J Appl Physiol 116: 1210–1219, 2014.
First published March 13, 2014; doi:10.1152/japplphysiol.00996.2013.
Acid-base balance in the developing marsupial: from ectotherm to endotherm
Sarah J. Andrewartha,1,2 Kevin J. Cummings,3 and Peter B. Frappell1,2
1
University of Tasmania, Hobart, Tasmania, Australia; 2CSIRO Marine and Atmospheric Research, Hobart, Tasmania,
Australia; and 3Department of Biomedical Sciences, University of Missouri, Columbia, Missouri
Submitted 3 September 2013; accepted in final form 6 March 2014
Tammar wallaby (Macropus eugenii); Sprague-Dawley rat; thermoregulation; ventilatory requirement; acid-base balance
is ectothermic, relying entirely on
the thermally stable pouch of its mother for thermal homeostasis (7, 22, 42, 52, 58). The development of endothermy
occurs as a continuum of events rather than a step-wise change
(see Fig. 2 in Ref. 19) with the transition beginning at least
halfway through pouch life (19, 42, 52, 58). The transition zone
between ectothermy and endothermy can be clearly defined by
examining the resting mass-specific rate of oxygen consumption (V̇O2) at pouch body temperature (Tb), which gradually
changes from values close to the predicted V̇O2 for an adult
ectotherm (e.g., lizard) of the same mass to values predicted for
an adult marsupial (see Fig. 2 in Ref. 19, 27). In the Tammar
wallaby, this transition occurs between postnatal days 55 and
200 at a mass of 70 –300 g (see Fig. 2 in Ref. 19). A myriad of
morphological, physiological, and behavioral changes that allow for endothermy and the maintenance of Tb occurs across
THE NEWBORN MARSUPIAL JOEY
Address for reprint requests and other correspondence: S. J. Andrewartha,
CSIRO, Marine and Atmospheric Research, Hobart, Tasmania, Australia
7000 (e-mail: [email protected]).
1210
this time period (21, 48). The marsupial joey is easily accessible via the external pouch and therefore provides an insightful model for the study of homeostatic mechanisms that develop in parallel with endothermy (see Ref. 62 for a review).
At pouch vacation, most joeys are able to successfully
defend Tb through increased shivering and nonshivering thermogenesis (NST) (19, 36, 42). Shivering in marsupials has
been first observed at approximately two-thirds of the way
through pouch development (54, 65), although quantitative
measurements are lacking. Brown adipose tissue (BAT) is the
major site of NST in newborn placental mammals and plays an
important role in the maintenance of Tb during Ta fluctuations
(11). The presence of BAT in marsupials and its role in NST is
an issue that has a contentious history (23, 29, 37, 49), and
there is evidence that skeletal muscle may be the primary NST
site in some marsupials (56). Although the precise thermogenic
mechanisms may differ between placentals and marsupials,
species from both orders of mammals are prone to hypothermia
during cold stress in the early stages when thermogenic ability
is still developing.
Changes in V̇O2 with temperature must be met with proportional changes in V̇E if arterial acid-base status is to be
maintained constant. Indeed, V̇E/V̇O2 is maintained constant in
postnatal day 18 Tammar wallaby joeys during Tb challenges
(38), suggesting that blood gases and pHa remain stable with
changing Tb, at least at this age (i.e., a “pH-stat” mechanism of
regulation) In contrast, many ectotherms increase pHa with
decreasing Tb. This strategy is hypothesized to maintain constant the fractional ␣-imidazole ionization, thereby conserving
charge status and protein conformation (i.e., ␣-stat regulation:
⌬pHa/⌬Tb ⬃ 0.16 U/°C) (see Refs. 14, 24, 45; for review, e.g.,
Ref. 50).
How neonatal endotherms regulate acid-base status during
Tb changes is still uncertain. Some studies show a strategy that
approaches ␣-stat (e.g., 66), whereas others suggest pH-stat
(e.g., 64). Discrepancies between different studies are likely
the result of methodological differences with respect to the
induction of hypothermia; some, for example, have used anesthetics (e.g., pentobarbital and halothane) with known effects
on V̇E, thermogenesis, and acid-base status (1, 2, 40, 57).
Further discrepancies arise with the use of heterothermic adult
mammals. Some torpid mammals adopt a pH-stat strategy,
maintaining constant PaCO2 and pHa over a wide Tb range
(e.g., 30, 32; see Ref. 39 for a review). Conversely, awake
ground squirrels show a hyperventilation (i.e., increased V̇E/
V̇O2) during hypothermia (8, 69) and a decrease in PaCO2 that
results in a ⌬pHa/⌬Tb of ⫺0.012 (9), approximating ␣-stat
regulation.
Whether unanesthetized marsupials adopt an ␣- or pH-stat
strategy of blood acid-base regulation over pouch life, when
the control systems regulating respiratory and metabolic homeostasis are undergoing rapid development, have not been
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Andrewartha SJ, Cummings KJ, Frappell PB. Acid-base balance in the developing marsupial: from ectotherm to endotherm. J
Appl Physiol 116: 1210 –1219, 2014. First published March 13, 2014;
doi:10.1152/japplphysiol.00996.2013.—Marsupial joeys are born ectothermic and develop endothermy within their mother’s thermally
stable pouch. We hypothesized that Tammar wallaby joeys would
switch from ␣-stat to pH-stat regulation during the transition from
ectothermy to endothermy. To address this, we compared ventilation
(V̇E), metabolic rate (V̇O2), and variables relevant to blood gas and
acid-base regulation and oxygen transport including the ventilatory
requirements (V̇E/V̇O2 and V̇E/V̇CO2), partial pressures of oxygen
(PaO2), carbon dioxide (PaCO2), pHa, and oxygen content (CaO2)
during progressive hypothermia in ecto- and endothermic Tammar
wallabies. We also measured the same variables in the well-studied
endotherm, the Sprague-Dawley rat. Hypothermia was induced in
unrestrained, unanesthetized joeys and rats by progressively dropping
the ambient temperature (Ta). Rats were additionally exposed to helox
(80% helium, 20% oxygen) to facilitate heat loss. Respiratory, metabolic, and blood-gas variables were measured over a large body
temperature (Tb) range (⬃15–16°C in both species). Ectothermic
joeys displayed limited thermogenic ability during cooling: after an
initial plateau, V̇O2 decreased with the progressive drop in Tb. The Tb
of endothermic joeys and rats fell despite V̇O2 nearly doubling with
the initiation of cold stress. In all three groups the changes in V̇O2
were met by changes in V̇E, resulting in constant V̇E/V̇O2 and V̇E/
V̇CO2, blood gases, and pHa. Thus, although thermogenic capability
was nearly absent in ectothermic joeys, blood acid-base regulation
was similar to endothermic joeys and rats. This suggests that unlike
some reptiles, unanesthetized mammals protect arterial blood pH with
changing Tb, irrespective of their thermogenic ability and/or stage of
development.
Marsupial Acid-Base Balance
investigated. This study aims to characterize the regulation of
metabolic, respiratory and blood acid-base variables in hypothermic Tammar wallaby joeys (Macropus eugenii) during the
transition from ectothermy to endothermy. We hypothesize
that acid-base regulation is linked with thermoregulatory ability with ecothermic joeys (in which Tb is essentially unregulated) utilizing an ␣-stat strategy for acid-base regulation (i.e.,
similar to some ectothermic reptiles) and endothermic joeys
adopting a pH-stat strategy similar to rat pups. To test this
hypothesis we will utilize two groups of joeys on either side of
the ectothermic-endothermic transition and compare their ventilatory, metabolic, and acid-base responses during forced hypothermia to those of the juvenile rat, a known endotherm.
•
Andrewartha SJ et al.
1211
(CO2) was determined on 10 ␮l of each sample using a galvanic cell
(Oxycon, University of Tasmania, Australia). Approximately 20 ␮l of
each sample was used to determine lactate concentration (Accusport
analyser, Boehringer Mannheim, Mannheim, Germany) and hemoglobin concentration (HemoCue, Ängelholm, Sweden).
Rodents: cannulation and blood variables. As detailed above for
the joeys, except recovery was at 26°C (within thermoneutral zone ⫽
A
METHODS
Animals
B
Cannulation and Blood Variables
Marsupials. Animals were removed from the pouch and anesthetized with halothane (5% induction, 2–1% maintenance, as appropriate). A small midventral incision (⬃1.5 cm) was made in the neck,
and the left internal carotid artery was occlusively cannulated with
cannula tubing (ID 0.50 mm, OD 0.90 mm, Microtube extrusions,
North Rocks, NSW). The tip of the cannula was positioned so that it
rested as close to the heart as possible. The cannula was sutured into
place and coiled to allow some movement, and a subcutaneous tunnel
used to externalize the cannula in a middorsal position on the neck,
where it was reinforced with a suture. The cannula was filled with
heparin (1,000 units/ml) to ensure it remained patent while the
animals recovered in the mother’s pouch for at least 24 h. All young
were observed to have reattached to the teat after surgery.
Arterial samples (0.5 ml) were collected at Tb ⫽ 36.5, 28, and
20°C and analyzed immediately (see protocol), and volume replacement with 0.5 ml heparin-saline (20 units/ml) ensured that the animals
did not dehydrate or hemodilute. PO2, PCO2, and pH were determined
from each blood sample using blood gas analyzers (models PHM 73
and BMS 3 Mk 2, Radiometer, Denmark) maintained at each experimental Tb and calibrated before and after every sample. pH was
immediately measured using ⬃70 ␮l of the sample to obtain an initial
reading, then subsequently moving ⬃10-␮l increments of the sample
through the electrode until the readings varied less than 0.005 units.
PO2 and PCO2 were measured using ⬃0.45 ml of the sample (including
the blood used for pH measurement, which was still hermetically
preserved) every 30 s over a 3- to 7-min period (temperature dependent) with PO2 regressed linearly back to time zero. Oxygen content
C
Fig. 1. A: rate of oxygen consumption (V̇O2, ml·min⫺1·kg⫺1) against changing
body temperature (Tb) in ectothermic (gray diamonds, n ⫽ 4) and endothermic
(}, n ⫽ 5) Tammar wallaby joeys and 27 ⫾ 0.5-day-old Sprague-Dawley rats,
initially in normoxia (e) and during exposure to helox (). Rate of carbon
dioxide production (V̇CO2, ml·min⫺1·kg⫺1) (B) and minute ventilation (V̇E,
ml·min⫺1·g⫺1) (C) in the same joeys and rats. n Values for rats as reported in
Table 3. Error bars are ⫾SE and some errors are too small to visualize.
*Significant difference (Dunnett’s) from the control values (where Tb ⫽
36.5°C for the joeys and Tb ⫽ 37.5°C in helox for the rats).
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Marsupials. Experiments were performed on four ectothermic
Tammar wallaby joeys (Macropus eugenii) (aged ⬃90 days, mean
body mass of 97.71 ⫾ 2.06 g) and five endothermic joeys (aged ⬃125
days, mean body mass of 253.64 ⫾ 49.79 g). Different animals were
used for endothermic and ectothermic joey measurements. Animals
were selected by mass to fall at the lower and upper ends of the
transition zone between ectothermy and endothermy (19). Animals
were obtained from an established colony maintained outdoors at La
Trobe University (Bundoora, Australia) with unrestricted access to
irrigated pasture and water. Experiments were conducted under La
Trobe University animal ethics permit [LTU AEC 04/37(3)].
Rodents. Experiments were performed on 13 Sprague-Dawley rats
(Rattus norvegicus; aged 27 ⫾ 0.5 days, mean body mass of 77.64 ⫾
3.22 g). One-week postweaned rats of ⬃80 g were targeted because
they were the smallest rats that could be cannulated and have blood
sampled repeatedly. Animals were obtained from a colony at La Trobe
University Central Animal House where they were maintained in a
temperature-controlled holding facility at 22°C with a 12:12-h light/
dark cycle and had access to water and rat pellets ad libitum.
1212
Marsupial Acid-Base Balance
26 –28°C in air; S. Andrewartha, personal observation; Ref. 12) for at
least 24 h.
Respiratory Variables and Aerobic Metabolism
Andrewartha SJ et al.
environment. Pressure oscillations caused by inspiration could be
measured by a differential pressure transducer (MP45–1 Validyne
Engineering, Northridge, CA). The system was calibrated by injecting
a known volume of air (0.1 ml) into the chamber and monitoring the
resulting pressure oscillation. The relatively large animal chamber
ensured that potential overestimations of calibration volumes were
avoided, because any potential pressure spike was absorbed by the
chamber volume.
Experimental Protocol
Marsupials. Young were removed from the pouch (after 24-h
postsurgery recovery), fitted with a mask (see above) and a thermocouple inserted into their urogenital sinus, and placed into a dark
constant-temperature (CT) chamber (modified freezer). A settling
period of at least 1 h was allowed to ensure complete equilibration of
Tb with Ta (Tb ⫽ 36.5°C). For both ectothermic and endothermic
joeys, Ta was adjusted to ensure that Tb reached 20°C in ⬃2 h. Thus,
for the endothermic joeys, the Ta was changed to 10°C immediately
after the 36.5°C blood sample was taken, and for the ectothermic
joeys, Ta was initially adjusted to 20°C after the 36.5°C blood sample
was taken and then to 10°C after the 28°C blood sample was taken. Ta
within the CT chamber declined at a rate of ⬃0.2°C/min after each
adjustment.
Arterial blood samples were collected at Tb ⫽ 36.5, 28, and 20°C.
V̇O2, V̇CO2, Tb, Ta, and ventilatory variables were monitored continuously throughout the experimental period and collected with a
Powerlab (AD Instruments, Bella Vista, NSW, Australia) at a rate of
1,000 Hz (see above). When Tb reached 20°C and blood samples had
been taken, the animal was removed from the experiment, warmed,
anesthetized, the cannula removed via surgical procedure, and the
joey returned to the pouch.
Rodents. Each animal (after at least 24-h postsurgery recovery at
26°C) was placed into the 1-liter respirometry chamber inside a
constant-temperature chamber (modified refrigerator) at 28°C and
allowed at least 30 min to settle. The incurrent air was then changed
to helox (80% helium, 20% oxygen), which facilitates heat loss
because helium has a conductance for heat ⬃6 times greater than
nitrogen (at 27°C; Refs. 31, 59). A further 0.5-h settling period was
allowed at 30°C (because of difference in lower critical temperature in
helox, see Ref. 12). The chamber Ta was then adjusted to 7°C (cooled
at a rate of ⬃0.2°C/min), and over ⬃2.5 h Tb decreased from 37.5 to
22°C. Arterial blood samples were collected at Tb ⫽ 37.5 (normoxia),
37.5 (helox), 30 and 22°C. The respiratory chamber was sealed for
ventilation measurements at Tb ⫽ 37.5 (normoxia), 37.5 (helox), 34,
30, 26, and 22°C. V̇O2, V̇CO2, Tb, and Ta were monitored continuously
Table 1. Respiratory variables during dynamic Tb changes in ectothermic (mass ⫽ 97.71 ⫾ 2.06 g) Tammar wallaby joeys
Variable
Tb ⫽ 36.5°C
Tb ⫽ 32°C
Tb ⫽ 28°C
Tb ⫽ 24°C
Tb ⫽ 20°C
TI, s†
TE, s†
TP, s
TTOT, s†
VT, ml/kg†
f, breaths/min†
TI/TTOT
VT/TI, ml/s†
V̇E, ml·min⫺1·kg⫺1†
V̇O2, ml·min⫺1·kg⫺1†
V̇CO2, ml·min⫺1·kg⫺1†
RER†
V̇E/V̇O2
V̇E/V̇CO2
0.32 ⫾ 0.07 (4)
0.57 ⫾ 0.15 (4)
0.24 ⫾ 0.24 (2)
1.02 ⫾ 0.26 (4)
5.26 ⫾ 0.58 (4)
58.48 ⫾ 10.75 (4)
0.34 ⫾ 0.04 (4)
1.24 ⫾ 0.19 (4)
362 ⫾ 101 (4)
8.01 ⫾ 0.42 (4)
5.87 ⫾ 0.46 (4)
0.74 ⫾ 0.06 (4)
45.35 ⫾ 11.86 (4)
59.32 ⫾ 11.90 (4)
0.26 ⫾ 0.02 (4)
0.40 ⫾ 0.04 (4)
0.13 ⫾ 0.08 (3)
0.76 ⫾ 0.07 (4)*
5.98 ⫾ 0.65 (4)*
83.47 ⫾ 8.42 (4)*
0.36 ⫾ 0.04 (4)
1.72 ⫾ 0.48 (4)
385 ⫾ 138 (4)
9.03 ⫾ 1.33 (4)
7.27 ⫾ 1.39 (4)
0.80 ⫾ 0.05 (4)
40.33 ⫾ 10.17 (4)
49.05 ⫾ 10.13 (4)
0.33 ⫾ 0.04 (4)
0.45 ⫾ 0.07 (4)
0.35 ⫾ 0.06 (4)
1.14 ⫾ 0.13 (4)
9.01 ⫾ 0.37 (4)*
58.11 ⫾ 6.39 (4)
0.30 ⫾ 0.02 (4)
2.10 ⫾ 0.56 (4)*
372 ⫾ 98 (4)
8.28 ⫾ 1.20 (4)
7.13 ⫾ 1.08 (4)
0.86 ⫾ 0.03 (4)
45.64 ⫾ 9.56 (4)
52.68 ⫾ 10.88 (4)
0.53 ⫾ 0.08 (4)
0.93 ⫾ 0.05 (4)
0.34 ⫾ 0.23 (3)
1.72 ⫾ 0.27 (4)
8.37 ⫾ 0.46 (4)*
38.69 ⫾ 5.64 (4)
0.32 ⫾ 0.02 (4)
1.25 ⫾ 0.30 (4)
212 ⫾ 48 (4)*
5.44 ⫾ 1.00 (4)*
4.61 ⫾ 1.08 (4)
0.85 ⫾ 0.05 (4)
42.92 ⫾ 10.07 (4)
51.74 ⫾ 13.87 (4)
0.67 ⫾ 0.05 (4)*
1.25 ⫾ 0.12 (4)*
0.27 ⫾ 0.15 (3)
2.12 ⫾ 0.13 (4)*
9.76 ⫾ 0.42 (4)*
29.61 ⫾ 1.88 (4)*
0.32 ⫾ 0.03 (4)
1.01 ⫾ 0.15 (4)
146 ⫾ 20 (4)*
3.37 ⫾ 0.39 (4)*
3.44 ⫾ 0.60 (4)*
1.01 ⫾ 0.08 (4)*
44.26 ⫾ 4.68 (4)
45.10 ⫾ 6.61 (4)
Values are means ⫾ SE (n). Tb, body temperature; TI, inspiratory time; TE, expiratory time; TTOT, total breathing time; TP, inspiratory pause; VT, tidal volume
of breathing; f, breathing frequency; TI/TTOT, inspiratory timing index; VT/TI, mean inspiratory flow; V̇E, minute ventilation; V̇O2, rate of oxygen consumption;
V̇CO2, rate of carbon dioxide production; RER, respiratory exchange ratio (V̇O/V̇CO2); V̇E/V̇O2and V̇E/V̇CO2, ventilatory requirement ratios. †Significant effect
of temperature on the variable; *difference from control, Tb ⫽ 36.5°C values (Dunnett’s comparison to a control).
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Marsupials. To obtain respiratory gases, a mask constructed from
either the base of a 50-ml centrifuge tube or 25-ml syringe was sealed
over the mouth and nostrils of the joey with a nontoxic polyether
material (Impregum F ESPE, Henry Schein Halas, Melbourne, Victoria, Australia). Air was pushed through the mask, via a Y-connector
sealed into the top of the mask, at a known rate of 0.2– 0.8 l/min (mass
dependent) and monitored by a mass-flow controller (840L-2, Sierra
Instruments, Monterey, CA). Air leaving the mask was subsampled,
dried (Drierite, W.A. Hammond drierite, Xenia, OH), and analyzed
for fractional O2 and CO2 concentration (S-3A/1 and CD-3A, respectively, Applied Electrochemistry, Pittsburgh, PA). V̇O2 and V̇CO2 were
calculated from airflow though the chamber and the difference between the incurrent and excurrent fractional concentrations of the dry
gases as detailed in Frappell et al. (18).
V̇E was determined using a pneumotachograph (LT10L, AD Instruments, Bella Vista, NSW, Australia) placed upstream from the
mask. Pressure changes resulting from alterations in respiratoryrelated airflow were measured using a differential pressure transducer
(MP45-1 Validyne Engineering, Northridge, CA) and integrated to
yield volume. The system was calibrated by injecting known volumes
of air into the sealed mask (before the animal was connected). Up to
60 breaths were analyzed at each target Tb for tidal volume (VT),
expiratory time (TE), inspiratory time (TI), inspiratory pause [TP,
present in young marsupials, see MacFarlane and Frappell (38) for
review], total breath time (TTOT ⫽ TI ⫹ TE ⫹ TP), breathing
frequency (f ⫽ 1/TTOT ⫻ 60), and minute ventilation (V̇E ⫽ VT ⫻ f)
as outlined by Ref. 18.
Rodents. Air was pushed through the 1-liter respirometry chamber
at a known rate of ⬃0.9 l/min and monitored by a mass-flow
controller (840L-2, Sierra Instruments, Monterey, CA). Air leaving
the chamber was subsampled, dried (Drierite, W.A. Hammond drierite), and analyzed for fractional O2 and CO2 concentration (S-3A/1
and CD-3A, respectively, Applied Electrochemistry, Pittsburgh, PA),
and V̇O2 and V̇CO2 were calculated as above. It was not necessary to
instantaneously correct V̇O2 and V̇CO2 for chamber washout considerations, because the average deviation of measured to corrected raw
values was less than 3%.
V̇E was obtained using the barometric method (for a comprehensive
review of the technique, see Ref. 44). The chamber was sealed by
activating solenoids for a duration of ⬃60 breaths at each target Tb
(see protocol below). A moist cloth in the chamber ensured a saturated
•
Marsupial Acid-Base Balance
•
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Andrewartha SJ et al.
Table 2. Blood gas variables during dynamic Tb changes in ectothermic (mass ⫽ 97.71 ⫾ 2.06 g) and endothermic
(253.64 ⫾ 49.79 g) Tammar wallaby joeys
Variable
Ectothermic joeys
PaO2, kPa
PaCO2, kPa
pHa
CaO2, mmol/l
[Hb]a, g/l
Endothermic joeys
PaO2, kPa
PaCO2, kPa
pHa
CaO2, mmol/l
[Hb]a, g/l
[La]a, mmol/l
Tb ⫽ 36.5°C
Tb ⫽ 28°C
Tb ⫽ 20°C
17.01 ⫾ 0.86 (4)
5.49 ⫾ 0.37 (4)
7.32 ⫾ 0.09 (4)
3.87 ⫾ 0.57 (4)
56.50 ⫾ 7.96 (4)
16.25 ⫾ 1.06 (4)
6.09 ⫾ 0.28 (4)
7.11 ⫾ 0.06 (4)
3.06 ⫾ 0.21 (4)
50.00 ⫾ 12.38 (4)
17.59 ⫾ 0.19 (4)
4.58 ⫾ 0.27 (4)
7.21 ⫾ 0.06 (4)
3.08 ⫾ 0.34 (4)
50.00 ⫾ 10.09 (4)
18.72 ⫾ 0.51 (3)
4.98 ⫾ 0.30 (3)
7.38 ⫾ 0.05 (3)
4.29 ⫾ 0.40 (2)
74.00 ⫾ 2.00 (2)
1.30 ⫾ 0.30 (2)
18.09 ⫾ 1.08 (3)
5.66 ⫾ 0.57 (3)
7.23 ⫾ 0.07 (3)
4.48 ⫾ 0.92 (2)
74.00 ⫾ 4.00 (4)
3.40 ⫾ 0.50 (2)
18.96 ⫾ 0.04 (3)
4.89 ⫾ 0.30 (3)
7.33 ⫾ 0.07 (3)
4.79 ⫾ 0.50 (2)
70.00 ⫾ 8.00 (2)
2.35 ⫾ 1.25 (2)
Values are means ⫾ SE (n). PaO2, arterial partial pressure of oxygen; PaCO2, arterial partial pressure of carbon dioxide; pHa, arterial pH; CaO2, oxygen content
of arterial blood; [Hb]a, arterial hemoglobin concentration; [La]a, arterial lactate concentration. Temperature exerted no significant effect on any variable.
Data Analysis and Statistics
Technical limitations prevented us from obtaining measurements
for some animals at certain Tb setting. Thus, rather than using general
linear models, a mixed model approach—valid when missing values
are at random (35, 68)—was adopted. The effects of Tb on ventilatory, metabolic, and blood gas variables were tested for using a mixed
model approach (PROC MIXED) with repeated measures (i.e., using
animal as a random factor) and Dunnett’s post hoc comparison (with
Tb ⫽ 36.5°C as control for joeys and Tb ⫽ 37.5°C in helox as control
for rats) using SAS (v 9.1) software (34). The variance structures were
determined following the recommendations for repeated-measures
analyses by Littell et al. (34), and in all cases an “unstructured”
variance structure was used that makes no assumption regarding equal
variances or correlations. Initially, all models were run with all
interaction terms included, but as no interactions were statistically
significant only the main effects are reported. Statistical significance
was assumed at P ⬍ 0.05.
RESULTS
Metabolic Variables
At the initial Tb of 36.5°C, V̇O2 and V̇CO2 in ectothermic
joeys were 8.01 ⫾ 0.42 and 5.87 ⫾ 0.46 ml·min⫺1·kg⫺1,
respectively (Fig. 1, A and B, Table 1). V̇O2 and V̇CO2 were
maintained at the start of cooling, but both reduced significantly compared with initial values when Tb reached 26°C and
23°C, respectively (P ⬍ 0.05 for both). Overall, there was a
significant effect of reducing Tb on both V̇O2 and V̇CO2 (P ⬍
0.001), with Q10 values of 2.41 ⫾ 0.28 and 2.47 ⫾ 0.39
respectively.
In contrast to ectothermic joeys, V̇O2 and V̇CO2 in endothermic joeys increased during initial cooling, from initial values
of 17.42 ⫾ 1.94 and 11.78 ⫾ 1.53 ml·min⫺1·kg⫺1, respectively, at Tb ⫽ 36.5°C to maximal values of 32.51 ⫾ 6.61 and
25.54 ⫾ 6.32 ml·min⫺1·kg⫺1, respectively, at Tb ⫽ 31°C (P ⬍
0.01 for both, Fig. 1, A and B, Table 1). From its maximum
value, V̇O2 returned to initial (36.5°C) value at Tb ⫽ 28°C,
falling slightly but significantly below initial value by Tb ⫽
20°C (P ⫽ 0.037). V̇CO2 changed in parallel to V̇O2 with falling
Tb, but was not significantly lower than the initial V̇CO2 (at
Table 3. Respiratory variables during a dynamic body temperature changes in 27 ⫾ 0.05-day-old Sprague-Dawley rats
Normoxia
Variable
Tb ⫽ 37.5°C
Tb ⫽ 37.5°C
TI, s†
TE, s†
TTOT, s†
VT, ml/kg†
f, breaths/min†
TI/TTOT
VT/TI, ml/s†
V̇E, ml·min⫺1·kg⫺1†
V̇O, ml·min⫺1·kg⫺1†
V̇CO2, ml·min⫺1·kg⫺1†
RER
V̇E/V̇O2
V̇E/V̇CO2
0.21 ⫾ 0.01 (13)
0.29 ⫾ 0.01 (13)
0.50 ⫾ 0.02 (13)
111.22 ⫾ 0.66 (13)
121.96 ⫾ 3.53 (13)
0.42 ⫾ 0.01 (13)
4.21 ⫾ 0.30 (13)
1383 ⫾ 106 (13)
37.14 ⫾ 1.62 (11)
28.09 ⫾ 1.57 (11)
0.76 ⫾ 0.03 (11)
38.04 ⫾ 3.35 (11)
51.92 ⫾ 5.69 (11)
Helox
Tb ⫽ 34°C
0.21 ⫾ 0.01(13)
0.15 ⫾ 0.01 (13)*
0.26 ⫾ 0.01 (13)
0.16 ⫾ 0.01 (8)*
0.47 ⫾ 0.01 (13)
0.30 ⫾ 0.01 (13)*
13.39 ⫾ 1.17 (13) 20.32 ⫾ 1.09 (13)*
130.72 ⫾ 4.25 (13) 201.16 ⫾ 6.44 (13)*
0.45 ⫾ 0.01 (13)
0.48 ⫾ 0.01 (13)
5.10 ⫾ 0.53 (13) 10.79 ⫾ 0.49 (13)*
1776 ⫾ 199 (13)
4022 ⫾ 164 (13)*
40.71 ⫾ 1.66(9)
87.97 ⫾ 3.98 (13)*
31.04 ⫾ 1.99 (9)
63.26 ⫾ 2.30 (13)*
0.76 ⫾ 0.02 (9)
0.73 ⫾ 0.02 (13)
41.10 ⫾ 5.07 (9)
46.59 ⫾ 2.29 (13)
53.57 ⫾ 5.40 (9)
64.37 ⫾ 3.15 (13)
Tb ⫽ 30°C
Tb ⫽ 26°C
0.14 ⫾ 0.01 (13)*
0.15 ⫾ 0.01 (12)*
0.15 ⫾ 0.01 (13)*
0.19 ⫾ 0.01 (12)*
0.28 ⫾ 0.01 (13)*
0.35 ⫾ 0.02 (12)*
18.62 ⫾ 1.00 (13)*
18.08 ⫾ 1.49 (12)*
219.33 ⫾ 10.28 (13)* 179.62 ⫾ 9.01 (12)*
0.47 ⫾ 0.01 (13)
0.44 ⫾ 0.01 (12)
11.06 ⫾ 0.43(13)*
8.89 ⫾ 0.46 (12)*
4089 ⫾ 199 (13)*
3137 ⫾ 203 (12)*
81.15 ⫾ 4.19 (13)*
58.31 ⫾ 3.71 (13)*
57.16 ⫾ 2.34 (13)*
40.63 ⫾ 1.63 (13)*
0.71 ⫾ 0.02 (13)
0.72 ⫾ 0.03 (13)
51.51 ⫾ 2.76 (13)
56.54 ⫾ 4.58 (12)
73.06 ⫾ 3.26 (13)
77.06 ⫾ 4.85 (12)
Tb ⫽ 22°C
0.21 ⫾ 0.01 (13)
0.29 ⫾ 0.02 (13)
0.50 ⫾ 0.03 (13)
14.93 ⫾ 1.49 (13)
131.76 ⫾ 7.20 (13)
0.43 ⫾ 0.01 (13)
5.52 ⫾ 0.39 (13)
1774 ⫾ 171 (13)
36.82 ⫾ 3.24 (13)
25.19 ⫾ 1.42 (13)
0.72 ⫾ 0.05 (13)
50.79 ⫾ 5.38 (13)
69.39 ⫾ 4.77 (13)
Values are means ⫾ SE (n). †Significant effect of temperature on the variable; *difference from control, Tb ⫽ 37.5°C helox values (Dunnett’s comparison
to a control).
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throughout the experimental period. All variables were collected by
Powerlab (AD Instruments, Bella Vista, NSW, Australia) at a rate of
1,000 Hz.
Sixty breaths at each target Tb were analyzed for VT according to the
equation described by Drorbaugh and Fenn (17). The same 60 breaths
were also analyzed for breathing frequency (f ⫽ 1/TTOT ⫻ 60) and
minute ventilation (V̇E ⫽ VT ⫻ f) as outlined by Frappell et al. (18).
1214
Marsupial Acid-Base Balance
30
34
26
22
37
28
32
20
•
Andrewartha SJ et al.
to overall V̇E, VT/TI more than doubled with initial cooling,
from 5.10 ⫾ 0.53 ml/s at Tb ⫽ 37.5°C to 10.79 ⫾ 0.49 ml/s at
Tb ⫽ 34°C. This was the result of a decrease in TI and an
increase in VT (P ⬍ 0.01 for both).
In all groups, V̇E/V̇O2 and V̇E/V̇CO2 remained unchanged as
Tb fell. V̇E/V̇O2 averaged 43.70 ⫾ 9.27 in ectothermic joeys,
51.45 ⫾ 4.25 in endothermic joeys, and 47.90 ⫾ 2.79 in rats.
V̇E/V̇CO2 averaged 51.58 ⫾ 10.68, 68.00 ⫾ 3.76, and 65.87 ⫾
3.17 in ecto- and endothermic joeys and rats, respectively,
across the Tb range (Fig. 2, Tables 1 and 3).
Blood Gases
36
Blood gases were unaltered by changes in Tb in all groups.
Ectothermic joeys were relatively hypoxic and hypercapnic
relative to the endotherms. PaO2 and PaCO2 averaged 16.95 ⫾
0.35 and 5.39 ⫾ 0.18 kPa in ectothermic joeys, 18.59 ⫾ 0.51
Tb ⫽ 36.5°C) at the coldest Tb. Q10 for V̇O2 and V̇CO2 from
Tb ⫽ 31–20°C was 1.83 ⫾ 0.13 and 1.78 ⫾ 0.15, respectively.
For rats, no difference was observed in V̇O2 or V̇CO2 measured in room air or helox at the initial Tb of 37.5°C (P ⬎ 0.10
for all; Fig. 1, A and B, Table 3). Both V̇O2 and V̇CO2 more than
doubled during cooling when exposed to helox; Tb fell from
37.5 to 34°C (P ⬍ 0.001 for both; Fig. 1, A and B, Table 3).
From Tb ⫽ 34 –25°C, both V̇O2 and V̇CO2 decreased, falling to
values initially observed at Tb ⫽ 37.5°C values. Q10 for V̇O2
and V̇CO2 from Tb ⫽ 34 –22°C was 2.42 ⫾ 0.22 and 2.45 ⫾
0.14, respectively.
A
B
Ventilation and Respiratory Requirement
In ectothermic joeys, V̇E fell significantly with progressive
hypothermia, from 362 ⫾ 101 ml·min-1·kg-1 at Tb ⫽ 36.5°C to
212 ⫾ 48 and 146 ⫾ 20 ml·min⫺1·kg⫺ 1 at Tb ⫽ 24 –20°C
(P ⫽ 0.011; Fig. 1C, Table 1). The decrease in V̇E with falling
Tb resulted from a significant fall in f (P ⬍ 0.001), whereas VT
progressively increased (P ⫽ 0.003). TI/TTOT was maintained
constant across the Tb range (Table 1). VT/TI was nearly twice
as great at Tb ⫽ 28°C (2.10 ⫾ 0.56 ml/s) compared with Tb ⫽
36.5°C (P ⫽ 0.004; Table 1).
In the endothermic joeys, V̇E doubled with initial cooling,
from 737 ⫾ 149 ml·min⫺1·kg⫺1 at Tb ⫽ 36.5°C to 1,553 ⫾
487 ml·min⫺1·kg⫺1 at Tb ⫽ 28°C (P ⫽ 0.011; Fig. 1C, Table 2).
The increased V̇E at Tb ⫽ 28°C was produced via an
increase in f (P ⫽ 0.038, Table 2). Paralleling the increase
in overall V̇E, VT/TI doubled as Tb was lowered to 28°C (P ⫽
0.041; Table 1).
Similar to endothermic joeys, V̇E more than doubled in rats
with initial cooling (parallel to changes in V̇O2 and V̇CO2),
reaching a maximum value of 4,089 ⫾ 199 ml·min⫺1·kg⫺1 at
Tb ⫽ 30°C (Fig. 1C, Table 3). Again, in parallel to the
metabolic effects, V̇E at Tb ⫽ 22°C was not different than the
initial level at Tb ⫽ 37.5°C (Fig. 1C). Changes in V̇E in the rats
were achieved with increases in VT and f (Table 3). In addition
Fig. 3. Arterial partial pressure of oxygen (PaO2, kPa) (A), partial pressure of
carbon dioxide (PCO2, kPa) (B), and pH (pHa) during dynamic body temperature (Tb) cooling in endothermic Tammar wallaby joeys (}, n ⫽ 3), ectothermic Tammar wallaby joeys (gray diamonds, n ⫽ 4), and Sprague-Dawley
rats (, n as reported in Table 5) initially in normoxia (e) and during exposure
to helox (). Error bars are ⫾ SE. A summary of the joey and rat data can be
found in Tables 4 and 5. The dashed regression in C indicates predicted ␣-stat
regulation (⌬pHa/⌬Tb ⬃ 0.16 U/°C) from pHa of ectothermic joeys at 37°C
(Tbstart).
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Fig. 2. V̇E (ml·min⫺1·kg⫺1) vs. V̇CO2 (ml·min⫺1·kg⫺1) during changes in Tb in
ectothermic (gray diamonds, n ⫽ 4) and endothermic (}, n ⫽ 5) Tammar
wallaby joeys and 27 ⫾ 0.5-day-old Sprague-Dawley rats, initially in normoxia (e) and when exposed to helox (). n Values for rats as reported in
Table 3. The numbers inside (or next to) the symbols represent Tb. Error bars
are ⫾SE. The isopleth represents the average V̇E/V̇CO2 value at control Tb
(36.5°C in joeys, 37.5°C helox in rats) for the 3 groups.
Marsupial Acid-Base Balance
•
1215
Andrewartha SJ et al.
Table 4. Respiratory variables during dynamic Tb changes in endothermic (mass ⫽ 253.64 ⫾ 49.79 g) Tammar wallaby
joeys
Variable
Tb ⫽ 36.5°C
Tb ⫽ 32°C
Tb ⫽ 28°C
Tb ⫽ 24°C
Tb ⫽ 20°C
TI, s
TE, s
TTOT, s
VT, ml/kg
f, breats/min
TI/TTOT
VT/TI, ml/s†
V̇E, ml·min⫺1·kg⫺1†
V̇O2, ml·min⫺1·kg⫺1†
V̇CO2, ml·min⫺1·kg⫺1†
RER
V̇E/V̇O2
V̇E/V̇CO2
0.67 ⫾ 0.10 (5)
0.97 ⫾ 0.27 (5)
1.64 ⫾ 0.27 (5)
18.37 ⫾ 2.57 (5)
40.12 ⫾ 4.86 (5)
0.44 ⫾ 0.09 (5)
9.50 ⫾ 4.23 (5)
737 ⫾ 149 (5)
17.42 ⫾ 1.94 (5)
11.78 ⫾ 1.53 (5)
0.68 ⫾ 0.05 (5)
40.79 ⫾ 4.72 (5)
62.31 ⫾ 10.18 (5)
0.44 ⫾ 0.03 (5)
0.52 ⫾ 0.12 (5)
0.96 ⫾ 0.14 (5)
8.96 ⫾ 4.26 (5)
71.51 ⫾ 10.27 (5)*
0.48 ⫾ 0.05 (5)
13.43 ⫾ 5.67 (5)
1264 ⫾ 264 (5)
32.51 ⫾ 6.61 (5)*
25.54 ⫾ 6.32 (5)*
0.77 ⫾ 0.04 (5)
39.60 ⫾ 3.88 (5)
52.72 ⫾ 7.03 (5)
0.44 ⫾ 0.07 (5)
0.49 ⫾ 0.08 (5)
0.96 ⫾ 0.14 (5)
22.52 ⫾ 6.03 (5)
76.13 ⫾ 16.52 (5)*
0.47 ⫾ 0.04 (5)
18.78 ⫾ 4.23 (5)*
1553 ⫾ 487 (5)*
26.80 ⫾ 6.71 (5)
21.93 ⫾ 6.72 (5)*
0.78 ⫾ 0.04 (5)
56.35 ⫾ 5.38 (5)
71.83 ⫾ 5.81 (5)
0.52 ⫾ 0.03 (5)
0.65 ⫾ 0.09 (5)
1.23 ⫾ 0.12 (5)
14.81 ⫾ 3.07 (5)
53.66 ⫾ 5.72 (5)
0.44 ⫾ 0.05 (5)
8.98 ⫾ 3.34 (5)
784 ⫾ 117 (5)
16.08 ⫾ 3.00 (5)
12.83 ⫾ 3.07 (5)
0.78 ⫾ 0.05 (5)
50.14 ⫾ 8.71 (5)
64.33 ⫾ 10.85 (5)
0.76 ⫾ 0.24 (5)
0.82 ⫾ 0.08 (5)
1.56 ⫾ 0.31 (5)
12.93 ⫾ 3.36 (5)
52.10 ⫾ 12.34 (5)
0.49 ⫾ 0.05 (5)
8.18 ⫾ 4.18 (5)
607 ⫾ 168 (5)
8.41 ⫾ 1.13 (5)*
6.67 ⫾ 1.23 (5)
0.80 ⫾ 0.09 (5)
71.39 ⫾ 15.69 (5)
88.62 ⫾ 16.58 (5)
Values are means ⫾ SE (n). †Significant effect of temperature on the variable; *difference from control, Tb ⫽ 36.5°C values (Dunnett’s comparison to a
control).
DISCUSSION
In contrast to our initial hypothesis, young Tammar wallaby
joeys with little ability to thermoregulate, did not utilize an
␣-stat strategy (see, for example, some reptiles) for pHa regulation. Instead, the strategy of the ecto- and endothermic joeys
and rats was similar, with V̇E remaining matched to V̇O2 and
V̇CO2 over a wide Tb range. Thus the ventilatory requirement
ratios (V̇E/V̇O2 and V̇E/V̇CO2) were preserved, resulting in
constant blood gases and pHa.
Methodological Considerations
During the initial stages of hypothermia, our “ectothermic”
joeys maintained V̇O2 (and V̇CO2), demonstrating some (albeit
very limited) ability to mount a thermogenic response, most
likely through NST because shivering was not observed in
these young animals (Fig. 4). Indeed, despite this plateau in the
V̇O2 response, these animals were unable to defend Tb to any
measurable degree during ambient cooling. Nevertheless, it is
possible that a transition from an ␣- to a pH-stat mechanism of
acid-base regulation occurs in younger animals and we may
have detected this if we measured even younger animals.
Given the preservation of V̇E/V̇O2 previously observed with
cooling in younger joeys (38), this seems unlikely. Rather, our
data suggest that marsupial joeys adopt a pH-stat strategy of
acid-base regulation during hypothermia, regardless of stage of
development and thermogenic ability,
Our resting V̇O2 values are comparable with previously
published values. Metabolic data obtained for ecto- and endothermic joeys at their normal, euthermic Tb of 36.5°C fall on
their prediction lines (Fig. 1, see Fig. 2 in Ref. 19). Similarly,
the resting values for V̇O2 and V̇CO2, V̇E, V̇E/V̇O2, and V̇E/V̇CO2
of normothermic (Tb ⫽ 37.5°C) rats (in normoxia and helox)
are expected for ⬃80 g rats (46). Importantly, although some
of these variables have been measured on cold-tolerant species
that undergo torpor (e.g., 69) this study provides simultaneous
respiratory, metabolic, and blood acid-base data that have been
obtained without anesthetics that have potent effects on thermoregulatory and respiratory control and has previously complicated data interpretation (e.g., 2, 40). The use of helox to
facilitate heat loss in the rats had no effect on initial metabolic,
ventilatory, or blood variables, as has been shown in other
species (26).
To assess the relatively small sample sizes, post hoc power
analysis (analysis of variance with repeated measures, within
factors) was conducted on V̇E, which was a factor that changed
significantly in all animal groups. The analysis revealed statis-
Table 5. Blood gas variables during a dynamic body temperature changes in 27 ⫾ 0.05 day-old Sprague Dawley rats
Normoxia
Helox
Variable
Tb ⫽ 37.5°C
Tb ⫽ 37.5°C
Tb ⫽ 30°C
Tb ⫽ 22°C
PaO2, kPa
PaCO2, kPa
pHa
CaO2, mmol/l
[Hb]a, g/l
[La]a, mmol/l
16.30 ⫾ 0.93 (7)
5.42 ⫾ 0.60 (8)
7.42 ⫾ 0.04 (7)
5.10 ⫾ 0.64 (6)
93.3 ⫾ 3.50 (4)
0.60 ⫾ 0.32 (4)
16.51 ⫾ 0.86 (7)
4.68 ⫾ 0.27 (8)
7.42 ⫾ 0.05 (8)
4.86 ⫾ 0.26 (6)
86.6 ⫾ 4.71 (4)
0.7 ⫾ 0.40 (4)
16.26 ⫾ 0.54 (8)
3.71 ⫾ 0.41 (8)
7.36 ⫾ 0.02 (8)
5.50 ⫾ 0.44 (7)
90.6 ⫾ 5.56 (4)
1.0 ⫾ 0.43 (4)
17.26 ⫾ 0.61 (5)
4.23 ⫾ 0.74 (5)
7.39 ⫾ 0.01 (5)
4.61 ⫾ 0.67 (5)
85.00 ⫾ 9.00 (2)
LO (2)
Values are means ⫾ SE (n). LO ⫽ below detectable levels.Temperature exerted no significant effect on any variable.
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and 5.17 ⫾ 0.29 kPa in endothermic joeys, and 16.17 ⫾ 0.53
and 4.60 ⫾ 0.36 kPa in rats (Fig. 3, Tables 2 and 5). pHa was
maintained constant in all groups during hypothermia, averaging 7.22 ⫾ 0.05, 7.31 ⫾ 0.01, and 7.40 ⫾ 0.03 in ecto- and
endothermic joeys and rats, respectively (Tables 4 and 5).
Similarly the other blood variables remained unchanged across
the Tb range (Tables 4 and 5). CaO2 averaged 3.34 ⫾ 0.29
mmol/l in ectothermic joeys, 4.52 ⫾ 0.01 mmol/l in endothermic joeys and 5.22 ⫾ 0.33 mmol/l in rats across the Tb range.
[Hb]a averaged 52.17 ⫾ 10.00 g/l in endothermic joeys,
72.67 ⫾ 4.67 g/l in ectothermic joeys, and 87.06 ⫾ 1.53 g/l (P ⫽
0.520) in rats. [La] averaged 2.35 mmol/l in endothermic joeys
and 0.57 ⫾ 0.24 in rats across the Tb range (Tables 2 and 5).
1216
Marsupial Acid-Base Balance
A
•
Andrewartha SJ et al.
the teat, surgically implant catheters, return the joey to the
pouch, reattach them to the teat, and trust the mother would not
reject the joey (as occurred in a number of cases).
V̇E, V̇O2, and pHa Regulation during Hypothermia:
Comparative and Developmental Aspects
Fig. 4. A: fractional changes in metabolic rate (expressed as change in V̇CO2
from the V̇CO2 at the initial starting Tb) during dynamic body temperature (Tb)
cooling in endothermic Tammar wallaby joeys (}, n ⫽ 3), ectothermic
Tammar wallaby joeys (gray diamonds, n ⫽ 4), and Sprague-Dawley rats (,
n as reported in Table 3) initially in normoxia (e) and during exposure to helox
(). B: same data presented as log V̇CO2 vs. (Tb ⫺ Tbstart)/10, which yields a
straight line with a slope of Q10, which ⫽ 2.45 ⫾ 0.04 (from Tb ⫽ 34°C) in
the rats, 1.78 ⫾ 0.15 (from Tb ⫽ 31°C) in endothermic joeys, 2.47 ⫾ 0.39
(from Tb ⫽ 31°C) in ectothermic joeys. Error bars are ⫾ SE. A summary of
the joey and rat data can be found in Tables 2, 3, and 4. The dashed regressions
indicate the predicted V̇CO2 for a similar sized lizard (calculated from Ref. 5
assuming a respiratory exchange ratio of 0.85 and a Q10 of 2.5).
tical power of 0.76 for an effect size of 0.70 for the ectothermic
joeys and 0.72 for an effect size of 0.65 in the endothermic
joeys (G*Power analysis software, Universität Kiel, Germany).
Using a priori power analysis and the observed effect sizes a
total of 5 ecto- and 5 endothermic (as measured) wallabies
would be required to reach the ideal statistical power level of
0.80 (12a). It is therefore unlikely that type II statistical error
has occurred because of the high statistical power of the post
hoc analysis and similarities of the sample sizes between the a
priori power analysis and experimental data. Furthermore, the
ease of obtaining animals needs to be considered. Tammar
wallabies breed seasonally and raise an individual joey that
remains permanently attached to the teat until near pouch
vacation. Our experiments required us to remove joeys from
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B
In euthermic conditions, an increase in the prevailing V̇O2
and V̇CO2 of mammals is generally met by an increase in V̇E for
the maintenance of blood gases (e.g., during moderate exercise,
see Ref. 67 for review). There are, however, exceptions, as is
the case during sleep when there is a relative hypoventilation
and subsequent increase in PaCO2 (e.g., 53).
In all three groups there was no significant effect of Tb on
V̇E/V̇O2 or V̇E/V̇CO2. Alterations in V̇E were achieved by
different means, however, among the different groups. In
parallel with the increase in V̇O2 with initial cooling, rats
increased V̇E via an increase in both VT and f, before VT fell
once Tb reached 22°C. This pattern is similar to immature
rodents, where the decrease in VT was attributed to failure at
the level of the central rhythm generator for breathing (60). In
contrast, endothermic joeys initially increased V̇E by increased
f only. Both VT and f decreased in ectothermic joeys during
hypothermia, similar to previous observations in younger joeys
during severe hypothermia (38). There was no alteration in
postinspiratory pause in ectothermic joeys in contrast to neonatal rats where postinspiratory (and postexpiratory) pauses
become larger during cooling (60).
Broadly speaking, whatever the thermoregulatory ability or
species, inspiratory flow rates (VT/TI ⬵ inspiratory drive) are
directly correlated with V̇E , i.e., an increase in V̇E was
associated with an increase in VT/TI. V̇E is tightly correlated
with V̇O2 and the increase in V̇O2 that accompanied thermogenesis occurred when Tb declined. Therefore, although direct
effect of Tb on inspiratory drive is expected because of the
direct effect of temperature on chemo- and stretch receptors
(41, 61), changes in drive were presumably masked by the
effects of thermogenesis and the need to ensure V̇E remain
matched to metabolic rate. A similar conclusion was drawn for
newborns, where the increase in metabolism during cold exposure masked the effects that Tb per se has on vagal ventilatory mechanisms (38). It thus appears that the predominant
driver in endothermic (and those that will become endothermic) animals is the preservation of V̇E, V̇E/V̇O2, and V̇E/V̇CO2
that results in constant blood gases (Figs. 2 and 3).
Unlike our animals, awake, nonanesthetized ground squirrels show a relative hyperventilation (and thus an increase in
pHa) during hypothermia (Tb ⫽ 37.5–31°C) when measurements were taken immediately after the animals were awakening from a daily torpor bout (9). During “forced” hypothermia
(used in the current study), the drop in Tb is unregulated,
whereas the Tb changes occurring in torpid animals is regulated. Furthermore, animals capable of torpor appear to be
more intrinsically cold tolerant, at least at the cellular level
(47), and therefore may not respond to hypothermia in a similar
manner to a homeotherm.
Despite their relative immaturity, V̇E/V̇O2 (and V̇E/V̇CO2)
was preserved across the Tb range in our younger joeys,
resulting in stable blood gases and pHa. The findings of this
study are supported by the constant V̇E/V̇O2 observed in ectothermic day 18 Tammar wallaby joeys (average mass ⫽ 2.66 ⫾
Marsupial Acid-Base Balance
Potential Clinical Relevance
These data potentially provide some insight into the acidbase regulation in mammals (including humans; e.g., newborns) that may be susceptible to hypothermia. In clinical
settings (e.g., during cardiopulmonary bypass and other surgeries), there is still much debate over the most appropriate
approach (i.e., ␣- or pH-stat) for regulating pH (see Refs. 4, 25
for reviews). A recent, comprehensive clinical review suggests
that age may be important and that adverse effects are best
avoided by adopting a pH-stat strategy for pediatric and an
␣-stat strategy for adult patients (6). Our data obtained in
Andrewartha SJ et al.
1217
unanesthetized animals suggest that a pH-stat strategy during
clinical hypothermia will result in an acid-base status that is
more similar to accidental hypothermia.
Conclusions
Constant ventilatory requirement resulted in constant blood
gases and hence, constant pHa in endothermic and ectothermic
joeys and rats. Thus, across the transition zone between ectothermy and endothermy, the regulation of blood gases and
blood-acid base balance in marsupial joeys is more akin to the
regulation seen in endothermic and heterothermic mammals
(constant pHa, e.g., rats in this study) than that often observed
for ectothermic vertebrates (␣-stat). Although young marsupial
joeys, similar to ectothermic vertebrates (e.g., reptiles), are
poikilothermic, the regulation of blood acid-base status is more
akin to that observed in homeothermic mammals (older joeys
and rats). Constant pHa regulation while the young marsupial
joey is still essentially poikilothermic (similar to a reptile)
suggests that pHa regulation is established early (and perhaps
independently) during the development of endothermy in marsupials. Furthermore, these data have potential implications for
clinical management of pHa of newborns with highly labile
body temperatures, situations of accidental hypothermia, and
clinical hypothermia that may occur during surgery.
ACKNOWLEDGMENTS
SJA was a recipient of an Australian Postgraduate Award and a Latrobe
University Institute for Advanced Study Postgraduate Writing-up Award.
Shannon Simpson, Lyndal Horne, Jonas Haag and Tegan Youdan are thanked
for surgical assistance. Tobie Cousipetcos and Eva Suric are commended for
animal husbandry.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: S.J.A. and P.B.F. conception and design of research;
S.J.A. performed experiments; S.J.A. analyzed data; S.J.A., K.J.C., and P.B.F.
interpreted results of experiments; S.J.A. prepared figures; S.J.A. drafted
manuscript; S.J.A., K.J.C., and P.B.F. edited and revised manuscript; S.J.A.,
K.J.C., and P.B.F. approved final version of manuscript.
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