This study was supported by the National Research, Development and Innovation Office of Hungary (grant 116912 to &#193;H), the COST office (Food and Agriculture COST Action FA1205: AQUAGAMETE), the Stipendium Hungaricum Scholarship Programme (grant to ZM) and the New Hungarian National Excellence Predoctoral Fellowship (grant to EK).

All methods described here have been approved by the Hungarian Animal Welfare Law.
1. Reagent Preparation
<ol>
	<li>Stock solutions
	<ol>
		<li>Prepare 1 M trehalose by adding 0.378 g of trehalose dihydrate into 1 mL of dH2O. Mix well until it dissolves completely.</li>
		<li>Prepare 1 M sucrose by adding 0.342 g of sucrose into 1 mL of dH2O. Mix well until it dissolves completely.</li>
		<li>Prepare 1 M HEPES by&#160;adding 0.238 g of HEPES to 1 mL of dH2O. Mix well until it dissolves completely.</li>
		<li>Prepare 20 mg/mL collagenase (290 U/mg) by&#160;adding 100 mg to 5 mL of PBS. Mix thoroughly until it dissolves. Sterile filter through 0.2 &#181;m filters. Aliquot 50 &#181;L into 0.2 mL PCR tubes and freeze at - 20 &#176;C.</li>
		<li>Prepare 15 mg/mL trypsin (~ 10000 U/mg) by&#160;adding 75 mg to 5 mL of PBS. Mix thoroughly until it dissolves. Sterile filter through 0.2 &#181;m filters. Aliquot 50 &#181;L into 0.2 mL PCR tubes and freeze at - 20 &#176;C.</li>
		<li>Prepare 1 mg/mL DNase I (~ 3000 U/mg) by&#160;adding 5 mg to 5 mL of PBS. Mix thoroughly until it dissolves. Sterile filter through 0.2 &#181;m filters. Aliquot 50 &#181;L into 0.2 mL PCR tubes and freeze at - 20 &#176;C.</li>
		<li>Prepare 0.4% trypan blue by&#160;adding 40 mg of trypan blue to 10 mL of PBS. Sterile filter through 0.2 &#181;m filters. Aliquot 1 mL into 1.5 mL tubes.</li>
		<li>Prepare 200 mg/L MS-222 (Tricaine methane sulfonate) by&#160;adding 200 mg of MS-222 into 1 L of dH2O. Mix well until it dissolves completely. Additionally, buffer the MS-222 solution with sodium bicarbonate to a neutral pH of 7.0.</li>
	</ol>
	</li>
	<li>Solutions for vitrification
	<ol>
		<li>Prepare Equilibration solution (ES): Prepare 2 mL of ES by mixing 121.5 &#181;L of methanol (MeOH; 1.5 M), 220 &#181;L of propylene glycol (PG; 1.5 M), 200 &#181;L of FBS (10%), 200 &#181;L of trehalose stock solution (0.1 M), 50 &#181;L of HEPES stock solution (25 mM) and 1208.5 &#181;L of L-15 into a 2 mL tube.</li>
		<li>Prepare Vitrification solution (VS): Prepare 2 mL of VS by mixing 439 &#181;L of PG (3 M), 426 &#181;L of dimethyl sulfoxide (Me2SO; 3 M), 200 &#181;L of FBS (10%), 200 &#181;L of trehalose stock solution (0.1 M), 50 &#181;L of HEPES stock solution (25 mM) and 685 &#181;L of L-15 into a 2 mL tube.</li>
	</ol>
	</li>
	<li>Solutions for warming
	<ol>
		<li>Prepare warming solution 1 (WS1): Prepare 1.5 mL of WS1 in a 2 mL tube by mixing 150 &#181;L of FBS (10%), 450 &#181;L of sucrose stock solution (3 M) and 900 &#181;L of L-15.</li>
		<li>Prepare warming solution 2 (WS2): Prepare 1.5 mL of WS2 in a 2 mL tube by mixing 150 &#181;L of FBS (10%), 150 &#181;L of sucrose stock solution (1 M) and 1200 &#181;L of L-15.</li>
		<li>Prepare warming solution 3 (WS3): Prepare 1.5 mL of WS2 in a 2 mL tube by mixing 150 &#181;L FBS (10%) and 1350 &#181;L of L-15.</li>
	</ol>
	</li>
	<li>Prepare digestion solution: for each sample prepare 500 &#181;L of the digestion solution by mixing 50 &#181;L of collagenase stock solution (2 mg/mL), 50 &#181;L of trypsin stock solution (1.5 mg/mL), 10 &#181;L of DNase I (20 &#181;g/mL) and 390 &#181;L of L-15 in a 2 mL tube.</li>
	<li>Obtain dissection tools: scissors, microscissors, tweezers, curved tweezers, and acupuncture needles.</li>
</ol>
2. Testes Collection
<ol>
	<li>Euthanize the fish by placing it into a dish containing 200 mg/L MS-222 (pH = 7). Before continuing with the dissection, make sure that the gills have stopped moving and that at least 10 min have passed since that moment to ensure death by hypoxia.</li>
	<li>Dry the fish by patting it on a paper towel and place it on its dorsal side on a dissecting mat.
	<ol>
		<li>Firstly cut off the pelvic and pectoral fins for easier dissection.</li>
		<li>Horizontally snip the skin on the belly of the fish between the pectoral fins and cut the skin and underlying muscle along the belly until the anal fin.</li>
		<li>Open the body cavity of the fish and pin the left and right body wall to the dissection mat with needles. 
		NOTE: Testes are located above the gastrointestinal organs on both sides of the swimming bladder (Figure 1).</li>
	</ol>
	</li>
	<li>To prevent contamination, do not take out the gastrointestinal organs, but gently push them to one side and remove the testis from the opposing side. Testes are connected to the body wall so remove them carefully and cut the connecting peritoneum by microscissors.</li>
	<li>Additionally, in order to prevent contamination, firstly sterilize the testes by putting them in 70% ethanol for 2 s, and then place them in L-15 on a 96-well plate on ice. 
	NOTE: If it is necessary, clean testes from fatty tissue and large blood vessels precisely under the stereomicroscope</li>
</ol>
3. Ultra-Rapid Cooling of the Testicular Tissue
<ol>
	<li>Mark needles with appropriate labels depending on the sample type and prepare the equilibration (ES) and vitrification solutions (VS).</li>
	<li>Transfer the testes to a larger well plate or dish and pin two to three testes (or more depending on the size of the needle) on a sterile acupuncture needle.
	<ol>
		<li>Firstly position the testis on top of curved tweezers. Place the point of the needle in the middle of the testis and carefully pull the testis upward with the tweezers.</li>
		<li>After puncturing the testis, gently pull it upwards toward the top of the needle. Make sure the testes are separated from each other and that they are not falling off or sliding down.</li>
		<li>Place the needles with pinned testes in a 2 mL tube filled with L-15 until vitrification (at 25 &#730;C; storage time should not exceed 1 h). 
		NOTE: Pinning the testes on the needle should be done fast to prevent the testes from drying out.</li>
	</ol>
	</li>
	<li>Transfer the needle with testes into the equilibration solution and incubate for 5 min at 25&#730;C.</li>
	<li>Transfer the needle into the vitrification solution and incubate for 30 s at 25&#730;C.</li>
	<li>Remove the needle from the vitrification solution, quickly and gently absorb the remaining solution from the tissue by a sterile paper towel. Use caution to avoid the testes sticking to the paper towel or falling off.</li>
	<li>Quickly place the needle into liquid nitrogen placed in a Styrofoam box. 
	NOTE: Do this step quickly to avoid exposure of tissue to the liquid nitrogen vapor. Use low volumes of liquid nitrogen to prevent cross-contamination of samples.</li>
	<li>Keep the needle in liquid nitrogen in a closed Styrofoam box for 5 - 10 min.</li>
	<li>Precool an opened 4.5 mL cryotube and its cap in the same box.</li>
	<li>After 5 - 10 min, transfer each needle into a separate cryotube under the surface of liquid nitrogen and close the cryotube. 
	Caution: Always use protective clothing (such as insulated gloves) when working with liquid nitrogen as exposure can lead to severe frostbite.</li>
	<li>Place the cryotube onto a metal cane and place it in the cryobank canister as fast as possible. Store the samples in a canister storage dewar until further use.</li>
</ol>
4. Warming Procedure
<ol>
	<li>Warm each needle separately. All warming solutions should be at 25 &#176;C.</li>
	<li>Detach the cryotube from the metal cane and plunge it into liquid nitrogen stored in a Styrofoam box.</li>
	<li>Open the cryotube in the liquid nitrogen vapor (using forceps) and release the needle into the liquid nitrogen.</li>
	<li>Transfer the needle very fast into the first warming solution and incubate for 1 min (at 25 &#176;C). Make sure to transfer the needle quickly to prevent its warming in the air during transfer.</li>
	<li>Transfer the needle into the second warming solution and incubate for 3 min (at 25 &#176;C).</li>
	<li>Transfer the needle into the third warming solution and incubate for 5 min (at 25 &#176;C).</li>
	<li>Release the testes from the needle by sliding them downwards with curved tweezers. Place each warmed testis individually into a separate well (96-well plate) filled with 250 &#181;L of L-15 supplemented with 10% FBS. Make sure that the wells are labeled according to the sample. Keep the well plate on ice until all samples are warmed and until further work.</li>
</ol>
5. Tissue Digestion
<ol>
	<li>Prepare 500 &#181;L of the dissociation solution for each sample in separate 2 mL tubes. Label the tubes according to the code of the sample. Incubate the prepared dissociation solution for 5 min at room temperature.</li>
	<li>Add the warmed tissue into the digestion solution and cut it into small pieces by at least 30 movements of small scissors.</li>
	<li>Incubate the cut tissue on a shaking plate for 90 min at 25 &#730;C.</li>
	<li>Stop the digestion process by adding 400 &#181;L of L-15 and 100 &#181;L of FBS (10% FBS v/v). Briefly shake the solution and incubate for 1 min at room temperature.</li>
	<li>Filter the obtained solution through 50 &#181;m filters into a 1.5 mL tube. Label the tubes accordingly.</li>
	<li>Centrifuge the filtered solution at 200 &#215;g for 10 min at 25 &#730;C.</li>
	<li>Carefully remove the supernatant and resuspend the pellet in 20 &#181;L of L15 supplemented with 10% FBS. Manually shake the tube until the pellet is completely dissolved. Keep the cell suspension on ice or at 4 &#730;C until further use.</li>
</ol>
6. Viability Evaluation and Cell Counting
<ol>
	<li>Mix 5 &#181;L of cell suspension and 5 &#181;L of 0.4% trypan blue solution in an appropriately labeled 0.2 mL PCR tube. If different volumes are used, make sure that the dilution ratio of 1:1 remains.</li>
	<li>Incubate this suspension for 1 to 3 min at room temperature (25 &#176;C).</li>
	<li>Check the viability of each sample in a hemocytometer under the microscope.</li>
	<li>Count the number of live cells (unstained by trypan blue) in 15 fields. Calculate the total number of cells in 20 &#181;m of the cell suspension. The suspension is now ready for any downstream application. 
	NOTE: When optimizing the procedure for other species with larger body size, use testis fragments of equal weight. Use testes from one individual for all experimental groups in order to avoid individual variability in the results. In the case of zebrafish, we took advantage of the fact that left and right testis should contain an equal (or very similar) number of germ cells13 (see the Representative Results). The left testis was kept as a control while the right testis was vitrified/warmed. Viability percentage was calculated the number of cells isolated from the vitrified/warmed testis compared to the number of cells isolated from the fresh testis.</li>
</ol>

<ol>
	<li>Mazur, P., Leibo, S. P., Seidel, G. R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+the+germplasm+of+animals+used+in+biological+and+medical+research:+Importance,+impact,+status,+and+future+directions.">Cryopreservation of the germplasm of animals used in biological and medical research: Importance, impact, status, and future directions.</a> Biol. Reprod. 78 (1), 2-12 (2008).</li><li>Carmichael, C., Westerfield, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+and+In+Vitro+Fertilization+at+the+Zebrafish+International+Resource+Center.">Cryopreservation and In Vitro Fertilization at the Zebrafish International Resource Center.</a> Methods Mol. Biol. 546, 45-65 (2009).</li><li>Yang, H., Carmichael, C., Varga, Z. M., Tiersch, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simplified+and+standardized+protocol+with+potential+for+high-throughput+for+sperm+cryopreservation+in+zebrafish+Danio+rerio.">Development of a simplified and standardized protocol with potential for high-throughput for sperm cryopreservation in zebrafish Danio rerio.</a> Theriogenology. 68 (2), 128-136 (2007).</li><li>Morris, J. P., Berghmans, S., Zahrieh, D., Neuberg, D. S., Kanki, J. P., Look, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+sperm+cryopreservation+with+N,N-dimethylacetamide.">Zebrafish sperm cryopreservation with N,N-dimethylacetamide.</a> Biotechniques. 35 (5), 956-968 (2003).</li><li>Yoshizaki, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogonial+transplantation+in+fish:+A+novel+method+for+the+preservation+of+genetic+resources.">Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources.</a> Comp. Biochem. Physiol. Part D: Genomics Proteomics. 6 (1), 55-61 (2011).</li><li>Gao, D., Critser, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cryoinjury+in+living+cells.">Mechanisms of cryoinjury in living cells.</a> ILAR J. 41 (4), 187-196 (2000).</li><li>Asturiano, J. F., Cabrita, E., Horv&#225;th, &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress,+challenges+and+perspectives+on+fish+gamete+cryopreservation:+A+mini-review.">Progress, challenges and perspectives on fish gamete cryopreservation: A mini-review.</a> Gen. Comp. Endocrinol. 245, 69-76 (2017).</li><li>K&#225;sa, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+sperm+vitrification+protocols+for+freshwater+fish+(Eurasian+perch,+Perca+fluviatilis)+and+marine+fish+(European+eel,+Anguilla+anguilla).">Development of sperm vitrification protocols for freshwater fish (Eurasian perch, Perca fluviatilis) and marine fish (European eel, Anguilla anguilla).</a> Gen. Comp. Endocrinol. 245, 102-107 (2017).</li><li>Cuevas-Uribe, R., Leibo, S. P., Daly, J., Tiersch, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+channel+catfish+with+sperm+cryopreserved+by+rapid+non-equilibrium+cooling.">Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling.</a> Cryobiology. 63 (3), 186-197 (2011).</li><li>Wang, Y., Xiao, Z., Li, L., Fan, W., Li, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+needle+immersed+vitrification:+A+practical+and+convenient+method+with+potential+advantages+in+mouse+and+human+ovarian+tissue+cryopreservation.">Novel needle immersed vitrification: A practical and convenient method with potential advantages in mouse and human ovarian tissue cryopreservation.</a> Hum. Reprod. 23 (10), 2256-2265 (2008).</li><li>Liu, J., Cheng, K. M., Silversides, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Live+Offspring+from+Testicular+Tissue+Cryopreserved+by+Vitrification+Procedures+in+Japanese+Quail+(Coturnix+japonica).">Production of Live Offspring from Testicular Tissue Cryopreserved by Vitrification Procedures in Japanese Quail (Coturnix japonica).</a> Biol. Reprod. 88 (5), 124 (2013).</li><li>Liu, J., Cheng, K. M., Silversides, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+needle-in-straw+vitrification+can+effectively+preserve+the+follicle+morphology,+viability,+and+vascularization+of+ovarian+tissue+in+Japanese+quail+(Coturnix+japonica).">Novel needle-in-straw vitrification can effectively preserve the follicle morphology, viability, and vascularization of ovarian tissue in Japanese quail (Coturnix japonica).</a> Anim. Reprod. Sci. 134 (3-4), 197-202 (2012).</li><li>Lee, S., Yoshizaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+cryopreservation+of+spermatogonia+in+critically+endangered+Manchurian+trout+(Brachymystax+lenok).">Successful cryopreservation of spermatogonia in critically endangered Manchurian trout (Brachymystax lenok).</a> Cryobiology. 72 (2), 165-168 (2016).</li><li>Marques, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability+of+zebrafish+(Danio+rerio)+ovarian+follicles+after+vitrification+in+a+metal+container.">Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container.</a> Cryobiology. 71 (3), 367-373 (2015).</li><li>Luji&#263;, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+successful+vitrification+of+salmonid+ovarian+tissue.">First successful vitrification of salmonid ovarian tissue.</a> Cryobiology. 76, 154-157 (2017).</li><li>Bono-Mestre, C., Cardona-Costa, J., Garc&#237;a-Xim&#233;nez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+on+cell+viability+of+three+zebrafish+testicular+cell+or+tissue+cryopreservation+methods.">Effects on cell viability of three zebrafish testicular cell or tissue cryopreservation methods.</a> CryoLetters. 30 (2), 148-152 (2009).</li><li>Marinovi&#263;, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosurvival+of+isolated+testicular+cells+and+testicular+tissue+of+tench+Tinca+tinca+and+goldfish+Carassius+auratus+following+slow-rate+freezing.">Cryosurvival of isolated testicular cells and testicular tissue of tench Tinca tinca and goldfish Carassius auratus following slow-rate freezing.</a> Gen. Comp. Endocrinol. 245, 77-83 (2017).</li><li>P&#353;eni&#269;ka, M., Saito, T., Rodina, M., Dzyuba, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+early+stage+Siberian+sturgeon+Acipenser+baerii+germ+cells,+comparison+of+whole+tissue+and+dissociated+cells.">Cryopreservation of early stage Siberian sturgeon Acipenser baerii germ cells, comparison of whole tissue and dissociated cells.</a> Cryobiology. 72, 119-122 (2016).</li><li>Lee, S., Iwasaki, Y., Shikina, S., Yoshizaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+eggs+and+sperm+from+cryopreserved+whole+testes.">Generation of functional eggs and sperm from cryopreserved whole testes.</a> Proc. Nati. Acad. Sci. USA. 110 (5), 1640-1645 (2013).</li></ol>

The authors have nothing to disclose.

The main purpose of this study was to adapt the needle immersed ultra-rapid cooling procedure developed for avian and mammalian species10,11,12 to the cryopreservation of fish testis (zebrafish as a model organism). Most of the previous studies regarding cryopreservation of zebrafish genetic resources were primarily focused on cryopreservation of zebrafish sperm2,3,...

Novel trends in science and biotechnology have led to the creation of thousands of new mutant lines of mice, Drosophila, zebrafish and other species used as model organisms in biomedical and other sciences1. Furthermore, as new technologies are developed and become available, the numbers of mutant lines steadily increase2. This leads to the necessity for a safe storage of genetic resources beyond the common practices of keeping breeding colonies. As a method which enables safe storage of genetic resources for an indefinite period of time, cryopreservation offers many advantages such as extension of reproductive season, circumvents the need for continuous maintenance of broodstock, and it is more cost- and labor-efficient2.
Protocols for sperm cryopreservation developed during the past several years2,3,4 offer the opportunity for successful storage of zebrafish male genetic material. However, cryopreservation of eggs or embryos in fish is not yet possible due to their complex structure and large amounts of yolk material. Recently, the practice of transplantation of primordial germ cells (PGCs) or spermatogonial stem cells (SSCs) offers a bypass to this barrier by developing into functional sperm and eggs after transplantation5. Therefore, cryopreservation of SSCs offers a new frontier in conservation of rare and valuable genetic resources.
Even though cryopreservation offers many advantages, the slow-rate freezing process generates several conditions that may lead to cell damage2. These include intracellular and extracellular ice formation, dehydration, cryoprotectant toxicity and others. Intracellular ice damages the cells, extracellular ice may lead to mechanical crushing of cells, while water diffusion from the cells during slow-rate freezing may lead to dehydration6. Recently, vitrification as a technique which prevents the negative effects of ice formation has been applied in the cryopreservation of fish gametes7,8,9. It presents an ultra-rapid cooling technique through which the internal and external media turn into an amorphous/glassy state without crystalizing into ice7,10. Successful vitrification of testicular and ovarian tissue has been evidenced in avian and mammalian species10,11,12, thus opening possibilities for its application in fish, as well.
In this study, we present the needle immersed vitrification (NIV) procedure for the cryopreservation of whole zebrafish testes. We demonstrate a reliable method for the isolation of zebrafish early-stage germ cells without contamination and a cryopreservation process that yields relatively high amounts of early-stage germ cells with a low presence of other cells, especially spermatozoa. To the best of our knowledge, this is the first study to demonstrate a detailed visualized protocol for ultra-rapid cooling of fish gonadal tissue and zebrafish germline cells. Additionally, repeatability of the method is demonstrated in five different zebrafish strains: AB wild type, casper (roy-/-; nacre-/-), leopard (leot1/t1), vasa [Tg(vas::eGFP)] and Wilms tumor [Tg(wt1b::eGFP 1)] transgenic line.

<table><tbody><tr><td>Leibovitz media (L-15)</td><td>Sigma-Aldrich</td><td>L1518</td><td>Supplemented with L-glutamine</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma-Aldrich</td><td>F9665</td><td></td></tr><tr><td>Tricaine methanesulfonate (MS-222)</td><td>Sigma-Aldrich</td><td>E10521</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Sucrose</td><td>Acros Organics</td><td>57-50-1</td><td></td></tr><tr><td>Trehalose</td><td>Acros Organics</td><td>99-20-7</td><td>Dihydrate</td></tr><tr><td>Methanol</td><td>Reanal</td><td>20740-0-08-65</td><td></td></tr><tr><td>Propylene glycol</td><td>Reanal</td><td>08860-1-08-65</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Reanal</td><td>00190-1-01-65</td><td></td></tr><tr><td>Collagenase</td><td>Gibco</td><td>9001-12-1</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T8003</td><td></td></tr><tr><td>DNase I</td><td>Panreac AppliChem</td><td>A3778</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma-Aldrich</td><td>T6146</td><td></td></tr><tr><td>Phospate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P4417</td><td>Tablets for preparation of 200 ml PBS solution</td></tr></tbody></table>

cryopreservation of zebrafish spermatogonia by whole testes needle immersed ultra-rapid cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

On average, the number of early-stage germ cells isolated from a single fresh zebrafish testis varied between 40,000 and 200,000 cells depending on the size of the fish. When digesting fresh zebrafish testes in all 5 strains, early-germ cells were not the only cells present in the cell suspensions (Figure 2). Beside the early-stage germ cells, numerous spermatozoa were found as well. On the other hand, there were far less spermatozoa after digestion of cryopr...

Watch this Scientific Journal Video about Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling at JoVE.com

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...

cryopreservation-zebrafish-spermatogonia-whole-testes-needle-immersed

Research

JoVE Journal

Biology

8.2K Views.  Szent István University. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

Video: Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Developmental Biology

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Genetics

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

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 ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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 ...

Vitrification-based Cryopreservation: An Ultra-rapid Cooling Technique to Preserve Biological Specimens at Very Low Temperatures for Long-term Storage

After euthanizing the mice, use sterile forceps and scissors to slowly isolate the tumor from the mice. Wash the tumor tissues in DPBS and a 10-centimeter dish. Use forceps and scissors to dissect and remove necrotic areas, fatty tissue, blood clots, and connective tissue.
Next, use a mold to cut the tumor tissues to a maximum thickness of 1 millimeter. Wash the tumor slices with DPBS in a 10-centimeter dish. To begin the vitrification process, use forceps to transfer the slices into tube V1 and incubate at 4 degrees Celsius for 4 minutes. Roll and invert the tube briefly and incubate at 4 degrees Celsius for another 4 minutes.
Next, pour the V1 solution and slices into a 10-centimeter dish and use forceps to transfer the slices into tube V2. Incubate at 4 degrees Celsius for 4 minutes. Roll and invert the tube briefly and incubate at 4 degrees Celsius for another 4 minutes. After this, pour the V2 solution and the slices into a 10-centimeter dish.
Transfer the slices into tube V3 and incubate at 4 degrees Celsius for 5 minutes. Roll and invert the tube briefly and incubate at 4 degrees Celsius for another 5 minutes.
Make sure that all of the slices sink to the bottom of the tube. If several slices remain floating, roll and invert the tube briefly and incubate at 4 degrees Celsius until all of the slices sink completely. Then, pour the V3 solution and slices into a 10-centimeter dish. Cut the tissue holders to the proper length and place them on sterile gauze. Transfer the slices onto the holders. Wrap the holders with gauze.
Using forceps, place the holders in liquid nitrogen and let them incubate for 5 minutes. After this, label the cryogenic vials with the tissue information. Transfer the holders with tissue slices into the vials, which will be stored in liquid nitrogen.

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Summary

Explore More Videos

Chapters in this video

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

Making Gynogenetic Diploid Zebrafish by Early Pressure

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Vitrification-based Cryopreservation: An Ultra-rapid Cooling Technique to Preserve Biological Specimens at Very Low Temperatures for Long-term Storage