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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.
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 However, methods for marking aquatic amphibian species are limited, with the existing resources being outdated, ineffective, or prohibitively costly.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.
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....
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).
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'
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.
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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.
....Name | Company | Catalog Number | Comments |
3 needle round liners | Worldwide Tattoo Supply | 1203RLB | Packaged sterile |
5 Needle Round Disposable ULTRA | Worldwide Tattoo Supply | HTIPRS5-U | Packaged sterile |
5 needle round liners | Worldwide Tattoo Supply | 1205RLB | Packaged sterile |
7 needle round liners | Worldwide Tattoo Supply | 1207RLB | Packaged sterile |
Clip Cord | Worldwide Tattoo Supply | N/A | |
Foot pedal | Worldwide Tattoo Supply | N/A | |
Inkpots | Worldwide Tattoo Supply | N/A | |
Kimwipes, delicate task wipes | Fisher Scientific | 06-666A | |
RCA Connection | Worldwide Tattoo Supply | N/A | |
Scream Ink Pitch Black, 1oz | Worldwide Tattoo Supply | SI101 | |
Sodium bicarbonate (NaHCO3) | Sigma-Aldrich | S5761 | |
Stainless steel grips | Worldwide Tattoo Supply | N/A | |
Stealth 2.0 Rotary Tattoo Machine | Worldwide Tattoo Supply | N/A | |
Stealth 2.0 Rotary Tattoo Machine Box Set | Worldwide Tattoo Supply | STEALTH2-SET | |
Styrofoam island | N/A | N/A | This is the lid of a styrofoam cold shipping container |
Tricaine (ethyl 3-aminobenzonate methanesulfate) | Sigma-Aldrich | E10521 | CAUTION: IRRITANT |
Unbleached paper towels | Grainger | 2U229 | Paper towels MUST be unbleached, bleach is toxic to amphibians |
Voltage Supply | Worldwide Tattoo Supply | N/A | |
Wash bottle (with frog-safe water) | Fisher Scientific | FB0340923T | Frog safe water is dechlorinated, pH 7.0-8.5, conductivity 1200-1800 uS |
X. laevis adult female | Xenopus1 | N/A | |
Zip-top plastic bag | N/A | N/A | This bag should be large enough to hold the styrofoam island |
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