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Refinement is perhaps the most elusive concept when considering alternatives to experimental procedures. Refinement requires the reduction of animal pain and distress to the minimum level consonant with the study under consideration. The most obvious example is the use of anesthetics, analgesics, and tranquilizers to reduce pain and distress chemically. However, the number of methods that can be employed to improve animal subjects' well-being and comfort within the confines of an experimental design is not limited to the chemical methods noted above.

Refinement methods begin with the housing and environment in which the animal spends most of its time. Adequate cage size and nutritious food are clearly mandated. However, the use of enrichment, exercise programs, and social groupings can all help to decrease stress and improve well-being among animal subjects. Preconditioning animals to test environments (mazes, restraint apparatus, etc.) both decreases stress and improves experimental results. Positive reinforcement is preferable to noxious stimuli but when painful or distressing stimuli must be used animals should be able to escape. Early euthanasia (euthanasia of an animal prior to the normal end of the study) must be performed if the animal stops eating, drinking, grooming, or ambulating as a result of illness or disability due to the experimental procedures. Death as an endpoint must be avoided unless there is a compelling scientific rationale since it is assumed that there is significant pain or distress when an animal is in extremis.

Even though antibody and hybridoma production seems relatively painless and of relatively low stress, inappropriate use of adjuvants or use of the incorrect adjuvant can cause lesions and other problems among experimental animals. Investigators should choose techniques and adjuvants which exhibit the least irritating effects on the animals used. An information source for adjuvants and antibody production is available for consideration by investigators.

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