rotary tattoo method for long-term marking of xenopus in research environments

Optimizing Amphibian Marking: A Guide to Tattooing &lt;i&gt;Xenopus&lt;/i&gt;

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 development5,6,7, basic molecular and cellular biology7,8,9,10, and for modeling human disease7,11,12,13.
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&#39; 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&#39; 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&#39;s jaw, toe, or hind limb17,18. 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 amphibians17,19. 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&#39;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&#39;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 chips17,19. In the glass bead method, glass beads are threaded onto a small suture and sewn into the frog&#39;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&#39;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&#39; 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 technique17,19. The authors warn that by disturbing the frog&#39;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.

Author Spotlight: Refining &lt;i&gt;Xenopus laevis&lt;/i&gt; Marking Techniques for Biomedical Studies

Methods for Tattooing Xenopus laevis with a Rotary Tattoo Machine

Our research is focused on investigating the organization of the cell cortex using Xenopus egg extracts and supported lipid bilayers. We are trying to understand how dynamic cytoskeletal patterns are regulated within the cortex and how these patterns support basic cell functions like cell division. This protocol outlines an effective and safe method for marking Xenopus laevis.This approach enables researchers to track genetic lines and experimental replicates and to monitor animal health. Tattooing is a cost-effective permanent marking technique for amphibians. It's much less invasive than other permanent methods, such as skin graft or tags, it's also more reliable than qualitative methods, like photos or pattern recognition.

Rotary Tattoo Method for Long-Term Marking of Xenopus in Research Environments

To begin, set up the equipment for tattooing. Place the anesthetized frog on its back on a dry, unbleached paper towel. Using a dry, lint-free wipe, clear the water and mucus from the frog's chest.Then find the sternum in the center of the frog's chest with fingers, and using the non-dominant hand, hold the skin taught. Position the assembled tattoo gun vertically relative to the working surface and dip the needle into the ink. Keeping the gun vertical, press the needle tip onto the frog's skin, followed by the foot pedal.Draw lines onto the frog's skin while applying even pressure. If the needle gets stuck, clear excess ink or skin from the needle with a wipe. If necessary, clear excess ink on the frog's chest with frog-safe water and a wipe, and then wick away moisture with a dry wipe.Continue to ink the same area until dark, legible numbers persist after wiping away excess ink. Fill a tank with 10 to 15 liters of fresh frog-safe water and add a styrofoam island to provide a surface for the frog to wake up without drowning. Wet a paper towel with frog-safe water and place it flat on the styrofoam island.Next, lay the frog belly down on the towel facing the water. Fold half of the paper towel over the back half of the frog, and using cupped hands, wet its top portion. After 24 hours, when the tattoo is healed, return the frog to its long-term housing unit.Of the frogs tattooed, 100%had visible tattoos immediately after tattooing, 100%had them 24 hours after tattooing, and 91%had them four months after tattooing.

rotary-tattoo-method-for-long-term-marking-xenopus-research

Research

JoVE Journal

Biology

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.

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 ...

Preparation of the Tattoo Gun to Mark Frogs for Research Purposes

To begin, connect the tattoo gun and foot pedal to the power supply. Position the foot pedal underneath the working surface. For assembling the tattoo gun, loosen the screws in the grip with an Allen wrench.Insert the plastic tip all the way into the grip and tighten the screw to hold it in place. Then insert the metal tube in the back of the grip to half of its length and tighten the screw while adjusting the length. Insert a tattoo needle through the back of the grip to secure it in the plastic tip at the front of the grip.Slide the back of the needle and metal tube through the tube clamp. After adjusting the metal tube, hand tighten the tube clamp until it cannot move. Remove the black O-ring, hook the needle onto the rotor arm, and replace the O-ring.Next, set the required voltage on the power supply. Once the power supply is plugged in, press the foot pedal to check if the machine works. Fill the plastic ink pot with black tattoo ink to about three quarters full before tattooing.