The University of New Mexico

1
The University of New Mexico The Office of
Animal Care and Compliance

• present
• Basic Biomethodology for Laboratory Rabbits
• A learning module developed and presented by the
  OACC

2
Introduction

• This module was developed to assist you in
  becoming proficient in performing common
  techniques in the Rabbit

3
General Information

• We all have an ethical responsibility to animals
  in terms of minimizing pain and distress
• This can be accomplished, in part, by using
  proper animal handling and experimental
  techniques
• The PHS requires that animal care and use is
  based on the Guide for the Care and Use of
  Laboratory Animals
• Personnel caring for animals should be
  appropriately trained

4
Entrance Procedures

• Access to most UNM animal facilities is by
  card-key
• Sharing of card-keys could result in termination
  of your access privileges
• Animal users are not provided with access to the
  animal facility until all training requirements
  are met

5
Personal Protective Equipment

• All animal facilities require some level of
  protective clothing in order to protect the
  animals housed within from contaminants that may
  enter the facility via the personnel and to
  protect the personnel from exposure to animal
  allergens or other potential hazards

6
Examples of Protective Clothing

• Lab Coats
• Jumpsuits
• Shoe Covers
• Hair Bonnets
• Masks
• Gloves

7
Animal Records

• All animals used in research require some kind of
  record and identification, even if it is only a
  cage card

• Cage cards should include protocol number,
  investigator, species, strain, age/wt, sex,
  vendor, and date received

8
Rabbit Cage Technique

• This is a typical rabbit caging rack used in
  research facilities today all stainless steel
  construction is another popular system

• The large cage area required for the animals
  well-being also allows the handler easy access to
  the animal
• Litter pans are located beneath the animal and
  may be changed without disturbing the animal

9
Rabbit Cage Technique

• Approach the animal with confidence, avoiding
  sudden movement and loud noises
• Sudden movement or loud noises will startle the
  animal they may thump their hind feet on the
  cage floor in protest

• Open the cage door fully to allow full access to
  the animal
• Slowly reach into the cage and place your hand on
  the rabbits neck just behind the ears

10
Rabbit Cage Technique

• Grasp the animal firmly but gently by the nape of
  neck DO NOT grasp by the ears
• Place your other hand under the animals hind
  quarters to support its weight

• Pull the rabbit from the cage and rotate your
  hands slightly so that the animals hind quarters
  are beneath it next to your abdomen
• Your hand holding the nape of the neck should be
  directly over the hindquarters and against your
  chest

11
Rabbit Cage Technique

• From this position, you can transfer the animal
  to another cage or restrain the animal for
  transport
• To transfer to another cage simply place the
  animal in the cage and remove your hand from
  under its hind quarters while still holding the
  nape of the neck

• Once the animals rear feet are on the cage floor,
  release the nape of the neck

12
Rabbit Cage Technique

• To restrain the animal for transport, tuck the
  head under your arm like a football
• Covering a rabbits eyes is the best way to calm
  them down

13
Assessing the General Health of Rabbits

• A brief assessment of the health of every animal
  should be conducted prior to performing any
  technical procedures

• The animal should be observed for signs of
  illness including
• Ocular or nasal discharge
• Rough hair coat
• Abnormal posture
• Uterine, rectal or penile prolapse
• Limb abnormalities
• Malocclusion
• Dehydration
• Dystocia
• Abnormal behavior

• Example of Middle Ear Infection, or Head Tilt

• Example of Malocclusion

These photos compliments of Cornell University
14
Assessing the General Health of Rabbits

• Any signs of pain or distress should be reported
  immediately
• Observe the feed and water supplies to ensure
  that there is evidence that the animal has been
  eating and drinking
• Stressed animals may perform self mutilation
  this can also result from an improper IM injection

• Ear Mites can also affect rabbits

These photos compliments of Cornell University
15
Sexing Rabbits

• It is important that every animal handler be
  properly trained to distinguish between male and
  female rabbits
• Restraining the animal correctly is very important

• For larger/older rabbits, you must cradle the
  animal in one arm while holding the nape of the
  neck

• Please note that you should be wearing gloves
  anytime you are handling an animal

• For smaller/younger rabbits, you can cradle the
  animal in the one hand that is holding the nape
  of the neck

16
Sexing Rabbits

• While restraining the animal in dorsal recumbency
  with one hand, you must gently palpate the
  genital area to expose the genitalia
• Apply gentle pressure to the area and the
  genitalia should protrude from the fur

FEMALE MALE

• Please note that you should be wearing gloves
  anytime you are handling an animal

17
Sexing Rabbits

• Young rabbits can be very difficult to sex, it is
  sometimes helpful to compare them side by side

• These are a 4 week old male and female (buck and
  doe)
• Note how similar they look

• In adult rabbits, the penis is more pronounced,
  as is the slit in the vagina

• These are a 9 month old male and female (buck and
  doe)
• Note the differentiation of the penis and the
  vaginal slit

• Please note that you should be wearing gloves
  anytime you are handling an animal

18
Rabbit Behavior

• Rabbits are gentle animals, and if care is taken,
  become accustomed to handling if they are not
  upset by the experience
• Inappropriate handling can result in toenail
  scratches to the handler from the toe nails on
  the powerful rear legs
• Rabbits are herbivores and have the unique
  characteristic of re-ingesting soft fecal pellets
  directly from the anus - this process of
  coprophagy" allows the acquisition of B vitamins
  that have been produced by microorganisms in the
  cecum
• Environmental enrichment devices should also be
  used as appropriate, but must be approved by the
  Attending Veterinarian prior to use

19
Restraint

• When attempting to restrain rabbits, sudden,
  jerky moves should be avoided to decrease the
  likelihood of injury
• Approaching with gentle confidence is best
• Select the appropriate method of restraint for
  the procedure you wish to perform
• If the rabbit begins to struggle violently, it
  should immediately be placed on a solid surface
  and calmed
• Struggling frequently leads to fracture of lumbar
  vertebrae and injury to the spinal cord that may
  necessitate euthanasia

20
Manual Restraint

• There is predominately ONE method of restraint in
  rabbits aside from mechanical or chemical
• The rules are
• Support the animals weight
• Cover the animals eyes
• Restrain the hind legs

• Restraining the hind legs can be accomplished by
  holding the animal tight against your body or
  placing the animal on a hard surface and applying
  firm but gentle downward pressure to the hind end
• If the hind legs are not secured, the animal can
  kick and injure itself or the handler

21
Precautions

• Monitor the condition of the animal the entire
  time it is restrained
• Observe the breathing rate and color of the ears,
  nose and oral cavity
• Release the animal immediately if there are any
  signs of gasping or change in coloring from pink
  to blue

22
Mechanical Restrainers

• Several types of devices are commercially
  available to restrain rabbits
• Quality devices prevent the animal from turning
  around or twisting yet allow easy access to the
  head and ears
• Care should be taken when placing a rabbit in a
  restraint device since struggling may result in
  damage to the spine
• Struggling is reduced if the device snugly
  secures both the head, back and hindlegs

23
Mechanical Restrainers

• Because of the potential for injury to the animal
  and the skill required to correctly place the
  rabbit in a commercial restrainer, this procedure
  must be demonstrated live by a trained individual
  
• Please contact the ARF to arrange for training in
  the procedure

24
Identification Methods

• It is important to select the appropriate
  identification method for your research purposes
• This should be based upon the age of the animal,
  the number of characters you wish to include, and
  the duration of the experiment
• You should record the identification information
  on the cage card in the event that clarification
  of the numbers or characters becomes necessary
  for any reason

25
Identification Methods

• Indelible markers can be used for short-term
  identification
• Ear tattoos are the most common form of permanent
  identification of rabbits
• There are 2 types of rabbit tattoo devices
• The Pen Type (electric/battery operated)
• The Manual Type (pliers)
• Microchip transponders can also be used

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2
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Temporary Identification

• Non-toxic, permanent markers can be used to
  temporarily mark the fur, ear, or skin of the
  animal
• This ink usually lasts 3-4 days without the need
  to remark

27
Microchip Transponders

• Often called Pit Tagging, they are implanted
  subcutaneously between the scapulae for permanent
  identification of individual animals
• Each microchip is encrypted with a unique,
  non-replicable number and are read with a
  portable, hand-held scanner

28
Microchip Transponder

• To implant these chips, the rabbit should be
  restrained in a mechanical restrainer
• The hair is removed from the insertion site by
  shaving
• The area is prepped with an iodophor, followed by
  alcohol
• The implantation needle, with the syringe
  attached, is purchased in a sterile package
• Make a tent from the loose skin at the implant
  site

29
Microchip Transponders

• Insert the needle subcutaneously, with the bevel
  up, and depress the plunger

Note that the bevel is facing up

• Once the needle is removed the injection site
  should be observed for bleeding
• If bleeding is noted, apply pressure with a
  sterile gauze pad, or a drop of super glue to the
  entry site

30
Tattooing

• Tattooing should be used on animals over 6 wks of
  age as a permanent method of identification
• Anesthesia is not required, but can be used to
  immobilize the animal
• A tattoo device can be used to write numbers or
  other characters in the ears of rabbits
• Tattooing can be done with either a pen type or
  manual type tattoo device

31
Tattooing

• The use of tattoo equipment requires further
  hands-on training
• All tattoo equipment should be disinfected prior
  to use and sterilized between animal rooms

32
Injections

• Various routes exist for injecting rabbits
• Discuss the appropriate route, volume, site and
  needle selection with the veterinarian
• All injections must be described in your approved
  protocol
• All injections must be performed using sterile
  needles and syringes
• A new needle and syringe should be used for each
  rabbit

33
Intramuscular Injections - IM

• Regardless of the method used for IM injections,
  it must be noted that the sciatic nerve runs
  along the length of the femur
• It is very important to avoid injuring this nerve
• This is best accomplished by only injecting into
  the quadriceps muscle
• It is imperative that the rabbit is properly
  restrained this procedure requires hands-on
  training by ARF personnel
• Swab the area with 70 ethanol before placing the
  needle and aspirate to look for blood before
  injecting

34
Intramuscular Injections - IM

• Target injection site the Quadriceps Femoris
  (lateral thigh)

• Because of the risk of injury to the sciatic
  nerve, injection into the Biceps Femoris (caudal
  thigh) can only be performed by EXPERIENCED
  PERSONNEL

35
Intramuscular Injections - IM

• Restrain the rabbit as described earlier
• Swab the area with 70 ethanol
• Insert the needle, bevel up, into the quadriceps
  at a 45 angle
• Aspirate to ensure that you have not entered a
  blood vessel then slowly inject the material

• A 1 inch, 22 or 23ga needle should be used

• This image demonstrates injection into the caudal
  muscle and is only to be performed by experienced
  personnel

36
Subcutaneous Injections SC or SQ

• Restrain the rabbit in a restrainer or as
  described earlier
• Prep the area with 70 ethanol
• Use the thumb and forefinger to make a tent of
  skin over the back
• Fold the skin over the needle
• Aspirate to ensure proper placement and inject
  the material
• Do not use the area between the scapulae as this
  is where you will regularly grasp the animal

The area most frequently used is along the back
or side. Clean the skin with alcohol, clip or
part the hair, and fold the skin over the needle
rather than thrusting the needle into the skin.
37
Intraperitoneal Injections IP

• A state of "hypnosis" or tonic immobility can be
  induced in rabbits and is used for the IP
  injection
• Proper hands-on training is required to learn IP
  injection in the rabbit
• Please contact the ARF to receive this training

38
Intravenous Injections IV

• The most common site for IV injections is the
  marginal ear veins
• Because the rabbits ear veins are so fragile it
  is recommended that this procedure be done under
  anesthesia
• Locate a marginal ear vein and remove the hair
• Gentle stroking and tapping of the ear may make
  the vein more visible

Marginal ear veins

• The animal can be restrained in a mechanical
  restrainer for this procedure

39
Intravenous Injections IV
Marginal Ear Vein
Central Artery

• Disinfect injection site and insert needle into
  the vein at a slight angle
• You will not be able to aspirate, instead inject
  slowly and watch for clearing of the lumen
• Incorrect positioning will result in a slight
  bulge in the ear - if this occurs, remove needle
  and repeat process proximal to previous site
• Upon completion remove needle and apply pressure
  to injection site

40
Intravenous Injections IV

• Prep the site with 70 ethanol
• Attempt the injection starting at the middle or
  slightly distal part of the ear
• With the ear under tension insert the needle,
  bevel up, approximately parallel to the vein and
  insert the needle at lest 3 mm into the vein
• DO NOT ASPIRATE this will cause the vein to
  collapse
• Inject the material in a slow, fluid motion

• Begin injection here

41
Intradermal Injections ID

• In order to perform ID injections the animal
  should be anesthetized
• Shave an injection site on the back of the animal
  to remove the hair
• Swab the site with 70 ethanol
• Insert the needle into the skin, bevel up,
  holding the needle nearly parallel to the plane
  of the skin
• Do not aspirate
• Inject the material
• A properly performed ID injection should result
  in a small, round skin welt

42
Injection Sites and Volumes

• SQ in the Back or sides Maximum 30-50 ml
  20-23ga needle
• IM Lateral Thigh (quadriceps) 0.5 ml
  22-23ga needle
• IP Lower Ventral Quadrants 50-100 ml
  19-20ga needle
• ID Lateral Abdomen/Thorax 0.05 ml 25ga
  needle
• IV Marginal Ear Vein 5 ml 20-25ga needle

43
Oral Gavage

• Gavaging is used to dose an animal with a
  specified volume of material directly into its
  stomach
• Only a specialized, commercially available
  gastric feeding tube should be used for this
  procedure
• A mouth speculum used in concert with a rubber
  tube (4 mm diameter) can be used to administer
  compounds
• Flexible or plastic tubes may be bitten or chewed
  and some care must be taken to prevent this - a
  tongue depressor with a centrally placed hole,
  when placed on end behind the diastema will serve
  as a satisfactory speculum
• Proper hands-on training is required to learn
  gavage dosing in the rabbit
• Please contact the ARF to receive this training

44
Oral Gavage

• First, measure the tube from the tip of the nose
  to the last rib - this is how far you must insert
  the tube
• Restrain the rabbit, ideally in a box restraint

Make sure you measure the gavage tube for proper
length
45
Oral Gavage

• Lubricate the tube with sterile lubricant
• In most cases, introduction of the tube toward
  the rear of the mouth will induce swallowing and
  the tube will readily enter the esophagus
• At this time straighten out the tube and continue
  to pass
• In the event resistance is met, DO NOT FORCE as
  this can cause serious injury
• The most common place to meet restriction is in
  the back of the throat the tube should pass
  freely when the rabbit swallows
• A violent reaction (coughing, gasping) usually
  follows accidental introduction of the tube into
  the larynx or trachea

46
Oral Gavage

• Check for proper insertion by injecting a few
  milliliters of water and aspirate for the
  presence of greenish-brown stomach contents
• If correct placement is achieved, inject solution
• Upon completion remove the tube by pinching the
  end of the tube and gently removing the tube
• Pinching the end of the tube prevents contents in
  the tube from draining into the oral-pharyngeal
  region
• When the procedure is complete, observe the
  rabbit for signs of distress such as gasping or
  frothing at the mouth

47
Blood Collection

• It is important to select the proper method of
  blood collection that corresponds to the volume
  required for your research purposes
• Some methods are intended for survival and others
  are not
• Consult the veterinarian for more information

Typical Blood Collection Sites Includes the
Following

• Marginal Ear Vein Survival
• Central Ear Artery Survival
• Cardiocentesis (cardiac puncture) Non-Survival

48
Marginal Ear Vein

• The marginal ear vein of the rabbit is useful for
  collection of small volumes of blood
• Sedation with acetylpromazine (1 mg) will allow
  handling of the animal and causes vasodilation
• Blood collection is fairly simple at this site
• The area is shaved and cleaned with alcohol

Marginal Ear Vein
49
Marginal Ear Vein

• The vein is occluded, the needle carefully
  inserted, and blood slowly withdrawn
• Use of a butterfly set may avoid damage to the
  vessel if the animal moves
• You may also nick the vessel with a scalpel
  blade and let the blood drip into the collection
  tube
• Gauze held with pressure over the venipuncture
  site for a few minutes will prevent hematomas
  from forming

50
Central Ear Artery

• The central ear artery can be used for collecting
  larger volumes of blood from the rabbit
• Phlebotomy from the central ear artery requires
  the use of a plain 20 ga. one inch needle
  attached to a silicone-coated tube
• Alternatively, cutting the hub off a 20 gauge
  needle and collecting the flowing blood into a
  tube will decrease the time till coagulation of
  the needle
• Ten ml of blood/kg body weight can be collected
  in this manner but the rabbit must be carefully
  restrained and hematomas must be prevented by
  direct pressure

51
Central Ear Artery

• Prior sedation of the rabbit will minimize
  distress and movement while promoting
  vasodilation

Central Ear Artery
52
Central Ear Artery

• Within 1 minute of administration of
  acetylpromazine (1 mg) the central ear artery
  should dilate
• The central ear artery has a bounding pulse and
  generally runs cranial to (ahead of) the central
  vein
• Swab the artery with alcohol and catheterize with
  20 or 21 gauge needle pointed towards the base of
  the ear

53
Central Ear Artery

• Advance the needle approximately 5 mm into lumen
  of artery
• When using the needle with hub removed the
  technician can release the needle which will
  remain in the vessel during phlebotomy

54
Central Ear Artery

• When blood collection is completed, withdraw the
  needled from the ear artery and apply firm
  pressure to the puncture site for at least one
  minute

55
Cardiocentesis - Cardiac Puncture

• This procedure must be performed under deep
  anesthesia and is a NON-SURVIVAL procedure
• There are several methods for performing this
  procedure and all require hands-on training
• Method 1 Syringe and needle rabbit in dorsal
  recumbency
• Method 2 Blood donor set and vacuum tubes
• Method 3 Bleeding needle and vacuum tube
  rabbit in lateral recumbency

56
Cardiac Puncture

• Syringe and needle with rabbit in dorsal
  recumbency is a very common method
• Once the animal is anesthetized, prep the chest
  with 70 ethanol
• Insert the needle at the base of the sternum,
  bevel up, into the thoracic cavity at a 15-20
  angle directed just to the left of midline

• Aspirate slowly
• If blood begins to flow into the syringe,
  continue to aspirate with steady, even pressure
• If no blood is seen reposition the needle and try
  again

57
Cardiac Puncture

• Once the required blood volume is collected, the
  rabbit is euthanized while still deeply
  anesthetized
• Up to 120 milliliters or more of blood may be
  collected from an adult rabbit using this method
• The problem with this method is removing the
  syringe to empty it into your collection tube
  the needle may become dislodged from the heart

58
Cardiac Puncture

• A blood donor set with vacuum tubes is the best
  method for this procedure but requires two
  persons to perform it
• Once the animal is anesthetized, prep the chest
  with 70 ethanol
• Insert the needle at the base of the sternum,
  bevel up, into the thoracic cavity at a 15-20
  angle directed just to the left of midline
• When you have entered the heart, blood will
  immediately begin to flow into the donor set
  tubing

59
Cardiac Puncture

• When the blood flow has reached the end of
  tubing, the second person must insert it into a
  vacuum tube
• As each tube fills the second person must move
  the donor set to the next empty tube
• Be sure to pinch off the tubing as you relocate
  the needle or blood will be lost

This needle is inserted into the vacuum
tubes Note the finger holds
This needle is inserted into the heart
60
Cardiac Puncture

• The person holding the needle in the heart can
  view the blood flow into the collection tubes and
  adjust the needle tip position slightly to
  maintain good flow
• This method is very much a collaborative effort

This needle is inserted into the heart
This needle is inserted into the vacuum tubes
61
Cardiac Puncture

• Once the required blood volume is collected, the
  rabbit is euthanized while still deeply
  anesthetized
• Up to 150 milliliters or more of blood may be
  collected from an adult rabbit using this method
• The problem with this method is that it requires
  two persons

62
Cardiac Puncture

• Bleeding needle and vacuum tube with rabbit in
  lateral recumbency is a less common, but equally
  effective method
• Once the animal is anesthetized, the point of
  maximum intensity of heartbeat should be
  determine while palpating the laterally recumbent
  rabbit
• Prep the side of the chest with 70 ethanol
• An 18 gauge 1.5 inch bleeding needle attached to
  a vacuum tube should be directed into the heart
  at the point of maximum intensity
• Blood will flow into the needle when the heart
  chamber is entered

63
Cardiac Puncture

• Once the required blood volume is collected, the
  rabbit is euthanized while still deeply
  anesthetized
• Up to 100 milliliters or more of blood may be
  collected from an adult rabbit using this method
• The problem with this method is changing
  collection tubes

64
Anesthesia/Analgesia

• This module will provide a brief introduction to
  analgesia and anesthesia in the rabbit
• Your veterinarian or ARF personnel should always
  be consulted for advice on selection and
  administration of analgesia or anesthesia
• The use of analgesics and/or anesthetics must be
  described in your approved animal use protocol
• There is a drug formulary on the OACC website
  that lists drugs, dosages, and uses for various
  species

65
Anesthesia/Analgesia

• Injectables used for anesthesia and analgesia
• Typically given IP or IM but may be given SQ
• It is important to weigh the rabbit prior to
  dosing with an injectable anesthetic to avoid
  over or under dosing the animal

66
Anesthesia/Analgesia

• Topicals as an adjunct to, or in lieu of
  injectable analgesics, topical anesthetics may
  also be used
• These long-acting agents are painted or dropped
  into the surgical wound before the skin is closed

67
Anesthesia/Analgesia

• Inhalants the most commonly used inhalant at
  UNM is isoflurane
• Isoflurane is administered in 10002 - induction
  concentrations of isoflurane are 3-4 and
  maintenance concentrations are 1.25-1.75
• Inhalant anesthetics must be used with a
  scavenging device
• Contact the veterinarian for further training in
  the appropriate use of the anesthesia machines
  available at UNM

68
Monitoring

• Anesthetized animals must be monitored closely
  during the procedure to assure that they are
  maintained in the proper anesthetic plane
• If the plane is too light the animals may move or
  struggle
• If the plane is too deep the animals may die
• The plane can be assessed by pinching the toe,
  tail, or ear of the animals
• Any reaction from the animal indicates that the
  anesthesia is too light and additional anesthesia
  should be given

69
Monitoring

• The respiration and color of the mucous membranes
  and exposed tissue of the animal should also be
  closely monitored
• The respiration rate should be even
• An increase in respiration indicates that the
  anesthesia is too light
• A deep, shallow, decreased or irregular
  respiration indicates that the anesthesia is too
  deep

70
Monitoring

• The color of the mucous membranes and exposed
  tissues should be bright pink to red
• Dusky grey or blue color is indicative of an
  anesthetic plane that is too deep
• Core body temperature can also be monitored in
  rabbits the most common anesthetic complication
  is hypothermia
• Measures must be taken to control the body
  temperature during and after anesthesia

71
Recovery

• Place the animal on a clean, dry pad or towel to
  avoid contact with the cage floor
• Once the animal has reached sternal recumbency
  and appears to making a normal recovery, it may
  be returned to the animal holding area
• Animals should be watched for several days
  following a procedure

72
Euthanasia

• The definition of euthanasia is the intentional
  induction of a painless death
• The veterinarian should always be consulted for
  advice on selection and administration of
  euthanasia agents
• The euthanasia method must be fully described in
  your approved animal use protocol

73
Euthanasia Injectable Inhalant

• Injectable anesthetics can also be used for
  euthanasia when administered at higher doses
• Barbituate anesthetics produce rapid and humane
  euthanasia when injected IP or IV
• The use of a commercially available euthanasia
  solution is the preferred method for euthanasia
  in rabbits
• Halothane is the most effective inhalant
  anesthetic for euthanasia, but isoflurane can
  also be used
• Inhalants can only be utilized with the use of an
  anesthesia machine

74
This Concludes Module 4 Basic Biomethodology
for Laboratory Rabbits

• Please download the exam, complete it, then
  e-mail it to KMirowsky_at_salud.unm.edu