We tested several different injections sites. Subcutaneous injections were tested on the ventral abdomen, and the ventral rear leg. We found the elastomer in both of these areas was very hard to visualize. Some of the material seemed to break up, resulting in a dispersed patch of material. In other cases, the skin pigment made the tag difficult to see, or the color difficult to distinguish. The tags were hard to see even with fluorescence. Also, the frogs are difficult to restrain during the subcutaneous injection, making it difficult to inject the material. Subcutaneous injections were not optimal.

 

 

We also tested injection of the material in the toe webbing (Picture 3). This method requires some practice and patience. The webbing is quite thin, and placing the needle within the tissue without puncturing through entirely is tricky. After some practice, we found this method to be superior to the subcutaneous injections. The material is visible to the naked eye, and is quite easy to visualize by shining the blue flashlight in the frog tank. Multiple webbing sites can be used in combination with the four colors for numerous unique identification codes. Also, we found it easier to inject in the webbing because one person can hold the frog (Burrito style) to prevent squirming, while another pulls out a leg and injects the toes. There are a few drawbacks with this method. The material tends to ooze back out of the injection site if the frog is very active after the injection, or if the material is injected too close to the point of needle entry. NMT recommends minimum handling for 10 days post-injection. The material may also break up into a patch of small colored spots, instead of one large spot. Depending on how many colors you injected in one web, this could be problematic.

It is important to periodically recheck the tag site post injection, as we have found the tags can move around and occasionally fall out even after the elastomer has cured. If the tag has fallen out, retagging can be performed.

The instructions provided by the manufacturer for mixing and using the elastomer are quite detailed. Mix the amount you need, according to the directions, keeping the recommended 1:10 ratio of curing agent to elastomer.

 

The minimum recommended amount to mix is 0.5 cc elastomer with 0.05 cc curing agent. This amount is enough to identify quite a few frogs with toe markers, possibly 50 or more, depending on how much is injected in each frog. Thus, one should plan to tag the maximum number of frogs at one time to avoid wasting the elastomer. The company states that mixing smaller amounts can present a problem in obtaining an accurate ratio of materials, which is important in getting the elastomer to cure properly. We have not tried mixing smaller amounts, but plan to try it in the future.

 

If the elastomer is mixed at room temperature, it has a usable life of about 45 minutes. However, with time the elastomer loses elasticity and starts to harden in the syringe making it difficult with which to work. However, a new formulation is being developed by NMT which performs better and can be mixed in smaller quantities. Cooling the two components before mixing, and keeping the mixture at 4 degrees C after mixing, is reported to extend the life of the material by as much as a week. We have tried cooling the materials in a freezer for an hour before mixing. This did seem to extend the working time from an hour to the several hours we spent tagging frogs. We did not test how much longer it was useful.

 

The injection procedure requires two people (wearing gloves) in our experience. One person should restrain the frog with the desired rear leg extended. We find the burrito method or the Iron Claw work well. The person injecting should dry the desired foot and webbing with a paper towel or gauze before injecting.This greatly eases the ability to maintain a grasp on the foot while injecting the material.

 

To inject the material, puncture the webbing with the elastomer syringe. We find the web tissue to be thicker midway or higher up the web, and closer to the toe, rather than in the middle of the web between toes (Picture 4). It is easier to insert the needle into this thicker tissue, rather than into the thin tissue in the middle of the web field. Slide the needle within the webbing as far proximal as possible before beginning to inject the elastomer. Slowly inject the material while withdrawing the needle. Stop injecting before you reach the exit hole, to minimize the risk of leakage.

 

The toe webbing method has proven reliable with some practice. The main drawback is the need to inject many frogs at once to prevent waste of the material. We will probably limit use of this material to tagging a large number of frogs to identify them as a group. For example, red elastomer between toes two and three could represent F8 inbred Nigerians.

 

 

We also use the “Visible Implant Alphanumeric Tags” as a means of individual frog identification. These are small flexible tags implanted under the skin. From the NMT website: "The tags have a coding scheme of three alphanumeric characters (one letter and two numbers) and 3 fluorescent colors (red, orange and yellow). They are available in two sizes; standard (1.0 mm x 2.5 mm) and large (1.5 mm x 3.5 mm).” The tags come with either a fluorescent background with black letters, or black background with fluorescent letters (Picture 6). The latter take longer to receive, as they are a special order. The tags are ordered in sets of 100, and the number set can be specified (numbers 0-99, etc.)

 

Picture 5: Tags as supplied on the card under plastic, with the tag injector, sold separately. We do not use the injector for inserting the tag.	Picture 6: Close up of the alphanumeric tags

 

 

We found the numbers on the small (standard) size tags to be far too small. Even with the amber viewing glasses, blue LED flashlight and a magnifying glass, the numbers were nearly impossible to decipher. The large size tags worked much better. We found the large tags with the black background and yellow fluorescent letters to be far superior to any other color combination. The amber glasses and flashlight are required to read the numbers. We did find some difficulty in distinguishing between the numbers “0” and “8”.

 

There is some time involved in getting the frog properly positioned to read the tag, especially with the flashlight and goggles. The frog must be grasped firmly and held to visualize the tag. If you needed to find a particular tagged frog in a large group of frogs, it could take a considerable amount of time to search through and find the desired animal. For this reason, we have decided to use these tags only for frogs that are being isolated. The tag then acts as secondary identification if the frog escapes its tank or is accidentally mixed up with another frog.

 

The best tag placement site we have found so far is the ventral surface of the rear lower leg (Picture 7). The tag is limited from moving very far by the knee joint, and the skin is generally a lighter color in this area. The tag still moves freely within the area, but can be easily manipulated with your fingers to move it to an area where it can be visualized.

 

Picture 7: Tag inserted on the underside of the leg, visualized with amber glasses and flashlight. Insertion point is visible below the tag.

 

Insertion Procedure

 

The alphanumeric tags come on a sheet, sandwiched between two layers of plastic material (Picture 5, 6). We found the applicator supplied by the company to be problematic for our use. The relatively thick skin of the frog makes inserting the syringe tip very difficult without first making a small “starter” incision. Loading the tags can also be difficult, in our experience. We now use a small pair of Extra fine Graebe forceps with a thin tip (Picture 8). The tag is removed from the plastic with the forceps, and is positioned at the end of the forceps as shown(Picture 9).

An assistant is necessary to hold the frog. Our assistant rolls the frogs up burrito style in a wet paper towel, and holds them upside down with the rear legs exposed. Take a dry paper towel and dap the skin where you will grasp it with the forceps. Drying the skin makes it a lot easier to hold with the forceps. The skin at the injection site is held up with forceps to form a tent (Picture 10). Forceps with teeth (we use Adson Brown, 1X2 teeth, 2 mm tip) are useful to grasp the slippery skin. The insertion site should be as far distal as possible. We use small sharp scissors to make a small hole (Picture 12). The forceps with the tag can then be passed through the hole (Picture 13). Be sure to firmly grasp the tag while inserting it to prevent it from being misplaced or turned upside down. Be sure you insert the tag correct side up. Place the tag as far from the opening as possible. To dislodge the tag from the forceps, use gentle external pressure with the second set of forceps or a finger over the tag while withdrawing the forceps.

 

Apply wound glue liberally to the opening with the syringe (Picture 14). (Do not inject the glue, simply use the syringe as an dropper type applicator.) We find the glue adheres better if the skin is first blotted dry. Allow the glue to dry for at least one minute before returning the frog to the tank. Be sure to clean the instruments and disinfect with alcohol between frogs.

 

 

 

Picture 8: Instruments we use to insert the tag: Extra fine Graebe forcepsto insert tag, Adson Brown forceps,1X2 teeth, 2 mm tip, to grasp the skin, and small surgical scissors	Picture 9: Grasp the tag with the forceps and position the tag at the end of the forceps. Be sure you insert the tag rightside up.
Picture 10: Lift a tent of skin on the ventral surface of the leg
Picture 12: Cut a small hole.	Picture 13: Insert the tag with the forceps.
Picture 14: Wound glue and a syringe to apply the glue.	Picture 15: The tag is easily visualized through light colored skin with amber goggles and blue light.

 

 

The opening takes 3 to 4 weeks to heal fully, so we refrain from breeding these frogs for a month after tag insertion. Two weeks post injection, we inspect the area for tag retention and wound healing. To date, we have had an 84% retention rate. We believe tag loss is mostly due to making an overly large hole, and not applying enough wound glue. In cases where the tags fall out, we wait a month and reinsert another tag. We plan to try using a suture to close the incision and hopefully prevent tag loss.

 

We have had some problem with the tags rolling up or folding over after they are inserted under the skin. In each case, we have been able to flatten the tag out successfully by manual manipulation from outside the body. We have also had a problem where a tag was accidentally inserted upside down. The remedy in this case is to remove the tag with forceps and start over, or try to turn it over with forceps. Both of these methods are difficult. In general, however, we have found the tags work well. We use this method to tag our important individual frogs. We are able to tag about 15 frogs per hour. This includes time involved in setting up and getting the frogs from their tanks.

 

 

Following is a table comparing different aspects of the Microchip versus the Alphanumeric tag. Overall, the microchip is easier to read and has a lower rate of loss (for us), but it’s much higher cost is a serious consideration.

 

Microchip	Alphanumeric Tag
Unit Cost: about $6/ chip	Unit Cost: about $1/tag
Addn’l Cost: chip reader (cost?), glue, instruments	Addn’l Cost: glue, instruments. Injector is $124, but we don’t use it
Code Identification: Simpler Need a reader to ID. Little manipulation of the frog is necessary beyond simple restraint. 10 digit code is a bit harder to record in a database with more potential errors but essentially neverending codes	Code Identification: More Complex Usually need brown glasses and blue light (both supplied) to fluoresce and read tag. Dark skin pigment interferes with reading code. Often requires manipulating frog to find/read the tag. Also may need to manipulate the tag under the skin to visualize the code. Fewer codes but easier to record the three digits.
Rate of Loss: Lower Tends not to fall out if it is placed on the side of the leg opposite the incision	Rate of Loss: Higher Tags tend to migrate under the skin and can be lost through an unhealed incision more easily
If chip falls out, it can usually be recovered from the bottom of the tank	If tag falls out, it is usually washed out of the tank, and cannot be recovered
Size: Larger May not work in legs of smaller (male) frogs	Size: Smaller Easily works with large or small frogs
Lifetime: life of frog (100+ years). In fact, the chip can be recovered from a dead frog, sterilized, and used again.	Lifetime: life of frog
Placement orientation: not a concern	Placement position: potential to be placed upside down or to curl/fold under the skin when inserting.

 

 

Contributed by Maura Lane

 Special thanks to Nicolas Pollet who alerted us about the elastomers