University of Wisconsin-Madison

Techniques for Animal Experimentation

Blood Collection

• anatomic sites vary for species and volume needed. Blood collection for various species is taught by RARC in its biomethodology classes (for classes offered, see Appendix)
• cardiac puncture is done only as a terminal procedure and always under anesthesia
• guidelines for maximum volumes of blood collection
  • up to 10% of blood volume collected once weekly
  • up to 20% of blood volume collected once monthly
  • over 20% of blood volume should only be collected as a terminal procedure, under anesthesia or consult with your Attending Veterinarian - can do this survival if you replace washed red cells and fluid
• in larger animals that may have repeated blood draws over time, the animal's red cell indices can be monitored to be sure overcollection is not a problem
• indwelling vascular catheters can make repeated blood sampling easier on the animal and the investigator when repeated sampling is needed
• factors to consider:
  • minimum volume required for experimental purposes
  • maximum volume safely withdrawn
  • repeat vs. terminal sampling
  • experience of personnel
  • personality, age, and health of animal
  • how blood will be used

Approximate Total Blood/Plasma Volumes of Different Species

Note: Estimations of total intravascular blood volume for a species can vary widely with the measurement technique employed, the age and the body condition (lean vs. obese) of the individuals being assayed. These values are approximations, so estimate blood volumes conservatively for the safety of the animal.

Source: Adapted from Formulary for Laboratory Animals, Hawk and Leary, 1999

For example: To estimate a safe volume of blood that can be collected weekly from a 25g mouse (assume a milliliter of blood weighs 1.0g)
25g x 79ml/1000g = 25g x 0.079ml/g = 1.98 ml total blood volume
1.98 ml x 10% ~ 0.2 ml of blood (200µl)

Recommended Blood Collection Sites

* preferred site
website with excellent pictures demonstrating the saphenous/metatarsal vein puncture technique at: http://www.uib.no/vivariet/mou_blood/Blood_coll_mice_.html
cardiac puncture is a terminal procedure and MUST be done under anesthesia if you don't see your species here, consult with your Attending Veterinarian Source: Adapted from Formulary for Laboratory Animals, Hawk and Leary, 1999

Injection Sites and Volumes

(these are guidelines only; research protocols may differ considerably) (G = gauge of needle)

Source: Adapted from Formulary for Laboratory Animals, Hawk and Leary, 1999

For all species, the recommended volume list for oral gavage is 5 ml/kg.

Surgery

Standards for Aseptic Surgery in Non-Rodents

• must have a dedicated surgical suite with functional components consisting of
  • surgical support (place to clean surgical packs, autoclave, etc)
  • animal preparation area (for shaving and scrubbing animal)
  • surgeon's scrub area
  • actual operating room
  • postoperative recovery area
• preparation of the animal for surgery includes
  • shaving or plucking of hair
  • scrubbing with surgical scrub
  • final "rinse" with alcohol/povidone iodine solution
• preparation of the surgeon includes
  • surgical scrub
  • mask, booties
  • sterile gown and gloves
• all instruments, supplies, and implanted materials must be sterile for each individual animal surgery
  • sterilization can be by autoclave, gas, liquid chemical sterilant
• aseptic technique must be used during the surgery to minimize the possibility of the introduction of infection
  • anything that touches the animal must be sterile
  • gentle tissue handling to minimize tissue damage
  • antibiotics do NOT replace aseptic techniques
• documentation of post-operative recovery monitoring is essential
• wound clips, sutures, staples, etc., must be removed from animals within 14 days of the surgery (document this too)
• exceptions or deviations from the above standards must be scientifically justified and approved by your ACUC
• contact your Attending Veterinarian for acceptable surgical facilities on campus, and for further help in developing your surgical protocols

Standards for Aseptic Surgery in Rodents

• dedicated surgery suite is not required, but a clean uncluttered area not used for other purposes during the surgery is necessary
• start with sterile instruments (autoclaved, gas-sterilized); maintain sterility of instruments using a glass bead sterilizer (available on loan from RARC--see Appendix) or by cold sterilization in appropriate solutions (i.e., Clidox), with a rinse in sterile water or saline
• preparation of the animal for surgery includes
  • shaving or plucking of hair
  • scrubbing with povidone iodine
  • "rinse" with alcohol, being careful not to chill the animal
• preparation of the surgeon includes
  • surgical scrub
  • gloves
  • mask (when possible)
  • clean lab coat or surgical scrubs
• anything touching the incision site or inside the incision site must be sterile
• maintenance of normal body temperature during surgery and recovery is vital to survival in small animals
• after any anesthetic procedure, animals must be monitored until normal righting reflexes return--make sure you DOCUMENT the monitoring!
• wound clips, staples, sutures, etc., must be removed from animals within 14 days postoperatively (document this too)
• exceptions or deviations from the above standards must be scientifically justified and approved by your ACUC

Surgically Modified Rodents

• available from many commercial sources, specific pathogen-free (can save time, money and aggravation)
• if your desired surgical modification is not listed below, contact the vendor--many of them can do procedures that are not specifically listed (see Appendix for phone numbers and website addresses)
  • Charles River Laboratories
  • Harlan Sprague Dawley
  • Jackson Laboratories
  • Taconic
• surgical modifications available include
  • thyroparathyroidectomy*
  • parathyroidectomy*
  • thyroidectomy with parathyroid transplant
  • adrenalectomy*
  • adrenal demedullation
  • hypophysectomy
  • pinealectomy
  • splenectomy
  • hepatectomy 70%
  • nephrectomy*
  • thymectomy (adult, adolescent, or neonate)
  • olfactory bulb ablation
  • subdiaphragmatic vagotomy
  • nodoal ganglionectomy (unilateral)
  • splanchnic nerve resection
  • superior cervical ganglionectomy
  • vasectomy
  • castration
  • oophorohysterectomy
  • hysterectomy
  • ovariectomy
  • ureter ligation
  • submaxillary salivary duct ligation
  • orbital enucleation
  • pituitary gland transplant to renal capsule*
  • subcutaneous implants
  • skin grafts *special care for transport and housing is necessary

Antibody Production

• the complete UW-Madison policy for antibody production in mammals is provided in the Appendix
• the use of Freund's complete antigen (FCA) should be avoided if at all possible; possible alternatives include:
  • incomplete Freund's adjuvant (IFA)
  • TiterMax® www.titermax.com800/345-2987
  • Ribi Adjuvant systems
  • 800/548-RIBI
  • Lipovant™
  • Adjuvax™
  • Alhydrogel™
• when FCA is used, it should be used only for the initial immunization. IFA or other adjuvants can be used for subsequent immunizations.
• injection volumes
  • intradermal or subcutaneous
    • <0.1 ml per site in rabbits
    • <0.05 ml per site in mice
  • intraperitoneal
    • up to 0.5 ml in mice
    • NOT an acceptable route in rabbits
• live animals should not be used for production of monoclonal antibodies unless in vitro techniques cannot be used (i.e., try in vitro first). For further information and links see http://grants.nih.gov/grants/olaw/references/dc98-01.htm, which is the text of the "Dear Colleague" letter sent out by OPRR (now OLAW) concerning antibody production. http://www.nal.usda.gov/awic/pubs/antibody/ and http://www.nal.usda.gov/awic/pubs/antibody/company.htm have valuable links for information concerning in vitro technologies
• ascites production
  • requires clinical observation of the animals be done at least daily
  • use of anesthesia and aseptic technique for abdominal paracentesis is recommended
  • a maximum of 2 or 3 abdominal paracenteses--euthanasia precedes the final paracentesis
  • an 18-20 gauge needle is recommended for paracentesis
  • fluid replacement for removed ascitic fluid is recommended to help prevent shock caused by fluid loss

Assessment and Alleviation of Pain and Distress in Animals

• "stress" is a normal feature of life for all animals (including us--just ask any graduate student), and serves important adaptive functions, such as for flight-or-fight, predation, or "social-climbing"
• "distress" occurs when the animal is unable to adapt completely to a stressor (scientific definition)
• distress may be manifested by some behaviors and by biochemical and physiological changes, although some animals "hide" fear and distress
• behavioral, biochemical, and physiological changes indicative of distress can markedly affect research data; therefore, preventing distress in research animals is of prime importance, from both a humane and a research standpoint
• there are many possible stressors for animals
  • inadequate space and overcrowding
  • social hierarchy
  • social deprivation
  • lack of environmental conditions needed to display species-specific behaviors (i.e., gnawing for rats, exercise for dogs)
  • inappropriate handling and restraint
  • noise
  • odors
  • pheromones
  • fear
  • high-intensity lighting
  • irregularities in temperature, humidity, and light cycles
  • weaning
  • diet and feeding schedules
  • disease
  • injury
  • surgery
  • experimental procedures
  • stuff we haven't thought of yet
• not all of these stressors can be "treated" with drugs, so planning experiments to reduce stress on the animals is of paramount importance. Something as simple as preconditioning a rat to accept handling can reduce stress considerably.
• response of an animal to a stressor can vary, depending on
  • age
  • sex
  • past experience (i.e., handling, previous experimental procedures)
  • genetic profile
  • physiologic state
  • psychological state
• ways to reduce or eliminate distress
  • careful attention to animal husbandry (i.e., light, temperature, humidity, caging, bedding, etc.)
  • provision of species-appropriate environmental enrichment (in some species, this is mandated by law)
  • social housing for social species
  • training (of people AND animals)
  • gentle, quiet handling
  • limiting the numbers of stressors imposed on an animal
  • anesthetics, analgesics, and anti-inflammatory drugs for intra- and post-operative or experimentally-induced pain
• although we cannot know exactly what an animal perceives as "painful," the rule of thumb is to consider any stimulus we humans would consider painful as also painful to animals
• use of anesthetics and analgesics
  • required by law, unless withholding them is scientifically justified and ACUC-approved
  • should be planned in consultation with your veterinarian while writing protocols
  • use of paralytic drugs is not permitted without anesthesia
  • "surgical plane of anesthesia" = unconsciousness, immobility, and analgesia
  • anesthetized animals must be monitored and the monitoring recorded
  • there is NO one "perfect" anesthetic for all animals
  • choice of anesthetic depends on
    • species
    • procedure
    • available equipment
    • expertise with anesthetic regimen
    • goals of the experiment

Contact Numbers

• many useful anesthetics and analgesics (and euthanasia compounds) are controlled substances, requiring licensure from the Wisconsin State Controlled Substances Board and the Drug Enforcement Administration (DEA) in order to purchase and possess them
  • Wisconsin State Controlled Substance Board
    • general information - 608/266-7586
    • request forms - 608/267-9883
    • State of Wisconsin, Department of Regulation and Licensing website - http://www.drl.state.wi.us/
  • DEA
    • Milwaukee area office - 414/297-3395; this is the Diversion Investigator number (make SURE you tell them you are a scientist, not a practitioner--the application forms are different)
• a downloadable text of the schedules of controlled substances is available at: http://www.usdoj.gov/dea/pubs/schedule.pdf
• your veterinarian is your single best resource for information about anesthetics and analgesics

Clinical and Physiological Signs of Pain in Laboratory Animals

Reprinted from Rollins and Kessel. The Experimental Animal in Research, Vol. 1., CRC Press 1990.

Species Specific Behavioral Signs of Pain

Reprinted from Rollins and Kessel. The Experimental Animal in Research, Vol. 1, CRC Press, 1990.

Alternatives to the Use of Live Vertebrate Animals

• in order to minimize the use of animals in research, UW-Madison is committed to "the Three R's"
  • Reduction--using the least number of animals necessary for statistically valid scientific results
  • Replacement--using non-animal alternatives (i.e., cell culture) or choosing a species lower on the phylogenetic tree (i.e., mice instead of monkeys)
  • Refinement--choosing methods and experimental procedures that minimize pain and distress in research animals (i.e., using laparoscopic techniques rather than laparotomy; defining endpoints as early in a disease process as possible)
• investigators must consider alternatives to animal use (mandated by the AWA) and we have a question in our animal use protocol reflecting this
• resources for alternative/refined experimental methods
  • remember to check other databases besides Medline (i.e., Agricola), and don't forget to actually search for "alternatives" as a keyword
  • Animal Welfare Information Center (AWIC) - http://www.nal.usda.gov/awic/
  • AltWeb--Alternatives to Animal Testing on the Web - http://altweb.jhsph.edu/
  • Canadian Council on Animal Care (CCAC) - http://www.ccac.ca/