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Due to the considerable range of adaptive diversity that is demonstrated by the 8,000 plus species of amphibians and reptiles, no concise or specific compendium on approved methods in field research is practical or desirable. Rather, these guidelines are intended to advise the investigator, who may already be an authority on the biology of the species under study, on the current field techniques that are considered to be humane and effective in amphibian and reptile research. The researcher has the ultimate responsibility for the ethical and scientific validity of his/her work and for the methods that are employed. To those who adhere to the principles of careful field research, these guidelines will simply be a formal statement of precautions already in place.
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Investigators have an obligation to identify and assess the consequences of their research activities on wild animals, populations and the environment. Whenever possible, action should be taken to avoid, alleviate or minimize any adverse effects. Research activities should include the collection of adequate samples to ensure valid research results, yet be balanced to minimize adverse effects. Investigators should always weigh potential gain in knowledge against the negative consequences of disturbance. Although short-term detrimental effects may result from research activities, research can ultimately yield long-term positive effects for the affected population.
Observer-related disturbance can be characterized in at least two ways. First, disturbances may create biases that affect both the gathering and analysis of data, if the actual process of measurement acts to alter the parameter being measured. Second, research activities might effect the status and well-being of the study subjects themselves. Both effects vary along a continuum from those that are overtly obvious to those that are subtle and difficult to detect. In addition, researchers should realize that disturbance also can occur to those species that are not under study.
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Whether one is collecting study animals for eventual release or for museum preparation, the same humane considerations should apply. These animals should not be exposed to excessive or inappropriate handling, conspecific aggression, predation, temperature extremes, or undue suffering.
Each investigator should observe and pass on to students and co-workers a strict ethic of habitat conservation. Because many essential details of life history may remain unknown until a study is well along, collecting should always be conducted so as to leave the population and habitat as undisturbed as possible.
The permanent removal of more than 50% of the animals from any breeding or hibernation aggregation should be avoided, unless justified for scientific reasons. Similarly, the collection of large numbers of females from any population for destructive sampling should be avoided unless justified for scientific reasons. When permanent, destructive human alteration of a specific habitat is imminent (construction, water impoundment, etc.), the removal of entire populations may be justified. Systematists should investigate extant collections for suitable specimens before conducting any field work. If the purpose of an experiment is to alter behaviour, reproductive potential or survivability, the interference should be no more than is determined necessary by the investigator to accurately test the hypotheses under question.
Investigators must obtain and comply with all permits required for the capture, handling and collection of those amphibians and reptiles of the correct species and in the appropriate jurisdiction. In addition, they must be familiar with the current list of threatened and endangered species and must comply with all rules and regulations pertaining to these and all other categories of amphibians and reptiles.
Field research on amphibians and reptiles frequently involves the capture of live specimens for data recording, marking, temporary confinement, or relocation. Investigators should be familiar with herpetological traps and trapping techniques and should choose a trap type that is best suited to the species and type of study. In some cases, the use of a variety of traps is required to obtain an unbiased sample, particularly when secretive, nocturnal or inactive species are being targeted. Capture techniques should prevent or minimize injury or damage to the animal. Care should be exercised to avoid accidental capture of non-target species. Trapping and handling equipment should be routinely inspected and maintained in good working order. At the end of each collecting period traps should be properly closed or removed.
Amphibians and reptiles are sensitive to heat, cold, dehydration, energy deprivation and stress, and investigators must make every effort to avoid trap deaths from causes such as exposure, drowning, shock, and desiccation. Animals should not be left in traps longer than is absolutely necessary, although this may vary with the species, weather, objectives of the study, and trap type. In general, traps should be checked at least once daily. Traps should be shaded or positioned to avoid exposure to direct sunlight and care should be taken to reduce predation in pitfall traps. Where possible, trapping should be avoided when weather conditions threaten the survival of trapped animals. Pitfall traps set during extremely dry periods should have some moisture provided in order to prevent desiccation of captured amphibians.
In some instances, research may necessitate the judicious collection and preservation of specimens in the field. This may be done to provide information on species identification, genetics, population structure and dynamics, comparative anatomy and physiology, evolutionary relationships, behaviour, parasites and diseases, economic factors, geographic and microhabitat distributions, and ecology in natural or disturbed habitats. The knowledge that results from these studies may be applicable to other biological sciences and may help to facilitate decisions involving management policies for all species, whether endangered or economically important, for the conservation of habitats, ecosystem analysis, pest and disease control, and predator control.
Many specimens removed from the field are deposited in the collections of natural history museums or biological data banks for future studies. Museum collections are curated repositories for whole specimens and their parts, whereas biological banks are collections of histologically or cryobionically preserved organs, tissues (including live cultures), cells (including sperm and ova) or embryos. Both kinds of repositories allow qualified researchers to study their collections. It is strongly recommended that, when appropriate, voucher specimens should be retained at the conclusion of field investigations, so that they will be available for use by future investigators. The collection of samples for museum preparation from natural populations is critical to:
1. Understanding the biology of animals throughout their ranges and over time.
2. Recording the biotic diversity over time and/or different habitats.
3. Establishing and maintaining taxonomic reference material essential to understanding the evolution and phylogenetic relationships of amphibians and reptiles.
Humane methods of kill trapping are those that kill the animal instantly while avoiding damage to the body parts that are required for the investigation.
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The nature of restraint will depend upon the procedure and the species involved. Restraint techniques can range from confinement in an enclosure, through various types of physical restriction, to chemical immobilization. Any decision to use physical or chemical restraint in a research project should be based upon an understanding of the behavioural and physical characteristics of the species to be restrained, the field conditions under which the procedure will occur, the knowledge and skill of those persons handling the animals, the goals of the investigation, and the availability of appropriate equipment and facilities. Investigators must use the least restraint that is necessary to do the job in a humane and effective manner, with minimal stress to the animal.
Because amphibians or reptiles, especially venomous species (including those with toxic skin secretions), may be capable of inflicting skin irritation or serious injury to those handling them, some form of restraint is usually required . The well-being of the animal under study is of paramount importance and it must be emphasized that improper restraint, especially of frightened or stressed animals, can lead to major physiological disturbances. In addition, the capture of some species of animals may alter their behaviour and cause death or predispose to predation.
Many species of amphibians and reptiles are intolerant of physical restraint and in some cases there is potential for injury to animal or handler. Investigators should not allow unsupervised, inexperienced persons to handle any animal species until adequately trained to restrain, manipulate and release the animals properly. The researcher should consult the current literature and seek the advice of experienced professionals before handling an unfamiliar species.
The following are general guidelines that must be considered when a researcher restrains a reptile or amphibian.
Most amphibians and reptiles are relatively small and slow moving, and can be restrained by hand or in a net. However, many small species are easily injured if the handler uses excessive force. Tail autotomy (tail shedding) can occur in most lizards if they are restrained by the tail. Although this is not a serious injury, it will influence future growth and reproduction by depriving the animal of fat stores as well as the integrity of the specimen. Tail loss may also affect the behaviour of the animal.
Because some reptiles may struggle excessively when manually restrained, the use of nets, hooks, tongs or handling bags may be required to reduce injury.
Venomous snakes(for example, rattlesnakes) and some large turtles are potentially dangerous and require special methods of restraint. Adherence to the following general guidelines is recommended when working with hazardous reptiles:
1. Procedures chosen should minimize the amount of handling time required, and reduce or eliminate the contact between handler and animal.
2. Those handling dangerous species should not work alone. A second person knowledgeable in capture/handling techniques and emergency measures, should be present whenever possible.
3. Only experienced personnel should handle venomous snakes . They should be familiar with the emergency procedures that are to be initiated in the event of an accidental bite or contact. A supply of the appropriate antivenin should be available at all times. In addition, a physician or medical facility should be made aware of the nature of the studies being undertaken so that proper arrangements can be made for emergency care and examination.
4. Whenever possible, an anaesthetic and/or physical restraint should be used before physical contact with the specimen.
Prolonged distressful restraint should not be undertaken. In some cases, it may be advisable to utilize general anaesthesia for restraint in the field. If so, the anaesthetic chosen should be one that permits a rapid return to a normal physiological and behavioural state and the animal should be monitored until complete recovery occurs. The unpredictable nature and prolonged recovery times that are associated with the use of some anaesthetic agents in reptiles and amphibians may negate their use under field conditions. Because of this uncertainty, some minor procedures may be less traumatic and deleterious when performed quickly without anaesthetics by experienced personnel. The pain perception of vertebrate species is not uniform; what causes pain and distress to a mammal may not have comparable effects on a reptile or amphibian. Consultation with an experienced herptile scientist or veterinarian is required to choose the appropriate agents and to establish experimental protocols. Many chemicals used for restraint or immobilization of amphibians or reptiles also have the potential for deleterious effects on humans.
Pertinent literature, experienced professionals and veterinarians should be consulted before a researcher initiates a project involving the immobilization of herptiles. Where possible, investigators should assess the effects of immobilization chemicals and procedures on captive individuals before incorporating them into field studies.
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Many studies require individuals to be marked for identification. Marking techniques can have a wide variety of 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:
1. The animal should experience no immediate or long-term hindrance or irritation from the mark or marking procedure.
2. The animal should suffer no adverse effects to its behaviour, physiology, longevity, social life, ecology or survival. The animal should not experience a greater susceptibility to predation or a reduced ability to breed.
3. The mark should be able to be applied quickly, easily and with minimal pain.
4. The mark should be readily visible to identify individuals or groups as required.
5. The mark should be effective in allowing the researcher to meet the objectives of the study and persist on the animal until research objectives have been fulfilled.
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 the 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 anesthetics) 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:
1. The dye must contrast with the normal skin pigmentation, and
2. Loss of tattoo legibility due to diffusion or ultraviolet degradation must be minimized.
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. In addition to this, turtles have 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 the limitations previously mentioned for such procedures.
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 Standardized Inventory Methodologies for Components of British Columbia's Biodiversity: Wildlife Radio Telemetry.
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.
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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.
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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.
1. In order to determine the husbandry requirements of a particular species the researcher should consult the current literature and experienced professionals.
2. When dealing with an unfamiliar species, the researcher should evaluate and compare several methods of housing in order to determine the method most appropriate for the needs of the animal and the objectives of the study.
3. Restraint and ease of maintenance by animal keepers should not be the primary determinant of housing design.
4. Normal field maintenance should incorporate, as far as possible, those aspects of the natural habitat that are deemed to be important to the survival and well-being of the animals.
5. The adequacy of captive conditions can be judged, relative to the natural environment, by monitoring a combination of factors such as changes in growth and weight, survival rates, breeding success, activity levels, general behaviour and appearance.
6. Consideration should be given to providing an environment that includes features such as natural materials, refuges, heat sources and water baths.
7. Natural foods should be duplicated as closely as possible, as should natural light and temperature conditions, unless alterations of these are factors under investigation.
8. Frequency of cage cleaning should represent a compromise between the level of hygiene necessary to prevent disease and the amount of stress that results from frequent handling and exposure to unfamiliar surroundings and bedding.
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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:
1. At the site of the original capture (unless conservation efforts dictate otherwise).
2. When the weather conditions and the season are conducive to survival.
3. If it is in the best interest of the study subjects, and if their ability to survive in nature has not been impaired.
4. When the released animals are not a health hazard or are otherwise detrimental to the existing populations in a specific geographic area (for example, when they do not jeopardise the genetic integrity of a population or carry a potentially infectious disease).
5. When the animals' release is compliant with federal, provincial or local laws.
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.
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