Frog Oocyte Harvest

Policy

The number of laparotomies on frogs to obtain oocytes should be conditional upon the relative health of an individual animal, quality of the oocytes obtained, the age of the animal, and probable duration of egg production. Up to a maximum of five recovery surgeries (with a final 6th terminal surgery) will be permitted. Adequate animal identification and record keeping is required to ensure that each frog does not receive more than the maximum number of recoverable surgeries. Exceptions to this policy may be approved by the IACUC for compelling scientific reasons. Surgeries must be performed by trained persons using appropriate anesthesia, such as tricaine methane-sulfonate (MS-222). Surgeries must be done using aseptic technique. Single housing or small group housing for several days after surgery should be provided as part of the post-surgical care of laparotomized animals. Frogs should be monitored daily during the post-operative period (at least one week) for appetite as well as any complications such as dehiscence or infection. The appearance of such adverse effects should provide justification for immediate euthanasia. Investigators must verify their personnel have adequate training to perform these procedures.

Adequate recovery time must be allowed between laparotomies. Investigators should alternate oocyte collection between right and left ovaries and rotate frogs so that the interval between surgeries is maximized. Recovery times should not be less than one month unless exceptions are granted by the IACUC for scientific reasons.

Xenopus Oocyte Harvest recommended protocol

Pre-operative considerations: Frogs should be fasted for 12 hours or less to prevent regurgitation, emesis, or stomach eversion during anesthesia.  The prevention of these events will significantly reduce potential distress.

Anesthesia and supportive care during surgery: MS-222 can be safely used on Xenopus sp. at a dosage range of between 0.5 and 3 g/L. Dosage selection is dependent upon the weight/size of the frog and the duration of anesthesia required. The added benefit of MS-222 use is that this compound has analgesic properties and removes the necessity of administration of post- surgical analgesia in most cases. The final solution of MS-222 should be buffered to the pH of the system or source water with sodium bicarbonate (NaHCO₃). Buffering will decrease induction time and reduce the anesthetic “excitation” phase. Once a surgical plane of anesthesia has been reached, (noted by a lack of response to deep pain, i.e. toe pinch) frogs should be placed in dorsal recumbency on the non-absorbent “blue” side of a clean/unused diaper pad.  The frog can be frequently exposed to water containing dissolved MS-222 to maintain current level of anesthesia for long procedures (>30 minutes) or moistened less frequently for brief procedures (<30 minutes). The frog’s skin must remain moistened throughout the procedure to prevent desiccation. Take care not to introduce anesthetic water into the incision as this will prolong anesthetic recovery.

Aseptic precautions

Instrument Sterilization: Surgical instruments should be washed or soaked post usage to remove gross debris. Instruments should be wrapped or packed appropriately, and steam autoclaved or vapor phase/gas plasma Hydrogen Peroxide sterilized. The autoclave or the Hydrogen Peroxide sterilizer must be appropriately and regularly evaluated to ensure proper function and that sterilization is actually occurring. Instruments should be stored in a dry place in which the integrity of the wrapping or packing material will be maintained for a defined period of time. If multiple surgeries are to be done on different animals during one day, then the tips of previously sterilized instruments can be re-sterilized using a glass bead sterilizer, after mechanical wiping to removed organic debris.

Surgical Skin Preparation: Skin asepsis in Xenopus sp. is not typically required for the most common surgical procedures. However, the removal of gross debris from the surgical incision site only (via brief rinse with sterile saline, moistened small gauze pad or cotton swab) is recommended. If sterile gloves, a clean lab coat or scrub top, and sterile instruments are utilized in an aseptic manner and surgery occurs in a dedicated /previously disinfected surgical area on a clean surface; then there is minimal potential for contamination of the surgical site and the development of subsequent post-operative infection.

If necessary, the recommended product for skin asepsis is 0.5% povidone-iodine or 2mg/L of benzalkonium chloride with a contact time of 10 minutes post removal of excess skin secretion from the surgical incision area. Disinfectants should be applied via moistened small gauze to the surgical incision site only.  The surgical site should be rinsed with sterile saline before the surgical incision is made. Products that contain soaps or detergents should be avoided due to the potential for toxicity post exposure.

Surgical Procedure: A small, paramedian, coelomic incision (0.5-2cm) through the skin and muscular layers should be made on either the right of left side of the coelom. A portion of the corresponding ovary is exteriorized and removed. Remaining ovarian tissue is replaced into the coelomic cavity and checked for excessive hemorrhage. The muscular and skin tissue layers must be closed separately using, either monofilament nylon, monofilament absorbable (Polydioxaone/PDS II), or braided absorbable (Poloyglactin 910/Vicryl) suture material (3-0 to 4-0) via a simple interrupted suture pattern. The use of absorbable suture material prevents the need to remove the sutures at a later time.

Post-surgical Recovery and Monitoring: Post surgery, the frog is allowed to recover 4 to 10 hrs. in a container with a level of water not to cover the nostrils of the frog (desiccation of the skin on the dorsum of the frog can be prevented by placing moistened gauze on any exposed surfaces). Once the frog is active and mobile the water level can be raised to a more normal level and the gauze removed. Recovery water, de-chlorinated tap water, should contain 25-50g/L of non-iodized salt to aid in the recovery from anesthesia (frogs should not remain in this type of water for > 12 hours) and this container should be covered or have a lid in place to prevent potential escape attempts. Frogs should be monitored daily for one-week post-surgery for evidence of excessive inflammation of the incision site, suture dehiscence, or behavioral abnormalities indicative of illness (anorexia, listlessness, or lethargy). If evidence of wound infection or illness is noted, then either vet services should be contacted for evaluation and treatment or the animal should be euthanized.