Breeding
Blood/tissue collection
Death as an endpoint
Behavioral studies
Administration of experimental agents
Major survival surgery
Antibody production/collection (monoclonal) using the ascites method
Use of hazardous agents
Multiple major survivor surgery
Antibody production/collection (monoclonal) without using the ascites method
Immunosuppression (chemical, irradiation, genetic)
Minor survival surgery
Antibody production/collection (polyclonal)
Tumor growth
Terminal surgery
Chronic disease studies
Transgenic/knockout mouse production
Other nonsurgical procedures
It is important to clarify with your IACUC department the definition of ambiguous or vaguely termed procedures. For example, our IACUC protocol makes clear the differences of the above types of surgery as well as provides additional references for surgical standards for non-rodent mammals in both survival and terminal surgery.
Relevance of Research
The IACUC will want to make sure that the sacrificing of animals will, of course, be intended for the overall benefits of human beings, other animals, or the advancement of scientific knowledge. In general, this should be brief and intelligible to the layman, take, for example, our description of the effects of shear stress on vascular endothelium and its relationship to the process of atherosclerosis and its role in the failure of bypass grafts:
“The force of the flowing blood, also known as shear stress, affects the cells lining the blood vessels (endothelium) in many complex ways. It is recently understood that the formation of atherosclerosis – hardening of the arteries – and proper healing of bypass surgery grafts may be affected by this shear stress. This laboratory attempts to understand the way in which shear stress acts on the cells of the blood vessel; specifically, we are trying to understand the cells’ signals that carry the message of shear stress within the cell. Ultimately, knowledge of these signals, and how they are deranged, will lead to prevention of atherosclerosis and longer working bypass grafts.”
This can often be quick and easy, as you likely have a detailed synopsis from a grant or research proposal handy. Keep it simple and you’ll save yourself rounds of revisions.
Animals and Their Care
Animals Requested
Detailed description of live, vertebrate animal(s) to be used in the protocol is required. Those details may include the scientific name of the animal; the common name of the animal; the strain or stock of the animal; the sex, age, and weight ranges to be used; if the animals used are genetically engineered in any way; and if the animals are to be used at more than one institution or on more than one protocol. Health and behavior are issues to consider. Ensure the animal is free from disease and that the animal is behaviorally suited for the research environment it will be subjected to.
Outlining Housing and Care of Animals
Depending on the institution, there may be already established housing and animal care facilities that are in keeping with federal requirements. It may be as easy as gaining access to your facility’s guidelines and becoming part of the community of animal researchers. If the facility is in such capacity, obtain this information early on and be knowledgeable of the regular proceedings to the housing and care of the animals you will be using. Use this information to your advantage when writing your proposal as to demonstrate basic understanding of the institution’s abilities and limitations.
Any uncommon care requirements or housing needs should be stated clearly. Have knowledge of the facility requirements prior to writing the proposal. For example, a satellite area or facility that may be used to house extra animals or for postoperative care and observation should be clearly outlined. Which staff and to what capacity that staff will be attending to the care of the animals in such remote places is important information to be included. Be detailed in this information; provide a formal standard operating procedure document to justify the need for alternative housing and care. The feeding schedule and type of food, cage type and size, cleaning schedule, veterinary care, and emergency intervention should be protocoled. A useful and important element to this care is implementing a daily log of activities to document proper care on a daily basis. A logged record will keep IACUC satisfied and, as importantly, maintain compliance with federal regulations.
Justification of Animal Quantity
The estimated total number of animals to be used during the project should be listed. If this is to change significantly, update the protocol with the IACUC. Describe the basis for how the total number of animals to be used was determined. You may be asked to provide specifics to how your experiments will be carried out, including the experimental and control groups, multiple time points, variables, and how all of this fits into the grouping and the size of the groups. See the example below:
“The majority of our experiments will be performed on rats and mice that are not genetically modified. These experiments will be performed on Fischer 344 rats that are predominantly aged (22–24 months), with a smaller number of younger rats (6 months) for control. Similarly, mouse experiments will generally be performed on aged mice (18 months) with a smaller number of experiments on younger mice (6 weeks) for control.
We estimate using 220 animals for the initial year. For training purposes, 10 animals will be needed to assure excellent operative technique, thus minimizing vein graft thrombosis and need to repeat surgeries. In addition, these animals can have tissue harvested for the control group (time 0), thus optimally using all animals. We have four groups (control, vein graft, low flow, high flow) of 20 animals each: 5 animals will be used for morphometric analysis of neointimal hyperplasia and immunohistochemistry at 1 week after implantation, 5 animals will be similarly used for 3 weeks, and 10 animals will be used to harvest tissue for reverse transcription polymerase chain reaction (RT-PCR) (n = 5) and protein (n = 5) extraction at 3 weeks. Group sizes were determined by power analysis (omitted here). We will want to perform these experiments with and without RNA interference (RNAi) inhibition. Thus 10 + (4 × 20 without RNAi) + (4 × 20 with RNAi) = 170 animals. In addition, we will perform control analysis on a smaller number of young animals; 10 animals for time 0, 20 without RNAi, and 20 with RNAi. Thus we estimate 170 + 50 = 220 animals per year.
After the vein graft group (year 1), additional experiments will be performed with arteriovenous fistula (AVF) (year 2), and angioplasty (year 3). Thus we will need 220 animals for each of these additional 2 years, i.e. 220 + (2 × 220) = 660 animals over all 3 years.”
A detailed description does not equate to a well-designed study, however. Statistical justification should be used when appropriate. Getting meaningful data with as few animals as possible is the aim, especially to avoid needing to repeat procedures or use extra animals. It may be helpful to seek advice from your institution’s biostatistician before starting to help with power analysis and the statistical methods to be used in analysis once data is obtained.
Justification for use of these animals, weighing the predicted scientific or educational value of the project against the potential detrimental effects on the well-being of the animals, is ultimately paramount to all other considerations. Demonstrate that investigators involved in the project will show responsibility to all matters relating to the well-being of the animals and vow obligation to treat the animals with respect. The more knowledge about the project that is revealed will show the investigators’ awareness of potential adverse effects to the animals being used and will ease the investigators’ ability to demonstrate ways to minimize these effects, to give specifics on monitoring and assessment of the animal well-being and appropriate actions to be taken if adverse effects are observed.
Pain Management and Special Circumstances
USDA Pain Category Assignment
The United States Department of Agriculture (USDA) has established guidelines for pain management for research protocols. Defined as “any procedure that would reasonably be expected to cause more than slight or momentary pain or distress in a human being to which that procedure is applied, that is, pain in excess of that caused by injections or other minor procedures” [2]. Each procedure involved in each specific animal in the proposed project must be classified based on these guidelines in Table 13.2 for IACUC documentation.
Table 13.2
United States Department of Agriculture (USDA) pain classification
USDA category | Category B | Category C | Category D | Category E |
|---|---|---|---|---|
Description | Used for breeding or holding animals where no research is being conducted | Procedures, routine injections of nontoxic, nonirritating substances, or venipuncture that produces minimal, transient, or no pain or distress | Procedures that would cause more than minimal or transient pain and/or distress but are performed using appropriate anesthetics, analgesics, or tranquilizers | Procedures that would cause more than minimal or transient pain and/or distress but cannot be performed using appropriate anesthetics, analgesics, or tranquilizers without adversely affecting the study |
Examples | Breeding colonies, newly acquired animals | Observation of animals under normal conditions; euthanasia for tissue harvesting; positive-reward studies. Ear punch biopsies or tail snips for genotyping | Diagnostic procedures; non-survival surgical procedures; any post procedure outcome resulting in pain, discomfort, or distress. Minor survival surgical procedures | Toxicological or microbiological testing; application of noxious stimuli; infliction of burns or trauma; extreme environmental conditions; any procedure for which anesthetics, analgesics, or sedatives must be withheld for study purposes |
Adapted from Yale University, Institutional Animal Care and Use Committee [8]
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