Methods for Tattooing Xenopus laevis with a Rotary Tattoo Machine

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 However, methods for marking aquatic amphibian species are limited, with the existing resources being outdated, ineffective, or prohibitively costly.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 South African doctors to conduct the first pregnancy tests². While modern pregnancy tests no longer require frogs, the role of Xenopus research persists. Advantages of using Xenopus for biomedical research include their well-annotated genomes³, year-round inducible ovulation of large clutches of eggs⁴, and externally laid eggs amenable to in vitro fertilization. These features make them a useful asset for vertebrate embryology and development^5,6,7, basic molecular and cellular biology^7,8,9,10, and for modeling human disease^7,11,12,13.

Reliable methods for tracking individual Xenopus animals are essential for recording biological replicates and improving rigor and reproducibility in research. As Xenopus are frequently housed in groups, animal marking allows researchers to easily track individual animals⁴. Maintaining an accurate record of animals can save time and resources and improve the ability to track animals' health. For example, individual identification of animals can improve organization workflows for generating transgenic Xenopus lines, as this requires multiple generations of frogs with specific genotypes verified by sequencing¹⁴, which requires organization and individual identification of animals. This is particularly true when these mutations lack easily discernible adult phenotypes. Similarly, the use of Xenopus oocytes and embryos to study basic cellular and developmental biology benefits from tracking individual animals. After inducing ovulation, animals need to rest for a minimum of 3 months to prevent health complications such as hyper-ovulation syndrome¹⁵. Individual identification methods ensure that animals are not induced to ovulate too frequently.

Marking and tracking animals also enables lab personnel to track animals' health concerns. Animals of the same genotype falling ill can indicate excessive inbreeding or unanticipated health concerns associated with the transgene. Similarly, animals falling ill after recent ovulation can indicate issues with reagents, materials, or techniques. Tracking animals and their health enables lab personnel and veterinarians to follow up when concerns resurface and take preventative measures to prevent future illness. In mammals, there are numerous identification methods. Permanent methods for mice include ear punching, ear tags, tattooing, and subcutaneous microchips¹⁶. These can clearly and reliably differentiate animals within a colony or cage and can be easily administered by laboratory personnel. Methods such as ear punching are minimally invasive, require only one piece of specialized equipment, and work for animals of most ages. While these systems are straightforward and useful for mice, their use in frogs presents a unique set of challenges. Frogs and other amphibians lack a pinna (external ear structure). Some researchers have attached tags to the animal's jaw, toe, or hind limb^17,18. This approach resulted in various problems: jaw tags caused irritation, and agitated frogs attempted to pull off tags with their forelimbs¹⁷. Toe tags pierced the webbing between toes, impairing movement and carrying the risk of becoming lost. As such, amphibians require their own methods for identification. Historically, toe-clipping has also been used to mark amphibians^17,19. The toe is clipped with a sharp pair of scissors, and the animal can be identified by the length of toes on the forefeet and hind feet or by the angle at which the toe has grown back (in salamanders). However, this method poses the ethical concern that toe clipping may impair the animal's movement¹⁷. In addition, this can cause bleeding and introduce a risk of infection. Another established marking system is skin autografts, in which skin is taken from one part of the frog and surgically attached to another part. For example, a method is described for marking a frog's back or shoulder using a light-colored skin graft from its chest²⁰. Skin grafts also come with limitations and risks: the procedure is invasive and introduces the risk of Aeromonas hydrophila infection, or red leg, a potentially fatal affliction; complete healing of the autograft takes up to 6 weeks; and with the methods described, only 6 frogs can be housed together because of limited places to put an autograft²⁰.

Less invasive marking approaches include glass beads and transponder chips^17,19. In the glass bead method, glass beads are threaded onto a small suture and sewn into the frog's skin. This provides greater variability than skin autografts, with at least 60 distinctive color combinations. There is, however, a risk that the suture can come out and result in the beads being lost. Alternatively, a microchip transponder can be implanted under the skin in the frog's dorsal lymph sac. This is considered the most permanent marking method and enables a potentially infinite number of animals to be individually identified and cataloged. However, this is also the most expensive method, as individual microchips are expensive, and a large colony would be costly to mark. Microchips also require a special scanner to read¹⁹. One common approach for Xenopus identification is referring to animals' natural coloring and patterning. This works especially well for frogs such as X. laevis, which have distinct patterns that remain throughout adulthood. However, these patterns can change over time with stress, and coloring can appear different when frogs are moved between transparent and tinted containers¹⁵. Additionally, this identification method is less useful for X. tropicalis, which has less distinct marking patterns compared to X. laevis, or for albino animals, which have no color markings²¹. Even for species with distinct markings, lab personnel can interpret the placement and size of markings differently, which can cause errors in identification. Because of this, photographing animals is most reliable in conjunction with an additional identification method. Therefore, we seek to mark and identify Xenopus animals using a technique that is easily discernable, permanent, and minimally invasive.

There are limited published resources describing methods for tattooing amphibians. Tattooing has been described alongside other branding techniques, including heat brands, silver nitrate brands, and freeze brands¹⁷. In the same resource, tattooing was done by drawing numerals with a 27G hypodermic needle, and the process was noted not to cause infection, in contrast to the other branding techniques, which used a wire shaped into a numeral or other mark. In another source, an electric tattooing machine (described as a vibrating needle) was used to mark frogs, but little detail was provided on the technique^17,19. The authors warn that by disturbing the frog's protective slime layer, this procedure increases the risk of red leg. While there is no marking or identification method that is both completely noninvasive (such as photographs) and permanent (such as microchips), tattooing provides an effective compromise. Tattooing is relatively straightforward compared to other techniques, such as skin autografts. Additional benefits include a smaller learning curve and relatively inexpensive equipment. Tattooing aquatic amphibians comes with certain challenges, which can intimidate researchers and impair successful animal marking. This paper aims to provide researchers with well-documented methods for tattooing adult Xenopus with a rotary tattoo machine.

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 hex screwdriver, loosen the screws in the grip (Figure 2A, encircled in red).
   2. Insert the plastic tip all the way into the grip. Tighten the screw to hold it in place.
   3. Insert the metal tube in the back of the grip (Figure 2A). This will be adjusted, so insert it to half its length. Tighten the screw to hold it into place.
   4. Insert a tattoo needle through the back of the grip so that it sits comfortably in the plastic tip at the front of the grip (Figure 2B).
   5. Slide the back of the needle and metal tube through the tube clamp. Adjust the metal tube so that the needle fits comfortably in the plastic tip (Figure 2B). Hand-tighten the tube clamp until the tube is unable to move (Figure 2C).
   6. Remove the black O-ring and hook the needle onto the rotor arm; replace the O-ring to secure the needle into place (Figure 2C).
3. Set the voltage on the power supply so that there is enough power to pierce the skin but not so powerful that it is difficult to control. Once the power supply is plugged in and connected, press the foot pedal to make sure the machine is working. Take care not to point the needle at yourself or other personnel.
   NOTE: The tattoo machine used in this protocol works best between 6.0 and 9.0 V, but this may vary between tattoo machines and voltage supplies and should be determined empirically.
4. Fill the cap of a 1.5 mL microcentrifuge tube or a provided plastic inkpot with black tattoo ink to about three-quarters full.

2. Anesthesia

1. Prepare anesthesia in a tank large enough to submerge one adult female X. laevis frog.
   1. Using frog-safe (chlorine-free) water, add tricaine to the final concentration of 1.5 g/L and sodium bicarbonate to the final concentration of 3.5 g/L. Mix to dissolve. The pH of this solution is 7.15.
      CAUTION: Tricaine is an irritant.
2. Submerge one frog in the anesthesia tank. Ensure that the frog stays submerged in the tricaine solution until it is anesthetized (7-8 min).
   1. To check if the frog is fully anesthetized, pick it up, hold it upside down, and give it a firm squeeze on one foot. If the frog does not flinch or respond, it can be tattooed.
   2. If frogs consistently take more than 10 min to be anesthetized, prepare fresh tricaine solution buffered with sodium bicarbonate.
      NOTE: Frogs should not spend more than 30 min in tricaine solution¹⁵.

3. Tattooing

NOTE: Before tattooing a live animal, it can be useful to practice on a piece of fruit with a firm peel (such as a lemon or banana).

1. Prepare a recovery tank for the frogs before tattooing. Fill a tank with 10-15 L of fresh frog-safe water and add a Styrofoam island. This will provide a surface for the frog to wake up without drowning.
   NOTE: A Styrofoam island can be made using the lid of a Styrofoam cold shipping container placed inside a zip-top bag.
2. Place the anesthetized frog onto its back on a dry paper towel or bench paper (Figure 3A). Use unbleached (brown) paper towels for all work involving frogs.
3. Using a dry lint-free wipe, wipe away the water and mucus from the frog's chest.
4. Find the sternum in the center of the frog's chest with fingers and, using the non-dominant hand, hold the skin taut.
5. Marking the frog
   1. Hold the assembled tattoo gun vertically relative to the working surface and dip the needle into the ink (Figure 3B).
   2. Keeping the assembled tattoo gun vertical, press the tip of the needle onto the frog's skin before pressing the foot pedal. Draw lines onto the frog's skin while applying even pressure.
      NOTE: Some minor bleeding or redness is normal, as is minor skin sloughing.
   3. If the needle is getting caught on the frog's skin, clear excess ink or skin from the needle with a wipe. Excess ink is normal during tattooing.
   4. If the frog's chest is covered in too much ink to clearly see the area being marked (Figure 3C), clear excess ink with frog-safe water and a wipe (Figure 3C'). Then, wick away moisture with a dry wipe and continue inking.
      NOTE: A squeeze bottle of frog-safe water can be prepared before tattooing.
   5. Continue to ink the same area until dark, legible numbers remain after wiping away excess ink (Figure 3D). Instead of drawing the entire marking or number in one stroke, repeatedly make smaller strokes, especially for curves.

4. Recovery

1. Wet a paper towel with frog-safe water and place it flat on top of the Styrofoam island (Figure 4A).
2. Returning frogs to standing tanks
   1. Lay the frog belly-down on the towel facing the water (Figure 4B). Fold half of the paper towel over the back half of the frog and, using cupped hands, wet the top of the frog. Once a frog emerges from anesthesia (about 1 h after tattooing), they will enter water independently and swim normally.
   2. Return frogs to long-term housing after 24 h, once the tattoo has fully healed.

5. Cleaning and equipment maintenance

1. Reusing and autoclaving tattooing needles
   1. Rinse needles in 100% isopropanol and deionized water to loosen the ink and scrub off the remaining ink that has not dissolved.
   2. After scrubbing, place the needles into a metal autoclaving case or wrap them with aluminum foil and autoclave for 30 min on a dry cycle.
2. If there is ink on pieces of the tattoo machine or other equipment, wipe it with 100% ethanol and a paper towel.
3. The anesthetic solution has a final tricaine concentration of approximately 0.5%. Dispose of this according to institutional hazardous waste management guidelines.
4. Equipment storage and maintenance
   1. If possible, store the tattoo machine in a dry room. Excess moisture will damage the electric components and reduce the longevity of the machine.
   2. To keep the tattoo machine in good working order, check the bearing on the rotor (Figure 2C) to make sure it has not come loose from vibrations²². This can be tightened with an Allen wrench or hex screwdriver.
   3. About once a month, lubricate the bearing on the rotor arm using a thick lubricating grease²².
   4. On some tattoo machines, the rotor arm can get stuck and will not vibrate. To correct this, twist the rotor arm or compress the spring so that the needle is in the lowered position (Figure 2C).

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'). Once a tattoo has faded, it can be touched up using the same technique.

Poor technique will not cause harm to animals but can result in tattoos that fade quickly (Figure 5B), leading to more frequent touch-ups. Overzealous tattooing can cause bleeding and redness, and placing a frog in an incorrect recovery position can cause unintentional drowning.

The quality of tattoos and efficacy of the technique can be assessed in the months following tattooing (Figure 5E). So long as there is ink on the frog after the recovery period, a tattoo is considered successful.

Figure 1: Protocol workflow and bench setup. (A) Workflow and timing. (1) Frogs spend 7-8 min being anesthetized in a solution of 1.5 g/L tricaine and 3.5 g/L sodium bicarbonate, pH 7.15, dissolved in frog-safe water. While one frog is being tattooed, a second frog can be placed on deck in the anesthesia tank. Only one frog can be in tricaine solution at a time. (2) Frogs spend about 10 min out of water being tattooed. Time will vary depending on the size of the tattoo and the lab personnel's experience. (3) Unconscious frogs are placed on Styrofoam for 30-45 min to prevent drowning and covered with a wet paper towel to prevent them from drying out. (4) After tattooing, frogs spend 24 h recovering in fresh frog-safe standing water. Once they have recovered, frogs can be returned to their colony. (B) Bench setup for an example workspace. The tattooing workspace, an unbleached paper towel, is centered on the bench. The tattoo machine and voltage supply are to the left of the paper towel. These are placed on the personnel's dominant side (the personnel is left-handed). An inkpot and tattoo ink are placed near the voltage supply and out of the way of the tattooing workspace. The foot pedal is on the same side as the voltage supply. Pressing this foot pedal controls rotary motion. Lint-free wipes and a squeeze bottle with frog-safe water are to the right of the paper towel. These are used to clean excess ink from the frog's skin during tattooing. An Allen wrench is used to tighten and loosen screws in the tattoo machine. This is used to assemble and disassemble the tattoo machine. The anesthesia tank and extra paper towels are also kept on the bench during tattooing. Please click here to view a larger version of this figure.

Figure 2: Tattoo machine assembly. (A) Image of the tattooing needle, grip, and related components disassembled. The screws on the metal grip are circled in red. (B) Image of the tattooing needle appropriately assembled into the grip. (C) Image of the tattooing needle and grip assembled into the tattooing gun. Please click here to view a larger version of this figure.

Figure 3: Positioning and reference images for frog tattooing. (A) Image of a frog laying face-up on a paper towel. The arrow points to the frog's sternum. (B) Image of lab personnel inking frog. (C) Images of numbers during inking, before (C), and after (C') excess ink has been washed away. (D) Image of frog's tattoo immediately after inking, before recovery. Please click here to view a larger version of this figure.

Figure 4: Post-tattoo recovery. (A) Styrofoam island. The Styrofoam ice box lid is placed inside of a zip-top bag. (B) Frogs in recovery position post-tattooing. Styrofoam islands can typically fit 1 or 2 frogs. Please click here to view a larger version of this figure.

Figure 5: Examples of representative results. (A) A fresh tattoo 24 h after being tattooed. (B) Poor quality techniques. In (B), the tattoo machine was not properly assembled but was still capable of rotary motion. This photo was taken 4 months after tattooing. In (B'), the tattoo machine was not assembled properly and was not capable of rotary motion. This photo was taken 6 months after tattooing. (C) Improperly assembled tattoo machine. The metal tube and plastic tip are not present, and the metal grip is not correctly secured to the rest of the tattoo machine. Rubber band impairs rotary motion (D) Before (D) and after (D') of a frog tattooed with good technique and proper equipment setup. (D) was taken immediately after tattooing, and (D') was taken 4 months after tattooing. (E) Quantitative data of frog tattooing results. Of the frogs tattooed, 100% had visible tattoos immediately after tattooing, 100% had them 24 h after tattooing, and 91% had them 4 months after tattooing. n = 58 frogs. Please click here to view a larger version of this figure.

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 benefit of tattooing amphibians is that tattoos heal within 24 h and good tattoos will last for several months (Figure 5D'). Tattooing provides multiple advantages for the housing and husbandry of amphibians. Tattoos allow researchers and veterinary staff to readily distinguish individual animals using numbers that are easy to see and interpret, even in tanks with darker tints¹⁹. This procedure can also be done on any adult frog, compared to skin autografts, which are not recommended for frogs over 2 years old¹⁵. Most importantly, tattooing allows for identifying a large number of animals, with at least three digits fitting comfortably on the chest of X. laevis (Figure 5A).

Compared to attaching beads or tags, tattooing does not leave anything that could fall off and causes limited irritation¹⁷. In addition, tattooing allows for more versatility than other branding techniques, which require shaping a wire into the brand. The procedure itself is much less disruptive than skin autografts because no tissue is removed. However, like autografts, tattooing is also partially invasive and introduces a small risk of injury or infection. This is a necessary part of marking any animal, but it is important for researchers to be wary of excessive bleeding during tattooing and monitor animals for A. hydrophilia infection. This can be prevented by maintaining a clean workspace before, during, and after tattooing and by ensuring tattoos have healed completely before animals are returned to their colony. In writing and developing this paper, none of the frogs have been permanently injured or died from tattooing. A limitation of this procedure is that it has only been tested on adult frogs greater than 1 year old. As of yet, no froglets have been tattooed with this protocol. In addition, this procedure is not optimized for X. tropicalis, which have dark coloration on their chest and will make tattoos less discernible²¹. In the future, this tattooing protocol may be updated to include considerations for X. tropicalis.

As the Xenopus habitat is aquatic⁴, it is normal for these tattoos to fade over time. The need to frequently replace tattoos is documented in human lip tattoos inside the mouth, which fade quicker than epidermal tattoos because of continuous contact with saliva and beverages²⁴. However, the longevity of a tattoo is largely dependent on the tattooing technique. Poor technique can stem from an improperly assembled tattoo machine (Figure 4C) or suboptimal voltage supply settings. Voltage set too low will prevent the needle from penetrating the skin, resulting in faint marks or lines. Setting the voltage too high will cause unnecessary harm to the animal. Another common source of poor tattooing comes from incorrect needle positioning or pressure as personnel hold the tattoo gun. Tattooing with too little pressure has the same effect as setting the voltage too low and will result in a faint tattoo that fades quickly (Figure 5B'). While tattooing, it can feel like the needle is skimming over the frog's skin instead of piercing it. Tattooing with too much pressure will result in noticeable or uncontrollable bleeding. The best tattooing is done patiently and methodically by inking in short sections and retracing lines with heavy, consistent pressure.

Overall, the benefits of animal marking by tattooing in amphibians outweigh the drawbacks. Individual tracking of Xenopus in biomedical research enables scientific rigor in tracking biological replicates, allows researchers and veterinary staff to assess the well-being of individual animals, and ensures appropriate frequency of ovulation to maintain the long-term health of the animal. Regarding the 3Rs of animal research-refinement, reduction, and removal-this protocol is most relevant to refinement and reduction¹. Marking frogs makes it easier for researchers and veterinarians to track health concerns, as well as make sure frogs are given sufficient resting time between experiments. This refines the research process by ensuring a higher quality of data and samples gathered from animals, as well as refining animal husbandry and care practices. It also helps improve reproducibility in experiments. In addition, marking animals enables them to be reused, which reduces the total number of animals required. Frogs that lay particularly high-quality eggs or are most useful in other experiments can be marked and identified as better suited for research. If frogs consistently are not useful for experiments, they can be identified and removed from the colony, which saves resources and space. The purpose of this paper is to document best practices in amphibian tattooing and provide Xenopus labs with a detailed, easily accessible tattooing protocol. This practice is not yet commonplace in the Xenopus field, in part because the existing published resources are limited. This resource will enable researchers to safely and effectively mark animals, improve record keeping for large colonies, and refine the use of amphibians in biomedical research.

Materials

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