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Proper identification of lab animals is a fundamental requirement of biomedical research. It is essential for investigators to be able to distinguish between individual animals so that the correct animal is used for experimental procedures and data collection. Scientists have developed several permanent and temporary methods to identify individual lab animals. Here, we are going to discuss the two most commonly used methods: ear tagging and ear punching.

Before delving into the procedure for ear punching, let's introduce the ear punch code, the types of punches available, and discuss the benefits and disadvantages of this method. As you might have guessed, ear punching involves punching a hole or a notch in the animal's external ear or pinna. In order to standardize this method, universal ear punch codes have been developed.

Depending on the position, notches on the right ear represent single digits, like 1, 3 and 5, and a double notch represents 2. Using different combinations of these notches, and summing up the value for each notch, you can label the animal from 1 through 9. Similarly, on the left ear, the same notches represent the tens. And again, different combinations will cover the range of 10 through 90. Holes in the center of the right and left pinna represent 100 and 200, respectively. And holes in both indicate 300. Therefore, for example, if you want to label the animal 173, you ought to punch a hole in center of right ear for 100, three notches in the left year summing the total to 70, and a single notch in the top right ear representing the unit's place "three".

Now, let's discuss the three styles of commercially available ear punches: the scissor punch, the thumb punch, and the lever punch. Each of these has their advantages and disadvantages.

The thumb punch requires a pinching motion, but is small enough to fit into an Eppendorf tube so that the ear tissue can be more easily collected. Ergonomically, the lever punch is easier to use than thumb punch, especially for the thicker ears of the older rats. The scissor punch does not require a pinching motion, thus avoiding carpal tunnel fatigue and injury.

In spite of being a quick and simple method of identification, ear punching does have some limitations. First, the ear punch code discussed above has an upper limit of 399, which may limit the use of this method in some large-scale studies. Another limitation is that most animals must be grasped at the scruff in order to read the code, thus increasing the times they are handled. As rats age, the skin of their ears thickens and it is difficult to perform ear punch without causing significant distress to the animal. Therefore, it is best to perform the ear punches on young rats as they are weaned. For adult rats that require identification, the use of inhalant anesthesia should be considered when performing ear punches.

Now that you have some background information about the ear punch code and the types of tools available, let's learn how to perform this technique in mice.

Before starting the procedure, it is recommended that punching tools be cleaned with a disinfectant solution,rinsed with alcohol, and dried thoroughly. Avoid autoclaving the ear punches, as it will eventually dull them.

To begin, decide the animal's experimental code and the punch to be used. Let's use the code 173 and scissor punch as examples here. The same procedure is applicable to any other code or punch. When you are ready, restrain the animal by grasping it at the scruff so that the ears are easily accessible. Start with the hole for 100. Place the right ear in the punch so that the hole of the punch is positioned away from the pinna margin. Next, apply pressure to close the scissor quickly and firmly to cut through the skin. Then, release the punch and remove it from the ear. Avoid pulling or twisting the punch to prevent tearing the ear. To make a notch, place the punch on the edge of the ear pinna at the correct spot, in this case at the top of the right ear for the "three" in the code's unit place, and do the same – apply quick and firm pressure and then open the scissor to remove it. Repeat the same procedure on the other ear to punch three notches – two for "20" near the outer corner and one for "50" near the inner corner, totaling the code to "70".

Confirm that the correct code was punched before adding the animal back to the cage. Again, clean the punch with a disinfectant, rinse with alcohol and dry it thoroughly to prevent rusting.

Now, let's move on to ear tagging, which is another inexpensive and easy-to-perform method for the individual identification of mice and rats. There are a variety of commercially available ear tags. These include: metal tags that have a series of numbers and/or letters etched on them, and plastic tags with characters or barcodes on brightly colored backgrounds.

One disadvantage of metal tags is that it is often necessary to pick up the animal in order to read the code. In contrast, plastic tags are easy to read without having to handle the animals. Another advantage to plastic tags is their compatibility with MRI imaging. While the tags provide an individual identifier for a mouse or rat, they are not infallible. Tags can be lost due to over-grooming, dermatitis or infection, self-trauma inflicted trying to remove tags due to intolerance, fighting, and aggressive breeding behavior. If several animals are housed in the same cage, the loss of an ear tag can be troublesome. To circumvent this problem, a secondary form of identification such as a punch code on the ear opposite of the tag should be utilized. A facility should standardize both the choice of ear to be tagged and the code to be used on the other ear, and all codes should be indicated on the cage card.

Now let's learn how to properly apply an ear tag, as incorrect positioning can have several unfortunate consequences. Following application, we will also discuss removal of the ear tag, which is usually performed in case of an infection due to the tag application.

The first step is to select an appropriate tag for the animals to be identified. Here, we will use a metal tag to demonstrate the procedure on mice. Be sure to order the applier that is appropriate for the specific size and style of ear tags being used. Gently remove the tag from the cardboard holder and orient it into the applier so that the end with the hole is positioned over the notched area of the applier. The pointed end of the tag should be opposite the notch.

When you are ready to apply the ear tag, restrain the animal so that the ears are easily accessible. Animals may be less likely to struggle if the hind limbs can rest on a surface such as the cage top or a counter top. Once the animal is restrained, position the point of the ear tag deeply in the concha of the ear so the tag numbers face the back of the animal. Then, firmly squeeze the applier closed; the ear tag will pierce the ear and lock together. When you release, the tagged ear will fall out of the applier. Confirm that the tag has been properly positioned and applied. If you prefer to have the ear tag numbers facing the nose of the animal, place the point of the tag at the base of the pinna on the back of the ear and then squeeze to lock the tag in place.

Tags must be placed such that they do not cause a bend in the ear, interfere with the animal's mobility, or catch on any part of the caging. If the tag is placed too close to the edge of the ear, it can easily be torn. If positioned too close to the head, the skin of the neck area can catch in the tag, creating irritation, pain, and restricted movement, which can then lead to self-trauma. Occasionally, metal tags can cause irritation and swelling of the ear. This often leads to infections of the pinna, and the immediate treatment should include the removal of the tag.

In order to remove the tag, moisten a cotton-tipped applicator with an antiseptic solution and use it to remove any debris from around the tag. In severe cases of infection, the animal may need to be anesthetized. Then, using wire cutters, cut across the top of the tag loop from edge to edge. Importantly, do not cut across the flat surface as that will compress the tag, pinch the ear, and cause damage to the ear. Using hemostats, gently guide the ear tag through and out of the ear. Slip the tag out so that the crimped end does not pass through causing trauma and enlargement of the piercing hole. Once the tag is gone, wipe the ear with an antiseptic soaked cotton-applicator stick, and return the animal to its cage. If there is a purulent discharge from the ear, follow-up treatment may be necessary and consultation with the veterinary staff for assessment is recommended.

Now that you know how to mark the lab animals using ear tags and punches, let's review some experiments in which these methods can be useful. One major advantage of using ear punch code is that the punches can double as tissue samples for genotyping, thus reducing stress to the animals caused due to repeat handling. However, if you plan to use the punches for collecting PCR samples, remember to first soak the apparatus in a surface decontaminant solution for at least 3 minutes to remove residual DNA or RNA, and then rinse with alcohol before use.

Identification technique's importance increases further if the experiment is lasting over several days. For example, in this experiment the scientists first labeled the animals using a metal tag and then they injected the sciatic nerve with a suspension of transgenic tumor cells expressing luciferase protein. Next, in order to quantify cancer progression, they imaged the animals using bioluminescence at one, ten and eighteen days post injection.

You've just watched JoVE's presentation demonstrating the two commonly used methods of identification in rodents: ear tags and ear punches. You should now understand the ear punch code, know the different types of commercially available rodent ear tags, and should be able to accurately perform these procedures without causing significant animal discomfort.

Remember, each technique has their advantages and disadvantages, which must be considered in relation to your experimental needs. In addition to considering cost, the ease of the technique and the level of pain to the animals should be some of the primary considerations when choosing the appropriate identification method. As always, thanks for watching!