LABORATORY: TEMPERATURE AND RESPIRATION OF TOADS AND
MAMMALS
Aerobic and Anaerobic Cellular Respiration
In most cells, metabolism is supported by oxidation of carbon fuels, including lipid,
carbohydrate and protein. The overall metabolic process consumes carbon fuels and
oxygen (O2), and produces heat, carbon dioxide (CO2) and water. Many physiological
questions revolve around changes in metabolic rate, and many approaches have been
developed to measure the substrates and products of metabolism. Direct calorimetry
measures the rate of heat production (but requires expensive, ultra-sensitive
instruments). Indirect calorimetry measures the rate of O2 consumption or CO2
production. O2 is consumed mainly by the mitochondrial electron transport system
(ETS) in an effort to build a proton gradient across the inner mitochondrial membrane.
Carbon dioxide is not produced in the ETS, but rather in a series of reactions, primarily
in the tricarboxylic acid cycle. There relationship between heat production, O2
consumption and CO2 production depends on the nature of the metabolic fuels. In
indirect calorimetry, the ratio of CO2 produced to O2 consumed is called the respiratory
quotient (RQ), and can be used to assess the major carbon fuel. An RQ near 1
signifies carbohydrate is the dominant fuel, whereas an RQ near 0.7 suggests fatty
acids are most important.
The metabolic rate of animals is influenced by numerous factors, but the beststudied factors are body size and temperature. For animals with similar physiology (e.g.
mammals), as body mass increases metabolic rate increases but not quite as much.
Metabolic rate (Q) usually varies with body mass (M) raised to an exponent (b).
Q=aMb
The value for b, known as the scaling coefficient, is typically between 2/3 and 3/4 If you
log transform the relationship, you obtain an equation for a straight line, where the
scaling coefficient (b) is the slope of the relationship.
log Q = b (log M) + log a
Temperature exerts complex effects on metabolic rate, with different mechanisms
operating in homeotherms versus poikilotherms. When homeotherms are placed in the
cold, they increase metabolic rate to produce more heat to maintain a constant body
temperature. Conversely, when poikilotherms are placed in the cold, their body
temperature decreases, slowing all of the metabolic processes within the animal. The
relationship between metabolic rate and temperature is described by the van’t Hoff
equation, in which you calculate a Q10 value.
 RMR1 

Q10  
RMR
2 

10
T2 T1
Where RMR1 and RMR2 are the routine metabolic rates at the respective temperatures
T1 and T2.
Questions: Where does CO2 arise in oxidative metabolism? What steps
consume oxygen? Why is heat produced? Under what conditions would oxygen
consumption be a poor index of metabolic rate? What might you predict if you
measured the RQ of a hummingbird throughout the day? Discuss how
homeotherms and poikilotherms cope with changes in temperature. For those
of you who love calculations, consider the pathways that lead to complete
oxidation of lipid (e.g. palmitate), carbohydrate (e.g., glucose) and protein
(e.g., alanine). Can you calculate how much (i) ATP is produced, (ii) O2
consumed, and (iii) CO2 produced?
In this exercise, you will measure O2 consumption and CO2 production in living animals
at rest under and observe how metabolic rateis influenced by temperature, and how the
response differs in homeothermic versus poikilothermic organisms. You will be given
access to toads and mice. The toad is a more temperate local animal. It avoids thermal
extremes by seeking refuge under leaf litter. It is easy to measure the rate of oxygen
consumption in these larger animals. A half of the class will conduct the experiment with
a toad and the other half of the class will conduct the experiment with a mouse. When
you arrive, the equipment will be set up with the animal chamber disconnected and the
pump unplugged. Half of the students will conduct the experiment with a toad and the
other half will conduct the experiment with a mouse.
Experimental Operating Protocol
Step # 1 - Determining the organisms mass
In physiological studies interested in the metabolic rate of an organism it is important to
determine the animals mass. This is because for animals with similar physiology (e.g.
mammals), as body mass increases metabolic rate increases but not quite as much
(allometric).
Record the mass of both the toad and the mouse so you will be able to determine mass
specific metabolic rate later.
Step # 2 - Establishing a baseline
Using the Gas Sensors (See Appendix for setup schematic explained)
Before setting up the LoggerPro to collect data the CO2 sensor and the O2 sensors must
be calibrated. It is important to calibrate the IRGA while running the calibrate function
within the Logger Pro Program. Similar setups will be used to measure oxygen
consumption and/or CO2 production using gas sensors. The setup, provided by Qubit
Systems, is shown in the Figure to the right.
Calibration of CO2 sensor; proceed as follows:
(1)
Fill a gas bag with compressed air from the laboratory compressed air supply
(orange taps on the side bench). Make sure that the IRGA is switched on, and that
the range switch on the side of the instrument is set on the 0 - 2000 ppm (part per
million) range.
(2)
With Logger Pro running and the IRGA file loaded, a graph will appear on the
screen showing CO2 concentration in units of ppm. Click on EXPERIMENT from
the main menu, and select ‘Calibrate’.
(3)
Click on ‘Sensor Set-up’ at the top of the dialogue box and then on the DIN 1 icon.
‘CO2 Analyzer’ should appear in the Sensor box.
(4)
Select ‘Calibrate’ at the top of the dialogue box, and click on the DIN 1 icon so that
‘CO2 Analyzer’ appears in the Sensor dialog box. Click on ‘Perform Now’. A box
labelled ‘Reading 1’ will appear showing the current voltage output from the IRGA
(Input 1) and the CO2 concentration this corresponds to (Value 1).
(5)
Refer to the diagram showing the configuration of the gas exchange system.
Attach the tubing from the outlet of the gas drying column to the IRGA input port.
Attach the tubing from the other end of the gas drying column to the outlet to the
pump i.e. by-pass the animal chamber.
(6)
Attach the inlet of the pump to the gas bag containing the air. To create a CO 2 free
air stream attach the white CO2 scrubber to the gas line immediately after the
pump and before the flow meter. Turn on the pump and flush CO 2-free air through
the IRGA. Observe the voltage change on the computer screen and wait until it
declines to a steady reading. At zero CO2, this reading should be approximately
0.8 volts. Ignore the digital display on the front of the IRGA if this does not read
zero, since you will be using the computer to collect your data
(7)
When a stable zero voltage reading is displayed, type 0 against ‘Value 1’ and then
click on ‘Keep’. A box will appear labelled ‘Reading 2’ showing the current voltage
output of the IRGA (Input 1) and a CO2 concentration (Value 2).
(8)
Remove the white CO2 scrubber from the line. You will not need it again. Attach
the bag containing compressed air to the inlet of the pump, and wait until a stable
voltage reading is attained. Compressed air contains approximately 400 ppm CO 2.
Enter this ppm value in the ‘Value 2’ box on the screen. Click on ‘Keep’. Again, do
not be concerned about the value shown on the IRGA’s digital display if this does
not read 400 ppm.
(9)
The screen will revert to the original calibration dialog box. Select DETAILS at the
top of the screen, and write down the slope and intercept values in a note-book. If
the computer crashes you will not have to re-calibrate if you know these values.
(10) Click on OK. The IRGA is now calibrated.
Calibration of the O2 sensor; proceed as follows:
Similarly to the CO2 sensor, it is important to calibrate the O2 sensor while running the
calibrate function within the Logger Pro Program.
(1)
Click on EXPERIMENT from the main menu, and select ‘Calibrate’.
(2)
Click on ‘Sensor Set-up’ at the top of the dialogue box and then on the DIN 2 icon.
‘O2 Sensor’ should appear in the Sensor box.
(3)
Select ‘Calibrate’ at the top of the dialogue box, and click on the DIN 2 icon so that
‘O2 Sensor’ appears in the Sensor dialog box. Click on ‘Perform Now’. A box
labelled ‘Reading 1’ will appear showing the current voltage output from the O 2
sensor (Input 1) and the O2 concentration this corresponds to (Value 1).
(4)
Turn on the pump so that room air is pumped through the gas exchange system.
Wait until a steady voltage appears in the Input 1 box, and then type 20.9 into the
value box. (Dry air contains 20.9% O2). Click on KEEP. A box will appear labelled
‘Reading 2’ showing the current voltage output of the IRGA (Input 1) and a CO 2
concentration (Value 2).
(5)
The O2 sensor has an output voltage of zero when it is exposed to O 2-free gas. To
simulate this situation, detach the O2 sensor DIN plug from DIN 2, and observe the
decline in the voltage signal. When this reaches a stable value close to zero, type
0 into the Value 2 box. Click on KEEP.
(6)
The screen will revert to the original calibration dialog box. Select DETAILS at the
top of the screen, and write down the slope and intercept values in a note-book. If
the computer crashes you will not have to re-calibrate if you know these values.
(7)
Click on OK. The O2 sensor is now calibrated.
Step # 3 - Setup the Pump
Begin the experiment by turning on the pump. Air will flow from the reservoir bag
through air pump, the flow meter, the dehydration column and the O 2 sensor then the
CO2 detector. After about 5 minutes, the readings should stabilize. This is the baseline
level of O2 and CO2 in the air in the reservoir. Note the air temperature at the time of the
experiment. Note the flow rate and do not change it during the experiment.
Step # 4 - Preparing the Animal Chambers
While the baseline data is being collected, select a toad or a mouse from the cage.
Place it headfirst into the chamber. Close the chamber, leaving the airport open. Try not
to disturb the animal too much, to ensure metabolism remains close to resting rates.
Weigh the animal at the end of the experiment so it is not disturbed too much during
your initial collection.
Step # 5 - Measuring CO2 production and O2 consumption
Once the gas baselines have stabilized, disconnect the tubing between the flow meter
and the dehydrating column and connect the chamber into the system. The chamber
should be orientated so that the pump that is in the chamber is closer to the drying
column than the flow meter. Make sure the pump is turned on by plugging it in. This
pump ensures a complete mixing of the air in the chamber. Close the airport so no lab
air can enter the chamber. After 30-40 minutes, the O2 and CO2 readings should
stabilize at new levels (CO2 higher, O2 lower). You can stop the program at this point.
Record the difference between the baseline O2 and CO2 concentrations and the final O2
and CO2 concentration. You can now calculate the rate of CO2 production (mg CO2
/min/ g) and O2 consumption (mg O2 /min/g).
Step # 6 - Determining the effects of temperature on metabolic rate
To determine the effects of temperature you will be repeating the experiment with an ice
pack cooling the animal chamber. Make sure you get a good base line reading before
you insert the chamber and the test subject. Again run the experiment for 30-40
minutes. Record the readings with and without the animals.
Carefully remove the animal and return it to its appropriate cage after the experiment.
Clean out the tube and ensure BOTH pumps are turned off before you leave.
If time permits, repeat the measurements on the same animals to get an idea of
reproducibility.
Questions: How does the total metabolic rate of the toad compare to that of the
mouse? What happens when you correct for body mass (i.e., their mass-specific
metabolic rates)? The toad is a poikilotherm, with a body temperature near
ambient temperature. Using a Q10 value of 2, calculate what the toad metabolic
rate would have been if the ambient temperature was 40oC (near that of the
mouse). Plot the metabolic rate of the toad versus temperature. Using the van’t
Hoff equation, calculate the Q10 value between the higher two temperatures. What
was the Q10 based on the lower two temperatures?
With the measurements of gas exchange, you will be able to calculate the metabolic
rate of the animal.
Total Oxygen Consumption
O2 consumption
(mg O2 / min)
=
Change in [O2]
(mg O2 / litre)
x
Air flow
(litre / min)
Total Carbon Dioxide Production
CO2 production
(mg O2 / min)
=
Change in [CO2]
(mg O2 / litre)
x
Air flow
(litre / min)
Mass-specific Oxygen Consumption
O2 consumption
(mg O2 / min/ g mass)
=
Change in [O2]
(mg O2 / litre)
x
Air flow
(litre / min)
/
animal mass
(g)
/
animal mass
(g)
Mass-specific Carbon Dioxide Production
CO2 production
(mg O2 / min/ g mass)
=
Change in [CO2]
(mg O2 / litre)
x
Air flow
(litre / min)
Questions:
-How does the total metabolic rate of the toad compare to that of the
mouse?
-What happens when you correct for body mass (i.e., their mass-specific
metabolic rates)?
-The toad is a poikilotherm, with a body temperature near ambient
temperature. Using a Q10 value of 2, calculate what the toad metabolic rate
would have been if the ambient temperature was 40oC (near that of the
mouse). Plot the metabolic rate of the toad versus temperature. Using the
van’t Hoff equation, calculate the Q10 value between the higher two
temperatures. What was the Q10 based on the lower temperatures?
-What are the respiratory quotients (RQ) of the experimental animal at two
different temperatures? What metabolic fuels are these animals using based on
your RQ values?
References and Recommended Reading
Principles of Animal Physiology CD Moyes and PM Schulte (2008)
 Box 2.2 Methods and Models Systems. Metabolic Rate (p 62)
 the basis of the respiratory quotient can be found in Chapter 2 (pages 62-63)
 Box 13.2 Mathematical Underpinnings (p 640)
 thermal strategies and the influence on metabolic rate are discussed in Chapter
13