Surgery Policy & Guidelines

Policy

Successful surgical outcomes in research animals of all species (including mice and rats) require the same techniques and procedures as in any veterinary practice. Researchers performing surgical procedures in all animals must adhere to the following:

  • All surgery must be performed in accordance with the researcher’s approved IACUC protocol.
  • All survival surgeries (either minor or major) must be performed using aseptic surgical techniques. Exception: If the survival period is very short (i.e., < 2 hrs), then aseptic technique may not be necessary.
  • Non-survival surgical procedures do not require aseptic techniques or dedicated facilities, but should be performed in a clean, clutter-free area. Exception: At least some elements of aseptic technique may be required if the non-survival surgery is prolonged (i.e., > 12 hrs) - Veterinary consultation is recommended.
  • Major survival surgical procedures on larger animals (non-rodents) must be performed in a dedicated large animal surgical facility.
  • A single animal may not undergo more than one major survival surgery unless the multiple procedures are required to meet the objective of a single animal research activity, justified for scientific reasons and approved by the IACUC.
  • All surgeries must be performed by qualified, trained personnel using techniques that minimize tissue trauma, maintain effective hemostasis, and use wound closure techniques that minimize trauma and promote healing.
  • Adequate anesthesia and analgesia must be used to prevent or mitigate pain, distress, and discomfort.
  • Post-surgical analgesic use must be followed as described in the approved IACUC protocol.
  • If using inhalant anesthesia, equipment must be properly set up, maintained, and serviced, with an appropriate Waste Anesthetic Gas scavenging system.
  • Research personnel must maintain adequate intra-operative (e.g., during the surgery) and post-operative monitoring records. IACUC members and/or veterinary staff may request copies of all such records for review without prior notice.
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Definitions

  • Aseptic Technique – Practices and procedures used to reduce microbial contamination to the lowest possible level.
  • Dedicated Large Animal Surgical Facility – A dedicated large animal surgery suite includes separate areas for animal preparation, surgeon preparation, and surgery.  These areas are set up to be cleaned and maintained in an aseptic condition (e.g., all room surfaces are non-porous and easily sanitized) prior to performing survival surgery.
  • Survival Surgery – An operative procedure after which the animal recovers from anesthesia.
  • Terminal (Non-Survival) Surgery – A procedure in which the animal is euthanized prior to recovery from anesthesia.
  • Major Surgical Procedure – Any surgical intervention that penetrates and exposes a body cavity OR any procedure which produces permanent impairment of physical or physiological functions1.  Some procedures, such as laparoscopic procedures, craniotomies and other relatively minor surgical procedures that may penetrate a body cavity will be reviewed by the IACUC on a case-by-case basis as to classification.
  • Minor surgical procedure – Any surgery other than those considered major surgical procedures.

Animal Surgery Guidelines

Personnel Training for Animal Surgery

Personnel Training for Animal Surgery

Regardless of an individual’s qualifications or educational background, all personnel performing surgery must have thorough knowledge and understanding of the approved IACUC protocol procedures and must be trained by someone who possesses a knowledge of surgery policies, proper surgical technique, and familiarity with the relevant surgical procedure and with the anatomy of the species. ULAR veterinarians are charged by the IACUC to confirm that the training is adequate.  It is the Principal Investigator’s and/or Lab Manager’s responsibility to ensure personnel are qualified to perform procedures and to maintain training records for all members of the research team.

At a minimum, training of surgical personnel must include:

  • A thorough knowledge of aseptic technique.
  • Administration and assessment of anesthesia
  • Appropriate tissue handling
  • Appropriate use of instruments
  • Effective methods of hemostasis.
  • Correct use of sutures and/or skin staples (see guidance for Wound Closure Selection (Table 4) under the Aseptic Surgical Techniques tab)
  • Post-surgical care and monitoring, including the ability to recognize and alleviate pain and distress.
  • Completion of the Aseptic Surgery chapter in the CITI Training is required for all personnel that will be performing surgical procedures.

Record-Keeping Requirements for Animal Surgery

Record-Keeping Requirements for Animal Surgery

Intra-Operative Records (i.e., records kept during the surgery) must include the following information:

  • Animal identification – species and animal identification.
  • Body weight at the start of the procedure (if using inhalant anesthetics alone, this can be an estimate for mice and rats, e.g., 25 or 30 gm).  This may be required for precise calculation of drug dosages (anesthetics, analgesics, antibiotics, etc.)
  • Name of the surgeon.
  • Date and description of surgical procedure.
  • Details of all medications given to the animal, including dosage (mg or mg/kg), route, and time of administration.
  • Notes concerning any complications encountered.
  • Euthanasia method (terminal surgeries only)
  • Researchers are encouraged to use or customize one of the following templates:

Post-Operative Monitoring Records must include the following:

  • Details of all care and monitoring provided to the animal such as wound cleaning, bandage changes, flushing of indwelling catheters, body temperature, heart rate, etc., as described in the approved IACUC protocol.
  • Any complications encountered-- e.g., delayed recovery from anesthesia, bleeding from incision site, excessive inflammation, lameness or head tilt, any indication of pain, etc.
  • Dose, route, and time of all medications or compounds administered to the animal, especially post-operative analgesia.
  • If postoperative records are maintained in the vivarium, contact information for all research staff responsible for daily assessment and care must be provided on the record.
  • All postoperative record entries must include time, date, and initials of personnel performing the procedure.
  • Researchers are encouraged to use or modify one of the templates listed below:

Areas Used for Animal Surgery

Areas Used for Animal Surgery

The IACUC reviews and approves all surgical areas. In addition, all surgical areas are subject to inspection during semi-annual facility inspections.
All areas used for surgery in animals must be designed and managed to achieve the following:

  • Separation of the animal/surgeon preparation, operating room and recovery areas.
  • Minimization of personnel traffic flow through the surgery area
  • Airflow should be away from the surgery area if possible (i.e., positive room pressure, use of filtered or laminar-flow air).
  • All surfaces must be non-porous and easily sanitized. Unsanitizable surfaces (e.g. cloth-upholstered chairs) should be removed to an area where live animals are not used.
  • A regular room-cleaning and disinfection schedule must be established (daily cleaning of floors and work surfaces, weekly to monthly cleaning of walls and cabinets).
  • The surgery area should be free from clutter.  Excess items not required for the procedure should be stored in cabinets or drawers.
  • A dedicated three-room (prep, operating and recovery) surgical suite is not required for the performance of surgical procedures in rodents.  However, the space must be designated/reserved exclusively for surgery while in use and appropriately managed to minimize contamination from other activities in the room while surgical procedures are being performed.

NOTE:  Investigators are responsible for cleaning shared surgical facilities after each use.

ULAR maintains several large-animal surgical suites in UCI vivaria; investigators are encouraged to contact Veterinary Services for more information.

Requirements for Animal Surgery

Requirements for Animal Surgery

  • Eyes should be lubricated with a sterile ophthalmic ointment to prevent corneal drying, especially for any survival surgery.
  • Supplemental heat should be provided during surgery and recovery to maintain body temperature, as animals lose their ability to regulate body temperature while under general anesthesia.
    • For large animal survival surgery, use of circulating water blankets or Bair Huggers should be used instead of electric heating pads, especially under the animal to prevent skin burns.
  • Use of supplemental fluids (saline or LRS, IV or SQ) should be considered, as well as post-operative nutritional support, such as by providing moistened food pellets on the cage floor.
  • Skin sutures or staples must be removed 10-14 days after surgery, once the incision has healed. Note: If the animal(s) will be euthanized within 28 days of the surgery, the sutures do not need to be removed.
  • Handle tissues gently –
    • Minimize the use of toothed or crushing instruments.
    • Hold the cut edge rather than grasping the middle of a tissue layer.
    • When tying off vessels, include a minimum of surrounding tissues.
    • Sparingly use electrocautery and electroscalpels, as these cause tissue necrosis.
    • Keep tissue moist during surgery.
  • Ablate “dead space” during closure - Any pockets or spaces remaining between tissue layers will fill with extracellular fluid or blood and increase the risk of developing abscesses.
  • Minimize the duration of surgery - Prolonged surgery times expose tissues to contaminants and dry out tissues, and lead to an increased risk of necrosis and postoperative infection.

Aseptic Surgical Techniques

Aseptic Surgical Techniques

The goal of aseptic technique is to reduce the possibility of microbial contamination to the lowest practical level. No single technique, practice, germicide, or piece of equipment will achieve this objective. Rather, proper aseptic technique is dependent on numerous practices that require input and cooperation of all personnel within the operating area. Components of successful aseptic technique include:

Selection and Preparation of the Surgical Area

Surgery should be conducted in a designated animal procedure space (i.e., a location within a procedure room or laboratory space free from clutter that promotes asepsis during surgery). During the surgery period, the area should be dedicated to the surgery so that cleanliness is ensured, and contamination is minimized. See guidance about Surgical Areas for more info.

  • Hard surfaces such as tabletops and other equipment (i.e. stereotax fames) should be disinfected prior to surgery – See Table 1. Recommended Hard Surface Disinfectants (below). 
  • Note: Major survival surgical procedures on larger animals (non-rodents) must be performed in a dedicated large animal surgical facility.

Sterilization of Instruments & Supplies

All instruments and supplies that come in contact with the surgical (incision) site must be sterile at the start of surgery and maintained as sterile throughout. When performing surgeries on rodents, a hot bead sterilizer may be used in between animals when doing more than one. There are a number of options available to sterilize surgical equipment and supplies:

  • Autoclave - Relies on pressurized steam, is extremely reliable, and cost-effective. However, instruments must be durable (e.g., stainless steel) and the process is relatively slow, from 15 to 60 minutes. Instruments are typically wrapped or sealed in packs that are opened as needed on the day of surgery
  • Ethylene oxide - A gaseous sterilant that requires specialized containment equipment. This is a good sterilization method for supplies that cannot tolerate high heat such as plastics and catheters. It is more costly than autoclaving and typically is performed overnight
  • Cold sterilant solutions (hypochlorite, glutaraldehyde, etc.) - Generally, cold sterilants must have prolonged contact time (15 - 60 minutes) to sterilize surgical equipment. In addition, the instruments must be rinsed completely with a sterile solution like saline to prevent tissue irritation.
  • Hot bead sterilizer - This device is a small tabletop unit, approximately 6 x 6 x 8 inches. The appliance heats a small container of Pyrex beads to approximately 250 C and can sterilize the tips of metal surgical instruments in 10-20 seconds. It is very useful for sterilizing instruments between rodents when performing surgeries on multiple animals.
  • Pre-sterilized items - Many instruments and supplies can be purchased in sterilized packaging. Such items must be used prior to the label expiration date.
  • Implanted Items - Special consideration needs to be made for items that will be implanted (i.e. left in the animal after surgery is completed). Many of these items are “sensitive and/or fragile” and can be difficult to sterilize. Possibilities include use of ultra-short autoclave cycle, ethylene oxide, or use of a cold sterilant. Note: Use of alcohol alone is not a sterilant.

See Table 2. Recommended Sterilants for Surgical Instruments & Equipment  (below)

Surgeon Preparation

Surgeon for Rodents

  • Clean covering such as a disposable gown or lab coat worn over work clothes
  • Mask
  • Hair cover
  • Gloves - Surgeons should wash and dry their hands before donning gloves
    • Using sterile surgical gloves allows you to touch all areas of the sterile surgical field and surgical instruments with your gloved hand.
    • Using clean exam gloves and a “tips-only” technique restricts you to using only the sterile working ends of the surgical instruments to manipulate the surgical field.
    • The gloved, but not sterile, hand must never touch the working end of the instruments, the suture, suture needle, or any part of the surgical field.

Surgeon for Large Animals

  • Clean scrubs
  • Mask
  • Hair cover
  • Shoe covers
  • Gloves and Gown
    • The surgeon must scrub the hands and forearms with a disinfectant soap for a minimum of three (3) minutes and then dry them with a sterile towel prior to survival surgery in all species.
    • After washing a sterile surgical gown followed by sterile surgical gloves must be aseptically donned.

Animal Preparation

  • Hair should be removed from the surgical site, preferably with electric clippers (# 40 blade) or depilatory rather than a razor. If a depilatory is used, thoroughly rinse the chemical from the animal’s skin or apply a neutralizing agent.
  • The skin should then be cleaned and disinfected with three alternating scrubs using a chlorhexidine or povidone iodine-based disinfectant followed with an alcohol rinse. See Table 2 below for specific recommendations. The site should be scrubbed by starting at the center of the site and working outward in a circular pattern.
  • If using a stereotaxic frame, the animal should be placed in the frame before the skin disinfectant is applied.
  • The surgical area prepared should be approximately twice that needed for the incision, in the event a larger incision than planned may be required.
  • A sterile surgical drape should be used whenever possible to isolate the disinfected area from surrounding tissue and hair and help maintain asepsis of instruments and supplies, such as suture material. To be effective, a drape needs to be impermeable to moisture.

Techniques to Maintain the Sterile Field

  • Sterile gloves, instruments, suture material, suture needle, etc. must never touch non-sterile fields.
  • When working alone and manipulation of non-sterile objects (e.g. anesthesia machines, microscopes, lighting, etc.) is required, it may be helpful to use sterile aluminum foil or sterile plastic covers to manipulate the objects.
  • When using “tips-only” technique, the sterility of the instrument tips must be maintained throughout the procedure. Hands, if using non-sterile gloves, must not come into contact with the surgical area. Recommend that a sterile towel or drape be used to place tips of instruments down when not in use.

Table 1. Recommended Hard Surface Disinfectants

AGENTEXAMPLESNOTES
Alcohols70% ethyl alcohol85% isopropyl alcoholContact time required is 15 minutes. Contaminated surfaces take longer to disinfect. Remove gross contamination before using.
Quaternary AmmoniumRoccal®, Quatricide®Rapidly inactivated by organic matter. Compounds may support growth of gram negative bacteria.
ChlorineSodium hypochlorite (Clorox ® 10% solution) ChlorineCorrosive. Presence of organic matter reduces activity. Chlorine dioxide must be fresh; kills vegetative organisms within 3 minutes of contact.
GlutaraldehydesGlutaraldehydes (Cidex® Cetylcide®, Cide Wipes®)Rapidly disinfects surfaces.
PhenolicsLysol®, TBQ®Less affected by organic material than other disinfectants.
ChlorhexidineNolvasan® , Hibiclens®Presence of blood does not interfere with activity. Rapidly bactericidal and persistent. Effective against many viruses.
Hydrogen peroxide Peracetic acid Acetic acidSpor Klenz, Virkon®Contact time 10 minutes.

Table 2. Recommended Sterilants for Surgical Instruments & Equipment

AGENTEXAMPLESNOTES
Steam Sterilization (moist heat)AutoclaveEffectiveness dependent upon temperature, pressure and time, e.g. 121°C for 15 min vs 131°C for 3 min. Appropriate sterilization indicators should be used to ensure sterility.
Dry HeatHot Bead Sterilizer Dry ChamberFast Instruments must be cooled before contacting tissue. Only tips of instruments are sterilized with hot beads.
Gas sterilizationEthylene OxideRequires 30% or greater relative humidity for effectiveness against spores. Gas is irritating to tissue; all materials require safe airing time. Appropriate sterilization indicators should be used to ensure sterility.
ChlorineSterilant Levels of Chlorine dioxide (Clidox®, Alcide®) Sodium hypochlorite (Clorox® 10% solution)Corrosive to instruments. Items must be clean and free of organic material. Instruments must be rinsed with sterile saline or sterile water before use.
GlutaraldehydesGlutaraldehyde (Cidex®, Cetylcide®, Metricide®)Several hours required for sterilization. Corrosive and irritating. Instruments must be rinsed with sterile saline or sterile water before use. Product expiration dates must be adhered to as per manufacturer’s instructions.
Hydrogen peroxide Acetic acidActril®, Spor-Klenz®Several hours required for sterilization. Corrosive and irritating. Instruments must be rinsed with sterile saline or sterile water before use.
AlcoholEthanol or IsopropanolAlcohol is neither a sterilant nor a high-level disinfectant.

Table 3. Skin Disinfectants

AGENTEXAMPLES NOTES
IodophorsBetadine®, Prepodyne®, Wescodyne®Reduced activity in presence of organic matter. Wide range of microbicidal action. Works best in pH 6-7.
ChlorhexidineNolvasan®, Hibiclens®Presence of blood does not interfere with activity. Rapidly bactericidal and persistent. Effective against many viruses. Excellent for use on skin.

Table 4. Wound Closure Selection

MATERIALCHARACTERISTICS & FREQUENT USES
Polyglactin 910 (Vicryl®), Polyglycolic acid (Dexon®)Multifilament, Absorbable in 60-90 days; 25-50% loss of tensile strength in 14-21 days. Ligate or suture subcutaneous tissues where an absorbable suture is desirable. Not routinely recommended for skin closure due to high capillarity.
Polydiaxanone (PDS®) or, Polyglyconate (Maxon®)Monofilament, Absorbable in 6 months; 40% loss of tensile strength in 30-42 days. Ligate or suture tissues especially where an absorbable suture and extended wound support is desirable.
Polypropylene (Prolene®)Monofilament, Non-absorbable. Inert.
Nylon (Ethilon ®)Monofilament, Non-absorbable. Inert. General closure.
SilkMultifilament, Non-absorbable. (Caution: Tissue reactive and may wick microorganisms into the wound, so silk is not recommended for skin closure). Excellent handling. Preferred for cardiovascular procedures.
Stainless Steel Suture/Wound Clips/Wound StaplesNon-absorbable. Requires instrument for removal.
Cyanoacrylate (Vetbond®, Nexaband®, Tissue Mend®)Skin glue. For non-tension bearing wounds.

Multiple Survival Surgery

Multiple Survival Surgery

Regardless of whether the surgery is categorized as major or minor, multiple survival surgical procedures on a single animal should be evaluated to determine their impact on the animal’s well-being.

Multiple major surgical procedures on a single animal are acceptable only if they are:

  • Essential components of a single IACUC-approved research project or protocol;
  • Scientifically justified by the investigator; or
  • Necessary for clinical reasons.2

Animals should not undergo surgical procedures under more than one protocol, except under a few limited exceptions, such as vendor-performed ovariectomy or neutering performed by campus veterinary staff.  All surgeries performed on a single animal must be interrelated components of one project.

Cost savings alone is not a justification for multiple surgical procedures; however, conservation of scarce animal resources may be considered as part of a scientific justification by the IACUC.

One exception to the general rule against multiple survival surgeries is the harvest of oocytes from Xenopus frog species – see guidelines for multiple oocyte harvest.

References

Surgery in Non-Mammalian Species

Surgery in Non-Mammalian Species

Aquatic Species

  • Surgical preparation of the incision site should minimize disruption of skin and mucus layer.
  • The skin at the incision site should be gently wiped with sterile gauze or cotton-tipped applicator to reduce gross contamination. If greater antimicrobial activity is wanted, the skin can be wiped with a dilute solution of povidone iodine (1:20) or chlorhexidine (1:40). Application of harsher chemical disinfectants and alcohol may irritate the skin and increase the risk of tissue damage and postoperative morbidity and mortality.
  • For larger fish species, removing large scales by extracting them caudally can facilitate a smooth incision.
  • A sterile clear plastic drape can be positioned over the animal to help isolate the incision site, create a sterile field and help retain moisture. A rim of petroleum jelly can be used to adhere the drape to the animal, if desired.
  • The animal's skin should be kept moist throughout the surgery, with care taken to prevent irrigating the incision site with contaminated anesthetic or tank water.
  • After the animal has been anesthetized, the animal should be positioned to allow easy access to the surgical site.
  • The feathers at the surgical site should either be parted for small incisions or plucked to expose the intended incision site. The skin should be exposed to create a space approximately twice the size of the intended incision. Tape can be applied to surrounding feathers to prevent them from entering the sterile field during surgery.
  • The skin should then be cleaned and disinfected with a chlorhexidine or povidone iodine-based disinfectant. The site should be scrubbed by starting at the center of the site and working outward in a circular pattern. Typically, one scrub with a disinfectant, followed by alcohol will suffice.
  • If possible, the use of a sterile surgical drape is recommended to help isolate the sterile field and reduce the risk of postoperative infection.
  • The skin should then be cleaned and disinfected with a chlorhexidine or povidone iodine-based disinfectant. The site should be scrubbed by starting at the center of the site and working outward in a circular pattern. Reptiles harbor significant pathogens on the skin, such as Salmonella, and a prolonged vigorous scrub with multiple applications of disinfectant followed by an alcohol wipe is recommended.

Avian Species

  • After the animal has been anesthetized, the animal should be positioned to allow easy access to the surgical site.
  • The feathers at the surgical site should either be parted for small incisions or plucked to expose the intended incision site. The skin should be exposed to create a space approximately twice the size of the intended incision. Tape can be applied to surrounding feathers to prevent them from entering the sterile field during surgery.
  • The skin should then be cleaned and disinfected with a chlorhexidine or povidone iodine-based disinfectant. The site should be scrubbed by starting at the center of the site and working outward in a circular pattern. Typically, one scrub with a disinfectant, followed by alcohol will suffice.
  • If possible, the use of a sterile surgical drape is recommended to help isolate the sterile field and reduce the risk of postoperative infection.

Reptiles

  • The skin should then be cleaned and disinfected with a chlorhexidine or povidone iodine-based disinfectant. The site should be scrubbed by starting at the center of the site and working outward in a circular pattern. Reptiles harbor significant pathogens on the skin, such as Salmonella, and a prolonged vigorous scrub with multiple applications of disinfectant followed by an alcohol wipe is recommended.1

Oocyte Removal from Xenopus Frogs:

One exception to the general rule against multiple survival surgeries is the harvest of oocytes from Xenopus species.  The IACUC acknowledges that the quality of oocytes varies a great deal from animal to animal; maximizing the productivity of a single “good producer” enhances the reproducibility of critical experiments and may reduce the overall number of animals used.  While the harvest of oocytes meets the definition of “major” surgical procedure, the procedure is quickly performed by appropriately trained personnel and the animals rapidly return to normal feeding and activity.

Guidelines for Multiple Oocyte Harvest:

  • The maximum number of surgeries allowed per animal is six, three on alternating sides, with the sixth surgery being a terminal procedure.
  • The interval between procedures should be not less than one month.
  • The protocol must include a written description of the method used to identify animals to ensure adequate time has lapsed between surgeries (e.g., skin marking or tattooing, tank rotation, etc.).
  • Aseptic technique appropriate for aquatic species must be used to reduce microbial contamination.
  • General anesthesia such as MS-222 must be used.
  • Recommendations in the IACUC Surgery Policy and Guidelines for aquatic species must be followed.
  • Animals should be housed singly and carefully monitored for 24-48 hours post-surgery.
  • Skin sutures and wound clips, if non-absorbable, must be removed 2-3 weeks after surgery.