Intact Ovary Harvest from Mouse: A Surgical Procedure to Isolate Complete Ovary from Murine Model

Begin with an anesthetized female mouse in a right lateral position on a surgical platform. Next, shave the mouse’s abdomen and sterilize the area. Make a paralumbar skin incision and dissect the abdominal muscles to expose the abdominal cavity.

Now, locate one of the ovarian fat pads - the adipose tissue surrounding the ovaries.  The oviducts connect the ovaries to long tubular structures called the uterine horns. Further, gently exteriorize the ovary along with the ovarian fat pad, placing it on a wet gauze to keep the tissue hydrated.

Now, identify the ovarian bursa - the thin membrane encapsulating the ovary and end of the oviduct. Incise the bursa opposite the ovarian hilum, which forms the border between ovary and ovarian ligament. The ovarian ligament structurally connects ovary to the uterine horn.

After exposing the ovary, gently remove it from the ovarian bursa and clamp at the ovarian hilum to excise the ovary. Transfer the excised ovary into a culture plate containing chilled saline.

Store the intact mouse ovary composed of functional units - the ovarian follicles, each containing an oocyte, until further use.

Before beginning the procedure, confirm the appropriate level of sedation by toe pinch, and apply ointment to the animal's eyes. Place the mouse in the supine position on a stack of heated paper towels, and starting just below the ribs and moving distally, use number 40 blade hair clippers to remove a 2 to 3-centimeter square of hair a few millimeters lateral to the midline on each side of the animal, with a 1 to 2 2-centimeter strip of hair covering the midline.

After administering analgesia, transfer the animal onto a heated sterile surgical field in the supine position, and use a 2 by 2 centimeter piece of 70% ethanol soaked gauze followed by a betadine scrub to disinfect the surgical area. Next, make a 1 to 1.5-centimeter paralumbar incision in one of the surgical areas, and two scissors to blunt dissect the skin from the underlying fascia.

Then, make another incision through the fascia, and blunt dissect laterally under the fascia as superficially as possible until the abdominal cavity is reached. Place a 2 by 2 centimeter gauze surgical drape over the incision, and wet the gauze with sterile saline.

Using small forceps, locate the adipose tissue that surrounds the ovary, and gently extract the fat pad and ovary onto the drape. The ovarian fat pad will appear whiter than the surrounding adipose tissue. Place a 1.5 inch Bulldog clamp onto the fat pad, taking care not to obstruct the ovarian bursa, to hold the tract in place during the procedure.

Use a dissecting microscope to identify the ovarian bursa. Then, grasp the bursa with two watchmaker forceps and incise the bursa on the opposite side from the ovarian hilum to expose the ovary.

Before deciding where to tear open the bursa, try to visualize a pocket in the bursa where you will place the new ovary prior to suturing. Only open the bursa as far as necessary to remove the endogenous ovary.

Gently remove the ovary from the bursa, and clamp the ovarian hilum with watchmaker forceps to excise the ovary and to prevent bleeding. Then, place the donor ovary in a watch glass containing 4 degrees Celsius sterile saline, and place a loose cover over the glass.