Name: intact ovary harvest from mouse: a surgical procedure to isolate complete ovary from murine model
Uploaded: 2023-04-30T14:51:17.000+00:00
Duration: 1 min 32 s
Description: Source: Mason, J. B. et al. Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in ...

intact ovary harvest from mouse: a surgical procedure to isolate complete ovary from murine model

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

Before beginning the procedure, confirm the appropriate level of sedation by toe pinch, and apply ointment to the animal&#39;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.

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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.
<ol>
	<li>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.</li>
	<li>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 &#39;patch&#39; approximately 2-3 cm square on either side of the prone mouse leaving a strip of hair approximately 1-2 cm covering the midline.</li>
	<li>Lightly wipe the clipped areas with 70% ethanol and apply betadine solution to the clipped site with a cotton-tipped swab.</li>
	<li>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&#39;s neck for postoperative pain relief.</li>
	<li>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.</li>
	<li>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</li>
	<li>Place a small surgical drape over the incision (cut from 2 cm x 2 cm gauze) and wet the drape with sterile saline.</li>
	<li>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 &#8216;whiter&#8217; than the surrounding adipose tissue.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the donor ovary in watch glass filled with 4&#176; C sterile saline, loosely cover, and set aside until ready for transplantation.</li>
</ol>

<table><tbody><tr><td>Dissecting microscope</td><td>Any dissecting scope with an adjustable working distance.</td><td></td><td></td></tr><tr><td>Warm Water Recirculator and appropriate pad for your size surgical/recovery field.</td><td>Stryker/Gaymar</td><td>TP700</td><td>Circulating water pads produce even and consistent temperature operating and recovery fields.</td></tr><tr><td>Surgical instruments-dissection instruments for access to the reproductive tract</td><td></td><td></td><td>Choice of specific surgical instruments is highly surgeonspecific</td></tr><tr><td>Suture material for ovarian bursa</td><td>eSutures</td><td>2809G: 9-0 or 2810G: 10-0 ETHILON BLACK 5&quot; BV130-5 TAPER</td><td>10-0 suture can be difficult to locate if dropped on the surgery field.</td></tr><tr><td>Absorbable gelatin sponge</td><td>PFIZER</td><td>Gelfoam- PFIZER 09-0315-08</td><td>cut into small (~2mm) pieces under sterile conditions.</td></tr><tr><td>Staples for skin closure</td><td>Becton Dickinson</td><td>MikRon Autoclip 9mm (BD 427631)</td><td></td></tr><tr><td>Eye ointment.</td><td>MWI Veterinary Supply Co.</td><td></td><td>Puralube sterile opthalmic ointment</td></tr><tr><td>Syringes for anesthesia, analgesia and epinephrine</td><td>MWI Veterinary Supply Co</td><td>BD 305536</td><td>27g x 3/8in,Intradermal Bevel 1/2ml</td></tr><tr><td>Syringes for saline.</td><td>MWI Veterinary Supply Co</td><td>BD 309657.</td><td>3ml syringe, luer-lok tip, 27g needle</td></tr><tr><td>Clippers/blade</td><td>Andis</td><td>Super AGR+&amp;trade; Cordless Detachable Blade Clipper and a CeramicEdge&amp;reg; #40 blade.</td><td>Any clippers with a fine blade.</td></tr><tr><td>Ethanol</td><td>University chemical supply</td><td></td><td>dilute to 70% for use.</td></tr><tr><td>Betadine solution</td><td>MWI Veterinary Supply Co.</td><td>3233</td><td></td></tr><tr><td>2x2 gauze pads</td><td>MWI Veterinary Supply Co.</td><td>3073</td><td>autoclaved with surgery packs</td></tr><tr><td>Borosilicate glass beads</td><td>Sigma-Aldrich Co</td><td>Z273619</td><td>Autoclave in sintilation vials</td></tr><tr><td>4-vinylcyclohexene diepoxide</td><td>Sigma-Aldrich Co.</td><td>94956</td><td></td></tr></tbody></table>

Source: Mason, J. B. et al. <a href="https://www.jove.com/v/56638">Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice.</a> J. Vis. Exp. (2018)
In this video, we describe a surgical procedure to harvest intact ovary from a mouse model.

Source: Mason, J. B. et al. Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in ...

Begin with an anesthetized female mouse in a right lateral position on a surgical platform. Next, shave the mouse&#8217;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.&#160; 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.

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

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