Knowledge Base

Detect the Subject

1. Choose Setup > Detection Settings. Open the default Detection Settings 1.
2. If you work with video files, under Video click Select Video and choose a video file. Play the video up to when the subject appears. If you work with cameras, release the animal in the arena.
3. Click Automated Setup. Choose the type of subject you test, and click Next.
4. Wait until the subject is free from contact with objects or walls and walks with a normal posture. When ready, draw a rectangle around the subject. Do this for all subjects in the arena.

Tip: For rodents, try to leave the tail out of the rectangle. In all other cases, include the animal's tail.

5. If the subject's body is well detected, click Yes. Otherwise, try moving the Finetune slider and check if the yellow blob covers the entire animal's body (excluding the tail).

If detection does not improve, try again a few times using another image from the video. If this does not help, click No and click Advanced. For details, see Configure Detection Settings > Advanced Setup in the EthoVision XT Help.

Tracking the Nose and Tail-Base

In all cases, make sure that the body of the animal is well detected.

If you use the Deep learning detection technique with one subject per arena, under Method click Define and select a box around the subject. Make sure that there is some space around the subject's body.

Notes

• Check that the subject is well detected in all parts of the arena. In the Detection Performance pane, check that the proportion of samples where the subject is not found is acceptable (for example, less than 1%).
• If you selected Activity analysis in the Experiment Settings to detect freezing, click Activity. Adjust the settings until most of the purple pixels only appear when the animal moves. When the animal freezes, the number of purple pixels should be minimized.

Prepare a List of Trials (Optional)

The Trial List

In EthoVision XT, a Trial is an uninterrupted recording session. The Trial List is a table listing the trials planned for an experiment. When you create a new experiment, a few planned trials are already present.

To Add More Trials

1. Choose Setup > Trial List. Click the Add Trials button.
2. Type the number of trials you plan to carry out and click OK.
3. For each row:
   • Specify the independent variables (optional; see the next section).
   • Specify which video file you want to use (when tracking from video files).
   • Select Arena Settings, Trial Control Settings, and Detection Settings (optional). If those columns are not present in the Trial List, click the Show/Hide button, click Variables and select them.

Note: Planning your trials in advance also allows you to acquire trials in batch mode. See Trial List in the EthoVision XT Help.

Independent Variables

Independent variables are either simple descriptive categories (such as the genotype or age of your rats or mice), or conditions manipulated by the researcher (such as the substance being tested and the dosage). You can use independent variables to create analysis groups or filter data. For example:

• Compare tracks of treated subjects with tracks of control subjects.
• Only analyze subjects of a specific age class.

Note: If you created a template experiment, one or more independent variables have been predefined in your Trial List, for instance, Animal ID and Treatment.

To Define an Independent Variable

1. Choose Setup > Trial List.
2. Click the Add Variable button. A new column appears on the right of the table.
3. Specify the Label (for example: Dose), Description, Type (for example, Text or Numerical), Format, Predefined values (for example, 0.01, 0.02 etc. mg/kg), and Scope (whether the values of the variable differ between subjects within the same arena, between arenas within the same trial, or between trials).

Acquire the Data

Methods

Choose Acquisition > Open Acquisition. Locate the Acquisition Settings window at the right side of the screen.

• If you track from pre-recorded video files, select Track next planned trial, click the button next to Video and open the video you want to use.
• If you track from the live camera image, select Track next planned trial. Select Save video if you want to save the camera footage to a video file.

For more acquisition methods, see Acquire Data in the EthoVision XT Help.

Procedure for Acquiring One Trial

1. Under Settings, choose the Arena Settings, Trial Control Settings, and Detection Settings, if they are not already selected in the Trial List.
2. If you track from video and do not score behaviors manually, make sure the DDS (Detection Determines Speed) checkbox is selected in the Playback Control window. With this option you ensure that every sample is analyzed.
3. To start a trial, click the Start trial button in the Playback Control window.
4. Release the subject in the arena.
5. To stop the trial, click the Stop trial button.
6. Return the subject to its home cage or container.
7. Prepare the next subject, or load a new video file to acquire the next trial.

More Options

• You can automatically start and stop your trial depending on the behavior or position of your subject.
• You can acquire data in batch mode. For details, see Batch data acquisition in the EthoVision XT Help.
• You can score behaviors manually during tracking. If you saved the video during tracking, you can review the video and add or edit existing behavioral data. For details, browse the EthoVision XT Help:
  • To define behaviors, see Set Up an Experiment > Manual Scoring Settings.
  • To score behaviors, see Acquire Data > Score behaviors manually.
• If no planned trial is left, click the Add Trial button in the Playback Control window.

Note: Sometimes EthoVision XT tracks a reflection instead of the subject, confuses the nose-point and the tail-base, or swaps subjects when tracking multiple animals simultaneously. Whenever that occurs, you can correct those errors. For more information, see Edit the Tracks in the EthoVision XT Help.

Source: EthoVision XT 18 - THC - Trial and Hardware Control, Noldus Information Technology