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 the size and shape of dorsal and anal fin, as described in42,47.
1. Instruments and solutions preparation
<ol>
	<li>Prepare anesthetic stock solution (0.6% Tricaine).
	<ol>
		<li>Dilute 0.6 g of Tricaine (MS-222) in 100 mL of 10x Phosphate Buffer Saline (PBS).</li>
		<li>Distribute 1 mL of the Tricaine stock solution into several 1.5 mL plastic tubes and store at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>Prepare recovery water (0.9% NaCl solution) by adding 18 g of NaCl into 2 L of aquarium water. Store the solution at room temperature until use.</li>
	<li>Prepare the incision tools by breaking a razor diagonally to get a sharp point (Figure 1A).</li>
	<li>Prepare blood anti-coagulant solution (0.05 U/&#181;L of sodium heparin) by diluting 25 &#181;L of sodium heparin into 500 &#181;L of 1x PBS. Store the anti-coagulant solution at 4 &#176;C until use.</li>
	<li>Prepare two glass needles from a 90 mm long glass capillary by pulling a glass capillary with a needle puller (Figure 1B) following the instructions of the manufacturer. 
	NOTE: The outer diameter of the glass needle is 1 mm, while the inner diameter is 0.6 mm.</li>
	<li>Prepare a 1.5 mL plastic tube lid by cutting the lid and make a hole that fits with the needle outer diameter (Figure 1C). To make the hole, heat one end of the 9 mm glass capillary and stab the heated glass capillary through the lid. Alternatively, use a needle to stab through the lid until the diameter of the hole fits with the 9-mm glass capillary.</li>
</ol>
2. Gonadectomy procedure
<ol>
	<li>Prepare 0.02% of anesthetic solution by diluting one tube of Tricaine stock (0.6%) in 30 mL of aquarium&#160;water.</li>
	<li>Prepare dissection tools including one ultra-fine and two fine forceps (one with relatively wide tip), small scissors, nylon thread and razor as described in step 1.3.</li>
	<li>Anesthetize the fish by putting it into the 0.02% anesthetic solution for 30-60 seconds. 
	&#8203;NOTE: The duration of the anesthesia depends on the size and weight of the fish&#160;and must be adapted. To ensure that the fish is fully anesthetized, the fish body can be pinched gently using forceps. If the fish does not react, the gonadectomy can be started.</li>
	<li>Take out the fish from the anesthetic solution and place the fish horizontally on its side, out-of-water under a dissection microscope.</li>
	<li>Ovariectomy (OVX) in females
	<ol>
		<li>Remove oviposited eggs (eggs hanging outside the female body) if any and scrape the scales in the incision area (Figure 2A).</li>
		<li>Gently make an incision about 2-2.5 mm long between the ribs, between the pelvic and anal fins (Figure 2A), using the razor blade. Then, pinch gently the fish abdomen while taking out the ovaries little by little using fine forceps with wide tip.</li>
		<li>Cut the end of the ovaries using fine forceps and place the ovaries aside (Figure 2B). 
		NOTE: Take care not to break the ovarian sac if possible. If the ovarian sac is broken, remove any gonad traces as completely as possible without leaving even any non-ovulated eggs.</li>
	</ol>
	</li>
	<li>Orchidectomy in males
	<ol>
		<li>Gently make an incision between the ribs above the anus (Figure 2A), and open up the incision slowly using fine forceps.</li>
		<li>Gently grab the testes using the fine forceps and slowly take out the testes. Afterwards, cut the end of the testes to completely remove the testes (Figure 2B). For male orchidectomy, all preparations are similar to in females until the incision part. When grabbing the testes, sometimes the fat resembling the testes is obtained. However, after restoring the fat, it is possible to try to find the testes again (Figure 2B). 
		NOTE: For both males and females, it is important to minimize the incision size in the abdomen to prevent excessive damage that can lead to mortality. Sometimes the intestines may also appear through the incision along with the gonads, so make sure they are properly returned inside the incision before closure. Prior knowledge on ovaries and testes location in medaka abdomen is essential.</li>
	</ol>
	</li>
	<li>Suture the incision similarly in males and females (Figure 3).
	<ol>
		<li>Place the nylon thread beside the incision area&#160;and stab the skin from the right side of the incision through inner body cavity using ultra-fine forceps to take the thread in with fine forceps (Figure 3; 1-2).</li>
		<li>Stab the skin from the left side of the incision through outer body cavity to take out the thread ( Figure 3; 3-4).</li>
		<li>Close the incision opening and make two knots and cut the excessive thread (Figure 3; 4-6). 
		NOTE: The suture must be adequately tight, and the remaining thread on the fish must be long enough to prevent loosening of the suture. The whole procedure from anesthesia until suturing commonly takes up to 6 minutes. Longer time&#160;may lead to&#160;mortality.</li>
		<li>Put the fish in the recovery water and leave them for at least 24 hours before transferring them to the aquarium system. 
		&#8203;NOTE: Gonadectomized fish usually show normal behavior after 1-2 hours in the recovery water. Therefore, depending on the experiment purpose, one can sample the fish after this time interval.</li>
	</ol>
	</li>
</ol>
3. Blood sampling procedure
<ol>
	<li>Prepare the tools: a glass needle, a silicone capillary, a plastic tube with a hole, an empty 1.5 mL plastic tube, a minicentrifuge and tape.</li>
	<li>Anesthetize the fish using 0.02% anesthetic solution as described in step 2.1 and place the fish under a dissection microscope in a vertical position (Figure 4A). Place the fish on a bright surface to ease visualization of the caudal puncture vein.</li>
	<li>Install the blood drawer by attaching a glass needle to the silicone capillary (Figure 4B). Break the tip of the needle with wide tip forceps and coat the inside of the needle with anti-coagulant solution by suctioning and blowing. 
	NOTE: The use of a sucker and a silicone capillary with at least 50 cm length are recommended for safety measures to avoid any direct contact of the blood when suctioning. In addition, make sure that the opening of the needle tip is sufficiently large to allow drawing the blood.</li>
	<li>Direct the needle toward the peduncle area of the fish, aim at the caudal peduncle vein (Figure 5A) and draw the blood using mouth until at least one fourth the total volume of the needle is filled (Figure 5B). 
	NOTE: It is important to stop suctioning before removing the needle from fish body.</li>
	<li>Release the needle and put a piece of tape at the proximity of the sharp side of the needle. Place the 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 (Figure 5C).</li>
	<li>Put the fish in the recovery water and leave them for at least 24 hours before transferring them to the aquarium system. 
	NOTE: To perform a second blood sampling from the same fish, sample the blood one week after the first blood sampling.</li>
	<li>Flash spin down the collected blood for 1-2 seconds with&#160;1,000 x g at&#160;room temperature to collect the blood in the tube.</li>
	<li>Proceed directly to downstream applications or store the blood at -20 &#176;C until use. 
	NOTE: Refer to the previous study for sex steroid extraction from the whole blood48.</li>
</ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>Glass capilary</td><td></td><td>GD1</td><td>Glass Capillary with Filament GD-1; Narishige</td></tr><tr><td>Heparin sodium salt</td><td></td><td>H4784-1G</td><td>Sigma-aldrich</td></tr><tr><td>Needle puller</td><td></td><td>P97</td><td>Flaming/Brown Micropipette puller Model P-97; Sutter Instrument</td></tr><tr><td>Nylon thread</td><td></td><td>N45VL</td><td>Polyamide suture, 0.2 metric; Crownjun</td></tr><tr><td>Plastic tube</td><td></td><td>T9661</td><td>Eppendorf Safe-lock microcentifuge tube 1.5 ml, Sigma-aldrich</td></tr><tr><td>Razor blade</td><td></td><td>-</td><td>Astra Superior Platinum Double Edge Razor Blades Green, salonwholesale.com</td></tr><tr><td>Silicone capillary</td><td></td><td>a16090800ux0403</td><td>Uxcell Silicone Tube 1 mm ID x 2 mm OD, amazon.com&amp;nbsp;</td></tr><tr><td>Tricaine</td><td></td><td>WXBC9102V</td><td>Aldrich chemistry</td></tr></tbody></table>

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 ...

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

Research

JoVE Journal

Biology

4.3K Views.  Norwegian University of Life Sciences. Het artikel beschrijft een snel protocol om bloed van de kleine teleostvis te gonadectomiseren en te bemonsteren, met behulp van Japanse medaka (Oryzias latipes) als model, om de rol van geslachtssteroïden in de dierfysiologie te onderzoeken. 

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

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

Developmental Biology

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Improved Procedure to Obtain Granulosa Cell-Free Stage I Zebrafish Oocytes

Rapid Isolation of Stage I Oocytes in Zebrafish Devoid of Granulosa Cells

The study of oocyte development holds significant implications in developmental biology. The zebrafish (Danio rerio) has been extensively used as a model organism to investigate early developmental processes from oocyte to embryo. In zebrafish, oocytes are surrounded by a single layer of somatic granulosa cells. However, separating granulosa cells from oocytes poses a challenge, as achieving pure oocytes is crucial for precise analysis. Although various methods have been proposed to isolate zebrafish oocytes at different developmental stages, current techniques fall short in removing granulosa cells completely, limiting the accuracy of genome analysis focused solely on oocytes. In this study, we successfully developed a rapid and efficient process for isolating pure stage I oocytes in zebrafish while eliminating granulosa cell contamination. This technique facilitates biochemical and molecular analysis, particularly in exploring epigenetic and genome structure aspects specific to oocytes. Notably, the method is user-friendly, minimizes oocyte damage, and provides a practical solution for subsequent research and analysis.

Author Spotlight: Enhanced Method for Isolating Pure Zebrafish Stage I Oocytes

This protocol describes a modified procedure for rapidly isolating clean stage I oocytes in zebrafish devoid of granulosa cells, thereby providing a convenient method for oocyte-specific research.

The study of oocyte development holds significant implications in developmental biology. The zebrafish (Danio rerio) has been extensively used as a model ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

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Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

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

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