Name: Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling (Video) | JoVE Journal
Uploaded: 2018-03-04T13:00:00.000+00:00
Duration: 13 min 32 s
Description: Learn needle immersed vitrification for cryopreserving whole zebrafish testes. This method enables safe germ cell storage and eliminates spermatozoa post-warming, simplifying downstream applications.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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/pubmed?term=((Cryopreservation+of+the+germplasm+of+animals+used+in+biological+and+medical+research%3A+Importance%2C+impact%2C+status%2C+and+future+directions%5BTitle%5D)+AND+%22Biol.+Reprod%22%5BJournal%5D)">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/pubmed?term=((Cryopreservation+and+In+Vitro+Fertilization+at+the+Zebrafish+International+Resource+Center%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">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/pubmed?term=((Development+of+a+simplified+and+standardized+protocol+with+potential+for+high-throughput+for+sperm+cryopreservation+in+zebrafish+Danio+rerio%5BTitle%5D)+AND+%22Theriogenology%22%5BJournal%5D)">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. IV, Berghmans, S., Zahrieh, D., Neuberg, D. S., Kanki, J. P., Look, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+sperm+cryopreservation+with+N%2CN-dimethylacetamide%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">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/pubmed?term=((Spermatogonial+transplantation+in+fish%3A+A+novel+method+for+the+preservation+of+genetic+resources%5BTitle%5D)+AND+%22Comp.+Biochem.+Physiol.+Part+D%3A+Genomics+Proteomics%22%5BJournal%5D)">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/pubmed?term=((Mechanisms+of+cryoinjury+in+living+cells%5BTitle%5D)+AND+%22ILAR+J%22%5BJournal%5D)">Mechanisms of cryoinjury in living cells.</a> ILAR J. 41 (4), 187-196 (2000).</li>
<li>Asturiano, J. F., Cabrita, E., Horváth, Á <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progress%2C+challenges+and+perspectives+on+fish+gamete+cryopreservation%3A+A+mini-review%5BTitle%5D)+AND+%22Gen.+Comp.+Endocrinol%22%5BJournal%5D)">Progress, challenges and perspectives on fish gamete cryopreservation: A mini-review.</a> Gen. Comp. Endocrinol. 245, 69-76 (2017).</li>
<li>Kása, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+sperm+vitrification+protocols+for+freshwater+fish+%28Eurasian+perch%2C+Perca+fluviatilis%29+and+marine+fish+%28European+eel%2C+Anguilla+anguilla%29%5BTitle%5D)+AND+%22Gen.+Comp.+Endocrinol%22%5BJournal%5D)">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/pubmed?term=((Production+of+channel+catfish+with+sperm+cryopreserved+by+rapid+non-equilibrium+cooling%5BTitle%5D)+AND+%22Cryobiology%22%5BJournal%5D)">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/pubmed?term=((Novel+needle+immersed+vitrification%3A+A+practical+and+convenient+method+with+potential+advantages+in+mouse+and+human+ovarian+tissue+cryopreservation%5BTitle%5D)+AND+%22Hum.+Reprod%22%5BJournal%5D)">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/pubmed?term=((Production+of+Live+Offspring+from+Testicular+Tissue+Cryopreserved+by+Vitrification+Procedures+in+Japanese+Quail+%28Coturnix+japonica%29%5BTitle%5D)+AND+%22Biol.+Reprod%22%5BJournal%5D)">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/pubmed?term=((Novel+needle-in-straw+vitrification+can+effectively+preserve+the+follicle+morphology%2C+viability%2C+and+vascularization+of+ovarian+tissue+in+Japanese+quail+%28Coturnix+japonica%29%5BTitle%5D)+AND+%22Anim.+Reprod.+Sci%22%5BJournal%5D)">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/pubmed?term=((Successful+cryopreservation+of+spermatogonia+in+critically+endangered+Manchurian+trout+%28Brachymystax+lenok%29%5BTitle%5D)+AND+%22Cryobiology%22%5BJournal%5D)">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/pubmed?term=((Viability+of+zebrafish+%28Danio+rerio%29+ovarian+follicles+after+vitrification+in+a+metal+container%5BTitle%5D)+AND+%22Cryobiology%22%5BJournal%5D)">Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container.</a> Cryobiology. 71 (3), 367-373 (2015).</li>
<li>Lujić, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+successful+vitrification+of+salmonid+ovarian+tissue%5BTitle%5D)+AND+%22Cryobiology%22%5BJournal%5D)">First successful vitrification of salmonid ovarian tissue.</a> Cryobiology. 76, 154-157 (2017).</li>
<li>Bono-Mestre, C., Cardona-Costa, J., García-Ximénez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+on+cell+viability+of+three+zebrafish+testicular+cell+or+tissue+cryopreservation+methods%5BTitle%5D)+AND+%22CryoLetters%22%5BJournal%5D)">Effects on cell viability of three zebrafish testicular cell or tissue cryopreservation methods.</a> CryoLetters. 30 (2), 148-152 (2009).</li>
<li>Marinović, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryosurvival+of+isolated+testicular+cells+and+testicular+tissue+of+tench+Tinca+tinca+and+goldfish+Carassius+auratus+following+slow-rate+freezing%5BTitle%5D)+AND+%22Gen.+Comp.+Endocrinol%22%5BJournal%5D)">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>Pšenička, M., Saito, T., Rodina, M., Dzyuba, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryopreservation+of+early+stage+Siberian+sturgeon+Acipenser+baerii+germ+cells%2C+comparison+of+whole+tissue+and+dissociated+cells%5BTitle%5D)+AND+%22Cryobiology%22%5BJournal%5D)">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/pubmed?term=((Generation+of+functional+eggs+and+sperm+from+cryopreserved+whole+testes%5BTitle%5D)+AND+%22Proc.+Nati.+Acad.+Sci.+USA%22%5BJournal%5D)">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

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...

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 offers the possibility of maintaining live cells and tissues at low temperatures for an infinite period of time, at least in human terms. Vitrification is a form of cryopreservation which involves ultra-rapid cooling into a glassy, amorphous state without the formation of crystalline ice and without the damages associated. In this video, we present the protocol for the vitrification of whole zebrafish testes, which allows their safe storage until their later use as a source of germ cells for transplantation into suitable recipients.The current vitrification protocol has one significant advantage over the slow-rate freezing protocols in regard to germ stem cell manipulations. And that is that after vitrification and warming, the spermatozoa die and they get adjusted so they are not present in the cell suspension. Therefore, there is no need for isolation of germ cells after warming.And that readily facilitates the downstream manipulations. Euthanize the fish by placing it into a dish containing 200 milligrams per liter of MS-222 at pH seven. Before continuing with the dissection, make sure that the gills have stopped moving, and at least 10 minutes have passed since that moment to ensure death by hypoxia.Dry the fish by patting it on a paper towel and place it on its dorsal side on a dissecting mat. Firstly, cut off the pelvic and pectoral fins for easier dissection. 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.Open the body cavity of the fish and pin the left and right body wall to the dissection mat with needles. Testes are located above the gastrointestinal organs on both sides of the swimming bladder. To prevent contamination, do not take out the gastrointestinal organs, but gently push them to one side and remove the testes from the opposing side.Testes are connected to the body wall, so remove them carefully and cut the connecting peritoneum by micro scissors. Additionally, in order to prevent contamination, firstly sterilize the testes by putting them into 70%ethanol for two seconds, and then place them in L-15 on 96-well plate on ice. Mark needles with appropriate labels, depending on the sample type, and prepare the equilibration and vitrification solutions.Transfer the testes to a larger-well plate or dish and pin three testes or more, depending on the size of the needle, on a sterile acupuncture needle. Firstly, position the testes 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.After puncturing the testis, gently pull it upwards towards the top of the needle. Make sure the testes are separated from each other and they are not falling off or sliding down. Place the needles with pinned testes in a Tubulator Eppendorf tube filled with L-15 until vitrification at 25 degrees.The storage time should not exceed one hour. Transfer the needle with the testes into the equilibration solution and incubate for five minutes at 25 degrees. Transfer the needle into the vitrification solution and incubate for 30 seconds at 25 degrees.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.Quickly place the needle into liquid nitrogen in a styrofoam box. Keep the needle in liquid nitrogen in a closed styrofoam box for five to 10 minutes. Pre-cool an opened 4.5 milliliter cryotube and its cap in the same box.After five to 10 minutes, transfer each needle into a separate cryotube under the surface of liquid nitrogen and close the cryotube. 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 drawer until further use.Warm each needle separately. All warming solutions should be at 25 degrees. Detach the cryotube from the metal cane and plunge it into liquid nitrogen stored in a styrofoam box.Open the cryotube in the liquid nitrogen vapor using forceps, and release the needle into the liquid nitrogen. Transfer the needle very fast into the first warming solution and incubate for one minute. Make sure to transfer the needle quickly to prevent it warming in the air during transfer.Transfer the needle into the second warming solution, and incubate for three minutes. Transfer the needle into the third warming solution and incubate for five minutes. Release the testes from the needle by sliding them downwards with curved tweezers.Place each warmed testis individually into a separate well filled with 250 microliters of L-15, supplemented with 10%FPS. 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.Prepare 500 microliters of the dissociation solution for each sample in separate two-milliliter tubes. Label the tubes according to the code of the sample. Incubate the prepared dissociation solution for five minutes at room temperature.Add the warmed tissue into the digestion solution, and cut it into small pieces by at least 30 movements of small scissors. Incubate the tissue on a shaking plate for 90 minutes at 24 degrees. Stop the digestion process by adding 400 microliters of L-15 and 100 microliters of 10%FPS.Briefly shake the solution and incubate for one minute at room temperature. Filter the obtained solution through 50-micron filters into a 1.5 milliliter tube. Label the tubes accordingly.Centrifuge the filtered solution at 200 G for 10 minutes at 25 degrees. Carefully remove the supernatant and re-suspend the pellet in 20 microliters of L-15, supplemented with 10%FPS. Manually shake the tube until the pellet is completely dissolved.Keep the cell suspension on ice or at four degrees until further use. Mix five microliters of cell suspension and five microliters of 0.4%trypan blue solution in an appropriately-labeled 0.2 milliliter PCR tube. If different volumes are used, make sure that the dilution ratio of one to one remains.Incubate this suspension for one to three minutes at 25 degrees. Check the viability of each sample in a hemocytometer under a microscope. Count the number of live cells unstained by trypan blue in 15 fields.Calculate the total number of cells in 20 microliters of the cell suspension. The suspension is now ready for any downstream application. On average, the number of early-stage germ cells isolated from a single fresh zebrafish testis vary between 40, 000 and 200, 000 cells, depending on the size of the fish.In all zebrafish lines used in this study, the current vitrification protocol yielded average viability rates of 50%and higher. This indicates favorable viability and repeatability of the presented protocol. When digesting fresh zebrafish testes in all five strains, besides the early-stage germ cells, numerous spermatozoa were found as well.After digestion of cryo-preserved testes, there were far less spermatozoa, indicating that they do not survive this vitrification protocol, and that they are most likely eliminated during the digestion process. The needle-immersed vitrification protocol presented in this study offers several advantages in the cryopreservation of testicular pieces. Firstly, several pieces can be pinned to one needle, and therefore they can simultaneously be exposed to cryoprotectants and to the liquid nitrogen.Furthermore, this method enables that very low amounts of cryoprotectants are attached to the tissue pieces. And therefore, cooling and warming rates can be higher. One significant advantage is that spermatozoa do not survive this protocol, and therefore in the end, the cell suspensions are enriched spermatogonia, which will facilitate the downstream applications.But one thing that has to be taken into account is the possibility of cross-contamination through nitrogen. Therefore when using this method, liquid nitrogen should not be reused.

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

Research

JoVE Journal

Biology

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.

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

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.

Making Gynogenetic Diploid Zebrafish by Early Pressure

In this video, we describe the procedure for vitrification of harvested rabbit embryos at desired development stage using ethylene glycol and dimethyl sulfoxide as cryoprotectants and cryogenic straw as the carrier system. The vitrified embryos have high survival rate and can be stored for an extended period until use.

Rabbit Embryo Vitrification Using Cryogenic Straw:  An Ultra-Rapid Cooling Technique to Preserve Rabbit Embryos for Long-term Storage

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.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

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.

Recovery of Adult Zebrafish Hearts for High-throughput Applications

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 for Blood Tests in Adult Zebrafish

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.

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

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.

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

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.

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

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.

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

Learn needle immersed vitrification for cryopreserving whole zebrafish testes. This method enables safe germ cell storage and eliminates spermatozoa post-warming, simplifying downstream applications.

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

Explore More Videos