This ovariectomy technique employs a recoverable surgical procedure for obtaining amphibian oocytes for our medical research or another application for overcoming the pathologic condition of retained eggs. It does not require euthanasia to obtain amphibian oocytes for research purpose. In fact, after a partial ovariectomy the amphibian subject can be reproductively active post recovery.
After confirming a lack of response to pelvic limb pinch, place an anesthetized female anuran in a dorsal recumbent position, and use moist, sterile gauze to wipe the surgical field with povidone-iodine solution for 10 to 15 seconds. Place a clear, sterile plastic drape over the surgical area and reduce evaporation by keeping the surrounding skin moist, before using a number 15 or 11 scalpel blade to make a 3 millimeter perimedian skin incision in the mid coelom between the shoulders and the cloaca. Use cotton-tipped applicators to apply localized pressure to any small vessels around the incision as necessary, and elevate the abdominal membrane for a successive membrane incision.
Use eyelid retractors to retract the ceolomic incisions, and excise a portion of the egg mass without ligating any blood vessels. For a complete ovariectomy, cauterize the surrounding blood vessels, and use a mono-filament suture to close the ceoliotomy incision with an interrupted everting suture pattern. Different amphibians species, such as these Necturus and the glass frog, exhibit varying degrees of skin transparency, affecting the ability to visualize their reproductive status transdermally.
In some species, morphological differences can be detected between females that are gravid, versus recently oviposited animals. In this example, a female Rana muscosa that has recently oviposited can be distinguished from a gravid female by the flaccidity of her skin and her thinner appearance. The behavioral stages of oviposition in Rana muscosa include courting, in which a male actively pursues a female and amplexus, during which the Rana muscosa male mounts and firmly clasps onto the female's back.
During amplexus, the male squeezes the female in a soft pumping manner. Female Rana muscosa move around and display intermittent handstand behaviors, eventually leaning up against surfaces that eggs can be stuck on to, while the male pumps her abdomen vigorously. Immediately after oviposition, female ovaries return to a stage one category.
During this time, follicles are hard to visualize when performing ultrasound. Stage two is represented by the appearance of echogenic dots dispersed throughout the ovary. Stage three is determined by the presence of larger, rounder echogenic dots with dark centers, indicative of yolked, mature follicles.
In this representative four year assessment of captive female Necturus, egg development was high, but oviposition was not ensured. Some females with retained eggs concomitant with fluid gain in the body cavity, showed excessive girth and swelling, along with some signs of burst blood vessels, such as that shown in these affected Rana and Necturus. Although the surgical approach can be used for managing ovarian pathology, this technique may also be involved in gamet and umbilical research.
After the surgery, the practitioner can return to the first protocol outlined in this manuscript for monitoring the ongoing overall health of the amphibian subject.
