According to the Animal Welfare Act, Subchapter A, 2.31 "The Principal Investigator has considered alternatives to procedures that might cause more than  momentary or slight pain or distress to the animals, and has provided a written narrative description of the methods and sources. e g., the Animal Welfare Information Center, used to determine that alternatives were not available. 

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Replacement

Replacement involves seeking alternatives that involve as little pain and distress, if any, as possible within the constraints of the project's goals. The most common replacement involves switching to a species lower on the phylogenic scale (e.g. dogs to mice).

Occasionally, computer simulation, chemical or mechanical models, or in vitro laboratory studies can supplant an animal model altogether. Investigators are expected to demonstrate their efforts to find reasonable alternatives through comprehensive literature searches and consultation with colleagues and other sources.

Reduction

Investigators are obligated to minimize the number of animals that may experience pain, distress, or discomfort and must justify these numbers upon application to the IACUC. The project should obtain the most information possible from the fewest number of animals. Methods of reduction are characterized by the following:

Appropriate statistical methods in the design of the study and the analysis of the data collected:

• Performing pilot studies to minimize potential complications in the project;
• Use an appropriate species to maximize data return;
• Consult a statistician in an effort to determine the minimum number of animals needed to show statistical significance;
• Use animals as their own controls;
• Reduce experimental variability by using animals of defined genetic or microbiological status;
• Control for factors such as stress, diet, housing, etc. that may affect results.
• Ensure, through comprehensive literature searches and consultation with colleague and other resources, that experiments are not unnecessarily duplicated; and
• Share tissues, blood, etc. or live animals with other investigators provided the sharing does not increase pain or distress caused to the animal.

Refinement

Once the basic study design is formulated, investigators should refine the protocols used, making every effort to minimize any potential pain or distress and provide appropriate means of management. Refinement includes, but is not limited to, the following:

• Proper assessment of potential pain and distress, including both behavioral signs (based on the animal's normal behavior patterns) and to physiological parameters (e.g. plasma cortisol and catecholamine levels, white blood cell counts, cardiovascular parameters).
  When in doubt, investigators should assume that the animal is in pain and take steps to alleviate it.
• Responsible use of anesthetics, analgesics, and sedatives when appropriate;
• Setting appropriate endpoints for the experiment or stages of the experiment;
• Using general techniques to reduce stress and minimize complications (e.g. proper handling and restraint methods, good surgical technique /post-operative care); and
• Seeking additional training for unfamiliar procedures or techniques to be used.