Title
University of North Carolina at Chapel Hill Standard on Rodent Identification
Introduction
Purpose
The purpose of this standard is to ensure that the advantages and disadvantages for animal identification methods in addition to the required training for certain techniques are communicated to animal users.
Scope
This Standard applies to all research personnel engaged in the identification process for rodents within their animal colony.
The University of North Carolina at Chapel Hill ("UNC-Chapel Hill") Institutional Animal Care and Use Committee (IACUC) expects that anyone involved in animal work at the university will comply with this Standard. Requests for exceptions to this Standard must be reviewed and approved by the IACUC.
Standard
There are several IACUC approved methods used to identify rodents. Each method has both advantages and disadvantages. In long-term studies it is important to choose a method that is permanent and easily read. All methods must be described in the approved protocol prior to use in animals. Some techniques require specific training and certification by either the Division of Comparative Medicine (DCM) Training Team, or by Laboratory Animal Coordinator (LAC). The IACUC Mouse Handling & Techniques and Rat Handling & Techniques packets should be referenced for acceptable techniques and methods.
Long Term Methods
Ear Notching/Punching
This method is frequently used in both mice and rats and should only be done on rodents that are 14 days of age or older due to the potential for tissue regrowth in animals that are younger than this age. Several inexpensive tools may be purchased to achieve this. Most ear punchers resemble a hole puncher and notchers allow for precision. There are previously created maps that serve as a numbering system, or the researcher may create a map. This technique requires certification prior to approval.
A) Advantages - Ear notching or punching can be done quickly causing very little pain or distress. The instruments are not costly and can be obtained easily. Tissue collected from ear notching or punching can be used for genotyping. Does not require anesthesia.
B) Disadvantages - This method cannot be applied until the ears are fully developed. This may be too late for those that use young rodents. This method may not be a reliable permanent way to identify individuals in strains that are prone to fighting. Rips or tears caused by fighting may leave the pattern indiscernible. Tools used to notch ears dull easily so they must be replaced frequently.
Ear Tagging
Ear tags can be purchased with numbers and/or letters. Correct placement of the tag makes them fairly easy to read. Traditional metal ear tags should be placed in the lower half and on the middle third of the ear, making sure to avoid the area with the highest concentration of blood vessels. This placement prevents the ear from folding over and minimizes the risk of the ear tag being ripped off, which may occur when the tag is placed too close to the outside edge of the ear. This technique requires certification prior to approval. Alternatives to metal bar tags are button & bar code ear tags. Use manufacturer recommendations for placement. (see References section.) There is a tattooing technique for identification that can be used as an alternative method to ear tagging. Please see “Tattooing” section for more details.
A) Advantages - Ear tags are inexpensive and are fairly easy to apply. This method does not require the use of anesthesia. Tagging can be done quickly and does not seem to cause pain and only minor distress. Alternative ear tags are often smaller than traditional ear tags and may provide integration with colony and/or data management software.
B) Disadvantages - Tags can fall out if not applied properly. They can also be lost if ears are ripped or torn in strains that fight. Different sized tags are available for different species. Traditional metal ear tags are relatively heavy for weanlings and may cause young mice to tilt their head even when the proper sized tag is applied or cause the ear pinna to fold over if placed too high. Some strains are prone to scratching the tagged area which can lead to infection, hematomas, and granulomas. Cancer-prone strains can develop tumors around the ear tags resulting in undesired study endpoints. DCM Veterinary Services have also reported seeing tag reactions with masses developing on ear pinna. Other issues seen have been folded pieces of ear pinna within the tag, body tissue included in the ear tag due to being placed too close to the base of the ear, and ear tags placed too close to the center of the ear. Therefore, it is important to remember that incorrect placement of ear tags can lead to health issues that can affect both the overall health of the animals as well as the study results.
Microchipping
Microchips, electronic transponders, are safe and reliable.
A) Advantages - Microchips may be applied without the use of anesthesia. Applying microchips seems to cause little or no pain. They are a reliable method for permanent identification. Animals can be identified (by reading the microchip/transponder) without handling and removing them from the cage. Some microchips are designed to provide other information such as core body temperature and heart rate.
B) Disadvantages - May be more expensive than other methods. Not all microchips are recommended for reuse by the manufacturer. In order to reuse the chips they must be sterilized by ethylene oxide. If not implanted properly there is a slight risk of infection. In a very limited number of cases, microchips may stimulate tumor growth. They may not be compatible with advanced imaging equipment.
Tattooing
This method seems to be growing in popularity. It is both permanent and fairly easy to apply. There is a tattooing technique for identification that can be used as an alternative method to ear tagging. (see JAALAS article in ingentaconnect and also SouthPointe Surgical tattooing system in the References section.)
A) Advantages - This method works in all strains,. Low maintenance costs (after initial purchase) Tattoos can be applied to rodents of any age. The markings are easily read, especially when applied to the tail of light colored rodents. When placed in the proper area, it is not necessary to handle the animal to read the tattoo. Tattooing causes only minor pain and distress and does not require the use of anesthesia. Tattooing provides permanent identification.
B) Disadvantages - May be difficult to read. This improves as the mice age. Larger up-frong cost. There is a small chance of inducing infection if the tattoo is not applied correctly.
Toe Clipping
Toe clipping must be scientifically justified in an approved IACUC protocol. Adequate justification does not include cost, convenience, or lack of formal training in other identification methods. As stated in the Guide for the Care and Use of Laboratory Animals, “As a method of identification of small rodents, toe-clipping should be used only when no other individual identification method is feasible and should be combined with genotyping.
It may be the preferred method for neonatal rodents up to 7 days of age as it appears to have few adverse effects on behavior and well-being at this age (Castelhano-Carlos et al. 2010; Schaefer et al. 2010).” The ideal timing is between 5-7 days of age when the toes are separate and the bone is not calcified and the toes are no longer webbed. Toe clipping beyond 7 days of age is considered painful and is not an acceptable procedure for either identification or genotyping. If tissue for genotyping is required beyond 7 days of age, alternative methods of collecting tissues should be considered. Contact DCM veterinarians at 919-962-5335 for additional information.
The IACUC allows toe clips on a maximum of 4 toes and no more than 2 toes per foot. It is preferable to remove toes from the hind feet rather than the front feet. Any identification numbering system should be designed to minimize the number of toes clipped per animal. The hallux (also referred to as dew claw or thumb) may not be cut as this may decrease the rodent’s grasping ability.
Procedures
- Obtain adequate training from Principal Investigator (PI) and/or Laboratory Animal Coordinator (LAC).
- Restrain the animal for the minimum amount of time required for the procedure. With a sharp instrument, remove the toe(s) at the most distal joint of the toe (i.e., remove the last phalangeal [toe] bone; P3).
- Sharp scissors are recommended for toe clipping in neonatal rodents.
- Instruments must be clean and disinfected initially, and blade surfaces should be cleared of debris and wiped with 70% alcohol between animals.
- Apply pressure to the exposed tissue with gauze or other clean and absorbable material to ensure hemostasis. Monitor animals continuously until bleeding has stopped.
- During the procedure, the neonatal rodents should be handled gently and then placed back with the mother as quickly as possible.
Krebs O, Schreiner CM, Scott WJ, Bell SM, Robbins DJ, Goetz JA, Alt H, Hawes N, Wolf E, Favor J. Replicated anterior zeugopod (raz): a polydactylous mouse mutant with lowered Shh signaling in the limb bud. Development. 2003; 10:6037–6047
A) Advantages - Toe clipping can be done at a very early age. The tissue can be used for genotyping. No anesthesia is required. This seems to cause little or no pain when performed at a very early age.
B) Disadvantages - Toe clipping may not be performed after seven days of age. The young show signs of distress when removed from their mother and siblings. This may cause a small amount of pain. There is a small possibility of infection. A reduction in the number of toes may reduce the ability to grasp objects.
Short Term Methods
Hair Clipping
Trim patterns in fur. Keep a record or picture to identify rodents.
A) Advantages - Causes no pain.
B) Disadvantages - This is very temporary. The hair will grow back within ten days and must be repeated. In animals as small as mice, removal of much hair can affect their ability to maintain body temperature.
Permanent Markers and Fur Dyes
It is easy to apply marks or dyes to different body parts. Temporary identification can be achieved with non-toxic stains or dyes that come in a variety of colors.
A) Advantages - This method is non-invasive. It causes only minor stress due to restraint.
B) Disadvantages - This method can be time-consuming because it must be redone soon after application. If you are working with nursing pups, the mothers will groom the neonates excessively and the markings may disappear overnight. This could result in a loss of identity.
If you would like to inquire about equipment used, or training provided, in the methods discussed above, please email the OACU Education & Oversight Team. You may send questions to the general IACUC email account iacuc@med.unc.edu.
Exceptions
Requests for exceptions to this Standard must be reviewed and approved by the IACUC.
Related Requirements
University Policies, Standards, and Procedures
References
- Chen, M., et al., "Tattooing Various Combinations of Ears, Tail, and Toes to Identify Mice Reliably and Permanently," Journal of the American Association for Laboratory Animal Science 55, no. 2 (March 2016): 189-198.
- SouthPointe Surgical, Rodent Tattoo System.
- University of Wisconsin-Madison, Research Animal Resources and Compliance, "Animal Identification."
- RapID Lab, Bar Code Tags, "RapID Tags® Product Info."
- Stoelting, "Small Animal Ear Tags."
Contact Information
Policy Contact
- Name: Michael Chi
- Title: Associate Director, Office of Animal Care and Use
- Email: mchi@unc.edu
Subject: Animal Health
- Contact: DCM Vet Services
- Telephone: 919-962-5335
Subject: Office of Animal Care and Use
Important Dates
- Effective Date and title of Approver: 04/2005; UNC IACUC
- Revision and Review Dates, Change notes, title of Reviewer or Approver: Revised 05/017; UNC IACUC; Revised 05/15, Revised 11/2018; Transferred to new standards template, updated toe clip description, ear identification options and updated contact information; UNC IACUC. Revised October 2023.
Approved by: UNC IACUC