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

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 animal’s 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 rabbit’s 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 animal’s hind quarters to support its weight • Pull the rabbit from the cage and rotate your hands slightly so that the animal’s 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 rabbit’s 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 animal’s weight • Cover the animal’s 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 1 2

26. 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 • A 1 inch, 22 or 23ga needle should be used • 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 • 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 rabbit’s 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