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INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCESDR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH

ANIMAL HOUSE

Animal care and use courses

INTRODUCTION TO RABBITS

Derived from

The American Association for Laboratory Animal Science(AALAS)

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Introduction to Rabbits

Lesson 1. Introduction

Welcome to the course Introduction to Rabbits. This is the rabbit module in a course series thatprovides an introduction to working with animals in a research environment.

Introduction - Goals

The goals of this course are to provide training for individuals working with this species in aresearch facility and/or to support the writing of an animal use protocol 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 regulatorysources, drug doses, practical tips, etc.

Lesson 2. Occupational Health Issues

Occupational Health and Safety

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

The IACUC, PI, supervisors, and personnel should all work together to ensure that everyonewho works with animals participates in the occupational health and safety program.

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Injuries and Allergies

Working with rabbits is associated with the following hazards:

Injuries

Personnel handling rabbits can be injured by bites (incisors) or scratches (especially by thehind 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 topeople and rabbits.

Allergies

Everyone working with laboratory animals should be enrolled in their institutionsoccupational health and safety program. This program will help you monitor anydevelopment of allergies, since it is possible for people working with rabbits to developallergies after some time.

It is essential that workers exposed to rabbits wear proper personal protective equipment(PPE), including scrubs or disposable gowns, gloves, and shoe covers. If you are alreadyallergic to rabbits a respirator (such as an N95 type) can be worn to protect from airborneallergens. Persons who develop allergy symptoms should seek medical counseling from theoccupational health and safety program. In severe cases, personnel may have to discontinueworking with this species.

Zoonoses

Zoonoses are diseases transmitted by animals to humans.

In general, transmission of zoonotic disease from laboratory animals is uncommon because ofongoing efforts by both facility staff and vendors to monitor and improve the health status ofanimals using reliable health surveillance programs. Experimentally infected animals are asource of zoonotic transmission to humans. Infected cell lines, animal tissues, and animal tissueproducts can also be sources. Also, contact with wild rabbits in field research may expose

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humans to zoonotic agents carried by this species. Health surveillance programs, routinesanitation, and personal protective equipment have important roles in preventing zoonoses.

Rabbits can be a reservoir of the following infectious agents which are transmissible to people.

Bacteria:

Francisella tularemia

Francisella tularensis (tularemia) typically causes sudden death in rabbits. The organism istransmitted by blood-sucking arthropods and the disease occurs mainly in wild rabbits, makingit a concern for wildlife researchers. Human transmission is by contact with blood and tissues ofaffected rabbits and through bites of infected ticks.

Tularemia is potentially fatal in humans. The disease manifests as cutaneous lesions,septicemia, and meningitis.

Leptospira spp.

Rabbits may be a reservoir for Leptospira spp. bacteria, which are shed in the urine.Transmission occurs by contact with urine and tissues, or inhalation or ingestion of aerosoldroplets.

Humans with leptospirosis may have influenza-like symptoms, orchitis, rash, skin and mucosalhemorrhage, hemolytic anemia, hepatorenal failure, jaundice, encephalitis, and pneumo

Yersinia pseudotuberulosis

Yersinia spp. occur in wild rabbits. These bacteria are transmitted by direct contact and thefecal-oral route.

Yersinia spp. are transmissable to humans and causes a gastroentercolitis syndromecharacterized by fever, diarrhea, and abdominal pain. Infection with Yersinia can causeulcerative mucosal lesions in the terminal ileum, mesenteric lymphadenitis, hepatosplenicabcesses, iritis, skin ulceration, osteomyelitis, septicemia, and postinfectious arthritis.

Fungi:

Microsporum spp., Trichophyton spp.

Dermatophytic fungi grow in the skin and hair follicles and cause a condition of reddened skinand patchy hair loss known as ringworm. The symptoms are the same in animals and humans.Infection may be inapparent in individual animals.

Dermatophytes are spread by direct contact. Fungal spores are long-lived and may become

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widely dispersed in the environment.

Lesson 3. Humane Standards

Humane Standards

All of the federal laws, regulations, policies, and guidelines applicable to animal research havea core requirement for 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 andafter a research procedure.

What is a procedure? A procedure is any activity carried out on the animal, such as bloodcollection, restricting food or water, administering experimental and control substances, orsurgery. This peri-procedural care requirement covers:

properly preparing the animal to humanely undergo the procedure;

supporting the animals physiological function during the procedure; and

providing additional supportive care to aid the animal in recovering from the procedure.

Staff Training

The investigator has the responsibility to see that staff working with the animals are properlytrained to not only perform the procedure humanely but also to provide the necessarysupportive care to the animals.

When performing any procedure, such as a blood collection, you should think through the steps

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that are necessary to protect the animal's welfare. For example, for blood collection, youshould limit the volume taken acutely or chronically. And, with any venipuncture, you shouldbe prepared to care for the animal in the event of trauma to the vein area or excess hemorrhage.

Lesson 4. Housing

Housing

Rabbits should be housed in pairs or groups in cages or pens. Adult animals may bedifficult to introduce for pair housing. Follow your institutional social housing SOP.

Minimum cage size requirements are specified by the Animal Welfare Regulations.Although metal cages can be used for rabbits, plastic cages may be preferred because theyprovide the animals a quieter, warmer environment. Rabbits are intelligent animals thateasily become bored when caged. Therefore they should be offered enrichment when thestudy allows.

Enrichment

Rabbits can be playful and will use a variety of environmental enrichment items. They seemto enjoy objects that they can nudge and toss around their cage, as well as hanging chains,triangles, or rattles. Nylon chew bones and rubber balls with bells are also an option.

Timothy hay cubes or autoclaved hay can be provided as a food supplement. Rabbits willalso eat a variety of fresh fruit, vegetables, and cereal.

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Housing and Disease Transmission

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

Other species, including guinea pigs, because of the potential for contagion acrossspecies. Example: Bordetella bronchiseptica is transmissible from rabbits to guineapigs.

Rabbits of different pathogen status. Specific pathogen free rabbits are at risk forinfections and parasitic infestations transmitted from rabbits obtained fromconventional sources.

Lesson 5. Acclimation & Quarantine

Acclimation

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

Quarantine

Routine quarantine procedures, if required, may prolong the holding of your animals inspecial facilities. Quarantine procedures are specific to the institution receiving the animals

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based on the origin of the animals, institutional policy, and health status of the animal coloniesat the institution. Quarantine aims to prevent transmission of diseases between new animalsand established colonies.

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

Lesson 6. Biological Features

Biological Features

Though rabbits share many anatomical and physiological features with humans, rabbits havemany unique biological characteristics. A knowledge of species-specific characteristics is helpfulto 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

Rabbits are very intelligent animals, a fact that is often overlooked when they are housed alonein cages. Rabbits often have a shy temperament and can be easily intimidated. When frightened,rabbits may freeze or hunch down, stomp their rear feet, or they may panic and bolt. However, iffeeling defensive when approached in the cage, a rabbit may attack and bite. Though often shy,rabbits can be forceful and will make noise by throwing toys around. Therefore, rabbits canbenefit from being housed in pairs and being provided with toys and objects to manipulate.

Aggression

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When you work with rabbits, be gentle and quiet; dont make sudden movements or speakloudly. If confronted with an aggressive rabbit in a cage, place a towel over the animal to cover itcompletely. This allows you to safely scoop the covered rabbit in your arms and remove it fromthe cage while supporting the hind end. Once out of the cage, most aggressive rabbits are easy tohandle and should be treated gently like any other rabbit.

Gastrointestinal

Teeth

Rabbits have teeth that are open rooted, meaning that these teeth grow continuously throughoutadult life. Particularly if the jaws are anatomically malaligned, a rabbits teeth have the potentialto overgrow; although the first incisors are most likely to overgrow, so may the second incisorsand the cheek teeth. Staff must be alert to detect any signs of this condition and to provideappropriate treatment.

Inability to vomit

Rabbits do not vomit because they lack the neurophysiological mechanisms for emesis.Presurgical fasting is not necessary in rabbits, as it is for larger laboratory species (carnivoresand omnivores).

Dietary Fiber

Rabbits are herbivores and hind gut fermenters, i.e., they digest plant materials in the cecum.Rabbits need higher dietary fiber than do rodents, especially for general maintenance (~22.5%).This is helpful also to reduce obesity in caged rabbits, to promote better digestion, to reduce theincidence of gastrointestinal disorders and inappetance. Supplementing with alfalfa or other haysis helpful to promote normal gut motility.

Low fiber diets will alter the production of cecotropes (a normal type of feces in rabbits) becauseof hypomotility of the hindgut and a prolonged retention time of material in the cecum.Hypomotility may lead to diarrhea or cecal impaction.

Obesity

Caged rabbits fed ad libitum commonly become overweight. To prevent obesity, rabbits aregenerally fed a high fiber diet in restricted amounts.

Coprophagy and digestive strategy

Rabbits are herbivores that have a unique strategy for digesting plant materials in the cecum. Therabbit excretes dietary fiber without expending energy to digest it. In the large intestine, dietaryfiber is separated from the digesta and excreted as pelleted feces, which accumulate on the cagepan. The nonfiber material (fluids and small particles) are returned to the cecum for further

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digestion and fermentation. After a period of hours (and generally at night), the cecum expelssome of its contents, which are passed as a type of feces known as cecotropes. The rabbitconsumes cecotropes directly from the anus. Stomach digestion and intestinal absorption of thisfecal material yields nutrients that are essential to the rabbit.

Elizabethan or cervical collars are sometimes used on rabbits to prevent them from reaching anddisturbing/dehiscing a surgical incision. Since a collar prevents the rabbit from bending over toreach the posterior area of the body, it also prevents the animal from consuming the expelledcecotropes. Therefore, the use of a collar should not be an automatic decision, and if used, acollar should be removed at the earliest opportunity.

Enteropathy

Rabbits are extremely vulnerable to microbial imbalances in the cecum and colon. Factors thatcan contribute toward developing an enteropathy are:

Infectious agents - Clostridium spiroforme and E. coli. Destabilization of gut microbialpopulations (e.g., due to pH changes or antibiotic treatment) leads to a proliferation of E.coli and Clostridium.

Dietary factors - Low fiber diets cause cecocolonic hypomotility. When fiber content islow, digestible carbohydrate overload causes an enterotoxemia. Associated factors areacidification of the cecum, overgrowth of E. coli and Clostridia, and reduced volatilefatty acid production.

Stress due to inhibition of intestinal motility. Common stress factors are environmentaltemperature change, dietary change, and transportation.

Infectious

SPF vs non-SPF

Specific pathogen free (SPF) rabbits are produced in isolation from specific rabbit pathogens andparasites. Rabbit sources vary in the pathogens that are absent from their animal stock, but SPFrabbits are commonly free of:

Bacteria -

Bordetella bronchiseptica, Pasteurella spp., Salmonella spp., Streptococcusmoniliformis, hemolytic Steptococci, Clostridium piliforme (Tyzzers disease).

Viruses -

Rabbit hemorrhagic disease virus, rabbit pox virus, rabbit rotavirus.

Because many facilities may have both SPF and non-SPF rabbit colonies, it is important that allstaff comply with facility procedures aimed at preventing contamination of the SPF rabbits.

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Metabolism

Albinism

The most common research rabbit is the New Zealand White, which is an albino breed. Albinismin rabbits is an inherited disorder of melanin metabolism caused by the lack of the enzymetyrosinase, which has an impact both on melanocytes and neurons. Neuronal morphologicalabnormalities and functional impairments involve the following sites: medial vestibular nucleus,cochlear nuclei, and retina. Studies comparing albino and pigmented animals have showndifferences even in pharmacotoxic kinetics in these tissue areas.

In the rabbit, there is a graded series of alleles at the C locus providing a step-wise reduction incolor. The genetics of albinism in rabbits has been used to study the physiological effects ofalbinism.

Drug clearances

The rabbit has a higher rate of metabolism than larger species, such as dogs, but less than thelaboratory rodents, such as mice and rats. Compared to these species, the rabbit has anintermediate rate for drug clearance from the body. Rabbits should receive drug doses that havebeen scaled to the rabbits metabolism. Through a discipline known as allometry, mathematicalformulas have been developed to adjust dose rates between species of disparate size.

In general, rabbit-specific dose rates have been determined and are widely published for drugsthat are commonly used in animal research, such as anesthetics, analgesics, sedatives, andantibiotics. Investigators are advised to obtain rabbit dose rates from laboratory animalreferences or from their institution's veterinary staff.

Respiratory System

Rabbits have a narrow oral cavity (glottis) that restricts visibility of the larynx for endotrachealintubation. A visual method of intubation is difficult because of the limited space for insertion ofa laryngoscope. However, the blind method of intubation is easy due to the simple anatomy ofthe rabbits oropharynx and laryngopharynx. Rabbits should be routinely intubated in the tracheawhen consistent with the anesthetic regimen.

Skeletal System

Spinal fracture

Rabbits housed in cages have bony hypoplasia and may have osteoporosis due to inactivityimposed by living in the restricted space of a cage. As a result, laboratory rabbits are known forthe fragility of their bones. The lumbar spine is most prone to injury and fracture. A forceful kick

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by the strong, muscular hind limbs is sufficient to cause a lumbar fracture (most commonly at theL7-S1 junction). Rabbits must be carefully handled when transported or restrained to preventthem from panicking and kicking out with their hind limbs. Rabbits with a spinal fracture willpresent as splayed and paralysed in rear limbs.

Note: This may be masked by the rabbit's habit of sitting motionless.

Lesson 7. Detecting Pain and Distress

Assessing Pain and Distress

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

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

Clinical Signs

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

Activity level

o Hypoactivityo Lethargyo Restlessness

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Behavior

o Vocalizationo Self-traumao Aggressivenesso Ataxia

Appearance

o Protruded third eyelid (nictitating membrane)o Hunched postureo Cyanosiso Pale mucous membraneso Fecal-stained fur

Vital signs

o Respiratory distress Body condition

o Weight losso Emaciationo Dehydration

Intake:

o Reduced intake of food and water

Chronic Pain and Distress

A chronic state of pain or distress may be more subtle and difficult to detect. A good knowledgeof the animals normal appearance and behavior is especially important to recognize chronic painor distress.

Lesson 8. Procedures for Injections and Blood Collection

Volume Recommendations

Below are volume recommendations for acute intravenous fluid administration and bloodcollection 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 producehypovolemic shock. Repeated collections of smaller amounts of blood will have the sameeffect. In such procedures, animals should receive warmed, physiological fluids to replace the

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volume of blood collected. In addition, monitor the animals hematocrit for anemia.

b Animals should be exsanguinated only under anesthesia.

From:

1. Hawk and Leary, Formulary for Laboratory Animals, 2nd Edn., Iowa State UniversityPress, 1999.

2. Wolfensohn and Lloyd, Handbook of Laboratory Animal Management and Welfare, 2ndEdn., Blackwell Science. 1998.

Peripheral Blood Vessels

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

Route ofVascular Access Comments

Ear vein

Ear artery

1. These methods may be performed withoutsedation, although sedation is helpful forvasodilation and chemical restraint.

2. Topical anesthetic formulations may be appliedto produce a local anesthesia.

Jugular vein

Lateral saphenous

Medial saphenous

1. These methods are typically used with sedationor anesthesia for chemical restraint.

2. If these veins must be accessed via a surgicalincision, the following methods will berequired: general anesthesia, aseptic technique,and peri-operative monitoring.

Cardiac puncture

1. Cardiac puncture requires anesthesia.2. Cardiac puncture is most often allowed only as

a terminal procedure. Check with yourinstitution for guidelines on this route of bloodcollection.

Nonvascular Injections

Below are the nonvascular routes of injection that are commonly used in rabbits. Included arevolume recommendations for the acute administration of fluids. Recommended needle sizes are

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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 UniversityPress, 1999.

2. Wolfensohn and Lloyd, Handbook of Laboratory Animal Management and Welfare, 2ndEdn., Blackwell Science. 1998.

Lesson 9. Polyclonal Antibody Production

Polyclonal Antibody Production: Antigens & Adjuvants

When using any animal species for polyclonal antibody production, the issues below shouldbe addressed within the animal protocol. For more detail, refer to the Institute for LaboratoryAnimal Research publication, ILAR Journal Volume 37(3) Adjuvants and AntibodyProduction, 1995.

Antigen Preparation

The antigen preparation should be free of extraneous microbial contamination and byproductssuch as polyacrylamide gel. The protocol should describe how the antigen-adjuvant emulsionwill be prepared.

Adjuvant UsedIf the use of Freund's complete adjuvant (FCA, also called CFA) is proposed, your IACUCmay require justification of this choice of adjuvant. FCA has been associated withgranulomatous inflammation, focal necrosis, ulceration of skin, fistulous tracts, muscleatrophy, self-induced trauma, hypersensitivity reactions, and weight loss. The USDA states

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that the injection of FCA may cause more than momentary or slight pain. This means thatFCA injections might place an animal into USDA pain category D (painful/stressful butrelieved), requiring the use of post-injection analgesics. Check with your IACUC todetermine 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.

Polyclonal Antibody Production: Immunizations

Booster Frequency

In common booster schedules, the initial and subsequent immunizations are spaced at intervalsof two to three weeks (minimum). Booster immunizations may be delayed if significantinflammatory 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 onAdjuvants and Antibody Production): "Anatomic sites used for grasping, handling, orrestraint...should be avoided when possible. Extension of granulomatous inflammation into thespinal cord following inadvertent injection of a FCA-antigen mixture into the paraspinalmusculature 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

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and how affected animals will be treated. Refer to the ILAR Journal for recommendations onpost-injection observations as follows:

ILAR Journal Volume 37(3) Adjuvants and Antibody Production, Institutional Policies andGuidelines on Adjuvants and Antibody Production, 1995:

"Investigators and veterinary staff should observe animals for evidence of pain or distress, andfor evidence of lesions such as swelling, abscess or fistula formation, and infection or ulcerationat the immunization sites. CCAC Guidelines recommend that observations should be made atleast 3 times weekly for a period of 4 weeks following immunization, or until all lesions havehealed. An immunization clinical incidence form which includes the agent, route, site or sites,volume, date of injection and the body weight of the animal on the injection date may be helpfulin maintaining careful data on individual animals. Veterinary follow-up, including clinicalobservations and palpations of the injected sites, and determination of the necessity for anysupportive therapy is strongly recommended. All guidelines should instruct investigators tocontact the veterinary staff if injection site lesions or evidence of pain or distress are identified inany animals. This will permit timely and appropriate assessment and institution of therapy whenrequired. Supportive therapy may include topical cleansing, antibiotic administration, analgesicadministration, or all three. Fluid replacement or nutritional supplements may also be required ifthe animals have sustained anorexia or decreased fluid intake.

"Although lesser in severity and frequency by comparison to Freund's adjuvant, inflammatorylesions can occasionally be observed following immunization with alternative adjuvants such asTiterMax or Ribi adjuvants. Inflammatory reactions, which include draining abscesses, havebeen observed 2-3 weeks following immunization with TiterMax adjuvant (Check et al., 1990).These lesions generally persisted for approximately 1 week and then gradually subsided. Itwould therefore seem prudent to recommend similar post-injection observations for animalsreceiving alternative adjuvants."

Lesson 10. Analgesics, Sedatives, and Anesthetics

Analgesics, Sedatives, and Anesthetics

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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 in thebloodstream when used in rabbits.

These agents are described below and rabbit dose rates for common drugs and drug regimensare provided. If you need to use other drug agents, check with your institutions veterinary stafffor assistance in determining a dose rate appropriate for use in rabbits.

Analgesics:

NSAIDs

Carprofen 4 mg/kg SC once daily Flunixin 1.1 mg/kg SC or IM twice daily Ketoprofen (NSAID) 1.0 mg/kg SQ once daily

Opioids

Buprenorphine 0.01 0.05 mg/kg SC q 8-12 hr Butorphanol 0.1 0.5 mg/kg IV q 4 hr

From:Wolfensohn and Lloyd, Handbook of Laboratory Animal Management and Welfare, 2nd Edn.,Blackwell Science, 1998.

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 anincreased frequency of administration.

Sedatives:

Acepromazine, diazepam, midazolam and zolazepam. Although categorized as a dissociativeanesthetic, ketamine in rabbits produces sedation and immobilization with negligible analgesia.

Acepromazine 1mg/kg SC (hypotensive) Diazepam or midazolam 1-2 mg/kg IV or SC Ketamine 50 mg/kg IM

These agents are commonly mixed with other drugs in an anesthetic cocktail. Refer to the pageaccessed from the Anesthetics link.

From:Wolfensohn and Lloyd, Handbook of Laboratory Animal Management and Welfare, 2nd Edn.,Blackwell Science, 1998.

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Sedatives have no effect on consciousness, the perception of pain or other sensations. Whencombined with general anesthetics, the result is a balanced anesthesia where muscle relaxation,unconsciousness, and analgesia are enhanced.

Sedatives + Analgesia:

Sedatives with analgesic effect: medetomedine and xylazine

Medetomidine 250 ug/kg SC or IMReverse with atipamezole 50 ug/kg IV

Xylazine 3 mg/kg SC or IM

For doses in combination with anesthetics, refer to the page accessed from the Anesthetics link.

From:Wolfensohn and Lloyd, Handbook of Laboratory Animal Management and Welfare, 2nd Edn.,Blackwell Science, 1998.These agents are commonly mixed with other drugs in an anestheticcocktail.

Some sedatives also have analgesic effects. When combined with general anesthetics, abalanced anesthesia is attained, and these sedatives enhance analgesia through specific effects.

Anesthetics:

Gaseous anesthetics provide the best means for long periods of anesthesia due to thecontinuous administration via inhalation. Since methoxyflurane is no longer available, and theuse of ether is not recommended, researchers have access only to those agents that are bestadministered via a vaporizer. Recommended agents: isoflurane and halothane.

Injectable anesthetics are generally used in a cocktail mixed with one or more sedatives andanalgesics. The anesthetic cocktails most commonly used on rabbits in the USA containketamine or tiletamine as the anesthetic agent. Bolus injections of anesthetic cocktail mayproduce a surgical level of anesthesia for periods ranging from 20 to 45 minutes, which is idealfor many surgical procedures commonly performed in rabbits, such as cannula implantation.The duration of surgical anesthesia depends on the drug agents. Repeated dosage of injectableagents to provide a long term anesthesia is not recommended because of the resultingfluctuations in systemic blood concentration and therefore in anesthetic effect. In suchsituations, the repeat doses of anesthetic cocktail are administered only when the animal beginsto show evidence of pain. This is a poor practice of anesthesia because a surgical plane ofanesthesia is not continuous throughout the procedure. Animal welfare mandates require anavoidance of such unnecessary animal pain and distress.

Examples:

1. Ketamine (10 mg/kg IM) and medetomidine (0.5 mg/kg IM)

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2. Ketamine (10 mg/kg IM) and xylazine (3 mg/kg IM)3. Ketamine (75 mg/kg IM) and acepromazine (5 mg/kg IM)4. Ketamine (25 mg/kg IM) and dizepam or midazolam (5 mg/kg IM)5. Propofol (0.2-0.6 mg/kg/min IV infusion)6. Induction agents for gaseous anesthesia:

o Propofol (10 mg/kg IV), oro Thiopental (30 mg/kg IV), oro Methohexital (10 mg/kg IV)

From:Wolfensohn and Lloyd, Handbook of Laboratory Animal Management and Welfare, 2nd Edn.,Blackwell Science, 1998

Because rabbits metabolize drugs so rapidly, many anesthetic agents have brief durations ofeffect. An anesthetic regimen should be chosen to match the duration of drug effects with thelength of the procedure. In particular, short acting agents (and regimens) should be not be usedfor long procedures because repeat drug administrations, necessary to prolong anesthesia, willproduce uneven blood concentrations and therefore periodically inadequate anesthesia. Forlong procedures, gaseous anesthesia is the often the most practical method to sustain uniformlyadequate levels of anesthesia.

Lesson 11. Surgery

Definitions

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

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

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

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Major Survival Surgery Requirements

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

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

The surgeon must wear a cap, mask, 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 vitalsigns through the procedure and afterward.

Note: Because rabbits cannot vomit, it is unnecessary (and usually not advised) to fast themprior to surgery.

Minor Procedures

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Minor survival surgery also requires the use of sterile instruments and aseptic technique. Minorsurgery does not expose a body cavity and causes little or no physical impairment. Minorsurgery on rabbits consists of procedures such as peripheral vessel cannulation, wound suturing,and percutaneous biopsy.

If you will be performing nonsurvival surgery, it may not be necessary to follow all thetechniques outlined above for major survival surgery. According to the Guide "at a minimum,the surgical site should be clipped, the surgeon should wear gloves, and the instruments andsurrounding area should be clean" (p 118).

Lesson 12. Supportive Care and Monitoring

Overview

Supportive care aims to:

Maintain the animal's physiological status as near normal as possible.

Minimize animal pain and distress.

Supportive care includes the monitoring of both physiological parameters and analgesia duringanesthetic and surgical procedures. Monitoring of vital signs and potential signs of pain shouldbe conducted throughout the procedure and the recovery period.

Key Concerns

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Keep in mind that:

General anesthesia causes disturbances of thermoregulation and other physiologicalfunctions. Some animals are unable to properly thermoregulate for a while after someprocedures, including anesthesia and surgery.

Warming devices (e.g., heated tables and pads) are recommended for routine use tomaintain the animal's body temperature.

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

Postoperatively, the animal may experience pain, discomfort, and distress unless treatedwith 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 theprocedure involves surgery.

Procedures During Anesthesia and Surgery

During anesthesia and surgery, the following procedures are strongly recommended.

Supportive care:

Provide a source of warmth to a rabbit from the onset of anesthesia to the end ofanesthetic recovery.

Infuse sterile physiological fluid (warmed) to compensate for blood loss during aprocedure and depressed fluid intake post-procedure. This should be performed in concertwith input from the veterinary staff.

These supportive measures should be included in the animal care and use protocol.

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)

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Procedures After Anesthesia and Surgery

After anesthesia and surgery, the following procedures are recommended.

Monitoring post anesthesia:

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

Monitoring post procedure:

Assess appearance, activity, and behavior as indications of pain and discomfort (seescreen Detecting Pain and Distress).

Assess food and water intake.

Assess wound healing.

Lesson 13. Euthanasia

Euthanasia

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The term euthanasia is derived from Greek and means "good death." Animals should beeuthanized when killed for any purpose, including research. To euthanize a rabbit, you must betrained 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 AmericanVeterinary Medical Association's document, the AVMA Guidelines for the Euthanasia ofAnimals. The inclusion of conditionally acceptable methods in your protocol may requirescientific justification and IACUC approval.

Methods

Acceptable:

Barbiturates:

Excerpted from the AVMA Guidelines for the Euthanasia of Animals -

"Intravenous injection of a barbituric acid derivative is the preferred method for euthanasia indogs, cats, and other small companion animals."

Acceptable with Conditions:

Inhalant Anesthetics

Excerpted from the AVMA Guidelines for the Euthanasia of Animals

"Inhaled anesthetics are acceptable with conditions for euthanasia of small animals (< 7 kg)where the following contingencies can be met: (1) In those species where aversion or overtescape behaviors have not been noted, exposure to high concentrations resulting in rapid loss of

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consciousness is preferred. Otherwise, gradual fill methods can be used, keeping in mind theeffect that chamber volume, flow rate, and anesthetic concentration will have on the timeconstant and rate of rise of anesthetic concentration. Inhaled anesthetics can be administered asthe sole euthanasia agent or as part of a 2-step process, where animals are first renderedunconscious through inhaled anesthetic agent exposure and then subsequently killed by asecondary method. (2) Order of preference is isoflurane, halothane, sevoflurane, enflurane,methoxyflurane, and desflurane, with or without N2O. Nitrous oxide should not be used alone.Methoxyflurane is acceptable with conditions only if other agents or methods are not available.Ether is not acceptable for euthanasia."

Carbon Dioxide

Reformatted from the AVMA Guidelines for the Euthanasia of Animals

The use of CO2 to euthanize mice is acceptable with conditions; precautions must be taken toensure the least amount of stress to the animals prior to death. The only source of CO2 that isacceptable is from a compressed gas cylinder (some institutions have CO2 piped in through theplumbing, which is acceptable since it originates from a compressed gas tank that is regulated).The tank must have a pressure-reducing regulator and flow meter. An optimal flow rate for CO2euthanasia systems should displace 10% to 30% of the chamber or cage volume/min.

When CO2 is introduced to an uncrowded chamber from a compressed gas cylinder slowly, itmixes with the room air and anesthetizes the animals before they are asphyxiated. Thisprevents the following situations, which should be avoided:

Climbing or jumping to avoid exposure to CO2 Severe irritation to the mucous membranes Prolonged distress due to fluctuating levels of CO2 in the chamber

After animals are anesthetized and asphyxiated, gas flow should be maintained for at least 1minute after apparent clinical death. It is important to verify that an animal is dead beforeremoving it from the chamber. If an animal is not dead another method of euthanasia should beused."

Potassium chloride in conjunction with prior general anesthesia

Reformatted from the AVMA Guidelines for the Euthanasia of Animals

"Saturated potassium chloride solutions are effective in causing cardiac arrest following rapidintracardiac or intravenous injection.

"It is of utmost importance that personnel performing this technique are trained andknowledgeable in anesthetic techniques, and are competent in assessing anesthetic depthappropriate for administration of potassium chloride intravenously.

"Administration of potassium chloride intravenously requires animals to be in a surgical plane

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of anesthesia characterized by loss of consciousness, loss of reflex muscle response, and loss ofresponse to noxious stimuli."

Cervical Dislocation

Excerpted from the AVMA Guidelines for the Euthanasia of Animals

"Manual cervical dislocation is acceptable with conditions for euthanasia of small birds, poultry,mice, rats weighing < 200 g, and rabbits when performed by individuals with a demonstratedhigh degree of technical proficiency. In lieu of demonstrated technical competency, animals mustbe unconscious or anesthetized prior to cervical dislocation. For heavy rats and rabbits, the largemuscle mass in the cervical region makes manual cervical dislocation physically more difficult."

"Those responsible for the use of this technique must ensure that personnel performing cervicaldislocation techniques have been properly trained and consistently apply it humanely andeffectively."

Penetrating captive bolt

Excerpted from the AVMA Guidelines for the Euthanasia of Animals

"The use of rabbit-sized penetrating captive bolts to euthanize rabbits in laboratory or productionfacilities is acceptable with conditions. The captive bolt must be maintained in clean workingorder, positioned correctly, and operated safely by trained personnel."

American Veterinary Medical Association, AVMA Guidelines for the Euthanasiaof Animals

Working Party Report (Europe), Recommendations for Euthanasia ofExperimental AnimalsParts 1 and 2