MOUSE METABOLISM

Objectives:

 

1) Understand the effect of body size on metabolic rate.

2) Understand the relationship between oxygen consumption and metabolic rate.

3) Understand relationship between surface area and volume.

Introduction

A Metabolism Chamber will be used to measure oxygen consumption in mice. The cylindrical plexiglass chamber is equipped with a removable cage to which a thermometer is clipped. Once a mouse is sealed in the chamber, it consumes oxygen and exhales carbon dioxide, which is absorbed by soda lime placed in the bottom of the chamber. Therefore, as oxygen is used, the pressure inside the chamber drops, which causes a soap film on the end of a calibrated tube to be drawn toward the chamber. The time required for the soap film to traverse a given distance of the tube is a measure of oxygen consumption rate (mls/sec). Since homeothermic (warm-blooded) animals, on the average, produce 4.8 calories of heat for each ml of oxygen consumed, metabolic rate can be determined from oxygen utilization rate.

In this experiment we will test one of two possible hypotheses: the first is to determine the effect of size on the metabolic rate of an organism.

You will calculate metabolic rate from your measurements of the oxygen consumption rate for mice (15-20 g) and for rats (100-150g). Oxygen consumption will be estimated by using the small animal metabolism chamber (the set up is shown below).

In order to determine the metabolic rates for each animal, you will need to calculate the metabolic rate using the oxygen consumption rate you measure in class. Each group will do one rat and one mouse.

 Lab Setup

We will pool the class data, so that we can use statistics to determine whether the differences that we have measured between the two groups is statistically significant.

Lab Setup---

Materials and Methods:

All experiments should be initiated by examining the apparatus and carefully testing it for leaks. The procedures for determining metabolic rate are given below:

1. Cover the bottom of the metabolism chamber with a thin layer of soda lime so that all exhaled carbon dioxide will be absorbed, differentially allowing for the measurement of oxygen consumption, only. CAUTION: Do not touch this caustic chemical or allow the animals to touch it.

2. Accurately weigh the wire cage, place a mouse in the cage, and reweigh both. Determine the mouse's weight by difference. (HINT: A spacing device, such as a cardboard tube, placed in the cage will confine the animal, minimizing physical activity.)

3. Clip a centigrade thermometer onto the cage and place the cage in the chamber. Wet the inside of the calibrated 5 ml tube with water. This procedure will later reduce the possibility of the soap-bubble seal drying out and breaking during migration. Wet the rubber stopper to insure a tight seal on the metabolism chamber. Insert the rubber stopper, which contains the tube, into the chamber. Allow the animal to remain in the chamber for about 10 minutes for temperature equilibration.

4. Seal the chamber by applying a drop of bubble solution to the end of the pipette. Often the foam from shaking a dishwashing detergent or toy soap bubble solution makes a satisfactory bubble.

5. Use a stopwatch to determine the time in seconds required for the soap bubbles to traverse a distance along the pipette equivalent to exactly 5 ml. Practice this technique until the measured time interval appears consistent. Repeat the procedure for a total of 3 trial runs. If inconsistencies occur, look for the following sources of error:

a. leaks in the system

b. insufficient or CO2-saturated soda lime

c. dirty or blocked pipette, or

d. failure to sufficiently wet the inside of the pipette.

e. calculate the total number of seconds required for the animal to consume 15 mL of oxygen (5 mL * 3 consistent trials). The following will serve to illustrate:

Measurement Number

Time Required in Seconds

1

105

2

95

3

100

Total time

300 seconds (5 minutes)

6. Remove the cage containing the animal from the metabolism chamber as soon as the necessary measurements have been obtained. Mice usually run out of the cage on their accord when the ends are removed and the cage is brought close to their original "home" cage.

 

7. The following equation, when entered into the appropriate cell in an Excel spreadsheet, will calculate metabolic rate for you. Once you enter the equation in the top field for metabolic rate of mice, click on that cell and highlight all the other appropriate cells beneath, then press "Ctl D" The equation will automatically be entered into the other cells. Once you have the equation in all of the cells for mice, select those cells, copy them, then paste in the metabolic rate column for rats. The "=" sign tells Excel that what follows is an equation. If you wish, you may copy the equation and paste it into Excel; however, you must supply all information in the square brackets "[red]". Be sure to remove the brackets, but not the parentheses. The best way to do this is to select everything in a given set of brackets (including the brackets), then click on the cell that contains the needed data.

=15/[total sec for 3 trials]*273/(273 + [cage temperature in degrees C]) * [Barometric Pressure]/760 * 60 * 60 * 4.8/1000 * 1/(0.437 + (2.143 * [wt of mouse])) * 10000

8.The first step is the measured time (in seconds) required for the animal to consume 15 mL of oxygen (5 mL * three trials).

 

9. In comparing results of experiments performed under different environmental conditions, it is usually necessary to calculate some corrections. All observed gas volumes (oxygen) should be corrected to the volume that would have been observed under conditions of standard temperature (degrees C) and pressure (760 mm Hg). This correction is made using the next two steps in the equation (steps 2 and 3). A more detailed explanation follows.

Vcorr = Vobs x 273/(273 + T degrees C) x (Barometric Pressure/760)

Where:

Vcorr = corrected volume in mLs  (indicates the mL of oxygen consumed by the animal under standard conditions.)

Vobs = observed volume of oxygen consumed in mLs

B.P. = barometric pressure in mm Hg

T degrees C = temperature measured in the cage in degree Celsius.

 

10. Since metabolic rate determinations are based on a one-hour time interval, the number of calories produced will be multiplied by 60sec/min x 60 min/hour (the next two steps; steps 4 and 5).

 

11. You may assume that each normal animal releases 4.8 gram calories (cal) of heat for each mL of oxygen consumed. Therefore heat production can now be calculated from oxygen consumption by multiplying the volume of oxygen used by 4.8 cal/mL of oxygen consumed (step 6). However, metabolic rate is typically expressed as Cal, and there are 1,000 gram cal/Cal, so divide by 1,000.

 

12. Finally, metabolic rate is related to that animal's surface area. You can find the surface area in square centimeters (cm2) (in this case) with the following equation, which can only be used for small animals, since the relationship is not truly linearly except for a small range of weights (step 7)

y = b + mx
where

y = surface area in cm2

b = 0.437 (the "y" intercept)

m = 2.143 (the slope)

x = weight of the animal

For example, for a 40 g animal, the surface area can be calculated: y = 0.437+ 2.143(40)

surface area in cm2 = 86.2 cm2

 

13. Metabolic rate is usually expressed as Cal/hr/M2, so we must convert cm2 to M2. There are 10,000 cm2/M2, so multiply by 10,000 (the last step; step 8).

 

14. Now complete the Lab Worksheet.

Link to metabolism write-up.