Welcome to the "Working with the IACUC" web-based course for non-VA staff. This course is designed primarily for staff performing research that involves the use of laboratory animals and IACUC members, but veterinarians, senior husbandry staff and anyone interested in learning more about how animal research must be conducted will likely find the course materials useful as well. If you are working at a VA Medical Center, you may be required by your adminstrator to take the VA version of the course and exam instead. If offered by your institution, the VA version of this course will appear in the course/exam menu.

This course was funded by the VA Office of Research and Development as part of the "Working with Laboratory Animals" instructional series, with production assistance by the VA Employee Education Service.

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This course is designed with the following specific goals in mind:

1. Help investigators fill out animal protocol forms and interact effectively with the Institutional Animal Care and Use Committee (IACUC);

2. Provide a review of basic issues with which IACUC members must become familiar to effectively review animal research proposals;

3. Provide information on important issues that must be addressed in a typical animal protocol form; and

4. Provide training required by the USDA Animal Welfare Act Regulations and Public Health Service Policy.

As you go through this program, you will not be reading many regulations or guidelines. However, on occasion you may want to know the actual regulatory basis for some of the material presented. To look at the actual regulatory language, just click on the underlined hypertext. The relevant regulatory text will be shown to you in another smaller window, and you can go on to view the actual agency web site if you wish. As an example, click on the underlined text in this sentence.

Self-assessment questions appear at the end of most lessons. Your responses to the questions are not recorded or tallied- the purpose of these questions is to help you retain the information presented in the course. After you have clicked on the correct answer(s), click on the "Check Answer" button below the question and responses. A green check will appear beside correct answers. Incorrect answers display a red "X".

Important: to get credit for this course, you must take and pass the exam (remember that the self-assessment questions in the course are not scored). To take the exam covering this course at any time, click on the Courses and Exams link above and to the right and choose the exam for this course.

Some of the questions at the end of the lessons will randomly appear in the final 20-30 question comprehensive exam. You must score around 85% to pass the exam. If you do not pass, you may take the exam again as many times as you wish, but the questions are randomly drawn from a large pool of questions, so the exam will change each time.

Once you pass the exam, you will be able to print a completion certificate using the printer connected to your computer. Make sure you submit a copy to your IACUC office for inclusion in your IACUC files. You may print out additional certificates any time you need them.

By law, an institutional committee must review all aspects of the animal care and use program. This committee is most commonly referred to as the "Institutional Animal Care and Use Committee", or IACUC.

Your institution probably has its own IACUC, but smaller institutions sometimes use the IACUC of an affiliate instead of having one of its own. Check with research administrators to make sure you know where to submit your animal protocol forms.

There are at least three people you should get to know when you are planning animal experiments.

• The first is the IACUC chairperson or alternately, the IACUC executive secretary or director. The IACUC is an invaluable source of information to help you obtain approval for your planned experiments.
• The second is the institutional veterinarian, who can help you plan experiments and provide medical and procedural guidance.
• The third is the animal facility manager, who can help you make arrangements to order and house animals after you receive IACUC approval.

The IACUC is responsible for making sure that all federal laws, regulations and policies are followed when investigators perform animal research. The IACUC has many jobs. Some of them include:

1. Reviewing and approving animal use protocols submitted by investigators.
2. Monitoring the animal care and use program by conducting thorough reviews of the program and inspections of the animal facilities semiannually.

Federal regulators regard the IACUC as an essential partner in ensuring compliance with animal welfare laws and guidelines. In effect, the IACUC functions as the self-regulating body for animal research on behalf of the institution. Because the IACUC is such a critically important component of the animal care and use program, evaluating the effectiveness of the IACUC often serves as a barometer of the quality of the entire animal care and use program.

An effective IACUC protects both the individual investigator and the institution, while inspiring confidence in the general public that animal research is being performed in an ethical manner. Research utilizing animals is a privilege, not a right, and the IACUC must do its part to make sure that animal research is performed according to the highest standards. By assuring that animal research complies with animal welfare laws and guidelines, the IACUC ensures that animals are not subjected to unnecessary pain and distress, and protects both the investigator and the institution.

The entire system is built on trust. However, a single incident of serious noncompliance with animal welfare regulation or guidelines can jeopardize the entire institution's privilege of conducting animal research.

In this course, you won`t be faced with large chunks of regulatory language, but you must be aware of the basic laws and guidelines that govern animal research in the United States. We will hit the highlights of the most important of these laws and guidelines.

The first important agency regulating animal research is the United States Department of Agriculture (USDA). Congress gave the USDA broad authority to regulate animal research when it passed the Animal Welfare Act. The USDA then established the regulations to enforce the Animal Welfare Act. These very detailed enforcement documents are known as the USDA Animal Welfare Act Regulations, and take precedence over regulatory documents produced by all other agencies.

The USDA has published a Policy Manual that clarifies how some of the language in the USDA Animal Welfare Act Regulations should be interpreted.

What institutions are covered by the USDA Animal Welfare Act Regulations? The Animal Welfare Act instructs the Secretary of the USDA to regulate any institution that fits the following criteria:

1. Uses live animals in research, tests, or experiments, and

2. Purchases or transports live animals in (interstate) commerce OR receives funds under a grant, award, loan, or contract from a department, agency, or instrumentality of the United States for the purpose of carrying out research, tests, or experiments.

Thus, the scope of the Animal Welfare Act is based upon the authority of the federal government to regulate interstate commerce, and its responsibility to make sure funds provided for animal research and testing are used appropriately. Dealers who sell animals are covered by the Animal Welfare Act Regulations, as are exhibitors such as traveling exhibits, carnivals, and zoos.

To what animals do the USDA Animal Welfare Act Regulations apply? Here is the definition of an animal given in the regulations:

"Animal means any live or dead dog, cat, nonhuman primate, guinea pig, hamster, rabbit, or any other warm blooded animal, which is being used, or is intended for use for research, teaching, testing, experimentation, or exhibition purposes, or as a pet. This term excludes: Birds, rats of the genus Rattus and mice of the genus Mus bred for use in research, and horses not used for research purposes and other farm animals, such as, but not limited to livestock or poultry used or intended for use for improving animal nutrition, breeding, management, or production efficiency, or for improving the quality of food or fiber. With respect to a dog, the term means all dogs, including those used for hunting, security, or breeding purposes."

The second important agency involved in regulating animal use is the Department of Health and Human Services, which is the home of the Public Health Service (PHS). Passed by Congress in 1985, the Health Research Extension Act directed PHS to provide guidelines for animal research. The Office of Laboratory Animal Welfare (OLAW; formerly known as "OPRR", or Office for Protection from Research Risks) is responsible for monitoring institutional compliance with PHS policy and guidelines. OLAW relies primarily on two documents for judging compliance, both of which are very important to animal research.

The first, fairly brief one, is the PHS Policy on Humane Care and Use of Laboratory Animals. It incorporates nine U.S. Government Principles For The Utilization And Care Of Vertebrate Animals Used In Testing, Research, and Training that must be considered when institutions receive support from U.S. Government agencies.

The second, lengthier document is the Guide for the Care and Use of Laboratory Animals (usually called the Guide). These two documents together provide important information sometimes collectively called "PHS Policy." Compliance with PHS Policy is a required condition for receiving PHS support for activities involving vertebrate animals.

Institutions that accept any PHS agency research funding must agree to follow PHS Policy for animal research. PHS agencies include the National Institutes of Health (NIH), the Centers for Disease Control and Prevention (CDC), and the Food and Drug Administration (FDA). Normally, if an institution does not accept any such funding, it is not subject to PHS guidelines, but many institutions not technically subject to PHS Policy follow it because it strongly promotes effective and ethical animal research programs.

PHS Policy covers all vertebrate species used for research, teaching, and testing. This is in contrast to the USDA AWA Regulations and Standards, which are not currently applied to laboratory mice and rats, birds, and animals used for agricultural research.

There is another organization that has played a critical role in ensuring high quality animal care and use in the U.S., but it has no regulatory authority whatsoever. The Association for Assessment and Accreditation of Laboratory Animal Care, International,or "AAALAC", is a non-profit organization that accredits animal facilities. Institutions participate in the AAALAC accreditation program by voluntarily submitting to AAALAC site visits every three years. AAALAC uses the USDA AWA regulations as well as PHS guidelines, plus other reference documents for evaluations. If an institution meets all applicable standards, then it is awarded AAALAC accreditation, a valuable symbol of institutional commitment to quality care and use of animals.

Participation in the AAALAC accreditation program is strictly voluntary. However, AAALAC accreditation has now become an accepted standard for quality animal research programs.

For research animals housed in agricultural settings such as farms, another guide, the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching, is often used. The recommendations in this so-called "Ag" guide are more applicable to agricultural settings than some of the recommendations in the Guide for the Care and Use of Laboratory Animals, (referred to as the Guide in this course). AAALAC often uses the Ag guide for evaluating animal care in farm settings.

The USDA also accepts the standards in the Ag guide for research animals housed in agricultural settings.

Keep in mind that biomedical research animals can be housed in farm settings, but a farm setting may not be appropriate for all animals. For instance, animals undergoing major surgery or other invasive procedures might do better in a traditional indoor facility, primarily because the "farm" environment is usually not as clean and as easily controlled.

Because it is so important, let`s emphasize the following recurring theme in all modern regulatory documents impacting animal research: Whether you are performing research or testing on animals, or using animals for teaching, you must receive IACUC approval before any use of animals begins.

Consulting with the veterinarian is an important part of filling out the animal forms. The USDA Animal Welfare Act Regulations stipulate that if procedures on animals are proposed that may cause more than momentary or slight pain or distress to animals, consultation with the attending veterinarian or his or her designee (a veterinarian with training or experience in laboratory animal medicine) must occur in the planning of those procedures. This is a good time to get advice on the latest medications and anesthetics, as well as tips on new procedures and ways to reduce pain or suffering. However, the AWA Regulations do not specify how the consultation has to be conducted.

In practice, different institutions meet this requirement in different ways. The consultation can be in the form of an actual meeting or a phone consultation between the principal investigator and the veterinarian, or it can be in the form of a review of the animal forms by the veterinarian before the IACUC meeting so that revisions to the animal form can be made prior to IACUC review.

You may be asked in your animal protocol form to name the veterinarian consulted and when. This documents compliance with the mandatory consult for the IACUC. Because of the benefit to the investigator, the animals, and the IACUC (in the form of "cleaner" animal protocol forms and less time spent correcting problems), many institutions require a veterinary consult during the planning stages for all projects involving animals, whether or not proposed procedures will cause more than momentary or slight pain or distress. Check with your IACUC or veterinarian to determine if you need a veterinary consultation for your particular project.

Some of the first items on an animal protocol form are often deceptively simple ones, such as "How will the proposed use of animals improve the health of people or animals?" and "What is the experimental design of the animal studies planned?" Although simple questions, it is very important to answer them completely to provide the IACUC with adequate information. Let's take them one at a time.

" How will the proposed use of animals improve the health of people or animals?." This question or a variant appears in almost all animal protocol forms. This is a very important issue because you are asking for the privilege of using animals for procedures that rarely will benefit them individually, and might result in their death. In general, there must be a compelling potential for benefit to human or animal health to warrant the use of animals. Points to consider:

1. If you are studying a human or animal disease or health concern, it is helpful to carefully explain the disease, what causes it, what therapy is currently used to treat it, and how the proposed animal experiments might better prevent human or animal pain and suffering.
2. Because there are non-scientists on the IACUC, in at least part of your response you should use language that a high school student would understand. Your response should be written so that members of the general public (including the lay member on the IACUC) would readily understand why it is important to use animals for your work.
3. Make sure you explain medical terms, and define abbreviations the first time they are used.

Another important question on protocol forms is "What is the experimental design of the animal studies planned?" In responding, keep in mind that the IACUC needs to understand the proposed use of animals. To perform an appropriate review of your proposed animal work, IACUC members must understand what combination of procedures will be performed on individual animals. Details of procedures such as surgery and euthanasia are usually requested elsewhere, but the general types of surgery and the final disposition of the animals should be clear from your response. Keep these points in mind:

1. For more complex experiments it is very helpful to provide a flow chart to make the experimental design clear.
2. The description of the animal procedures should stand by itself. The IACUC should not have to read another document such as a grant application to understand what you propose.
3. Once again, define all abbreviations the first time they are used to facilitate comprehension.
4. Do not use technical language that only specialists in your field would understand. Not only is it difficult for trained professionals to navigate through technical jargon outside their fields, by law, at least one member of the IACUC must be a lay member who is not a scientist.

Somewhere on your forms you will also have to justify your choice of species.

The central theme evaluated by the IACUC is this - assuming that animals are indeed necessary, the least sentient ("aware") species capable of providing the needed data should be used. The hierarchy of sentient species can be a subject of disagreement, but generally it goes as follows:

• Apes (chimps, orangutans, gorillas)
• Monkeys (baboons, rhesus monkeys, marmosets, tamarins)
• Larger animals commonly kept as pets such as dogs and cats
• Larger animals such as pigs and goats commonly used as farm animals
• Rabbits
• Rodents (guinea pigs, hamsters, rats, mice)
• Non-mammalian vertebrates (poultry, amphibians, reptiles, fish)
• Invertebrates (crustaceans, slugs, insects, arachnids, worms, etc.)
• Single cell organisms (yeast, bacteria, etc)

Justifications for using a particular species may include:

1. The presence of previous work in the biomedical literature that validates the use of a particular species in an animal model of a human disease.

2. The existence of a large body of previous laboratory data that would have to be repeated if another species was used instead.

3. Characteristics of the species that render it uniquely suited to the proposed research.

4. Size, availability and cost.

5. Availability of reagents or research tools unique to that species.

Cost savings alone is not an adequate justification for using a particular species! The justification should be based on sound scientific reasoning.

You will also be asked to request a certain number of animals, and justify why you need that number. The IACUC realizes that it can be difficult to provide such information in advance, but the law requires them to review the number of animals to be used.

Some important points:

According to the Guide, a statistical analysis should be used to justify animal numbers whenever possible. Commonly a power analysis is the most appropriate tool for justifying group sizes. Although it is always wise to consult a biostatistician, the VA will provide a software "wizard" for animal studies and a basic statistics web course to help you complete basic power analyses in 2005.

Surprisingly, you might even find out that you need to ask for more animals per group than you thought would be necessary.

It is acceptable to ask for animals that will be used to perfect surgical or other techniques prior to initiating planned experiments. This is preferable to beginning a large experiment that will experience technical problems that might cause pain or distress to the animals.

Studies on cadavers from other approved protocols in advance of any procedure on a live animal are strongly encouraged. By doing this, techniques can be perfected as much as possible before any live animals are used.

It is also acceptable to ask for animals that will be used in pilot experiments in addition to animals requested for more robust experiments. Pilot experiments can be used to perfect technique, demonstrate feasibility, or provide a justification for proceeding with larger, more tightly controlled experiments.

Now is a good time to address the very important concept of alternatives. We've already touched upon it during the discussion of the importance of using the least sentient animal species possible, and during our discussion of justifying animal numbers requested.

In 1959, two British scientists, William Russell and Rex Burch, urged all animal researchers to follow a policy of minimizing animal pain and use. They described three important concepts now known widely as the "three R's":

The purpose of these concepts is to minimize animal use and pain or distress while still achieving the critical scientific objectives that lead to advances in health and medicine.

The first "R" is replacement. Replacement is simply replacing the use of animals with non-animal techniques. Examples of non-animal techniques that can sometimes replace animals include:

- Computer models.

- Cell culture or tissue culture systems.

- In vitro assays.

Unfortunately, it is not very common for any of the above alternate systems to adequately replace animals in experiments. However, it does happen, and consideration should always be given to non-animal systems.

Practical examples of "replacement" include the use of cell culture techniques to replace animals as incubators for cell lines, the use of immunologic bench assays to replace bioassays involving animals, and the use of computer software to model the pharmacokinetics of drugs in place of animal studies. Later on, we will discuss the important issue of using in vitro techniques instead of animals to grow monoclonal antibodies- another example of replacement.

Some people also consider the use of less sentient animal species (discussed last lesson) to be another example of replacement, but others consider such use to be "refinement" instead (the third "R").

The second "R" is reduction. Reduction is simply reducing the number of animals used. Examples of reduction include:

- Limiting group sizes to the minimum needed to obtain statistically significant data.

- Performing multiple experiments simultaneously so that the same control group can be used for all the experiments.

- Sharing tissues with other investigators so that additional animals are not needed.

- Designing experiments so that animals serve as their own controls.

- Using newer instrumentation that improves precision and reduces the number of animals needed per data point.

Reduction is usually more feasible than replacement. However, when considering how to reduce animal use, you must find a balance between causing more pain or distress on fewer animals and causing less pain or distress in more animals. For instance, if an investigator proposes to double the number of invasive surgical procedures on animals so that fewer animals are used, the increased pain and distress experienced by the remaining animals may not be justified by a simple reduction in animal use. This is a difficult area, and you should seek advice from your veterinarian and IACUC as needed.

The last "R" is refinement. As opposed to reducing the number of animals used, refinement refers to changing experiments or procedures to reduce pain or distress in those animals that must be used. Refinements in anesthesia, surgery, analgesia, and many procedures occur frequently.

Examples of refinements include:

- New anesthetics that allow rapid induction and reduced recovery times.

- New analgesics that provide more extended pain relief postoperatively with less frequent administrations.

- New bleeding and injection techniques that cause less tissue damage or distress.

- Improved surgical techniques that minimize trauma and the length of anesthesia.

Now that we have gone through the "3 R's", we have to discuss the legal mandate for considering alternatives. The USDA Animal Welfare Act Regulations require the IACUC to do two things regarding alternatives:

The first is to ensure that the principal investigator has considered alternatives if painful or distressing procedures are proposed.

The second is to evaluate a written narrative provided by the principal investigator that describes which source or sources were used to determine that alternatives were not available.

The bottom line is that the IACUC must evaluate a written account of how you determine that alternatives to painful or stressful procedures are not available. A USDA policy states that the following must be part of the written alternatives narrative:

1. The databases searched (Index Medicus, Medline, Current Contents, etc) or other sources used, such as colleagues at scientific meetings, journal articles read, and presentations attended.
2. The date that any database searches were performed.
3. The years of citations covered by database searches.
4. The key words and/or search strategy used when searching a database.
5. A search for reduction and refinement, not just replacement of animals.

When responding to the question or questions on your animal protocol forms about alternatives, make sure the mandated information above is included in your response(s). It is a best practice to save copies of your database search "hits" with abstracts to document that you made a good faith effort to find alternatives.

If you need help with database searches for alternatives, there are several good options now.

• ALTBIB: Bibliography on Alternatives to Animal Testing. This NIH website is specifically designed to help you perform database searches on alternatives. The site is available to all users at http://toxnet.nlm.nih.gov/altbib.html. It helps to properly format searches in PubMed, provides links to live PubMed searches on selected animal testing topics. It also displays links to other important sites on alternatives. Click here for brief instructions on using the site.


• University of California Center for Animal Alternatives. This website offers a variety of pre-formatted searches of multiple databases. Click here for the home page or here for the page listing the pre-formatted searches.


• Altweb. The Altweb website is run by the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and provides extensive information on alternatives. The site includes searches that address pain management and humane endpoints.


• Animal Welfare Information Center (AWIC). AWIC was formed as part of the USDA by a Congressional mandate to ensure that scientists using animals could receive help in searching for alternatives. AWIC has trained professionals that can help you decide which databases are most appropriate, and which search strategy works best for each database. AWIC also has extensive literature on alternatives, and strategies to reduce pain and distress in research animals. Click here to see a variety of useful white papers on specific procedures commonly used in animal research.


• Medical Librarian. You can also consult your local medical librarian for assistance. Some institutions even include a medical librarian on the IACUC to provide expert guidance in this area.

There are a number of organizations that have active research programs into alternatives to animal use. They include the Johns Hopkins Center for Alternatives to Animal Testing(CAAT), the Institute for In Vitro Sciences, and the European Centre for Validation of Alternative Methods.

There is another regulatory mandate we have to cover that is similar to the alternatives mandate. The USDA Animal Welfare Act Regulations state that IACUCs must evaluate a written assurance that the proposed animal studies do not unnecessarily duplicate previous studies. You will be asked to document that your proposed work is not unnecessarily duplicative somewhere in your forms.

The form of the written documentation is not specified by the Animal Welfare Act, but typically the same types of documentation used for the alternatives mandate do double duty here. Although not mandated by the AWA, experience has shown that database searches are effective ways to document that work proposed is not unnecessarily duplicative. For this reason, many institutions require that a database search of the research and medical literature be completed to help document a lack of unneccesary duplication, even though such a search is not required by the AWA.

If you are experienced in searching literature databases, don`t forget the new Altweb website, http://www.altwebsearch.org, that provides simultaneous searches of multiple medical and research databases.

Note that the critical concept is that unnecessary duplication is not allowed. Acceptance of new ideas in science is often dependent upon the ability of other scientists to duplicate published reports. The IACUC can allow duplication of previous work if you can convince them that it is important scientifically to do so.

Many institutions require the Principal Investigator (PI) to assign animals to pain/distress categories somewhere on the animal protocol form. The goal of this lesson is to help you correctly classify experimental procedures on laboratory animals into USDA pain/distress categories, upon which most institutional categories are based. The name given to the pain/distress categories may differ from the "BCDE" category system used by USDA, but very likely the category definitions are similar if not identical those used by USDA.

The USDA AWA regulations stipulate that the number of research animals used by an institution must be reported annually to the USDA. The animals must be placed by species into one of four USDA pain/distress categories. To help collect accurate information, VA IACUCs ask an investigator to categorize the animals requested using the same system.

To do this properly, you must understand how animals are assigned into the four USDA pain/distress categories. The category labels (B through E) come from the column labels used on the USDA annual report form. The categories will be discussed in order from no pain/distress (B) to most pain/distress (E).

A simple yet useful definition of a painful or distressful procedure on an animal is this:

"A procedure that would cause pain or distress in a human."

It is important to understand that if multiple procedures will be performed on an animal, the animal is placed in the category appropriate for the most painful/distressful procedure. One animal cannot be placed in multiple categories.

Category B animals are those that are being "bred, conditioned, or held for use in teaching, testing, experiments, research, or surgery but not yet used for such purposes." These animals have not been used for any research procedure, however minor. Category B is the place to put breeders and other animals that are not undergoing any experimental procedures.

Category C animals are not subjected to procedures that involve pain or distress or would require the use of pain-relieving drugs. Routine procedures such as injections and blood sampling from veins that produce only mild, transient pain or discomfort are reported in this category. Another example of category C procedures is an observational study of animal behavior. Animals that are euthanized before tissue collection or other manipulations are also commonly placed in this category, if no other procedures performed that put them in a higher pain/distress category.

Category D animals are those subjected to potentially painful procedures for which anesthetics, analgesics, or tranquilizers will be used. The important concept is that animals are given appropriate anesthesia and/or pain relief to limit their pain and distress as much as possible.

Examples of category D procedures are

Surgery conducted with appropriate anesthesia and postoperative analgesia; Rodent retroorbital eye bleeding performed under anesthesia; Primate tattooing performed for identification under anesthesia; Removal of a small tumor under local or general anesthesia, and use of analgesia after an animal's skin is exposed to ultraviolet light to cause a "sunburn"; and Terminal exsanguination (euthanasia by removal of blood) under anesthesia

Category E animals are those that are subjected to painful or stressful procedures without the use of anesthetics, analgesics, or tranquilizers. Withholding of anesthetics, analgesics, or tranquilizers can only be allowed if it is scientifically justified in writing and approved by the IACUC. Examples of category E procedures are lethal dose studies (e.g. LD50 studies) that allow animals to die without intervention, pain studies that would not be possible if pain-relieving agents were administered, and psychological conditioning experiments that involve painful stimuli such as a noxious electrical shock that cannot immediately be avoided by an animal.

Category E studies are given increased scrutiny by IACUCs because they must be satisfied that less painful or stressful alternatives are not available, or that less painful/stressful endpoints cannot reasonably be used. By law, the institution must annually report all category E procedures to the USDA ( on the new draft ACORP) and include a scientific justification supporting the IACUC's decision to approve them. Often, the justification given by the researcher on the animal forms submitted to the IACUC is used for the annual report.

It is important for information on category E procedures to be complete and accurate. Once submitted to the USDA, this information will likely be available to the public through a Freedom of Information Act request.

The criteria used for intervention in research studies to prevent unnecessary pain and distress are called "endpoint criteria" because they describe when it is time to:

- euthanatize an animal to prevent suffering;

- Discontinue a painful procedure; or

- Remove an animal from a study.

Many institutions have default endpoint criteria that must be used unless an investigator receives approval from the IACUC for different criteria. Check with your IACUC to see if default endpoint criteria have been developed for your institution. Endpoint criteria should ideally apply to animals before, during, and after experimental procedures. As an example, this rabbit is suffering from an inner ear infection unrelated to any experimental manipulations, and can no longer eat or drink normally. Euthanasia is a humane option in this case.

Common examples of endpoint criteria include a limit on weight loss as a percentage of body weight, anorexia for an extended time (e.g. three days), sudden pain or distress that cannot be controlled with analgesics, sedatives or tranquilizers, or severe medical conditions that cannot be controlled with appropriate therapy (e.g. severe systemic infections, kidney or liver failure, heart disease).

More specific criteria are often used for certain types of studies. For example, endpoint criteria used for rodent cancer studies involving the growth of tumors under the skin (as shown in the photo) often include maximum tumor volumes or tumor weight as a percentage of body weight, skin ulceration over the tumor, interference with normal gait or movement, and interference with normal feeding and drinking behaviors.

Endpoint criteria for mice used to produce ascites fluid rich in monoclonal antibodies will be discussed later in the course.

Surgery on animals requires highly trained, conscientious individuals, and appropriate prior planning. To understand the issues involved, some important terms and concepts must be addressed. Surgery will undoubtedly be addressed in some detail in your animal protocol forms.

Sterile or Aseptic Technique. This refers to a series of practices followed to prevent the contamination of the surgical site by microbes during surgery. If an animal will recover from surgery, sterile technique must be used.

General anesthesia. Simply stated, general anesthesia is a state of unconsciousness characterized by a complete lack of pain and sensory perception.

Regional anesthesia. In contrast to general anesthesia is regional anesthesia, which refers to preventing pain and sensory perception in one small part or a region of the body, such as a section of skin or an entire limb.

Prior to beginning surgery, you must ensure that the animal will not feel pain during the procedure. General or regional anesthesia must be provided.

It is important to distinguish between survival and non-survival surgery.

Survival surgery is surgery in which the animal regains consciousness after anesthesia. If an animal undergoes survival surgery, aseptic (sterile) technique must be used to prevent postoperative infections, no matter what vertebrate species is involved. The incision site must be properly prepared prior to the incision. The hair must be clipped and the skin must be disinfected, often with chlorhexidine or iodine solutions.

Non-survival surgery is surgery in which the animal is euthanized while under anesthesia, and does not regain consciousness. If an animal undergoes non-survival surgery, sterile technique may not be required. Even though the animal will not survive beyond the end of surgery, at a minimum, the surgeon should wear gloves, the surgical site should be clipped, and the instruments and work area should be clean.

Major surgery is defined as surgery that penetrates and exposes a body cavity such as the chest or abdomen, or surgery that produces substantial physical or physiological impairment. Examples of major surgeries include laparotomy, thoracotomy, craniotomy, joint replacement, and limb amputation.

Minor surgery is less invasive surgery that does not meet the criteria for major surgery above.

Multiple major survival surgeries on one animal are discouraged unless they can be justified scientifically by the investigator and approved by the IACUC. You are sure to be asked about this issue on your forms if you are proposing survival surgery. There are a number of justifications commonly accepted by IACUCs, based on guidance provided by the Animal Welfare Act Regulations and the Guide. These include:

Scientific justification- if more than one major survival surgery must be performed to achieve research objectives;

Conservation justification- if the animal species under consideration is rare, endangered, or in short supply;

Clinical justification- if the animal must undergo multiple major survival surgeries to treat a medical condition, in consultation with a veterinarian;

Other unusual justifications- if approved by the USDA Chief Administrator.

Cost savings alone is not an adequate justification for multiple major survival surgeries. For instance, animals cannot be subjected to multiple major survival surgeries by being traded between investigators just to save money.

The rooms that can be used for surgery vary depending on:

A dedicated surgical suite is required for major survival surgery on all non-rodent mammals (this includes rabbits).

In contrast, a clean area or portion of a room (along with the use of aseptic technique) is acceptable for:

Major survival surgeries on rodents and lower vertebrates. All non-survival surgeries. All minor survival surgeries.

Postoperative care must be provided after survival surgeries. The animal should be monitored to make sure it is recovering properly. If the surgical procedure would be expected to cause pain in a human, then it should be assumed that the procedure will be painful in an animal , no matter what the species, and appropriate postoperative analgesia should be provided unless nonuse is approved by the IACUC. The agent, dose, route, frequency, and duration of postoperative analgesia provided should be discussed with and approved by a veterinarian , preferably during the planning stages of the experiments.

Documentation of postoperative care is very important. A simple rule to follow is this- "if it isn't written down, it didn't happen." The USDA requires that health care records be maintained in a manner consistent with prevailing professional veterinary practice standards.

For animals larger than rodents, individual health care records are usually maintained, with records of daily observations and treatments during the postoperative care period.

For smaller animals like rodents, group records instead of individual records are usually kept. Depending on the institution, the veterinary staff or the research staff may maintain the records, but the records should always be accessible to the veterinary staff should complications arise. The records should be maintained at least a year after the death of the animal to meet USDA policy.

In the absence of complications, the postoperative period traditionally ends 7-10 days after surgery, when skin sutures are often removed. After that, routine daily monitoring can be resumed, and routine entries in the health records discontinued.

The USDA does not allow changes in animal ownership during the postoperative recovery period, and does not allow movement of the animal between facilities during recovery from anesthesia unless the IACUC approves it. These prohibitions are meant to help ensure continuity of care during the postoperative period. Appropriate health records must be maintained regardless of the animal's location.

Animals are often fasted prior to surgery so that the risk of aspiration pneumonia is minimized. Aspiration pneumonia can occur if an animal vomits, then breathes (or "aspirates") the vomit into the lungs. For this reason, fasting is often recommended.

However, rodents and rabbits are unable to vomit because of their esophageal physiology, and thus they should not be fasted prior to surgery unless there are other medical or scientific reasons for doing so.

The production of polyclonal and monoclonal antibodies in animals has been critical for biomedical research progress for many years. This section will help you understand the ethical and procedural concerns that must be addressed when planning antibody production in animals and filling out IACUC forms.

Polyclonal antibodies are produced when a large number of B lymphocytes produce antibodies against different parts of the same antigen or multiple antigens. Typically, animals are immunized multiple times to elicit a strong antibody response, then bled so that immune serum can be collected and used in experiments. Two important considerations in producing polyclonal antibodies in animals are proper immunization technique, and proper bleeding technique.

When producing polyclonal antibodies, adjuvants are usually mixed with antigens to augment the antibody response. The classic adjuvant is Freund's adjuvant, which is available in two forms:

• "Complete" (Complete Freund's Adjuvant, or "CFA"). CFA is a mixture of oils and water plus killed Mycobacterium tuberculosis. It typically elicits a very strong immune reaction. If used more than once, the immune reaction usually progresses to intense inflammation and sterile abscesses.
• "Incomplete" (Incomplete Freund's Adjuvant, or "IFA"). IFA is similar to CFA, but is missing the killed mycobacteria. This renders the IFA less effective as an immune stimulant, but also less inflammatory to the animal. IFA can be used safely multiple times without causing intense inflammation.

To prevent inflammation and pain, CFA must only be used once. IFA is less inflammatory, and can be used multiple times. Typically CFA mixed with antigen is given to an animal the first time, then IFA mixed with antigen is given second, then either IFA mixed with antigen or antigen alone for subsequent immunizations.

The USDA states that the injection of CFA may cause more than momentary or slight pain. This means that CFA injections might put an animal into USDA pain category D (painful/stressful but relieved), requiring the use of post-injection analgesics or sedatives.

You may be asked to list any possible medical complications resulting from the use of adjuvants during polyclonal antibody production in your animal protocol forms.

To reduce inflammation when using CFA, consider the following measures:

1. Choose or make preparations of CFA with a lower mycobacterial concentration, i.e., 0.05 to 0.1 mg/ml, rather than 1 mg/ml.
2. Add a concentrated antigen solution to the adjuvant to obtain a more antigen-rich emulsion, thereby reducing the volume of emulsion injected.
3. Use multiple injection sites to limit the volume injected at any one site.
4. Separate the injection sites to avoid fusion of inflammatory lesions.
5. Maintain sterility of the antigen solution.

The quantity of CFA or IFA adjuvant injected should be limited. Institutional guidelines vary, but typical limits on adjuvant use are around 1 ml of combined adjuvant/antigen per immunization for rabbits (typically up to ten divided 0.1 ml injections), and around 0.25 to 0.5 ml combined adjuvant/antigen per immunization for smaller animals (up to ten divided 0.05 ml injections). These amounts have been shown to produce high titer antibodies, yet limit inflammation.

Beware: CFA is a health hazard to humans.

If you are already sensitized to mycobacterial antigens by a previous exposure to CFA or through a natural infection of tuberculosis, you are likely to experience severe inflammation if you splash CFA into your eye or accidentally inject yourself with it. The inflammation and pain may be so severe that surgical removal of the site may be necessary. Protect your eyes and prevent accidental injection of yourself or a colleague when using CFA!

Less inflammatory alternatives to CFA and IFA are now available and in use. Examples are the block copolymer adjuvant Titermax®, and the lipid A-derivative adjuvant MPL® by RIBI. Other promising alternative adjuvants are also on the market. Such alternatives can be considered as a means of further reducing inflammation induced by Freund`s adjuvant.

The route of immunization should be chosen to limit pain and inflammation. Regardless of the adjuvant used, the subcutaneous route typically provides a strong immune response, and is recommended. The intravenous route is not appropriate if adjuvant is used because the thick consistency of the adjuvant can result in lethal emboli in the blood stream.

There are several other routes of immunization that are usually discouraged because there is little evidence that they offer any advantage over the subcutaneous route:

Intradermal (ID): Causes more pain because the skin itself cannot stretch much as body fluids and white blood cells enter the immunization area, resulting in increased pressure and pain. Some institutions allow the ID route if less volume of adjuvant and antigen are injected.

Intraperitoneal (IP): Inflammation on surfaces of abdominal organs can result in peritonitis, granulomas, and pain.

Foot pad: Injections can cause pain and lameness. When allowed by an IACUC, usually only one foot may be injected. Foot pad injections are usually discouraged in rodent species, and deemed inappropriate in larger species. Rabbits will often chew on their own feet after foot pad injections, presumably because of intense pain or irritation.

Some evidence indicates that immunization injections should be spaced 3-6 weeks apart to elicit an optimal polyclonal antibody response, and the highest possible titer. There may be a temptation to hurry the process and use shorter intervals, but a reduction in antibody titer may result. This is because circulating antibody from the previous immunization can remove antigen from circulation and thus limit its ability to induce a strong immune response.

Blood collection is obviously essential for collecting the immune sera from immunized animals. Often a "pre-bleed" is performed prior to immunization to determine if specific polyclonal antibody is already present in the animal (this could complicate some subsequent antibody studies). Periodic blood collections are needed thereafter to determine when a good antibody response is present. Once a good titer has been produced, serum for present and future experiments needs to be collected.

The amount of blood that can be collected and the interval between collections are covered in more detail in the next lesson.

Monoclonal antibodies are secreted by clones of a single B cell. To produce them, animals (typically rodents) are immunized with an antigen, then spleen cells or lymph node cells are collected after euthanasia and fused with an immortal cell line. The fused cells are placed in a special medium that allows only hybrid cells to grow.

These hybrid cells, or hybridomas, are expanded in number, and the clones that produce antibody against the antigen of interest are saved.

If adjuvant is used during the immunization process, the same principles apply as described in the polyclonal antibody section.

Thus, the first use of animals in generating monoclonal antibodies is to create the hybridoma cell line.

The second common use of animals in generating monoclonal antibodies is to grow the hybridoma cell line on the peritoneal lining of histocompatible animals, and collect the antibody-rich ascites fluid.

Over the past years, there have been a number of in vitro techniques introduced that can sometimes replace the use of animals for expanding hybridoma cell lines, and for collecting purified monoclonal antibody. Consequently, non-animal alternatives for generating purified monoclonal antibodies must be considered, and found to be unsuitable before the IACUC can approve animal use for that purpose.

When requesting approval to use animals for expanding hybridoma cell lines, be prepared to explain why in vitro techniques will not work. In 1999, The Committee on Methods of Producing Monoclonal Antibodies (sponsored by the Institute for Laboratory Animal Research and the National Research Council) suggested the following guidelines for IACUCs to use when evaluating the need for using animals for hybridoma expansion (Recommendation 4):

-When a supernatant of a dense hybridoma culture grown for 7–10 days (stationary batch method) yields a monoclonal antibody concentration of less than 5 mg/ml, or if other systems are used and concentrations obtained are less than 500 mg/ml (hollow fiber system) and 300 mg/ml (semi-permeable membrane system).

-When more than 5 mg of monoclonal antibody produced by each of five or more different hybridoma cell lines is needed simultaneously. It is technically difficult to produce this amount of monoclonal antibody since it requires more monitoring and processing capability than the average laboratory can achieve.

-When analysis of monoclonal antibodies produced in tissue culture reveals that a desired antibody function is diminished or lost.

-When a hybridoma cell line grows and is productive only in the animal.

-When more than 50 mg of functional monoclonal antibody is needed, and previous poor performance of the cell line indicates that hollow-fiber reactors, small-volume membrane-based fermentors, or other techniques cannot meet this need during optimal growth and production.

These same criteria can help you decide if in vitro methods will suffice. The burden of proof is now on the investigator to show that in vitro methods of obtaining purified monoclonal antibody do not work, or are not effective in providing the amount of antibody needed.

If in vivo methods are needed because in vitro methods cannot replace them, consideration must be given to minimizing the amount of pain and suffering involved. The following parameters should be considered when animals are used to expand hybridomas using the ascites collection technique:

1. The amount of pristane used to “prime” the peritoneal cavity and make it better able to support hybridoma growth should be minimized.
2. The degree of abdominal distension should be monitored at least daily and should distension begin to interfere with breathing, the ascites fluid should be removed.
3. The number of peritoneal “taps” used to collect ascites fluid should be minimized.
4. The needle used should be as small as possible (20 gauge or higher). Because mice with ascites are not good anesthetic risks, ascites fluid is usually collected with a needle and syringe without anesthesia, and smaller bore needles cause less pain.
5. Endpoint criteria tailored to collecting ascites should be developed. In addition to typical endpoint criteria such as weight loss and extended anorexia, additional criteria to consider include a limit on the number of abdominal taps allowed, the presence of dyspnea (difficult breathing) unrelieved by a tap, and the development of solid hybridomas instead of more diffuse neoplasms producing ascites. Usually you will be asked about endpoint criteria somewhere on your forms.

Don`t forget to describe blood collections planned in conjunction with monoclonal antibody production somewhere on your form. More information on blood collection is provided in the next lesson.

When collecting blood samples, the volume and frequency of collection must be carefully limited so that neither shock nor anemia result.

How much blood can be collected at one time? Your institution may have specific guidelines, so check with your veterinarian or IACUC. One simple guideline is to collect no more than 1% of the body weight as blood at one time. Assume that a ml of blood weighs 1 gram. For example, for a 4,000 gram rabbit, 40 ml (0.01 x 4,000= 40 ml) could be safely collected. For a 20 gram mouse, 0.2 ml (0.01 x 20= 0.2 ml) could be safely collected.

How often can blood be collected? Once again, check with your veterinarian or IACUC. In general, multiple blood collections should be spaced far enough apart to prevent anemia and distress in animals. Blood collections of no more than 1% of body weight can usually be performed every 2-4 weeks without harm. More frequent collections might require hematocrit monitoring to prevent severe anemia in an animal.

Blood collection from rodents can be challenging. Consider the following points:

1. Retroorbital (also termed "periorbital" or "eye") bleeding of mice and rats should only be performed under anesthesia, unless the IACUC approves its use without anesthesia for scientific reasons. To perform it properly so that tissue trauma is minimized, good training is essential. If you are not skilled in this procedure, find someone who can demonstrate how to do it with minimal trauma to the animal.
2. Alternate methods of bleeding rodents might give adequate blood volume with no need of anesthesia. Examples include blood collection from the lateral saphenous and jugular veins.
3. Blood collection from the heart should only be performed on anesthetized animals, usually as a terminal procedure (this is true of all animals).

Both PHS policy and the USDA Animal Welfare Act Regulations and Standards stipulate that personnel must be trained so that they are qualified to perform research on animals. The institution is given the responsibility for providing training, and as the self-regulating unit of the institution for animal research, the IACUC must ensure that personnel are qualified to perform the procedures proposed in animals as part of the review process.

The USDA Animal Welfare Act Regulations and PHS Policy require an institution to cover a number of specific topics as part of its training program (one of the purposes of this course is to help satisfy these USDA and PHS training requirements):

1. Humane methods of animal maintenance and experimentation, including:
   1. The basic needs of each species of animal;
   2. Proper handling and care for the various species of animals used by the facility;
   3. Proper pre-procedural and post-procedural care of animals;
   4. Aseptic surgical methods and procedures.
2. The concept, availability, and use of research or testing methods that limit the use of animals or minimize animal distress;
3. Proper use of anesthetics, analgesics, and tranquilizers for any species of animals used by the facility;
4. Methods whereby deficiencies in animal care and treatment are reported, including deficiencies in animal care and treatment reported by any employee of the facility. No facility employee, Committee member, or laboratory personnel shall be discriminated against or be subject to any reprisal for reporting violations of any regulation or standards under the Act; and
5. Utilization of services (e.g., National Agricultural Library, National Library of Medicine) available to provide information:
   1. On appropriate methods of animal care and use;
   2. On alternatives to the use of live animals in research;
   3. That could prevent unintended and unnecessary duplication of research involving animals;
   4. Regarding the intent and requirements of the Act.

To comply with the law, the IACUC must assure itself that appropriately trained personnel will be conducting studies on animals. Consequently, you will be asked to state your experience and training in performing the proposed procedures. Although academic degrees are useful indicators of educational experience, they are not often useful by themselves in evaluating an individual's experience in animal research.

It is perfectly fine to admit that you do not have the necessary training or experience to perform a procedure proposed in your animal protocol forms- just make sure you identify experts who can train you or your staff before the animal procedures begin.

Because there are allergy, disease, and physical risks associated with animal contact, each institution must have an occupational health and safety program for personnel who come in contact with animals. Although receiving certain immunizations or medical treatment in the program may be voluntary, PHS Policy requires an institution to offer an occupational health and safety program to employees who "work in animal facilities or have frequent contact with animals."

Details of occupational health and safety programs vary by institution, but typically, services include rabies immunizations, TB testing, and tetanus immunizations. Offered services usually depend on the type and amount of exposure to animals, and individual risk of contracting zoonotic diseases or developing allergies.

Although personnel may decline to accept any services, many institutions require personnel to enroll in the local Occupational Health and Safety Program before they begin work with animals. This is for their safety, and the safety of the animals as well. For instance, tuberculosis can not only be passed from primates to humans (zoonotic transfer), but also from humans to non-human primates. To protect the non-human primates, humans with primate contact must be screened for tuberculosis.

Some institutions require participation in the Occupational Health and Safety Program by linking it to IACUC approvals or access to the animal facility. Make sure you and your staff enroll, even if optional services are declined.

A National Research Council publication is available to aid institutions in designing their occupational health and safety programs.

If your animal work requires the use of hazardous or toxic agents, there are many important considerations. Such agents can be categorized in the following way:

Infectious diseases

Toxic chemicals, including carcinogens, mutagens, biological toxins, and organic chemicals

Radioactive substances

Recombinant DNA

Some points to consider when using such agents in animals:

The risk of accidental human infection or exposure is usually reduced if animals are anesthetized or sedated before they are injected with agents using a hypodermic needle. When anesthetized, animals will not struggle unpredictably- this helps prevent accidental redirection of the needle toward personnel handling the animals.

If using an infectious agent, an antibiogram or other appropriate therapeutic panel should be developed on infectious strains before they are used in animals. If an accidental human exposure occurs, physicians will know immediately which antibiotic or other therapeutic agent to use to best treat the infection. Consult with your occupational health staff in advance of experiments.

If using a toxic agent, know in advance what antidote or action to take if accidental exposure through an injection, spill, or break in the skin occurs. Have any necessary antidotes, decontamination kits, or spill kits readily available. Consult with the appropriate safety officer in advance of experiments.

When administering hazardous agents to animals, it is best for personnel to work in pairs. If one person becomes contaminated, the second person can help decontaminate the person and the area quickly.

The IACUC usually is charged with evaluating safe practices for using hazardous agents in animals, many times with the assistance of a biosafety or other similar committee and appropriate safety officers. Check with your IACUC staff to find out the correct process for receiving approval for work with hazardous agents in animals.

Let`s start with infectious agents. Guidelines for performing infectious disease work with animals are found primarily in the CDC/NIH publication entitled Biosafety in Microbiological and Biomedical Laboratories, or "BMBL". The BMBL has guidelines for working with a wide variety of infectious agents in both research laboratories and the animal facility.

There are four levels of containment procedures for infectious agents recognized in the BMBL. The levels are designated Biosafety Level 1, Biosafety Level 2, Biosafety Level 3, and Biosafety Level 4. The containment and handling safeguards become more stringent as the biosafety level (or “BSL”) number increases. For each of the four biosafety levels, there are corresponding Animal Biosafety Levels (or "ABSL") (Animal Biosafety Level 1, Animal Biosafety Level 2, Animal Biosafety Level 3, Animal Biosafety Level 4) that provide guidelines for housing and manipulating animals infected with agents that require that level.

Before beginning any animal studies involving infectious agents, both research staff and personnel in the animal facility must understand how to safely conduct the study in the animal facility. Standard Operating Procedures (SOPs) should be written and approved by appropriate committees before any infectious work begins, and you may be required to provide a detailed SOP prior to the IACUC review. An SOP should describe how animals will behandled and housed in the animal facility after they are infected. It is very important that the veterinarian, biosafety officer, biosafety committee, and animal facility manager be involved in the planning as needed to minimize the risk of exposing humans or other animals to the agent.

The CDC and USDA enforce additional regulations for certain infectious agents and biological toxins. These “select agents” are given additional regulatory oversight because of their potential use in biological (“germ”) warfare. The list of select agents includes infectious agents like hemorrhagic fever viruses, and plague, brucellosis, and anthrax bacilli. It also includes a number of biological toxins such as aflatoxin and botulinum. An investigator must register with the Centers for Disease Control and Prevention (CDC) or USDA and obtain approval before possessing or beginning any work with agents on the “select agent” list. Local approval from the Institutuional Biosafety Committee is also required.

Both CDC and USDA maintain websites with information on how to apply for permits to maintain or use select agents.

As with infectious disease studies, the use of toxic chemicals or radioisotopes in animals requires careful coordination between many people, including:

The research staff.

The veterinarian and animal facility supervisor.

The appropriate institutional committees (biosafety, radiation safety) responsible for use of hazardous agents.

The IACUC should make sure that all research and animal facility personnel are trained to properly minimize the risk of accidental human or animal exposure. SOPs describing containment and handling procedures should be written and approved by appropriate committees before any animal work begins. It is critically important to also train the animal caretakers who will clean, feed, and water the animals. If highly specialized training is required to handle animals safely, then the research staff might have to assume husbandry duties for infected animals.

There are additional guidelines to consider if your work in animals includes:

1. The inoculation of infectious agents with recombinant DNA into animals, or
2. The use of recombinant molecules in an animal’s genome.

The purpose of the NIH Guidelines for Recombinant DNA and Gene Transfer are to specify practices for constructing and handling recombinant deoxyribonucleic acid (DNA) molecules and organisms and viruses containing recombinant DNA molecules.

Although the recombinant DNA inserted into many transgenic mice may not be covered, it is wise to check with your local Biosafety Committee before you produce transgenic mice to determine what committee approvals will be necessary.

Experiments involving recombinant DNA must usually be reviewed by a local recombinant DNA, biosafety or other similar committee. In some cases an NIH committee called the "Recombinant DNA Advisory Committee" must also approve the experiments. Experiments involving very high risk, such as introducing novel antibiotic resistance into human pathogens, may also require NIH approval before initiation. Guidance can be obtained from the Office of Biotechnology Activities (OBA) at the NIH.

The NIH Guidelines for Recombinant DNA and Gene Transfer document describes different levels of agent containment practices very similar to the levels described in the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories manual. We will not go into great detail, but the basic risk classifications in the NIH Guidelines for Recombinant DNA and Gene Transfer will be covered.

If recombinant nucleic acid is introduced into infectious agents, the level of laboratory and animal facility containment required is primarily based upon the "Risk Group" assigned to the agent involved. The Risk Groups are based upon disease potential in healthy humans and availability of therapy. They do not account for instances in which an individual may have increased susceptibility to such agents, e.g., preexisting diseases, medications, compromised immunity, pregnancy or breast feeding (which may increase exposure of infants to some agents). For each of them, there is a corresponding set of animal biosafety guidelines that must be used, as follows:

1. Risk Group 1 (RG1)- Agents that are not associated with disease in healthy adult humans. Generally, if an RG-1 agent is used in animals, Biosafety Level 1-N (BL1-N) animal containment measures are used.
2. Risk Group 2 (RG2)- Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available. Generally, if an RG-2 agent is used in animals, BL2-N animal containment measures are used.
3. Risk Group 3 (RG3)- Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk). Generally, if an RG-3 agent is used in animals, BL3-N animal containment measures are used.
4. Risk Group 4 (RG4)- Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk). Generally, if an RG-4 agent is used in animals, BL4-N animal containment measures are used.

The containment practices recommended for each of the animal biosafety levels (BL1-N through BL4-N) are found in Appendix Q of the NIH Guidelines for Recombinant DNA and Gene Transfer.

Somewhere in your animal forms you will have to list what hazardous agents you will use, and probably additional information of the type covered in this lesson. Advances in infectious disease, carcinogenesis, toxicology and other fields are often dependent on animal research. Make sure that such studies are conducted safely so that risks to the research staff, husbandry staff, and other animals are minimized.

(Photo: Dasypus novemcinctus, the nine-banded armadillo, the only known nonhuman natural host of the leprosy bacillus, Mycobacterium leprae)

The Guide states that social animals should be housed in pairs or groups whenever possible unless scientific, health, or behavioral considerations prevent it. The IACUC is expected to address this issue during protocol review, and if you want to house animals singly, you may have to provide a justification on the protocol form. Some justifications for single housing include:

1. Behavioral problems. For instance, adult male C57BL/6 and BALB/c mice will fight if not raised together from birth, hamsters are usually solitary by choice, both male and female rabbits will sometimes fight if housed in groups, and individual animals of most species will sometimes not be compatible with other cagemates.
2. Health problems. For instance, animals with health problems may not be able to compete for food in a pair housing or group situation, they may be singled out for aggressive behavior by others in the group, and they may need medications that can only be given if they are singly housed.
3. Scientific reasons. If single housing must be used to achieve scientific objectives, then the IACUC may approve single housing. Examples include dietary, water consumption, or metabolic studies in which data on a single animals must collected, controlled feeding experiments in which food or water consumption would be altered by the presence of more dominant animals, and behavioral studies that would be altered by the presence of other animals.

The Guide recommends that solid bottom caging with bedding be used preferentially for rodents. This Guide addresses this issue because there is some evidence that rodents prefer solid bottom caging with soft bedding over wire mesh flooring, and some limb pathology has been associated with prolonged housing of rodents on wire mesh floors. The IACUC is expected to address this issue during protocol review, and if you want to house animals on wire mesh flooring, you might be asked to provide a scientific justification on the protocol form.

Some toxicology projects in rodents are performed on wire mesh floors so that animals do not remain in contact with metabolites in urine and feces. Rodents in metabolism cages must usually be on wire mesh floors so that urine and feces can be collected under the cage.

The Animal Welfare Act regulations and standards require that institutions adopt a program of dog exercise. Dogs must be exercised outside of their cages if they are housed singly in an area less than two times the minimum space required. If the runs in your facility do not provide singly housed dogs with enough space, the dogs must be exercised unless a dog is exempted:

-By the attending veterinarian for health reasons, or

-By the IACUC based upon a scientific justification provided by the investigator.

Exemptions must be evaluated by the attending veterinarian every 30 days or less to comply with the Animal Welfare Act Regulations.

If you have concerns that exercise outside of the runs would adversely impact your dog studies, discuss the issue with your veterinarian and the IACUC. If applicable, you may have to justify why dogs need to be exempted from the dog exercise program in the animal protocol forms.

The Animal Welfare Act regulations and standards also require that institutions adopt a program of psychological enrichment for nonhuman primates. Just as dogs can be exempted from the exercise program, nonhuman primates can be exempted from the enrichment program:

1. By the attending veterinarian for health reasons, or
2. By the IACUC based upon a scientific justification provided by the investigator.

If you have concerns that the psychological enrichment program would adversely impact your studies with nonhuman primates, discuss the issue with your veterinarian and the IACUC. Just like the dog exercise program, you may have to address this issue on your animal protocol form.

Once granted, exemptions must be evaluated at least every 30 days by the attending veterinarian.

The Guide also has special language addressing prolonged restraint of animals while they are conscious. In general, restraint for all animals should be the least restrictive and for the shortest time necessary to complete research objectives. Prolonged restraint should be avoided unless it is essential for achieving research objectives. Examples of prolonged restraint include primate chairing, rodent restraint in inhalation chambers, and swine and dogs restrained in slings. Consider the following guidelines:

1. Restraint devices are not to be considered normal methods of housing.
2. Restraint devices should not be used simply as a convenience in handling or managing animals.
3. The period of restraint should be the minimum required to accomplish the research objectives.
4. Animals to be placed in restraint devices should be given training to adapt to the equipment and personnel.
5. Provision should be made for observation of the animal at appropriate intervals, as determined by the IACUC.
6. Veterinary care should be provided if lesions or illnesses associated with restraint are observed. The presence of lesions, illness, or severe behavioral change often necessitates temporary or permanent removal of the animal from restraint.

If you are proposing primate chairing for more than 12 hours at a time, you are required to provide at least one hour of unrestrained activity during that 12-hour period, unless continuous restraint is required by the research proposal and approved by the IACUC.

Euthanasia literally means a "good death". A more appropriate simple definition is a "gentle death".

The USDA AWA defines euthanasia as "the humane destruction of an animal accomplished by a method that produces rapid unconsciousness and subsequent death without evidence of pain or distress, or a method that utilizes anesthesia produced by an agent that causes painless loss of consciousness and subsequent death."

The Guide defines euthanasia simply as "the act of killing animals by methods that induce rapid unconsciousness and death without pain or distress."

There are at least three verbs in common use that refer to the act of euthanasia - "euthanize" and "euthanatize" are frequently encountered in American English and "euthanase" is often used in British English. Some argue that "euthanatize" is more grammatically correct as a verb of Greek origin because it retains more of the root word "thanatos". That is arguable since the noun form is "euthanasia" rather than "euthanatasia". In spite of much passionate opinion, there is currently no generally accepted "proper" verb form. You may see both "euthanatize" and "euthanize" used in this course because both are in common use in American English.

Because it is necessary to euthanatize most animals as part of experimental protocols, it is very important to use appropriate euthanasia techniques.

Because improper technique can cause pain and suffering to animals during euthanasia, you must be trained to properly and humanely perform euthanasia. Of course, the IACUC is interested in your training and ability to humanely perform any procedure on animals, but proper training for euthanasia is an area of emphasis because of the increased potential for harm to animals.

Do not perform euthanasia, or any other procedure on an animal until a person experienced with the procedure has trained you, and you feel confident performing the technique.

PHS Policy and the Guide state that methods of euthanasia should be consistent with the recommendations of a panel sponsored by the American Veterinary Medical Association (AVMA), unless the IACUC approves deviations for scientific reasons. This Report of the AVMA Panel on Euthanasia contains many guidelines used by IACUCs to evaluate methods of euthanasia.

To begin, it is important to know that euthanasia methods can be broadly separated into physical and nonphysical (or pharmacologic) methods.

Physical methods rely on trauma to the head or spine to cause rapid death, or death due to fatal loss of blood. Examples include cervical dislocation, decapitation, captive bolt pistols, and exsanguinations ("bleeding an animal out").

Non-physical or pharmacologic methods rely on drugs to cause loss of consciousness and death.

The brown bottle shown at left contains a barbituate-base injectable euthanasia solution commonly used for larger animals. The pinkish-purple color is meant as a warning that it is not safe to use as an anesthetic.

Administration of injectable euthanasia agents into the heart provides rapid loss of consciousness and death. But intracardiac injections should only be performed in heavily sedated, anesthetized, or comatose animals, unless the IACUC approves it after considering an extraordinary justification.

The same holds true for blood collection from the heart.

There are two especially important issues regarding euthanasia of rodents and small rabbits.

The first is the use of decapitation alone. The primary justification for using decapitation without sedation or anesthesia is the need to recover tissues and body fluids that are chemically uncontaminated by sedatives or anesthetic agents. Special commercial guillotines are designed to accomplish decapitation in a uniformly instantaneous manner are available, as shown to the right.

The advantages of decapitation are that it may induce rapid unconsciousness, it does not chemically contaminate tissues, and it is rapidly accomplished. The disadvantages are that the handling and restraint required to perform this technique may be distressful to animals, the guillotine blade is a hazard to personnel performing the technique, the technique may be aesthetically displeasing to personnel, and there is some experimental evidence that brain activity and sensory capabilities do not end immediately.

Consequently, the use of decapitation without prior anesthesia or sedation should be used in research settings only when scientifically justified by the user and approved by the IACUC.

The second issue is cervical dislocation. Cervical dislocation is used to euthanatize poultry, other small birds, mice, and immature rats and rabbits.

For mice and rats, the thumb and index finger are placed on either side of the neck at the base of the skull or, alternatively, a rod is pressed at the base of the skull. With the other hand, the base of the tail or hind limbs are quickly pulled, causing separation of the cervical vertebrae from the skull.

For immature rabbits, the head is held in one hand and the hind limbs in the other. The animal is stretched and the neck is hyper-extended and dorsally twisted to separate the first cervical vertebra from the skull. In poultry, cervical dislocation by stretching is a common method for mass euthanasia, but unconsciousness may not be instantaneous.

Advantages of cervical dislocation are that it may induce rapid unconsciousness, it does not chemically contaminate tissue, and it is rapidly accomplished.

Disadvantages are that it may be aesthetically displeasing to personnel, its practical use is limited to poultry, other small birds, mice, and immature rats and rabbits, and that there is some experimental evidence that brain activity and sensory capabilities do not end immediately after dislocation.

When animals are euthanized, other animals should not be present because vocalization and release of pheromones in urine and feces can occur during euthanasia that induce anxiety in other animals. Similarly, euthanasia chambers should be cleaned well between uses to reduce animal anxiety caused by exposure to alarm pheromones in urine and feces.

Exsanguination, or the near-complete withdrawal of blood from an animal, can be used to ensure death in unconscious animals. Because anxiety is associated with hypoxia resulting from very low blood pressure, exsanguination should not be used as a sole means of euthanasia.

Neonates are not covered in detail by the AVMA Panel on Euthanasia report. In general neonates are resistant to low oxygen levels (hypoxia), and accordingly carbon dioxide is not very effective as a euthanasia agent unless there is prolonged exposure. Neonates of larger species (dogs, cats, pigs) can be given intravenous euthanasia agents.

Rodent neonates offer special challenges. Due to the difficulties of performing intravenous injections and the difficulty of properly anesthetizing rodent pups, IACUCs may allow quick decapitation with scissors, with or without hypothermia prior to decapitation. Check with your IACUC for local policy.

"Cold-blooded" animals like fish, reptiles, and amphibians can be euthanized with barbiturates by the intravenous, intraabdominal, or intrapleuro-peritoneal route, depending on anatomic features.

Tricaine methanesulfonate (TMS, MS-222) may be placed in the water to euthanatize fish, amphibians or flushed directly over the gills to euthanatize large fish.

Species such as snakes, lizards, turtles, frogs, and toads may be euthanized by overexposure to gaseous anesthetics such as halothane or methoxyflurane in a chamber or via face mask.

Note that carbon dioxide gas may not be very effective for reptiles, who can stop or reduce their breathing for long periods without overt ill effects, and may not die even after prolonged exposure.

Pithing is the destruction of the central nervous system by mechanical means. Either the brain, the spinal cord, or both may be destroyed, depending on the species and additional methods of euthanasia used. Pithing is a physical means of euthanasia, and thus should be used only if nonphysical methods are not appropriate.

Accordingly, pithing is generally used as an adjunctive procedure to ensure death in an animal that has been rendered unconscious by other means. For some species such as frogs, with anatomic features that facilitate easy access to the central nervous system, pithing may be used as a sole means of euthanasia, but anesthetic overdose is a more suitable method. Alternate methods of euthanatizing amphibians are given next.

Pithing requires knowledge of anatomic landmarks and requires great skill. Like all methods of euthanasia, it should only be performed by trained personnel.

It is very important that you make sure an animal is really dead before placing it in a bag and disposing of the bag. It is easy to mistake a deeply anesthetized animal for a dead animal, and you do not want the animal to experience the terror of waking up in a closed bag and slowly suffocating to death.

OLAW and USDA recently emphasized the importance of ensuring that euthanatized animals are really dead, and further stated that unintended recovery of animals after euthansia represents 1) serious noncompliance with the PHS Policy and 2) a serious deviation from the provisions of the Guide for the Care and Use of Laboratory Animals. Such incidents must be reported to OLAW by the IACUC with a full explanation of the circumstances and actions taken to prevent recurrence.

Check with your veterinarian or IACUC to determine the best means of assuring that an animal has died, consistent with your institutional policies.

Given the hospital settings of some institutions, it is very important that any animal use of human clinical areas be properly planned, properly performed, discreet, and approved in advance by the IACUC as well as clinical professionals in charge of the area.

Some institutions (e.g. VA medical centers) use a special approval form to help the IACUC and investigators ensure that human clinical areas such as radiology suites and MRI and PET scanners are properly used and decontaminated after use. Check with your institution to see what approvals you must obtain before using human clinical areas for animal research.

It is very important to thoroughly clean patient care areas after using them for animal procedures to prevent any odor, transmission of zoonotic diseases, or pests such as fleas. Although most people would agree that the use of sophisticated human clinical equipment for animal research is warranted, most would wonder about cleanliness if obvious evidence of an animal`s presence remained after the animal was gone!

Think carefully before using explosive agents in research settings. In most animal facilities, the use of ether to anesthetize or euthanatize animals, and the use of other explosive agents are often prohibited unless there are compelling scientific reasons for not using non-explosive alternatives. Check with your safety officer, veterinarian, or IACUC before using any explosive agents in the animal facility.

If you are approved to use explosive agents such as ether to euthanatize animals, DO NOT put the bagged carcasses in a refrigerator or freezer UNLESS you are absolutely certain that all of the agents have evaporated from the carcasses, and then only if the refrigerator or freezer is certified as explosion-proof. Sparks produced by non-explosion-proof refrigerators and freezers can ignite the fumes given off by the carcasses and cause a tremendous, deadly explosion.

Also, you must be aware that ether stored in metal cans will form highly unstable peroxides around the can lid over time. These peroxides can become so unstable that they can detonate if the can is jarred. If you have old cans of ether in the chemical storage vault in your laboratory area, consult with the appropriate safety officer at your institution to make sure that they do not represent an explosion hazard.

If you observe misuse or mistreatment of animals, or you see procedures that you don`t think comply with federal regulations or guidelines, report it immediately to your veterinarian or the IACUC.

Once an allegation of mistreatment, misuse, or noncompliance is received, the USDA Animal Welfare Regulations and Standards and PHS Policy require the IACUC to review and if warranted, investigate the allegations, whether made by the public or an employee of the institution.

The IACUC then makes recommendations to a high-ranking "Institutional Official" at the institution, who reviews the report and decides if additional action is needed.

If the IACUC decides that any animal activities need to be stopped to protect either animals or people, they are given clear regulatory authority to do so. In fact, if an IACUC votes to suspend a protocol or animal activity, that decision cannot be overturned by any administrator at the institution.

If an institution accepts PHS funds, any protocol suspension must be reported by the Institutional Official to the Office of Laboratory Animal Welfare.

Some other considerations:

1. If you see that animals are in danger or in pain, take immediate steps to remove animals from the threat, and notify the animal care staff or veterinarian immediately.
   
   
2. The vast majority of IACUCs take their job of investigating and correcting problems very seriously. However, if you are not satisfied with the final actions taken by the local IACUC or institutional administrators, and remain concerned that animals are being misused, mistreated, or are otherwise not being used for IACUC-approved research, contact the Office of Laboratory Animal Welfare (OLAW) or the USDA Animal Care Section.

Our society needs animal research and the accompanying medical advances that have reduced suffering and increased the quality of our lives. By understanding more about animal research, you help your IACUC and the research community assure the American public that animal research is conducted according to the highest standards.

Iron lungs are a relic due to animal research that resulted in polio vaccines. The public continues to look to researchers for the next medical advances that will cure their diseases, reduce their suffering, and give them good quality lives. Animal research will be necessary for many of these advances. Work with your IACUC to make sure you do your part to inspire confidence in our commitment to high quality animal research programs.

If you found any errors or would like to suggest improvements in the course, please send an e-mail message to Dr. Michael Fallon (michael.fallon@med.va.gov).

You should now be ready to complete animal forms with confidence.

Move to the last screen for final instructions.

Thanks for taking the Working with the IACUC web course!

If you are ready to take the examination that covers the content in this course, click on the Courses and Exams link at the top and right of this screen to go back to the page that shows the available courses and exams.

You will be able to see when you passed each exam to document your participation in this training. If your institution is registered with the site, your IACUC administrator will be able to see this information as well. Regardless, we recommend that you print a copy of your completion certificate and ask that it be placed in your IACUC files.

You may now review any lesson by choosing it on the menu to the left, or choose another option by clicking on the appropriate link above and to the right.