Knowledge Base

Setting up the experiment in EthoVision XT

create the experiment

1. Start EthoVision XT and choose File > New from template.
2. In the Select a template option window, select Apply a pre-defined template. Next, follow the instructions in the guided setup.

For more information, see Set Up an Experiment in the EthoVision

XT Help. For details on connecting the camera, see Camera

Installation in the EthoVision XT Help, or watch the video tutorial

Set Up the Camera (Help > Video Tutorial). 3. Enter the name of the experiment and click OK. 4. Before you can actually acquire data, check the settings listed in the rest of this section.

experiment settings

Choose Setup > Experiment Settings.

Under Video Source, and Tracked Features, make sure that the options

selected corresponds to your needs. To adjust the camera settings, click the video icon in the camera row.

note The option Deep learning is only available if your PC has a graphic

processor (GPU) that supports additional software (CUDA). See Deep

learning: Requirements and Limitations in the EthoVision XT Help.

arena settings

Choose Setup > Arena Settings > Open Arena Settings 1. General Information 1. Click Grab to grab an image of the empty enclosure from the

camera image, or click Browse to select a video file and then click

Grab. 2. Click 1. Draw Scale to calibrate and calibrate your arena. 3. Click 2. Select Shape and Draw Arena. Check that the arena covers the whole area in which you want to track the animal. For open fields and other enclosures, remember to include enough of the walls so that the animal is still tracked when it rears, but exclude any bright reflective rims that might interfere with tracking. Make sure the label stays inside the arena.

tip If you need to rotate the outline of the arena, drag

around all elements (1), and then click the Rotation mode

button and move the outline until it matches the arena in the video image (2). General Information

tip If you need to reshape the outline of the arena, click

the Point edit mode button and move the vertices of the

outline. 4. To draw zones, click 3. Select Shape and Draw Zones. See more details in the chapter about your test. For details, see Arena Settings in the EthoVision XT Help.

trial control settings

In the Trial Control Settings you can define conditions for the start and stop of data acquisition. For details, see the EthoVision XT Help. Choose Setup > Trial Control Settings.

detection settings

Choose Setup > Detection Settings > Detection Settings 1. 1. If you work with pre-recorded video, select a video file. If you work with the live camera image, release the animal in the arena. 2. Click Automated Setup. General Information 3. Select: - Rodent for tracking rats and mice. - Adult fish for tracking fish from the top view. - Other for any other case like tracking insects, crustaceans, or when tracking fish from the side view. 4. When the subject does not move a lot, and is far from the enclosure walls and other objects, draw a rectangle around it. EthoVision XT detects the subject. If the subject's body is well detected (marked in yellow), click Yes.

In other cases move the Finetune slider or click No and adjust

the Advanced detection settings.

Note

The Automated Setup may not work in all situations. If the subject is

not detected correctly, see below.

Advanced detection settings

1. Under Video, choose the sample rate.
2. Under Method, check that either Dynamic subtraction or

Differencing is selected as the detection method. Differencing is the

preferred option if other methods do not work well, or if you work with hooded animals. 3. Select the tracking method. The options here are depending on your experiment settings, including the number of body points you want General Information to track. The optimal method is automatically selected after choosing the type of animal in the Automated Setup.

For more information, see Tracking methods in the EthoVision XT

Help. 4. When using Dynamic subtraction, move the slider to define the animal's contrast. The animal must be fully detected in all parts of the arena and the noise must be minimal.

When using Differencing, set the Sensitivity slider. The slider

determines what difference in contrast from the background is seen as the animal.

For more information

Choose Help > EthoVision XT Help and search for Advanced

Detection Settings.

Choose Help > Video Tutorial and watch the videos under How

Subject Detection Works.

trial list

Choose Setup > Trial List. Enter your independent variables such as rat ID, first or second phase of the experiment, treatment (with values drug vs. control), dose, type of object, name of the experimenter, etc. If you want, you can pre-define all your trials here, or you can enter the independent variable values as General Information you carry out the trials. You can also prepare the trials in Excel, randomize them and then paste the values into your Trial List. Furthermore, you can define a list of trials for batch acquisition. See Acquire a series of trials in the EthoVision XT Help. Figure 1.1 An example of the Trial List with four planned trials. General Information

Acquire the data

protocol

Each test described in this manual has one or more specific protocols. See the chapter that applies.

important Always record your video with EthoVision XT or Media

Recorder. Do not record video with Pylon Viewer or other software! The resulting video file may not be compatible with EthoVision XT.

acquire the tracks

Procedure

1. Check that you have selected the correct Video Source in the Experiment Settings (see page 15).
2. Choose Acquisition > Open Acquisition.
3. optional Skip this step if you have entered the values of the independent variables already in the Trial List.

Open the Independent Variables tab of the Analysis Results and

Scoring pane at the bottom of your screen and enter the values of

the independent variables. You can also do this during acquisition. 4. If you did not plan any trials in the Trial List, click the New trial button in the Playback Control window (Ctrl+F3). General Information 5. If you planned two or more trials: select in the Acquisition Settings window whether you want to track only the next planned trial, or all planned trials. If you carry out batch acquisition and track live, also specify the Inter-trial interval. 6. optional In the Acquisition Settings window, select Auto-start data analysis. Do this to start calculation of analysis results immediately after acquisition. If you choose this option, first create an Analysis profile and optionally a Data profile and a Track Smoothing profile (see page 24). Choose whether to carry out analysis only for the profiles highlighted in blue in the Experiment Explorer, or for all possible profile combinations. In the latter case, first delete all profiles you do not need for analysis. 7. Start the trial (either choose Acquisition > Start Trial or click the Start trial button in the Playback Control window or press Ctrl+F5). 8. If you do live tracking, release the animal in the arena. 9. Data acquisition starts when the Start track condition in your Trial Control Settings becomes true.

10.The trial stops automatically when the Stop track condition specify

in the Trial Control Settings becomes true or when the time defined

in the Trial Control Settings under Maximum trial duration has

expired. If you planned a series of trials, the next trial will start.

To stop the trial manually, either choose Acquisition > Stop Trial or

click the Stop trial button in the Playback Control window or press

Ctrl+F6. General Information If you chose to simultaneously record a video file, the video file that

has been created is stored on your PC in the Media Files folder of

your experiment.

After testing

After each test, remove any debris (bedding, feces, etc.) from the arena and clean all interior surfaces, walls and floor thoroughly, first using water and soap, or a perspex cleaner (see also Table 1), then with water. Allow at least 5 minutes between tests to ensure that any residual odor will have dissipated before the next animals are introduced into the test apparatus.

Do not use wet ethylene oxide, steam, strong acids, hydrocarbons,

ethanol, methanol, or solvents containing acetone.

Table 1 Acceptable techniques and substances for cleaning acrylic

(perspex) Electronic beam High intensity visible light Gamma radiation Alkaline substances (in low concentrations) Ethylene oxide gas Low-temperature hydrogen peroxide gas plasma Hydrogen peroxide up to 3%) Diluted solutions of isopropyl alcohol UV radiation General Information

Analyze the data

data preparation

Track editing

Choose Acquisition > Edit Tracks. You can fix tracking errors and swap back nose-points and tail-points that have been swapped in tracking. Normally you do not need to edit your data.

Track smoothing

Choose Acquisition > Track Smoothing Profile. Select one or both options to remove noise and artifacts due to for

example body wobbling. For more information, see Smooth the Tracks

in the EthoVision XT Help.

Select data for analysis

Choose Analysis > Data Profile. The data you select on this screen will

be analyzed or visualized (see the next sections).

To analyze data per zone, in the Result box, click Settings and select

Results per zone. Use this function for example to analyze the

velocity of the subject in the different zones.

To analyze some tracks, not others, under Filter, choose one of the

variables available. For example, Treatment to analyze Treated

animals only. Insert the box in the Data profile sequence. To analyze segments of tracks based on the subject's behavior,

under Nesting choose one of the options. For example, to select all

data points when the subject was moving, choose Movement. Insert the box in the Data profile sequence. To analyze the tracks split in segments of equal duration, in the

Result box, click Settings and select Time bins. Use this function for

General Information example to analyze a 10-minute recording in ten segments of one minute each.

To create groups of tracks and compare results, under Common

Elements click Result and add a Result box. You can link each Result

box with a specific Filter (see above). This way you can compare the results of different selections, or visualize groups of tracks that result from different treatments, etc.

For more information

See the EthoVision XT Help.

visualize the data

You can visualize your tracks in three ways:

Plot the tracks

Choose Analysis > Results > Plot Tracks. You can view your tracks on a still image of the background. Tracks can be shown in different colors according to the values of independent variables (for example, blue for animals treated with saline and red for drug-treated animals). Sample points can be shown in different colors General Information according to the values of dependent variables, for example red when the animal was moving fast.

To set the track colors, in the Track Plot Settings pane, click the Colors

tab.

Plot integrated data

Choose Analysis > Results > Plot Integrated Data. Look at a track with the video file in the background. When you plot integrated data you can also view Time Event plots of your independent variables. Just like with plotting tracks, you can show tracks or sample points in different colors.

Plot heatmaps

Choose Analysis > Results > Plot Heatmaps. Make heatmaps of the location of your animals during the tracks. For more information, see Visualize Data in the EthoVision XT Help.

calculate the statistics

General procedure

1. Choose Analysis > Analysis Profile > New. Select the variables you are interested in. For information about the variables suggested, see the chapter about the test of your choice and the EthoVision XT Help.
2. Choose Analysis > Results > Statistics & Charts, then click Calculate.

The Trial Statistics tab shows the results per trial. The Group Statistics

tab shows the statistics and charts from the results over all trials or the groups defined in your Data profile (see page 24).

Adjust the table layout (optional)

Sometimes you need to adjust the layout of your results table, for

example to export the results to a statistics software. Click the Layout

button on the toolbar and specify which headers should be on the rows General Information and which on the columns. For details and examples, search for table layout in the EthoVision XT Help.

Batch calculations

It is also possible to carry out multiple calculations at once with different filters, data profiles or analysis profiles. To do so: 1. In the Statistics & Charts window, click Batch. 2. Select the profiles from the lists and click Add. 3. Repeat the previous step for other combinations. 4. Click Calculate. Choose the profiles from the lists on the toolbar to view the analysis result for that combination.

For more information, see Calculate Statistics in the EthoVision XT

Help.

Advanced analysis with JavaScript custom variables

In the Analysis profile you can now create fully custom dependent variables with JavaScript code. With JavaScript you have an almost endless range of possibilities to extract information from your tracks. JavaScript variables can be of type continuous, event, or state. See JavaScript in the EthoVision XT Help.

note If you are not familiar with programming, we can create custom

analysis variables for you. Please contact Noldus for information.

export the results

To find out if your independent variables (like treatment, dose, etc.) have a significant effect on the dependent variables, you can export your data to a statistical package. 1. Choose Analysis > Export > Statistics. 2. Choose Trial Statistics or Group Statistics.

You can also export the raw track data. Choose Analysis > Export > Raw

Data.

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Source: EthoVision XT 17.5 Application Manual, General Information