Surgery - Xenopus Oocyte Harvest (Guideline)

Guidelines: The IACUC has provided a set of guidance documents (Policies, Guidelines, and Informational Sheets) for use when planning animal procedures at the University of Iowa. An exception to a Guideline must be described and justified in the Animal Protocol and approved during the normal review process.

Purpose:  The purpose of these guidelines is to provide direction for personnel conducting surgery to harvest oocytes from Xenopus frogs at the University of Iowa. These guidelines are intended for use by properly trained personnel listed on an IACUC-approved Animal Protocol who will be performing approved surgical procedures on Xenopus, or assisting with those procedures. Deviation from these guidelines must be described and justified in an IACUC-approved Animal Protocol.

Definitions

• Best Practice
  • Technique or method that consistently shows superior results
  • Items in Guidelines labeled as "best practice" are strongly recommended, but not required

Instrument and Equipment For Surgery

• It is best practice to start each surgery session using sterile instruments, surgical supplies and wound closure materials At a minimum start with clean instruments (free of gross debris) that have had their tips sterilized with a dry bead sterilizer
  • It is not recommended to use chemical sterilants for equipment used for frog surgeries
    • improperly rinsed instruments can negatively impact the health of the frogs
  • If a sterile instrument or sterile glove comes in contact with a non-sterile item, it is NO LONGER STERILE
  • Re-sterilize or replace contaminated items before continuing aseptic procedures
  • Re-sterilize or replace items before continuing aseptic procedures between animals
• Refer to sterilization methods for further information on material sterilization methods

Surgery Location

• Designate a clean uncluttered work area away from laboratory traffic, ventilation ducts and open windows
  • Dedicate the area solely to surgical procedure(s) when in use
  • Utilize a separate room used primarily for aseptic procedures, if possible

Area Preparation

• Clean work surface with  a disinfectant (see table below)
• Apply a clean drape over the working surface where the surgery will be performed
• Establish a sterile field near the animal for placement of sterile instruments

Examples of hard surface disinfectants

Animal Preparation

• Anesthetize the animal in accordance with the approved Animal Protocol
  • Refer to the IACUC Guidelines on Anesthesia for details of anesthetic agents, procedures, and monitoring requirements
• Wash gross debris from surgical site of frog's skin with saline
• Cover the frog, around the incision site, with a drape soaked in the anesthetic water to maintain anesthesia and skin moisture
  • This DOES NOT provide a sterile barrier but will keep mucus off the instruments and suture material
  • Dry drapes will stick to the mucus layer and disrupt its natural protective barrier and SHOULD NOT be used

Aseptic Surgical Technique (suggested best practice)

• Put on a clean lab coat or scrub top, surgical mask and tie back or cover long hair
• Unwrap sterile instruments making sure to only touch the outer surface of the wrap
  • Do not touch the interior of the packaging or instruments as this will compromise the sterility of the instruments
• Perform a surgical scrub of the surgeon’s hands
• Put on sterile gloves without touching the exterior of the glove surface
  • The interior of the sterile glove packaging may be used as a sterile field for instruments
• Dedicate instruments used to incise skin solely for that purpose
  • Separate these instruments from those used to manipulate exposed tissue and organs
• Maintain sterility of gloves and instruments throughout the surgery
• Maintain sterile suture material within the sterile field at all times
  • Avoid pulling sterile suture across non-sterile areas (e.g., across animal’s body, areas surrounding the sterile field)

Aseptic Tip Technique

• Put on a clean lab coat or scrub top, surgical mask and tie back or cover long hair
• Start with clean instruments (free of gross debris) that have had their tips sterilized with a dry bead sterilizer
• Wash hands or perform surgical scrub
• Put on sterile or procedural gloves
  • The interior of the sterile glove packaging may be used as a sterile field for instruments if sterile gloves are used
• Arrange the instruments so that the tips are within a sterile field and the handles are outside the sterile field
  • Do not contaminate the sterile tips of the instruments during this process
  • Instrument tips must be maintained within this sterile field throughout the surgery
• Utilize only the sterile tips of the instruments inside the body cavity
• Use a dry bead sterilizer to sterilize the tips of surgical instruments in between multiple surgeries.  
• Maintain sterile suture material within the sterile field at all times
  • Avoid pulling sterile suture across non-sterile areas (e.g., across animal’s body, areas surrounding the sterile field)

Wound closure

• Close incision in two layers (muscle then skin)
  • Close muscle with absorbable suture
    • Include the white fascia membrane over the muscle layer
  • Close skin with absorbable or non-absorbable suture
    • DO NOT use gut suture as it may dissolve in water prior to wound healing
    • PDS or monofilament Vicryl suture can be used
• Utilize an interrupted suture technique when closing the skin

Anesthetic Recovery and Post-Surgical Monitoring

• Recover the animal from anesthesia according to the IACUC Guidelines on Anesthesia
• Place frog in fresh non-chlorinated water with head elevated above the water line to prevent drowning until conscious
• Return frog to animal housing only once it has fully recovered (i.e., swimming and diving)
• Assess the animals at least daily (including weekends and holiday) for at least three days post-operatively or as justified in the Animal Protocol
  • Monitor animals for signs of pain (Pain Recognition by Species) and contact OAR Veterinary Staff if signs are observed
  • Monitor incision site for the following:
    • Incisional integrity (i.e. sutures intact and wound is closed)
    • Incision site infection: redness, excessive swelling or discharge
    • Excessively tight sutures
• Remove suture in the skin layer at 10-14 days post-surgery if non-absorbable suture was used or if absorbable suture is still present
• Consult with Office of Animal Resources Veterinary staff if problems arise

Multiple Oocyte Harvest Surgeries

• Maintain surgical records (see below) identifying how many surgeries have been performed on each frog
  • Up to five survival surgeries may be performed on each individual frog
  • The sixth surgery must be performed as a terminal procedure
• Perform surgery on alternating sides of the abdomen with a minimum interval of two weeks between surgeries
  • A minimum of four weeks should occur between surgeries if you must harvest/implant oocytes on the same side

Record Keeping

• Record the following information in addition to the anesthesia record requirements  Note: the anesthesia record may be combined with the surgical record; click here for the template
  • Procedure
  • Date, time, dose and route of each analgesia administration (if applicable)
  • Date when wound closures are removed/are no longer present
  • Post-operative monitoring observations, including the date and time of each observation and a brief description of the animal’s health status and incision site appearance
  • Reflect the animal’s health status by commenting on animal’s behavior:
    • Normal behavior
    • Abnormal behavior
• Keep surgery, anesthesia and post-operative records readily accessible for review

Last Reviewed by the IACUC 12/11/2019