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Approval Date: November 1, 2017

Purpose

The purpose of this policy is to provide guidance to researchers regarding acceptable methods for marking animals to identify individuals and tissue collection for the purpose of rodent genotyping.

Background

Individual animal identification is important for animal colony management, animal health/medical records, and for research data interpretation. There are several identification and genotyping methods, including temporary markings, tattoos, ear punch/notch, ear tags, tattoos, and tail biopsies. Genotyping is the process through which the genetic make-up of an animal is determined using a tissue sample. The nature and quantity of tissue required varies with the type of DNA test being performed.  Common DNA collection methods include ear punches and tail biopsies. Toe clipping is another method used for both identification and obtaining DNA for genotyping; however, it can only be used when no other alternative method can be utilized as detailed below.

Temporary Markings

Use of an indelible (permanent) marker to write numbers or other distinguishable markings on the fur, tail, or skin of the animal does not require the use of anesthesia and does not need to be included in the IACUC protocol.

Ear Tags

Tags must be appropriately sized for the species and age. Proper placement is necessary to prevent ear irritation or trauma. Use of ear tags does not require the use of anesthesia and does not need to be included in the IACUC protocol.

Ear Punches

The IACUC recommends the use of ear punches over tail biopsies to obtain material for genotyping. Ear punches can be used for both identification of the animal and genotyping of the collected tissue. They do not require the use of anesthesia and do not need to be included in the IACUC protocol.

Tattoos

Tattoos are an effective method of permanent identification. An electric tattoo machine may be used or a needle and syringe. All needles used for tattoos must be sterile and changed between each group of animals or when the needle becomes blunted. Machines must be maintained according to manufacturer’s recommendations. Tattooing only needs to be included in the protocol if the use of anesthesia is necessary.

Investigators may consult with LAMS or DLAM Rodent Breeding Services (RBS) for tattooing technical services or training on this method.

Tail Biopsies

A conventional procedure for genotyping entails snipping 5-15 mm of the distal portion of the animal’s tail. Many strains of mice have mature vertebrae within 5 mm of the distal tail by day 17 of age. Since evidence exists that pre- and post-weanling rodent pups perceive pain, the IACUC recommends an anesthetic is used for all ages. Tail biopsies must be included in the IACUC protocol.

Only individuals with demonstrated competence in tail cutting and proper restraint may perform tail biopsies. The following requirements must be followed when tail biopsies are performed:

  • Tail biopsies in rodents 17 days or older must be conducted under general anesthesia. Isoflurane is the preferred anesthetic. The use of an analgesic is recommended.
  • Tail biopsies in rodents less than 17 days of age does not require general anesthesia.
  • Tail biopsies must be conducted using clean, sharp instruments such as surgical blades, razor blades, or sharp scissors. If individual blades cannot be used per sample, then instruments should be cleaned between individuals to remove all tissue. The operator must change instruments when dullness of the cutting edge is detected.  Changing instruments after every ten uses will ensure a sharp edge.
  • Following the procedure, the biopsy site must be observed for active bleeding and hemostasis ensured before returning animals to the housing facilities. The following methods can be used to stop bleeding: digital pressure, cautery, tissue adhesives, coagulation powder, or coagulation sticks.

* If using cautery with vaporized isoflurane, caution should be used to prevent a fire. A fire can be ignited if the cauterizer comes into contact with the oxygen-enriched atmosphere streaming from the anesthetic equipment.

Toe Clipping

Toe clipping is both a method used for identifying mice and obtaining DNA for genotyping.  IACUC subscribes to the recommendations of The Guide for the Care and Use of Laboratory Animals and the Interagency Research Animal Committee of the federal government regarding the use of toe clipping as a means of identification in small rodents. In particular, The Guide (p.75) stipulates that “toe-clipping should be used only when no other identification method is feasible.” In brief, because toe clipping is viewed as a potentially painful procedure, this method of identifying an animal must be justified in the protocol and must be performed in the most painless and humane way consistent with current veterinary practice and standards.

Therefore, the IACUC requires the following when the use of toe clipping as a method of rodent identification is necessary:

  • Justification must be provided in the protocol regarding why no other alternative method of identifying the animal can be utilized. If the same animal must be genotyped, then the animal may only undergo one procedure for both tissue harvest for genotyping and permanent identification. Toe clipping for the sole purpose of obtaining DNA for genotyping is not acceptable justification. The toe clip procedure must be described in detail in the IACUC protocol submission.
  • The procedure can only be conducted on pre-weanling pups up to 13 days of age.
  • The procedure should be performed using sharp disinfected scissors that are disinfected between animals.
  • The use of vapocoolant as a local anesthetic should not be used for toe clipping procedures given its propensity to cause signs of distress in pups undergoing this procedure.
  • The procedure may be performed on a maximum of one toe per foot on the hind limbs only. The amount of toe removed should be kept to a minimum.
  • Assure adequate hemostasis. Observe the site post-procedurally for active bleeding. Apply pressure and/or hemostatic agents.

Any deviations from this policy must be justified and approved by the IACUC prior to use.

References

  • Hankenson FC, Garzel LM, Fischer DD, Nolan B, Hankenson KD. 2008. Evaluation of Tail Biopsy Collection in Laboratory Mice (Mus musculus): Vertebral Ossification, DNA Quantity, and Acute Behavioral Responses. JAALAS 47:10-18.
  • Kasanen, H.E., H-M. Voipio, H. Leskinen et al. 2011. Comparison of ear tattoo, ear notching and microtattoo in rats undergoing cardiovascular telemetry. Lab Animal 41:19-29.
  • Guide for the Care and Use of Laboatory Animals. 8th ed. Washington, DC: National Academic Press; 2011.
  • Castelhano-Carlos MJ, Sousa N, Ohl F, Baumans V. Identification methods in newborn C57BL/6 mice: a developmental and behavioural evaluation. Lab Anim. 2010;44(2):88-103. doi:10.1258/la.2009.009044.
  • Norecopa A, Mj C, Dc S, Spangenberg E. Supplementary statement on toeclipping in rodents. 2010;(March):2-5.
  • Paluch L-R, Lieggi CC, Dumont M, Monette S, Riedel ER, Lipman NS. Developmental and behavioral effects of toe clipping on neonatal and preweanling mice with and without vapocoolant anesthesia. J Am Assoc Lab Anim Sci. 2014;53(2):132-140.
  • Bonaparte D, Cinelli P, Douni E, et al. FELASA guidelines for the refinement of methods for genotyping genetically-modified rodents: a report of the Federation of European Laboratory Animal Science Associations Working Group. Lab Anim. 2013;47(3):134-145. doi:10.1177/0023677212473918.