Rodent Identification II

Lab Animal Research

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Overview

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN

Animal records must be accurately maintained to ensure that data collection is correct. Records range from maintaining information on cage cards to having a detailed database with all of the relevant information on each animal. The primary component of recordkeeping is the individual identification of research animals. There are a variety of methods suitable for identifying mice and rats. This video describes the procedural techniques for tattooing, microchip placement, and temporary identification methods, and also explores the benefits of each.

Cite this Video

JoVE Science Education Database. Lab Animal Research. Rodent Identification II. JoVE, Cambridge, MA, (2017).

Principles

The use of tail tattooing is beneficial in many research protocols. Tail tattoos are ideal for an animal that is to be imaged in an MRI machine, or one that is genetically predisposed to ulcerative dermatitis. For quick identification of an animal in a cage, without having to restrain it, the tail tattoo is easily visualized. However, this technique requires skill, practice, and a steady hand; specialized equipment is also required.1

Experimental protocols may require the genotyping of neonates as early as day one. As a result, it is essential that these pups be permanently identified. At this young age, the ear pinnae are not developed enough to insert an ear tag, and an ear punch cannot yet be used on them. In the past, it was common for a researcher to use a toe removal code to identify neonates. However, this was stressful to both to the young animal and the dam, and thus has been considered inhumane.2 The use of toe tattooing allows the animals to be permanently identified until they are large enough for ear tagging or ear punching.3

Although the process for implantation of a microchip is relatively easy, there are several factors that limit its usefulness for identification in mice. The sterile microchips are the size of a large grain of rice, and require a 10 to 12 gauge needle for delivery. Thus, they are very large for a mouse. The cost of microchips and the reading device can also be prohibitive, as the life span of the mouse is short and the number of animals is usually extensive. Animals that are destined to be imaged in an MRI machine cannot have implants in them. However, if there is a group of valuable animals that must be permanently identified, microchips can be used.4

Acute studies that require animals to be identified for hours, or a few days, do not necessitate the use of permanent identification. Nontoxic markers are made specifically for coloring the fur of animals, and are available in several colors. These marker dyes can remain visible for several weeks. The markers can also be used in conjunction with other methods to easily spot specific animals in a group.

Procedure

1. Tail tattoo

Tail tattoos are easily read without the need to handle the animal.

Figure 1
Figure 1. Tail tattooing on adult mice

  1. Prepare the tattoo equipment according to the manufacturer's instructions. A small amount of ink is required. Black dye should be used for albino mice or rats, and green dye for pigmented mice or rats.
  2. Prepare the animals for identification.
    NOTE: To minimize apprehension or distress, tail tattooing should be performed in a procedure room rather than in an animal housing area.
    1. Appropriately restrain the animal for tattooing so that movement is minimal.
    2. Weanling rats are restrained using a plastic-notched cup, with the lip of the cup secured under the bolts on the tattoo platform, and the tail extending through the notch.
    3. Adult mice can be restrained by means of a cup restrainer, with the tail pulled taut from the opening and the flange of the cup held in place via a platform or other device.
    4. Clean the tail with a small amount of diluted tissue cleaner applied with a cotton-tipped swab. Adult rats generally have a buildup of scale and debris on the tail that must be cleaned prior to applying the tattoo. A soap and water scrub of the tail will remove the majority of the dirt and debris. Dry the tail well before proceeding.
    5. Apply tissue oil to the skin with a cotton-tipped swab just prior to tattooing. The tissue oil is necessary to minimize tissue damage by softening the skin and lubricating the tattoo needle. In addition, the oil dissolves any dirt not removed by the cleaning process and prevents ink staining of nontattooed skin.
  3. Apply the tattoo.
    1. Dip the tip of the needle into the pigment with the machine off.
    2. Bring the tip of the needle to the tattoo site with the gun activated by the foot pedal.
    3. Make short, even strokes in one direction. Numerals and letters should be tattooed in such a way so that they are constructed in segments with minimal angles and curves (when possible).
    4. Penetrate deeply enough into the dermis with the needle for permanent pigment deposition. A change in the sound of the activated needle can help discern the depth.
    5. Approach the skin with the needle at a 90o angle for adequate deposition of pigment.
  4. Post-tattoo cleanup and care
    1. Blot (do not wipe) the completed tattoo of excess pigment with a paper towel.
    2. The presence of blood on the tattoo or the paper towel indicates that the tattoo was made too deep, and is therefore unlikely to be permanent.
    3. Reinforce thin areas of tattoo characters with additional pigment. Characters are reinforced with pigment deposited parallel to, but not on top of, already-deposited pigment.
    4. To add additional pigment, dip the tip of the needle into the dye reservoir for every two characters for mice and every one character for rats.
    5. Reasons for fading tattoos include inadequate amounts of pigment, tattoos made too shallow, tattoos made too deeply, an inappropriate angle of the needle to the skin, blunt or hooked needles being used, or the tattoo being made too quickly.
    6. Thoroughly clean the tattoo equipment after each use according to the manufacturer's instructions.

2. Toe tattooing of neonates

Experimental protocols can require the genotyping of neonates as early as day one, which makes it essential that these pups be permanently identified. The use of toe tattooing allows the animals to be identified until they are large enough for ear tagging or ear punching.

Figure 2
Figure 2. The proper restraint technique for tattooing a neonate mouse. The lancet has the green dye on the tip.

  1. Restrain preweaned pups.
    1. Cup the neonate in the hand on a gauze pad. Position the foot between the thumb and crook of the index finger to expose the chosen toe, or toes, for tattooing.
    2. Hold the foot close to the toes so that the toe has a solid surface behind it, preventing it from bending away.
    3. Do not overtwist the leg when positioning the foot. Pups that are restrained may wiggle, but no vocalization is necessarily heard.
  2. Prepare the equipment.
    1. Select a 4.5 mm Goldenrod animal lancet.
    2. Place a small spot of green tattoo paste on a nonporous surface; aluminum foil works well.
    3. Dip only the tip of the lancet in the tattoo paste.
    4. Use only a small amount of paste.
  3. Apply the tattoo.
    1. Poke the desired toe with the Goldenrod lancet according to an identification code.
    2. Puncture the skin to introduce the paste into the skin, leaving a mark.
    3. Poke the same spot three times to insure proper penetration.
    4. Avoid poking so deeply that the toe bleeds; this can result in a fainter tattoo.
  4. Post-tattoo cleanup and care
    1. Gently blot the spot with an absorbent wipe/towel to remove excess paste.
    2. Do not attempt to clean excess paste from the foot or body.
    3. Return the animal to its cage.
    4. When nursing pups are tattooed, it is advisable to also tattoo the dam. This may prevent her from overreacting to the paste on the pups, which could lead to excessive grooming of the tattoo.
    5. Use a new lancet for each litter.
    6. The tattoos should be verified on the day after the procedure. Redo the tattoo if the dots are faint or not visible. If the dots are visible the day after tattooing, they should remain visible for the lifetime of the animal.

3. Toe tattooing of adult rats and mice

When a litter of neonates is tattooed, it is suggested that the mother also be tattooed so that the paste on her pups is not foreign to her.

  1. Restraint of the animal.
    1. Restrain the animal using either a plastic cone or a Plexiglass restrainer that allows access to the hind feet.
    2. Extend the foot from the restraint device.
    3. Position the foot between the thumb and crook of the index finger to expose the chosen toe, or toes, for tattooing
    4. Hold the foot close to the toes so that the toe has a solid surface behind it, preventing the toe from bending away.
    5. Do not overtwist the leg when positioning the foot. Adults that are restrained may try to withdraw the foot.
  2. Prepare the equipment.
    1. Select a 5 mm Goldenrod animal lancet.
    2. Place a small spot of green tattoo paste on a nonporous surface; aluminum foil works well.
    3. Dip only the tip of the lancet in the tattoo paste.
    4. Use only a small amount of paste.
  3. Apply the tattoo.
    1. Poke the desired toe with the Goldenrod lancet according to an identification code.
    2. Puncture the skin to introduce the paste into the skin, leaving a mark.
    3. Poke the same spot three times to insure proper penetration.
    4. Avoid poking so deeply that the toe bleeds; this can result in a fainter tattoo.
  4. Post-tattoo cleanup and care
    1. Gently blot the spot on an absorbent wipe/towel to remove excess paste.
    2. Do not attempt to clean excess paste from the foot or body.
    3. Return the animal to its cage.
    4. Use a new lancet for each animal.
    5. The tattoos should be verified on the day after the procedure. Redo the tattoo if the dots are faint or not visible. If the dots are visible the day after tattooing, they should remain visible for the lifetime of the animal.

4. Microchipping

The implantation of an RFID chip is a commonly used method for identifying animals.

  1. Load the microchip into the applier, or select a preloaded syringe.
  2. Restrain the animal using a Plexiglass restraint tube that allows grasping of the skin over the shoulders.
  3. Tent the skin, creating a pocket.
  4. Place the needle through the skin, bevel up and parallel to the spine, and directed it toward the tail.
  5. Eject the microchip subcutaneously.
  6. Withdraw the needle and pinch the skin closed at the point of injection. This prevents the microchip from following the needle out of the skin. Continue to apply pressure to provide hemostasis for any skin bleeding.
  7. Remove the animal from the restraint device and scan the chip to confirm the identification code.

Figure 3
Figure 3. Microchip placement in adult rats.

5. Temporary identification for mice

Nontoxic dyes and animal markers can be used for temporary identification of mice.

  1. Restrain the mouse such that the area between the scapulae is accessible for markings on the fur.
  2. Place a marker, spot, or streak of dye on the fur in this area. This region is used to prevent the animal from removing the marking while grooming.
  3. Check the marking periodically if using it as the sole identifier for more than a few days.
  4. The tail can also be used for short durations. However, the animal may remove a mark from it while grooming.

6. Temporary identification for rats

  1. Restrain the rat so that the area for dye or marker application is easily accessible. In some cases, when only a stripe or dot is needed, the animal will not need to be restrained but is allowed to move freely in the cage.
  2. Manual restraint by grasping the tail and holding it taut while applying the identification is commonly done.
  3. Temporary immobilization using inhalant anesthetics can be done for fractious animals.
  4. Check the marking periodically if using this method as the sole identifier for more than a few days.

The ability to identify individual research animals is an essential aspect of scientific recordkeeping and data collection. Distinguishing between animals ensures that the correct subject is used for the intended experimental procedure.

There are several permanent and temporary methods that scientists use to identify individual lab animals. In this video, we'll discuss methods involving tattooing adult rodent tail, neonate toes and adult toes. Next, we'll touch upon the microchip placement technique commonly performed in adult rats, followed by the universal temporary identification method that utilizes non-toxic dyes.

Before delving into protocols for these methods, let's review the considerations, benefits, and shortcomings of each of these techniques. Tail tattooing is beneficial in many research protocols, such as experiments involving MRI imaging or the ones where animals are genetically predisposed to skin conditions including ulcerative dermatitis. Another advantage of tail tattoos is that they are easily visualized without having to manually restrain the animal.

Certain experimental protocols require genotyping of neonates as early as day one, which makes it essential that these pups be permanently identified. The use of toe tattooing allows such animals to be identified until they are large enough for ear tagging or ear punching. It is a good idea to apply a similar toe tattoo on the adult mother to prevent her from over-reacting and excessively grooming her pup's new tattoo. Although there are several advantages to using this method, the disadvantage is that the procedure requires skill, practice, a steady hand and specialized equipment.

The next permanent identification method under consideration is microchipping. Although, implanting microchips is a relatively easy process, there are several factors that limit its usefulness. First, these chips are approximately the size of a large grain of rice and require a 10-12 gauge needle for implantation, which is too large for a mouse, thus limiting the method to adult rats. Second, microchips are expensive and are therefore not ideal for identifying large numbers of animals. Lastly, microchips cannot be used in the experiments involving MRI. However, if there is a group of valuable animals like breeder animals with unique genetics that must be permanently identified, microchips can be used.

The last method that we'll discuss is temporary identification using non-toxic markers. The non-toxic markers are made for coloring the fur of research animals. This method is ideal for short-term studies that require that animals be identified for only a few hours or days, although the dyes can remain visible for several weeks. Some researchers use these markers in conjunction with other identification methods to easily spot specific animals in a group.

Now that we have discussed the background, let's learn the procedures, starting with tail tattooing. Here, we will demonstrate the procedure on adult mice, but the same can be applied for weanling and adult rats.

To minimize any apprehension or distress to the animals, tail tattooing should be performed in the procedure room rather than in the animal housing area. Before applying the tattoo, restrain the mouse using a cup restrainer, with the tail pulled taut from the opening. The flange of the cup is held in place via a specialized glass platform. Next, clean the tail with a small amount of diluted tissue cleaner on a cotton-tipped swab. This step is important to remove any buildup of scale and debris that might interfere with the tattoo. Next, apply tissue oil to the tail skin with another swab. This minimizes tissue damage by softening the skin and providing lubrication for the tattoo needle. In addition, the oil dissolves dirt not removed by the cleaning process and prevents ink staining on the non-tattooed skin. Now you're ready to apply the tattoo.

While the machine is off, dip the tip of the tattoo needle, which is either a lancet or hypodermic needle, into the pigment. Next, activate the gun using the foot pedal, place the tattoo tool on the tail, and bring the tip of the needle to the tattoo site. Approach the skin with the needle at a 90° angle and penetrate deeply enough into the dermis such that pigment is permanently deposited. A change in sound of the activated needle can help discern the depth. Make short even strokes in one direction, forming numerals and letters. Try to use minimal curves and angles when applying the tattoo.

To add additional pigment, dip the tip of the needle into the dye reservoir for every one-two characters for mice and every one character for rats. Blot the completed tattoo with a paper towel to remove excess pigment. The presence of blood on the tattoo or the paper towel indicates that the tattoo was made too deep, and is therefore unlikely to be permanent. Reinforce thin characters with additional pigment by placing the needle parallel to, not on the top of, the already deposited pigment. Thoroughly clean the tattoo equipment after each use, and a new or sterilized needle and a fresh supply of ink should be used between cages to prevent disease transmission.

Next, we will learn how to apply toe tattoos to neonates and adults. Here, we will demonstrate the procedure in mice, but the same steps are applicable for rats.

The neonates are tattooed on their toes as the tailbones are not ossified and the length of the tail is too small. Also as the tail grows, the skin would be stretched and the numbers may become distorted, faint, and unreadable. Start by placing a spot of green tattoo paste on a non-porous surface such as aluminum foil.Next, cup the pre-weaned pup in your hand using a gauze pad. Position the foot between your thumb and the crook of your index finger to expose the chosen toes for tattooing. Hold the foot close to the toes so that the selected toe has a solid surface behind it to prevent it from bending away.

Now, dip the tip of a 4.5 mm lancet into the tattoo paste. Then, poke the desired toe as per the pre-determined code. To ensure proper permeation, poke the same spot three times. Penetrate the skin enough so that the paste leaves a mark, but avoid poking so deeply that the toe bleeds. Gently blot the new tattoo with the gauze to remove any excess tattoo paste, and return the animal to its cage. Use a new lancet for the next litter. One day after the procedure, verify that the tattoo is visible.

When nursing pups are tattooed, it is advisable to also tattoo the dam. This may prevent her from overreacting and excessively grooming her pup's tattoos. To apply a toe tattoo to an adult, again, first place a small spot of green tattoo paste on an aluminum foil. Then, restrain the animal using either a plastic cone or a Plexiglass restrainer that allows access to the hind feet. Extend the foot from the restraint device and position the foot between your thumb and the crook of your index finger to expose the chosen toes for tattooing.

Now, dip the tip of a 5mm lancet in the tattoo paste. And, like before, apply the tattoo by poking and puncturing the skin of the desired toe at least three times to ensure proper penetration. Next, gently blot the tattoo to remove any excess paste, and return the animal to its cage. As with the neonates, verify the tattoo one day after the procedure. If the mark is visible the day after tattooing, it should remain visible for the animal's lifetime. If it is faint or not visible, then redo the tattoo as described previously.

The next identification method that we'll discuss involves implanting microchips into rodents. The chip commonly used for labeling lab animals is a Radio-frequency Identification, or RFID, chip. Usually, the chip is preloaded in needles, which can then be placed into the applier.For chip insertion, use a Plexiglass tube to restrain the animal making sure that you can still grasp the skin over the shoulders. First, tent the skin to create a pocket. Then place the needle through the skin, bevel up, parallel to the spine, and directed toward the tail. Eject the microchip subcutaneously and withdraw the needle. Pinch the skin closed at the injection site to prevent the chip from following the needle out of the skin. Continue to apply pressure to provide hemostasis for any skin bleeding. Remove the animal from the restraint device and scan the chip using the reader provided by the manufacture to confirm the identification code.

Lastly, let's learn how to apply temporary dyes to rodents for identification. Let's start with mice. First, restrain the animal such that the area between the shoulder blades is accessible. This part of the body is safe from self-grooming. Place a spot or streak of the dye on the fur in this area and place the animal back into its cage.

For rats, restrain the animal manually by grasping the tail and holding it taut. This ensures that the area for dye application is easily accessible, which could be head, shoulders, or rump. Place a spot or streak of the dye on the fur. It is sometimes possible to apply non-toxic dye without restraining the animal. Check the markings on these animals periodically, especially if you are using them as the sole identifier for more than a few days.

Now that we have reviewed the procedures of these different identification methods, let's see how scientists are using them in different research studies today.

The use of rodents in behavior experiments is pretty common. Here, the researchers used the tail tattooing to mark the animals permanently and then observed them for a several days in an artificially constructed home-cage environment. The data collected revealed differences in behaviors, like distance moved, between the control and the experimental animals, over a 10-day post surgery period.

In another behavior experiment, the investigators performed toe tattooing in neonates and placed them in a recording box with special microphones to record ultrasonic vocalizations. The purpose of this experiment was to look at the differences in call rate, that is number of vocalizations per minute, in wild type and Shank2 deficient mice--a model of autism-over 12-day neonatal period and adult hood.

Lastly, as discussed earlier, the use of non-toxic dye is an efficient way to mark animals for short-term experiments. Here, the scientists were interested in transient expression of a plasma protein. So they marked the animals with temporary markers, injected the plasmid DNA intravenously, and collected the blood sample two days later to determine protein expression using the western blot technique.

You've just watched JoVE's introduction to tattooing, microchip placement, and temporary identification methods for laboratory mice and rats. Animal identification is a primary component of recordkeeping in research. Therefore, when choosing the appropriate identification method, many factors must be deliberated. This includes the level of discomfort to the animal, the ease of the technique, the experimental needs, and finally the cost associated with the method. As always, thanks for watching!

Summary

When choosing the appropriate identification method, many factors must be deliberated. Each technique has advantages and disadvantages that must be considered in relation to experimental needs. While the costs must be weighed along with other factors, the ease of the technique and the level of discomfort to the animals should be the primary considerations.4,5

References

  1. Robinson, V., Morton, D.B., Anderson, D., Carver, J.F.A., Francis, R.J., Hubrecht, R., Jenkins, E., Mathers, K.E., Raymond, R., Rosewell, I., Wallace, J., and Wells, D.J. 2003. Refinement and reduction in production of genetically modified mice. Laboratory Animals. 37:S1-S50.
  2. Schaefer, D.C., Asner, I.N., Seifert, B., Bürki, K., and Cinelli, P. 2010. Analysis of physiological and behavioural parameters in mice after toe clipping as newborns. Laboratory Animals. 44:7-13
  3. Castelhano-Carlos, M.J., Sousa, N., Ohl, F., and Baumans, V. 2010. Identification methods in newborn C57BL/6 mice: a developmental and behavioural evaluation. Laboratory Animals. 44: 88-103.
  4. Danneman, P.J., Suckow, M.A., and Brayton, C.F. 2013. The laboratory mouse. Second edition. New York, NY: CRC Press.
  5. Institute for the Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press.

1. Tail tattoo

Tail tattoos are easily read without the need to handle the animal.

Figure 1
Figure 1. Tail tattooing on adult mice

  1. Prepare the tattoo equipment according to the manufacturer's instructions. A small amount of ink is required. Black dye should be used for albino mice or rats, and green dye for pigmented mice or rats.
  2. Prepare the animals for identification.
    NOTE: To minimize apprehension or distress, tail tattooing should be performed in a procedure room rather than in an animal housing area.
    1. Appropriately restrain the animal for tattooing so that movement is minimal.
    2. Weanling rats are restrained using a plastic-notched cup, with the lip of the cup secured under the bolts on the tattoo platform, and the tail extending through the notch.
    3. Adult mice can be restrained by means of a cup restrainer, with the tail pulled taut from the opening and the flange of the cup held in place via a platform or other device.
    4. Clean the tail with a small amount of diluted tissue cleaner applied with a cotton-tipped swab. Adult rats generally have a buildup of scale and debris on the tail that must be cleaned prior to applying the tattoo. A soap and water scrub of the tail will remove the majority of the dirt and debris. Dry the tail well before proceeding.
    5. Apply tissue oil to the skin with a cotton-tipped swab just prior to tattooing. The tissue oil is necessary to minimize tissue damage by softening the skin and lubricating the tattoo needle. In addition, the oil dissolves any dirt not removed by the cleaning process and prevents ink staining of nontattooed skin.
  3. Apply the tattoo.
    1. Dip the tip of the needle into the pigment with the machine off.
    2. Bring the tip of the needle to the tattoo site with the gun activated by the foot pedal.
    3. Make short, even strokes in one direction. Numerals and letters should be tattooed in such a way so that they are constructed in segments with minimal angles and curves (when possible).
    4. Penetrate deeply enough into the dermis with the needle for permanent pigment deposition. A change in the sound of the activated needle can help discern the depth.
    5. Approach the skin with the needle at a 90o angle for adequate deposition of pigment.
  4. Post-tattoo cleanup and care
    1. Blot (do not wipe) the completed tattoo of excess pigment with a paper towel.
    2. The presence of blood on the tattoo or the paper towel indicates that the tattoo was made too deep, and is therefore unlikely to be permanent.
    3. Reinforce thin areas of tattoo characters with additional pigment. Characters are reinforced with pigment deposited parallel to, but not on top of, already-deposited pigment.
    4. To add additional pigment, dip the tip of the needle into the dye reservoir for every two characters for mice and every one character for rats.
    5. Reasons for fading tattoos include inadequate amounts of pigment, tattoos made too shallow, tattoos made too deeply, an inappropriate angle of the needle to the skin, blunt or hooked needles being used, or the tattoo being made too quickly.
    6. Thoroughly clean the tattoo equipment after each use according to the manufacturer's instructions.

2. Toe tattooing of neonates

Experimental protocols can require the genotyping of neonates as early as day one, which makes it essential that these pups be permanently identified. The use of toe tattooing allows the animals to be identified until they are large enough for ear tagging or ear punching.

Figure 2
Figure 2. The proper restraint technique for tattooing a neonate mouse. The lancet has the green dye on the tip.

  1. Restrain preweaned pups.
    1. Cup the neonate in the hand on a gauze pad. Position the foot between the thumb and crook of the index finger to expose the chosen toe, or toes, for tattooing.
    2. Hold the foot close to the toes so that the toe has a solid surface behind it, preventing it from bending away.
    3. Do not overtwist the leg when positioning the foot. Pups that are restrained may wiggle, but no vocalization is necessarily heard.
  2. Prepare the equipment.
    1. Select a 4.5 mm Goldenrod animal lancet.
    2. Place a small spot of green tattoo paste on a nonporous surface; aluminum foil works well.
    3. Dip only the tip of the lancet in the tattoo paste.
    4. Use only a small amount of paste.
  3. Apply the tattoo.
    1. Poke the desired toe with the Goldenrod lancet according to an identification code.
    2. Puncture the skin to introduce the paste into the skin, leaving a mark.
    3. Poke the same spot three times to insure proper penetration.
    4. Avoid poking so deeply that the toe bleeds; this can result in a fainter tattoo.
  4. Post-tattoo cleanup and care
    1. Gently blot the spot with an absorbent wipe/towel to remove excess paste.
    2. Do not attempt to clean excess paste from the foot or body.
    3. Return the animal to its cage.
    4. When nursing pups are tattooed, it is advisable to also tattoo the dam. This may prevent her from overreacting to the paste on the pups, which could lead to excessive grooming of the tattoo.
    5. Use a new lancet for each litter.
    6. The tattoos should be verified on the day after the procedure. Redo the tattoo if the dots are faint or not visible. If the dots are visible the day after tattooing, they should remain visible for the lifetime of the animal.

3. Toe tattooing of adult rats and mice

When a litter of neonates is tattooed, it is suggested that the mother also be tattooed so that the paste on her pups is not foreign to her.

  1. Restraint of the animal.
    1. Restrain the animal using either a plastic cone or a Plexiglass restrainer that allows access to the hind feet.
    2. Extend the foot from the restraint device.
    3. Position the foot between the thumb and crook of the index finger to expose the chosen toe, or toes, for tattooing
    4. Hold the foot close to the toes so that the toe has a solid surface behind it, preventing the toe from bending away.
    5. Do not overtwist the leg when positioning the foot. Adults that are restrained may try to withdraw the foot.
  2. Prepare the equipment.
    1. Select a 5 mm Goldenrod animal lancet.
    2. Place a small spot of green tattoo paste on a nonporous surface; aluminum foil works well.
    3. Dip only the tip of the lancet in the tattoo paste.
    4. Use only a small amount of paste.
  3. Apply the tattoo.
    1. Poke the desired toe with the Goldenrod lancet according to an identification code.
    2. Puncture the skin to introduce the paste into the skin, leaving a mark.
    3. Poke the same spot three times to insure proper penetration.
    4. Avoid poking so deeply that the toe bleeds; this can result in a fainter tattoo.
  4. Post-tattoo cleanup and care
    1. Gently blot the spot on an absorbent wipe/towel to remove excess paste.
    2. Do not attempt to clean excess paste from the foot or body.
    3. Return the animal to its cage.
    4. Use a new lancet for each animal.
    5. The tattoos should be verified on the day after the procedure. Redo the tattoo if the dots are faint or not visible. If the dots are visible the day after tattooing, they should remain visible for the lifetime of the animal.

4. Microchipping

The implantation of an RFID chip is a commonly used method for identifying animals.

  1. Load the microchip into the applier, or select a preloaded syringe.
  2. Restrain the animal using a Plexiglass restraint tube that allows grasping of the skin over the shoulders.
  3. Tent the skin, creating a pocket.
  4. Place the needle through the skin, bevel up and parallel to the spine, and directed it toward the tail.
  5. Eject the microchip subcutaneously.
  6. Withdraw the needle and pinch the skin closed at the point of injection. This prevents the microchip from following the needle out of the skin. Continue to apply pressure to provide hemostasis for any skin bleeding.
  7. Remove the animal from the restraint device and scan the chip to confirm the identification code.

Figure 3
Figure 3. Microchip placement in adult rats.

5. Temporary identification for mice

Nontoxic dyes and animal markers can be used for temporary identification of mice.

  1. Restrain the mouse such that the area between the scapulae is accessible for markings on the fur.
  2. Place a marker, spot, or streak of dye on the fur in this area. This region is used to prevent the animal from removing the marking while grooming.
  3. Check the marking periodically if using it as the sole identifier for more than a few days.
  4. The tail can also be used for short durations. However, the animal may remove a mark from it while grooming.

6. Temporary identification for rats

  1. Restrain the rat so that the area for dye or marker application is easily accessible. In some cases, when only a stripe or dot is needed, the animal will not need to be restrained but is allowed to move freely in the cage.
  2. Manual restraint by grasping the tail and holding it taut while applying the identification is commonly done.
  3. Temporary immobilization using inhalant anesthetics can be done for fractious animals.
  4. Check the marking periodically if using this method as the sole identifier for more than a few days.

The ability to identify individual research animals is an essential aspect of scientific recordkeeping and data collection. Distinguishing between animals ensures that the correct subject is used for the intended experimental procedure.

There are several permanent and temporary methods that scientists use to identify individual lab animals. In this video, we'll discuss methods involving tattooing adult rodent tail, neonate toes and adult toes. Next, we'll touch upon the microchip placement technique commonly performed in adult rats, followed by the universal temporary identification method that utilizes non-toxic dyes.

Before delving into protocols for these methods, let's review the considerations, benefits, and shortcomings of each of these techniques. Tail tattooing is beneficial in many research protocols, such as experiments involving MRI imaging or the ones where animals are genetically predisposed to skin conditions including ulcerative dermatitis. Another advantage of tail tattoos is that they are easily visualized without having to manually restrain the animal.

Certain experimental protocols require genotyping of neonates as early as day one, which makes it essential that these pups be permanently identified. The use of toe tattooing allows such animals to be identified until they are large enough for ear tagging or ear punching. It is a good idea to apply a similar toe tattoo on the adult mother to prevent her from over-reacting and excessively grooming her pup's new tattoo. Although there are several advantages to using this method, the disadvantage is that the procedure requires skill, practice, a steady hand and specialized equipment.

The next permanent identification method under consideration is microchipping. Although, implanting microchips is a relatively easy process, there are several factors that limit its usefulness. First, these chips are approximately the size of a large grain of rice and require a 10-12 gauge needle for implantation, which is too large for a mouse, thus limiting the method to adult rats. Second, microchips are expensive and are therefore not ideal for identifying large numbers of animals. Lastly, microchips cannot be used in the experiments involving MRI. However, if there is a group of valuable animals like breeder animals with unique genetics that must be permanently identified, microchips can be used.

The last method that we'll discuss is temporary identification using non-toxic markers. The non-toxic markers are made for coloring the fur of research animals. This method is ideal for short-term studies that require that animals be identified for only a few hours or days, although the dyes can remain visible for several weeks. Some researchers use these markers in conjunction with other identification methods to easily spot specific animals in a group.

Now that we have discussed the background, let's learn the procedures, starting with tail tattooing. Here, we will demonstrate the procedure on adult mice, but the same can be applied for weanling and adult rats.

To minimize any apprehension or distress to the animals, tail tattooing should be performed in the procedure room rather than in the animal housing area. Before applying the tattoo, restrain the mouse using a cup restrainer, with the tail pulled taut from the opening. The flange of the cup is held in place via a specialized glass platform. Next, clean the tail with a small amount of diluted tissue cleaner on a cotton-tipped swab. This step is important to remove any buildup of scale and debris that might interfere with the tattoo. Next, apply tissue oil to the tail skin with another swab. This minimizes tissue damage by softening the skin and providing lubrication for the tattoo needle. In addition, the oil dissolves dirt not removed by the cleaning process and prevents ink staining on the non-tattooed skin. Now you're ready to apply the tattoo.

While the machine is off, dip the tip of the tattoo needle, which is either a lancet or hypodermic needle, into the pigment. Next, activate the gun using the foot pedal, place the tattoo tool on the tail, and bring the tip of the needle to the tattoo site. Approach the skin with the needle at a 90° angle and penetrate deeply enough into the dermis such that pigment is permanently deposited. A change in sound of the activated needle can help discern the depth. Make short even strokes in one direction, forming numerals and letters. Try to use minimal curves and angles when applying the tattoo.

To add additional pigment, dip the tip of the needle into the dye reservoir for every one-two characters for mice and every one character for rats. Blot the completed tattoo with a paper towel to remove excess pigment. The presence of blood on the tattoo or the paper towel indicates that the tattoo was made too deep, and is therefore unlikely to be permanent. Reinforce thin characters with additional pigment by placing the needle parallel to, not on the top of, the already deposited pigment. Thoroughly clean the tattoo equipment after each use, and a new or sterilized needle and a fresh supply of ink should be used between cages to prevent disease transmission.

Next, we will learn how to apply toe tattoos to neonates and adults. Here, we will demonstrate the procedure in mice, but the same steps are applicable for rats.

The neonates are tattooed on their toes as the tailbones are not ossified and the length of the tail is too small. Also as the tail grows, the skin would be stretched and the numbers may become distorted, faint, and unreadable. Start by placing a spot of green tattoo paste on a non-porous surface such as aluminum foil.Next, cup the pre-weaned pup in your hand using a gauze pad. Position the foot between your thumb and the crook of your index finger to expose the chosen toes for tattooing. Hold the foot close to the toes so that the selected toe has a solid surface behind it to prevent it from bending away.

Now, dip the tip of a 4.5 mm lancet into the tattoo paste. Then, poke the desired toe as per the pre-determined code. To ensure proper permeation, poke the same spot three times. Penetrate the skin enough so that the paste leaves a mark, but avoid poking so deeply that the toe bleeds. Gently blot the new tattoo with the gauze to remove any excess tattoo paste, and return the animal to its cage. Use a new lancet for the next litter. One day after the procedure, verify that the tattoo is visible.

When nursing pups are tattooed, it is advisable to also tattoo the dam. This may prevent her from overreacting and excessively grooming her pup's tattoos. To apply a toe tattoo to an adult, again, first place a small spot of green tattoo paste on an aluminum foil. Then, restrain the animal using either a plastic cone or a Plexiglass restrainer that allows access to the hind feet. Extend the foot from the restraint device and position the foot between your thumb and the crook of your index finger to expose the chosen toes for tattooing.

Now, dip the tip of a 5mm lancet in the tattoo paste. And, like before, apply the tattoo by poking and puncturing the skin of the desired toe at least three times to ensure proper penetration. Next, gently blot the tattoo to remove any excess paste, and return the animal to its cage. As with the neonates, verify the tattoo one day after the procedure. If the mark is visible the day after tattooing, it should remain visible for the animal's lifetime. If it is faint or not visible, then redo the tattoo as described previously.

The next identification method that we'll discuss involves implanting microchips into rodents. The chip commonly used for labeling lab animals is a Radio-frequency Identification, or RFID, chip. Usually, the chip is preloaded in needles, which can then be placed into the applier.For chip insertion, use a Plexiglass tube to restrain the animal making sure that you can still grasp the skin over the shoulders. First, tent the skin to create a pocket. Then place the needle through the skin, bevel up, parallel to the spine, and directed toward the tail. Eject the microchip subcutaneously and withdraw the needle. Pinch the skin closed at the injection site to prevent the chip from following the needle out of the skin. Continue to apply pressure to provide hemostasis for any skin bleeding. Remove the animal from the restraint device and scan the chip using the reader provided by the manufacture to confirm the identification code.

Lastly, let's learn how to apply temporary dyes to rodents for identification. Let's start with mice. First, restrain the animal such that the area between the shoulder blades is accessible. This part of the body is safe from self-grooming. Place a spot or streak of the dye on the fur in this area and place the animal back into its cage.

For rats, restrain the animal manually by grasping the tail and holding it taut. This ensures that the area for dye application is easily accessible, which could be head, shoulders, or rump. Place a spot or streak of the dye on the fur. It is sometimes possible to apply non-toxic dye without restraining the animal. Check the markings on these animals periodically, especially if you are using them as the sole identifier for more than a few days.

Now that we have reviewed the procedures of these different identification methods, let's see how scientists are using them in different research studies today.

The use of rodents in behavior experiments is pretty common. Here, the researchers used the tail tattooing to mark the animals permanently and then observed them for a several days in an artificially constructed home-cage environment. The data collected revealed differences in behaviors, like distance moved, between the control and the experimental animals, over a 10-day post surgery period.

In another behavior experiment, the investigators performed toe tattooing in neonates and placed them in a recording box with special microphones to record ultrasonic vocalizations. The purpose of this experiment was to look at the differences in call rate, that is number of vocalizations per minute, in wild type and Shank2 deficient mice--a model of autism-over 12-day neonatal period and adult hood.

Lastly, as discussed earlier, the use of non-toxic dye is an efficient way to mark animals for short-term experiments. Here, the scientists were interested in transient expression of a plasma protein. So they marked the animals with temporary markers, injected the plasmid DNA intravenously, and collected the blood sample two days later to determine protein expression using the western blot technique.

You've just watched JoVE's introduction to tattooing, microchip placement, and temporary identification methods for laboratory mice and rats. Animal identification is a primary component of recordkeeping in research. Therefore, when choosing the appropriate identification method, many factors must be deliberated. This includes the level of discomfort to the animal, the ease of the technique, the experimental needs, and finally the cost associated with the method. As always, thanks for watching!

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