Welcome to the course Writing an Animal Protocol for Research on Rabbits.

This is the rabbit module in a course series on the preparation of an animal use protocol. Each course in this series refers to a different animal species. Every course offers information that is both pertinent to all research animals and specific to the one animal species presented.

• The first 7 lessons are similar in all courses of this series. Differences in these lessons relate mainly to the regulatory coverage, housing requirements, and the zoonotic hazards of each animal species.

• The remaining lessons present information that is more specific to each animal species, such as biological features, anesthetic doses, and biomethodologies.

The goal of this course is to cover important information about using rabbits in biomedical research settings. If you are responsible for handling rabbits or if you must write an animal use protocol, this course will be useful by providing you with:

• Information on key regulatory issues.
• Guidance on searches for alternatives in the care and use of animals.
• Highlights of unique biological features of these animals.
• Overviews of acceptable basic methodologies.
• Requirements for supportive care procedures.

Hypertext links in this course provide you with supporting information, such as regulatory sources, drug doses, practical tips, etc.

This course will not provide you with detailed information on how to conduct the methods and procedures described. For this, you should use other courses offering in-depth information and hands-on instruction from your institution's animal facility staff.

Click to view the credits for this course.

To ensure the humane treatment of laboratory animals, research involving rabbits is regulated by two federal agencies:

• The United States Department of Agriculture (USDA) / Animal Care; and
• The Public Health Service (PHS) / Office of Laboratory Animal Welfare(OLAW).

If your institution receives any funding from the PHS or is voluntarily accreditated by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC), then your research must also comply with the National Research Council publication, the Guide for the Care and Use of Laboratory Animals. This document will simply be referred to as the Guide in this course.

The Public Health Service Policy requires institutions to have an occupational health and safety program for individuals working with laboratory animals. This requirement is also reiterated in the Guide.

It is the responsibility of principal investigators to assure that their laboratory staff are informed of and participate in their institution's occupational health and safety program.

Elements of an occupational health and safety program, including institutional responsibilities, are described in the guideline, Occupational Health and Safety in the Care and Use of Research Animals (shown at right), published by the National Research Council.

Working with rabbits is associated with the following hazards:

Injuries
Personnel handling rabbits can be injured by bites (incisors) or scratches (especially by the hind feet). Generally, these are caused by a lack of knowledge in how to handle, transport, and restrain a rabbit. Likewise, poor technique in handling, etc., can cause injury to the rabbit. Training staff to work effectively and humanely with rabbits is essential to prevent injuries to people and rabbits.

Allergies
People can develop an allergy to rabbits after having contact with them for some time. Persons who develop allergy symptoms should seek medical counseling and may have to discontinue working with this species.

Zoonoses
In general, transmission of zoonotic disease from naturally infected laboratory animals is uncommon because of the ongoing efforts to improve the health status of animals by vendors of specific pathogen free animals and by facility staff. Experimentally infected animals are a source of zoonotic transmission to humans. Also, contact with wild rabbits in field research may expose humans to zoonotic agents carried by this species. Health surveillance programs, routine sanitation, and personal protective equipment have important roles in preventing zoonoses.

Rabbits can be a reservoir of the following infectious agents which are transmissable to people:

Bacteria

1. Francisella tularensis
2. Leptospira spp.
3. Yersinia pseudotuberulosis

Fungi

1. Microsporum spp., Trichophyton spp.

For more information, refer to Occupational Health and Safety in the Care and Use of Research Animals, published by the National Research Council.

Your protocol form should ask you for an assurance that you have considered alternatives to the use of animals if painful or distressing procedures are proposed. This is to satisfy mandates by the Animal Welfare Act and PHS Policy to avoid or minimize discomfort, pain, and distress consistent with sound scientific practices. Alternative procedures are those which may replace animals with nonanimal methods, reduce the number of animals used, or refine the methodology to minimize animal pain or distress. For more information on what is meant by alternatives to the use of animals, please refer to the course Working with the IACUC, which is part of this series.

The assurance must take the form of a written narrative that describes which sources were used to determine that alternatives were not available. Typically, you may be asked to provide the results of a database search including information on:

1. The databases searched.
2. The date the search was performed.
3. The years of citations covered by database searches.
4. The key words and/or search strategy used when searching a database.

It is strongly recommended that this information be sought during development of a protocol.

Organizations that can assist you in performing an alternatives search are:

• ALTWEB, Center for Alternatives to Animal Testing, Johns Hopkins University
• Animal Welfare Information Center, National Agricultural Library
• Center for Animal Alternatives, University of California - Davis

The following is a case study of alternatives searches that may guide you in the development of a search strategy that is pertinent to your own research.

Click on each database below for a sample search on key terms for this example.
You can follow the links to the database for a search in real time.

Example Search:
Study of an intervertebral disc degeneration model in rabbits.

PubMed

Agricola

More Example Searches:
Additional search routines are available elsewhere in this course to provide you with guidance on performing searches on alternatives in the care and use of animals related to the lesson topic.

All of the federal laws, regulations, policies and guidelines applicable to animal research have as their core intent the humane treatment of the animals involved in a study. Accordingly, your IACUC will have requirements for the proper care of your animals prior to, during and after a research procedure.

What is a procedure? A procedure is any activity carried out on the animal, such as venipuncture or surgery. This peri-procedural care requirement includes:

• Properly preparing the animal to humanely undergo the procedure;
• Supporting the animal's physiological function during the procedure; and
• Providing additional supportive care to aid the animal in recovering from the procedure.

The investigator has the responsibility to see that staff working with the animals are properly trained not only to perform the procedure humanely but also to provide the necessary supportive care to the animals.

When performing any procedure, such as a blood collection, you should think through the steps that are necessary to protect the animal's welfare. For example, for blood collection, you should limit the volume to the minimum that may be taken safely. That amount may vary depending upon whether a single sample is taken or multiple, chronic collections are done and, with any venipuncture, you should be prepared to care for the animal in the event of trauma to the vein area or excess hemorrhage.

Refer to your institution's IACUC and attending veterinarian for specific guidelines.

Rabbits may be housed singly or in pairs or groups, and in cages or pens. Minimum cage size requirements are specified by the Animal Welfare Act Regulations. Although metal cages can be used for rabbits, plastic cages may be preferred because they provide the animals a quieter, warmer environment. Rabbits are intelligent animals that easily become bored when caged. It is common, therefore, to provide rabbits with enrichment devices, e.g., nontoxic chew toys.

For reasons of disease transmission, rabbits should be separated from contact with:

• Other species, including guinea pigs, because of the potential for contagion across species. Example: Bordetella bronchiseptica is transmissable from rabbits to guinea pigs.
• Rabbits of different pathogen status. Specific pathogen free rabbits are at risk for infections and parasitic infestations transmitted from rabbits obtained from conventional sources.

Please refer to your animal facility staff for procedures to protect the health status of your rabbits.

Example Alternatives Search:

For additional information on types of rabbit housing, please refer to the examples of "alternatives" searches on rabbit housing.

Upon arrival at your facility, your rabbits should have an acclimation period before they are used in research studies. This period of time allows animals to adapt to a new environment. Effects of transportation stress include alterations in various blood parameters, immune cell function and animal behavior. The period of time necessary for biological stabilization will depend on the parameters to be studied. Refer to your institution's attending veterinarian for recommendations that are appropriate for your project. Typically, acclimation periods range from 4 days to 1 week.

Example Alternatives Search:
For citations on acclimation of rabbits, please refer to the example of "alternatives" searches provided.

Routine quarantine procedures may prolong the holding of your animals in special facilities. Quarantine aims to prevent transmission of diseases between new animals and established colonies.

Acclimation and quarantine periods run concurrently, although they serve different purposes. Most institutions do not allow experiments on animals while quarantined.

Though rabbits share many anatomical and physiological features with humans, rabbits have many unique biological characteristics. A knowledge of species-specific characteristics is helpful to effectively manage these animals and to plan experimental procedures for their use.

The photo shows a rabbit with jaw malalignment and incisor overgrowth.

Researchers should be aware of the following practical features of rabbit anatomy and biology. Click on the following items for a brief description and some practical tips.

Behavior

• Normal Behavior
• Aggression

Gastrointestinal

• Teeth
• Inability to vomit
• Dietary Fiber
• Obesity
• Coprophagy and digestive strategy
• Enteropathy

Infectious

• SPF vs non-SPF
• Sensitivity to antibiotics

Metabolic

• Albinism
• Drug clearances

Respiratory

• Endotracheal Intubation

Skeletal

• Spinal fracture

If your proposed study involves a painful procedure, the protocol form may ask for a method of assessing whether the rabbits are experiencing pain or distress.

Assessing pain and distress in rabbits is difficult at times because rabbits, like many other species, commonly conceal outward signs of even moderate pain and distress. In this case, the behavioral changes that reveal a rabbit's pain and distress may be subtle and detectable only with specialized behavioral methods.

Rabbits may also exhibit overt clinical signs of pain and distress, the more so when pain is more intense. Laboratory staff working with rabbits should be trained to recognize these abnormalities in:

• Activity level – hypoactivity, restlessness.
• Behavior – e.g., vocalization, self-trauma, aggressiveness, ataxia.
• Appearance – e.g., protruded third eyelid (nictitating membrane), hunched posture, cyanosis, pale mucous membranes, fecal-stained fur.
• Vital Signs – e.g., respiratory distress.
• Body Condition – e.g., weight loss, emaciation, dehydration.
• Intake – e.g., reduced intake of food and water.

A chronic state of pain or distress may be more subtle and difficult to detect. A good knowledge of the animal’s normal appearance and behavior is especially important to recognize chronic pain or distress.

Example Alternatives Search:
For citations on detecting pain and distress, please refer to the example of "alternatives" searches provided.

Below are volume recommendations for acute intravenous fluid administration and blood collection in rabbits.

IV fluid volume (ml) max. acute admin.	Tot. blood volume (ml)	Safe bleed volume (ml)a	Bleed-out volume (ml)b
10 ml/kg	57-65 ml/kg	7.7 ml/kg	31-310 ml

^a Removing greater quantities of blood (exceeding 10% of total blood volume) can produce hypovolemic shock. Repeated collections of smaller amounts of blood will have the same effect. In such procedures, animals should receive warmed, physiological fluids to replace the volume of blood collected. In addition, monitor the animal's hematocrit for anemia.

^b Animals should be exsanguinated only under anesthesia.

From:

1. Hawk and Leary, Formulary for Laboratory Animals, 2nd Edn., Iowa State University Press, 1999.
2. Wolfensohn and Lloyd, Handbook of Laboratory Animal Management and Welfare, 2nd Edn., Blackwell Science. 1998.

Below are peripheral vessels that are commonly accessed for blood collection or fluid administration. Recommended needle sizes are 23 to 25 gauge. Larger needles may be necessary for injecting large volumes or viscous materials.

Below are the nonvascular routes of injection that are commonly used in rabbits. Included are volume recommendations for the acute administration of fluids. Recommended needle sizes are 23 to 25 gauge; larger needles may be necessary for injecting viscous materials.

• Subcutaneous (SQ or SC): 30 - 50 ml (scruff, flank);
  limit 20 ml per injection site

• Intraperitoneal (IP): 50 - 100 ml

• Oral (PO): 5 ml

• Intradermal (ID): 0.1 ml/site

• Intramuscular (IM): 0.5 - 1.0 ml per site

From:

1. Hawk and Leary, Formulary for Laboratory Animals, 2nd Edn., Iowa State University Press, 1999.
2. Wolfensohn and Lloyd, Handbook of Laboratory Animal Management and Welfare, 2nd Edn., Blackwell Science. 1998.

For citations of injection and blood collection procedures in rabbits, please refer to the examples of "alternatives" searches provided.

When using any animal species for polyclonal antibody production, the issues below should be addressed within the animal protocol. For more detail, refer to the Institute for Laboratory Animal Research publication, ILAR Journal Volume 37(3) Adjuvants and Antibody Production, 1995.

Antigen Preparation
The antigen preparation should be free of extraneous microbial contamination and byproducts such as polyacrylamide gel. The protocol should describe how the antigen-adjuvant emulsion will be prepared.

Adjuvant Used
If the use of Freund's complete adjuvant (FCA) is proposed, your IACUC may require justification of this choice of adjuvant. FCA has been associated with granulomatous inflammation, focal necrosis, ulceration of skin, fistulous tracts, muscle atrophy, self-induced trauma, hypersensitivity reactions, and weight loss. The USDA states that the injection of FCA may cause more than momentary or slight pain. This means that FCA injections might place an animal into USDA pain category D (painful/stressful but relieved), requiring the use of post-injection analgesics. Check with your IACUC to determine your institution's policy.

Recommendations for FCA from the Institute of Laboratory Research are as follows:

1. FCA should be used only once, usually for the initial immunization.
2. Formulations of FCA should not exceed 0.1 mg dry mycobacterial cell mass/ml.
3. Less inflammatory alternatives to Freund's adjuvant are available and should be considered.

Booster Frequency
In common booster schedules, the initial and subsequent immunizations are spaced at intervals of two to three weeks (minimum). Booster immunizations may be delayed if significant inflammatory reactions are still present from the initial immunization.

Injection Site Selection and Preparation
Excerpted from the ILAR Journal, vol 37, issue 3 (Institutional Policies and Guidelines on Adjuvants and Antibody Production): "Anatomic sites used for grasping, handling, or restraint...should be avoided when possible. Extension of granulomatous inflammation into the spinal cord following inadvertent injection of a FCA-antigen mixture into the paraspinal musculature has been associated with posterior paresis in guinea pigs (Kleinman et al., 1993). Care therefore should be taken when making injections near the dorsal spinal column. Granulomas can also be noted in other organs after injections with FCA (Schiefer and Stunzi, 1979)."

Post-injection Observations
Your animal protocol should describe how animals will be monitored for post-injection lesions and how affected animals will be treated. Refer to the ILAR Journal for recommendations on post-injection observations.

Because rabbits have a high rate of metabolism, drugs are rapidly eliminated from their bodies. Dose rates appropriate for larger species produce ineffective drug concentrations when used in rabbits.

The next screen describes these agents and provides rabbit dose rates for the common drugs and drug regimens. If you need to use other drug agents, check with your institution’s veterinary staff for assistance in determining a dose rate appropriate for use in rabbits.

Click on the drug types for rabbit doses of common agents and drug regimens:

Analgesics:
Available in two drug types – the opioids and the nonsteroidal anti-inflammatory drugs (NSAIDs). The rapid clearance of many of these drugs in rabbits results in the need for an increased frequency of administration.

Sedatives:
Sedatives may obtund consciousness but in normal doses do not do so sufficiently to ablate the perception of pain or other sensations. When combined with general anesthetics, they may be used to induce a "balanced" anesthesia where muscle relaxation, unconsciousness, and analgesia are enhanced.

Sedatives + Analgesia:
Some sedatives also have analgesic effects. When combined with general anesthetics, a balanced anesthesia is attained, and these sedatives enhance analgesia through specific effects.

Anesthetics:
Because rabbits metabolize drugs so rapidly, many anesthetic agents have brief durations of effect. An anesthetic regimen should be chosen to match the duration of drug effects with the length of the procedure. In particular, short acting agents (and regimens) should be not be used for long procedures because repeat drug administrations, necessary to prolong anesthesia, will produce uneven blood concentrations and therefore periodically inadequate anesthesia. For long procedures, gaseous anesthesia is the often the most practical method to sustain uniformly adequate levels of anesthesia.

For additional information on analgesia and anesthesia in rabbits, including the physiological impact of these agents on rabbits, please refer to the examples of "alternatives" searches provided.

Aseptic technique should be used when performing major survival surgery on rabbits. This is required by the Animal Welfare Act (federal law). The standards described here are consistent with the Guide for the Care and Use of Laboratory Animals.

• Survival surgery means that the animal regains consciousness after anesthesia. In nonsurvival surgery, the animal is euthanized before recovery from anesthesia.

• Major surgery means penetrating and exposing a body cavity such as the chest or abdomen; or producing substantial physical or physiological impairment.

If you will be performing major survival surgery on rabbits, federal requirements are that:

• The surgery be carried out in a dedicated surgical facility.

• The surgeon must wear cap, mask, and sterile gown and gloves.

• The incision site is appropriately clipped, scrubbed, disinfected, and draped.

• Instruments and surgical materials are sterile.

• The animals receive proper supportive care and monitoring for anesthesia and vital signs through the procedure and afterward.

If you will be performing minor survival surgery, requirements are less stringent than outlined on the preceding page. According to the Guide, page 62, requirements still include: "aseptic technique and instruments and appropriate anesthesia. Although laparoscopic procedures are often performed on an 'outpatient' basis, appropriate aseptic technique is necessary if a body cavity is penetrated."

If you will be performing nonsurvival surgery, it may not be necessary to follow all the techniques outlined above for major survival surgery. According to the Guide, also on page 62, "at a minimum, the surgical site should be clipped, the surgeon should wear gloves, and the instruments and surrounding area should be clean."

Supportive care aims to:

• Maintain the animal's physiological status as nearly normal as possible.
• Minimize animal pain and distress.

Supportive care includes the monitoring of both physiological parameters and analgesia during anesthetic and surgical procedures. Monitoring of vital signs and pain should be conducted throughout the procedure and the recovery period.

Keep in mind that:

• General anesthesia causes disturbances of thermoregulation and other physiological functions.

• Warming devices (e.g., heated tables and pads) are recommended for routine use to maintain the animal's body temperature. Care should be taken to avoid devices that may cause thermal injury.

• During surgery, the animal may experience pain if anesthesia is inadequate at any time during the procedure.

• Postoperatively, the animal may experience pain, discomfort, and distress unless treated with analgesics and appropriate supportive care measures.

Due to the interaction of metabolic factors and drug effects that can cause animal mortality, rabbits should receive good supportive care and monitoring during anesthesia, whether or not the procedure involves surgery.

During anesthesia and surgery, the following procedures are recommended.

Supportive Care:

• Provide a source of warmth to a rabbit from the onset of anesthesia to the end of anesthetic recovery.
• Infuse sterile physiological fluid (warmed to body temperature) to compensate for blood loss during a procedure and depressed fluid intake post-procedure.

Monitoring during Anesthesia:

• Analgesia - toe pinch, ear pinch.
• Respiration - gross changes in rate, character of breathing.
• Color of mucous membrane and skin – poor oxygenation (blue), poor blood perfusion (pale)

After anesthesia and surgery, the following procedures are recommended.

Monitoring post Anesthesia:

• Rabbits must be monitored until fully recovered from anesthesia as indicated by the ability to ambulate and maintain core body temperature.

Monitoring post Procedure:

• Assess appearance, activity, and behavior as indications of pain and discomfort (see screen Detecting Pain and Distress).
• Assess food and water intake.
• Assess wound repair. Routine use of antibiotics is not indicated after uncomplicated aseptic surgery.

The term euthanasia is derived from Greek and means "good death." Animals should be euthanatized when killed for any purpose, including research. To euthanatize a rabbit, you must be trained in the concepts of euthanasia, the method to be used, and the proper handling of rabbits.

Methods are classified as acceptable or conditionally acceptable, as set by the American Veterinary Medical Association. The inclusion of conditionally acceptable methods in your protocol may require scientific justification and IACUC approval.

Click on each method of euthanasia below for recommendations on its use in euthanatizing rabbits.

Acceptable Methods:

• Barbiturates
• Inhalant anesthetics
• Carbon dioxide
• Carbon monoxide
• Potassium chloride in conjunction with general anesthesia

Conditionally Acceptable Methods:*

• Nitrogen
• Argon
• Cervical dislocation (rabbits < 1 kg)
• Decapitation
• Penetrating captive bolt

* The inclusion of conditionally acceptable methods in your protocol may require scientific justification and IACUC approval.

Example Alternatives Search:

For additional information on euthanasia of rabbits, including the impact of euthanasia agents on tissues, please refer to the examples of "alternatives" searches provided.

Federal Laws, Regulations, Policies:

1. Animal Welfare Act, as Amended (7 USC, 2131-2156)
2. Animal Welfare Act Regulations and Standards, Code of Federal Regulations, Title 9, Part 2-Regulations, Sections 2.31-2.33
3. Health Research Extension Act of 1985, Public Law 99-158, November 20, 1985, "Animals in Research".
4. Public Health Service Policy on Humane Care and Use of Laboratory Animals, Revised September, 1986, Reprinted March, 1996
5. USDA Animal and Plant Health Inspection Animal Care Policy Manual
   Policy #11 - Painful/Distressful Procedures
6. U.S. Government Principles For The Utilization And Care Of Vertebrate Animals Used In Testing, Research, And Training, Interagency Research Animal Committee

Guidelines:

1. Guide for the Care and Use of Laboratory Animals, National Research Council, 1998.
2. Occupational Health and Safety in the Care and Use of Research Animals, National Research Council, 1997.

Texts:

1. Peter R. Cheeke, Rabbit Feeding and Nutrition, Academic Press, 1987.
2. Hawk and Leary, Formulary for Laboratory Animals. 2nd Edn., Iowa State University Press, 1999.
3. Hrapkiewicz, Medina, and Holmes, Clinical Laboratory Animal Medicine: An Introduction, 2nd Edn., Iowa State University Press, 1998.
4. Suckow and Douglas. The Laboratory Rabbit. CRC Press, 1997.
5. Weisbroth, Flatt and Kraus, The Biology of the Laboratory Rabbit, Academic Press, 1974.
6. Wolfensohn and Lloyd, Handbook of Laboratory Animal Management and Welfare. 2nd Edn., Blackwell Science. 1998.