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INTRODUCTIONDuring the past 20 years, the use of fish in research has been increasing. The development of fish as a food source has also grown. Aquaculture is one of the most rapidly expanding new food industries partly because fish are seen as a low-fat food source and as a replacement for meats that are fat-rich. Consequently, an increasing emphasis is placed on the amount of fish consumed, the quality of fish produced, and the efficiency of fish growth. Research into the health and husbandry of food fishes is advancing with the development of the aquaculture industry and an increasing interest in maintaining fish as pets. At some point, the U.S. Department of Agriculture (USDA) or a similar group might inspect fish to insure their quality as a food source. Research on fish is also expanding and intensifying as a result of a greater interest in establishing new aquaria for entertainment and educational purposes. Although aquarists are interested in a wider range of species than aquaculturalists are, they are in a unique position to solve special husbandry problems with special species with particular biological properties. The attention that fish have received from both hobbyists and the fish industry has increased awareness of fish health as a factor that im Fish research and more specifically research in aquaculture helps address questions regarding environmental pollution, conservation, and protection of the freshwater estuarine and marine environment. Outside of the laboratory, fish are also subjected to environmental stresses (man-made and otherwise) that can harm their health and well being. All of these factors should also act to stimulate field research, that is, research performed directly on fish in their natural habitat, even though the number of experimental variables is much greater. Recently, as a result of greater concerns about the humane use of higher vertebrates in research, fish have been evaluated as a replacement in toxicologic, pharmacologic, and genetic studies that might otherwise employ mice or other mammalian species. All of this will increase the use of fish in research and expand the knowledge base on the care and use of these species in the laboratory. Fish research in laboratory animal science explores and uses their incredible diversity; the 20,000 species of fish worldwide constitute about half of all living vertebrates. In addition to size, which varies from a few to 15 meters, fish vary significantly in their taxonomy, morphology, genetics, behavior, physiology, and ecology. PERMITS AND LICENSES Wild-caught fish for laboratory use, whether captured by an investigator or a collector, may require scientific collecting permits. These include the federal and state scientific collectors permits and import permits. Obtaining a state scientific collection permit requires that one contact the appropriate governing agency (such as the department of natural resources), and request an application. The permits are not difficult to complete, last for 1 to 2 years, and are usually approved, although collecting protected or regulated species (such as striped bass, trout, or certain shellfish) may require special justification. The species sought will need to be listed by the applicant. Special collecting techniques such as electrofishing, use of certain types of nets, or use of chemicals may also call for a separate letter of authorization. The state office will require the collector to notify it where and when the collecting will take place, as well as to submit a year-end report on collection activity. Federal permits are not needed unless one is working outside state boundaries, in federal waters, or with federally protected species. A telephone call to the Federal Wildlife Permit Office will help to determine if a permit is required, as well as through which regional office the application should be routed. Having a federal permit for an endangered species will not automatically cover state requirements, however. The appropriate state agency will be able to determine if a special state license is needed in addition to the federal one. A federal listing of endangered and threatened wildlife and plants can be obtained from the U.S. Fish and Wildlife Service. Title 50 of the U.S. Fish and Wildlife Service restricts movement of certain food or sport fishes or their products from different parts of the world. Although the legislation applies only to food and sport fishes (including live and dead fish, fish product, and fish eggs), it is wise to be aware of diseases and their potential for transmission even when importing ornamental fishes for research. When importing fish from outside the U.S., a special Declaration for Importation of Exportation of Fish or Wildlife (USFWS Form 3-177) must be completed and filed with the local U.S. Customs agent. Fishes that appear on the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) lists are strictly controlled, and without the proper authorization, importation is prohibited. Appendix I of CITES lists animals that are threatened with extinction, and their acquisition is highly discouraged except under the most exceptional circumstances. CITES permits commercial trade in species listed in Appendix II, provided that the country of origin has issued an export permit. Appendix III, species may be traded; listings are made in order to gain recognition and protection for species that are in danger of becoming threatened. SUMMARY OF APPLICABLE ANIMAL WELFARE LAWS AND GUIDELINES Any vertebrate research performed or sponsored by the U.S. government is covered by the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (PHS Principles). For guidance in following these principles, investigators are directed to the Guide (NRC 1996), which recommends that an “appropriate environment be provided” for nontraditional species and that “expert advice on the natural history and behavior of nontraditional species” be sought (NRC 1996). Except for a short bibliography, fish-specific information is not included in the Guide. Other useful guidelines include The Care and Use of Amphibians, Reptiles and Fish in Research by the Scientists Center for Animal Welfare (SCAW) and Guidelines for The Use of Fish in Field Research by American Society of Ichthyologists and Herpetologists, the American Fisheries Society, and the American Institute of Fisheries Research Biologists. Also useful is the Canadian Council on Animal Care’s Guide to the Care and Use of Experimental Animals (CCAC Guide). The Animal Welfare Act does not cover fish or any of the cold-blooded vertebrates. The PHS Principles recommend selecting the appropriate species of the appropriate quality, as well as using the minimum numbers of animals required for valid results. Non-animal alternatives, discomfort, distress, and pain must also be considered. Living conditions should contribute to the health and well being of the animal; and trained and experienced personnel, inclusive of veterinary care should care for animals. Investigators and personnel should be qualified, experienced, and receive training if necessary. Any exceptions to the PHS Principles must be reviewed by the IACUC. All of the above conditions are directly applicable to research with fish. In addition to consulting with the attending veterinarian of the facility, it is frequently valuable for the Institutional Animal Care and Use Committee (IACUC) to have access to regular ad hoc members that are full-time specialists in the maintenance of fish as research animals. Individuals with relevant backgrounds could be scientists, aquarists, curators, directors of husbandry, or veterinarians working in aquariums or aquatic research centers. ZOONOSES Aquatic animals live immersed within an environment of potential pathogens. The presence of microorganisms alone is a danger to caretakers. In closed systems, however, the concentration of microorganisms may be amplified increasing the risk of human infection. Typically, bacteria associated with lesions are gram-negative organisms such as Aeromonas hydrophila in fresh water and Vibrio spp. in saltwater. Other organisms can be acquired from aquatic animals (Table 1). Once exposed, infection or disease will occur depending on the virulence of the organism and the susceptibility of the host. Following several common sense guidelines can minimize the likelihood of a serious illness. First, and most important, is the practice of proper hygiene. Hands should always be washed with an antimicrobial soap after handling animals or working in their environment. Second, any open wounds should be covered to prevent inoculation. Third, immunosuppressed individuals should avoid exposure to potential pathogens. Fourth, ill employees should not come in contact with animals or their environment. Finally, if an injury does occur while handling an animal or working in its environment, proper first aid must be applied. Working with all animals presents some level of danger from injury or zoonotic infection. The likelihood that an injury or infection will occur is dependent upon the individual’s ability, proper hygiene, and common sense. Therefore, it is essential to adopt proper husbandry practices, use equipment designed for the specific task, train inexperienced personnel, and develop emergency protocols tailored to the animals being used. INTRODUCTIONDuring the past 20 years, the use of fish in research has been increasing. The development of fish as a food source has also grown. Aquaculture is one of the most rapidly expanding new food industries partly because fish are seen as a low-fat food source and as a replacement for meats that are fat-rich. Consequently, an increasing emphasis is placed on the amount of fish consumed, the quality of fish produced, and the efficiency of fish growth. Research into the health and husbandry of food fishes is advancing with the development of the aquaculture industry and an increasing interest in maintaining fish as pets. At some point, the U.S. Department of Agriculture (USDA) or a similar group might inspect fish to insure their quality as a food source. Research on fish is also expanding and intensifying as a result of a greater interest in establishing new aquaria for entertainment and educational purposes. Although aquarists are interested in a wider range of species than aquaculturalists are, they are in a unique position to solve special husbandry problems with special species with particular biological properties. The attention that fish have received from both hobbyists and the fish industry has increased awareness of fish health as a factor that impacts the environment and as a measurement of environmental health.
Fish research and more specifically research in aquaculture helps address questions regarding environmental pollution, conservation, and protection of the freshwater estuarine and marine environment. Outside of the laboratory, fish are also subjected to environmental stresses (man-made and otherwise) that can harm their health and well being. All of these factors should also act to stimulate field research, that is, research performed directly on fish in their natural habitat, even though the number of experimental variables is much greater. Recently, as a result of greater concerns about the humane use of higher vertebrates in research, fish have been evaluated as a replacement in toxicologic, pharmacologic, and genetic studies that might otherwise employ mice or other mammalian species. All of this will increase the use of fish in research and expand the knowledge base on the care and use of these species in the laboratory. Fish research in laboratory animal science explores and uses their incredible diversity; the 20,000 species of fish worldwide constitute about half of all living vertebrates. In addition to size, which varies from a few to 15 meters, fish vary significantly in their taxonomy, morphology, genetics, behavior, physiology, and ecology. PERMITS AND LICENSES Wild-caught fish for laboratory use, whether captured by an investigator or a collector, may require scientific collecting permits. These include the federal and state scientific collectors permits and import permits. Obtaining a state scientific collection permit requires that one contact the appropriate governing agency (such as the department of natural resources), and request an application. The permits are not difficult to complete, last for 1 to 2 years, and are usually approved, although collecting protected or regulated species (such as striped bass, trout, or certain shellfish) may require special justification. The species sought will need to be listed by the applicant. Special collecting techniques such as electrofishing, use of certain types of nets, or use of chemicals may also call for a separate letter of authorization. The state office will require the collector to notify it where and when the collecting will take place, as well as to submit a year-end report on collection activity. Federal permits are not needed unless one is working outside state boundaries, in federal waters, or with federally protected species. A telephone call to the Federal Wildlife Permit Office will help to determine if a permit is required, as well as through which regional office the application should be routed. Having a federal permit for an endangered species will not automatically cover state requirements, however. The appropriate state agency will be able to determine if a special state license is needed in addition to the federal one. A federal listing of endangered and threatened wildlife and plants can be obtained from the U.S. Fish and Wildlife Service. Title 50 of the U.S. Fish and Wildlife Service restricts movement of certain food or sport fishes or their products from different parts of the world. Although the legislation applies only to food and sport fishes (including live and dead fish, fish product, and fish eggs), it is wise to be aware of diseases and their potential for transmission even when importing ornamental fishes for research. When importing fish from outside the U.S., a special Declaration for Importation of Exportation of Fish or Wildlife (USFWS Form 3-177) must be completed and filed with the local U.S. Customs agent. Fishes that appear on the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) lists are strictly controlled, and without the proper authorization, importation is prohibited. Appendix I of CITES lists animals that are threatened with extinction, and their acquisition is highly discouraged except under the most exceptional circumstances. CITES permits commercial trade in species listed in Appendix II, provided that the country of origin has issued an export permit. Appendix III, species may be traded; listings are made in order to gain recognition and protection for species that are in danger of becoming threatened. SUMMARY OF APPLICABLE ANIMAL WELFARE LAWS AND GUIDELINES Any vertebrate research performed or sponsored by the U.S. government is covered by the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (PHS Principles). For guidance in following these principles, investigators are directed to the Guide (NRC 1996), which recommends that an “appropriate environment be provided” for nontraditional species and that “expert advice on the natural history and behavior of nontraditional species” be sought (NRC 1996). Except for a short bibliography, fish-specific information is not included in the Guide. Other useful guidelines include The Care and Use of Amphibians, Reptiles and Fish in Research by the Scientists Center for Animal Welfare (SCAW) and Guidelines for The Use of Fish in Field Research by American Society of Ichthyologists and Herpetologists, the American Fisheries Society, and the American Institute of Fisheries Research Biologists. Also useful is the Canadian Council on Animal Care’s Guide to the Care and Use of Experimental Animals (CCAC Guide). The Animal Welfare Act does not cover fish or any of the cold-blooded vertebrates. The PHS Principles recommend selecting the appropriate species of the appropriate quality, as well as using the minimum numbers of animals required for valid results. Non-animal alternatives, discomfort, distress, and pain must also be considered. Living conditions should contribute to the health and well being of the animal; and trained and experienced personnel, inclusive of veterinary care should care for animals. Investigators and personnel should be qualified, experienced, and receive training if necessary. Any exceptions to the PHS Principles must be reviewed by the IACUC. All of the above conditions are directly applicable to research with fish. In addition to consulting with the attending veterinarian of the facility, it is frequently valuable for the Institutional Animal Care and Use Committee (IACUC) to have access to regular ad hoc members that are full-time specialists in the maintenance of fish as research animals. Individuals with relevant backgrounds could be scientists, aquarists, curators, directors of husbandry, or veterinarians working in aquariums or aquatic research centers. ZOONOSES Aquatic animals live immersed within an environment of potential pathogens. The presence of microorganisms alone is a danger to caretakers. In closed systems, however, the concentration of microorganisms may be amplified increasing the risk of human infection. Typically, bacteria associated with lesions are gram-negative organisms such as Aeromonas hydrophila in fresh water and Vibrio spp. in saltwater. Other organisms can be acquired from aquatic animals (Table 1). Once exposed, infection or disease will occur depending on the virulence of the organism and the susceptibility of the host. Following several common sense guidelines can minimize the likelihood of a serious illness. First, and most important, is the practice of proper hygiene. Hands should always be washed with an antimicrobial soap after handling animals or working in their environment. Second, any open wounds should be covered to prevent inoculation. Third, immunosuppressed individuals should avoid exposure to potential pathogens. Fourth, ill employees should not come in contact with animals or their environment. Finally, if an injury does occur while handling an animal or working in its environment, proper first aid must be applied. Working with all animals presents some level of danger from injury or zoonotic infection. The likelihood that an injury or infection will occur is dependent upon the individual’s ability, proper hygiene, and common sense. Therefore, it is essential to adopt proper husbandry practices, use equipment designed for the specific task, train inexperienced personnel, and develop emergency protocols tailored to the animals being used. Table 1: Aquatic microorganisms associated with zoonotic infections Aeromonas hydrophila
Campylobacter spp. EXAMPLES OF USES OF SPECIES IN BIOMEDICAL RESEARCH Fish have been used in biomedical research for many years. With their diverse sizes and their myriad of anatomical variations, fish offer the scientists opportunities to explore novel organs and structures. These studies can have profound implications for understanding mammalian biology and physiology. For example, one of the first investigations demonstrating the role of renal tubular secretion in the excretion of xenobiotics was accomplished using the aglomerular toadfish. Until then it was almost heresy to suggest that substances appearing in the urine had come from anything but glomerular filtration. More recently, nephron neogenesis following toxication-induced injury, not found in mammals, has been demonstrated in goldfish kidneys. Other specialized features of interest to biomedical researchers include antifreeze-like molecules in the blood of arctic species, electrical activity in muscles of the electrical eel, survival of dehydration in the African lungfish, and copper accumulation in white perch. Fish are also extensively studied as models for research on aging, vision, locomotion in cells, and leukemia. Species are also evaluated for pharmacologically active compounds such as Indian catfish venom, and angiogenic inhibitors and antineoplastic agents in shark tissues. Fish are also studied as indicators of environmental pollution using parameters such as neoplasias, and immunological function. Small species of fish have been used in many studies because their size allows large numbers to be kept in a limited space and their short life cycles provide the opportunity to examine multiple generations. Fish have also been used to investigate carcinogenicity and toxicity of various compounds. Japanese medaka and zebrafish transgenic specimens are being used to evaluate the roles of multiple genes in development. The zebrafish has long been the favorite organism in many scientific disciplines. Although its attributes as a model were expounded for many years and thus were no secret, the zebrafish sat in the wings while other more popular vertebrates such as chick, amphibians, and mouse were examined at length. An explosion of research utilizing the zebrafish began in the late 1970s when investigators at the University of Oregon began using it as their model in neuroscience. Prior to this, the zebrafish was one of the significant organisms in the study of teratology and toxicology, development, and, to some extent, behavior. Recently, however, the field of zebrafish genetics has gained immense popularity and success, in part owing to the fact that zebrafish are diploid and are amenable to genetic manipulations. In addition, treating them with copper and measuring their clotting function has developed an artificial hemophilia in zebrafish by a newly developed sensitive clotting time assay. The clotting function can be detected rapidly and reliably in 30 hr larvae and in adult fish by measuring the blood clotting time. Zebrafish are also being used in carcinogenicity, mutagenesis, and reproductive studies. In mutagenicity studies, adult fish are subjected to the mutagens and their haploid embryos can be screened for mutagenicity during the first generation. Because the embryo has fewer cells than most vertebrate species, it can serve as a model for more complex vertebrate species. Additionally, because the embryo is transparent, one can easily observe pathologic changes in the developing embryo. There are two websites where information on zebra fish research and genomics can be found. These are: www.zfin.org and www.zebra.sc.edu. Blood flow patterns have been studies in the hearts of zebra fish embryos and found to be comparable to developing human embryos. The transparency of the zebra fish embryo makes viewing through a microscope very easy for this type of research. Researchers have been able to measure the velocity of blood flow in the heart using this model. Altered blood flow in the developing heart can lead to heart defects, and this research may assist in being able to recognize and correct problems in developing embryos, including in the human.
The zebrafish has recently been used as a model for embryonic development in vertebrates. Researchers from the University of Pennsylvania School of Medicine have demonstrated, that a maternal factor is critical in the proper development of the embryo before the embryonic genome is initiated. Mutant female zebra fish were selectively bred to wild type males. The subsequent offspring were analyzed for mutations in their genetic code that were associated with birth defects. This investigation has assisted researchers in understanding the formation of birth defects as well as abnormalities associated with human fertility and sterility (Dosch et al, 2004), (Roland et al, 2004). Zebrafish is also being used to understand eye disease in higher animals, including man. Researchers first identified a family of eyeless fish and then discovered the gene that controls eye development and the mutations that lead to eyelessness. In other studies, a cell known as Muller glial cells have been found to allow zebrafish to regenerate their retinas, even when there has been extensive damage to the retina. The same cell also exists in humans and other species. These cells have been found in all ages of humans–from young children to people in their 90's. It is possible that these stem cells could be used to regenerate damaged retinas in humans. Before doing the tests in humans, additional studies will be performed in rats. Platy fishes and Swordtails are two additional species that are being increasingly used in research. A number of years ago, fish fanciers were performing hybridizations between species of these fish and found increased incidence of tumors in the hybrid fish. As a result, these animals are being used in studies of spontaneous and induced neoplasia. The Gordon-Kosswig Melanoma Model is one example of the use of these animals in cancer research. Oncogenes and tumor-suppressor genes play a role in this model. Another example is the Pu2 model, which is results from altered melanocytes in certain hybrid fish. These species are also being used in genetics research, particularly in study of multigenetic phenotypes, behavioral research, toxicology, parasitology, and immunology. The number of studies using these animals has increased dramatically during the past 10 years. The starfish is becoming popular in research because they are perfect for live cell microscopy studies. The oocytes of the starfish are transparent and develop in seawater, making them perfect for easy observation of the stages of development and cell division. Starfish oocytes make good models for observing cell division in living animal eggs. Also, starfish are evolutionary close to vertebrate animals, allowing connections to be made between starfish and vertebrates cell division. Researchers studying starfish oocytes found an actin network that moves the chromosomes during cell division, preventing chromosome loss. Movement of chromosomes by actin has not previously been shown. These important biological mechanisms are likely to apply to mammalian oocytes (Lenart et al, 2005).
The Armed Forces Institute of Pathology (AFIP) in A Handbook: Animal Models of Human
Disease (AFIP 1989) lists the following species as models: multiple schwannomas of bicolor
damselfish, type I diabetes in carp, DNA damage in the Amazon molly, Wilson’s disease in the
white perch, hepatocellular carcinoma in rainbow trout, and malignant melanoma in
platy/swordtail hybrids. Methods for Fish Biology presents an excellent overview of the concept and design of research methods employing fish including field experiments, fish genetics, systematics, and taxonomic methods using morphology and electrophoresis, chromosome analysis, histology, anesthesia, surgery, and hematology. It also includes specific areas of study on respirometry, growth, biogenetics, the nervous system, stress and acclimation, aquatic toxicology, reproduction, behavior, autecology (the study of single-species ecology), community ecology, as well as a section on maintaining fish for research and teaching. The Laboratory Fish (Academic Press) provides information on managing fish in the laboratory setting and includes information on conduct of common experimental procedures that are often needed in fish. An electronic version of the text is available. Marine Fish Schools, universities and educational institutes are increasingly organizing their own field trips to the coast to teach many aspects of marine biology. On such trips living fish may be collected for use in the classroom and it is important that the persons in charge have a good understanding of how to keep them. It is therefore necessary to know both the best and most humane way of capturing and transporting marine fish as well as keeping them in the “captive environment”. Departments of Fisheries need to keep marine fish for a wide variety of research as their value in commercial terms is considerable and their indirect use in pollution and monitoring studies is of importance. Increasingly it is found how little is known about even common shallow marine fish and how profitable research on their ecology and behavior can be. Research into fish behavior, biochemistry, physiology, nutrition, genetics, ontogeny, functional morphology and many other topics all demand that the research worker has access to fresh or living material (see table 2) and it is important that they should be kept humanely with the minimum of stress. There is no single marine fish which serves the same role as the goldfish or trout for fresh water research, but the dogfish (of which there are several species), does have a special place among marine fish for it is used widely in courses of zoology as dissection material to demonstrate the segmental arrangement of primitive vertebrates. It is also increasingly used in physiological research on the vertebrate nervous system and neuromuscular control systems. Vibration detection through the lateralis system and chemoreception by the greatly enlarged olfactory lobes are important in the dogfish but relatively few studies have been made on the behavior of intact animals. Table 2: Marine Fish Used in Biomedical Research
Valentine's Sharpnose Puffer Fish and Their Genes There have been some interesting developments in the use of fish as animal models of human disease. The human FRG1 gene maps to human chromosome 4q35 and has been identified as the gene potentially causing a type of muscular dystrophy. The Japanese puffer fish Fugu rubripes also has this gene, and this species may serve a model for understanding how this gene influences changes in the muscles. The puffer, also known as the fugu, is highly poisonous and is considered a delicacy in Japan. Chefs prepare them and the gourmet diners take the risk that they may be poisoned and die!! This fish has recently become a focus of the genome project and scientists are rapidly decoding its genome, because it has a highly compact genome. The fish has approximately the same number of genes as mankind, but lacks the junk DNA that clutters the human genome. Using the fugu’s genetic code as a basis for looking at the human genome, researchers have been able to identify and additional 900+ previously unknown human genes. A derivative of Tetrodotoxin, the toxin produced by the Puffer fish, is being tested in human clinical trials as a pain reliever. Tetrodin, originally used in Asia to help heroin addicts through withdrawal, has been found to be 3000 times more potent than morphine as a pain killer. A study in Asia showed that cancer patients treated with the drug had significant pain relief for 20 days following two injections a day for three days. Trials are now underway in Canada. Other models have been found in a variety of fish and its relatives of the shark family. Antithrombin, a major coagulation inhibitor in mammals has recently been cDNA cloned from Atlantic salmon Salmo salar, which may serve as a model for understanding the role of antithrombin in coagulation. The study of ancient forms of cytokins is underway in species such as the rainbow trout, Mediterranean sea bass and carp. Opioid receptor sequences have been found in bullfrogs, striped bass, thresher shark, and Pacific hagfish, all of which may be added to the list of animal models for pain perception. Serendipity and the Dogfish: A substance derived from dogfish sharks has recently been found to suppress appetite and decrease body weight in rodents ( a double animal model!). This research may lead to a safe method of weight control in human beings. The compound (MSI-1436) is derived from cholesterol and is obtained from the liver of dogfish sharks. Obese mice fed a diet of the shark-product, lost weight and lost FAT, not muscle. Often dieters lose muscle mass instead of the desired adipose tissue. An interesting side-bar to the discovery of the new anti-obesity treatment, is that the compound from dogfish was first tested to be a preventative treatment for HIV. In the process of testing animals infected with retroviruses, the researchers discovered that the animal subjects quit eating. This ruled out the substance as an HIV treatment–but ruled in its ability to fight fat. Serendipity has often been a factor in research discoveries. Next year, MSI-1436 will enter into Phase I human trials to see if the compound will work in humans. Squalamine, another product from dogfish livers has been shown to have anti-cancer properties. Sharks and Cancer Links: Scientists have determined that something in shark and skate immune cells inhibits the growth of cancer; however, the actual substance in these primordial fish has not, as of yet, been identified. Elasmobranchs, that is sharks, skates, and sting-rays, rarely get cancer; whereas, other types of fish often have cancer. Studies, utilizing chemical carcinogens, have no effect on elasmobranchs, and this finding led to the conclusion that these species have something inherent in their physiology, that prevents cancer. Using immune cells from these species in cell culture, scientists have found that cancer cell growth is inhibited and, in some cases, the cancerous cells are killed. AMPHIBIANS
Amphibians include over 4500 species within 3 major lineages - caecilians, salamanders, and anurans. These lineages are linked by several unique physiological traits. The most prominent is the indirect life-style of many amphibians - the aquatic gill-breathing larval stage and the aquatic or terrestrial lung or skin-breathing adult stage. A second feature of amphibians is the presence of 2 types of skin glands: mucous and granular. Mucous glands keep the skin surface moist to allow transcutaneous respiration, but they also put the animals at risk of desiccation. The granular glands secrete toxins and function primarily for defense. The more than 160 species of caecilians are not diverse, all being legless and inhabiting tropical regions. This poorly understood group of amphibians is used in research relatively infrequently, and, therefore, will not be discussed further. The salamanders are represented by nearly 400 species, some of which have a four-legged aquatic larval stage, while others (primarily the Plethodontidae) pass the larval stage within eggs that are laid in moist places on land. Some salamanders retain the larval gills through their adult stage and depend on oxygenated water throughout their lives. While most salamanders have legs, they have become vestigial in some. The anurans, or frogs and toads, are the most successful lineage of amphibians with approximately 4,000 species distributed around the world in a multitude of habitats. The larval stage of most, but not all, anurans includes a legless tadpole or polliwog. The degree of dependency on water varies - species range from purely aquatic to completely terrestrial. Frogs are not only the most diverse group of amphibians; they also are by far the most commonly used amphibians in research. Housing for newly acquired amphibians should be functional prior to their arrival, so that the animals can be quickly unpacked. Newly arrived individuals should be given a thorough visual evaluation and housed separately from individuals already present in the colony. A minimum 30-day quarantine period is suggested. During quarantine, animals should be carefully monitored for morbidity and mortality and evaluated for endoparasites. Any clinical disease should be properly diagnosed and aggressively treated. It is best to locate recently arrived amphibians, particularly ranis frogs, in a room with low traffic since they are easily agitated. When frightened, these frogs will jump excessively and may traumatize themselves on the cage lid. Using a deep opaque cage will minimize the trauma to the frogs. Placing opaque plastic over cages housing newly acquired frogs will also minimize the amount of jumping. ANIMAL WELFARE REGULATIONS AND POLICIES Being “lower vertebrates,” amphibians are exempt from U.S. Department of Agriculture regulations. The Guide for Care and Use of Laboratory Animals (Guide) (NRC 1996) does not provide specific recommendations for the maintenance of amphibians. However the Guide does state, “An appropriate environment should be provided for nontraditional species...Expert advice on the natural history and behavior of nontraditional species should be sought when such animals are to be introduced into a research environment.” To assure that humane issues are being properly addressed, one must use available guidelines; possess a good understanding of amphibian biology; and apply, with adaptations, procedures used for traditional species. Institutional animal care and use committees (IACUCs) need to establish institutional guidelines for dealing with the use of amphibians, both in the field and the laboratory. A cooperative process, incorporating input from written guidelines, committee members, faculty using amphibians, campus veterinarians, and consultants should develop these guidelines. Guidelines contrived in such a positive manner tend to be stronger and hold up better in this complex field where much is still not well understood. USES IN BIOMEDICAL RESEARCH The species most frequently used as laboratory animals can be considered in two groups: • Wholly aquatic species, particularly the platannas (Xenopus species); X. laevis (South African clawed toad); X. borealis (Kenyan clawed toad); X. tropicalis; X. epitropicalis (Nigerian clawed toad). • Species in which the adults are mostly terrestrial: Frogs: Rana pipiens (leopard frog); R. japonica (Japanese Frog); R. niigromaculata; R. catesbeiana (bull frog); R. clamitans; R. lessonae; R. ridibunda (hybrid R. esculenta); R. temporaria (common European frog); Discoglossus pictus Toads: Bombina species: Bufo bufo; B. marinus; B. arenarum Amphibians have long been used as subjects for studies in ecology, ethology, and evolution. More recently, declining amphibian species throughout the world have stimulated an interest in these animals as environmental sentinels. Amphibians are frequently used as models in comparative medicine, with the predominant model species being the African clawed frog (Xenopus laevis). Xenopus is used in studies of development and differentiation at both cellular and molecular levels. One of the primary uses of Xenopus is to obtain oocytes for molecular biology research. The frog’s embryos are also being used to study specific genes and their functions. For example, genes that play a role in the signaling pathways for movement. Additionally, amphibians are frequently used in biomedical research to study vision, hearing, respiratory physiology, endocrinology, analgesia, toxicology, tissue freezing, and tissue regeneration. Numerous peptides of frog origin have received considerable attention as potential therapeutics for bacterial infections, allergies, asthma, central nervous disorders, and analgesics. Metamorphosis in frogs and toads is a set of complex developmental programs controlled by thyroid hormone (TH). The pervasive and dramatic changes that occur as a tadpole turns into a frog have captured the attention of life scientists from many disciplines. For evolutionary biologists, the extent to which related organisms incorporate metamorphosis in their life cycle is of paramount interest. For specialists who study cell death, a burgeoning field of research interest, apoptosis (programmed cell death) is a major event in metamorphosis. TH can induce it in many tissues, even in whole organs. For endocrinologists, TH-induced metamorphosis is a model for the general problem of the molecular basis of TH action and the interaction of the hypothalamus, the pituitary and the thyroid glands. Axolotls are unique among vertebrates in their ability to regenerate lost appendages as adults. They provide the opportunity to study the mechanism of regeneration in vertebrates and are an inspiration to pursue the goal of appendage regeneration in humans. As a group, the homeobox (a class of highly conserved DNA sequences encoding a protein domain involved in binding to DNA)-containing genes are important in pattern formation in the primary body axis, developing limbs and regenerating limbs. To date, a total of 22 homeobox genes have been identified as being expressed in regenerating limbs. Nearly all of these are also expressed during limb regeneration, further supporting the view that limb development and regeneration involve similar regulatory mechanisms. REPTILES
INTRODUCTION The taxon Reptilia encompasses more than 15,000 species of vertebrates, of which more than 9,000 are birds and not dealt with herein. This leaves roughly 300 species of lizards, 175 species of amphibians, and 2,700 species of snakes that are linked by ectothermy and certain other retained primitive characteristics, yet are highly divergent among themselves. In the rest of this paper, they are referred to collectively by the traditional term “reptiles.” That birds (whose closest relatives are crocodilians) are reptiles in exactly the same way that primates are mammals underscores the wide range of morphology, physiology, and behavior achieved by those evolutionary lineages that even many biologists still anthropocentrically call “lower vertebrates.” This extreme diversity among and within reptile groups is widely underappreciated yet central to their welfare in captivity. COMPOSITION AND CHARACTERISTICS Beyond the trademark shell, turtles (also known as chelonians) range from strictly terrestrial, dome-bodied, herbivorous tortoises to highly aquatic, flat-bodied, carnivorous soft-shelled turtles. Most species used in laboratory research are omnivorous and semi-aquatic. Crocodilians (alligators, caimans, crocodiles, and gavials) are semi-aquatic carnivores and infrequently maintained in captivity outside of zoological parks. The 2 species of tuataras (Sphenodon) are confined to certain islands off the coast of New Zealand, very rarely maintained in laboratory settings, and not dealt with further here. The approximately 6,000 species of lizards, amphibians, and snakes are each others’ closest relatives, collectively termed “squamates” or “squamate reptiles,” and characterized as a group by the presence of 2 copulatory organs (called hemipenes) in males. Many species of lizards and snakes are widely used in research, resulting in thousands of publications annually. The best entries to the voluminous primary literature on reptiles are the annual volumes of Zoological Record. With few exceptions (for example, some brooding females pythons), reptiles are ectothermic; this means that they depend upon an external heat source to maintain body temperatures above ambient (note that some reptiles routinely maintain fairly constant high temperatures for hours, such that the term “cold-blooded” is inappropriate). Heating is typically accomplished by basking, although some secretive species heat themselves conductively under rocks and other warm cover objects. Ectothermy and the generally low metabolic rates associated with that life-style have numerous important consequences for proper captive maintenance; for example, a variable thermal environment is crucial for good health, periods of torpor are acceptable, and frequent feeding is not necessarily imperative. Most reptiles also rely substantially on chemosensory information in dealing with their environments, meaning that anthropocentric perspectives may be misleading in terms of proper care. Reptiles vary in size from the 1 g adults of certain gecko lizards (such as Sphaerodactylus) to several species of turtles, crocodilians, lizards, and snakes that exceed 100 kg in mass. Although all turtles, crocodilians, and tuataras lay eggs, several groups of lizards, amphibians, and snakes have independently evolved viviparity; crocodilians and some squamates exhibit various forms of postnatal parental care. Natural lizard diets range from insects for most small species to leaves for green iguanas (Iguana iguana) and mammals for some large monitors (Varanus). All snakes are carnivores; some vipers consume individual prey items in nature weighing up to 156% of their own weight. SAFETY CONSIDERATIONS
Toxins and Other Weaponry The vast majority of turtles and squamates are harmless to humans. Claws and jaws of even moderate to large reptiles can be injurious; an adult green iguana (Iguana iguana) and even a small crocodilian or monitor can deliver an extremely painful and scarring bite. Heavy gloves are appropriate for handling these animals. Large constricting snakes (such as pythons more than 2 m in total length) are potentially capable of killing an adult human by constriction in a matter of a few minutes. At least 2 people should always be present when large constrictors are managed.
Zoonoses Some turtles and lizards carry Salmonella. Although the transfer of disease-producing organisms from reptiles to humans is not regarded as likely, routine hygiene, especially washing hands after handling the animals, is advisable. USES OF REPTILES IN THE LABORATORY Because of their remote relationship to man and his domestic animals, reptiles are little used in laboratories. The criteria for their selection are that they are easy to obtain and maintain, will breed readily in captivity, and that they are not endangered in the wild. The last proviso is particularly important, now that so many species of reptile are becoming scarce, following destruction of their habitat, and in some cases over-collection for commercial purposes. Species that are on Appendix I of the CITES Convention should not be used, and their import or export is unlikely to be permitted. It is hardly surprising that the behavior of reptiles in captivity is often a travesty of that in nature. They are liable to become sluggish and refuse to feed, eventually dying of starvation. Reptiles seldom breed, so that stock can only be replenished by purchase or by the collection of fresh specimens. These are some of the reasons why reptiles are used in experimental work less often than other vertebrates. Nevertheless, many species can be maintained in the laboratory, although the animal house that is assigned primarily for housing small mammals may not be suitable for the accommodation of reptiles. Chelonia
Chelonia are not commonly used as laboratory animals, the majority of experimental subjects being turtles, which have been used in studies on the effects of temperature change on heart rate, respiratory rate and gut passage time. The physiological effects of diving have also been studied including such parameters as heat exchange rates. Small turtles also make ideal subjects for the construction of heating and cooling curves, neuromuscular, and behavioral studies. Tortoises are less commonly used in the laboratory. An anatomical peculiarity shared by members of the Chelonia is that the scapulae are inside the rib cage. The embryology and especially the development of the brain of the tortoise has been intensively studied. Many Chelonians hibernate during the cooler portions of the year and seasonal changes in many biochemical and physiological parameters have been described. The series of books entitled Biology of the Reptilia of which Carl Gans is series editor (Academic Press, London 1970 to date) should be consulted before any study is undertaken using Chelonia.
Table 3: Areas of Research Interest Involving Snakes
The Gila Monster: Recently researchers have found the saliva of the Gila Monster has an effect on Type II diabetes. Scientists have synthesized a synthetic version of the hormone found in the Gila’s saliva and used it in a trial with 1000 human patients. The medicine helps control insulin and body weight. Patients report a decrease in appetite when taking the hormone REFERENCES Andersen, O, Flengsrud, R, Norberg K, et al. 2000. Salmon antithrombin has only three carbohydrate side chains, and shows functional similarities to human beta-antithrombin. European Jo Biochem. 267:1651-7. Brierley, C. Fish Eyes Could Hold Clue to Repairing Damage Retinas in Humans. 7/31/07 http://www.eurekalert.org/pub_releases/2007-07/wt-fec07310.php. Capen, C.C., Johnson, L.K., and O’Neill, T.P. Animal Models of Human Diseases. Registry of Comparative Pathology, Washington, DC. DeNardo, D. 1995. Amphibians as laboratory animals. ILAR Journal 37(4):173-181. DeTolla, L.J., S. Srinivas, B. Whitaker, et al. 1995. Guidelines for the care and use of fish in research. ILAR Journal 37(4):159-173. Dosch, R., Wagner, D.S., Mintzer, K.A., Runke, G., Wiemelt, A.P., Mullins, MC. Maternal Control of Vertebrate Development before the Midblastula Transition: Mutants from the Zebrafish I. Developmental Cell. 6(6): 771-780. Gans, C. Biology of the Reptilia. Wiley, New York, NY. Gardiner, D.M.. and S.V. Bryant. 1996. Molecular mechanisms in the control of limb regeneration: the role of homeobox genes. International Journal of Developmental Biology 40(4)797-805. Greene, H.W. 1995. Nonavian reptiles as laboratory animals. ILAR Journal 37(4):182-186. Grewal, PK, Todd, Lc, et al. 1998. FRG1, a gene in the FSH muscular dystrophy region on human chromosome 4q35, is highly conserved in vertebrates and invertebrates. Gene. 216:13-9. Guide to the Care and Use of Experimental Animals, 1993. Canadian Council on Animal Care, Ottawa, ON. Guidelines for Use of Fishes in Field Research, 1988. Fisheries 13(2):16-23. Guidelines for Use of Live Amphibians and Reptiles in Field Research, 1987. American Society of Ichthyologists and Herpetologists, Austin, TX. Guinn, E.: Making Blind Zebrafish See. Conway Institute of Biomolecular and Biomedical Research. Innovations Report: Forum for Science, Industry, and Business. 1/7/04. Guy Chaumette - Photographer and award winning filmmaker. A Photographic experience website. Link: http://www.divemad.com/index.htm Jagadeeswaran, P. and Liu, Y.C. 1997. A hemophilia model in zebrafish: analysis of hemostasis. Blood Cells, Molecules, and Diseases 23(1):52-7. Kanamori, A. and D.D. Brown. 1996. The analysis of complex developmental programs: amphibian metamorphosis. Genes to Cells 1(5):429-35. Kazianis, S, Walter, R: Use of Platyfishes and Swordtails in Biological Research. Lab Animal: 31,3: 46-52. Li, X, Keith, DE Jr, Evans, CJ. 1996. Mu opioid receptor-like sequences are present throughout vertebrate evolution. Jo of Molec Evol. 43:179-84. Lollar, K: 2004: Mote Scientists Research Shark, Cancer Link. Www.news-press.com/news/local_state/040808cancer.html. Matthews, M., Trevarrow, B., Matthews, J: A Virtual Tour of the Guide for Zebrafish Users. Lab Animal: 31,3: 34-40. Mulvihill, K. 2003. Bolld Flow in Fetal Heart Key to Normal Development. Reuters Health, New York, January 9. Poole, T.: The UFAW Handbook on the Care and Management of Laboratory Animals, 1987. Longman Scientific and Technical, Essex, England. Poepsel, Mark. 2004. Healing Medicine from Gila Monsters. www.kold.com/Global/story.asp?S 1965442&nav=14RTOA5e. Rollin, B.E. and Kesel, M.L.: The Experimental Animal in Biomedical Research, Volume II, 1995. CRC Press, Boca Raton, FL. Scapigliati, G, Bird, S, Secombes, CJ. 2000. Invertebrate and Fish Cyokines. European Cytokine Network. 11:354-61. Schreck, C.B. and Moyle, P.B.: Methods for Fish Biology, 1990. American Fisheries Society, Bethesda, MD Shaeffer, D., Kleinow, K., and Krulisch, L.: The Care and Use of Amphibians, Reptiles, and Fish in Research, 1992. Scientists Center for Animal Welfare, Greenbelt, MD. Sipp, D.: NRH1 and Wnt Signaling Come Together in Convergent Extension. EurekAlert. 7/20/04. *Stoskopf, M.: Fish are Here to Stay. Lab Animal: 31,3: 9. Stoskopf, M.:The Laboratory Fish. Lab Animal: 31,3: 28.* U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. In Public Health Service Policy on Humane Care and use of Laboratory Animals, 1996. Vascotto, S.G., Beckham, Y., and Kelly, G.M. 1997. The zebrafish’s swim to fame as an experimental model in biology. Biochemistry and Cell Biology 75(5):479-85. *Wade, N., Fish Genes Aid Human Discoveries, New York Times, 7/26/02.* Wagner, D.S., Dosch, R., Mintzer, K.A., Wiemelt, A.P., Mullins, M. C. Maternal Control of Development at the Midblastula Transition and beyond: Mutants from the Zebrafish II. Developmental Cell 6(6): 781-790. Zoological Record. Zoological Society of London.
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