.4, but only 2% at pH 7.0. Blood lactate concentration does not increase significantly during dives of emperor penguins, even during dives longer in duration than the previously measured aerobic dive limit [ADL; duration beyond which blood lactate concentration increases above resting levels (Kooyman et al., 1983); 5.6 min for the emperor penguin(Ponganis et al., 1997b)] for this species (Ponganis et al.,2009). The rise in blood lactate does not occur until the post-dive period in this species (Ponganis et al., 1997b; Ponganis et al., 2009). Based on lactate, PCO2 and pH measurements obtained from emperor penguin blood samples drawn at various time points during diving (Ponganis et al.,2009), a pH of 7.4 was deemed most representative of conditions at the end of the dive.
The percentage of net O2 content depletion in either the arterial or venous system (dependent on the site of insertion) for each dive of each penguin was calculated [% O2 content depletion=(initial O2 content–final O2 content)/initial O2content×100]. The rate of O2 content depletion [(initial O2 content–final O2 content)/dive duration] was also calculated for the arterial and venous systems. This provides an overall depletion rate, not an instantaneous measure of O2 consumption. Data from penguins with arterial PO2 electrodes provided an estimate of the net depletion of O2 in the arterial system; those with venous probes provided estimates of net venous O2 depletion.
An example of oxygen–hemoglobin (O2–Hb) dissociation curves from (A) one penguin at pH 7.5, 7.4 and 7.3, and (B) the emperor penguin, the bar-headed goose (Anser indicus)(Black and Tenney, 1980) and the domestic duck (Anas platyrhynchos, forma domestica)(Hudson and Jones, 1986) at pH 7.4. Note that as for the bar-headed goose, the O2–Hb dissociation curve of the emperor penguin is significantly left-shifted as compared with the domestic duck (and most birds). The bar-headed goose photo is courtesy of Graham Scott; the domestic duck photo is by Maren Winter(licensed under the terms of the GNU Free Documentation License, Version 1.2 or any later version); the penguin photo is by J.M.
An example of oxygen–hemoglobin (O2–Hb) dissociation curves from (A) one penguin at pH 7.5, 7.4 and 7.3, and (B) the emperor penguin, the bar-headed goose (Anser indicus)(Black and Tenney, 1980) and the domestic duck (Anas platyrhynchos, forma domestica)(Hudson and Jones, 1986) at pH 7.4. Note that as for the bar-headed goose, the O2–Hb dissociation curve of the emperor penguin is significantly left-shifted as compared with the domestic duck (and most birds). The bar-headed goose photo is courtesy of Graham Scott; the domestic duck photo is by Maren Winter(licensed under the terms of the GNU Free Documentation License, Version 1.2 or any later version); the penguin photo is by J.M.
Data analysis and statistics
As outlined above, the dissociation curve, calculated using linear regression, was applied to PO2 values to yield SO2 values, and calculations of O2depletion rate and percentage of O2 depletion were made. Differences between arterial and venous results were determined with one-way ANOVA. Correlation between dive duration and the variables of final SO2, pre-dive SO2, percentage O2 content depleted and depletion rate was addressed with Spearman rank order correlation tests. Statistical significance was assumed at P<0.05 and the significance level is quoted in the text. Values are expressed as means± s.d. PO2 values are expressed in mmHg(as measured). Figures include the corresponding values in kPa, assuming 1 mmHg=0.133 kPa.
RESULTS
Dive behavior of emperor penguins at the Penguin Ranch for the dives associated with the PO2 profiles in this study has been described previously (Ponganis et al., 2009; Ponganis et al.,2007). Dive duration and depth data are given in Table 1.
Dive, SO2, % O2 content depletion and depletion rate of the arterial and venous blood for the emperor penguins in this study
PO2 electrode location . | Duration (min) . | Maximum depth (m) . | Pre-dive SO2 (%) . | Initial SO2 (%) . | Maximum SO2 (%) . | Final SO2 (%) . | Δ SO2 (initial SO2 – final SO2) . | % O2 content depletion . | Depletion rate (ml O2 dl–1 min–1) . |
---|---|---|---|---|---|---|---|---|---|
Venous (N=9 penguins, 130 dives) | 6.1±4.6 | 38.6±24.7 | 74.1±13.7 | 75.6±13.0 | 80.9±12.7 | 46.6±30.2 | 29.0±30.3 | 37.8±40.0 | 0.9±1.4 |
(0.8–23.1) | (4–155) | (29.7–95.8) | (38.4–95.3) | (49.5–98.8) | (0–97.0) | (–27.8–+92.3) | (–58.0–+100) | (–6.2–+4.7) | |
Arterial (N=6 penguins, 71 dives) | 5.1±2.1 | 29.1±17.3 | 96.2±2.3 | 96.5±2.3 | 99.1±0.8 | 75.6±13.4 | 20.9±14.1 | 21.6±14.5 | 0.9±0.6 |
(1.1–11.9) | (11–91.5) | (85.7–99.3) | (85.7–99.2) | (93.5–100) | (47.4–98.9) | (–5.0–+50.7) | (–5.5–+51.7) | (–0.4–+2.0) |
PO2 electrode location . | Duration (min) . | Maximum depth (m) . | Pre-dive SO2 (%) . | Initial SO2 (%) . | Maximum SO2 (%) . | Final SO2 (%) . | Δ SO2 (initial SO2 – final SO2) . | % O2 content depletion . | Depletion rate (ml O2 dl–1 min–1) . |
---|---|---|---|---|---|---|---|---|---|
Venous (N=9 penguins, 130 dives) | 6.1±4.6 | 38.6±24.7 | 74.1±13.7 | 75.6±13.0 | 80.9±12.7 | 46.6±30.2 | 29.0±30.3 | 37.8±40.0 | 0.9±1.4 |
(0.8–23.1) | (4–155) | (29.7–95.8) | (38.4–95.3) | (49.5–98.8) | (0–97.0) | (–27.8–+92.3) | (–58.0–+100) | (–6.2–+4.7) | |
Arterial (N=6 penguins, 71 dives) | 5.1±2.1 | 29.1±17.3 | 96.2±2.3 | 96.5±2.3 | 99.1±0.8 | 75.6±13.4 | 20.9±14.1 | 21.6±14.5 | 0.9±0.6 |
(1.1–11.9) | (11–91.5) | (85.7–99.3) | (85.7–99.2) | (93.5–100) | (47.4–98.9) | (–5.0–+50.7) | (–5.5–+51.7) | (–0.4–+2.0) |
Values are means ± s.d. and range. Pre-dive, initial and maximum SO2 were determined at pH 7.5, final SO2 at pH 7.4
O2–Hb dissociation curve
Complete O2–Hb dissociation curves were determined with blood from 12 penguins, with additional P50 values at specific pH levels and fixed acid Bohr effects obtained in an additional two penguins. The P50 was 28±1 mmHg (N=12 penguins) at pH 7.5 (Fig. 1A). The CO2 Bohr effect (slope of log P50vs pH) was –0.45 (y=–0.45x+4.81, r2=0.98, P=0.008). The fixed acid Bohr effect(addition of HCl or lactic acid) was not significantly different from that of CO2.
The resulting regression equations from the plots of log[SO2/(100–SO2)]vs log(PO2) (all saturation points,all penguins combined) were:
pH 7.5:log[SO2/(100–SO2)]= 2.92589 × log(PO2) – 4.24338(N=43, r2=0.98, P<0.0001),
pH 7.4:log[SO2/(100–SO2)]= 2.94767 × log(PO2) – 4.39858(N=70, r2=0.98, P<0.0001),
pH 7.3:log[SO2/(100–SO2)]= 3.04945 × log(PO2) – 4.72019(N=38, r2=0.99, P<0.0001),
pH 7.2:log[SO2/(100–SO2)]= 3.15958 × log(PO2) – 4.97618(N=9, r2=0.99, P<0.0001).
Samples from other species (chicken and pinnipeds) run for dissociation curve method validation agreed with previously published values(Bartels et al., 1966; Hirsowitz et al., 1977; Lenfant, 1969; Lenfant et al., 1969a). Mixing technique verification tests confirmed that no hemolysis occurred while mixing blood samples in the syringes, based on clear plasma color in hematocrit tubes after centrifugation. The mean hemoglobin concentration in this study(18.3±1.1 g dl–1, N=17) was equivalent to that of previous studies (Kooyman and Ponganis, 1998; Ponganis et al., 1997a). Oxygen content analyses performed with 100%O2-saturated blood (Tucker chamber) agreed within 5% of the maximal O2 content calculated using the equation in this study[O2 content (ml O2 dl–1 blood)=(1.34 ml O2 g–1 Hb)×[Hb](g dl–1)×SO2+(0.003×PO2)].
PO2 profiles
In addition to the arterial PO2 data collected in previous studies (Ponganis et al., 2009; Ponganis et al.,2007), an arterial PO2 and dive profile was obtained for one more emperor penguin in the 2008 Antarctic field season (Fig. 2A). This increased the arterial data sample size to six penguins (71 dives in total). The venous data were from nine penguins (130 total dives). These profiles have been adequately described in previous studies(Ponganis et al., 2009; Ponganis et al., 2007).
SO2 and blood O2depletion
In vivo arterial Hb saturation (Sa,O2) often remained near 100% for much of the dive, reflecting the large increase in arterial PO2 at the beginning of the dive and the fact that despite a subsequent decrease, PO2 remained relatively high until the later dive phase (Fig. 2A)(Ponganis et al., 2007). A rapid decline in Sa,O2 generally began during the final ascent phase of the dive (Fig. 2A). Sv,O2 (venous Hb saturation)profiles reflected venous PO2 profiles(Ponganis et al., 2007) in that they were quite variable among dives(Fig. 2B, Fig. 3), with marked fluctuations, transient increases during the dive, and a large range of final Sv,O2 values(Fig. 4). Re-oxygenation occurred rapidly upon return to the surface, as demonstrated by the return in both Sa,O2 and Sv,O2 to values consistent with those at rest within approximately 2 min post-dive(Fig. 2). Pre-dive and initial Sv,O2 values(Table 1, Fig. 2B, Fig. 3) were often higher than those corresponding to the mean venous PO2 of emperor penguins at rest (Ponganis et al.,2007).
Final Sv,O2 was ≤20% in 28% of dives,≤5% in 15% of dives and ≤2% in 6% of dives. With the exception of only one dive, Sv,O2 decreased below 20% only in dives greater than the previously measured ADL(Ponganis et al., 1997b)(Table 1, Fig. 4).
Max SO2 (the peak SO2 during the dive), pre-dive SO2, initial SO2, final SO2,Δ SO2 (initial SO2 minus final SO2), percentage O2 content depletion, and blood O2 store depletion rate data are given in Table 1. Max SO2, initial SO2, final SO2,Δ SO2 and percentage O2 content depletion were all significantly different between the arterial and venous compartments [one-way ANOVA: P≤0.001 for all exceptΔ SO2 (P=0.035); F=144.40, 180.42, 59.14, 4.53, 10.93, respectively]. The blood O2 store depletion rates between the two compartments, however,were not significantly different (one-way ANOVA: P=0.94; F=0.00627).
Both final Sa,O2 and Sv,O2 demonstrated a strong and significant negative correlation to dive duration (Spearman rank order correlation, Table 2, Fig. 4). The percentage of O2 content depleted during the dive showed a strong and significant positive correlation to dive duration for both compartments (Spearman rank order correlation, Table 2). The pre-dive Sv,O2 had a significant, but weak positive correlation to dive duration, whereas pre-dive Sa,O2 was not significantly related to dive duration(Spearman rank order correlation, Table 2). Blood O2 store depletion rate had a significant,but weak, positive relationship to dive duration in both the arterial and venous compartments (Spearman rank order correlation, Table 2).
Spearman rank order correlation results
. | . | Final SO2/duration . | . | Pre-dive SO2/duration . | . | % O2 content depletion/duration . | . | Depletion rate/duration . | . | ||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Location . | N . | Spearman R . | P . | Spearman R . | P . | Spearman R . | P . | Spearman R . | P . | ||||
Venous | 130 | –0.722* | <0.001 | 0.216* | 0.007 | 0.804* | <0.001 | 0.272* | 0.001 | ||||
Arterial | 71 | –0.677* | <0.001 | 0.174 | 0.074 | 0.667* | <0.001 | 0.210* | 0.040 |
. | . | Final SO2/duration . | . | Pre-dive SO2/duration . | . | % O2 content depletion/duration . | . | Depletion rate/duration . | . | ||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Location . | N . | Spearman R . | P . | Spearman R . | P . | Spearman R . | P . | Spearman R . | P . | ||||
Venous | 130 | –0.722* | <0.001 | 0.216* | 0.007 | 0.804* | <0.001 | 0.272* | 0.001 | ||||
Arterial | 71 | –0.677* | <0.001 | 0.174 | 0.074 | 0.667* | <0.001 | 0.210* | 0.040 |
Correlation is significant at the 0.05 level
The SO2 profile during (A) 1 h of diving of bird 1 (2008) (arterial SO2) with Pa,O2 superimposed. Note that Pa,O2/Sa,O2 are at low levels at the start of the measurements because a dive had occurred just before the series recorded, and (B) the current record dive (23.1 min) of an emperor penguin[bird 19 (Ponganis et al.,2007), venous SO2]. Note the arterialization of the venous blood O2 store in this dive, as Sv,O2 before the dive is as high as 95% and the initial Sv,O2 of the dive is 91%. Sv,O2 decreased to 1% by the end of this long dive. SO2 was determined at pH 7.5 throughout the entire dive to maintain consistency and to provide a conservative estimate of continuous SO2.
The SO2 profile during (A) 1 h of diving of bird 1 (2008) (arterial SO2) with Pa,O2 superimposed. Note that Pa,O2/Sa,O2 are at low levels at the start of the measurements because a dive had occurred just before the series recorded, and (B) the current record dive (23.1 min) of an emperor penguin[bird 19 (Ponganis et al.,2007), venous SO2]. Note the arterialization of the venous blood O2 store in this dive, as Sv,O2 before the dive is as high as 95% and the initial Sv,O2 of the dive is 91%. Sv,O2 decreased to 1% by the end of this long dive. SO2 was determined at pH 7.5 throughout the entire dive to maintain consistency and to provide a conservative estimate of continuous SO2.
DISCUSSION
O2–Hb dissociation curve
As hypothesized because of its potential to contribute to tolerance to low O2 in this species (Ponganis et al., 2007), the O2–Hb dissociation curve of the emperor penguin is indeed left-shifted relative to most birds, similar to that of other penguin species and the high-flying bar-headed goose(Black and Tenney, 1980; Lenfant et al., 1969b; Milsom et al., 1973; Petschow et al., 1977). Left-shifted hemoglobin (higher Hb–O2 affinity) is advantageous for the diving emperor penguin, as it implies that more O2 is available at any given PO2. This becomes particularly relevant for a breath hold diver that often experiences low PO2 values during diving. For example, at a PO2 of 20 mmHg, a pekin duck would be stripped of all its O2, whereas the hemoglobin of the emperor penguin, with its left-shifted dissociation curve, is still 27%saturated (pH 7.5; 21.5% at pH 7.4) at this same PO2 (Fig. 1B). This advantage could also assist in the prevention of deleterious effects such as shallow water blackout if Pa,O2 drops to low levels during diving.
Comparisons of the Sv,O2 profile [based on PO2 profiles from a prior study(Ponganis et al., 2009)] in dives of four emperor penguins (EP). SO2 was determined at pH 7.5 throughout the entire dive to maintain consistency and to provide a conservative estimate of continuous SO2. Note the variability of the Sv,O2 profile among different dives.
Comparisons of the Sv,O2 profile [based on PO2 profiles from a prior study(Ponganis et al., 2009)] in dives of four emperor penguins (EP). SO2 was determined at pH 7.5 throughout the entire dive to maintain consistency and to provide a conservative estimate of continuous SO2. Note the variability of the Sv,O2 profile among different dives.
An increased Hb–O2 affinity also allows for more complete depletion of the respiratory O2 store. As opposed to marine mammals whose lungs do not constitute a significant portion of total O2stores, the respiratory store of the diving emperor penguin remains a significant contributor to O2 stores while diving [19% of total O2 stores (Kooyman and Ponganis, 1998)], and this species dives upon inspiration(Kooyman et al., 1971). The increased O2 affinity of emperor penguin Hb allows for continued extraction of this O2 from the respiratory O2 store for use during diving. By contrast, a pekin duck forcibly submerged to the point of `imminent cardiovascular collapse' was left with 25% of its respiratory O2 store unused, as the blood contained no O2 at an air sac PO2 near 30 mmHg(Hudson and Jones, 1986).
Presumably, the biochemical adaptation underlying the increased Hb–O2 affinity is achieved with specific amino acid substitution(s), similar to that of other species. In two species of high-altitude birds with increased Hb–O2 affinity (bar-headed and Andean geese), the same α1β1 intradimer contact site is disrupted, accomplished by two different amino acid substitutions (on the α-chain in one of the species, and on the corresponding β-chain site in the other)(Perutz, 1983; Weber and Fago, 2004). This same intradimer contact site is altered in high O2 affinity fish Hb, and even reconstituted human Hb demonstrates significantly higher affinity for O2 when this single site is altered(Jessen et al., 1991; Weber and Fago, 2004). Upon examination of the published amino acid sequence of emperor penguin hemoglobin(Tamburrini et al., 1994), it is revealed that this specific site is not altered in emperor or rockhopper(Eudyptes crestatus) penguins. However, as compared to human Hb,there are changes in six α-chain and five β-chainα 1β1 contact sites, and one α-chainα 1β2 contact site. It is possible that one of these modifications accounts for the emperor penguin's increased Hb–O2 affinity, although other structural features might also be responsible.
The final SO2vs dive duration from the compiled PO2 profiles of all dives [venous and arterial; Ponganis et al. and current study(Ponganis et al., 2009; Ponganis et al., 2007)]. Note the significant amount of overlap between arterial and venous values.
The final SO2vs dive duration from the compiled PO2 profiles of all dives [venous and arterial; Ponganis et al. and current study(Ponganis et al., 2009; Ponganis et al., 2007)]. Note the significant amount of overlap between arterial and venous values.
We hypothesize that the small Bohr effect (–0.25) found in the previous study of isolated emperor penguin Hb may be secondary to experimental conditions and the sensitivity of the isolated Hb to co-factors(Tamburrini et al., 1994). It was hypothesized in that study that the low Bohr effect prevents `stripping'of O2 during the long dives of emperor penguins(Tamburrini et al., 1994). However, such a low Bohr effect for Hb in whole blood was not found in this study, nor is it present in other diving penguins and marine mammals(Lenfant et al., 1970; Lenfant et al., 1969b). A small Bohr effect is a potential disadvantage in terms of unloading O2 to the tissues in a diving animal, especially in those with relatively high Hb–O2 affinities. Blood pH data during dives have also demonstrated that emperor penguin blood does not become very acidotic during the dive, indicating that the effects of the Bohr shift may not be particularly relevant during diving in this species(Ponganis et al., 2007).
SO2 extremes
Final Sv,O2 reached very low levels, ≤5%in 15% of dives (corresponding to O2 contents <1.2 ml O2 dl–1 blood) and approached 0% in some dives,particularly in those with durations longer than the previously measured ADL(Ponganis et al., 1997b)(Table 1, Fig. 2B, Figs 3 and 4). This resulted in a large percentage venous O2 content depletion during the dive(Table 1). The wide range of final Sv,O2 values for dives of similar duration (Fig. 4) and the fluctuations in the venous PO2 and SO2 profiles during dives(Fig. 3) suggest significant variability in tissue O2 extraction and blood flow patterns during dives. As previously suggested (Ponganis et al., 2009), this may reflect differences or changes in the peripheral vascular response including regulation of blood flow to muscle and other organs as well as through arterio-venous (a-v) shunts.
Final Sa,O2 reached only as low as 47%(Table 1, Fig. 4; corresponding to an O2 content of 11.5 ml O2 dl–1 blood) in dives as long as 12 min. Such high values contrast with the lower values found in elephant seals (Mirounga angustirostris)(Meir et al., 2009) and,again, are considered secondary to the size of the respiratory O2store in emperor penguins and to the maintenance and efficiency of respiratory gas exchange during these dives. Such high Sa,O2 may well minimize the risk of shallow water blackout in these birds(Fig. 2A). It should be noted that many of these final Sa,O2 values are greater than would have been predicted from final air sac PO2 values for dives of similar duration(Ponganis et al., 2009; Ponganis et al., 2007). It is unclear why final Pa,O2 values are higher than those recorded in the air sacs, especially since air sac PO2 prior to dives and during dives is greater than Pa,O2 (Ponganis et al., 2009). Sample size, type of dive, circulatory lag time,and the location of the sampled air sac may contribute to these differences. Regardless of the explanation, it is apparent that emperor penguins can perform dives as long as 12 min in which final arterial PO2 and SO2 are relatively well preserved.
It should also be noted that there is considerable overlap between final arterial and many final venous SO2 values of the dives in this study (Fig. 4). Such overlap may be secondary to the previously suggested intra-dive a-v shunting (Ponganis et al.,2009) and the close equilibration of air sac, arterial and venous PO2 in the latter parts of such dives. Equilibration of arterial and venous PO2 and SO2 values toward the end of long breath holds has been reported in seals (Elsner et al.,1964; Stockard et al.,2007). As the sample size for arterial records was smaller than that of venous records, and because there were no dives longer than 12 min for arterial data (compared with >23 min for venous data), we believe that final Sa,O2 values in longer dives may well be lower than those documented in this study.
Both Sa,O2 and Sv,O2returned to values equivalent to those at rest within about 2 min post-dive(Fig. 2), consistent with PO2 profiles of this species(Ponganis et al., 2009; Ponganis et al., 2007). In the previous study, no relationship was shown between surface interval duration and the time to return to the PO2 value at rest(Ponganis et al., 2009). Thus,re-oxygenation occurred very rapidly upon return to the surface, and does not represent a limitation to the onset of the subsequent dive.
SO2 profiles and implications for O2 store utilization
As previously discussed (Ponganis et al., 2009), the initial transient increase in Pa,O2 during dives reflected the compression hyperoxia observed during diving in the air sacs of emperor penguins(Stockard et al., 2005) and demonstrated the maintenance of pulmonary gas exchange and transfer of O2 from the respiratory to the blood O2 store. Even as Pa,O2 declined from the early peak value, its value was relatively high for much of the dive (Fig. 2A). As a consequence, Sa,O2 remained near 100% for much of the dive (Fig. 2A), preserving a high O2 content in the arterial system for critical organs such as the brain. Sa,O2generally did not decrease significantly until the final ascent phase of the dive, consistent with the decline in ambient pressure and decrease in both air sac and arterial PO2 during ascent(Ponganis et al., 2007; Stockard et al., 2005)(Fig. 2A). The net transfer of O2 from the lungs to the blood also resulted in some final Pa,O2 values greater than the Pa,O2 of birds at rest and some final Pv,O2 values not only greater than that at rest, but even greater than Pv,O2 at the start of the dive (Ponganis et al.,2007). The corresponding SO2 values resulted in negative values for ΔSO2,percentage O2 content depleted and blood O2 depletion rates (i.e. an overall increase in SO2 during the dive) in such dives (Table 1, Fig. 5). These findings are consistent with the significant role of the left-shifted dissociation curve and with the contribution of respiratory O2 to total O2 stores in this species.
By contrast, although there was also a transient increase in Pa,O2 and consequently Sa,O2 in diving elephant seals at the beginning of dives, max Pa,O2 in the emperor penguin was nearly twice that in the elephant seal [mean max Pa,O2=88±20 mmHg for the elephant seal(Meir et al., 2009) and 157±52 mmHg in the emperor penguin]. As in the emperor penguin, Sa,O2 peaked near 100% in the elephant seal, but this high level of Sa,O2 was not maintained and instead decreased rapidly after the peak (Meir et al.,2009). These findings reflect the difference in the magnitude of the respiratory O2 store between these two species. Unlike emperor penguins, elephant seals dive upon expiration and the lungs do not constitute a significant portion of total O2 stores(Falke et al., 1985; Kooyman and Ponganis,1998).
It should be noted that maximum SO2 values in this study were probably slightly underestimated, because it was not possible to determine a true, critical PO2value at 100% SO2. In order to ensure complete saturation in the tonometry of the 100% O2-saturated blood for O2–Hb dissociation curve determination, a high PO2 (>200 mmHg) was used for that sample. Thus, the actual inflection point in PO2 at which SO2 first reaches 100% was not determined(Fig. 1A). The lack of this data point in the regression equations results in a slightly lower calculated SO2 for PO2values that approach the inflection point of 100%SO2. In turn, this implies that net depletion values and O2 depletion rates are slightly underestimated.
The blood O2 contribution to metabolic rate vs dive duration for all venous and arterial dives. Note that since SO2 was sometimes higher at the end of the dive than at the initial time point, negative values resulted in some cases for this variable.
Arterio-venous shunts
Pre-dive and initial Sv,O2 values were often higher than those corresponding to Pv,O2 of emperor penguins at rest (Ponganis et al.,2007), reaching as high as 95%(Table 1, Fig. 2B, Fig. 3). Although maximum Sv,O2 during a dive was much more variable than Sa,O2 (Table 1), the mean of maximum Sv,O2 was also quite high at 81%, and, in some dives, max Sv,O2 values were almost 100%(Table 1, Fig. 2B, Fig. 3). These `arterialized'venous values imply some degree of arterio-venous (a-v) shunting or at least a lack of tissue O2 uptake from the blood. In addition to previous findings in these species, including increases in Pv,O2during the descent period, the lack of blood lactate accumulation during the dive, muscle temperature profiles, dramatic bradycardias and a lack of association between heart rate and stroke frequency during diving, these results further support the concept that muscle is isolated from the circulation during dives of emperor penguins(Meir et al., 2008; Ponganis et al., 2009; Ponganis et al., 2007; Ponganis et al., 2003).
Such high values during surface intervals may be secondary to an increased gas exchange rate, enhanced reloading of O2 stores and improved ventilation perfusion matching due to a more pronounced hyperventilation and tachycardia before such dives. In addition to the maximization of the blood O2 store by the arterialization of its venous blood, these birds often made full use of this component of their O2 store by decreasing Sv,O2 to near 0% in longer dives, as discussed above (Table 1, Fig. 2B, Fig. 3). For example, in the longest recorded dive to date for an emperor penguin (23.1 min), pre-dive SO2 was 91–95%, initial SO2 was 91%, and final SO2 decreased to as low as 1%, demonstrating a near complete utilization of the venous blood O2 store in this remarkable dive (Fig. 2B).
Based on pre-dive SO2 values, the magnitude of a peripheral a–v shunt prior to the dive can be estimated with a shunt equation, as follows, assuming a 5 ml O2dl–1 a-v O2 difference at rest(Ponganis et al., 2007):{venous O2 content=[arterial O2 content×(% a-v shunt)]–[(arterial O2 content–5 ml O2dl–1)×(1–(% a-v shunt))]}. For example, the maximum pre-dive Sv,O2 in this study was 95.8%(Table 1). Using O2contents calculated for this level of Sv,O2(23.5 ml O2 dl–1) and the maximal pre-dive Sa,O2 (24.4 ml O2 dl–1) an arterio-venous shunt of 82% would be necessary to reach this venous SO2. For the pre-dive Sv,O2of 91.3% (22.4 ml O2 dl–1) prior to the 23.1 min dive (Fig. 2B), the magnitude of the a-v shunt would be 60%. These calculations should be considered slight overestimates since the Sv,O2 values are vena caval and not true mixed venous samples (myocardial O2 extraction would decrease the mixed venous Sv,O2further).
Intrapulmonary shunts
As discussed in previous studies, the mean Pa,O2 of emperor penguins at rest (68±7 mmHg) is less than two-thirds of the mean value in the air sac (Ponganis et al., 2009; Ponganis et al.,2007), probably mainly because of ventilation–perfusion mismatch (Powell, 2000; Powell et al., 1989). Using the O2–Hb dissociation curve and the classic pulmonary shunt equation [% shunt=(capillary O2 content–arterial O2 content)/(capillary O2 content–venous O2 content)], calculations can be made to estimate the size of the pulmonary shunt in the emperor penguin both at rest and in the pre-dive period. Calculating SO2 from the previously measured mean Pa,O2 and Pv,O2 values at rest (Ponganis et al.,2007) and assuming capillary SO2=100%, based on air sac PO2 measurements of 120 mmHg from previous studies (Stockard et al.,2005), and a [Hb]=18.3 g dl–1 (this study) to calculate O2 content, the extent of the intrapulmonary shunt in a resting emperor penguin is 28%. Again, this value may be slightly overestimated since mixed venous values were not used in the calculation. Nonetheless, shunting of pulmonary blood flow is usually very small in birds,less than 1–2.7% of cardiac output in anesthetized artificially ventilated geese and ducks [representing the true intrapulmonary shunt(Burger et al., 1979; Powell and Wagner, 1982)], or 6.3–8% in ducks [representing intrapulmonary plus extrapulmonary shunts(Bickler et al., 1986)]. The high percentage shunt value in emperor penguins may also be due in part to an overestimation of the capillary O2 content because capillary PO2 and saturation may be decreased secondarily to the thickened parabronchial capillary blood-to-air barrier reported in emperor penguins (Welsch and Aschauer,1986). Using pre-dive Sa,O2 and Sv,O2 values (mean values for pre-dive Sv,O2 and Sa,O2 in this study; Table 1) instead of values at rest, however, the intrapulmonary shunt is reduced to 14.3%. This suggests that the hyperventilation and tachycardia characteristic of the pre-dive period in this species (Kooyman et al., 1971; Kooyman et al.,1992; Meir et al.,2008) improves ventilation–perfusion matching prior to the dive.
Blood O2 store contribution to metabolic rate
Since the blood volume of an emperor penguin of a given mass can be calculated (based on 100 mlkg–1)(Kooyman and Ponganis, 1998; Ponganis et al., 1997a), a calculation of the contribution of the blood O2 store to overall metabolic rate was made: blood O2 store contribution=[(final O2 content–initial O2 content)/dive duration×blood volume], with the assumption that one-third of blood volume is arterial, and two-thirds venous in distribution(Kooyman, 1989). Using the mean values for all arterial and all venous dives combined, the blood O2 store contribution alone to metabolic rate from the arterial compartment is 0.3±0.2 ml O2 kg–1min–1 (range=–0.1–+0.7) and from the venous compartment is 0.6±0.9 ml O2 kg–1min–1 (range=–4.1–+3.1). These values are only approximately 5% (arterial) and 10% (venous) of the resting metabolic rate measured for this species (Kooyman and Ponganis, 1994; LeMaho et al.,1976; Pinshow et al.,1977). If dives in which SO2increased during the dive are excluded from the analysis, the blood O2 store contribution alone to metabolic rate from the arterial compartment is 0.4±0.2 ml O2 kg–1min–1 (range=0.0–+0.7) and from the venous compartment is 0.9±0.6 ml O2 kg–1min–1 (range=0.1–+3.1). In comparison, respiratory O2 store depletion rates were 2.3 and 5.3 times that in the venous and arterial blood O2 store compartments, respectively[mean=2.1±0.8 ml O2 kg–1min–1 (Stockard et al.,2005)].
As previously discussed, since SO2 was sometimes higher at the end of the dive than at the initial time point,negative values resulted for ΔSO2,percentage O2 content depletion, and depletion rates(Table 1, Fig. 5). For example, the venous O2 store contribution to metabolic rate was –1.8 ml O2 kg–1 min–1 for a 1.6 min dive. Assuming a gain in the venous blood O2 store of 1.8 ml O2 kg–1 min–1 in this 1.6 min dive, this indicates a minimum loss of 2.9 ml O2kg–1 from the respiratory system during the dive. If the respiratory system initially has 20% O2 at 69 ml kg–1 (Ponganis et al.,1999), it contains approximately 14 ml O2kg–1 at the beginning of the dive. For the 1.6 min dive, a minimum of 21% (2.9/14) of the respiratory O2 was transferred into the blood. This represents a minimum value, as it does not include respiratory O2 that entered the blood and replaced any O2 taken up by tissue from the blood. However, such calculations are useful to illustrate the maintenance of gas exchange during the dive and the potential magnitude of the transfer of respiratory O2 into the blood.
These calculations illustrate the complexity of estimating the contribution of the net blood O2 store to metabolic rate as the blood O2 depletion rate in a penguin (or any diver with a large respiratory O2 store) is a function of both the transfer of O2 into the blood and the uptake of O2 from blood by the tissues. Simultaneous air sac and blood PO2data would allow calculation of the net contribution of these O2stores to diving metabolic rate, but such measurements are not currently feasible. However, because the separately determined O2contributions from the lungs and blood are both low, these data are consistent with (1) a significant contribution from the exceptionally large muscle O2 store to diving metabolic rate in emperor penguins(Kooyman and Ponganis, 1998; Ponganis et al., 1997a), and(2) the low field metabolic rate and the true diving bradycardia (heart rate while diving significantly lower than that of heart rate at rest) exhibited by emperor penguins at the isolated dive hole(Meir et al., 2008; Nagy et al., 2001).
Conclusions
This investigation has revealed enhanced O2 affinity of emperor penguin hemoglobin, similar to that of high-altitude geese and other penguin species. These species are distantly related (Sphenisciformes and Anseriformes) (Hackett et al.,2008) and have evolved in different environments under different constraints. The conditions of hypoxia that high-altitude birds and penguin species encounter are also different, ranging from that of extended hypoxia during sustained flight for the bar-headed goose, to transient, though perhaps frequent hypoxia experienced by diving penguins. Despite these differences,both bar-headed geese and emperor penguins have evolved hemoglobins with remarkably similar O2 affinities. As neither of the two amino acid substitutions responsible for the increased Hb affinity in high-altitude birds is present in emperor penguin hemoglobin, the mechanisms underlying this shared trait are also different. Perhaps this level of O2 affinity is closer to the physiological limit of maximizing O2 uptake during hypoxia. As this is also the range of O2 affinity optimized by mammalian hemoglobins, including those of diving mammals, it demonstrates the compromise between O2 uptake from the respiratory system and O2 unloading to the tissues intrinsic to the O2–Hb dissociation curve.
SO2 profiles during diving, based on the O2–Hb dissociation curve, demonstrated (1) the maintenance of Sa,O2 levels near 100% throughout much of the dive, (2) a wide range of final Sv,O2 values and optimization of the venous blood O2 store resulting from arterialization and near complete depletion of venous blood O2during longer dives, and (3) that estimated contribution of the blood O2 store to diving metabolic rate was low and highly variable. This pattern is due, in part, to the influx of O2 from the lungs into the blood during diving, and variable rates of tissue O2uptake.
LIST OF ABBREVIATIONS
FOOTNOTES
The authors are indebted to the Penguin Ranch teams (Torre Stockard, Ed Stockard, Cassondra Williams, Katherine Ponganis, Robert `Red' Howard, Cory Champagne, Matt Tulis, Kozue Shiomi, and Brendan Tribble) and McMurdo Station personnel for invaluable logistical and field support. We would like to thank Frank Powell and Jeff Graham for use of the tonometers and gas mixing pumps(and Jeff Struthers for the fastidious refurbishment of this equipment); Judy St. Leger and Sea World staff for sea lion and harbor seal blood samples,Markus Horning and Jo-Ann Mellish for Weddell seal blood samples, and the San Diego County Vet lab for chicken blood samples. We thank Jeremy Goldbogen and Megan McKenna for useful comments on the manuscript. This work was supported by National Science Foundation grants 02-29638 and 05-38594. J. Meir was supported by an NDSEG fellowship, a Los Angeles ARCS fellowship made possible by Ed and Nadine Carson, and a Philanthropic Educational Organization (P.E.O.) Scholar Award.