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

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Transplantation of intact ovaries to aged mice

NOTE: This protocol describes the procedure used during the transfer of ovaries from a young mouse (60 days of age) to a post reproductive female (12 months of age) of the same inbred strain. The same procedure is used for ovarian transplantation between any two female mice.

1. Weigh and anesthetize the donor animal by intraperitoneal (I.P., 27 ga needle) injection, for example using a cocktail of ketamine, xylazine and acepromazine (65 mg/kg ketamine, 13 mg/kg xylazine 2.0 mg/kg, acepromazine; cocktail dose – 6.5 ml/kg). Return the animal to the heated home cage until the anesthesia has taken effect.
2. Once anesthetized, remove the animal from the home cage and place on heated paper towels. Using clippers (#40 blade preferred), remove the hair a few mm lateral to midline on each side, starting just below the ribs and moving distally, preparing a clipped 'patch' approximately 2-3 cm square on either side of the prone mouse leaving a strip of hair approximately 1-2 cm covering the midline.
3. Lightly wipe the clipped areas with 70% ethanol and apply betadine solution to the clipped site with a cotton-tipped swab.
4. Apply antibiotic gel for eye protection and administer meloxicam (2.0 mg/kg) subcutaneously (S.C., 27 ga needle) on the lateral aspect of the animal's neck for postoperative pain relief.
5. Place the animal in prone position on a heated sterile surgical field and wipe the clipped area with a 70% ethanol-soaked 2 cm x 2 cm cotton gauze.
6. Make a 1.0-1.5 cm paralumbar incision and bluntly dissect the skin from the underlying fascia. Make another incision through the fascia. Bluntly dissect laterally under this fascia as superficially as possible until reaching the abdominal cavity
7. Place a small surgical drape over the incision (cut from 2 cm x 2 cm gauze) and wet the drape with sterile saline.
8. Using small forceps, locate the adipose tissue that surrounds the ovary in the abdominal cavity and gently extract the fat pad and ovary and place it on the wetted gauze drape. The ovarian fat pad will appear ‘whiter’ than the surrounding adipose tissue.
9. Place a 1.5-inch Bulldog clamp on the fat pad (taking care not to obstruct the ovarian bursa) to hold the tract in place during the procedure.
10. Move the dissecting microscope into place and clearly identify the ovarian bursa. Grasp the bursa with two watchmaker forceps and incise the ovarian bursa opposite the ovarian hilum to expose the ovary. If the intended access area of the bursa is highly vascularized, apply a drop of epinephrine (1:100,000) with a syringe to the bursal surface to reduce bleeding at the time of tearing.
11. Gently remove the ovary from the ovarian bursa and excise the ovary by clamping at the ovarian hilum with a watchmaker forceps to prevent bleeding.
12. Place the donor ovary in watch glass filled with 4° C sterile saline, loosely cover, and set aside until ready for transplantation.