BACKGROUND

Individual animal identification is important for colony management (to track breeding crosses and genotype, etc.), for surgical and medical records, and for tracking individualized data of animals on chronic studies.  There are several methods available for identifying rodents, including ear punches/notches, ear tags, tattoos, and microchip transponders. Ear punching and toe-clipping can also provide tissue for genotyping.  Toe-clipping may only be used only when no other individual identification method is feasible and requires strong scientific justification. Please see the WSU IACUC Rodent Toe-Clipping Policy.

Genotyping of genetically modified rodents is critical for research reproducibility and reduction of animal numbers. Genotype is most often determined by polymerase chain reaction (PCR) analysis of DNA extracted from tissues of young rodents⁵. DNA is most commonly obtained from a tail biopsy, but may also be collected from ear punches, blood², hair³, buccal swabs⁴, and feces.

IACUC Guidelines

Identification Methods

Marker:

Procedure:

1. An indelible (permanent) marker is used to write on the tail, skin of hairless strains or neonates, or light-colored haircoats.
2. Reapplication is recommended every 2-3 days, as needed.

Recommendations:

• While ethanol-based inks in permanent markers have bactericidal properties^6,7, markers may be a source of cross-contamination. A colony-dedicated marker should be used for identification only.

• Makers should be replaced when soiled or dried-out as they can harbor pathogens.^{6, 7}
• Products:
  • Sharpie permanent marker (ear, tail, coat, feet; lasts 2-3 d)
  • Animal Markers (coat only; lasts 6-12 wks); Fine Tip Animal Markers

Ear tag:

Procedure:

1. Application tools are tag-specific and cannot be used interchangeably
2. Load ear tag in the applicator
3. Manually restrain the rodent so that the ear is accessible
4. Apply the tag to the lower/ventral 1/3 of the pinna, next to the cartilage ring
5. Quickly apply firm pressure to the applicator so that the tag pierces the ear and tag and folds on the other size
6. Incorrectly placed tags must be removed before replacing on the opposite ear

Recommendations:

• Either ear may be tagged, but no more than one tag should be on a rodent
• Use a 1 mm rodent ear punch to create a guide hole for application of metal ear tags
• Standard rodent metal ear tags:
  • National Band & Tag Company
• Alternative ear tags to metal bands:
  • RapID Tags®
  • Stoelting© Mouse & Rat Ear Tags
• Tools for removing ear tags:
  • Wire Cutting Scissors
  • Wire Cutters

Tattoo:

1. Micro-tattoo (ear(s), toe(s)/foot, tail):
   1. Can be performed without anesthesia at any age
   2. Swab the location to be tattooed with 70% ethanol
   3. Dip a sterile, small gauge needle (25-30g) in tattoo ink (see recommendations below) and pierce the skin to make small dots on the ear(s), toe(s), or tail.¹¹
      1. A new needle should be used for each animal
   4. Tattoo ink may also be injected intradermally using a small gauge needle and syringe (28-31 g insulin syringe + needle recommended)
   5. Remove excess ink with 70% ethanol
   6. Commercial micro-tattooing forceps can be purchased, and tattoos are applied following the manufacturer's instructions
2. Electric Tattoo Machine (ear, tail):
   1. Cannot be performed on neonates; complexity of identification scheme limited by age and size of the ear or tail
   2. Anesthesia is required
   3. Tattoo needles must be sterile and sharp, and should be changed regularly in accordance with the manufacturer's recommendations, or once the needle becomes dull
   4. Equipment should be disinfected between each use
   5. Procedure:
      1. Anesthetize the rodent
      2. Swab the ear or tail with 70% ethanol
      3. Using an electric tattoo machine, free-hand tattoo identification symbols on the tail or ear
      4. Remove excess ink with 70% ethanol
      5. Recover the animal and return it to its cage
3. Somark® Labstampâ„¢ Tattoo Machine (tail only): 
   1. Available for rental through DLAR VTS with purchase of ink cartridges
   2. For mice older than 3 weeks of age
   3. Does not require anesthesia

Recommendations:

• Ketchum Animal Tattoo Ink Paste
  • Available in green (visible on black mice) or black only
  • Thicker paste consistency is less messy and may be easier to work with
• India Ink
• Human Tattoo Ink

Subcutaneous transponders, a.k.a. Microchips or RFID transponders:

Procedure:

1. Follow the manufacture's recommendations for the minimum age/size requirements for placement
2. Ensure the microchip is functional prior to implantation by scanning with the reader
   1. This is especially important with reuse, but new microchips occasionally fail and should not be placed
3. Prepare the interscapular region with 70% ethanol and part the hair to visualize the skin and minimize translocation of hair into the subcutaneous space
4. Implant the microchip by injecting it using a sterile trocar under the skin
5. Withdraw the needle slowly and flatten the skin at the injection site to re-appose the injection site edges
6. Medical grade cyanoacrylate adhesive may be applied to close the injection site; this is recommended to reduce loss of microchips, but not required
7. Verify chip placement by rescanning the animal with the microchip reader

Recommendations

1. While not required, clipping the hair can improve visualization and proper placement
2. Microchip identification systems:
   1. Kent Scientific Corporation RFID Transponder System
   2. Somark® Digitailâ„¢ System (implanted in the tail)

Identification and Genotyping Method

Ear Punch:

Procedure:

1. Restrain rodents by gently scruffing
2. Use an ear punch instrument to remove punches of tissue on one or both ears
3. Punches may be used on outermost edge of the pinna to create or notch or within the center to create a hole
4. Avoid the area of the ear closest to the head where the cartilage is thicker and more vascularized as it may be painful and bleed
5. The ear punch instrument should be disinfected between cages or individual animals if the tissue is to be used for genotyping to prevent DNA contamination

Recommendations:

• Ear punch instruments are available in different sizes, 0.5 to 2 mm diameter punches.
  • 2 mm diameter punches are recommended to reduce hole closure and healing as well as to ensure sufficient sample size for DNA analysis
  • 1 mm diameter punches are recommended for generating holes for ear tagging
• Ear punch tools may dull with regular use and should be replaced as need
• There are a variety of punch instruments available:
  • Fine Science Tools (Plier-Style, Finger Loop, Ear Punch with Replaceable Tip)
  • Kent Scientific (Nail-Clipper Style, Scissor Style)
  • VWR®Animal Ear Punches (Plier-Style, Scissor Style, Thumb-Style)
  • World Precision Instruments (Simple Ear Punch, Scissor Style)
  • Stoelting (Scissor Style, Thumb-Style)
• DLAR Training owns many of the ear punch instrument styles listed above. Please feel free to test different models during training before purchasing one for your lab. Instrument selection is largely based on user preference.

Genotyping Method

Tail Biopsy:

General Requirements:

• Anesthesia is required for mice and rats 21 days of age and older and must be described in your IACUC protocol
• Tail biopsy length should be limited to the smallest amount possible
  • ~2 mm yields sufficient DNA for multiple PCR reactions
  • If larger samples are required, it should be described with a justification in the protocol
• Rodents should undergo tail biopsy as early as possible to minimize pain associated with vertebral ossification
  • Tail biopsy of rodents > 21 days of age should be limited to reevaluation of erroneous or ambiguous results and not standard practice unless justified in your protocol
• Tail biopsy of younger mice yields more DNA than that of adult mice⁵

Procedure:

• Sterile surgical scissors, scalpel or straight blades may be used to biopsy tails.
  • Surgical scissors must be disinfected after each animal or use a new blade for each animal.  This helps ensure correct genotyping.
• 2-3 mm of the tail tip is removed
• Bleeding should be controlled with gentle fingertip pressure
  • The use of styptic powder with benzocaine (Kwik-Stop) is recommended for hemostasis and analgesia.
• Cessation of bleeding must be confirmed prior to returning animals to their cage

Recommendations:

• Best practice is to use a new sterile scalpel or straight blade for every animal; they must be replaced after 5 uses or sooner if they become dull
• Analgesics should be considered and described in the protocol for biopsy of rodents > 21 d age

References

1. NIH Office of Intermural Research. (2022). Guidelines for Tissue Collection for Genotyping of Mice and Rats https://oacu.oir.nih.gov/system/files/media/file/2022-01/b3-rodent_genotyping.pdf.
2. Campbell DB, Hess EJ. (1997). Rapid genotyping of mutant mice using dried blood spots for polymerase chain reaction (PCR) analysis. Brain Res Brain Res Protoc. 1:117-123.
3. Schmitteckert EM, Prokop CM, Hedrich HJ. (1999). DNA detection in hair of transgenic mice a simple technique minimizing the distress on the animals. Lab Anim 33:385-389.
4. Meldgaard M, Bollen PJ, Finsen B. (2004). Noninvasive method for sampling and extraction of mouse DNA for PCR. Lab Anim 38:413-417.
5. Picazo MG, García-Olmo DC. (2015). DNA from tissues of young mice is optimal for genotyping. Electron. J. Biotechnol 18: 83-87.
6. Coll AR. (2007). Using Marker Pens on Patients: a Potential Source of Cross Infection with MRSA. Surg Engl. 89: 661-664.
7. O'Bryan E, Pollock M, Joseph S. (2019). Comparing the sterility and visibility of surgical marking pens available in Australia. ANZ J Surg 89: 1114-1118.
8. Kitagaki M, Hirota M. (2007). Auricular chondritis caused by metal ear tagging in C57BL/6 mice. Vet Pathol 44: 458-466.
9. Baron BW, Langan G, Huo D, et al. (2005). Squamous cell carcinomas of the skin at ear tag sites in aged FVB/N mice. Comp Med 55: 231-235.
10. Cover CE, Keenan CM, Bettinger GE. (1989). Ear tag induced Staphylococcus infection in mice. Lab Anim 23: 229-233.
11. Chen M, Kan L, Ledford BT, et al. (2016). Tattooing Various Combinations of Ears, Tails, and Toes to Identify Mice Reliably and Permanently. JAALAS 55: 189-198.
12. Bains RS, Cater HL, Stewart M et al. (2020). The effects of microchipping C57BL/6N mice on standard phenotyping tests [version 2; peer review: 2 approved] F1000Research. 9:20 (https://doi.org/10.12688/f1000research.21633.2)
13. Elcock LE, Stuart BP, Wahle BS, et al. (2001). Tumors in long-term rat studies associated with microchip animal identification devices. Exp Toxicol Pathol 52: 483-491.
14. Roughan JV, Tatum S. (2019). Welfare and Scientific Considerations of Tattooing and Ear Tagging for Mouse Identification. JAALAS 58: 142-153.
15. Hankenson FC, Garzel LM, Fischer DD, et al. (2008). Evaluation of Tail Biopsy Collection in Laboratory Mice (Mus musculusa): Vertebral Ossification, DNA Quantity, and Acute Behavioral Responses. JAALAS 47: 10-18.

Approved: December 2012

Revisions Approved: 5/2015, 4/2016, 6/2018, 1/2023