Guidelines on Anesthesia and Analgesia in Rats

Unit for Laboratory Animal Medicine
May 19, 2023 12:00 am

This document has been designed by the ULAM veterinary personnel as a guideline for sedation, anesthesia, and analgesia of laboratory rats. This is not intended to be an inclusive tutorial on all possible drug combinations that can be used in rats. The following guidelines are also general recommendations and consequently do not include reference to specific research associated concerns.

All surgical procedures, anesthetics, analgesics, antibiotics or other medications used on animals must be approved by the IACUC, described in the animal use protocol and performed by personnel listed on the protocol and appropriately trained for the surgical procedure. Any techniques or drug protocols deviating from this document must be justified and approved in the IACUC protocol prior to implementation.

  • Responsibility

    1. Principal Investigator: Responsible to ensure appropriate anesthesia and/or analgesia is provided for all rodents undergoing painful procedures including rodent survival surgery unless otherwise indicated in the relevant approved protocol.
  • Glossary Definitions

    Acclimation Period

    The time period provided to an animal after shipment to allow physiological and psychological stabilization prior to any experimental manipulation.


    This encompasses both of the following definitions:

    1. Local Anesthesia: Temporarily induces loss of sensation to a specific part of the body. May provide pain relief.
    2. Systemic Anesthesia: Temporarily induces loss of sensation with loss of consciousness. Only provides pain relief due to or during loss of consciousness.


    Central depression causing stupor where the animal is unaware of its surroundings but still responsive to painful procedures.

    Surgical Plane of Anesthesia

    The stage of anesthesia in which the animal is at an appropriate anesthetic depth and the surgical procedure can begin.

  • Procedures

    1. Prior to Anesthetic/Analgesic/Sedative Event

    1. Newly arrived animals should have an acclimation period of at least 3 days prior to anesthesia or sedation.
    2. Age and body weight should be considered when designing an A/A plan.
    3. Pre-anesthetic fasting is usually not necessary in rodents. If pre-anesthetic fasting is required:
      1. The fasting period must be limited to 2-3 hours and no longer due to the higher metabolism in rats.
      2. Water should NEVER be restricted.
    4. Apply sterile non-medicated ophthalmic ointment to the eyes to prevent corneal drying during anesthesia or sedation.

    2. Routes of Administration

    1. More detailed information regarding injection techniques and maximum quantities safely administered to rats can be found in Guidelines on Administration of Substances to Laboratory Animals.

    3. Normal Monitoring Parameters

    1. More information on anesthetic/sedation monitoring requirements can found in Anesthesia and Sedation Monitoring Guidelines.
      1. Respiratory rate should be 70 - 110 breaths/min.
        1. A drop in respiratory rate of 50% can be normal during anesthesia.
        2. Respiratory pattern can be used to monitor anesthesia.
          1. Deep and < 70 breaths/min: the animal may be too deep.
          2. Shallow and >110 breaths/min: the animal may be too light.
      2. Pulse rate should be 260 - 500 beats/min.
      3. Normal temperature ranges while under anesthesia fall between 35.9°C and 37.5°C (96.6°F - 99.5°F).
      4. Mucus membrane color should be pink. Never pale white or blue.
        1. Normal capillary refill time (CRT) is < 2 seconds.

    4. Recovery

    1. Visibly observe and monitor animals every 15 minutes during recovery from anesthesia until the animal is fully ambulatory.
      1. Animals that have received alpha-2 agonists (dexmedetomidine, xylazine) as part of their anesthetic protocol can receive a reversal agent to expedite recover.
        1. See Table 6 below for recommended doses.
      2. Recover rodents:
        1. Individually until fully ambulatory to avoid cannibalism by cage mates; the recommended method.
        2. In clean cages without bedding to limit the possibility of tracheal foreign body obstruction or aspiration pneumonia.
      3. Monitoring parameters and thermal supplementation should be continued throughout the recovery period.
        1. See Anesthesia and Sedation Monitoring Guidelines and Guidelines on the Performance of Surgery in Rodents for more information on post-operative monitoring and appropriate thermal support devices.
    2.  Maintain animals in the surgery area to facilitate appropriate monitoring during the recovery period.
    3. Provide nutritional support following anesthesia as soon as the animal is recovered to facilitate the recuperation process.
      1. Moist chow, regular chow, or diet gel should be provided on the cage floor to encourage eating as soon as possible.

    5. Anesthetics

    1. Detailed information on all approved anesthetics and sedatives can be found in Anesthesia and Analgesia Drug Descriptions.
    2. Table 1: Anticholinergics  


         Dosage and Routea   

         0.05 mg/kg SC   
         0.01-0.02 mg/kg SC   
       a Subcutaneous (SC)
    3. Table 2: Inhalant Anesthetics Used in Rats 


      Dosage and Route


      To effect. Typically:
      • 4 - 5% for induction
      • 1 -2% for maintenance
      300µL on gauze placed in a 500ml container
      • Chamber induction for brief anesthesia for procedures
      • Gauze must be protected so animal cannot come into contact with isoflurane
      • Use a container that allows animals to be visible during induction
      •  Use a container made of a material that can be sanitized 
      To effect. Typically:
      • 4 - 7% for Induction
      • 2 - 4% for maintenance
      Requires use of a calibrated vaporizer
    4. Table 3: Injectable Anesthetics Used in Rats 


      Dosage and Route a

      Duration of Anesthesia






      + Xylazine
      40 - 90 mg/kg ket IP
      + 5 - 10 mg/kg xyl IP
      45 - 90 minutes
      Re-dose with 1/3 of ketamine dose only.
      + Xylazine
      + Acepromazine
      31.25 mg/kg ket IP or IM
      + 6.25 mg/kg xyl IP or IM
      + 1.25 mg/kg ace IP or IM
      Welberg et al. 2006
      + Dexmedetomidine   
      75 mg/kg ket
      + 0.25 - 0.5 mg/kg dex IP
      for non-premedicated animals.
      75mg/kg ket
      + 0.03 - 0.1 mg/kg dex IP
      for animals premedicated with 
      buprenorphine or other opioids.
      120 minutes





      40 - 50 mg/kg IP
      80 - 95 minutes
      Respiratory depression/ poor analgesia
      • Avoid buprenorphine co - administration.  Buprenorphine and pentobarbital will result in cardiorespiratory depression. Administer buprenorphine after full recovery.





      10 mg/kg IV
      5 - 7 minutes
      Titrate as needed
       a Intramuscular (IM), Intraperitoneal (IP), Intravenous (IV)
    5. Table 4 Injectable Sedatives Used in Rats 


      Dosage and Route a

      Duration of Sedation   


      + Diazepam
      40-80 mg/kg ket IP
      + 5-10 mg/kg dia IP
      20 - 30 minutes
      Sedation / immobilization
      + Midazolam
      75 mg/kg ket IP
      + 5 mg/kg mid IP
      20 - 30 minutes
      + Acepromazine   
      75-80 mg/kg ket IM or IP
      + 2.5 mg/kg ace IM or IP
      20 - 30 minutes
      50 - 100 mg/kg IP
      Poor muscle relaxation / mild analgesia
       a Intramuscular (IM), Intraperitoneal (IP)
    6. Table 5: Injectable Anesthetics Requiring Scientific Justification and IACUC Approval 


      Dosage and Routea

      Duration of


      Alpha- Chloralose
      (5% w/v concentration)
      Not Recommended
      31 - 65 mg/kg IP
      • Poor analgesia, possible convulsions, metabolic acidosis. Do NOT use for survival surgical procedures.
      Alpha- Chloralose
      + Urethane
      Not Recommended
      50 - 60 mg/kg chl IP
      + 500 - 800 mg/kg ure IP
      (administer urethane 20-30 min.
      prior to a- chloralose)
      500 minutes
      • Do NOT use for survival surgical procedures. Can be used for prolonged non-recovery monitoring procedures.
      Chloral hydrate
      (5% concentration)
      Not Recommended
      300 - 450 mg/kg IP
      60 - 136 minutes
      • Concentration above 5% produces peritonitis. Anesthetic dose is near the lethal dose. Do NOT use for surgical procedures. Can be used for sedation for monitoring procedures.
      (ethyl carbamate)
      (50% w/v concentration 
      Not Recommended
      1000 - 1500 mg/kg IP
      Up to 24 hours
      • Carcinogenic and mutagenic DO NOT use for survival surgical procedures.
      • For non-recovery use only due to progressive acidosis, hyperosmolality of body fluids, and osmotic toxicity to mesenteric vasculature.
       a Intraperitoneal (IP)   
    7. Table 6: Injectable Reversal Agents Used in Rats 


      Dosage and Route a

      Reversal Agent For


      0.1 - 1.0 mg/kg IP, IM or SC
      Dexmedetomidine or Xylazine
      Preferred reversal agent for alpha-2 agonists
      1.0 - 2.0 mg/kg IP or SC
      Less effective than atipamezole
       a Subcutaneous (SC), Intramuscular (IM), Intraperitoneal (IP)

    6. Analgesia

    1. Unrelieved pain can have profound negative physiologic consequences, which may alter research results.
      1. Rats show a variety of responses to pain, some of which may be fairly subtle and easily missed on casual examination.
      2. Pain assessment in rats consists of evaluating behavioral and physiologic parameters (see Table 7).
    2. The IACUC requires the use of preemptive analgesia (analgesics given prior to the first skin incision) for all survival surgical procedures.
      1. Requirements for analgesic coverage differ depending on the classification of surgery as Type I, II, or III.
        1. See the Policy on Analgesia in Animals Undergoing Surgery for updated surgical classifications and analgesia requirements.
      2. The use of buprenorphine as a pre-emptive analgesic may decrease the amount of required anesthetic drugs, due to buprenorphine's sedative and respiratory depressant effects.
      3. See Table 8 for rat analgesics.
    3. Table 7: Pain Evaluation Parameters

      Behavioral Signs

      Physiologic Indicators

      Reluctance to move/ decreased activity
      Elevated blood pressure
      Abnormal posture/ hunched
      Elevated heart rate
      Back arching
      Elevated respiratory rate
      Social isolation
      Changes in body temperature
      Decreased appetite
      Dilated pupils
      Ungroomed hair coat
      Facial expressions (see Appendix A)
    4. Table 8: Analgesics Used in Rats 


      Dosage and Route a


      0.01 - 0.05 mg/kg SC, IP
      6 - 8 hours
      Buprenorphine extended-release
      (Ethiqa XR®)b
      0.65 mg/kg SQ
      72 hours
      5 mg/kg SC
      24 hours
      2.5 mg/kg SC
      12 hours
      1 - 2 mg/kg PO or SC
      12-24 hours
      5 mg/kg SC
      24 hours
      a Subcutaneous (SC), Intraperitoneal (IP), Orally (PO)   
      b This formulation of extended-release buprenorphine is known to cause pica (a desire to eat non-food substances) in rats. Ingestion of bedding is common. Rats dosed with Ethiqa XR® must be housed on paper bedding for 72 hours after dosing in order to minimize the impact of potential bedding ingestion.    
      c Monitor rats closely for signs of gastrointestinal complications, e.g. gastrointestinal bleeding, erosion, ulceration, and, in extreme cases, perforation, within 24-48 hours after administration. See References 17. Shientag 2012 below.

    7. Local Anesthetics

    1. Lidocaine and bupivacaine are the two most commonly used local anesthetics.
    2. Table 9: Local Anesthetics Used in Rats 


      Dosage and Route a

      Duration of Anesthesia


      4 mg/kg SC (0.4 ml/kg of a 1% solution)
      1.5 - 2 hours
      Rapid onset (1 - 2 min)
      1 - 2 mg/kg SC (0.4 - 0.8 ml/kg of a 0.25% solution)
      4 - 12 hours
      Slower onset (5 - 10 min)
       a Subcutaneous (SC)   
    3. These doses can be diluted in sterile water to provide a larger injection volume. These injectable anesthetics are most routinely administered in subcutaneous tissues near the site of the incision to be made.
      1. Administration can be performed in a "line block," in which the subcutaneous tissue proximal to the incision site is infiltrated with anesthetic in a linear fashion
      2. Administration can be performed in a "ring block," where subcutaneous tissue around the incision site is infiltrated circumferentially.
    4. High plasma concentrations of lidocaine or bupivacaine can cause cardiovascular effects (e.g., hypotension, dysrhythmias) and central nervous system depression followed by seizures. To avoid these adverse consequences:
      1. Weigh each animal individually and only give the maximum safe dose calculated for that individual.
      2. Aspirate the syringe prior to injection to ensure that IV injection is avoided.
    5. Local anesthetics are available in a variety of concentrations with or without epinephrine.
      1. Epinephrine causes vasoconstriction and prolongs the action of the local anesthetic.
      2. Epinephrine should not be used in animals that are suspected to have compromised cardiac function, or in locations that have poor collateral blood flow (distal tail, paw, etc.).

    8. Neonatal Rodent Anesthesia

    1. A rat neonate is a rat <10 days of age. There are several anesthetic methods currently presented in the literature for use in neonatal rodents and include:
      1. Injectable
      2. Inhalant
      3. Physical methods
    2. Injectable anesthetics have been associated with a high mortality in neonatal rodents. Do not use injectable anesthetics in neonatal rats <7 days old.
    3. Inhalant anesthetics in neonatal rodents have been associated with longer induction and recovery time than adult rodents with inhalant anesthetics.
    4. Hypothermia is the primary physical method utilized in neonatal rodent anesthesia.
      1. It is believed to provide anesthesia/analgesia by decreasing neural conduction and synaptic transmission.
      2. Hypothermia can only be performed in neonatal rodents <6 days old and should not be used for procedures lasting longer than 30 min.
      3. Ensure a barrier is between the neonate and the cooling agent (e.g. bed of crushed ice or chilled cold pack) at all times to prevent direct damage to the tissues; examples include:
        1. Placing a latex covering over the cooling agent.
        2. Placing the neonate in a cut off finger of a latex glove.
        3. Placing the neonate in a paper-lined test tube.
      4. Check the neonate for pedal reflex indicating proper plane of anesthesia.
      5. Maintain the neonate on a cooling agent for the procedure.
      6. Use fiber optic lighting for the surgical field as incandescent bulbs may warm the neonate.
      7. Re-warm the neonate slowly following hypothermia anesthesia.
        1. Rapid warming can cause tissue damage.
        2. Use of a circulating water heating pad set at 40 ºC (104ºF) or in an incubator set at 33 ºC (91.4ºF) is recommended.
      8. Return neonate to dam once are able to crawl.
    5. Parental cannibalism can occur with neonates after anesthesia. Follow the below steps to reduce the occurrence of cannibalism in anesthetized neonatal pups, if possible:
      1. Ensure the neonate is fully recovered before returning to the dam.
        1. Smear the neonate with soiled bedding from the mother's cage.
        2. Place the neonate back in the middle of the litter.
    6. Table 10: Inhalant Anesthetics in Neonatal Rats 

      Stage of Anesthesia   


      Oxygen (L/min)

      Isoflurane (%)

      Mask or Chamber
      0.5 - 1
      4 - 5
      0.5 - 1
      1 - 2
      Note: Neonates typically require a higher inhalant anesthetic dose than that observed in adults.   
    7. Table 11: Injectable Anesthetics in Neonatal Rats 


      Dosage and Route

      Duration of Action


      + Xylazine   
      40 - 90 mg/kg ket
      + 5 - 10 mg/kg xyl
      IP a or SC b
      20 - 40 minutes   
      Only to be used in rats greater than 7 days old   
      a Intraperitoneal (IP): < 25g needle, 1 ml syringe; maximum volume 1 ml   
      b Subcutaneous (SC): < 25g needle, 1 ml syringe, maximum volume 1 ml

    9. Neonatal Rodent Analgesia

    1. There is little information regarding the efficacy and dose ranges for the use of analgesics in neonates.
    2. Some data suggest that unalleviated pain in neonates can alter responses to pain and stress later in life.
    3. Investigate the literature on neonatal analgesia when a project involving neonatal surgeries and procedures is started, if possible. (LaPrairie and Murphy, 2010; Sternberg et al., 2005; Victoria et al., 2013a,b, 2014; LaPrairie et al., 2008; Walker et al., 2009).

    10. Emergency Resuscitation

    1. Attempts at resuscitating rats that have received an excessive dose of anesthetic or are experiencing cardiac or respiratory arrest for any reason, are generally unrewarding.
      1. Small chest compressions between the thumb and forefinger can be attempted.
      2. Respiratory depression can be treated by the administration of doxapram 5-10mg/kg IV or IP.
        1. If respiratory depression reoccurs, doxapram should be administered repeatedly at approximately 10-15 minute intervals.
      3. Supportive care for animals which reach too deep a level of anesthesia includes:
        1. Decreasing or discontinuing inhalant anesthetic.
        2. Stimulating the animal by gentle manipulation of the body.
        3. Raising the body temperature to normal.
        4. Providing supplemental oxygen through a facemask or nose-cone
      4. Administering reversal agents of anesthetic drugs, if applicable. (See Table 6 above)
  • Appendix A: ULAM Assessment of Pain in Rats

  • References

    1. Arras M, Autenried P, Rettich A, Spaeni D, Rulicke T. 2001. Optimization of intraperitoneal injection anesthesia in mice: drugs, dosages, adverse effects, and anesthesia depth. Comp Med 51:443-456.
    2. Baumans V, Coke C, Green J, Moreau E, Peterson-Kane E, Reinhardt A, Reinhardt V, Van Loo P eds. Making Lives Easier for Animals in Research Labs, Animal Welfare Institute, Washington, DC, 2007.
    3. Clowry, Gavin (2000).The successful use of fentanyl/fluanisone (Hypnorm) as an anesthetic for intracranial surgery in neonatal rats. Laboratory Animals34, 260-264.
    4. Danneman, Peggy (1997).Evaluation of Five Agents/Methods for Anesthesia of Neonatal Rats. Laboratory Animal Sciences47, 386-395.
    5. Dobromylskyj P, Flecknell PA, Lascelles BD, Pascoe PJ, Taylor P, Waterman-Pearson A. 2000. Postoperative and acute pain. In: Flecknell PA, Waterman-Pearson A, editors. Pain management in animals. London: W.B. Saunders. p 81-145.
    6. Flecknell, P. Laboratory Animal Anesthesia, 2nd Edition. Academic Press, 2015.
    7. Flecknell P, Waterman-Pearson eds. Pain Management in Animals, WB Saunders. London England, 2000.
    8. Fox JG, Anderson LC, Loew FM, Quimby FW eds. Laboratory Animal Medicine 2nd Ed. Academic Press, London England, 2002.
    9. Gaynor J, Muir W, Handbook of Veterinary Pain Management, Mosby, St. Louis Missouri, 2002.
    10. Gotoh, Hideo (2004).General Anesthesia of infant mice by isoflurante inhalation for medium-duration surgery. Experimental Animal53, 63-65.
    11. Hawk CT, Leary SL, Morris TH editors. Formulary for Laboratory Animals, 3rd Edition, Blackwell Publishing, Ames, Iowa, 2005.
    12. Hrapkiewicz K, Medina L, Holmes D, Clinical Laboratory Animal Medicine 2nd ed. Iowa Sate University Press, Ames, Iowa, 1998.
    13. Lieggi CC, Fortman JD, Kleps RA, Sethi V, Anderson JA, Brown CE, Artwohl JE, "An evaluation of preparation methods and storage conditions of tribromoethanol" Contemporary Topics in Laboratory Animal Science. 44.1 (2005) Pg 11-16.
    14. Mayer J., Mans C. (2018). ‘Rodents’ in Carpenter J.W., Mario C.J. (ed.). Exotic animal formulary, 5th ed. St. Louis: Elsevier Saunders. Pp. 459-493.
    15. Meyer RE, Fish RE. "A review of tribromoethanol anesthesia for the production of genetically engineered rats and mice." Lab Animal. 34.10 (2005).
    16. National Institutes of Health, Office of Animal Care and Use: "Pain and Distress in Mice, Rats, and Rabbits: Responsibilities, Recognition and Alleviation" updated 7/2004.
    17. Phifer, C.B. (1986).Use of Hypothermia for General Anesthesia in Preweanling Rodents. Physiology and Behavior38, 887-890.
    18. Shientag, LJ, et al. 2012. A therapeutic dose of ketoprofen causes acute gastrointestinal bleeding, erosions, and ulcers in rats. JAALAS 51(6):832-841.
    19. Sharp, P, Villano, J, The Laboratory Rat 2nd ed. CRC Press, Boca Raton, Florida, 2013.
    20. Wixon, SK and Smiler, KL. Anesthesia and Analgesia in Rodents, Chapter Nine. In: Anesthesia and Analgesia in Laboratory Animals. DJ Kohn, SK Wixon, WJ White, GJ Benson, Eds., Academic Press, 1997.
    21. Wright-Williams SL, Courade JP, Richardson CA, Roughan JV, Flecknell PA. 2007. Effects of vasectomy surgery and meloxicam treatment on faecal corticosterone levels and behavior in two strains of laboratory mouse. Pain. 130(1-2): 108-18.
    22. Zeller W, Meier G, Burki K, Panoussis B. "Adverse effects of tribromoethanol as used in the production of transgenic mice." Laboratory Animal Science. 32.4 (1998) Pg 407-413.
    23. Additional websites describing guidelines for Avertin use:
    24. Additional Websites regarding neonatal rodent anesthesia:
Species: Rats

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