Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

This method will help answer key questions concerning the physiological function of gonads, including the role of sex steroids in neuroendocrine system plasticity and regulation in teleosts and to a larger extent in vertebrates. This protocol makes it possible to perform gonadectomy at a high successful rate in small model teleost medeka, which is widely used in the research community. In order to be successful and achieve a high survival rate, this technique requires proper training, particularly in assessing the gonads and suturing the fish.

Begin by taking the fish out of the anesthetic solution and placing it horizontally on its side under a dissection microscope. To perform the ovariectomy in females, scrape the scales in the incision area. Gently make a two to 2.5 millimeter incision between the ribs and the pelvic and anal fins using a razor blade.

Pinch the fish abdomen gently while taking out the ovaries using fine forceps with a wide tip. It is important to avoid breaking the ribs, which can cause death. Cut the end of the ovaries with the fine forceps and set them aside.

To perform the orchidectomy in males, make an incision between the ribs above the anus and open the incision slowly using fine forceps. Gently grab the testes with the forceps and slowly take them out. Then cut the end of the testes to completely remove them.

After performing the procedure, suture the incision similarly for males and females. Place the nylon thread beside the incision area and stab the skin from the right side of the incision through the inner body cavity, using ultra-fine forceps to take the thread in. Then, puncture the skin from the left side of the incision through the outer body cavity to take the thread out.

Close the incision opening, make two knots, and cut the excess thread. Place the fish in the isotonic recovery water, and leave them there for at least 24 hours before transferring them to the aquarium system. Assemble the blood drawer by attaching a glass needle to the silicone capillary.

Break the tip of the needle with wide tip forceps and coat the inside of the needle with anticoagulant solution by suctioning and blowing. Direct the needle toward the peduncle area of the fish, aiming at the caudal peduncle vein, and draw the blood using the mouth until at least 1/4 of the total volume of the needle is filled. Release the needle and put a piece of tape near the sharp side.

Place a lid with a hole on a collection tube and put the needle inside the tube through the hole with the needle tip on the outside. Place the fish in the recovery water and leave them there for at least 24 hours before transferring them to the aquarium system. Flash spin the blood for one to two seconds at 1, 000 times G to collect the blood in the tube.

The survival rate after ovariectomy, or OVX, in females was 100%and 94%in males after orchidectomy. All fish survived the blood sampling procedure. Sham-operated females showed OV position.

All eggs were fertilized and proceeded to embryonic development. When the gonadectomy procedure was performed correctly, the body shape of the fish slightly changed, and no pieces of gonad remained. The incision and suture completely disappeared four weeks post gonadectomy.

After four months, all fish still showed a healthy phenotype and no gonadal tissue was found. Blood analysis in females revealed that the E2 level in OVX fish was significantly lower than in sham-operated fish 24 hours after surgery. After four months, the E2 level in OVX fish was still significantly lower than in sham-operated fish.

Partly OVX fish, where only 1/3 to 1/2 of the gonad was removed, showed significantly lower E2 levels than sham-operated fish, and significantly higher E2 levels than fully OVX fish. The 11-KT concentration in orchidectomized males was significantly lower than in sham-operated fish 24 hours after surgery. After four months, the 11-KT level was still lower than in sham-operated fish.

Partly orchidectomized fish showed lower levels of 11-KT than sham-operated fish and higher levels than fully orchidectomized fish. To ensure that the procedure is successful, it's essential to remove the gonad as carefully and completely as possible, without damaging other organs inside the fish. Since it's known that sex steroids play an important role in brain and pituitary cell plasticity, this technique provides a powerful tool to investigate underlying mechanisms in small teleost models.