Guidelines

Guidelines for Acceptable Methods of Identification and Genotyping of Rodents and Zebrafish

Unit for Laboratory Animal Medicine
Mar 15, 2016 12:00 am

To describe procedures to safely and appropriately identify and genotype animals used in laboratory research at the University of Michigan.

  • Responsibility

    1. Investigative personnel are responsible for implementing the approved identification and genotyping methods with oversight and assistance from ULAM personnel, as needed.
    2. The Principal Investigator is responsible for ensuring that all methods of animal identification and genetic sampling that requires removal of tissue are explicitly listed and approved by the Institutional Animal Care and Use Committee (IACUC) in the applicable animal use protocol. The Principal Investigator or designee should consult with ULAM veterinarians if needed, especially if methods other than the ones described in these guidelines are utilized.
  • Definitions

    1. Genotyping: The process through which an animal's genetic make-up is determined using a sample taken from the animal.
    2. Identification: The process of uniquely marking an animal so it can be differentiated from other animals within a group.
  • Procedures

    1. General Procedures

    1. For assistance learning how to perform any of the procedures listed in this document, training is available from the ULAM Training Core (ulam-trainingcore@umich.edu / 734-763-8039)
    2. IVAC Technical Services is available as fee-for-service to perform these techniques (ulam-techservices@umich.edu / 734-647-0731)
    3. See Appendix A for comparison of all methods of identification
    4. See Appendix B for comparison of all methods of genotyping

    2. Methods of Cage Identification

    1. Cage Cards
      1. All cages must have a visible cage card at all times. Cage cards are an acceptable means of identifying individually housed rodents or groups of rodents for studies where individual identification is not necessary. However, if cage cards are misplaced, animals may not be distinguishable.
      2. Include the following required information on the cage card:
        1. Strain or stock, and source of the animal,
        2. Principal Investigator
        3. Pertinent dates (date of birth and/or date of arrival)
        4. Protocol number
        5. Account number
        6. Lab contact information
      3. The required information may be provided by a pre-printed, electronically generated, bar-coded cage card.

    3. Methods of Rodent Identification

    1. Temporary Markings
      1. A temporary marking may be used for short-term individual identification, and must be repeated or re-applied as often as necessary to maintain visible markings.
      2. There are no restrictions on age of animals.
      3. Methods of temporary marking include:
        1. Clipping or shaving fur.
        2. Non-toxic dyes or stains may be used as temporary markings. A small area of fur that is deeply stained is the best method.
          1. Note that certain stains wear off quickly and reapplication is necessary.
        3. A non-toxic "permanent" marker may be used to write numbers, bars, or other distinguishable markings on the tail, fur, or ears.
    2. Implanted Microchips
      1. Microchips provide a permanent method of individual identification. They are easy to implant and the animal does not need to be restrained to read the microchip.
      2. Rodents should be at least 3 weeks old for microchips to be implanted.
        1. Refer to each product's manufacturers' specific recommendations for implantation.
      3. The cost of each microchip can be high, making microchips one of the more expensive methods of rodent identification.
        1. Different chips may require specific readers, depending on the manufacturer. A compatible reader is necessary in order to accurately read the chip once it is implanted.
        2. The appropriate reader needs to be readily available to confirm successful implantation of the microchip and to identify the rodents.
      4. Prior to performing the procedure, read the manufacturer's instructions and follow them appropriately and accurately.
        1. Microchips may fall out if not properly placed.
        2. Inject subcutaneously on the dorsal aspect of the neck with use of the appropriate injection apparatus provided by the manufacturer.
        3. Read chips before and after implantation to ensure that they are working properly.
        4. Microchips can be reused after proper cleaning and sterilization, following manufacturer's recommendation.
    3. Tattoo
      1. Tattoo application provides a permanent method of identification, and can be performed with an electric tattoo machine or microtattoo equipment.
        1. An electric tattoo machine can be used to write numbers or symbols on the tail, ear, or footpads.
        2. Microtattooing can be used to quickly and easily mark animals, especially neonates.
      2. Anesthesia is not required, but is recommended, as it may aid in the ease of this procedure.  Anesthetics must be described in the appropriate protocol.
      3. If a rodent is tattooed as a neonate, the animal may need to be re-tattooed when older, as the ink may fade due to the small amount originally applied.
      4. Poor technique can render the identification difficult to read.
      5. Although the original cost of the tattoo gun may be expensive, the cost associated with applying tattoos is low.
      6. All tattoo equipment must be disinfected between cages of animals.
        1. For electric tattoos, follow manufacturer recommendations for cleaning the needle and remaining tattoo equipment.
      7. Apply a local anesthetic (such as EMLA cream which takes 15 minutes to be effective) to the skin prior to the procedure.
      8. Needles must be sterile and sharp. Clean needles between cages animals and change needles when they become blunted.
      9. Follow manufacturer's guidelines for procedures and recommended age for both electric tattooing and microtattooing. Electric tattooing may require special training.
      10. See Appendix C for suggestions of tattooing equipment to purchase.
    4. Ear Tags
      1. Ear tags provide a quick and easy method of identification. Ear tags can fall out if not applied properly or can be lost if ears are ripped or torn.
      2. The tags are easy to apply, and restraint is necessary to read the ear tag number.
      3. It is recommended that ear tags are placed when rodents are approximately 2-4 weeks of age. If the animals are too young, the ears may not be able to support the weight of the tags, causing the ear to rip. The eyes or face may also be injured if the tag rubs in this area. Older animals receiving ear tags may experience increased discomfort in placement that could be avoided by placing the ear tags at this earlier age.
      4. The cost of the applicator and tags are relatively inexpensive.
      5. Ear tags are available in metal and plastic. Metal ear tags are commonly made from a nickel-copper alloy, and can be associated with inflammatory and proliferative reactions of the surrounding skin. Neoplasia, specifically squamous cell carcinomas, can also be associated with metal ear tags.
    5. Ear Notch or Punch
      1. Ear notching or punching is a quick and easy method to identification, although the ID pattern may be lost if the ear is injured or mutilated. If animals are grouped between cages, then animals with similar markings may become indistinguishable.
      2. It is recommended that ear notching is performed after 2 weeks of age, which is when the pinnae (ears) are generally large enough to notch or punch.
      3. Disinfect equipment with Spor-Klenz or another suitable disinfectant before use and between cages of animals.
      4. If possible, use a simple ear notching scheme to limit the number of notches or punches. It is important not to group animals from different cages, since this may cause two animals with the same ear notch to be in the same cage.
      5. It is often possible and recommended by ULAM veterinary personnel to use the excised tissue as a sample for genotyping, if needed, eliminating the need for a tail biopsy.
    6. Toe Amputation (Toe Clipping)
      1. See Procedures section 4.b.iv below

    4. Methods of Collecting Genetic Material for Genotyping

    1. Non-Invasive Methods
      1. Feces
        1. The use of fecal samples to obtain DNA for genotyping is a practical alternative to invasive methods.
        2. Fecal samples can easily be collected from rodents by collecting samples from the cage bottom or by collection of a fresh sample directly from the animal, as rodents routinely defecate when handled.
          1. The rodent can be restrained until a fecal pellet is excreted. This usually requires 30 seconds or less.
          2. The rodent can be placed in a clean cage or other appropriate device and the fecal pellet subsequently collected.
        3. This method allows for repeat sampling.
        4. See References sections 6 and 13 for descriptions of genotyping procedures using fecal samples.
      2. Hair Bulb
        1. The use of hair bulbs to obtain DNA for genotyping is a practical alternative to invasive methods.
        2. A tuft of hair is plucked from the ventral body using a pair of forceps.
        3. To avoid cross-contamination, a fresh pair of forceps is used for each animal. Due to electrostatic forces, wiping off the hair is not practical.
        4. This method allows for repeat sampling.
        5. See References sections 5 and 12 for descriptions of genotyping procedures using hair bulb samples.
      3. Saliva
        1. A small amount of saliva collected from mice can be used to obtain DNA for genotyping.
          1. Oral wash using a plastic pipet tip will yield enough oral epithelial cells and lymphocytes for sufficient DNA analysis.
        2. This method allows for repeat sampling.
        3. See References section 8 for a description of genotyping procedures using saliva samples.
    2. Invasive Methods
      1. Blood
        1. Blood can be drawn from any approved site (see Guidelines on Blood Collection). 
          1. This procedure is easiest to perform on mice and rats greater than 8 days of age.
          2. Dried blood spots can be used for analysis.
          3. For assistance with blood collection procedures, contact the ULAM Training Core.
      2. Ear Notch or Punch
        1. Ear notching or punching for identification yields tissue that may be collected and used to assess the rodent's genotype. See Procedures section 3.e for information on use for identification.
        2. The ear punch or notching instrument is disinfected between animals to avoid sample contamination.
      3. Tail Biopsy
        1. The tail biopsy procedure involves cutting through bone or cartilage, blood vessels, nervous tissue and skin.
        2. In the mouse, the distal tail is completely ossified and innervated between 17-21 days of age. Thus, tail biopsy is done on mice and rats less than 21 days of age to avoid undue stress and discomfort to the animals. The optimum age at which to perform biopsy is between 12 to 16 days, before the distal tail completely ossifies.
        3. For rodents greater than 21 days of age, tail biopsy requires anesthesia and pre-emptive analgesia. See Guidelines on Anesthesia and Analgesia in Mice or Guidelines on Anesthesia and Analgesia in Rats for recommendations.
          1. Any situation where general anesthesia and analgesia cannot be used for tail biopsies in mice or rats over 21 days of age must be scientifically justified in the animal use protocol and approved by the IACUC.
          2. This procedure is not considered a surgery, so sterile gloves and post-operative monitoring records are not required.
          3. After anesthesia, the animal is recovered individually in a clean cage. The mouse or rat must be fully ambulatory before it is returned to the original cage or co-housed with other rodents.
        4. For most genotyping procedures, 2 mm provides an adequate tissue sample. If necessary, up to 5mm can be removed at one time.
        5. If it is necessary to take more than two tail samples from a single animal, the ULAM faculty veterinarian should be contacted for consultation.
        6. Sterile tools are required at the start of this procedure, and it is recommended to re-sterilize, disinfect, or use a new instrument between animals. Disposable scalpel blades or razor blades can be used as a sterile method as long as a new, sterile blade is used for each mouse or rat.
          1. Scissors can be sterilized using a hot bead sterilizer.
        7. The tail biopsy site will bleed following tissue collection. Hemostasis can be achieved using compression, styptic pencils, silver nitrate, tissue adhesives (e.g., Nexaband®), or cautery.
      4. Toe Amputation (Toe Clipping)
        1. Toe amputation involves the removal of the toe at the most distal joint, and is used as both a method of identification and tissue collection for genotyping.
        2. According to the "Guide for the Care and Use of Laboratory Animals," toe clipping can be used only when no other identification method is feasible. For some research, it may be necessary to individually identify and genotype the same animal. Toe clipping is a method under which both of the above goals can be reached; however, it is a stressful procedure that can only be performed if no other identification and genotyping options are feasible. For example, rats have reduced grip strength at weaning following the procedure, and therefore it is not recommended for them.
          1. If genotyping is required, the tissue yielded from toe clipping should be used. A second method of tissue collection (e.g., tail biopsy) must be scientifically justified.
        3. Conditions for Approval: Toe clipping will only be permitted when the following conditions are met:
          1. The Principal Investigator provides a scientific justification with documentation to the IACUC explaining why each of the alternate identification methods listed in Procedures section 3 of this document is not feasible. Cost alone is NOT considered an adequate justification.
          2. The list of pertinent questions to be answered during IACUC protocol review is provided in Appendix D.
        4. Once the procedure is approved, all investigative personnel designated to perform the procedure must undergo training and demonstrate proficiency to the Principal Investigator or ULAM Training Core.
        5. The rodent must be less than or equal to 7 days of age at the time of the procedure. Ideally, toe-clipping is performed on neonates 5-7 days of age.
          1. No more than one toe is amputated per foot.
          2. Only the most distal segment (phalanx) is amputated.
          3. Toes are amputated from the hind paw preferentially.

    5. Methods of Zebrafish Genotyping

    1. Invasive Methods
      1. Fin Clip
        1. Zebrafish will regenerate caudal fin tissue that is removed.  Caution must be used to only remove the fin tissue, and not damage the peduncle or bleeding and permanent tissue damage may occur.
        2. Anesthetize the fish.
        3. With a sterile scalpel or scissors, cut up to ½ to 2/3 of the caudal fin.
        4. Recover the fish in fresh system water.
  • Related Documents

  • Appendix A: Advantages and Disadvantages of Rodent Identification Methods

       Type   

       Advantages   

       Disadvantages   

       Cage Cards   

    • Pertinent information is readily available
    • Card can be lost or misplaced

       Dye   
       (Non-toxic, waterproof)   

    • Easy to apply
    • Non-invasive
    • Identifies individual animals
    • Temporary

       Ear Punch / Notch   

    • Easy to perform
    • Pattern can identify individual animals
    • ID lost if ear is injured or mutilated
    • Punch / notch can become less readable over time
    • Animals can become indistinguishable if mixed between cages

       Ear Tags   

    • Easy to apply
    • Permanent
    • Identifies individual animals
    • Tags can be lost
    • Animal usually must be restrained to read tag

       Tattoo   

    • Permanent
    • Identifies individual animals
    • Anesthesia recommended
    • Animal may need to be restrained to read tattoo
    • Equipment can be expensive
    • Equipment must be disinfected between use
    • Training required to operate the tattoo instrument

       Microchip   

    • Easy to apply
    • Permanent
    • Identifies individual animals
    • Not necessary to restrain animal to read
    • Expensive
    • Microchip may migrate within the body

       Toe Clip   
       (requires scientific justification)   

    • Permanent
    • Pattern can indentify individual animals
    • Requires amputation of a body part
    • Requires anesthesia
    • Usually must restrain animal to read

     

  • Appendix B: Advantages and Disadvantages of Rodent Genotyping Methods

       Type   

       Advantages   

       Disadvantages   

       Ear notching / punching   

    • Can also serve as a method of identification
    • Easy to perform
    • Repeat samples cannot be taken if this method is also
      used for identification

       Tail biopsy   

    • Can be performed at an early age
    • Provides a high quality sample for genotyping
    • Requires anesthesia and analgesia for older animals
    • Limited amounts can be taken
    • Repeat samples are discouraged
    • Need alternate means of identification

       Fecal sample   

    • Repeat samples can be taken
    • Easy to perform
    • Requires specialized processing methods

       Blood sample   

    • Repeat samples can be taken
    • Requires specialized processing methods

       Saliva sample   

    • Repeat samples can be taken
    • Collection procedure can be difficult
    • Requires specialized processing methods

       Hair bulb sample   

    • Repeat samples can be taken
    • Easy to perform
    • Requires specialized processing methods

       Toe amputation   
       (requires scientific justification)   

    • Can also serve as a method of identification
    • Repeat samples cannot be taken if this method is also
      used for identification
    • Requires amputation of a body part

     

  • Appendix C: Tattoo Equipment Options

  • Appendix D: Required Principal Investigator Justification of Toe Clipping in Rodents

    1. What is the reason for performing toe clipping? (identification, genotyping, etc.)?
    2. Have you determined that all of the below listed methods of identification will not meet your needs? If so, please provide a written explanation with evidence, as to why these identification methods are not feasible.
      1. Cage cards
      2. Markings (clipping/shaving or permanent marker)
      3. Microchips
      4. Ear Tags
      5. Ear notching/punching
      6. Tattooing, including micro-tattooing
    3. If genotyping, do you intend on using the tissues removed from the foot as the sole tissue sample for genotyping or will additional samples be collected?
    4. If using the toe tissue for both identification and genotyping, please explain why none of the below listed of genotyping methods can be used in combination with another long term identification methods (microchipping, ear tag, ear notch/punch, micro-tattooing):
      1. Tail clipping
      2. Ear punching/notching
      3. Buccal swabs
      4. Fecal samples
      5. Blood Samples
      6. Hair samples
    5. Will toe clipping only be done in mice less than or equal to 7 days of age?
  • References

    1. Bonaparte D, et al. 2013. FELASA guidelines for the refinement of methods for genotyping genetically-modified rodents. Laboratory Animals 47(3) 134-145
    2. Campbell DB, et al. 1997. Rapid genotyping of mutant mice using dried blood spots for polymerase chain reaction (PCR) analysis. Brain Res Protoc. 1(2):117-23.
    3. Castelhano-Carlos MJ, et al. 2010. Identification methods in newborn C57BL/6 mice: a developmental and behavioural evaluation. Laboratory Animals. 1-16
    4. Dahlborn K, et al. 2013. Report of the Federation of European Laboratory Animal Science Associations Working Group on animal identification. Laboratory Animals 47: 2-11
    5. Gagiliene B, et al. 2014. Direct PCR on Hair: A New Animal-Friendly Genotyping Method. Thermo Fisher Scientific, Inc. Available at: https://www.thermofisher.com/content/dam/LifeTech/global/brands/Documents/1114/App_Note_Direct_PCR_on_Hair_2014_June09_final.pdf
    6. Hamann M, et al. 2010. Non-Invasive Genotyping of Transgenic Mice: Comparison of Different Commercial Kits and Required Amounts. ALTEX. 27(3):185-90.
    7. Hankenson FC, et al. 2008. Evaluation of tail biopsy collection in laboratory mice (Mus musculus): vertebral ossification, DNA quantity, and acute behavioral responses. JAALAS 47(6):10-18
    8. Irwin MH, et al. 1996. Identification of transgenic mice by PCR analysis of saliva. Nat Biotechnol. 14(9):1146-8
    9. Jones CP, et al. 2012. Evaluation of Common Anesthetic and Analgesic Techniques for Tail Biopsy in Mice. JAALAS 51(6): 808-814
    10. NIH Guidelines for Toe Clipping of Rodents. Dec 2013. Available at http://oacu.od.nih.gov/ARAC/documents/Toe_Clipping.pdf
    11. Schaefer DC, et al. 2010. Analysis of physiological and behavioural parameters in mice after toe clipping as newborns. Laboratory Animals. 44:7-13
    12. Schmitteckert EM, et al. 1999. DNA detection in hair of transgenic mice--a simple technique minimizing the distress on the animals. Lab Anim. 33(4):385-9
    13. Symonds EL, et al. 2012. A method for non-invasive genotyping of APCmin/+ mice using fecal samples. Biol Proced Online. 14: 1.