Special Considerations

Physical Restraint	Anesthesia Analgenics
Ventilation	Ascites Production
Surgery	Death as an Experimental Endpoint
Adjuvents	Euthanasia of Animals Used in Teaching and Research

Physical Restraint
Brief physical restraint of animals for examination, collection of samples, and a variety of other clinical and experimental manipulations can be accomplished with devices such as restraint boxes, squeeze chutes or catch pens. Such devices shall be suitable in size and design for the animal being restrained and operated properly to minimize stress and avoid injury to the animal. Any animal injured during restraint must be reported to the IACP at once.Restraint devices will be cleaned and disinfected after animals with infectious diseases have been in the device.

Restraint of animals for longer than four hours shall be avoided unless essential for research or teaching objectives and approved by the IACUC. Less restrictive restraint systems will be used when compatible with research objectives. Animals placed in restraint equipment for periods greater than one hour should be conditioned to such equipment prior to initiation of the research. This conditioning can be as simple as putting the animal in the restraint device for short periods of time before they are started on the research.

Attention must be paid to the possible development of lesions or illness associated with restraint including contusions, decubital ulcers, dependent edema, and weight loss. If these problems occur, veterinary care must be sought and provided for the animal and the animal shall be removed from the device immediately upon recommendation of the clinical or attending veterinarian.

Ventiliation
The Guide for the Care and Use of Laboratory Animals and the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching will be used to help determine ventilation requirements for research and teaching animals.

Strong animal odors in an animal room indicates inadequate ventilation. This requires a reduction in the number of animals, more frequent cleaning of cages, or eventually improved ventilation.

Surgery
Major Multiple Surgical Procedures - Multiple surgical procedures on a single surviving animal are discouraged. Multiple surgical procedures will only be approved when justified and related to specific components of a research project. This practice will be evaluated on an individual project basis by the IACUC. Cost savings alone is not an adequate reason for performing multiple surgical procedures on an animal.

For rodents (rats and mice), a clean uncluttered area will suffice as a surgery area. This must be capable of being wiped down with a sanitizing agent and kept clean.

A surgery suite for non-rodent vertebrate animals must be capable of being maintained as aseptic surroundings in which to do surgery.The surgery suite must consist of an induction prep area, surgeon scrub area, the surgery room proper, and recovery area. All traffic into the surgery room must be gowned, gloved, have shoe covers or dedicated shoes or boots, and masks.

The surgery facility must have a scavenger system in operation before inhalation anesthetics are used. Aseptic surgery suites for small animals are available in the ARF and Animal Sciences building and use can be scheduled through appropriate facility managers..

A Standard Operating Procedure (SOP) may be written and approved for the surgery, or details must be included in the Application to Use Animals form. There are many good tapes and books on operation of an aseptic surgery. These should be used as reference material.

Anesthesia Analgesics
Due to the explosive and flammable nature of ether and the carcinogenicity of chloroform, these chemicals will only be approved for use when nothing else will work and explosion proof hoods are available for safe use is in place.

Exposure to inhalation anesthetics constitutes a human health hazard; therefore, human exposure levels for volatile anesthetic (isoflurane) should be less than 2 ppm, and less than 25 ppm for nitrous oxide. Effective procedures must be employed to protect personnel from anesthetic vapors. "Guidelines for Anesthesia in Research and Teaching" is available from IACP.

Adjuvants
Improper use of Freund's adjuvant causes inflammation, induration or necrosis of tissue in animals. The IACUC has adopted the following guidelines intended to eliminate, or reduce to a minimum, animal discomfort associated with the use of this agent in research. Departure from these guidelines will require adequate justification to the Committee.

• Before using Freund's complete adjuvant (FCA), consider the use of Freund's incomplete adjuvant (FIC) or preferably another adjuvant.

• FCA should be used only for the first (priming) antigenic dose. Use of two or more doses of complete Freund's adjuvant is rarely warranted, and requires written scientific justification. The interval of at least 3 weeks must be allowed between doses.

• Injections containing FCA should be given subcutaneous only. Intraperitoneal, interdermal, intramuscular, or intraveneous use of FCA is not allowed and usually result in permanent lameness. Intraperitoneal injections produce peritonitis. Intravenous causes painful death.

• Intravenous injections can damage the lungs by rapid embolism. Use of footpad injections will not be approved unless the committee is convinced that footpad injection is the only way to immunize the animals or conduct the test.

• For subcutaneous injections, the inoculums containing FCA should be divided into fractions so that no more than 0.1 ml is injected per site for rabbits and 0.05 ml for mice, and that injections are at least 2 cm away from other injection sites.

• Injection sites should be clean and free of debris and contamination likely to result in infection, but do not require surgical site preparation.

Ascites Production
High doses of pristane injected intraperitoneally to induce peritoneal fluid production are associated with weight loss, hunched appearance and lethargy. When clinical signs are observed, the volume injected should be reduced to the minimum necessary to produce ascites.

The rate of ascites fluid production is variable; therefore, animals must be observed daily and the peritoneal fluid drained as necessary to prevent excessive accumulation and resultant pain or distress. A maximum of three abdominal taps of ascites fluid will be allowed in any single animal.

The following general guidelines should be followed:

• ascites fluid should be collected before the abdomen reaches the size of a near-term pregnant animal

• no more than two collections made per animal before euthanization during the third collection.

• the fluid should be withdrawn using a 21g or smaller needle, preferably on an anesthetized animal

• an individual animal must be euthanatized if its condition begins to deteriorate (piloerection, emaciation, lethargy or restricted movements or before the third and final collection).

Administration of saline subcutaneously or intraperitoneally after collection of ascites may help prevent shock.

Death As An Experimental Endpoint
Death is not a priori an acceptable experimental endpoint. In studies where death would be the anticipated outcome of an experimental treatment, investigators are required to define a point short of death where the animal may be humanely terminated, prior to its experiencing severe distress. Investigators must clearly justify and document any protocol in which animals must die as a direct result of experimental treatment, other than euthanasia. The written statement should include, but not be limited to, an explanation of why alternative experimental designs could not be used, procedures that will be followed to ascertain whether animals are in serious distress and steps taken to alleviate this condition, including possible euthanasia. Documentation might include appropriate journal articles, letters from knowledgeable colleagues, editorial reviews, etc.

Euthanasia of Animals Used in Teaching and Research
For the purposes of this document, euthanasia is the procedure of humanely killing animals rapidly and painlessly. It should be carried out by trained personnel using acceptable techniques in accordance with institutional policies and acceptable regulations. Techniques for euthanasia should follow current guidelines established by the American Veterinary Medical Association (AVMA) panel on Euthanasia (JAVMA, March. 1, 2001). For animals not included in the AVMA guidelines or for more detail on euthanasia, contact the IACP.