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Theme B WorkshopsWB1: Refinement/Reduction through Design of the Experiment WB1: Pilot Investigation of the Use of General Anesthesia to Refine the Statutory Mouse Bioassay for Paralytic Shellfish Poison Paralytic shellfish poison (PS) is a potentially lethal toxin that is found in varying concentrations in shellfish. Worldwide, food safety laws require shellfish to be tested by the mouse bioassay. Shellfish extracts are injected into mice and if PSP is present, the mice die. The time between injection and the last breath is used to calculate the amount of toxin in the sample, utilizing a calibration factor derived by injecting mice with known amounts the reference substance, saxitoxin, to create a dose-response curve. There is currently no in vitro alternative. To achieve a significant decrease in the severity of the bioassay, this study investigated the feasibility of generating a comparable dose-response curve in anaesthetized mice. Four levels of saxitoxin were used. Prior to receiving the saxitoxin, mice (n = 9 per dose level) were given a general anesthetic whilst control mice (n = 9 per level) remained unanesthetized, as is current practice. Time until death was recorded. A linear relationship was observed between median time to death of anaesthetized and unanesthetized mice for the levels of saxitoxin used. Although further data are required for validation of the technique, refinement of PSP testing using general anesthesia appears feasible. WB1: Refinement and Reduction Through the Control of Variation Poorly designed animal experiments are ethically unacceptable. Inter-individual variation creates "noise" that may obscure differences among treatment groups. Specific pathogen-free animals of uniform age or weight are used largely to reduce this variability. However, genetically and phenotypically heterogeneous outbred rodents continue to be widely used even though geneticists have advocated the use of isogenic strains for more than 60 years, and no case for using outbred stocks has been published recently. Should funding organizations and ethical review committees take a more active part in questioning the scientific validity of each experiment? This workshop will discuss variation and its implications for Reduction and Refinement in animal research. WB2: Refinement in Generation and Maintenance of Transgenics: Local Anesthesia, Cryopreservation, and Clinical Assessment of Phenotype WB2: Refinement in Transgenic Management Given the exponential growth in the production and use of transgenic animals in research throughout the world and the uncertainty of the impact of such transgenesis and knock-out procedures on the animals, there is an increased awareness of the need for a detailed phenotype assessment of each strain. The establishment of a detailed phenotype will not only be important for animal welfare, but will add to the scientific information base. This could be particularly important when models of human diseases are being generated, as it would be useful to correlate the phenotype with the clinical signs seen in humans with that disease. Moreover, there is an increasing trend to cross genetically modified animals of different strains, as well as with mutant strains. Each genetically modified and hybrid/mongrel animal should have a welfare passport so that the receiving institution is aware of the likely adverse effects, the time at which to expect these effects, as well as how to avoid or alleviate the effects. This workshop aims to provide some approaches and to draw on the experience of all those attending. WB2: Genotyping Mice with Care: Buccal Cell Collection on Mice Results in a Gentler, Cheaper, More Simplified Protocol New methods of altering the genetic makeup of animals have resulted in large numbers of genetically engineered animals being bred. The most common way to screen for genetically engineered mice (Mus musculus) is to use the polymerase chain reaction) (PCR) amplification of a DNA sample. Most commonly, the sample is obtained from a tail biopsy; this method is considered to be invasive. Here, a comparative study of current techniques, using ear punch biopsies, hair samples, and buccal epithelial cells, for obtaining a DNA sample has led to the development of a refined new protocol. The use of a Nasopharyngeal swab for buccal cell collection, along with proteinase K in a PCR compatible buffer for the DNA extraction, yielded results that indicated no increase in unreliability, and, therefore, the results were as dependable as those from current methodologies for genotyping. WB2: The Management of Animal Welfare by Clinical Observations and an Integrated Information System Ethical considerations and legal requirements prompted the in-house development of an integrated animal management system. First, the principles of humane endpoints should be applied in a systematic manner to prevent any more discomfort than is inevitable to complete the experiment. Second, investigators are required by Dutch law to acquire a special license for the genetic modification of animals. These licenses have terms on the keeping of animal welfare logs. Third, the legally required annual reports on the use of animals should contain reliable information on animal welfare. The integrated data management system allows investigators to plan the work in a detailed manner. Feedback information on clinical signs (general appearance, behavior, response to stimuli, changes in body weight) is recorded, and the severity and duration of signs generate automated alerts to animal caretakers and investigators. To avoid age-related pathology, the system monitors the mice not to be bred when older than six months of age and to be killed humanely when they reach an age of eight months, unless the ethical committee has allowed aging as part of the protocol. WB3: Assessing and Managing Pain and Distress for Ethics Committees WB3: Physiological And Behavioral Assessment of Pain In Ruminants Pain as a stressor elicits a range of physiological and behavioral responses that are commonly used to assess the impact of pain-causing stimuli on animals to determine whether or not significant pain is caused and to devise strategies for alleviating pain. This paper will outline a range of principles and caveats to guide the evaluation of physiological and behavioral responses to painful stimuli, so that they can be better used to minimize pain in the experimental context. Although most examples are drawn from studies of farm animals responding to painful husbandry practices, the guidance provided is more generally applicable. Physiological indices considered include heart rate, arterial blood pressure, and plasma concentrations of ACTH, cortisol and catecholamines. Behavioral indices considered include normal or abnormal activity, posture, demeanor, responsiveness and temperament. Where possible, coincident physiological and behavioral responses will be described and advice on pain alleviation provided. WB3: Genomic Approaches to Pain Assessment and Alleviation
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