Animal Physiology

Biology 4321

Lab Sec: Tue Aft Thur Aft Name  

Partners:

METABOLISM

We will measure the metabolic rate of a rodent by measuring the pressure changes it causes in a chamber as it uses oxygen.

1. Put fresh NaOH or KOH (10-20 pellets) in the bottom of the Plexiglas urimeter chamber. Sodium hydroxide is used to absorb CO2 exhaled by the animal. Why do we need to do this? Avoid getting the strong base on yourself or the animal; if you feel a soapy sensation on your hands then wash immediately. Put the wire cage over the NaOH. Insert the black rubber stopper into the opening of the Plexiglas chamber to make a tight seal. Running a little water around the stopper before sealing often helps make a tight seal.

2. Check that the urimeter/syringe setup is hooked up correctly. Ask the instructor.

3. Check that the ≥Biology Gas Pressure≤ transducer is plugged into channel 1 (CH1) of the Lab Pro analog to digital converter which should be plugged into the USB port of the computer.

4. Start Logger Pro3 by double clicking the alias file ≥Open LP3.cmbl≤ on the desktop. If a ≥Sensor Conformation≤ screen appears, select OK in the lower right corner. If all is ok, you should see a green box with a white arrow at the upper right.

5. We want to convert the voltage readings of the sensor to millimeters of gaseous oxygen. We will do a two point calibration:

a. Unplug the tubing from Pressure Sensor. Push the syringe all the way in to a reading of 0 ml.. Reconnect the tubing to the Pressure Sensor.

b. Under menu item ≥Experiment≤ slide down to ≥Calibrate≤ and then to the sensor identification to the right.

c. Select ≥Calibrate Now≤

d. Change the units to ≥ml≤.

e. Note that it is telling you the current voltage that it is reading under ≥Channel Input≤. (Mine reads 0.96 volts).

f. Your syringe is set at 0 ml so your enter value should be set to 0 ml.

g. Select ≥Keep≤.

h. For the second point calibration, pull out the syringe to a reading of 10 ml. Your voltage reading should change (mine read 0.83 volts). If you see no change, you have a leak. Get help form the instructor.

i. Type in 10 ml for the ≥Enter value≤ and select ≥Keep≤ and then select ≥Done≤.

6. Change the label ≥Potential≤ to ≥Oxygen≤ by going to menu item Data => Column Option => Potential. Change the word ≥Potential≤ to ≥Oxygen≤, the Short Name to O2 and select ≥Done≤

7. In the upper left look at the reading for oxygen. Slide the syringe to different values to see if your calibration was correct. Plus or minus 10% accuracy is fine.

8. Adjust the Y and X axis scales: Click once on the graph and then select Options => Graph options and then select tab ≥Graph Options≤. Set the range of the x-axis to 0 to 5 min and the range of the y axis to 0 to 10 ml and select Done. (Alternatively you can do this by selecting the numbers right on the graph and putting in the desired values.)

9. Set up the proper data collection rate: Select Experiment => Data Collection. Set ≥Length≤ to 5 minutes and ≥samples per min≤ to 120.

10. Try a trial run without a rodent to see that you get a steady signal and that when you move the syringe that you get the expected changes. To collect data, select the arrow in the green box in the upper right corner.

Measuring the basal metabolism of your animal:

1. Acquire a mouse or small or medium rat (careful, they can and do bite) and set it into the chamber. It should not be able to touch the NaOH. (Note: it is easier to let the animal crawl up into a chamber at a 30∞ angle rather than trying to drop it into the chamber).

2. With either the syringe or the transducer tube removed (to avoid creating a negative pressure), insert the black rubber stopper to make a tight seal. Reattach the tube that you removed.

3. Record. (The animal will move, but if it seems in distress, then remove immediately.)

4. When done, measure the weight of the rodent plus the chamber. Remove the animal and weigh the chamber. Subtract to determine the mass of the animal.

5. Determine the temperature (why?)

Calibrate system for fish metabolism measurement:

1. Suck out the fluid in the oxygen electrode and put in deionized water.

2. Check that the oxygen electrode is plugged the amplifier, the amplifier is plugged into channel 1 (CH1) of the Lab Pro analog to digital converter, and the LabPro is plugged into a usb port of the computer. The amplifier should be set to 0-20 mv mv.

3. Start Logger Pro3 by double clicking the alias file ≥Open LP3.cmbl≤ on the desktop. If a ≥Sensor Conformation≤ screen appears, select OK in the lower right corner. If all is ok, you should see a green box with a white arrow at the upper right.

4. We want to convert the voltage readings of the sensor to micromolar of oxygen. We will do a two point calibration:

a. Pull out the red connection banana plug wire going into the amplifier.

b. Under menu item ≥Experiment≤ slide down to ≥Calibrate≤ and then to the sensor identification to the right.

c. Select ≥Calibrate Now≤

d. Change the units to ≥µM≤ (Pressing option m gives you µ).

e. Note that it is telling you the current voltage that it is reading under ≥Channel Input≤. (Should be around 0 volts).

f. Your electrode is unplugged, so your enter value should be set to 0 µM.

g. Select ≥Keep≤.

h. For the second point calibration, plug back in the red connection banana plug wire going into the amplifier. Your voltage reading should change. If you see no change, there is a problem. Get help form the instructor.

i. Deionized water has a concentration of 246 µM O₂. Type in 246 µM for the ≥Enter value≤ and select ≥Keep≤ and then select ≥Done≤.

5. Change the label ≥Potential≤ to ≥Oxygen≤ by going to menu item Data => Column Option => Potential. Change the word ≥Potential≤ to ≥Oxygen≤, the Short Name to O2 and select ≥Done≤

6. In the upper left look at the reading for oxygen. It should read 246 µM O₂ plus or minus 10% accuracy is fine.

7. Adjust the Y and X axis scales: Click once on the graph and then select Options => Graph options and then select tab ≥Graph Options≤. Set the range of the x-axis to 0 to 5 min and the range of the y axis to 0 to 250 µM and select Done. (Alternatively you can do this by selecting the numbers right on the graph and putting in the desired values.)

8. Set up the proper data collection rate: Select Experiment => Data Collection. Set ≥Length≤ to 5 minutes and ≥samples per min≤ to 120.

9. Try a trial run without a fish to see that you get a steady signal and that when you you turn off the stirrer the signal drops. To collect data, select the arrow in the green box in the upper right corner.

MEASURE THE OXYGEN UPTAKE OF THE MINNOW.

1. Put in a SPECIFIED amount of deionized water in the cuvette. Record that amount

2. Find a SMALL minnow and add it into the cuvette.

3. Put the plunger in so that there are no bubbles (good luck).

4. Start the Data Logger data gathering and charting. Go until the system is depleted of oxygen or until the minnow is no longer utilizing oxygen (Note that the animal is under a different kind of stress as the experiment goes on, i.e., reduced levels of oxygen. It is only at the beginning when the animal has a 'normal' oxygen supply, therefore the basal metabolism is best measured at the beginning.)

5. Remove the minnow, carefully dry it, and weigh it.

6. As the oxygen concentration goes down, the minnow will take in less. Therefore, for your calculations of the basal rate, you want the initial highest rate. Do this by drawing a tangent through the oxygen utilization curve where it appears you have the steepest slope in the beginning and measure the slope to determine the rate. (If you want, I can show you how to do this using LoggerPro, but also make sure you know how to do it manually).

7. Determine the temperature (why?)

You may find it useful to dig out the 'yeast' experiment you did in Cell Biology for conversions etc. It is also on the Cell Bio web site.

III. DO ANY ONE OF THE FOLLOWING THINGS:

1. Measure the respiratory quotient of rodent.

2. Measure the basal metabolism of another animal.

3. Measure the basal metabolism of a minnow at different temperatures.

4. Measure the metabolism of rodent at a high and at a low temperature.

5. Plot the metabolic rate of a minnow as a function of the oxygen concentration.

6. Use air oxygen electrode

7. Use carbon dioxide air electrode

8. Any other creative idea.

FOR YOUR REPORT:

SHOW ALL CALCULATIONS:

Hand in your chart for the minnow recording as a group.

Do your report as a group or as individuals. You can checked off for correctness of questions (like was done in Cells) befoe turning in anything.

(Assume that an average human (you) utilizes 2000 Kcal per day.)

You only need to do what is asked below - e.g. you do not need a separate purpose, introduction, methods section.

1. WRITE DOWN the chemical reaction that would allow NaOH to 'absorb' CO2.

2. Determine the rate of oxygen utilization for the minnow. (Remember that your electrode is measuring in micromolar and you first need to convert to micromoles.) Turn in your computer chart showing how you made calculations from the chart or the computer determined slope.

3. Report in A TABLE the basal metabolic rate of the minnow, rodent and human in all of the following different units:

a) ml O2 min.-1 b) ml O2 min.-1 Kg-1

c) Kcal day-1 d) Kcal day-1 Kg-1

e) J hr-1

Also include the masses in your table. Identify the temperature(s) that were used.

Use footnotes directly below the table to note any assumptions that you made in any calculations. (there are quite a few)

4. DISCUSS the differences and/or similarities that you see between human, rodent, and minnow basal metabolism data that you have obtained. DISCUSS whether or not these comparisons are what you expect from what you have learned in class and from your text.

Compare actual numbers using the appropriate equations, tables or graphs presented in the text.

5. For the one extra thing that you did, appropriately PRESENT THE DATA (include as part of the table under #3 if it is appropriate) and DISCUSS it.