Protocols for Animal Research

How to Submit a Protocol

1. Protocols requesting approval to use animals in research are submitted on-line using the University Research Compliance Office website located at: http://www.ksu.edu/research/comply/

2. Specific detailed instructions for the completion of the forms can be found on that web page.

For questions regarding completion of the form, or to inquire about the status of a protocol that is currently under review, please contact the URCO office at 532-3224, or by email at comply@ksu.edu

Protocol Review Process

1. The ACUC meets twice each month on the first and third Thursday. Protocols for each meeting's agenda must be received by the close of business on Friday two weeks before the scheduled meeting. This submission deadline is necessary to process the proposal, distribute it to committee members, and arrange for review.

2. The following procedures do not require an animal protocol

   1. When animal tissue is obtained from another investigator who is conducting research with animals under an approved protocol.

   2. Experiments involving eggs or embryonic birds

   3. Experiments involving invertebrates

ARF Policies for Specific Procedures

Euthanasia as an alternative to death as an end point

1. Legal, regulatory, and moral guidelines require that animal pain, distress, and suffering be minimized in any experiment. For these reasons animals should be euthanized in death-end-point experiments prior to their actual death if experimental validity will not be compromised. If death is a required end point, the investigator may receive approval to conduct such studies by providing appropriate scientific justification in writing to the IACUC. The KSU IACUC has developed a policy covering this issue, which can be viewed at: http://www.ksu.edu/research/comply/iacuc/research/iacuc01.html

2. The scientific rationale should answer the following questions: What alternatives were considered and why can you not use morbidity instead of death as an endpoint? Can pain-relieving measures be used, and if not, why not? How many animals will be used this way and how is this the minimal number required? What additional information will be gained in the interval between morbidity and death? How will animals be monitored and how will this be recorded? What arrangements are available for prompt removal of dead animals?

3. In all cases investigators are expected to continue to monitor experimental animals at least twice daily (including weekends and holidays), to euthanize any animals which they judge to be moribund, to use alternative end points to death when possible, and to minimize animal numbers within statistical constraints in general, but especially in death end-point protocols. Investigators are also expected to provide the IACUC with a sample of the pain and distress form that they plan to use to record observations and actions. These forms will be posted near the animals in the animal room for inspection by the ARF veterinary staff.

4. The following are signs and symptoms for judging morbidity (disease/illness) and moribund condition (state of dying) in animals:
   Morbidity

   1. Rapid breathing rate

   2. Breathing rate very slow, shallow, and labored (preceded by rapid breathing)

   3. Rapid weight loss

   4. Ruffled fur (rough hair coat)

   5. Hunched posture

   6. Body temperature less than 30°C (hypothermia)

   7. Hyperthermia

   8. Ulcerative dermatitis or infected tumors

   9. Anorexia (loss of appetite)

   10. Diarrhea or constipation

   Moribund Condition
   Signs and symptoms for morbidity plus:

   1. Impaired ambulation (unable to reach food or water easily)

   2. Evidence of muscle atrophy or other signs of emaciation (body weight is not always appropriate)

   3. Any obvious illness including such signs as lethargy (drowsiness, aversion to activity, lack of physical or mental alertness), anorexia (loss of appetite, especially when prolonged), bleeding, difficulty breathing, CNS disturbance, or chronic diarrhea

   4. Inability to remain upright

Multiple survival surgical procedures

1. Multiple survival procedures may be permitted only with prior approval of the IACUC. Cost alone is not an adequate justification for performing more than one survival surgery on an animal. The KSU IACUC has developed a policy covering this issue, which can be viewed at: http://www.ksu.edu/research/comply/iacuc/research/iacuc07.html

2. Any proposed multiple survival surgery must be described in detail on the animal care and use form. The following are examples of multiple survival procedures that may be approved:

   1. Serial surgeries essential to the design of a given study.

   2. Entry into a body cavity which is done by a probe or needle, e.g. a *laparoscopic examination or needle electrode placement, which does not result in any injury likely to produce pain.

   3. Clinical procedures, which are not related to the research, project, but which may be necessary for the health and well being of the animal.

   4. Procedures, which are commonly performed multiple times in a clinical setting, such as multiple hysterotomies for delivery of the contents of a pregnant uterus.

   5. Species preservation such as commonly occurs with non-human primates and difficult to obtain animal models.

   6. Tumor reduction can be performed only on subcutaneous tumors and the procedures can only be done twice.

3. In all cases, the usages must be scientifically justified in the context of the overall purpose of the research project.

Major Procedures

1. Major procedures are those that enter a body cavity, or those that have the potential of inducing a handicap. For example, placement of a stent in the carotid through a percutaneous route does not enter a body cavity, but it does have the potential of inducing a carotid occlusion and causing a stroke. Thus it would be considered a major procedure.

2. Major procedures, in non-rodent species must be done in a dedicated sterile operating room, with a separate facility for preparation of the animal. Sterile surgical packs and sterile gloves are required for each procedure.

3. Rodent surgery and non-major survival surgery in non-rodents should be performed under aseptic conditions, meaning that: (1) the surgical area is clean (wiped down with a disinfectant (i.e., 70% ethanol, roccal, etc..), (2) fur is clipped from surgical site and skin is cleaned with a betadine solution or chlorhexadine solution, (3) instruments are sterilized either by autoclaving instrument pack or by soaking in a chemical sterilant (Instracal, Amerse) for at least 15 min, or by using a hot bead sterilizer and (4) the surgeon wears a mask and sterile gloves or latex gloves thoroughly cleaned with 70% ethanol, Alcare, or another germicidal agent.

Production of Monoclonal and Polyclonal Antibodies

1. The ARF provides an antibody production service for investigators on the KSU campus. ARF technicians are capable of producing monoclonal antibodies using ascites production in mice or polyclonal antibody production in rabbits or goats. The ARF maintains an IACUC approved protocol for antibody production. Investigators wishing to utilize this service should contact the ARF Director or Facility manager for additional details.

2. If a commercial company is making antibodies for you, you do not need an animal protocol. However, if you are using federal funds, you need to be sure that the company has an Animal Welfare Assurance Number.

Retro-Orbital Bleeding

1. Retro-orbital bleeding must be done with an anesthetic.

2. The frequency and number of times this procedure can be done will depend upon the merits of the proposal and the skill of the investigator.

Tumor Burdens in Animals
Although these standards refer specifically to rats and mice, they are applicable to any tumor bearing animals. The standards are as follows:

1. The maximum tumor burden on any one animal should not exceed an estimated 10% of the animal's body weight.

2. The maximum measurable total tumor burden on any one animal should not exceed one solitary mass of 1.5 centimeters in diameter on a mouse or 2 centimeters on a rat.

3. Multiple masses with a combined total diameter of 1.5 cm for mice and 2 cm for rats are permitted.

4. If an investigator requires tumor burdens larger than those specified above, or requires generation of partially necrotic or ulcerated tumors, a written scientific justification must be submitted to and approved by the IACUC.

5. Fluid neoplasia (e.g. hybridomas) and nonneoplastic ascites are addressed under hybridoma production standards.

Tagging Animals

Clipping of toes as a routine means of identification is not an accepted procedure. If investigators are unable to use ear notches, they should contact ARF to discuss alternatives.