Metabolic Rate Lab. 1 Introduction. 2 Background Information. Bi253 PSU. April 2013

Transcription

1 Metabolic Rate Lab 1 Introduction Bi253 PSU April 2013 In this lab we will examine the difference in metabolic rate between an endothermic animal (mouse, Mus musculus) and two ectothermic animals, a vertebrate (the goldfish, Carassius auratus) and an invertebrate (the crayfish Procambarus clarkii). The idea of the experiment is to change the ambient temperature and see how that affects the metabolic rate of the animal. 2 Background Information Endothermic animals use both metabolic and behavioral means to maintain a constant body temperature. Under most circumstances there is a nominal speciesspecific preferred body temperature. In mice this is between 30 and 31.5 C. At this temperature the mouse is comfortable and does not have to expend any excess energy to maintain its preferred body temperature. If the ambient temperature is lowered the mouse will begin to shiver, a type of muscle activity that does not perform any useful work, but does make waste heat. If the mouse is warmed, it will try to cool off by spreading saliva on its fur. The evaporating saliva takes away quite a bit of heat. At an even higher ambient temperature the mouse will spend much time hunting for a cooler spot. Saliva spreading and moving to a cool spot both take muscle activity, and so cost energy. Increased muscle activity results in an increased metabolic need, thus there is an energetic cost to the mouse to stay warm or cool when the ambient temperature changes from the preferred temperature. The energetic cost is is reflected in an increased metabolic rate, i.e., a mouse at 25 C or at 35 C will consume more oxygen and produce more carbon dioxide than one between 30 and 31.5 C. The range where the mouse expends the least amount of energy is the thermo-neutral zone. Contrasting with the mouse is the change in metabolic rate with temperature for an ectotherm. The crayfish and goldfish are both ectothermic and poikilothermic. That means that they do not have a metabolic means to maintain constant body temperature and furthermore, they make little or no attempt to maintain a constant body temperature by behavioral means. Thus you might expect that such animals would exhibit a relatively lower metabolic rate for a low ambient temperature and a progressively higher metabolic rate for increasing ambient temperature. In other words, the body temperature of the animal will track the environmental temperature. Higher temperatures should lead to greater oxygen consumption and carbon dioxide production. Larger animals will consume oxygen and produce carbon dioxide at a higher rate than smaller animals, 1

2 because they have more cells and tissues to support. Thus in comparing different animals it is important to normalize the measure of metabolic rate to body mass. A typical amount of oxygen consumption for a 25 g mouse is 0.41 liters per hour. To compare this to another kind of animal, or even to a mouse of a different mass, we would report the oxygen consumption as (this is a typical value) 1.65 liters CO 2 /kg-h, or 1.65 liters per kilogram of mouse per hour. As part of your lab report you will get to look up the metabolic rate for the ectotherms. 3 Overall Method dioxide production, you can save the experimental data file. The CO 2 level will steadily rise, and the slope of this line is the CO 2 production rate, i.e., per unit time. 6. Between experiments it will be necessary to flush out the animal container and the CO 2 probe with fresh air. 7. After all the experiments are conducted you should be able to make a graph of CO 2 production per minute per gram of animal (CO 2 min 1 gram 1 ) versus ambient temperature, for each type of animal. Each of the three animals will be tested under six different temperature regimes; two at 35 C, two between C, and two at C. The overall concept of the experiment is as follows: 1. You will take a mouse, goldfish, and crayfish and, in turn, weigh them. 2. Then you will place the animal in a container and attach the lid. The container will be made airtight after both temperature and CO 2 probes are inserted through holes in the container s lid. Thus all the respiratory CO 2 made by the animal will be trapped in the container and read by the CO 2 probe. 3. The container is kept at either room temperature or in a water bath that is either warmed with hot water or cooled with ice. 4. Only after the ambient temperature has stabilized will you be ready to insert the carbon dioxide probe into the container through the lid. 5. After some minutes using the computer to simultaneously record both temperature and carbon 4 Detailed Experimental Procedure There are some important things to keep in mind for this experiment to run smoothly. First, the CO 2 probes are a bit temperamental. If you give the probe a heavy dose of CO 2, for example by breathing on it, you will have prolonged unstable readings - the probe takes a long time to clear. The easiest way to clear the probe is to wave it around in the room air. You will want to make sure that you have a stable baseline before testing an animal. Both the crayfish and the goldfish should be in water! The amount of water should be the minimum amount to submerge the animal, and it should be aquarium water, not distilled water and not tap water. The CO 2 probe should not come into contact with the water. On the other hand, mice hate to be in water. So you will want to dry out the container before adding the mouse, or just do the mouse first. Mice like a bit 2

3 of structure in their environment - paper towels, cotton balls and a place to hide will make the mouse more likely to be calm. The more active any of the animals become, the less their metabolic rate depends on temperature and the more it depends on muscle activity and the rapid metabolism induced by stress, anxiety and fear. Under no circumstances should you treat any of the animals to promote a state of fear or anxiety. 1. Open the (metabolism) icon on the desktop; the parameters are already set. If not present, ask your TA to show you how to get started with the Vernier software. Observe the CO 2 value at the bottom right hand corner of the window to ensure that it produces a value in the range of ppm. 2. The different temperature regimes will be achieved using water baths adjusted to keep water at a particular temperature. The water baths take a while to adjust and stabilize so you should not change their temperature settings. The procedure for the fish and the crayfish is slightly different than for the mouse. Please read through carefully before starting any work. 3. MOUSE PROCEDURE. (a) Place the testing chamber on a balance and then tare the balance to zero the reading. (b) With the assistance of your TA, drop in a mouse and record the value. (c) Transfer the testing chamber with the mouse to the appropriate water bath - cold, room temperature or warm. Make sure the lid is sealed properly and make sure the probe is sealed into the lid. Any air leaks will interfere with obtaining good data values. (d) Start the Vernier recording and wait for the temperature to stabilize. At this point you can start watching the rise in CO 2. You do not want to leave the mouse for very long: It will use up the O 2 and generate too high levels of CO 2 if left. Mind your TA about the maximum time. (e) After recording, your TA will assist in returning the mouse to its home box, and you can begin to analyze the data. 4. FISH AND CRAYFISH PROCEDURE. The goldfish cannot be transferred from water of one temperature to water of another temperature instantly. The shock is not good for it. You must bring the goldfish to the desired temperature slowly, and then return it to the home tank temperature the same way. The crayfish is more tolerant, but we will use the same technique for both aquatic organisms. (a) Use a small container to scoop up some aquarium water from the home tank holding the fish or crayfish. Put this container on the balance, and tare. (b) Use a dip net or other means (for the crayfish) and transfer from the home tank to the small container. Record the balance reading. (c) Add a few inches of home aquarium water to the metabolic testing chamber and pour the fish from the small container into the testing chamber. All of this water will be at the same temperature: Room temperature of about 21 C. Thus you will always start your test with the goldfish or crayfish at their home tank temperature. (d) Put the testing chamber with the fish into the appropriate water bath and start the Vernier 3

4 recording. You will wait for the temperature to stabilize. Once the temperature has stabilized you can mark the time for starting your observation of the increase in CO 2 over time. The goldfish and crayfish should be left in the testing chamber for longer than the mouse, and there is little concern they will use up all the O 2. (e) After the test trial is over, pour the fish with the water into an empty small container. The fish and water are at the test temperature which may be quite different from the home tank temperature. Float the small container in the home tank for at least 20 minutes, or until the water temperature in the small container matches that in the home tank. Then tip the small container so that the fish can swim out. 5. After the data are collected, obtain the slope of the line of CO 2 - the slope is the rate of production using the slope function in the veneer software package. Record the slope into the appropriate data table. 6. Follow the same procedures as outlined above for a different temperature regime. Before continuing with the next experiment, it is necessary to flush out the clear testing chamber and the CO 2 probe with fresh air until readings return to the normal baseline value. 5 Data Analysis To compare endotherm and ectotherm CO 2 production rates, you must normalize the values. To do this, you must take the slope of CO 2 production per unit time and divide this by the animal s mass (CO 2 min 1 gram 1 ). This information is most easily interpreted when graphed. Produce a graph where the dependent variable (y-axis) is the normalized CO 2 production values (CO 2 min 1 gram 1 ) and the independent variable (x-axis) is the temperature. You should know that it is a lot more energetically expensive to be an endotherm than to be an ectotherm. For your report you will want to compare the graphs of CO 2 min 1 gram 1 for an endotherm versus an ectotherm and explain the differences in the shape of the graphs. Search the scientic literature for some basal metabolic rate values for the animals tested, or for similar animals and compare them to your values in your discussion section as well. 6 Discussion Questions 1. Define the following: (a) ectotherm (b) endotherm (c) poikilotherm (d) homeotherm. 2. Why do large endotherms have lower metabolic rates, that is, they generate less CO 2 g 1 min 1, compared with small endotherms. 3. Give some costs and benefits for ectotherms versus endotherms. Think of this in terms of types of environments each can exploit, length of activity each can endure, thermoregulation, energetic costs and opportunity for reproduction. 4. Define thermo-neutral zone. 4

5 5. Why was it necessary to convert metabolic rate (CO 2 min 1 ) into metabolic rate per kilogram of animal (CO 2 g 1 min 1 )? A parenthetical comment on heating up goldfish. The question may arise, just how hot can you make a goldfish? Of course we would not want to stress or kill the animals in this lab, so we make sure the temperatures are not so high... but how do we know? The maximum temperature for goldfish is not a fixed value, but depends on acclimation of the animal. In one study (Ford & Beitinger 2004) it was found that acclimation temperature accounted for 90% of the variance in temperature tolerance. The ultimate thermal minimum and maximum temperatures were found to be 0.3 and 43.6 C, respectively. The formula to determine the critical thermal maximum, CT max is: CT max = *(acclimation temperature) For goldfish acclimated to room temperature water, CT max would be *20 = 37 C. 7 Data Tables Use the tables below to record your data from each animal species tested. 5

6 ANIMAL: Temperature Animal Mass Slope Normalized CO 2 Production ANIMAL: Temperature Animal Mass Slope Normalized CO 2 Production 6

7 ANIMAL: Temperature Animal Mass Slope Normalized CO 2 Production 7