Procedures Involving Pain or Distress

Current federal animal use legislation emphasizes the need for the minimization of pain or distress imposed on animals during the course of a research study. The methods used to prevent, relieve, or minimize this pain or distress must be stated by the principal investigator. Also, any animal use causing pain or distress, which cannot be relieved by anesthetics, analgesics, tranquilizers, or sedatives must be justified for scientific reasons by the principal investigator in the animal study proposal. All efforts should be made to find alternatives to these procedures and avoid using these procedures, unless absolutely necessary for the outcome of the study. For guidance contact the Lab Animal Sciences Program - Bethesda at 301-496-1866.

The following procedures and techniques may cause significant pain or distress:

  • Physical Restraint - Prolonged physical restraint of animals is distressful and may cause pain. To minimize distress, the period of restraint should be the minimum required to accomplish the research objectives. Animals placed in restraint equipment should be conditioned to the equipment prior to the start of the study and physically monitored throughout the restraint period.
  • Deprivation - Deprivation of food or water, or feeding a diet deficient in an essential nutrient can significantly interfere with the normal physiology of an animal and is considered stressful. Fasting or water deprivation for 24 hours or less for most species is tolerable and should not have a detrimental effect. (Also see ARAC Guidelines for more information on Guidelines for Diet Control in Behavioral Studies)
  • Death as an Endpoint - In the past, radiation, toxicologic, or tumor research studies used the death of an animal as a necessary endpoint for data interpretation. This endpoint can result in unnecessary pain and distress, which may not be defendable. Alternative endpoints for studies with potentially lethal outcomes have been developed and have been shown to be scientifically sound. When death as an endpoint is required scientifically, all efforts should be made to alleviate the pain or distress by the pre-moribund use of analgesics, anesthetics, tranquilizers, or sedatives. When relief of pain or distress is not possible, it must be justified in the animal study proposal. (Also see ARAC Guidelines for more information on Endpoints in Animal Study Proposals)
  • Use of Complete Freund's Adjuvant (CFA) - Complete Freund's Adjuvant, a water-in-oil emulsion containing killed Mycobacterium butyricum, is used to potentiate immunologic responses to antigens. CFA by its nature, can cause severe pain, abscess formation, fever, and permanent organ damage. These side effects can be reduced or eliminated by using appropriate routes of administration, adequate separation of injection sites, and small volumes of inoculum per site. Favorable results with minimal side effects have been obtained by using 0.05 ml intradermally in rabbits, 0.1 ml subcutaneously in rodents, and up to 0.5 ml intramuscularly per site in large farm animals. CFA is necessary for only the initial immunization. Subsequent immunizations should be done using incomplete Freund's or other such adjuvants. Non-inflammatory adjuvants or adjuvants known to produce less intense inflammatory responses, yet capable of eliciting a humoral response, should be considered whenever possible. (Also see ARAC Guidelines for more information on Recommendations for Consideration in the Research Use of Inflammatory Agents - Complete Freund's Adjuvatn (CFA)
  • Multiple Major Survival Surgery - Multiple major survival surgeries on a single animal are discouraged. However, there are instances when investigators will have a scientific need for the performance of multiple major survival surgical procedures. Such procedures must be described in the protocol and scientifically justified.

Other procedures that may cause significant pain and/or distress:

  • Use of noxius stimuli.
  • Skin or corneal irritancy testing.
  • Allowance of excessive tumor burden.
  • Intracardiac or orbital sinus blood sampling.

The following procedures or techniques when performed properly by skilled personnel are not considered painful or distressful, but certain standards should be followed when these procedures are undertaken (For guidance contact the Lab Animal Sciences Program-Bethesda at 301-496-1866):

  • Ascites Tumors for Monoclonal Antibody Production - In view of the wide availability of in vitro methodology, the use of animals for monoclonal antibody production should be justified in the animal study proposal. Animals should be observed daily and relieved of abdominal distension as necessary, or euthanized. Ascitic burden should not usually exceed 10% of normal body weight in rats and mice. Animals which become moribund, cachectic, or are otherwise unable to obtain feed or water must be euthanized. Scientific studies have determined that 0.1 ml of Pristane for peritoneal cavity priming is as effective as 0.5 ml pristane with less side effects observed. Ascites fluid should be harvested in vivo once with recovery. Subsequent harvesting should be terminal.
  • Subcutaneous Tumors - Animals with subcutaneous tumors must be observed daily after observable tumor growth is noted. Animals should be euthanized when tumors become large or when they otherwise become moribund, cachectic, or are unable to obtain feed or water. Alternative endpoints should be used in studies that could lead to lethality.
  • Multiple Site Tumor Inoculation or Metastasis - Euthanize when symptoms become clinically significant, i.e. abdominal distension or organ system symptomatology such as labored breathing and neurologic signs. Animals which become moribund, cachectic or are otherwise unable to obtain feed or water must be euthanized. Alternative endpoints should be used in studies that could lead to lethality.
  • Blood Collection - Blood collection is the most common interventional procedure conducted on laboratory animals and is an essential component for many studies. When animal survival is required following a blood collection procedure, no more than 10 % of the animal's blood volume should be collected at any one time. Replacing lost blood volume can reduce the physiologic stress associated with blood loss. When multiple samplings are required, a bi-weekly limit of 7.5 % of blood volume is recommended. Blood collection parameters are provided for the mouse, rat, hamster, and rabbit.
  • Parenteral Injections - Should be performed with knowledge of the chemical and physical characteristics of the injectate. Consideration should be given to the pH, viscosity, concentration, sterility, pyrogenicity, and the presence of other noxious substances. Failure to do so may result in harmful reactions. Excess injectate volume can be painful and cause unnecessary tissue damage. Maximal injection volumes for mice, rats, hamsters, and rabbits vary with the route of administration.
  • Euthanasia - Euthanasia must be performed by trained personnel using appropriate techniques, equipment, and agents in order to affect a death, satisfactory to both research requirements and humane considerations. The humaneness of any euthanasia method depends on the rapid onset of unconsciousness and its maintenance until death. Upon completion of the procedure, confirmation by either ascertaining cardiac arrest or noting an animal's fixed and dilated pupils is necessary. Euthanasia should not be performed in the animal room or in the presence of other animals. A summary of the 2000 Report of the AVMA Panel on Euthanasia provides suggested euthanasia methods for laboratory animals. Click here for more information. Also see Guidelines For The Euthanasia of Mouse and Rat Fetuses and Neonates.
Transgenic Animals - Animals exhibiting phenotypic expressions which result in undue pain and distress must be euthanized. Examples of such symptomatology include: inability to obtain food or water, moribundity, cachexia, necrotic tumors, tumor burden exceeding 2 cm diameter in mice, or signs of severe organ or systemic involvement. If, for scientific reasons, euthanasia is not elected, the principal investigator must notify the Animal Care and Use Committee in writing, defining what measures will be taken to provide relief to the animals.