Many studies require individuals to be marked for identification. Marking techniques can cause effects ranging from short-term discomfort, to long-term influences on breeding and survival. It is essential to the welfare of the animal and the integrity of the research results, that the marking procedure not adversely affect the normal behaviour, physiology, ecology or survival of individuals. In choosing an acceptable marking technique, the investigator must consider the nature and duration of the restraint that is required for its application, the amount of tissue that is removed or damaged, the amount of momentary or prolonged pain that is involved and the potential risk for infection.
In many cases it is possible to reduce the harmful effects of the mark by careful design, with special consideration to colour, attachment position, mass, and the season in which it is applied. In general, investigators should not assume that marking procedures will have no adverse effects on their study subjects and should make efforts to evaluate any such influences. Where possible, investigators should assess the effects of new marking procedures on captive individuals before incorporating them into field studies. It is desirable for the scientific community to encourage ancillary research that specifically addresses the effects of marks and devices. Preferably, such research, which makes comparisons between marked individuals and controls, can be initiated in association with current studies. Because of the difficulty of providing appropriate controls, there are few systematic studies that assess the potential adverse effects of marking procedures. Many of the cautions mentioned here are based on anecdotal or unpublished observations.
In general, for a marking procedure to be effective, it should meet as many of the following criteria as possible:
In most amphibians and reptiles with normally developed limbs, the removal of toes has proven to be a useful method of permanent marking that is claimed to have little, if any, adverse effect on the behaviour and survival of most marked individuals. In general, no more than two non-adjacent toes per foot should ever be clipped. In addition, the specialized toes that are essential for survival activities such as burrowing, climbing, amplexus, nest excavation, or propulsion should not be interfered with. In salamanders, digit regeneration may be prevented by an application of phenylmercuric acetate solution after removal of the toe. Clipped toes should be kept for determination of age by skeletochronology, to maximize yield of data.
It is recommended that if other, less painful, permanent marking methods are available they should be used, as long as they do not influence survival in nature. If there are no feasible alternatives for toe clipping in the study species, it is recommended that the effects of digit removal be evaluated on captive individuals in order to determine whether or not animals are impaired by this technique.
When toe clipping is proposed as the method of marking amphibians and reptiles in the field, its use should be reviewed and approved by an institutional animal care committee.
Removal of subcaudal or ventral scutes according to a standardized numerical code provides a good permanent marking system for snakes, which does not appear to increase mortality or impair locomotion. The scute is removed with small surgical scissors or by rapid cauterization. Healing is usually rapid and infection is rare. A comparable method of marking is the electrocauterization of a number or letter on the skin. In order to be effective, the deep layers of the skin must also be cauterized to prevent regeneration. In amphibians, these brand marks may not be visible after a few months. The use of a local anaesthetic (aerosols containing benzocaine, such as Cetacaine®, or injectable local anaesthetics) is strongly recommended with branding or electrocauterization. However, the skin of reptiles is relatively impermeable and this reduces the effectiveness of topical products.
Tattooing has been used with success on both amphibians and reptiles. There are two potential problems that should be resolved prior to applying tattoos:
Paint should not be used to mark the moist and permeable skin of amphibians. Although reptile skin is less permeable, this varies among species, and some paints or paint solvents may be absorbed and kill the animal. Paints with non-toxic pigments, bases and solvents must be used. When the toxicity of an agent is unknown, it should be reviewed in the literature , or evaluated in laboratory trials, before being applied in the field. Very tenacious paints may, if applied across shell sutures, severely distort the normal shell growth of turtles, especially in sub-adults. In general, paint should not be applied to the sutures of turtle shells.
The size, shape and placement of tags should allow normal behaviours in the animal that is marked. Bands and tags that project from the body may impair physical activities or cause entanglement in undergrowth or aquatic cover. In addition, projecting markers may be torn as a result of the animal's movements. Brightly coloured tags may compromise an animal's camouflage or act as predator attractants. Petersen disc-type tags have been placed in the web between the hind toes of some frogs, but only large frogs are able to accommodate even small disc tags. Peterson discs have been associated with mortality in freshwater turtles. Coloured mylar ribbon tags 1-2" long have been considered an acceptable alternative.
In most species of turtles, the bony shell can be marked by cutting notches or drilling small holes in the marginal scutes of the carapace. Shell marking is permanent. Turtles have also been marked with disc-type tags and clamp-on-ear-type tags applied to the webs between the toes. However, these methods are subject to limitations previously mentioned under "Banding and Tagging".
The use of radioisotopes as markers in natural systems is a valuable study tool and may be the only means of adequately gathering information on the movements of very small species. However, the application of these agents should be undertaken only with caution. Researchers are required by law to have special training and to observe special precautions. In addition, a license is required which outlines the safety procedures, the disposal of waste material, and the release of isotopes into natural systems. The potential use of these agents must be evaluated with consideration to the deleterious effects that these agents can have on the study animal, as well as on its predators and the public.
Passive integrated transponders or microchips have been used to mark study animals permanently and can be used as an ancillary method of identification. These devices are implanted subcutaneously or intraperitoneally with complications rarely reported. However, animals must be recaptured and special equipment used to identify marked individuals. In addition, the transponders can migrate if applied subcutaneously, and this can make reading them more difficult.
The attachment of small radio transmitters to free-roaming vertebrates has become a routine method of monitoring the location and movement of individuals. Transmitters vary in size, mass, longevity and range characteristics, and are available from several commercial outlets. There is little direct evidence that when transmitters are of appropriate size, and are properly attached, they adversely affect research subjects. The researcher should choose a transmitter and method of attachment that harmonizes with the anatomy and behaviour of the study animal.
A wide variety of attachment methods for transmitters are currently in use and are reported in the literature. It is mandatory that investigators who are intending to utilize telemetry investigate those sources relevant to their study species. A review of the pertinent literature will help to identify any potential adverse effects that transmitters may have upon the behaviour, survival and well-being of their study animals. It is strongly recommended that prior to being used in the field, new attachment techniques be evaluated on captive individuals.
Many amphibians and reptiles are unsuitable for radio telemetry studies due to their small size and their habit of living in confined spaces below the ground surface. Although there is no firm consensus, it is generally recommended that the ratio of transmitter weight to animal weight should not exceed 5 %. In those cases where long transmitter life is required, most of this mass is a result of the battery. The continued miniaturization of transmitters will undoubtedly continue, and this will facilitate the future use of radio telemetry in reptile and amphibian studies, particularly with internally implanted transmitters.
Researchers intending to apply radio transmitters to amphibians or reptiles should consider the general guidelines and comments listed below. For more information on this topic consult the RIC (Resources Inventory Committee) manual entitled Wildlife Radio-telemetry, Standards for Components of British Columbia's Biodiversity No. 5.
Force-fed or implanted transmitters should be coated with an impervious, biologically inert coating. Transmitters of suitable size have been sealed in an inert plastic coating and force-fed to snakes. Force-fed packages are small enough to pass through the gut without obstructing the passage of food. Residence time of up to several days in the gut has been long enough to provide useful information on movement and body temperature. However, this method can influence behaviour of study animals and is now discouraged.
Transmitters also may be surgically implanted into the body cavities of large snakes. These transmitters should not interfere with the function of the organs surrounding them. In coelomic and subcutaneous implants, it may be necessary to suture the transmitter package in place to prevent its movement and interference with vital organs. The implantation of transmitters should only be performed under aseptic conditions by properly trained and experienced investigators.
Transmitter attachments that will impair reproduction, locomotion, behavioural interactions, thermoregulation or other normal activities should be avoided. The transmitter should be shaped and attached so as to eliminate or minimize the risk of entanglement with vegetation or other obstructions.
Amphibians and reptiles, including adults, continue to grow throughout their life. Therefore, consideration should be given to the eventual removal or release of an external transmitter so it does not cause constriction or irritation.
External attachment of transmitters on a number of species is possible by using various attachment harnesses and other techniques. However, the external device may alter the appearance of an individual enough to affect its behaviour and interactions with conspecifics and predators.
Radio telemetry has also been used in studies on larger turtles. The transmitter was attached to the dorsal surface of the shell by clamps over the edges of the carapace. Telemetry signals have been received both in and out of water.
It may be necessary to transport animals as part of an experimental protocol or to move them from capture sites to holding facilities. Transport containers and methods of shipping reptiles and amphibians will vary widely from species to species. The live traps that are used for capture are usually adequate for the transport of animals over short distances. However, if the animals are to confined for a longer period, these traps may not be suitable. Proper arrangements should be made to ensure that animals arrive at destinations during normal working hours, rather than on weekends or holidays.
Wild animals used in captive studies should be as healthy and free of trauma as possible. Some exceptions to this rule include investigations into the effects of environmental stress and disease. Because of the wide variation in husbandry requirements of amphibians and reptiles, and the diversity in study objectives, only the most general recommendations on housing can be made in these guidelines.
Whenever it is practical, researchers should release field trapped animals following the completion of their studies. However, there are exceptions to this and as a general rule, field trapped animals should be released only:
Captive animals that cannot be released should, whenever possible, be distributed to colleagues for further study. However, if the animal is in chronic distress or pain, or if release or rehabilitation is neither feasible nor likely to succeed, then euthanasia may be the only alternative. If animals must be destroyed subsequent to a study, then it should be done using a method of euthanasia which is humane, instantaneous and considered to be acceptable (see Reference section). In addition, the method of euthanasia should not interfere with any future research potential of the carcass or any specific post mortem analyses. In both the field and the laboratory, the investigator must be careful to ensure that euthanized animals are dead before disposal. Disposal of carcasses must be in accordance with acceptable practices as required by municipal or institutional regulations. Animals containing toxic substances or drugs should not be disposed of in areas where they may be scavenged or become part of the natural food chain.