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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we describe methods for tattooing adult Xenopus laevis (African clawed frog) with a rotary tattoo machine. Proper tattooing results in dark, easily legible numerals that last for several months and make animals easily distinguishable for research and recordkeeping purposes.

Abstract

Animal models expand the scope of biomedical research, furthering our understanding of developmental, molecular, and cellular biology and enabling researchers to model human disease. Recording and tracking individual animals allows researchers to reduce the number of animals required for study and refine practices to improve animal wellbeing. Several well-documented methods exist for marking and tracking mammals, including ear punching and ear tags. However, methods for marking aquatic amphibian species are limited, with the existing resources being outdated, ineffective, or prohibitively costly. In this manuscript, we outline methods and best practices for marking Xenopus laevis with a rotary tattoo machine. Proper tattooing results in high-quality tattoos, making individuals easily distinguishable for researchers and posing minimal risk to animals' health. We also highlight the causes of poor-quality tattoos, which can result in tattoos that fade quickly and cause unnecessary harm to animals. This approach allows researchers and veterinarians to mark amphibians, enabling them to track biological replicates and transgenic lines and to keep accurate records of animal health.

Introduction

Animal models are useful tools for investigating questions pertaining to human health. In practice, biomedical research using animal models requires careful organization and maintenance of a healthy animal colony. Best practices for ethical animal handling and husbandry aim to reduce the number of animals needed for experimentation and refine practices to ensure animal welfare1. The clawed frog genus, including Xenopus laevis (X. laevis; African clawed frog) and Xenopus tropicalis (X. tropicalis; Western clawed frog), have been used in biomedical research since the 1930s when X. laevis were used by S....

Protocol

All animal procedures described were approved by Dartmouth College's Institutional Animal Care and Use Committee.

1. Equipment setup

NOTE: A workflow for the procedure and an example bench setup are included (Figure 1).

  1. Connect the tattoo gun and foot pedal to the power supply. Position the foot pedal underneath the working surface.
  2. Assembling the tattoo gun
    1. Using an Allen wrench or.......

Representative Results

High-quality tattoos will have dark, legible strokes on the frog's chest and can be clearly differentiated from several feet away (Figure 5A). In general, larger numbers and markings are better for readability, but longer names and numbers can be made smaller to fit comfortably on the frog's chest. Tattoo longevity is more difficult to judge, but high-quality frog tattoos should remain dark and legible for at least 3-6 months (Figure 5D-D'

Discussion

Tattooing humans is an art form dating back thousands of years, and for as long as humans have tattooed themselves, they have also tattooed or branded animals23. The equipment and techniques for marking animals, particularly mammals, are well-established, well-documented, and widely accessible. While marking animals was originally for distinguishing livestock and theft deterrents23, its role in biomedical research has become just as important.

A .......

Acknowledgements

We thank Dartmouth College's Center for Comparative Medicine and Research for providing daily husbandry for the animals used in this protocol. We also thank Leah Jacob and Adwaita Bose for their help in testing the protocol and photographing animals. Lastly, we thank Ann Miller's lab for training in the practice of tattooing. This work was supported by NIH grant R00 GM147826 to J.L.

....

Materials

NameCompanyCatalog NumberComments
3 needle round linersWorldwide Tattoo Supply1203RLBPackaged sterile
5 Needle Round Disposable ULTRAWorldwide Tattoo SupplyHTIPRS5-UPackaged sterile
5 needle round linersWorldwide Tattoo Supply1205RLBPackaged sterile
7 needle round linersWorldwide Tattoo Supply1207RLBPackaged sterile
Clip CordWorldwide Tattoo SupplyN/A
Foot pedalWorldwide Tattoo SupplyN/A
InkpotsWorldwide Tattoo SupplyN/A
Kimwipes, delicate task wipesFisher Scientific06-666A
RCA ConnectionWorldwide Tattoo SupplyN/A
Scream Ink Pitch Black, 1ozWorldwide Tattoo SupplySI101
Sodium bicarbonate (NaHCO3)Sigma-AldrichS5761
Stainless steel gripsWorldwide Tattoo SupplyN/A
Stealth 2.0 Rotary Tattoo MachineWorldwide Tattoo SupplyN/A
Stealth 2.0 Rotary Tattoo Machine Box SetWorldwide Tattoo SupplySTEALTH2-SET
Styrofoam islandN/AN/AThis is the lid of a styrofoam cold shipping container
Tricaine (ethyl 3-aminobenzonate methanesulfate)Sigma-AldrichE10521CAUTION: IRRITANT
Unbleached paper towelsGrainger2U229Paper towels MUST be unbleached, bleach is toxic to amphibians
Voltage SupplyWorldwide Tattoo SupplyN/A
Wash bottle (with frog-safe water)Fisher ScientificFB0340923TFrog safe water is dechlorinated, pH 7.0-8.5, conductivity 1200-1800 uS
X. laevis adult femaleXenopus1N/A
Zip-top plastic bagN/AN/AThis bag should be large enough to hold the styrofoam island

References

  1. Hubrecht, R. C., Carter, E. The 3Rs and humane experimental technique: Implementing change. Animals (Basel). 9 (10), 754 (2019).
  2. Elkan, E. R. The Xenopus pregnancy test. British Med J. 2, 1253-1256 (1938).
  3. Vi....

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BiologyRotary Tattoo MachineAnimal ModelsBiomedical ResearchMarking TechniquesAmphibian TrackingAnimal WellbeingHigh quality TattoosPoor quality TattoosTransgenic LinesBiological ReplicatesAnimal Health Records

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