Promethion Metabolic Treadmill – Experiment Protocol

Experimental protocols often center around a key question as part of a more comprehensive research study. Assuming we’re working on a larger exercise physiology study, one question could be “when running at a steady speed, what is the effect of inclination on animal energy expenditure?” This is a clear, narrowly focused question that can be addressed by a well-designed protocol.

In short, the experiment will measure mouse metabolic rates at rest, measure mouse running metabolic rates, measure running metabolic rates at a 20-degree incline and measure post-running metabolic rates. Do we see any oxygen recovery?

These steps give a basic protocol outline. This will be refined in light of the treadmill procedures. We will use the treadmill’s Profile feature to generate a series of timed command steps to automate the protocol.

• Start with a fully assembled CGF integrated Sable Systems Metabolic Treadmill.
• Turn the treadmill on and insert an SD card in the front slot.
• Press SETUP and PROFILE to navigate to the profile edit page. We’ll use this to generate a template profile.
• Press the right arrow twice to add two more steps.
• Press ENABLE to activate the profile and then press BACK twice to return to the main page
• Press FILES and right arrow to select Profile 5.
• Press SAVE PROFILE to save the template profile to the SD card then hit BACK again.
• Remove the SD card and transfer it to a computer to edit the template profile in a spreadsheet.

REFINING THE PROTOCOL

We’ll demonstrate this schematically by adding steps and step details. Everything can be edited in the profile template. The research question for this example is “when running at a steady speed, what is the effect of inclination on animal energy expenditure?” Structure the questions as steps in a profile. A breakdown of the question requires us to essentially measure mouse metabolic rate at various states.

Based on those criteria, step 1 will be an empty treadmill monitored for 5 minutes or however long you like, at zero meters per minute with an inclination of zero degrees.

Step 2 is where we put the mouse in the treadmill and give it some time to equilibrate in this new environment. Because of the air disturbance caused in the treadmill chamber by inserting the mouse, we can pause the profile at step 2 until air parameters CO₂ and O₂ stabilize. Then resume the profile and allow the mouse 5 minutes to equilibrate at zero meters per minute and zero inclination.

In step 3, we start the belt running. This immediate transition between a stationary step 2 and a running step 3 means we utilize the treadmill’s default acceleration for belt running. If the default acceleration rate doesn’t suit your needs, you could insert a new step between steps 2 and 3 to specify the acceleration rate required. For this initial run in step 3, we choose a duration of 2.5 minutes at a speed of 15 meters per minute and zero inclination.

Return to your research question. The key part asks about the effects of inclination on energy expenditure. So, in step 4, we keep the mouse running at 15 meters per minute and add an inclination of 20 degrees. Continue this for 5 minutes. To begin the test subject’s cooldown, in step 5 we keep the mouse running at 15 meters per minute and return the inclination of zero degrees. Maintain this again for 2.5 minutes. Then in step 6, we let the mouse rest a bit before removing it from the treadmill. We used a trial period of rest for 5 minutes at zero meters per minute and zero inclination. In this time, we can also assess the extent of oxygen recovery after 10 minutes of variable activity.

To end the profile, we’ll add another reading of the empty treadmill after the mouse has been removed. That makes step 7 an empty treadmill monitored for 5 minutes at zero meters per minute and zero inclination, and again we can also pause the profile at step 7 to allow CO₂ and O₂ to stabilize after the disturbance caused by removing the mouse. Resume the profile once the air measure is stabilized.

To reiterate, in this profile we transition immediately from rest zero meters per minute to running 15 meters per minute between step 2 to step 3. In this case, we use the treadmill internal default acceleration to transition between running speeds. This saves a step. If you need a custom acceleration, it can be done by adding an additional step and entering the start and end speed across an appropriate time period to generate the required acceleration.

Now that we have a clearly defined profile, we can edit the template in a spreadsheet.

• In the spreadsheet application, open Files and access the SD card.
• Select to open All Files to access the template profiles csv files.
• Open both PROF05.csv and STIM05.csv.
• The STIM05.csv file shows all the treadmill stimulus settings.
• The stimuli encouraged the mouse to run. These can be edited or left in the default settings.

Now to edit the template profile according to our refined protocol schematic. Each of the 7 steps and its parameters will be replicated in the csv template.

• Step 1: Set 5 minutes, no speed nor inclination, for the empty chamber.
• Step 2: Introducing the mouse. Five minutes, no speed nor inclination.
• Step 3: Starting the mouse run for 2.5 minutes at 15 meters per minute, no inclination. Take note, the speed is set directly to 15 meters per minute, thus the transition uses the default acceleration.
• Step 4: Running the mouse at a 20-degree incline for 5 minutes; the speed remains at 15 meters per minute.
• Step 5: Returning the mouse to a zero-degree incline for 2.5 minutes and still at 15 meters per minute.
• Step 6 (the recovery step): Stop the belt and allow for 5 minutes of oxygen recovery.
• Step 7: Removing the mouse and measure another 5 minutes of an empty chamber.

Using an empty chamber at the start and the end is a good way to verify the treadmill’s respirometry measures. Once the new profile’s edits are complete, save it on the SD card in csv format. Reinsert the SD card to load the profile on the treadmill.

• Click FILES and select the profile number (5 in this case).
• Then select SD card to load the profile.
• The info bar will display “Profile and stimulus #05 loaded, 7 steps.”
• Hold the BACK button to return to home screen and click NO PROFILE. It will now state “Using Profile 5, steps: 7, 30 minutes.”

EXECUTING THE EXPERIMENTAL RUN INCLUDING A CGF AND A TREADMILL DATA RECORDING

• To customize the treadmill data file name, click FILES.
• Press DATA to edit.
• The keyboard will appear. Delete the default name DATA and enter a custom name (example “test”); limit it to 6 characters.
• All treadmill recorded data files will be named testxxxx (the latter four characters will be numerals). Hold BACK to return to Home Screen.

STARTING THE EXPERIMENTAL RUN

Make sure to capture all the relevant events. Also make sure all systems and settings are in good order. To ensure all events are captured, follow this sequence: 1) start a CGF recording first – verify the recording is active 2). Start the treadmill data recording, and 3). Start the profile.

Ensure that the treadmill and the CGF are in good order and you’re ready to go.

Start the CGF recording.

• In Promethion Live homepage, click RECORD, customize the file name, and click SAVE. The Promethion Live state will change from READY to HARVESTING DATA.
• And data acquisition info will be displayed. The CGF recording is in progress.
• Click the RECORD button to start the treadmill data recording. This will change the home screen by omitting some menu items and displaying “Recording SD file” and the file name.
• Press the large green RUN button to start profile 5. This will immediately activate step 1 and the PAUSE and STOP buttons will appear.
• The interface will display duration, the active recording, and the running profile step info.
• Step 1 measures respirometry from the empty chamber. As the five-minute mark is reached press pause to halt the profile. This will allow time to insert the mouse without disrupting profile time.
• Use the tunnel handling method to insert the mouse in the treadmill running chamber.
• Ensure the lid is secured and wait about a minute for the mouse to settle – and for potential operator air contamination to flow out of the chamber.
• Then press RESUME to continue with step 2: mouse respirometry at rest.
• During the rest time, the mouse will familiarize itself with the chamber, and the interface will show the progression of step 2 time will accumulate distance remain at zero. Then at 10 minutes, step 3 will commence, starting the running metabolic rate measure. During step 3, speed changes from 0 to 15 meters per minute accelerating at the default setting.
• The duration for this step is 2.5 minutes.
• At 12 minutes 30 seconds, the treadmill inclines to 20 degrees.
• This measures running metabolic rate and an incline for 5 minutes.
• Increased effort and possible gait changes may increase metabolic rate.
• At 17 minutes and 30 seconds, step 4 ends with the treadmill returning to horizontal. Running speed is still maintained at 15 meters per minute for another 2.5 minutes.
• Twenty minutes into the profile, the belt is stopped. This allows a 5-minute post-run measurement. This step examines if 5 minutes rest is sufficient for oxygen recovery after incline running.
• At 25 minutes (the end of step six) press pause to halt the profile. Use the tunnel to remove the mouse.
• After mouse removal, leave the profile paused and wait for CO₂ levels to return to ambient.
• Press resume to complete the final empty chamber measurement.
• At 30 minutes, step 7 will complete and profile 5 will terminate automatically. Stop the treadmill data recording by pressing QUIT, then stop the CGF recording.
• In the Promethion Live homepage, click STOP. The Promethion Live state will change from HARVESTING DATA to READY.
• Navigate to RECORDINGS to access the data file.

This concludes the general protocol design and basic treadmill data acquisition. Make sure to watch the next video in the series “General Treadmill Data Extraction and Data Transformation via Macro.”