I: Background and Purpose

This policy is designed to ensure that operative procedures involving small vertebrates (< 500g) used for research comply with UCR's OLAW Assurance, NIH policies, and AAALAC International guidelines, as outlined in the Guide for the Care and Use of Laboratory Animals. The policy offers investigators performing surgical procedures the best practices and procedural guidelines aimed at minimizing adverse clinical outcomes that may arise as a result of surgery. 

II: Related Policies and Guidance

• Policy 529-227: Agents for Anesthesia, Analgesia and Euthanasia
• Policy 529-251: Policy on Training of Animal Users
• Policy 529-303 Use of Volatile Anesthetics in Laboratory Animal Research
• Guidance 529-349: Guidelines for Rodent Anesthesia and Analgesia

III: Definitions

• Aseptic Technique: A set of practices used in surgery to prevent the spread of microorganisms and contamination. 
• Disinfection: The chemical or physical process that involves the destruction of pathogenic organisms. All disinfectants are effective against vegetative forms of organisms, but not necessarily spores.
• Major surgery: Major surgery penetrates and exposes a body cavity or significantly impairs physical or physiological functions (such as laparotomy, thoracotomy, craniotomy, joint replacement, and limb amputation).
• Minor surgery: Minor survival surgery does not expose a body cavity and causes little or no physical impairment (such as wound suturing, peripheral-vessel cannulation, vascular catheter implantation, subcutaneous pump implantation, subcutaneous implantation of tumor cells, and most procedures routinely done on an "outpatient" basis in veterinary clinical practice).
• Sanitation: The process of containing and preventing the spread of potentially harmful organisms using antiseptic agents.
• Sterilization: The process whereby all microorganisms, including spores, are      eliminated or destroyed. The criterion of sterilization is the failure of organisms to grow if a growth-supporting medium is supplied.
• Survival / Non-survival surgery: Non-survival surgery involves procedures where the animal is euthanized without recovery from anesthesia. All other surgeries are considered survival surgery

IV: Policy

A. Multiple Survival Surgery

a) Multiple survival surgical procedures on a single animal are discouraged but may be permitted if scientifically justified in an approved AUP. 

i. For example, multiple survival surgical procedures can be justified if they are related components of a research project, if they will conserve scarce animal resources, or if they are needed for clinical reasons. 

ii. If multiple survival surgeries are approved, particular attention must be paid to animal well-being, and outcomes must be evaluated continuously. 

iii. Cost savings alone do not justify performing multiple survival surgical procedures.

B. Animal Use Protocol (AUP)

a) The AUP must clearly state whether the surgical procedures should be classified as non-survival or survival, minor or major, and whether there will be multiple major survival surgeries. 

b) AUPs must describe the procedure that addresses the use of aseptic technique, anesthetics, recovery, and analgesia.

c) A rigorous justification for performing multiple survival surgeries must be included if applicable.

d) All protocols involving major survival surgery will be discussed at a convened meeting of the IACUC.

e) All personnel expected to perform surgery must be identified in the approved AUP and adequately trained on the surgical procedure by the PI or their designee. 

f) IACUC training for small mammal and rodent survival surgery is required for all surgeons expected to perform surgery. The PI is responsible for performing initial training on species other than small mammals (e.g., amphibians, reptiles, etc.). 

C.  Surgical Facilities

a) Locations where surgeries are performed on rodents and non-mammalian vertebrates do not require special facilities and may be done in a laboratory under appropriate conditions if approved by the IACUC. 

b) The area or benchtop where surgeries are performed should be made of material that can withstand repeated sanitation and should not be used to store items that cannot be sanitized (e.g., cardboard, untreated wood). 

c) Surgeries should not be performed in areas where winds can contaminate the sterile field, such as high traffic or vents. 

V. Surgical Procedure

A. Non-Survival Surgery:

Aseptic technique is generally not required for non-survival surgical procedures, provided the animal procedures, provided the animal is not anesthetized long enough to develop infection, and the study data is not affected by the lack of strict aseptic precautions. A clean, clutter-free area and adherence to basic veterinary sanitation practices are still recommended. 

B. Survival Surgeries: 

Aseptic technique is required for survival surgeries. General requirements for such surgeries are described in the sections below:

a) The surgeon

i. Surgeons should wear a clean lab coat, head covers, sterile gloves, and face mask.   

ii. Surgeons must wash their hands before aseptically donning sterile surgical gloves.    

b) The location

i. A sterile field must be created by sterilizing instruments, supplies, and implanted materials.   

ii. Operative techniques must be used to reduce the likelihood of contamination.

c)  The animal

i. Fur, hair, and feathers must be removed from the area where the incision will be made, leaving a clear border of at least 1 cm around the incision site. 

ii. The clipping of fur or plucking of feathers should be done in an area separate from where the surgery is to be conducted.

iii. For non-mammalian aquatic species such as amphibians, rinse visible      debris from surgical site using sterile saline or another sterile isotonic fluid and maintain continuous skin moisture throughout surgery. If skin disinfection is pursued, use a 10% povidone iodine solution, but do not use chlorohexidine or scrubs containing soaps or detergents, as these may damage the skin.

iv. The skin around the incision area must be prepared with an appropriate skin disinfectant.

v. Apply disinfectant from the center outward. The standard procedure is to alternate between surgical soap (e.g., betadine) and alcohol scrubs three times. Alcohol, by itself, is not an adequate skin disinfectant. 

vi. For non-aquatic species, care should be taken not to wet the animal excessively, which may contribute to increased heat loss (hypothermia).    

C. Anesthetics and Analgesics

a) The choice of anesthetics and analgesics depends on the species. For non-mammalian species, the PI and the attending veterinarian should determine the best anesthetic and analgesic regimen, noting that the PI is the potential subject matter expert. A thorough evaluation should be considered when determining the appropriate anesthetics and analgesics. A local anesthetic such as bupivacaine should be considered at incision sites. Administration of analgesics prior to the beginning of surgery (preemptive analgesia) can enhance their effectiveness. Contact the Campus Veterinarian for more information on the use of various anesthetics and analgesics.
b) The dose (mg/kg), vehicle, and volume of anesthetic must be listed on the AUP, including potential supplemental dosing information. 

c) The AUP must outline a system for monitoring the anesthetic depth, physiologic function, and assessment of clinic signs and conditions and employ it during surgical procedures. 

d) Pain is a stressor that, if not relieved, can lead to unacceptable levels of stress and distress in animals. Properly using anesthetics and analgesics in research animals is an ethical and scientific imperative. 

i. Preoperative or intraoperative administration of analgesics should be considered as they may enhance postsurgical analgesia. 

ii. Subsequent animal monitoring must include administering analgesia in response to behavioral manifestations of pain or distress unless a scientific justification is provided in the AUP and approved by the IACUC.

iii. Indicators of pain and distress are species-specific, but they can include vocalization, abnormal appearance or posture, and immobility. iv. Unless the contrary is established, it should be assumed that procedures that cause pain in humans also cause pain in animals.

D. Preoperative, Intraoperative, and Postoperative Care

a) Preoperative care:

i. All animals should receive a pre-anesthetic assessment. The assessment should include a thorough visual assessment for any signs of illness or abnormal behavior. An accurate weight should be obtained for appropriate drug dosing.

ii. Following anesthesia and analgesic administration, an ophthalmic ointment should be applied to the eyes to protect the corneas from drying.

iii. Because anesthetic agents cause cardiovascular and respiratory disturbances in most species. For most warm-blooded species, the equipment needed to maintain normal body temperature should be obtained, and its use should be delineated in the AUP. 

iv. The first dose of analgesics should be given prior to the first incision to ensure the pain-relieving properties are present during the animal's recovery.

b) Intraoperative Care

i. Procedures must be in place to maintain the animal's body temperature with supplemental heat for most warm-blooded species. This can be supplied with a warm water-circulating heating pad. Electric heating pads and heat lamps are not recommended due to burn risk.

ii. Anesthetized animals must be continuously monitored until fully recovered. Anesthetic monitoring depends on species but can include responsiveness to painful stimuli, the character of respiration, the color of skin, and skin or mucous membrane color as seen by observing the ears, tail, and oral mucosa or foot pads. A pedal withdrawal reflex (toe or footpad pinch on the hind foot) is recommended for assuring adequate depth of anesthesia prior to the first incision and at least every 15 minutes during the procedure. 

iii. For aquatic non-mammalian species such as amphibians, animals should remain wet during procedures outside of water. Water can be sprayed, dripped, or poured on exposed areas of the animal. Use water from the home tank wherever possible; clean, non-chlorinated water is also acceptable.

iv. Other monitoring may be indicated, depending on the surgical procedure, such as body temperature and tissue oxygenation. Tissues exposed for long periods of time should be kept moist with warm saline or lactated Ringer’s solution. If the surgery is prolonged or the animal has lost more than a small amount of blood, these same fluids should be administered subcutaneously or intraperitoneally to prevent dehydration and volume depletion.

v. The surgical incision should be closed using the appropriate technique that is consistent with the species. Wound clips, sterile absorbable or non-absorbable suture material, and tissue glue are examples of appropriate wound closure materials.

c) Postoperative Care

i. During the anesthetic-recovery period, attention should be given to thermoregulation, cardiovascular and respiratory function, and postoperative pain or discomfort. 

ii. Animals must be continuously monitored during recovery until they can right themselves or ambulate. The AUP must clearly outline a system that defines the parameters to be monitored. Typically, this includes response to stimuli, depth and quality of breathing, skin/mucous membrane oxygen saturation/color, and temperature monitoring via external support. 

iii. During recovery, small mammals and some reptiles should be placed in a clean, empty cage or on a paper towel on top of the bedding substrate to help protect against a suffocation hazard.

iv. The surgical incision should be monitored for postsurgical infections, bandaged as appropriate, and skin sutures, clips, or staples removed in a timely manner.

v. Semi-aquatic non-mammalian species should be partially submerged in freshwater with the head and nares held above water or placed in a closed but not airtight container with a moist paper towel on the bottom during recovery. Other methods are also acceptable if the skin is kept moist and the relative humidity in the immediate environment is maintained at > 70%.

VI. Surgical Records

A. Surgical Cage Cards 

a) Surgery cards can be obtained from Vivarium Operations. PIs should contact the facility manager where their animals are housed to obtain these cage cards.

b) The surgical cage card must annotate analgesics, the type of surgery, the surgeon, antibiotic administration, and the animal's condition.

c) Cards should remain with the cage until the animal is euthanized. Once sutures or wound clips are removed, the surgery card can be placed behind the primary cage card. 

B. Procedural Documentation

a) Both survival and non-survival surgical procedures must be documented. 

b) Template surgical records are available for the lab in the Forms section of the IACUC webpage.

c) Records must document the AUP number, all drugs administered (dose, route, time of administration, etc.) intraoperative monitoring details, surgery description, surgeon name or initials, post-op monitoring, suture/staple removal if applicable, and any adverse outcomes. 

d) All documentation of surgery is subject to review by the IACUC and other regulatory agencies that govern the use of animals for research purposes.