Veterinary Medical Care


Preventive Medicine

Animal procurement: Newly acquired animals can introduce disease into established colonies. In addition, production colonies maintained by suppliers occasionally experience outbreaks of disease. The Veterinary Service Center (VSC) monitors animal health quality from different suppliers and maintains quality control data provided by vendors. This information can be provided to investigators to assist in choosing appropriate sources of animals.

Quarantine and stabilization: With some species of laboratory animals, quarantine is necessary to minimize the introduction of disease into established colonies. The extent of the quarantine period is determined by the species and by knowledge of the animal's source and previous history as well as by regulatory requirements. For example, nonhuman primates must undergo a state mandated quarantine period of no less than 33 days. Arriving animals, regardless of source, should be allowed a stabilization period before use. Such a period allows the animal to recover from shipping stress, adapt to its new surroundings and become physiologically stable. A minimum acclimation period of 72 hours is suggested for all species.

Separation of species: Physical separation of animals by species is generally recommended to reduce the possibility of transmission of latent diseases and to prevent possible inter-species aggression or distress. This separation is usually accomplished by housing different species in separate rooms. Even when animals of the same species are obtained from multiple sources, their microbiological status may differ, in which case separate housing as provided by barrier caging or separate rooms may be advisable.


Surveillance, Diagnosis, Treatment and Control of Disease

A comprehensive veterinary medical program is in effect for all animals maintained at Stanford University. VSC personnel check animals in VSC maintained rooms daily, including weekends and holidays, for signs of illness, injury, or abnormal behavior. In cases where such observation will interfere with experimental objectives, prior arrangements must be made with the VSC to ensure adequate monitoring of animals and environmental systems.

To ensure the health status of animals maintained on campus, general medical surveillance procedures have been developed for each species. For example, in-house health monitoring is done for rodents as part of a sentinel monitoring program and is in effect in all VSC managed facilities. Serology, bacteriology, and parasitology samples are periodically taken from each sentinel cage and submitted to our in-house diagnostic laboratory for mouse or rat comprehensive testing. If evidence of murine viruses, parasites, or bacterial pathogens are discovered and confirmed in the sentinel animals, investigators are notified.

Health surveillance information, including information provided to us by our vendors, is maintained on file and available to investigative staff upon request. For more information on the surveillance, diagnosis, treatment, and control of animal diseases, including the possible affects of disease on experimental animal models, contact the Veterinary Service Center (723-3876).


Emergency Care

Veterinary care is provided 24 hours a day, 7 days a week. Any health problem noted by any animal user at any time, including evenings, weekends and holidays, should be reported immediately to the veterinary staff (723-4408). If the veterinary technician is not immediately available, a detailed message including the animal's identification, room number where it is located, the species, nature of the clinical problem, and the telephone number of the person making the report, should be left. In the case of an emergency, please listen to the message for paging instructions.


Anesthesia and Analgesia

Animal procedures are reviewed by both the VSC and the Administrative Panel on Laboratory Animal Care (A-PLAC) to ensure that proposed anesthetics and/or analgesics are appropriate for the species and research objectives. The VSC veterinary staff is available upon written request to provide assistance with, or training in the proper administration and use of anesthetics.

Written documentation of all surgical procedures, including the types, amounts, and time of administration of anesthetic, analgesic or tranquilizing drugs used and the physiologic parameters (i.e., heart rate, respiratory rate, body temperature, etc.) monitored during the procedure, is required. This documentation is subject to inspection by the USDA veterinary inspectors and the A-PLAC during its semiannual inspections of animal facilities and animal study areas. In addition, all manipulations and drug use should be recorded in the individual animal's record or the investigator's experimental notebook.

The ILAR Guide for the Care and Use of Laboratory Animals requires that any proposal to conduct painful procedures without anesthesia or analgesia must be scientifically justified by the investigator and approved by the institutional animal care and use committee. Such procedures must be directly supervised by the responsible investigator.

Obtaining Anesthetics/Analgesics

Research use of selected drugs and precursor chemicals are regulated by the Federal Drug Enforcement Administration (DEA) and California Department of Justice (CA-DOJ). Some of the drugs commonly used in animal research, such as Ketamine, are controlled substances and fall under the jurisdiction of DEA and CA-DOJ regulations. The Stanford Controlled Substances & Chemical Precursor Chemicals Program is managed by the office of Environmental Health and Safety (EH&S). For more information about the program, instructions on how to order controlled drugs, and to download order forms or recordkeeping documents, go to the EH&S web site:

Other drugs used for animal research, such as isoflurane and xylazine, are not controlled substances, and can be obtained through the Veterinary Service Center (VSC) Pharmacy. To place an order, complete a Pharmacy Request Form found on the door to the VSC Pharmacy located in RAF AF03C&D, and leave it in the bin on the door. For questions about VSC pharmacy orders, contact the Veterinary Care Unit at 725-3887.

Personnel Safety When Working With Anesthetic Gases

Exposure to anesthetic gases (e.g. Isoflurane, Halothane) can cause adverse health effects. These include behavioral modification, physical impairment, or headaches from exposure to sub-anesthetic concentrations, as well as reproductive or hepatotoxic effects from chronic exposure. Therefore, it is important that when working with anesthetic gases personnel exposure is reduced by ensuring the following:

  • Working in a vented fume hood or use proper gas delivery (i.e. precision vaporizer) and scavenging equipment (e.g. F-Air charcoal canister). Gas must not be vented into the room. All equipment must be maintained according to the manufacturers recommendations and documentation of proper maintenance retained in the laboratory. For recommendations regarding the proper equipment for gas anesthesia please contact the VSC.
  • Ensure a tight seal around the animal’s face when using an anesthetic mask.
  • Inspect and clean anesthetic masks, nose cones, and induction chambers immediately before and after use to ensure a proper fit and working condition with a suitable disinfectant (e.g. Alcide) to prevent cross-contamination.
  • All personnel must be properly trained in the use of anesthetic gases.

If you have concerns about exposure to anesthetic gases you should contact Environmental Health and Safety (723-7487) to arrange to have your laboratory and personnel monitored.

Suggested dosage for pre-anesthetics, tranquilizers, anesthetics, and analgesics are provided at the end of this section. Portions of the format and content of these dosage tables are based on a document originally compiled and edited by Pam Eisele, D.V.M. of the University of California, Davis. Additional guidelines on rodent survival surgery, surgical anesthetic monitoring, etc., are available from the A-PLAC (723-4550).



Surgery and Postoperative Care

Survival surgery: Survival surgery is defined as any surgery from which the animal recovers consciousness. Major surgery is defined as any surgical intervention that penetrates a body cavity or has the potential for producing a permanent handicap in an animal that is expected to recover. Minor surgery is any operative procedure in which only skin or mucous membrane is incised (e.g., vascular cutdown for catheter placement or implanting pumps in subcutaneous tissue). Because they are minimally invasive, gonadectomies on rodents and lower vertebrates are usually considered minor surgical procedures. Multiple major survival surgery is defined as two or more major survival surgical procedures performed at separate times on a single animal. It is permitted by the A-PLAC only under special circumstances, such as when the surgeries are essential and related components of a single scientific study. Cost alone is not an adequate reason for performing multiple major survival surgeries on an animal.

Major surgical procedures on mammals other than rodents must be conducted in VSC and A-PLAC approved surgical facilities intended for that purpose, using aseptic techniques. These techniques include wearing sterile surgical gloves, gowns, caps and face masks; using sterile supplies and instruments; and maintaining an aseptically prepared surgical field.

Minor surgical procedures on mammals other than rodents may be performed in a suitably located and equipped laboratory area, subject to approval by the VSC and the A-PLAC. Appropriate aseptic technique for these procedures includes a clean uncluttered work area, preparation of the surgical site including clipping of the hair, disinfection of the skin and draping of the surgical site with sterile drapes; the use of sterile supplies and instruments; and the use of sterile gloves and a surgical mask by the surgeon and any assistants working in the surgical field.

Surgical Procedures on rodent and non-mammalian species may also be conducted under the above conditions in laboratories or animal facility procedure rooms. The A-PLAC has written guidelines on rodent survival surgery and surgical monitoring. Contact the A-PLAC (723-4550) for copies of these guidelines.

Pre- and Postoperative care: Animals (other than some rodents) should generally be fasted prior to anesthesia and surgery to prevent vomiting, aspiration, and problems associated with a distended intestinal tract. Animals should be evaluated by performing a brief physical examination and recording baseline physiologic measurements of such parameters as body temperature, heart rate, and respiratory rate prior to the administration of an anesthetic agent. Animals should be weighed and dosages of agents administered calculated individually according to body weight measurements.

Postsurgical care includes clinical observation of the animal to ensure uneventful recovery from anesthesia and surgery. An intensive care unit is maintained by the VSC for the purpose of recovering anesthetized animals, and VSC postoperative care is available for a minimal fee. Once the animal has been returned to its normal housing area, subsequent care may be necessary. This may include supportive fluids, analgesics, and other drugs as required; monitoring of the animal to include daily body temperatures, clinical observations for signs of pain, abnormal behavior, appetite and excretory functions, and providing adequate care of surgical incisions. The investigator is responsible for supportive care unless arrangements have been made to contract VSC veterinary staff for these services. Written post-operative records including date, time, person making the observations, condition of animals, and any treatments/procedures performed should be maintained for inspection by USDA, A-PLAC, VSC, or other regulatory inspections. For examples of VSC surgical record forms and postoperative regimens, contact 723-4408.

Non-survival surgery: Non-survival surgery is defined as any surgery in which the animal will not regain consciousness. Such procedures may be performed in a suitably located and equipped laboratory, subject to VSC and A-PLAC evaluation and approval.

For more information regarding the use of VSC survival surgical and intensive care facilities, contact the Animal Health Technician Office (725-3887). In addition, the A-PLAC has available written guidelines on a variety of topics including rodent survival surgery and surgical monitoring. Visit the APLAC web site ( http://labanimals.stanford.edu/) and select "Guidelines" to download copies of these guidelines.


Euthanasia

Euthanasia is generally performed at the end of a project or, if possible, during a procedure in which animals experience severe or chronic pain or distress that cannot be relieved (9 CFR 2.31; PHS, 1986). Since there may be a need to euthanize animals for unanticipated reasons even on protocols that do not include euthanasia as part of the planned project, at least one method must be documented for each species used in a protocol. The euthanasia method chosen must be appropriate for the species and research use described by the protocol, and must be consistent with the recommendations of the American Veterinary Medical Association Panel on Euthanasia (2000, or succeeding revised editions). If the method deviates from AVMA recommendations, the deviation must be justified scientifically and approved by the A-PLAC. Euthanasia should be performed quickly and efficiently in a nonpublic area but generally not in rooms in which animals are housed.

This document describes the methods of animal euthanasia that are recommended for Stanford research projects. Additional methodologies may be approved under certain circumstances. VSC staff are available to demonstrate and/or discuss these techniques. Part 1 lists methods by species, while Part 2 includes a general discussion of each method. Conditionally acceptable methods must be scientifically justified by the Principal Investigator and approved by the A-PLAC.


Part 1
Recommended Agents and Methods of Euthanasia Listed by Species

AMPHIBIANS

  • Inhalant anesthetics
  • CO2
  • Barbiturates
  • Tricaine methane sulfonate (MS222)
  • Double pithing
  • Benzocaine
  • Conditionally acceptable - Single pithing; stunning and decapitation; decapitation and pithing

BIRDS

  • Inhalant anesthetics
  • CO2
  • Barbiturates
  • Conditionally acceptable: cervical dislocation; decapitation

CATS

  • Inhalant anesthetics
  • CO2
  • Barbiturates
  • Potassium chloride (under general anesthesia)

DOGS

  • Inhalant anesthetics
  • CO2
  • Barbiturates
  • Potassium chloride (under general anesthesia)

FISH

  • Tricaine methane sulfonate (MS222)
  • Benzocaine
  • Barbiturates
  • Inhalant anesthetics
  • CO2
  • 2-phenoxyethanol
  • Conditionally acceptable: stunning followed by decapitation/pithing; decapitation and pithing

NONHUMAN PRIMATES

  • Barbiturates
  • Potassium chloride (under general anesthesia)
  • Conditionally acceptable: inhalant anesthetics; CO2

RABBITS

  • Inhalant anesthetics
  • CO2
  • Barbiturates
  • Potassium chloride (under general anesthesia)
  • Exanguination (under general anesthesia)
  • Conditionally acceptable: cervical dislocation (< 1 kg); decapitation

REPTILES

  • Barbiturates
  • Inhalant anesthetics (in appropriate species)
  • CO2 (in appropriate species)
  • Conditionally acceptable - stunning and decapitation; decapitation and pithing

RATS, MICE AND OTHER SMALL MAMMALS

  • Inhalant anesthetics (halothane, isoflurane)
  • CO2
  • Barbiturates
  • Conditionally acceptable: methoxyflurane; cervical dislocation (< 200 g); decapitation

RUMINANTS

  • Barbiturates
  • Potassium chloride in conjunction with general anesthesia

SWINE

  • Barbiturates
  • Potassium chloride in conjunction with general anesthesia
  • Conditionally acceptable: inhalant anesthetics

Part 2
Comments on Recommended Agents and Methods of Euthanasia
  • INHALANT ANESTHETICS - Because in the liquid state most inhalant anesthetics act as topical irritants, animals should be exposed to the vapors of the anesthetic only. Air or oxygen must be provided during the induction period. All agents are given "to effect" until respiratory and cardiac arrest occurs.

    • Halothane and isoflurane have the most rapid action, and since halothane is better tolerated, it is preferred. Methoxyflurane is less suitable, due to its slow effect and poor market availability. Care should be taken to minimize personnel exposure to vapors.
    • Ether is acceptable but not recommended because it poses an explosive hazard and is a respiratory irritant that is considered stressful to animals. It cannot be used in the Stanford centralized facilities, and special precautions must be taken when used elsewhere. Administration should be performed in a fume hood, and signs indicating that ether is present or in use should be posted conspicuously. To avoid explosions, the carcasses of ether-killed animals should be stored in explosion-safe refrigerators or freezers, and should not be incinerated until the ether is removed by aeration in a hood. Methoxyflurane is a similar but non-explosive and less irritating agent which is recommended as a substitute for ether.
  • NON-ANESTHETIC GASES - Most agents in this category require the use of special equipment.
    • CO2 - Carbon dioxide is the preferred technique for euthanizing rodents and other small laboratory animals. Use of a sealed chamber filled by a compressed gas cylinder is required. CO2 generated by other methods, such as from dry ice, is unacceptable because gas flow can't be regulated precisely. Chambers should not be overcrowded. CO2 concentration of 70% or more should be utilized for euthanasia. Because CO2 can act as a reversible anesthetic, it is imperative that the animals be kept in the chamber for several minutes after respiratory arrest. Where possible, death should be verified by absence of a palpable heart beat. Due to physiologic characteristics, neonates require prolonged exposure to the gas. For more information, see the A-PLAC guideline on using CO2 for rodent euthanasia.
    • Nitrogen or carbon monoxide may be acceptable but are not recommended and require special equipment.
  • PHARMACOLOGICAL AGENTS - Use of these agents requires adequate restraint and mastery of appropriate injection techniques.

    • Barbiturates such as pentobarbital are acceptable for mammalian species and birds. These drugs should be administered intravenously (IV) except in rodents where intraperitoneal (IP) administration is an acceptable alternative. Sodium pentobarbital (Nembutal) is the most common barbiturate agent for euthanasia. The dosage is usually at least twice that required for anesthesia, and ranges from 85 mg/kg for larger species to 200 mg/kg for some rodents. A dosage of 120 mg/kg is sufficient for most species, but more should be given if death does not ensue. Commercial barbiturate euthanasia formulations as are also appropriate, and should be used following label directions (e.g., 1 ml/lb for Beuthanasia-D*). Sodium pentobarbital is a Class II drug which is regulated by the Drug Enforcement Agency. Personnel using this agent are required to store it in a locked location and maintain records which include the date and amount of use.
    • Chloral hydrate is not recommended, but may be used in ruminants and swine when administered I.V. at 900 mg/kg, but only after sedation with another drug.
    • Neither magnesium sulfate nor potassium chloride (KCl) can be used as a sole agent of euthanasia. Overdose with KCl is permissible in an anesthetized animal. Concentrated KCl should be given rapidly IV until rising serum potassium levels result in cardiac arrest.
    • Tricaine methane sulfonate (MS222) can be used either as an injectable agent (200-300 mg/kg of a 1% buffered solution) or as an immersion bath (2 mg/ml in H2O) for amphibians and fish. The immersion time needed to assure death can range from 20 minutes to three hours, so it may be advantageous to use MS222 as an anesthetic followed by a physical method of euthanasia. Benzocaine immersion (100-200 mg/liter H2O) is also acceptable. Note: Cutaneous exposure to MS222 can cause retinal toxicity. Gloves should be worn at all times when handling fish and amphibians, and in particular when using MS222.
    • Neuromuscular blocking drugs are absolutely condemned for use as euthanasia agents.
  • PHYSICAL METHODS - These methods require that the user have experience and skill in the techniques to be used.

    • Exsanguination is acceptable for all species if animal is first rendered unconscious by another agent.
    • Cervical dislocation is acceptable for mice, birds, rats (< 200 gm) and rabbits (< 1 Kg), but proper technique is essential. It is therefore recommended that animals be first sedated with another agent (carbon dioxide, pentobarbital or halothane are suggested). Its use as a sole means of euthanasia requires scientific justification and A-PLAC approval. For more information, see the A-PLAC guideline on the use of cervical dislocation for euthanasia of rodents.
    • Decapitation with proper equipment may be performed on small mammals or birds after the animal has been sedated or lightly anesthetized (carbon dioxide, pentobarbital or halothane are suggested). Decapitation of fish, amphibians and reptiles should be followed by pithing. Use as a sole means of euthanasia in any species requires scientific justification and A-PLAC approval. Decapitation should generally be used only when study design requires it due to the potential hazard to personnel. Many species react adversely to the smell of blood, so animals should not be decapitated in the presence of other animals and the person performing decapitation should change gloves and/or wash hands between animals.
    • Pithing of both the brain and the spinal cord (double pithing) may be used as the sole means of euthanasia in frogs of the genus Rana or other amphibians with anatomic features that facilitate easy access to the central nervous system. In all other amphibian and reptile species pithing should be followed by decapitation.
    • Under very specialized circumstances, stunning, rapid freezing or air embolism (under anesthesia) may be allowed in small species if research needs make it necessary and there are no available alternatives.