Comparative Biochemistry and Physiology Part A 126 (2000) 223 – 231
www.elsevier.com/locate/cbpa
Respiratory responses to short term hypoxia in the snapping
turtle, Chelydra serpentina
Sebastian Frische, Angela Fago, Jordi Altimiras *
Department of Zoophysiology, Danish Center for Respiratory Adaptation, Uni6ersity of Aarhus, DK-8000 A, rhus C, Denmark
Received 1 January 2000; received in revised form 22 March 2000; accepted 27 March 2000
Abstract
Among vertebrates, turtles are able to tolerate exceptionally low oxygen tensions. We have investigated the
compensatory mechanisms that regulate respiration and blood oxygen transport in snapping turtles during short
exposure to hypoxia. Snapping turtles started to hyperventilate when oxygen levels dropped below 10% O2. Total
ventilation increased 1.75-fold, essentially related to an increase in respiration frequency. During normoxia, respiration
occurred in bouts of four to five breaths, whereas at 5% O2, the ventilation pattern was more regular with breathing
bouts consisting of a single breath. The increase in the heart rate between breaths during hypoxia suggests that a high
pulmonary blood flow may be maintained during non-ventilatory periods to improve arterial blood oxygenation. After
4 days of hypoxia at 5% O2, hematocrit, hemoglobin concentration and multiplicity and intraerythrocytic organic
phosphate concentration remained unaltered. Accordingly, oxygen binding curves at constant PCO2 showed no changes
in oxygen affinity and cooperativity. However, blood pH increased significantly from 7.50 90.05 under normoxia to
7.72 9 0.03 under hypoxia. The respiratory alkalosis will produce a pronounced in vivo left-shift of the blood oxygen
dissociation curve due to the large Bohr effect and this is shown to be critical for arterial oxygen saturation. © 2000
Elsevier Science Inc. All rights reserved.
Keywords: Turtle; Respiration; Hypoxia; Heart rate; Blood pH; Organic phosphates; Bohr effect; Shunt
1. Introduction
In air-breathing vertebrates, the response to
environmental hypoxia includes hyperventilation,
an increase in the oxygen carrying capacity of the
blood by increasing the number of red blood cells
and changes in the oxygen affinity, either through
* Corresponding author. Present address: Zoological Institute, University of Göteborg, Medicinaregatan 18, P.O. Box
463, S-405 30 Göteborg, Sweden. Tel.: + 46-31-7733693; fax
+46-31-7733807.
E-mail address: [email protected] (J. Altimiras)
alteration of the ratio between different isohemoglobins or by modification of the intra-erythrocytic organic phosphate concentration (Weber
and Wells, 1989).
In turtles, cardiorespiratory adjustments during
hypoxia are rapid, including an increase in total
expired volume (reviewed in Glass and Wood,
1983), increased heart rate and increased pulmonary blood flow (Burggren et al., 1977). Additionally, a slow erythropoietic response after
hypobaric hypoxia has been described in painted
turtles (Chrysemys picta) (Meints et al., 1975),
increasing the oxygen carrying capacity through
an increased hematocrit.
1095-6433/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S 1 0 9 5 - 6 4 3 3 ( 0 0 ) 0 0 2 0 1 - 4
224
S. Frische et al. / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 223–231
In snapping turtles (Chelydra serpentina), oxygen uptake is maintained relatively constant even
when the inspired oxygen fraction is as low as 2%,
which is partly achieved via cardiorespiratory adjustments (Boyer, 1966). However, it is at present
not known whether the well-known ability of this
and other turtle species to withstand hypoxia
(Ultsch, 1985) could also be due to adjustments at
the molecular level, altering blood oxygen binding
properties, as known for other vertebrates. Moreover, the effect of changes in the blood oxygen
dissociation curve in animals exhibiting cardiac
shunting is determined by the interplay with other
respiratory variables, such as ventilation and the
magnitude of the right-to-left shunt (Rossoff et
al., 1980; Wood, 1984; Wang and Hicks, 1996).
Thus, the aim of this study was to characterize
the respiratory response of snapping turtles to
hypoxia and to determine whether changes in
blood oxygen transport play a role in the response. First, we characterized the cardiorespiratory response to increasing levels of hypoxia at
25°C, to select an appropriate hypoxic stimulus
for the longer hypoxic exposure, since an effect on
the blood oxygen transport at the molecular level
would only be expected if ventilation and heart
rate are also affected. Second, we measured the
effect of 4 days exposure to 5% O2 on several
blood variables such as hematocrit, hemoglobin
multiplicity and concentration, organic phosphates concentration in red blood cells and whole
blood oxygen binding properties. Finally, the
quantitative effect of these changes on blood oxygenation was evaluated using a simple model of
oxygen transport.
2. Material and methods
2.1. Animal care and handling
Three snapping turtles C. serpentina (L.) (3.4 –
4.4 kg) were housed in individual tanks (100× 50
cm, 30 cm water level) with a sand bottom and
fed regularly. Water temperature was maintained
at 28 91°C. The recording tank was identical to
the housing tank, but the animals were restrained
to a 50 ×40-cm portion (40 cm water level). To
facilitate the comparison with previously published values, the experiments were carried out at
25°C.
2.2. Series I: heart rate and 6entilatory 6ariables
during progressi6e hypoxia
To record the electrocardiogram and measure
heart rate, two holes were drilled on the plastron,
8 cm apart, along the longitudinal axis of the
animal. Two stainless steel screws (2 mm diameter) were housed in the holes and fixed with
cyanoacrylic two-component glue. Stainless steel
insulated wires were soldered to the screws, glued
to the plastron and lead to the top of the carapace, where they were connected to a miniature
three-pin connector. The electrode configuration
is similar to the standard mammalian lead II,
which maximizes the signal strength due to the
marked verticalized character of the turtle heart
(Altimiras, 1995). Heart rates were obtained from
the ECG trace. Implantation of electrodes was
carried out under halothane anaesthesia and the
animals were allowed to recover for at least 1
week before beginning the experiments.
The ventilation trace was obtained via a pneumotachograph (Godart, model 17212) connected
to a cylindrical 500 ml ventilation chamber placed
on the lid of the tank (Glass et al., 1983). The
system was calibrated as previously described
(Funk et al., 1986).
The experimental protocol consisted of a
graded decrease in inspired oxygen concentration.
Exposure to each level of inspired oxygen lasted
24 h and always followed the same order: 21 kPa
(21% O2), 15 kPa (15% O2), 10 kPa (10% O2) and
5 kPa (5% O2), followed by a final day of recovery. Thus, the entire protocol took 5 days. Gas
mixtures were delivered by a Wösthoff M201
pump.
The electrocardiogram and ventilation were
recorded continuously and stored digitally in a
computer using a customized LabView program
and a Data Translation acquisition card (DT2801A). Sampling frequency was 100 Hz.
2.3. Series II: determination of blood 6ariables
under normoxic and hypoxic conditions
Animals were repeatedly exposed to 4 days of
hypoxia at 5% O2, with a resting interval of at
least 4 weeks between trials. Repeated exposures
were conducted to compensate for the low number of animals. Animal 1, 2 and 3 were exposed to
hypoxia one, four and three times, respectively.
Blood samples (2–5 ml) were taken in heparinized
S. Frische et al. / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 223–231
syringes by caudal puncture on the ventral side of
the tail before and after 4 days of exposure to
hypoxia.
The following hematologic variables were determined: plasma pH, hematocrit (Hct), blood
hemoglobin concentration ([Hb]), erythrocyte organic phosphate concentrations and oxygen tenand
cooperativity
(n50)
at
sion
(P50)
half-saturation.
Hematocrit was measured in glass capillary
tubes in duplicate by standard methods. Blood
hemoglobin concentration was measured spectrophotometrically, using the reported extinction
coefficients for human hemoglobin of 14.37 and
15.37 mM − 1 cm − 1 at 542 and 577 nm, respectively (Van Assendelft and Zijlstra, 1975). Mean
corpuscular hemoglobin concentration (MCHC)
was calculated by the ratio [Hb]*Hct/100.
Erythrocyte adenosine and guanosine mono-,
di- and tri-phosphate concentrations were measured by reverse phase HPLC (Waters, model
515) on a Nucleosil C18 column (4×250 mm,
Macherey–Nagel). Samples were prepared by
mixing 100 ml of blood with 200 ml 12%
trichloroacetic acid, left on ice for 30 min and
centrifuged at 10 000 rpm for 2 min. A total of
200 ml of the supernatant was added to 400 ml of
a freshly prepared 3:1 mixture of Volasil 244
(BDH) and tri-n-octylamine (Sigma) at room temperature. After mixing, the mixture was centrifuged for phase separation. The top oily phase
was discarded and the aqueous phase at the bottom was diluted with an equal volume of HPLC
solvent A (0.065 M KH2PO4, 1 mM PIC reagent
1, Waters, as ion-pairing agent). A total of 30 ml
were injected on a 20-ml loop. Separation of the
organic phosphates was achieved in 20 min with a
10 – 25% linear gradient of methanol (solvent B).
The flow rate was 0.7 ml min − 1 and the absorbance was monitored at 254 nm. Calibration
of the column was performed with standard mixtures containing AMP, ADP, ATP, GMP, GDP,
GTP (Sigma) ranging from 7.5 to 45 ng ml − 1.
Blood oxygen binding values were determined
using the Tucker technique (Tucker, 1967). The
blood was placed in tonometers at 25°C connected to Wösthoff pumps and equilibrated with
different gas mixtures for at least 30 min before
measurement of the oxygen content. A total of
4% CO2 was used in all gas mixtures.
Plasma pH was measured at 25°C with the
capillary glass electrode of a Radiometer BMS II
connected to a Radiometer PHM64 pH-meter.
225
Hemoglobin multiplicity under normoxia and
hypoxia was determined by analytical isoelectric
focusing in a 7.25% polyacrylamide gel (pH range:
3.5–10) as described previously (Fago et al.,
1995), after hemolysis of aliquots of washed red
blood cells with a 3-fold volume of 10 mM
HEPES buffer pH= 7.8.
2.4. Data analysis, calculations and statistics
The following ventilatory variables were obtained from the pneumotacograph trace: tidal volume, ventilation volume and ventilation
frequency. Although the signals were sampled
continuously for 24 h, only four 1-h long recordings were analyzed to obtain tidal volume, ventilation volume and heart rate (those recorded after
3, 9, 15, and 21 h from the beginning of hypoxia).
Ventilation frequency and the descriptors of the
ventilatory pattern for each animal and treatment
were determined on the longest pneumotachographic trace free of recording artifacts, an average of 16 h (range: 11–24 h) of uninterrupted
ventilatory recordings. Heart rate was analyzed
distinguishing apneic heart rate from ventilatory
heart rate.
The analysis of the ventilatory pattern was
based on a double log-normal model (Tolkamp
and Kyriazakis, 1999). The method was preferred
over the classic log-survivor plot previously used
to characterize the breathing pattern in Xenopus
lae6is (Boutilier, 1984). The log-survivorship analysis implicitly assumes the independence between
the length of the interval between events and the
probability of the event to occur. This assumption
is erroneous when studying breathing patterns
because the longer the breath-hold, the more
likely it is for breathing to resume.
Intermittent ventilation is characterised by consecutive breaths occurring in succession followed
by an apneic period of variable length. The distinction between burst breathing (breath-holding
occurs under water) and bout breathing (breathholding occurs on the surface) (Boutilier, 1984) is
not applied in our analysis and all breaths are
treated as being part of a breathing bout that, in
turn, consists of one or more breaths.
The application of the double log-normal
model to the bimodal distribution of the logarithm of the time interval between breaths rendered a critical time (Tc), which was used to
distinguish between breaths within a bout and
226
S. Frische et al. / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 223–231
separate bouts. A detailed description of the
method can be found in the literature (Tolkamp
and Kyriazakis, 1999). Interbreath intervals
shorter than Tc were considered part of a bout
and those longer than Tc defined a new bout. In
Fig. 1, for instance, the 1-h pneumotacogram
during normoxia (trace A) included three breathing bouts with six, one and four breaths in each,
Fig. 1. Ventilation pattern during normoxia (A) and hypoxia
5% O2 (B) in turtle c2. V: , pneumotachograph air flow.
while the hypoxic pneumotacogram (trace B) consisted of numerous bouts with a single breath
each.
The concentration of organic phosphates
(PRBC) in the red blood cells (1 mg ml − 1) is given
by:
PRBC =
PHPLC (1−Vc)
0.33·0.5 Hct
where PHPLC is the amount (mg ml − 1) detected in
the sample (20 ml), 0.33 and 0.5 are the dilution
factors with trichloroacetic acid (TCA) and
HPLC solvent A, respectively, Hct is the hematocrit and Vc the fraction of precipitate after addition of TCA (measured in capillary tubes as
described for the Hct). The factor (1 − Vc) corrects for the concentrating effect of the supernatant due to formation of precipitate (Nielsen and
Lykkeboe, 1992).
Oxygen affinity (P50) and cooperativity (n50)
were interpolated from the Hill-plots of the data
between 30 and 70% saturation. R-values of the
linear regressions of the Hill-plots were all \ 0.96.
Statistical analysis was carried out using
ANOVA of repeated measurements for the cardioventilatory variables using STATISTICA ’98
Ed. (v.5.1).
Paired t-test’s were used to analyse the blood
measurements. The repeated measurements of
each animal under each experimental condition
were pooled to provide a single value per animal
per treatment, implying a true N value of 3.
All data are shown as mean9 S.E. Significant
differences were all taken at the fiduciary level
PB 0.05.
3. Results
Fig. 2. Ventilatory variables at different levels of hypoxia (as
shown by dotted line referred to on right axis). VT, tidal
volume; fR, respiratory frequency;V: E, expired ventilation;
FI,O2, inspired oxygen fraction. Values as mean 9 S.E.M. (N=
3). Mean values that are significantly different (PB 0.05) from
the mean during normoxia are marked with an asterisk.
The ventilatory responses to progressive hypoxia are shown in Fig. 2. Tidal volumes averaged 30.6 ml kg − 1, without significant changes
during hypoxia. Ventilation frequency averaged
15.69 7.0 breaths per hour in normoxia and rose
significantly (PB 0.05) to 28.59 11.9 breaths per
hour at 5% O2. Expired ventilation averaged
0.409 0.06 l h − 1 kg − 1 in normoxia and was
maintained at similar values at 15 and 10% O2,
but it significantly increased at 5% O2 to 0.709
0.14 l h − 1 kg − 1. On return to normoxia, expired
ventilation decreased to normoxic values.
S. Frische et al. / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 223–231
227
Table 1
Inter-individual differences in the cardiorespiratory variables
in normoxic conditionsa
fR (breaths h−1)
VT (ml BTPS kg−1)
fH,apnea (breaths h−1)
fH,ventilation (breaths
h−1)
Turtle 1
Turtle 2
Turtle 3
29.2
19.9
7.7
15.4
11.6
32.9
11.5
25.5
6.0
59.1
3.8
19.0
a
fR, respiration frequency; VT, tidal volume; fH,apnea, heart
rate during apnea; fH,ventilation, heart rate during ventilation.
A large difference in the breathing pattern of
the three turtles was noticed, as reported in Table
1. While turtle c1 took short and frequent
breaths, turtle c 3 took deeper breaths less often.
Despite this variation, the cardioventilatory response to hypoxia was consistent and similar for
all animals.
The ventilation pattern changed markedly at
5% O2 for all animals (see Fig. 1 for turtle c 2).
Each ventilation bout dropped from an average of
three to five breaths in normoxia and hypoxia at
15 and 10% O2 (Table 2) to practically a single
breath in each bout at 5% O2. The time interval
between bouts also decreased (19 94 min in normoxia vs. 492 min at 5% O2, Table 2). The
similarity in the response of all animals indicates
that the more regular ventilation pattern during
hypoxia is typical.
Fig. 3 shows the effect of hypoxia on heart rate.
In normoxia, heart rate averaged 8.792.3 beat
per minute and rose significantly to 18.393.2
beat per minute at 5% O2. This was due to the
increase in heart rate during apnea, which
changed from 7.69 2.3 to 15.7 92.7 beat per
minute, whereas heart rate during ventilation
episodes was not statistically different between the
different levels of inspired oxygen.
The hematological variables measured under
normoxic conditions and after 4 days exposure to
Fig. 3. Heart rate at different levels of hypoxia (as shown by
dotted line referred to on right axis). (A) Heart rate during
ventilation (closed bars) and during apnea (open bars). (B)
Average heart rate (over 4 h). Values as mean 9 S.E.M. (N=
3). Mean values that are significantly different (PB 0.05) from
the mean during normoxia are marked with an asterisk.
5% O2 are shown in Table 3. Blood pH rose from
7.509 0.05 (PB 0.05) at normoxia to 7.729 0.03
at hypoxia. No significant differences in hematocrit, hemoglobin concentration, blood oxygen
affinity (P50) or cooperativity (n50) were observed
between normoxic and hypoxic samples. Of the
organic phosphates examined, only ATP was detected in the blood of snapping turtles (Fig. 4).
Similar to the other blood variables examined,
ATP concentrations remained unchanged upon
exposure to hypoxia (Table 3).
Analytical isoelectrofocusing on polyacrylamide
gels indicated the presence of one major and two
minor hemoglobins (:5–10% of the total) in the
blood of snapping turtles (data not shown). No
differences in Hb multiplicity were found after 4
days at 5% O2 compared to the normoxic
condition.
Table 2
Interbreath intervals split according to the critical time criterion as described in the texta
O2 (%)
Time interval between breaths in bout (s)
Time interval between bouts (min)
Number of breaths in bout
21
15
10
5
29 96
32 99
71 932
79 967
19 9 4
159 6
17 9 7
492
4.5 9 1.1
3.5 9 1.5
3.3 9 0.9
1.1 9 0.1
a
Values as mean 9S.E.M. (N= 3).
228
S. Frische et al. / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 223–231
Table 3
Blood parametres in normoxia and after 4 days at 5% O2a
Hematocrit (%)
P50 (kPa) (4% CO2)
n50
pH50 (4% CO2)
Plasma pH
[ATP]RBC (mmol
l−1)
Hb (mmol l−1 heme)
MCHC (mmol l−1
heme)
Normoxia 21%
O2
Hypoxia 5% O2
27.8 94.7
3.69 0.4
1.79 0.1
7.53 90.06
7.50 90.05
2.0690.26
29.39 4.7
3.59 0.4
1.89 0.2
7.57 9 0.03
7.72 90.03*
2.3890.25
4.08 91.14
14.3 90.09
3.8691.01
13.2 90.39
Values as mean 9S.E.M. (N= 3).
* Significant difference (PB0.05) from the normoxia value.
a
4. Discussion
Significant differences in ventilation, heart rate
and blood pH occurred following exposure to
hypoxia. Snapping turtles exposed to different
degrees of hypoxia displayed the typical increase
in total ventilation previously reported in other
chelonian species when inspired oxygen tensions
dropped below a certain threshold (Boyer, 1966;
Jackson, 1973; Benchetrit et al., 1977; Burggren et
al., 1977; Glass et al., 1978, 1983; Vitalis and
Milsom, 1986a), which in snapping turtles appears
to be between 5 and 10 kPa O2.
The increase in total expired volume (V: E) is
almost exclusively mediated by increases in the
respiration frequency. The preferential modulation of ventilation frequency in determining total
expired volume in turtles is thought to be an
adaptive strategy to minimize the cost of breath-
Fig. 4. HPLC chromatographic analysis of organic phosphates. The elution profile of a standard (thin line) and a
representative sample (thick line) are shown. The gradient of
solvent B (methanol) is shown on the right axis.
ing (Vitalis and Milsom, 1986b) and is subjected
to vagal control from pulmonary stretch receptors
(Milsom, 1990). On the basis of the values for
oxygen uptake previously reported (Boyer, 1966),
and our data for ventilation volume, pulmonary
oxygen extraction can be estimated to increase
from 0.19 in normoxia to 0.39 at 5% O2. Similar
increases in extraction have been observed in Trachemys scripta (Jackson, 1973), and are probably
common in all turtles.
As shown in Fig. 1 and Table 2, at 5% O2 there
is a switch to a more regular ventilatory pattern
consisting of single breaths spaced by shorter
apneas. The regularity of the ventilatory pattern
during 5% O2 is likely related to a lower PO2 and
PCO2 of the blood stimulating different chemoreceptive areas. The shortening of the interval between breaths is likely due to lower arterial PO2,
while the decreased number of breaths per breathing bout (Table 2) results from a drop in the PCO2
at the end of the preceding ventilatory period
(West et al., 1989) caused by the increased V: E/
V: O2ratio (Kinney et al., 1977). The drop in PCO2 is
also evidenced by the relative alkalinization of the
blood during hypoxia (Table 3).
Heart rate increases 2.2-fold in agreement with
earlier studies (Boyer, 1966). An increase in the
cardiac output and/or a decrease in the R–L
shunt determines an increase in the pulmonary
blood flow (Burggren et al., 1977; Crossley et al.,
1998). During hypoxia at 5% O2 heart rate and
consequently pulmonary blood flow appear to
remain constant during ventilation and to increase
during apnea (Fig. 4), indicating that considerable
gas exchange occurs between breaths. Under these
conditions, a regular ventilatory pattern such as
that displayed during hypoxia (Fig. 1) would be
beneficial in maintaining a high lung PO2 under
limiting oxygen availability.
In contrast to ventilation and heart rate, most
of the hematological parameters remained unaltered after 4 days exposure 5% O2. Hematocrit,
hemoglobin concentration and multiplicity were
unaffected, i.e. there were no newly synthesised
hemoglobins upon hypoxia and no chemical modifications (e.g. oxidation, polymerisation) that
could be detected by isoelectrofocusing. Similarly,
ATP-concentrations in the red blood cells remained unchanged, in contrast to what happens
in fish (Wood and Johansen, 1972) and other
vertebrates (Weber and Wells, 1989) after expo-
S. Frische et al. / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 223–231
Table 4
Model analysis of arterial PO2 and Hb-saturation (see text for
details)a
Scenario
I
II
III
IV
% O2
V: A (ml min−1 kg−1)
P50 (mmHg)
Q: sys (ml min−1 kg−1)
Q: pul (ml min−1 kg−1)
PaO2 (mmHg)
Hb-saturation (%)
21
7.0
29.0
50
25
83.8
87
5
11.6
29.0
50
25
8.1
9.7
5
11.6
29.0
60
60
10.9
15.3
5
11.6
18.7
60
60
10.9
28
V: A, alveolar ventilation; P50, oxygen tension at half-saturation; Q: sys, systemic blood flow; Q: pul, pulmonary blood flow;
PaO2, arterial oxygen tension. Constants utilized in the model
analysis: Hill coefficient= 1.75 (this study); blood oxygen carrying capacity=3.98 mmol l−1 (this study); lung diffusion
deficit= 10 mmHg (Wang and Hicks, 1996); tidal volume =
30.6 ml BTPS kg−1 (this study); lung anatomical dead
space =0.7 ml BTPS kg−1 (Vitalis and Milsom, 1986a) and
rate of oxygen uptake= 0.25 ml min−1 kg−1 (Boyer, 1966).
Alveolar ventilation and shunt flow were obtained by calculation. P50 values were calculated using the Bohr factor of
−0.95 for snapping turtle blood (West et al., 1989).
a
sure to hypoxic conditions. Interestingly, red cell
triphosphate concentrations are also unchanged
after anemic stimuli in the painted turtle (Chrysemys picta) (Wang et al., 1999), suggesting that
such a common response in other vertebrates does
not play a role in modulating the oxygen affinity
of hemoglobin in turtles. Accordingly, in vitro
experiments showed that at a constant CO2 tension, neither the shape (n50) nor the position (P50)
of the oxygen dissociation curve changed in response to hypoxia. Thus, no adjustment at the
molecular level seem to occur in the blood of
snapping turtles after short exposure to low oxygen tensions.
However, the relative alkalinization of the
blood during hypoxia (Table 3) has important
consequences on the oxygen dissociation curve in
vivo. The position of the oxygen dissociation
curve (P50) relative to lung PO2 determines
whether increased ventilation or changes in shunt
patterns will increase PO2 of the arterial blood
most efficiently (Wood, 1984). Based on a steadystate model of gas exchange, it has been recently
proposed that arterial PO2 levels are maximized
under hypoxia by simultaneously eliminating cardiac R–L shunting and increasing ventilation
(Wang and Hicks, 1996). This conclusion was
reached assuming constant blood oxygen affinity
229
(implying constant blood pH), which is shown not
to be the case in this study.
Using the same model and taking into account
the blood pH changes, it is possible to estimate
arterial PO2 and hemoglobin saturation in snapping turtles during normoxia and hypoxia at 5%
O2 (Table 4). Normoxia results in an arterial PO2
at steady state of 11.2 kPa (83.8 mmHg), corresponding to a Hb-saturation of 87% (scenario I).
During hypoxia, different scenarios were considered in order to estimate the relative contribution
of each compensatory adjustment (increases in
ventilation, pulmonary blood flow and blood pH)
to arterial PO2 and O2 saturation.
In scenario II, the increase in ventilation alone
(according to the values measured in this study)
would determine an arterial PO2 and Hb-saturation of only 1.1 kPa (8.1 mmHg) and 9.7%,
respectively. Increasing pulmonary blood flow
and eliminating the R–L shunt together with the
increased ventilation (scenario III) further increases arterial PO2 and Hb-saturation to 1.5 kPa
(10.9 mmHg) and 15%, respectively, assuming a
constant P50 of 3.9 kPa (29.0 mmHg). Interestingly, the largest changes to blood oxygenation
are observed when the increase in blood pH is
taken into account (scenario IV). P50 will decrease
from 3.9 kPa (29.0 mmHg) in normoxia to 2.5
kPa (18.6 mmHg) (calculated from the Bohr-factor of − 0.95 reported for the blood of C. serpentina (West et al., 1989)). As a result, the
Hb-saturation increases 1.8-fold, whereas arterial
PO2 remains unchanged.
In conclusion, our study shows that respiratory
adaptation to environmental hypoxia in snapping
turtles is essentially based on the increased ventilation and on the consequent increase in blood
pH. The alkalinization of the blood results in a
left-shift of the oxygen dissociation curve through
the large Bohr-effect. This seems to be the major
factor in maintaining sufficient oxygen uptake
and supply to metabolizing tissues during hypoxia
at 25°C. In contrast, the regulation at a molecular
level of the blood oxygen transport (such as
change in isohemoglobin pattern or intraerythrocytic ATP-concentration) does not seem to be
necessary under the conditions investigated. The
simplicity in the respiratory response is probably
responsible for the well-known tolerance of low
oxygen tensions typical of this and other turtle
species.
230
S. Frische et al. / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 223–231
Acknowledgements
Rufus M.G. Wells for helpful advice for the
chromatographic analysis of organic phosphates
and Dorte Olsson for technical assistance.
Michael Axelsson provided constructive criticism
to the manuscript. The financial support of The
Danish National Research Council is gratefully
acknowledged.
References
Altimiras, J., 1995. Heart Rate Variability. Its Significance in Lower Vertebrates. Ph.D.Thesis. Department of Biochemistry and Physiology, University of
Barcelona.
Benchetrit, G., Armand, J., Dejours, P., 1977. Ventilatory chemoreflex drive in the tortoise, Testudo
horsfieldi. Resp. Physiol. 31, 183–191.
Boutilier, R.G., 1984. Characterization of the intermittent breathing pattern in Xenopus lae6is. J. Exp.
Biol. 110, 291–300.
Boyer, D.R., 1966. Comparative effects of hypoxia on
respiratory and cardiac function in reptiles. Physiol.
Zool. 39, 307–316.
Burggren, W.W., Glass, M.L., Johansen, K., 1977.
Pulmonary ventilation: perfusion relationships in
terrestrial and aquatic chelonian reptiles. Can. J.
Zool. 55, 2024–2034.
Crossley, D., Altimiras, J., Wang, T., 1998. Hypoxia
elicits an increase in pulmonary vascular resistance
of anaesthetised turtles (Trachemys scripta). J. Exp.
Biol. 201, 3367–3375.
Fago, A., Carratore, V., Di Prisco, G., Feuerlein, R.J.,
Sottrup-Jensen, L., Weber, R.E., 1995. The cathodic
hemoglobin of Anguilla anguilla. Amino acid sequence and oxygen equilibria of a reverse Bohr
effect hemoglobin with high oxygen affinity and
high phosphate sensitivity. J. Biol. Chem. 270,
18897–18902.
Funk, G.D., Webb, C.L., Milsom, W.K., 1986. Non-invasive measurement of respiratory tidal volume in
aquatic, air-breathing animals. J. Exp. Biol. 126,
519–523.
Glass, M.L., Wood, S.C., 1983. Gas exchange and
control of breathing in reptiles. Physiol. Rev. 63,
232–260.
Glass, M., Burggren, W.W., Johansen, K., 1978. Ventilation in an aquatic and a terrestrial chelonian
reptile. J. Exp. Biol. 72, 165–179.
Glass, M.L., Boutilier, R.G., Heisler, N., 1983. Ventilatory control of arterial PO2 in the turtle Chrysemys
picta bellii: effects of temperature and hypoxia. J.
Comp. Physiol. 151, 145–153.
Jackson, D.C., 1973. Ventilatory response to hypoxia in
turtles at various temperatures. Resp. Physiol. 18,
178 – 187.
Kinney, J.L., Matsuura, D.T., White, F.N., 1977. Cardiorespiratory effects of temperature in the turtle,
Pseudemys floridana. Resp. Physiol. 31, 309 – 325.
Meints, R.H., Carver, F.J., Gerst, J.W., McLaughlin,
D.W., 1975. Erythropoietic activity in the turtle: the
influence of hemolytic anemia, hypoxia and hemorrhage on hemopoietic function. Comp. Biochem.
Physiol. A. 50, 419 – 422.
Milsom, W.K., 1990. Mechanoreceptor modulation of
endogenous respiratory rhythms in vertebrates. Am.
J. Physiol. 259, R898 – R910.
Nielsen, O.B., Lykkeboe, G., 1992. In vitro effects of
pH and hemoglobin-oxygen saturation on plasma
and erythrocyte K+ levels in blood from trout. J.
Appl. Physiol. 72, 1291 – 1296.
Rossoff, L., Zeldin, R., Hew, E., Aberman, A., 1980.
Changes in blood P50. Effects on oxygen delivery
when arterial hypoxemia is due to shunting. Chest
77, 142 – 146.
Tolkamp, B.J., Kyriazakis, I., 1999. To split behaviour
into bouts, log-transform the intervals. Anim. Behav. 57, 807 – 817.
Tucker, V.A., 1967. Methods for oxygen content and
dissociation curves on microlitre blood samples. J.
Appl. Physiol. 23, 410 – 414.
Ultsch, G.R., 1985. The viability of nearctic freshwater
turtles submerged in anoxia and normoxia at 3 and
10 degrees C. Comp. Biochem. Physiol. A. 81,
607 – 611.
Van Assendelft, O.W., Zijlstra, W.G., 1975. Extinction
coefficients for use in equations for the spectrophotometric analysis of haemoglobin mixtures. Anal.
Biochem. 69, 43 – 48.
Vitalis, T.Z., Milsom, W.K., 1986a. Mechanical analysis of spontaneous breathing in the semi-aquatic
turtle, Pseudemys scripta. J. Exp. Biol. 125, 157 –
171.
Vitalis, T.Z., Milsom, W.K., 1986b. Pulmonary mechanics and the work of breathing in the semiaquatic turtle, Pseudemys scripta. J. Exp. Biol. 125,
137 – 155.
Wang, T., Hicks, J.W., 1996. The interaction of pulmonary ventilation and the right-left shunt on arterial oxygen levels. J. Exp. Biol. 199, 2121 – 2129.
Wang, T., Brauner, C.J., Milsom, W.K., 1999. The
effect of isovolemic anaemia on blood O2 affinity
and red-cell triphosphate concentrations in the
painted turtle (Chrysemys picta). Comp. Biochem.
Physiol. A. 122, 341 – 345.
Weber, R.E., Wells, R.M.G., 1989. Hemoglobin structure and function. In: Wood, S.C. (Ed.), Comparative Pulmonary Physiology. Marcel Dekker, New
York, pp. 279 – 310.
S. Frische et al. / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 223–231
West, N.H., Smits, A.W., Burggren, W.W., 1989.
Factors terminating nonventilatory periods in the
turtle, Chelydra serpentina. Resp. Physiol. 77,
337–350.
Wood, S.C., 1984. Cardiovascular shunts and oxygen
.
231
transport in lower vertebrates. Am. J. Physiol. 247,
R3 – R14.
Wood, S.C., Johansen, K., 1972. Adaptation to hypoxia
by increased Hb-O2 affinity and decreased red cell
ATP concentration. Nature New Biol. 237, 278 – 279.