LABORATORY: TEMPERATURE AND RESPIRATION OF TOADS AND MAMMALS

Transcription

1 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 (O 2 ), and produces heat, carbon dioxide (CO 2 ) 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 O 2 consumption or CO 2 production. O 2 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, O 2 consumption and CO 2 production depends on the nature of the metabolic fuels. In indirect calorimetry, the ratio of CO 2 produced to O 2 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 = a M b 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 Q 10 value.

2 Q 10 RMR RMR T T 1 Where RMR 1 and RMR 2 are the routine metabolic rates at the respective temperatures T 1 and T 2. Questions: Where does CO 2 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) O 2 consumed, and (iii) CO 2 produced? In this exercise, you will measure O 2 consumption and CO 2 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.

3 Step # 2 - Establishing a baseline Using the Gas Sensors (See Appendix for setup schematic explained) Before setting up the LoggerPro to collect data the CO 2 sensor and the O 2 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 CO 2 production using gas sensors. The setup, provided by Qubit Systems, is shown in the Figure to the right. Calibration of CO 2 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 ppm (part per million) range. (2) With Logger Pro running and the IRGA file loaded, a graph will appear on the screen showing CO 2 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. CO 2 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 CO 2 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 CO 2 concentration this corresponds to (Value 1).

4 (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 CO 2 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 CO 2, 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 CO 2 concentration (Value 2). (8) Remove the white CO 2 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 O 2 sensor; proceed as follows: Similarly to the CO 2 sensor, it is important to calibrate the O 2 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. O 2 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 O 2 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 O 2 concentration this corresponds to (Value 1).

5 (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% O 2 ). 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 O 2 sensor has an output voltage of zero when it is exposed to O 2 -free gas. To simulate this situation, detach the O 2 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 O 2 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 CO 2 detector. After about 5 minutes, the readings should stabilize. This is the baseline level of O 2 and CO 2 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 CO 2 production and O 2 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 minutes, the O 2 and CO 2 readings should stabilize at new levels (CO 2 higher, O 2 lower). You can stop the program at this point. Record the difference between the baseline O 2 and CO 2 concentrations and the final O 2 and CO 2 concentration. You can now calculate the rate of CO 2 production (mg CO 2 /min/ g) and O 2 consumption (mg O 2 /min/g).

6 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 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 Q 10 value of 2, calculate what the toad metabolic rate would have been if the ambient temperature was 40 o C (near that of the mouse). Plot the metabolic rate of the toad versus temperature. Using the van t Hoff equation, calculate the Q 10 value between the higher two temperatures. What was the Q 10 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 O 2 consumption = Change in [O 2 ] x Air flow (mg O 2 / min) (mg O 2 / litre) (litre / min) Total Carbon Dioxide Production CO 2 production = Change in [CO 2 ] x Air flow (mg O 2 / min) (mg O 2 / litre) (litre / min) Mass-specific Oxygen Consumption O 2 consumption = Change in [O 2 ] x Air flow / animal mass (mg O 2 / min/ g mass) (mg O 2 / litre) (litre / min) (g) Mass-specific Carbon Dioxide Production CO 2 production = Change in [CO 2 ] x Air flow / animal mass (mg O 2 / min/ g mass) (mg O 2 / litre) (litre / min) (g)

7 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 Q 10 value of 2, calculate what the toad metabolic rate would have been if the ambient temperature was 40 o C (near that of the mouse). Plot the metabolic rate of the toad versus temperature. Using the van t Hoff equation, calculate the Q 10 value between the higher two temperatures. What was the Q 10 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