This guide was developed for the purpose of providing UCSB researchers with an easily accessible reference of the drugs that will facilitate handling, and alleviate or minimize pain and distress in laboratory animals. This is not intended to be a comprehensive reference, but instead focuses on the drugs that are commonly used in our animal care and use program. All persons involved in research using animals are morally obligated and legally required to provide appropriate anesthesia and analgesia to minimize pain and distress to the animals. To that end, Principal Investigators (PIs) must consult with the Campus Veterinarian when creating, designing, or amending a protocol that will cause more than slight or momentary pain or distress to the animals. Additionally, in order to receive approval from the Institutional Animal Care and Use Committee (IACUC) to conduct a study that has the potential to produce more than momentary or slight pain or distress in laboratory animals, the PI must address the following concerns in the protocol application.

  • Describe the drugs or techniques that will be used to ameliorate pain or distress, or provide scientific justification why these drugs or techniques would adversely affect the procedures, results, or interpretation of the results and therefore withholding them is necessary. Pain alleviating drugs or techniques should be withheld only for the necessary period of time.

Proper use of pain alleviating drugs involves sophisticated control of all key physiological systems (nervous, cardiovascular, and respiratory systems). General anesthesia is a finely controlled near-death state for the anesthetized animal. A plethora of factors influence the process each time it is performed. Health of the animal can have a major impact. For example, an animal that appears normal on initial inspection may have pneumonia that can lead to uncontrolled anesthesia and death. A procedure area that is cold can dramatically change the manner that an animal will react to administered drugs. An individual animal's response to an anesthetic drug will vary during a procedure in response to the research manipulations as well as its own changing physiological state. It is known that age, sex, strain, previous drug exposure, and even time of day of exposure can have important impacts on anesthetic drug responses. These effects can be dramatic; different strains of mice given 40mg/kg pentobarbital IP display sleep times varying from 10 to 300 minutes!

The knowledge of physiology and pharmacology required for precisely managed anesthesia is enormous. This required expertise is recognized in clinical medicine by highly trained and specifically certified physicians and veterinarians. By contrast, in the research environment, persons administering anesthetics may have only cursory training in these areas. Fortunately through the use of modern drugs with wide safety margins and the availability of naïve purpose-bred and healthy animals from commercial vendors and safe anesthesia in the research setting is generally quite routine. However, always remember that anesthesia is a complex process and many factors can abruptly crop up to cause serious problems.

In order to facilitate the selection and administration of the appropriate chemical agent, this guide provides information regarding many of the commonly referenced regimens; however, there are undoubtedly other regimes that are satisfactory for which references were not located. The primary sources used for compilation of this guide were Laboratory Animal Anaesthesia (2nd edition) by P.A. Flecknell, Laboratory Animal Medicine (2nd edition) by Fox, Anderson, Loew, and Quimby (eds.), Anesthesia and Analgesia in Laboratory Animals (2nd edition) by Fish, Brown, Danneman, and Karas (eds.), Veterinary Pharmacology and Therapeutics (8th edition) by Adams (ed.), Pain Management in Animals by Flecknell and Waterman-Pearson (eds), and Formulary for Laboratory Animals (3rd edition) by Hawk and Leary. These references, in addition to others that may be of benefit to researchers are listed at the end of this monograph. Anesthesia guidelines from the American College of Laboratory Animal Medicine, Stanford University, and the University of California San Francisco were also very useful, and their availability was greatly appreciated.



The most important factor in carrying out a successful anesthetic protocol is to start with a healthy, well-conditioned animal. In order to obtain a predictable response to the selected anesthetic agent and valuable, meaningful data from your experimental preparation, everything possible should be done to select the best experimental animal for your study. Whenever possible and practical (e.g. non-rodent species), a complete blood panel should be performed on the animal to include at least a CBC, BUN, ALT, glucose, and total protein. In general, purchase-bred, specific-pathogen free rodents from approved animal vendors, are in excellent health and do not require a clinical blood work up. That said, all animals should  be examined and determined to be free of clinical disease prior to anesthesia and surgery. A qualified or trained individual should perform this assessment. The Campus Veterinarian should be consulted if there are any questions/doubts about to the health of the animal.

Selection of the most appropriate anesthetic protocol is also important. The anesthetic protocol must be compatible with: 1) The experimental protocol; 2) the species of animal to be used; 3) the age and physical state of the experimental animal; and 4) the technical expertise of the personnel involved in anesthetic administration and monitoring (to include needs for special equipment). The remainder of this guide should aid you in preparing and carrying out an appropriate anesthetic protocol, but consultation with a Campus Veterinarian is required by the IACUC for approval of an anesthetic and surgical protocol.


A period of fast sufficient to empty the stomach should be implemented prior to anesthesia to help prevent regurgitation and aspiration of gastric contents. Twelve hours is sufficient for most mammals. Rodents, do not regurgitate, and hence there is no need to fast rodents to prevent regurgitation and aspiration of gastric contents. However, fasting rodents for six to twelve hours helps to ensure consistent absorption of intraperitoneally administered injectable anesthetics. Very small mammals or juveniles should be subjected to a much shorter fast, usually from 2 to 4 hours, due to their high metabolic rate. Water need not be withheld in most cases.

Handling the Patient

The animal should always be handled gently and calmly. Struggling, fear, and anxiety should be avoided. Prolonged excitation will disturb the circulatory and metabolic status of the animal and may induce shock. Furthermore, attempts to anesthetize a struggling animal present a physical problem in addition to enhancing the likelihood of an abnormal response.

Pre-Anesthetic Treatments/Conditioning

Sedatives, analgesics, anticholinergics agents, and antibiotics may be used prior to anesthesia. Sedatives calm the animal, and reduce the dose of anesthetic agent needed. Similarly, analgesics may also reduce the dose of the anesthetic agent. Anticholinergic drugs block parasympathetic stimulation of the cardiopulmonary system and reduce salivary secretion, and are frequently used in combination with sedatives and analgesics as pre-medication to general anesthesia. Atropine is the most commonly used anticholinergic agent. In general, however, anticholinergics (e.g. atropine) are not required for most rodent surgical procedures. Similarly, sedatives are not used in rodent anesthesia due to the additional stress of administering a second injection. Contact the Campus Veterinarian to discuss your specific surgery and anesthetic protocol, and whether or not pre-anesthetics are required.

Patient/Anesthetic Monitoring

Animals respond in generally predictable ways to individual anesthetic agents, however, much individual variation from animal to animal can be seen in the depth of response to a given dose. For this reason, most anesthetic doses are given as ranges across which the ideal dose for each individual is most likely to fall. It should always be remembered that it is easy to supplement an animal with additional anesthetic agent, but once given, an injectable anesthetic agent cannot be removed. It is therefore recommended that dosing is done initially at the lower end of the anesthetic dose range.

It is important to understand the signs of anesthetic depth in the animal you are working with to ensure that a proper and humane, yet safe, level of anesthesia is continuously maintained. Anesthetic depth is determined by observing and categorizing the progressive loss of function seen with increasing doses of anesthetic agents. Observations should be made on a frequent basis. Not all observations are practical in all species. You must tailor your techniques of anesthetic monitoring to the species on which you are working. It must be remembered that no single observation is infallible. One must observe all signs possible in each animal and with the available information make the best clinical judgment possible regarding anesthetic depth.

    Observations to make include:

    Cardiovascular   system Respiratory System Muscle tone Eye
    • Heart rate and rhythm
    • Mucous membrane color
    • Capillary refill time
    • Arterial pulse and pressure
    • Body temperature
    • Respiratory rate
    • Depth of breathing
    • Character of breathing
    • Blood gases
    • Hemoglobin oxygen saturation (SpO2)
    • Jaw or limb tone
    • Response to painful stimulus (toe or tail pinch)
    • Position of eye (presence or absence of movement)
    • Size of pupil
    • Responsiveness of pupil to light
    • Palpebral reflex
    • Corneal reflex
    • Lacrimation


    In general, the signs of pain perception and light anesthetic depth are:

    • Tachycardia, arrhythmias
    • Pupillary dilation, tearing, globe movement
    • Presence of palpebral reflex
    • Tachypnea (or breath holding)
    • Movement in response to stimulus
    • Arterial hypertension
    • Coughing, swallowing, phonation

    The signs of deep anesthesia are:

    • Bradycardia, arrhythmias, cardiac arrest
    • Dry cornea, centrally fixed eyes, dilated pupil
    • Cessation of breathing (not breath holding)
    • Hypotension

    If you are ever in doubt about anesthetic depth, it is always better to decrease the rate of anesthetic administration. It should also be kept in mind that anesthetic depth is influenced by:

    • Drugs (pre-medications, anesthetics, neuromuscular blocking agents, etc.)
    • Species and strain of animal
    • Respiration (and resulting changes in arterial blood gases, e.g. PCO2 and PO2)
    • Stimuli

    The level of surgical stimulation is especially important. A quiet animal with good vital signs and evidence of moderate anesthesia may quickly show evidence of light-to-insufficient anesthesia in the presence of intense visceral stimulation.

    While monitoring a patient for anesthetic depth, it is extremely important to understand how an anesthetic agent affects the cardiovascular, respiratory, and thermoregulatory systems. Every effort should be made to maintain circulation, respiration, and body temperature within normal physiologic limits.

    • Circulation may be maintained through proper anesthetic dosing, maintaining proper body temperature, and the use of I.V. fluids when needed.
    • Respiration is aided with the use of an endotracheal tube which ensures that the airway remains patent and free from obstruction. When spontaneous ventilation is not adequate due to patient positioning, an open thoracic cavity, or use of neuromuscular blocking agents, a mechanical ventilator should be used. (Normal ventilation values are 15 ml/kg tidal volume and 15 breaths per minute for large animals).
    • Hypothermia will occur during exposure to anesthetic gases and in abdominal or thoracic surgery, particularly in small animals (e.g. rats and mice). This may result in a greatly prolonged anesthetic recovery or even death. Hypothermia may be counteracted to some extent by placing the animal on a warming pad, keeping the animal covered and/or administering warm IV fluids during the procedure, and until fully recovered.


    Surgery is not successful until the animal has fully recovered from anesthesia without any unintended physical or physiological impairments. The time that it takes until an animal is fully recovered from anesthesia will vary depending on the anesthetic agent, the type and duration of the surgery, and the physiological imbalances induced by the surgery or anesthesia. During this period the animal must be closely (e.g. at least every 15 minutes) and carefully monitored. Furthermore, since in rodents hypothermia and dehydration are two of the most common complications that are encountered, it is very important to provide supplemental heat and fluids.

    The following are recommendations for caring for an animal during the post-anesthetic phase:

    • The animal should be continuously monitored until it is conscious, or can be aroused when handled, and has stable vital signs (e.g. regular breathing pattern and rate).
    • Prevent hypothermia by placing the animal on a clean dry and warm surface. For example, the animal's cage may be placed on a padded surface supplied with supplemental heat (e.g. water circulating heating pad). Be cautious with supplemental heat sources; hyperthermia can be as detrimental as hypothermia.
    • Replacement fluid therapy is not usually required for most rodent surgeries, because they do not involve prolonger operative times or result in significant blood loss. However, fluid therapy can be beneficial and aids in the postoperative recovery of the animal. How much fluid to administer depends on numerous variables, but in most uncomplicated (i.e. no bleeding, short duration) rodent surgeries, warmed (97 – 100°F) fluids (saline, or Lacated Ringer's Solution) administered subcuntaneoulsy at a volume of 10 ml/kg per treatment with a daily maximum of 20-50 ml/kg is recommended.
    • As a general rule, animals should not be returned to the animal room until they can stand and move about their cage. To prevent cannibalism or suffocation, house rodents individually until they can stand and move around.
    • After the animal is returned to the animal room, a member of the investigator's staff or other individual to whom postoperative care has been delegated should monitor the animal at least once a day. The animal should be observed for signs of pain, and surgical complications, including infection or dehiscence of the surgical site(s). Pain assessment guidelines are included below.
    • Analgesic treatment should be administered as described in the IACUC protocol.
    • The Campus Veterinarian should be contacted if there are any unexpected complications.
    • External wound clips or sutures should be removed 7-14 days after the surgery.

    Pain Assessment

    The response of animals to pain, and hence any clinical or behavioral signs of pain, will vary depending on the animal species and the surgical procedure. The best way to identify signs of pain is to closely observe the appearance and behavior of the animal prior to surgery, and note any changes after surgery. The following are a number of signs associated with persistent pain.

    Sign Explanation
    Guarding The animal alters its posture to avoid moving or causing contact to a body part, or to avoid the handling of that body area.
    Abnormal appearance Different species show different changes in their external appearance, but obvious lack of grooming, changed posture, and a changed profile of the body are all observable signs.
    Altered behavior Behavior may be depressed; animals may remain immobile, or be reluctant to stand or move even when disturbed. They may also exhibit restlessness (e.g., lying down and getting up, shifting weight, circling, or pacing) or disturbed sleeping patterns. Animals in pain may also show altered social interactions with others in their group.
    Vocalization An animal may vocalize when approached or handled or when a specific body area is touched or palpated. It may also vocalize when moving to avoid being handled.
    Mutilation Animals may lick, bite, scratch, shake, or rub a painful area.
    Inappetence Animals in pain frequently stop eating and drinking, or markedly reduce their intake, resulting in rapid weight loss.




    Sedatives and Tranquilizers

    Sedatives and tranquilizers are generally used in animals to produce a psychological calming to facilitate handling, however, an important fact to remember is that analgesia is not produced. They are most often used as pre-anesthetic agents to reduce fear and anxiety and facilitate handling. When used as pre-anesthetic agents the amount of general anesthetic agent needed is decreased. Upon painful stimulation, arousal may occur and thus, these drugs should be used with caution on animals that are intractable or overtly aggressive. Loud noises may also cause arousal and unpredictable behavior. High doses may cause ataxia, cardiovascular and respiratory depression and hypotension without necessarily producing greater sedation. Depression may be even more pronounced in combination with a general anesthetic.

    The physiological state of the animal prior to administration of tranquilizers may markedly affect the degree of sedation achieved. Animals that are overtly aggressive, intractable and excited may not become manageable except with very high, respiratory-depressing doses.

    1. Phenothiazines
      • Common agents: acepromazine, chlorpromazine, promazine
      • Desirable Effects
        • Potentiates effects of anesthetics, hypnotics and narcotic analgesics (reduces dosages, extends duration, provides smooth recovery from anesthesia)
        • Anti-emetic effect
        • Facilitates skeletal muscle relaxation
        • Causes vasodilation of rabbit ear vessels
      • Undesirable Effects
        • Moderate hypotension
        • May produce hypothermia
      • Potential research complications
        • Dopamine blockade in basal ganglia
        • Decreased spontaneous motor activity
        • Blocks peripheral actions of catecholamines
        • Hypothermia as a result of depletion of catecholamine substances within hypothalamus
        • High doses block release of FSH and LH
        • Increased levels of prolactin in rats, sheep and goats
        • Hyperglycemia due to release of epinephrine via the adrenal medulla
        • In the cat, small doses reduce vasopressor action of epinephrine; large doses reverse the vasopressor action.
        • Reduces the hematocrits of animals
        • Antagonizes apomorphine-induced emesis in the dog
        • May produce cardiac arrhythmias in the dog
        • High doses of chlorpromazine in the cat produce tremors and rigidity.
        • May produce cleft lip and cleft palate in mouse fetuses
        • May produce ocular lesions after prolonged use
    2. Benzodiazepines
      • Common agents: diazepam, midazolam
      • Desirable effects
        • Sedation variable among species (minimal in dogs, marked in rabbits and rodents)
        • Potentiates most anesthetics and narcotic analgesics
        • Good skeletal muscle relaxation
      • Undesirable effects
        • None at normal dosages
        • Controlled substance
      • Potential research complications
        • Principal site of CNS depression is in the brain stem reticular formation
        • Blockade of spinal polysynaptic reflexes
        • Decreases synthesis of dopamine in the limbic and striatal areas of the rat brain
        • Potentiates GABA-mediated inhibition in the CNS
        • Transient arterial hypotensive effect occurs in the dog after IV administration
        • In humans, cleft palate has been associated with maternal intake during pregnancy
      • Miscellaneous
        • Diazepam should be used intravenously, because systemic absorption is variable after IM or SQ injection. Diazepam is also incompatible in solution with other drugs, therefore it should never be mixed in the same syringe with other drugs (except ketamine).
        • Midazolam works well after IM or SQ injection and may be mixed with certain other drugs
    3. Alpha-2-adrenergic agonist
      • Common agents: xylazine, medetomidine, detomidine
      • Desirable effects
        • CNS depressant, analgesic muscle relaxant, non-narcotic sedative
        • Potentiates action of most anesthetics
      • Undesirable effects
        • Animals may react to pain when used as the primary anesthetic
        • May produce hyperglycemia and diuresis
        • Variable effects on the cardiovascular system, hypotension is common
        • Ruminants require one tenth the dosage of carnivores
        • Bradyarrhythmias and reduced cardiac output
      • Potential research complications
        • Induces emesis occasionally in dogs due to direct stimulation of the emetic center in the CNS
        • Depresses cardiac contractility and decreases cardiac output
        • Sensitizes heart to epinephrine resulting in ventricular arrhythmias, including fibrillation
        • In the dog following IV administration, a transient increase in arterial pressure is produced followed by a decrease; arterial pH, PaO2, and PaCO2 do not change.
        • Little to no therapeutic effect in swine
        • In dogs, bloat from aerophagia may develop
        • May precipitate abortion
      • Miscellaneous:
        • Xylazine is a potent sedative and central nervous system depressant. It is used in combination with other agents to produce anesthesia. Its muscle relaxation and analgesic effects are excellent. It does, however, cause potent cardiovascular depression. Medetomidine and detomidine are newer and more specific central alpha-2 agonists, resulting in longer, more profound sedation and analgesia than xylazine and fewer adverse cardiovascular side effects.

    Injectable Anesthetic Agents

    1. Ketamine, the most common drug in this class, produces a state of chemical restraint and a type of anesthesia characterized by muscle rigidity and an apparent dissociation of the mind from the environment. Depth of anesthesia is dose related. It produces activation of the limbic system and depression of the thalamoneocortical system rather than dose related general CNS depression. It is rapidly metabolized to norketamine (demethylated ketamine) in the liver via the hepatic P-450 microsomal system. The metabolites are mainly excreted in both urine and bile.
      • Desirable effects
        • Safe and effective in most mammalian species
        • No significant respiratory or cardiovascular depression or change in hepatic or renal function
        • Increased sedation and analgesia in combination with alpha-2-adrenergic agonist (xylazine)
        • Many reflexes left intact: corneal blink reflex, laryngeal and pharyngeal reflexes
        • Provides mild-to-moderate analgesia
      • Undesirable effects
        • Not adequate for surgical anesthesia alone, must be used in combination with other anesthetic agents.
        • Salivary secretions are increased and may obstruct airways, preventable with atropine use
        • Spontaneous movements may occur with stimulation
        • The eyes remain open and corneal drying may result, preventable with application of a bland ophthalmic ointment
        • Side effects may include convulsions and disorientation and excitement during recovery
        • Muscle rigidity can be reduced with benzodiazepines (diazepam)
      • Potential research complications
        • Depresses thalamoneocortical system in conjunction with activation of the limbic system
        • Potent inhibitor of GABA binding in the CNS
        • Blocks neuronal transport processes for monoamine transmitters such as serotonin, dopamine, and norepinephrine
        • Exerts selective positive influence on heart muscle
        • Increases cardiac output, mean aortic pressure, pulmonary arterial pressure, central venous pressure, and heart rate
        • Elevates myocardial oxygen consumption
        • Pharyngeal and laryngeal reflexes remain intact; leads to increased incidence of laryngospasm, bronchospasm, and coughing secondary to secretions or manipulation of the pharynx or larynx
        • Increases cerebral blood flow in the dog by 80%
        • Effects of xylazine, when used in combination with ketamine, are antagonized by yohimbine, an alpha-2-adrenergic blocking agent
    2. Tiletamine/zolazepam (Telazol®), Tiletamine is an injectable anesthetic agent chemically related to ketamine. Zolazepam is a diazepinone minor tranquilizer.
      • Desirable effects
        • Safe, effective anesthesia for up to 25 minutes
        • Useful for minor surgical procedures but not abdominal surgery
        • Normal pharyngeal and laryngeal reflexes
        • Provides mild-to-moderate analgesia
      • Undesirable effects
        • Produces cataleptoid anesthesia; cranial nerve and spinal reflexes remain active which may result in some movement that is not indicative of the depth of anesthesia
        • Eyes remain open and pupils dilated; necessitates use of a bland ophthalmic ointment to prevent corneal drying
        • Nociceptive stimuli recommended to monitor depth of anesthesia
        • Corneal and pedal reflexes remain intact, and are unreliable indicators of depth of anesthesia
        • Produces copious salivation which is controlled by pretreatment with atropine
        • May produce pain on injection
      • Potential research complications
        • In dogs, produces persistent tachycardia, decreased stroke volume with minor change in net cardiac output
        • Increased systolic and diastolic blood pressure
        • Doubles respiratory rate and decreases tidal volume to less than half; arterial pO2 levels decrease
        • Crosses placental barrier and depresses respiration of newborns
    3. Barbiturates differ from tranquilizers and narcotics in that increasing the dose progressively increases the depth of depression until a state of general anesthesia is attained, however, at intermediate doses excitation may be induced. The primary use of barbiturates is in the induction and/or maintenance of general anesthesia. There are four basic groups of barbiturates which vary according to duration of action after IV administration.
      1. Ultra-short acting: methohexital (duration 5-15 minutes)
      2. Short-acting: thiamylal sodium, thiopental sodium (duration 10-45 minutes)
      3. Intermediate: pentobarbital sodium (duration 1-3 hours)
      4. Long-acting: phenobarbital, inactin (duration 8-24 hours)
      • Desirable effects
        • Smooth and rapid induction following IV injection in most species
        • Generally administer 1/3-1/2 of the calculated dose IV as a bolus, then titrate to desired surgical plane
      • Undesirable effects
        • Dosage to attain surgical anesthesia is usually close to that which causes respiratory failure
        • Cardiovascular and respiratory depression common
        • Intraperitoneal bolus injection in rodents associated with high variability between strains
        • Poor analgesic activity
        • Perivascular injection may cause local tissue necrosis (keep below 2%)
        • Prolonged recovery with pentobarbital and short-acting agents if repeated doses are administeredTransient apnea after IV injection is common
        • Administration of concurrent glucose, epinephrine and chloramphenicol may prolong recovery
        • Controlled substances
      • Potential research complications
        • Decreases cerebral oxygen consumption as much as 55%
        • Decreases sensitivity of polysynaptic junctions to the depolarizing action of acetylcholine
        • Raises the threshold of spinal reflexes
        • In dogs, the hemodynamic effects of pentobarbital are as follows: mean arterial pressure initially decreases, then returns to control levels and finally drops slightly; systolic arterial pressure is significantly depressed; cardiac output initially rises then gradually declines; total peripheral resistance drops initially, then rises above control; stroke volume is significantly depressed; myocardial contractility decreases significantly.
        • Increased incidence of ventricular fibrillation
        • Intra-arterial injection produces arterial wall spasms and may result in gangrene.
        • In rats, pentobarbital blocks release of LH and FSH if administered prior to normal gonadotropin release.
        • Hypothermia due to increased peripheral vasodilation
        • Resistance may develop after repeated use due to increased rate of metabolism by hepatic microsomal enzymes.
    4. Tribromoethanol (TBE) is an injectable anesthetic that causes generalized CNS depression, including both the respiratory and cardiovascular centers. It is metabolized by the liver, and excreted in the urine as TBE glucoronate. Pharmaceutical-grade TBE (e.g. Avertin) is no longer available, and is instead prepared from non-pharmaceutical-grade TBE dissolved in tertiary-amyl alcohol. This agent has no application in clinical veterinary medicine, and its continued routine use for rodent anesthesia is controversial, since effective pharmaceutical-grade alternatives are available. Use of TBE in laboratory animals must be reviewed and approved by the IACUC. Approval may be granted if there is sufficient scientific necessity, and the PI can demonstrate that the drug will be prepared, stored and used in a manner that ensures it stability, sterility, and efficacy (see preparation and handling recommendations below). 
      • Desirable effects
        • Produces surgical anesthesia in mice
        • Good muscle relaxation
      • Undesirable effects
        • Inconsistent and variable anesthetic duration
        • Serious adverse effects (peritoneal inflammation or death) when administered by the IP route, which is the most common route. Animals receiving TBE should be carefully and closely monitored for several days, and any morbidities or mortalities should be reported to the Campus Veterinarian.
        • The solvent used to prepare the stock solution, tertiary-amyl alcohol, and the stock solution need to be kept away from sources of ignition and stored in an explosion-proof refrigerator
        • TBE causes skin, eye, and respiratory irritation, therefore it should be prepared in a fume hood and research staff preparing the TBE stock solution should wear gloves, respirator, safety glasses and lab coat when handling TBE.
        • Narrow margin of safety
      • Preparation and Handling
        • Components, 2, 2, 2 Tribromoethanol and tertiary amyl alcohol, are mixed to prepare a stock solution. Numerous stock solutions are described in the literature. The concentration of TBE in tertiary-amyl alcohol in the pharmaceutical-grade Avertin was 66.7% (w/w), which corresponds to 1 gm of TBE per ml. The stock solution should be stored in a refrigerator protected from light (i.e. amber vial).
        • An aqueous working solution should be prepared fresh prior to use, and then discarded. Numerous concentrations of working solution are described in the literature, most describe the solution of the TBE in tertiary-amyl alcohol with sterile saline. The aqueous working solution must be heated to dissolve the stock solution, but care must be taken to ensure that it is not heated above 40°C. The working solution must be filtered through a 0.2m syringe-topped filter prior to administration to animals.

    Inhalant Anesthetic Agents

    Inhalant anesthetics provide a relatively rapid onset and recovery. The high degree of control over anesthetic depth and the relative constant response over a wide variety of species are advantages. However, specialized equipment and constant monitoring of the patient are required with use of inhalant anesthetics.

    The depth and duration of effect in inhalation anesthesia can be controlled by the anesthesiologist through the manipulation of anesthetic concentration and pulmonary ventilation. Differences in anesthetic solubility determine the rate at which the anesthetic concentration increases in the arterial blood. As highly soluble agents require more time to attain a significant concentration in the blood, they result in a more prolonged induction and recovery. The reverse is true for the highly insoluble agents which are therefore more controllable as their blood concentration can be rapidly changed; however, for this reason they are more hazardous.

    Use of inhalant agents requires the following special equipment: a source of carrier gas (usually oxygen or air); a vaporizer for the volatile anesthetics; a breathing system from which the anesthetic mixture is inhaled and a mask or endotracheal tube for connecting the breathing system to the animal. Numerous simple apparatuses have been devised and reported in the literature and are commercially available for use in small laboratory animals for these purposes.

    Regardless of the system, proper scavenging of waste anesthetic gas is required to avoid personnel exposure.

    1. Isoflurane
      • Desirable effects
        • Induction, recovery and changes in level of anesthesia are rapid
        • Undergoes very little metabolism and is almost completely eliminated in exhaled air, therefore, produces minimal interference in drug metabolism and toxicology studies
        • Neither flammable nor explosive
        • Indicated for neurological procedures because it maintains an isoelectric EEG and decreases the metabolic oxygen requirement
      • Undesirable effects
        • Produces respiratory depression
        • High vaporization pressure requires calibrated, precision vaporizer; not suitable for open-drop technique
        • Greater decrease in systemic vascular resistance than with other agents
      • Potential research complications
        • Increases arterial CO2 concentration and decreases arterial pH
        • Produces no effect upon AV conduction
        • Does not effect CNS irritability

    Analgesic Agents

    In general, experimental and surgical procedures that cause pain in humans should be expected to cause pain in laboratory animals. However, uncertainty exists regarding the degree to which the psychological (interpretive) component of pain might be a determinant of postoperative discomfort or suffering in animals. In determining which procedures require postoperative analgesia and which analgesic agents may be useful, several factors should be considered. Among these are:

    The invasiveness of the procedure:

    • Are body cavities invaded and exposed?
    • Are especially sensitive tissues involved (e.g., bones)?
    • Is significant tissue destruction or inflammation produced?

    The degree or severity of pain expected:

    • Comparison to similar procedure in people; would a reasonably stoic person tolerate the postoperative period without analgesia?
    • Behavior of the animal during postoperative period, e.g., level of activity, appetite, roughness of hair coat compared to sham anesthetized controls.

    Postoperative analgesia is desirable for all surgical procedures involving penetration deeper than skin and subcutaneous tissues. For procedures involving invasion of bones, joints, teeth or significant destruction or inflammation in other tissues, it is the responsibility of the Principal Investigator to provide sufficient justification in his/her animal use protocol if analgesics cannot be used.

    The following categorical examples may be useful to investigators in determining the necessity for supplementary postoperative analgesia in procedures involving experimental or instructional use of animals:

    Minimal to Mild Pain Moderate Pain Moderate to Severe Pain
    • Catheter implantation
    • Tail clipping in rodents
    • Ear notching
    • Superficial tumor implantation
    • Superficial lymphadenectomy
    • Multiple ID antigen injections
    • Intracerebral implantation
    • Vasectomy
    • Vascular access port implantation
    • Minor laparotomy incision
    • Thyroidectomy
    • Orchidectomy
    • C-section
    • Embryo transfer surgery
    • Hypophysectomy
    • Thymectomy
    • Major laparotomy/organ incision
    • Thoracotomy
    • Heterotopic organ transplantation
    • Vertebral procedures
    • Trauma models
    • Orthopedic procedures
    1. Opioid agonists: morphine
      • Desirable effects
        • Potent hypnotic and analgesic effects
        • Codeine/acetaminophen elixir is useful for mild to moderate pain relief in most animals, convenient administration to rodents and rabbits in water bottle
        • Epidural administration provides excellent analgesia without systemic adverse effects.
        • Effects reversible with pure opioid antagonists (i.e., Naloxone)
      • Undesirable effects
        • Morphine stimulates the gastrointestinal tract of dogs (meperidine does not)
        • Cardiovascular and respiratory depressants, alter thermoregulatory mechanism
        • Short duration of action, morphine, oxymorphone 2 to 4 hours, others less than 2 hours
        • Undesired excitation may occur in cat, mouse and farm animals
        • May cause sedation, especially in dogs and primates
        • May stimulate histamine release which can produce peripheral vasodilation and hypotension
        • Controlled substances
        • Increase CSF pressure, must be used cautiously in post-op procedures, including the brain
      • Potential research complications
        • Morphine
          • Increases release of anti-diuretic hormone, growth hormone, and prolactin from the pituitary by action in the hypothalamus
          • Inhibits release of LH
          • Causes emesis in dogs
          • Effectively inhibits coughing
          • Produces hypothermia in rabbits, dogs, and monkeys through release of serotonin
          • Produces hyperthermia in goats, cattle, and horses
          • Low doses produce hyperthermia in guinea pigs, rats, and mice; high doses induce hypothermia.
          • Hypothermia decreases in rabbits after chronic administration.
          • Produces a variable effect upon the size of the pupil, i.e., increases size in sheep, and horses; decreases size in dogs, rats, and humans
          • Initially stimulates the respiratory system in dogs, then depresses respiratory activity
          • Cheyne-Stokes type respiration may occur
          • In the conscious dog, induces coronary vasoconstriction, decreased coronary blood flow, and increased vascular resistance (opposite effects occur in humans)
          • In the dog, initially causes GI tract emptying, followed by constipation
          • Use morphine for epidural administration
    2. Opioid - partial agonists: buprenorphine
      • Desirable effects
        • Potent analgesic - 30 times more potent than morphine
        • Few side effects compared with morphine, including less sedation
        • Effective in all common laboratory animal species
        • Schedule V (low abuse potential)
        • Long duration of action of between 6-12 hours
      • Undesirable effects
        • Possible respiratory/cardiovascular depression; rare at analgesic doses
      • Potential research complications
        • Agonist activity at MU receptor only
        • May decrease or rarely increase heart rate and blood pressure
        • Respiratory depression
        • May increase pressure within the biliary tract
        • Produces mild sedation
        • Elevates cerebrospinal fluid pressure
    3. Opioid - agonists/antagonists: butorphanol
      • Desirable effects
        • Less potent analgesics than buprenorphine
      • Undesirable effects
        • Butorphanol is a class IV controlled substance
        • Non-human primates extremely susceptible to respiratory depression with butorphanol
      • Potential research complications
        • Analgesic potency is four times that of an equivalent quantity of morphine
        • Reduces aortic blood pressure for 15 to 30 minutes after IV administration
        • Does not cause histamine release like other opioid agonists
        • Potent anti-tussive activity
        • Produces mild sedation
        • Rarely produces anorexia, nausea, and diarrhea
        • Agonist activity at the kappa and sigma receptors, antagonist activity at the MU receptor
    4. Non-steroidal anti-inflammatory drugs: ketoprofen, flunixin meglumine, carprofen, meloxicam
      • Desirable effects
        • Peripherally acting
        • Anti-inflammatory properties
        • Newer agents (carprofen, meloxicam) have excellent analgesic activity
        • May be combined with opioids
      • Undesirable effects
        • Gastrointestinal side effects, including ulcers and gastrointestinal bleeding
        • May promote hemorrhage, therefore should not be used preoperatively or in patients with bleeding disorders
        • May adversely affect renal function, therefore should use only in well-hydrated patients
      • Potential research complications
        • Flunixin Meglumine
          • Antipyretic activity
          • Greater analgesic potency than phenylbutazone, pentazocine, meperidine, and codeine
          • If administered intra-arterially, produces ataxia, rapid breathing, muscle weakness, and hysteria

    Local Anesthetic/Analgesic Agents

    Local anestheticsare are used to produce desensitization and analgesia of the skin, local tissues, or regional structures. These agents may be used topically, by local infiltration, infiltration around a major nerve, or epidurally. They block the generation and transmission of nerve impulses by interfering with nerve cell membrane permeability. These agents may be combined with a vasoconstrictor (such as epinephrine) to increase the intensity and the duration of analgesia. They must not be injected intravenously.

    The use of these agents is becoming more common in laboratory animal medicine. They are frequently infiltrated along incision lines to produce local anesthesia. This practice frequently reduces dosage requirements for the primary anesthetic agent and postoperative analgesic, minimizing adverse effects these agents may cause. Adverse side effects of local anesthetics are rare, but one must use caution when administering them to small rodents.

    Local Anesthetic Agents


    • Intermediate potency
    • Immediate onset of effect
    • Use 0.5% to 2% for infiltration and nerve block
    • Duration of effect up to 2 hours
    • Metabolized by the liver
    • Maximum dose for mice = 17.5 mg/kg; local infiltration


    • Four times more potent than lidocaine
    • Use 0.25% for infiltration, 0.5% for nerve block
    • Intermediate onset of effect
    • Duration of effect up to 6 hours
    • Maximum dose for mice = 38 mg/kg; local infiltration


    • EMLA cream is a eutectic mixture of lidocaine and prilocaine in a 1:1 weight ratio
    • Provides dermal anesthesia when applied to skin under occlusive dressing
    • Onset, depth, and duration of analgesia depends on duration of application. Requires a minimum time of one hour. Variable effectiveness.
    • Analgesia persists up to 1 to 2 hours after removal

    Undesirable Effects of Local Anesthetics

    • Cardiovascular and respiratory depression and arrhythmias
    • Convulsions or seizures
    • Occur following IV administration in mice at concentrations >55 mg/kg for Bupivacaine, and >110 for Lidocaine
    • Occur following IV administration in rats at concentrations 22.7 +/- 7 mg/kg for Bupivacaine


    Injectable Anesthetic Agents

    (Doses in mg/kg & Route)

    Drug Mouse Guinea Pig Rat Rabbit
    Ketamine - - - 20-60
    Ketamine (K) +
    Xylazine (X)
    90-150 K
    7.5-16 X
    40-75 K
    5-10 X
    IP, IM
    40-75 K
    5-10 X
    , IM
    22-50 K

    2.5-10 X
    Ketamine (K) +
    Diazepam (D)
    - - - 60-80 K
    5-10 D
    Ketamine +
    Xylazine +
    Acepromazine (A)
    60-100 K
    7-20 X
    0.6-3 A
    61 K
    7 X
    0.6 A
    30-40 K
    5-6 X
    1-2 A
    IP, IM
    Pentobarbital 30-90
    (5 mg/kg in neonates)
    IP, IV
    Thiopental - 20-40
    - -


    1. Inhalant anesthetic agents are recommended for Guinea Pigs.


    • IM: intramuscular
    • IP: intraperitoneal
    • IV: intravenous
    • SQ: subcutaneous
    • PO: per os, given by mouth, orally
    • q: every

    Analgesic Agents

    (Doses in mg/kg; Route; Frequency)

    Drug Mouse Guinea Pig Rat Rabbit
    Buprenorphine - HCl 0.1 - 2 mg/kg
    q 3-8 hrs
    0.01 - 0.05 mg/kg
    q 8-12 hrs
    0.01 - 0.05 mg/kg
    q 8-12 hrs
    0.01 - 0.05 mg/kg
    IM, SQ
    q 6-12 hrs
    Buprenorphine - SR 0.6 - 1 mg/kg
    - 1.0 - 1.2 mg/kg
    Flunixin Meglumine(Banamine) 2.5 mg/kg
    q 12 hrs
    to 3 days
    1.1 - 2.5 mg/kg
    q 12 hrs
    to 3 days
    1 – 2 mg/kg
    q 12 hrs
    Up to 3 days
    1.0 mg/kg
    q 12 hrs
    Up to 3 days
    Meloxicam 2 mg/kg
    q 24 hrs
    - - -


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    This document is a compilation of anesthesia and analgesia guidelines prepared and revised by numerous laboratory animal veterinarians. We acknowledge their contribution below:


    • 1995 - Brent Martin, D.V.M.
    • 2009 - Manuel A. Garcia, D.V.M., Ph.D.


    • 1989 - Victor Lukas, D.V.M.
    • 1990 - Greg Bates, D.V.M.
    • 1991 - Peg Murphy-Hackley, D.V.M.
    • 1992 - Victor Lukas, D.V.M.
    • 1996 - Michael Larson, D.V.M.
    • 2001 - Nancy Poy, D.V.M.
    • 2001 - Gary Cain, D.V.M., Ph.D.
    • 2003 - Nancy Poy, D.V.M.
    • 2008 - Manuel A. Garcia, D.V.M., Ph.D.


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