The overall goal of this procedure is to demonstrate the surgical removal of murine testes and ovaries in a fast, safe, and effective manner. Murine orchiectomy is performed by a ventral midline incision into the scrotal sac, where the testes are isolated and removed, followed by closure of the skin incision. Murine ovariectomy is performed by a dorsolateral incision into the abdomen where the ovary is isolated and removed, followed by closure of the skin incision, with the option to repeat this procedure on the other side to perform a bilateral ovariectomy.
To begin the murine orchiectomy, first start by recording the weight of a male mouse and prepare to administer the appropriate pain medication and anesthetics per your institutional guidelines. Once the mouse is under anesthesia, cover the eyes with a lubricating eye gel to protect them from drying out during the procedure. Before proceeding, ensure that the mouse is at an appropriate plane of anesthesia by performing a toe-pinch response test.
Next, shave the inguinal and scrotal areas of the mouse. Using a cotton-tipped applicator, apply a thin layer of depilatory hair removal cream to the surgical site and surrounding areas. After 30 seconds, remove the depilatory cream with the clean gauze sponge.
Transfer the mouse to a surgical area and make sure a source of heat is available. Use soft adhesive tape to adhere the mouse's feet to the surgical mat. If the testes have ascended into the abdomen, use gentle pressure on the abdomen to help them descend.
Scrub the skin with Betadine solution, starting in the center over the surgical site and working readily outwards towards the border of the shaved area. Follow the Betadine scrub with an alcohol scrub performed in a similar manner. Repeat this process three times to ensure proper skin disinfection.
Cover the mouse with the surgical drape. Grasp the skin and make a midline incision about 1 to 1 1/2 centimeters in length. Grasping the cut border of the skin, use a blunt-tipped instrument to separate the skin from the underlying tissue.
Take time to do this on the lateral, superior, and inferior borders of the incision. Next, isolate one testicle by sliding the skin incision to be directly atop it. Use a pair of forceps to grasp the thin, transparent cremaster muscle that surrounds the testicle.
Identification of the correct tissue can be confirmed by its transparency and its ability to move independently from the underlying tissues enclosed within it. Next, incise the cremaster muscle by making an incision into the apex of the tissue. Place a locking clamp on the posterior border of the cut cremaster muscle to hold tension on the tissue.
Use a pair of forceps to grasp the testicle and exteriorize it from its cremaster sheath. Once the testicle is out, roll the tissue laterally and localize the cremaster insertion point that connects the cremaster muscle to the testicle and epididymis on the posterior side of the testicle. Clamp and tear this tissue to sever this connection.
Exteriorize the inguinal fat pad by slowly pulling it out. While doing this, watch for signs of bleeding and be sure to not harm any of the blood vessels in the process. Next, use a pair of locking hemostats to clamp the spermatic cord and blood vessels.
While holding slight tension on the testicle, cauterize the spermatic cord and blood vessels distal to the hemostat clamp. Slowly release the hemostat and ensure the tissue stump has good hemostasis and does not continue to bleed. Applying a slight outward pressure on the cremaster muscle will allow this tissue stump to retract into the body cavity.
Bring the two sides of the cremaster muscle together. Using 4-0 or 5-0 absorbable sutures, place a suture to join the two ends of the cremaster muscle. Once the suture is finished, trim the ends of the suture material to be 1/2 a centimeter in length.
Repeat this process for the other testicle by sliding the skin incision to be atop the other side, and follow the same steps used to remove the first testicle. Once both testicles have been removed and both sides of the cremaster have been sutured, evert the skin upwards and prepare to apply a wound clip to the area. Be sure the suture ends will not be caught in the wound clip.
The mouse is now ready to continue with postoperative monitoring. To prepare for murine ovariectomy, weigh a female mouse and administer pain medication and anesthesia per your institutional guidelines. Administer lubricating eye gel and check to make sure the mouse has no toe-pinch response.
Shave the dorsolateral regions of the mouse's back to prepare the surgical site. Using a cotton-tipped applicator, apply a layer of depilatory hair removal cream to the shaved area. After 30 seconds, remove the cream with a clean gauze sponge.
Transfer the mouse to a surgical mat and ensure that a source of heat is available. Scrub the skin with Betadine solution, starting in the center and working readily outwards towards the border of the shaved area. Follow this by scrubbing the skin with 70%alcohol solution applied in a similar manner.
Repeat this process for a total of three times to ensure proper skin disinfection. Plan the surgical incision as detailed in the written protocol. Place a surgical drape over the mouse's surgical site.
Grasp the skin with forceps and incise the skin with a one-centimeter incision. Use a blunt-tipped instrument or a plain pair of hemostats to separate the skin from the underlying tissue. Take time to do this on the lateral, superior, and inferior borders of the incision.
Grasp the abdominal wall and incise with a 1/2-centimeter incision. Use a pair of forceps to locate the ovary within the incision and pull to exteriorize it. Once the tissues have been identified, use a pair of hemostats to clamp and crush the distal uterine horn.
Use a second pair of hemostats or a pair of forceps to clamp just distal to the first clamp. Holding the bottom hemostat still, pull the top clamp up and away from the body to perform a crush and tear of the distal uterine horn. Slowly release the hemostat and watch to make sure there is not excessive bleeding coming from the surgical site.
Bring the two sides of the abdominal wall together and suture the incision closed, using a single suture with 4-0 absorbable suture material. Once the suture is secured, trim the ends of the suture material to be 1/2 a centimeter in length. Bring the two sides of the skin back together and prepare to place a wound clip.
While everting the skin, place a wound clip over the incision site. The mouse is now ready to continue with postoperative monitoring. Document the procedure date and time and analgesic administration per your institutional guidelines.
Once a mouse has undergone surgical orchiectomy or ovariectomy, transfer them to a clean, heated cage. Once a mouse has regained sternal recumbency, place them in a clean cage and provide moist chow and water. During their recovery, frequently check on the mouse's surgical site and look for any signs of infection or bleeding.
To prevent damage to the wound clip, remove any rodent igloo habitats from the cage of an ovariectomy mouse for one week following surgery. After 14 days, remove the wound clips from the mouse's skin. The demonstrated protocol details how to perform an orchiectomy and ovariectomy in a mouse to generate a sex-hormone-depleted model.
This involves removing the testicles through a midline incision in the scrotum, followed by exteriorizing the testicles and severing the spermatic cord in blood vessels, resulting in the removal of the testicle, epididymis, and inguinal fat pads. Surgical ovariectomy can be performed by a dorsolateral incision into the dorsum and exteriorizing the ovary, oviduct, and distal uterine horn. This is followed by severing the distal uterine horn, leading to removal of the ovary and oviduct.
Careful surgical technique and proper postoperative care will result in well-healed surgical sites. These surgical procedures can be further validated by analyzing hormone levels in mouse serum samples one week after orchiectomy. Ovariectomy can be confirmed by the observation of uterine atrophy following the removal of the ovaries.
This protocol enables the rapid and effective surgical removal of ovaries and testes from mice to generate a sex-hormone-depleted model. This can have many uses to study how depletion of these hormones affects various organs and physiological processes.
