The authors thank Ms Lourdes Carreon G Tan for her assistance in the fish husbandry. This work was funded by NMBU, Grants-in-Aid from Japan Society for the Promotion of Science (JSPS) (Grant number 18H04881 and 18K19323), and grant for Basic Science Research Projects from Sumitomo Foundation to S.K.

All experimentations and animal handling were conducted in accordance with the recommendations on the experimental animal welfare at Norwegian University of Life Sciences. Experiments using gonadectomy were approved by the Norwegian Food Safety Authority (FOTS ID 24305).
NOTE: The experiments were performed using adult male and female (6-7 months old, weight ca. 0.35 g, length ca. 2.7 cm) Japanese medaka. The sex was determined by distinguishing the secondary sexual characteristics, such as th...

As reported in previous literature, gonadectomy and blood sampling have long been used in other model species to investigate questions related to the role of sex steroids in regulation of the BPG axis. However, these techniques seem to be amenable only for bigger animals. Considering the small size of the commonly used teleost model, Japanese medaka, we provide a detailed protocol for gonadectomy and blood sampling that is feasible for this species.
The fact that the survival rate of gonadecto...

In vertebrates, sex steroids, which are mainly produced by the gonads, play important roles in the regulation of the Brain-Pituitary-Gonadal (BPG) axis through various feedback mechanisms1,2,3,4,5. In addition, sex steroids affect the proliferation and activity of neurons in the brain6,7,8 and endocrine cells, including gonadotropes, in the pituitary9,10, and thus serve crucial roles in brain and pituitary plasticity. Despite relatively good knowledge in mammals, the mechanism of BPG axis regulation mediated by sex steroids is far from being understood in non-mammalian species, leading to poor understanding of evolutionary conserved principles11. There is still a limited number of studies documenting the role of sex steroids on brain and pituitary plasticity, thus raising the need for further investigations of the role and effects of sex steroids on diverse vertebrate species.
Among vertebrates, teleosts have become powerful model animals in addressing numerous biological and physiological questions, including stress response12,13, growth14,15, nutritional physiology16,17 and reproduction2. Teleosts, in which sex steroids are mostly represented by estradiol (E2) in females and 11-ketotestosterone (11-KT) in males18,19, have&#160;long been reliable experimental models for investigating the general principle of reproduction across species. Teleosts show uniqueness in their hypothalamic-pituitary connection20,21 and distinct gonadotrope cells22, which are sometimes convenient for the elucidation of regulatory mechanisms. Moreover, due to their amenability to both laboratory and field experiments, teleosts offer many advantages compared to other organisms. They are relatively inexpensive to purchase and maintain23,24. In particular, small teleost models such as zebrafish (Danio rerio) and the Japanese medaka (Oryzias latipes), are species with very high fecundity and a relatively short life cycle enabling rapid analysis of gene function and disease mechanisms23, thus providing even greater advantages in addressing a plethora of biological and physiological questions, considering the numerous well-developed protocols and genetic toolkit available for these&#160;species25.
In numerous studies, the removal of gonads (gonadectomy) along with blood sampling techniques have been used as a method for investigating many physiological questions, including its impact in vertebrate reproductive physiology in mammals26,27,28, birds29 and amphibians30. Although the gonadectomy effect on reproductive physiology can be alternatively mimicked by sex steroid antagonists, such as tamoxifen and clomiphene, the effect of the drugs appears to be inconsistent due to bimodal effects31,32. Chronic exposure to a sex steroid antagonist may lead to ovarian enlargement33,34, which may disable observation of its effects for long-term purposes due to an unhealthy phenotype. In addition, it is impossible to perform a recovery experiment after sex steroid antagonist treatment, to warrant the specific effect of certain sex steroids. Along with those aforementioned points, other trade-offs of sex steroid antagonist use have been extensively reviewed31,32. Therefore, gonadectomy still appears today as a powerful technique to investigate the role of sex steroids.
While gonadectomy and blood sampling techniques are relatively easy to perform in bigger species, such as European sea bass (Dicentrarchus labrax)35, bluehead wrasse (Thalassoma bifasciatum)36, dogfish (Scyliorhinus canicula)37 and catfish (Heteropneustes fossilis and Clarias bathracus)38,39, they raise challenges when applied in small fish as medaka. For instance, the use of Fish Anesthesia Delivery System (FADS)40 is less feasible and appears to be prone to excessive physical damage for small fish. In addition, a gonadectomy procedure that is commonly used for bigger fish40 is not suitable for small fish that requires high precision to avoid excessive damage. Finally, blood sampling is challenging due to the limited access to blood vessels and the small amount of blood in those animals. Therefore, a clear protocol demonstrating every step of gonadectomy and blood sampling in a small teleost is of importance.
This protocol&#160;demonstrates the step-by-step procedures of gonadectomy followed by blood sampling in Japanese medaka, a small freshwater fish native to East Asia. Japanese medaka have a sequenced genome, several molecular and genetic tools available25, and a genetic sex determination system allowing for investigation of sexual differences before secondary sexual characteristics&#160;or gonads are well developed41. Interestingly, Japanese medaka possess fused gonads contrary to many other teleost species42. These two techniques combined take only 8 minutes in total and will complete the list of video protocols already existing for this species that included labeling of blood vessels43, patch-clamp on pituitary sections44 and brain neurons45, and primary cell culture46. These techniques will allow the research community to investigate and better understand the roles of sex steroids in feedback mechanisms as well as brain and pituitary plasticity in the future.

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gonadectomy and blood sampling procedures in the small size teleost model japanese medaka (oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive&#160;strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

This protocol describes every step for performing gonadectomy and blood sampling in a small size model teleost, the Japanese medaka. The survival rate of the fish after ovariectomy (OVX) in females is 100% (10 out of 10 fish) while 94% (17 out of 18 fish) of the males survived after orchidectomy. Meanwhile, after the blood sampling procedure was performed, all (38 fish) fish survived.
Sham-operated females show oviposition (<str...

Watch this Scientific Journal Video about Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes at JoVE.com

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

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

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

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...

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.

gonadectomy-blood-sampling-procedures-small-size-teleost-model

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JoVE Journal

Biology

4.2K visualizações.  Norwegian University of Life Sciences. 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, particu...