Knowledge Base

Individual Marking for Two-Subject Tracking

This section refers to tracking two subjects per arena with Deep Learning (up to four arenas). If you track one subject per arena, you do not need to mark the animals. For successful application of Deep Learning-based discrimination between two individuals, the interacting animals must look different, or be marked.

Important: EthoVision XT does not detect differences in the subject's body size. Toe clipping, ear punching, or tagging will not work. Differences in fur color and markings (also on the tail base) are essential for successful individual discrimination with EthoVision XT.

For information about non-invasive marking techniques, see Klabukov et al. 2023. Animals 13(22): 3452.

Different Fur Color

If the two subjects are of different fur color, for example one white and the other brown or black, you do not need to mark them.

Tail Marks

Tail marks only work if they are close enough to the abdomen of the subject. Only mark one subject, not the other. Make one ring leaving some space between the mark and the abdomen. Note that if the tail mark is too far from the abdomen, individual discrimination will not work.

• Advantages: The procedure is painless and easy.
• Disadvantages: It may require weekly marking, which could cause stress. Furthermore, droppings left in the arena could be detected as tail marks and affect individual discrimination. This could especially occur in long trials.

Shaving Marks

You can use a hair trimmer to shave a patch of fur from one of the subjects. The patch must be located along the spine of the subject. If you decide to shave both subjects, they must exhibit different patterns. Such marks generally last one to four weeks, depending on the stage of the hair cycle.

• Advantages: The procedure is painless; grooming and manipulation do not remove shaving marks; it may be performed on rodents of all colors.
• Disadvantages: The marking is temporary due to hair regrowth; you need to monitor the animals regularly to assess the condition of the mark. In long-term experiments, you need to renew the mark as hair regrows. The marking may also look too irregular, reducing the identification rate.

Fur Staining

White rodents can be marked with non-toxic dye or felt-tip pens. When using infrared illumination, make sure that the marks are visible under infrared light. Mark only one of the subjects.

• Advantages: The procedure is easy and painless, and may be used with rodents of all ages.
• Disadvantages: Dyes may fade over time or due to grooming; daily monitoring is needed to assess the condition of the mark; it may be used predominantly on white-furred animals; there is a potential adverse response to solvents and odor.

Do not make V-shape markings or separate markings that converge on the back of the subjects, as these could interfere with the detection of the nose point.

Bleaching

• Advantages: The procedure is painless and bleach marks are not removed during grooming.
• Disadvantages: It may only be used on dark-furred rodents. The software will not work optimally if the bleached area is irregular and so large that it breaks the subject's contour.

Important: Remove bleach solutions to avoid skin damage.

Color Marking

Color marks only work when they exhibit different light intensity in grayscale, for example light pink and blue. Different colors may look too similar in grayscale. A safer option is to mark one subject and not the other. For more details about color marking, see Tips for Color Tracking.

Marker Location

Make sure that the marks are visible in most cases, including when the animal is sitting curled up. Marks on the shoulders that are too small, combined with the effect of direct lighting (which should be avoided in all cases), may cause the marks to disappear within a few frames, making individual recognition unreliable.

Size of Marks

The mark must be at least 4 x 4 pixels after the arena has been rescaled to 256 x 256. This applies to tail marks and marks on the back of the animal. If in doubt, follow these steps:

1. Open the video file at zoom level 1:1 (Original Size).
2. Make a screenshot of the video window.
3. In a paint program, crop the image to include approximately only the arena.
4. Rescale the arena to 256 x 256 pixels. For rectangular arenas, rescale so that the longest side is 256 pixels, maintaining the original aspect ratio.
5. After rescaling, verify that the marker is at least 4 x 4 pixels.

Hooded Rats

The neural network for two-subject tracking and identification has not been trained with the color patterns of hooded rats, so acceptable results cannot be guaranteed when tracking two hooded rats. Perform tests before the actual experiments to verify that the software provides acceptable results.