The black mouse marking challenge: Technical note for black mouse identification in behavioural studies

Have you ever wondered how ethologists discriminate animals in social groups? Indeed, ethologists have to be able to identify animals that are sometimes very similar between each other, at least for us, humans. Moreover, when ethologists record behaviour of animals on videos, they also cannot turn around the scene and change their point of view. Hence, they have to find salient markings for quick identification. One of the biggest challenges for those scientists is also to find markings that do not disturb animals and modify their behaviour in order to ensure the studies reliability.

 

  1. The search of a good marking

It sounds simple but finding the good marking can sometimes be tricky. It was the case for our study with black mice. For example, some may not recommend ear tags in behavioural studies with mice since tags are big according to the size of the animals and mice often scratch and even rip the tags off their ear. Aside from modifying the behaviour of the mice (they often scratch), animals can lose the marking and hurt themselves. Ear tags are also too small to be discriminated on video recordings.

ear tag mouse

Even the small ear tags conceived for rodents are very big and heavy for the small mice. Photo credit: https://commons.wikimedia.org/wiki/File:Lab_mouse_mg_3308.jpg

I also heard about the use of bleach but I did not even want to try since it is a toxic substance, very smelly, distasteful and irritating.  It also requires a pause of several minutes before rinsing so it would entail to sedate animals in order to prevent from licking, a procedure we strictly wanted to avoid since it may affect behaviour and imply a recovery period.

Ethologists often use non-toxic colour marking conceived to identify animals. Blue, green, pink, red, you just have to choose the colour. We thus decided to buy some non-toxic colourful ink in tube and marker pens to compare. About the ink tube, I had a negative preconceived idea about the texture and the feeling on skin contact but I still wanted to try since it is supposed to adhere better on the black fur than the ink from marker pens. As expected, the ink stuck the hairs together, which may not be comfortable for mice at all! We had to test the other option.

 

Although not painful, some markings can disturb animal behaviour and perturb social relationships, especially in species where physical features are important for individual recognition. Don’t go overboard on markings and check the impact on the behaviour. Photo credits: https://www.alibaba.com/showroom/animal-marking-spray-paint.html and https://inforesist.org/kreativnye-instagram-akkaunty-zhivotnye-znamenitostej/

None of the marker pens we found on the  internet was conceived to be visible on black fur… I was surprised not to find a special white marking for black fur. In addition, marker pens are designed to adhere on the fur, and not on the skin. We even tried to apply marking colour on the tail and ears and we saw that marking was visible on videos during the light phase until 4 to 7 days, depending on the individuals, but was very salient only on the first day. I remember that I had the same issues when trying to identify piglets with colour spray in another project; the colour only remained some days on the naked skin of piglets and we had to refresh it weekly.

However, the tail marking of mice is still an easy way to identify black mice in experimental design in which cameras record the animals from above and during the light phase, especially if the colour is applied some hours before testing. May I remind that it is not always detectable from a horizontal plane since the bedding material, the environmental items or even the mouse body can cover the tail, but it can be used in complement of another marking.

mouse marking4Tail marking is an easy and unpainful procedure to identify black mice for the short-term in behavioural studies. Photo credit: ©FCT.

Fur clipping was the next alternative to test. Black mouse skin is often pink (sometimes with some black patches) giving a good contrast with black fur. After running some tests, we found that fur clipping was the best way to identify our black mice on video-recordings. The next chapter addresses the criterion considered by a question-and-answer series.

 

      2. Fur clipping: a good way to identify black mice

Is it possible to shave animals without using sedatives? Is it easy to handle animals and proceed quickly?

After some trials, we have found a very good way to handle the animals in order to shave them: we just have to use their natural behaviour of gripping the grid lid when pulling the tail to hold them (be careful to hold the base and not the tip of the tail since it is painful and the mouse is not correctly maintained). Only 30 seconds are necessary to hold and shave the mouse. We do not have to restrain the animal body using this technique and we assume that the procedure is less stressful. It also prevents us from biting since mice do not release the grid and cannot reach our hand on the tail.

 

Fur clipping is an easy and unpainful procedure to identify mice for several days in behavioural studies. Photo credit: ©FCT

Does it modify the behaviour of animals?

There is no apparent effect of fur clipping on the behaviour of the mice. For example, we did not see them licking or scratching the skin.

 Is the marking visible on the cameras?

We have to shave large skin surface in order to be able to detect it on video-recordings; we shaved animals on either the left side, the right side or the back to be able to discriminate three different mice on the cameras. There are still some limitations with this technique. For example, the occurrence of barbering (associated with patches of hairless skin on some parts of the body) can be frequent in C57BL/6 mice that can lead to confounding effects with the shaving marking. In addition, some of the mice have black spots on the skin, making the identification more difficult both because black skin can be confounded with black fur and fur re-grow faster on black skin spots than on pink skin (see pictures below).

mouse marking5Mouse with a black spot on the skin detected the day of fur clipping; this mouse will be more difficult to identify on video recordings. Photo credit: ©FCT

 

Another mouse with a black spot on the skin 20 days after fur clipping. The fur re-grown faster on the black spot than on the pink skin. Although fur clipping is visible when the tail is pulled (picture above), it is almost impossible to see it when not pulled (picture below); this animal cannot be identified on video recordings. These pictures also highlight the importance of shaving a big skin surface. Photo credit: ©FCT

How long time it takes before the fur re-grow and make the marking invisible on the cameras?

The fur clipping can be effective for visual identification at least 14 days, sometimes more depending on the mouse. Because we had to track mice from 5 to 10 days after marking, this type of marking was ideal for our purpose.

In conclusion, animal identification is not always an obvious task and this kind of anecdotic task in a research project can quickly become a real mission. In the future, ethologists could directly use fur clipping as a marking technique to identify black mice instead of going through all of the steps of mark testing and determination.

Sophie B.