Name: A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization (Video) | JoVE Journal
Uploaded: 2009-07-06T19:03:00.000+00:00
Duration: 11 min 1 s
Description: Learn a high-throughput zebrafish sperm cryopreservation protocol achieving 25% fertility in vitro. This method enables long-term genetic storage for reproductive biology research.

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5 minutes

This work was supported by NIH R01 HG002995 "TILLING the Zebrafish Genome: a Reverse Genetics Approach" to CBM.

Part A: Sperm Freezing Solutions
1. Ginsberg Fish Ringers (Make fresh every 3 days ;Ginsburg, 1963)
NOTE: ORDER OF ADDITION IS IMPORTANT TO PREVENT PRECIPITATION
In 450 ml sterile ddH2O dissolve:
<ul>
<li>NaCl 3.25 g</li>
<li>KCl 0.125 g</li>
<li>CaCl2•2 H2O 0.175 g 
 Then add:</li>
<li>NaHCO3 0.10 g</li>
</ul>
Bring final volume to 500 ml with sterile ddH2O and store at 4°C.
2. Freezing Medium (Make fresh daily)
<ol style="list-style-type:lower-roman">
<li>WITHOUT Methanol
<ul><li>Ginsburg Fish Ringers 10 ml (room temp.)</li>
<li>Powdered Skim Milk 1.5 gm</li>
</ul></li>
<li>WITH Methanol
 NOTE: ORDER OF ADDITION IS IMPORTANT TO PREVENT PRECIPITATION 
<ul>
<li>Ginsburg Fish Ringers 9 ml (room temp.)</li>
<li>Methanol 1 ml</li>
<li>Powdered Skim Milk 1.5 g
</li></ul>
 After assembling freezing media, mix well for 20 min. on orbital shaker or rocker. Before using, aliquot into 1 ml eppendorf tubes, avoiding surface bubbles. 
</li></ol>
Part B: Materials needed for sperm freezing
Materials needed for sperm freezing
<ol>
 <li>10 μl disposable pipettes (Fisherbrand cat# 22-358697) </li>
 <li>250ml beakers for fish water containing MESAB/Tricaine </li>
 <li>watch glasses (Pyrex cat# 9985-75) </li>
 <li>P20 pipetman (or equivalent) and tips </li>
 <li>Sponge fish holder (to hold male while squeezing) </li>
 <li>Dissecting microscope with above stage lighting </li>
 <li>Flat forceps (e.g. Millipore type) </li>
 <li>2.0ml cryogenic vials (Corning cat# 430488) </li>
 <li>10X10 cryoboxes (Nalgene cat#03-337-7AA) </li>
 <li>Styrofoam container filled with ~ 15 cm of finely crushed dry ice1 </li>
 <li>15 ml conical tubes (Falcon # 352099) </li>
 <li>Dewar flask containing liquid nitrogen </li>
 <li>Long tongs (e.g. CMS Fisher Health Care cat# 10-316B) </li>
 <li>Cryogloves </li>
 <li>Recovery tank for males </li>
 <li>One eppendorf tube filled with freezing media without methanol </li>
 <li>One eppendorf tube filled with freezing media with methanol</li>
 <li>Cryofreezer (e.g. Taylor-Warton K-series)</li>
</ol>
1 A simple method for making finely crushed dry ice is to wrap it in a towel and pulverize it with a hammer. A more efficient method is to use a dry ice grinder, e.g. Clawson model RE-2.
Part C: Sperm Freezing Method
<ol>
<li>MARK CAPILLARY TUBES: Prior to beginning, mark the 10μl capillary tubes with a lab pen at the level of 3.33 μl (i.e. 16.7 mm from bottom; see Fig. 1.1).</li>
<li>ANESTHETIZE MALE: Place male(s) in beaker containing MESAB/tricaine diluted in fish water (recipe in "The Zebrafish Book")</li>
<li>DRY FISH: Once anesthetized, lift fish out of anesthetic using a plastic spoon and gently blot fish dry on a paper towel, paying special attention to drying the ventral side. Water activates sperm so it is important to thoroughly dry around the cloaca. Position fish on sponge holder, ventral side up (Fig. 1.2). If region around anal fin is still moist, gently dry with a kimwipe. Take care to not squeeze sperm prematurely!</li>
<li>Place marked capillary tube in rubber mouth pipette adapter that is included with capillary tubes. The adaptor is a rubber hose that has a mouthpiece on one end and a capillary holder on the other. Alternatively, the capillary tube can be connected to a 50μl Hamilton syringe via a short piece of tygon tubing of the appropriate diameter.</li>
<li>Place male under the scope and expose the urogenital opening by carefully spreading the pelvic fins apart using the end of the capillary tube.</li>
<li>COLLECT SPERM: Expel sperm by gently stroking the sides of the fish with smooth forceps (e.g. Millipore), or by gently squeezing the sides of the fish between your index finger and thumb. Collect sperm in capillary tube as it is expelled using gentle suction (Fig. 1.2). Avoid feces that may be expelled with sperm.</li>
<li>NORMALIZE SPERM VOLUME TO 3.3 μl: If the volume of sperm reaches, or exceeds pen mark on capillary tube (i.e. 16.7 mm or greater), then proceed to step 8. If sperm volume does not reach target volume, then normalize volume to pen mark using Freeze Medium WITHOUT Methanol (Fig. 1.3). Minimum amount of sperm that is acceptable varies with quality of sperm. Good sperm is white and opaque. Poor sperm looks watery. In general, we accept as minimums: 1 μl good sperm (or 1/3 target) and 2 μl poor sperm (or 2/3 target). Volume is now normalized to 3.3 μl.</li>
<li>ADD CRYOPROTECTANT TO SPERM : Suck up Freezing Medium WITH Methanol to the orange mark (approx. total volume is now 20 μl; Fig. 1.4). Expel sperm and cryoprotectant mixture onto clean area of a watch glass, paying special attention to not introduce bubbles. Gently mix by pipetting up and down ~ 4 times (avoid introducing bubbles).</li>
<li>PLACE 10μl SPERM INTO EACH OF 2 CRYOVIALS: Pipette 10 μl of the sperm:cryoprotectant solution into the bottom of two separate cryovials that have been labeled with relevant information (Fig. 1.5). Moving quickly, cap vials and drop them into the bottom of room temperature 15 ml Falcon tubes- one cryovial/tube. Cap Falcon tube and insert the pair of cryovial-containing Falcon tubes into crushed dry ice.</li>
<li>FREEZE SPERM FOR 20 MIN. ON DRY ICE: Dry ice should be in fine powder form, not pellets. The tubes should be inserted into the dry ice deep enough that only their caps show (Fig. 1.6). In order to keep track of tubes in dry ice, give each pair of Falcon tubes a number from 1-20 (written on the caps) and record the time they go into the dry ice.</li>
<li>PLACE CRYOVIALS INTO LIQUID NITROGEN: After the sperm has been frozen on dry ice for 20 min., transfer them to a liquid nitrogen- containing dewar flask. Sperm is stored here until time to place into liquid nitrogen freezer. When placing in the freezer boxes, place freezer box in a bath of liquid nitrogen so that samples do not heat up. Use long metal tongs to recover vials from dewar flask and handle with cryogloves. Alternatively, cryovials can be transferred directly from dry ice into the freezer box if the freezer box is kept immersed in liquid nitrogen in a Styrofoam container.</li>
<li>Store cryovials long term in a cryogenic liquid nitrogen freezer. To maintain viability of sperm, it is important that vials are stored immersed in liquid nitrogen. In our experience, vials stored in the vapor phase lose viability over time. Speed is important! The time between adding the cryoprotectant and placing sperm vials in dry ice (i.e. steps 6-10) should be no more than 30 sec. </li></ol>
Part D: In Vitro Fertilization Method Using Cryopreserved Sperm
We find that this works best with two people. One person squeezes eggs from females while the other person thaws the sperm.
<ol><li>Set a water bath to 33°C.</li>
<li>Remove cryovial from liquid nitrogen freezer and transfer to liquid nitrogen-containing dewar flask until ready to thaw.</li>
<li>Squeeze eggs from females into 35mm plastic petri dish. If possible, try to obtain 3 clutches of eggs and combine into one dish. If you cannot get three good clutches within 1 min of your first clutch, then proceed to step 4 (one good clutch is sufficient). Definition of good clutch: &gt;150 uniformly nice looking eggs (i.e. yellowish, with no white debris indicative of degradation). Keep dish covered while sperm is thawed.</li>
<li>Remove vial from liquid nitrogen and remove cap. Quickly immerse vial ~1/2 way into 33°C water bath for 8-10 sec.</li>
<li>Quickly add 70 μl room temperature Hanks to vial and mix by pipetting up and down. Immediately add to eggs and mix gently with pipette tip.</li>
<li>Without delay, activate sperm and eggs by adding 750 μl fish water. Swirl to mix. Incubate 5 min at room temperature.</li>
<li>After 5 min, fill dish with fish water and incubate dish at 28°C. After 2-3 hrs, count and transfer fertile embryos to 100mm dishes (50/dish). Count infertile eggs so that fertilization frequency can be determined.</li>
<li>Take good care of the larvae! For first 5 days, change water daily and remove dead embryos. Put only larvae that have swim bladders into nursery on day 5.</li>
</ol>
Part E: Representative Results
We used this crypreservation methodology to make a library of 8600 frozen sperm samples for TILLING between the years 2001 and 2003. We have thawed 106 of these samples and determined their fertility. Fertility is the ratio of viable embryos produced in an in vitro fertilization divided by the total number of eggs that were fertilized with the frozen sperm. Fresh (non cryporeserved) sperm typically has &gt;95% fertility; cryopreserved sperm has much lower fertility: an average of 29% (n=106 vials). The vial-to-vial variability in fertility is large, ranging from less than 1% to as high as 62%. This variability is represented by the large error bars representing standard deviation in Fig. 2. However in 106 sperm samples thawed to date we have only once experienced 0% fertility in both sperm samples and failed to recover the corresponding TILLING mutation.
By thawing sperm vials from our library over the course of several years (2001-2008) we have found that the average fertility of sperm samples frozen by this method remains stable over time (Fig. 2). The average fertility of frozen sperm samples thawed in 2001 was 23.5% (based on 11 sperm samples) and in 2008 was 25% (based on 21 sperm samples).
A number of other investigators are now using this sperm freezing protocol in their labs. Some have reported good success with fertility rates and sample-to-sample variability similar to what we have observed. Others have reportedly failed to make this protocol work in their hands. We believe that likely sources of problems are:
<ol>
<li>Failure to use the exact cryovials or conical tubes we have suggested in the protocol, leading to a different rate of cooling when in dry ice;</li>
<li>Failure to use powdered dry ice, leading to a sub-optimal cooling rate;</li>
<li>Using male fish with poor fertility: you should get at least 1 μl of sperm from a single male. You can also test male fertility by doing an in vitro fertilization with fresh sperm, however this can be misleading because it takes very little fresh sperm to get near 100% fertility.</li>
<li>Using sub-standard quality eggs in the in vitro fertilization. Using anything but perfect clutches always leads to near 0% fertility, even from sperm samples that actually have good fertility (based on the fertility of the duplicate vial).</li>
</ol>
<img src="/files/ftp_upload/1395/1395fig1.jpg" alt="Zebrafish sperm cryopreservation process with diagrams detailing volume measurement and cryovials." data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1. Schematic of sperm freezing methodology.
<img src="/files/ftp_upload/1395/1395fig2.jpg" alt="Library fertility trends bar chart, 2001-2008, average percent fertility analysis." data-altupdatedat="1787630408000" data-alt="Figure 2"> Figure 2. Cryopreserved sperm fertility remains stable over time. Percent fertility is the number of viable embryos produced in an in vitro fertilization divided by the total number of eggs that were fertilized with the frozen sperm. "n" refers to the number of cryopreserved sperm vials whose fertility was tested in the corresponding year. Error bars represent standard deviation. Since each sperm vial is from a different male, vial-to-vial variability is observed.

<ol><li>Ginsburg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sperm-egg+association+and+its+relationship+to+activation+of+the+egg+in+salmonid+fishes%5BTitle%5D)+AND+%22J.+Embryol.+Exp.+Morphol%22%5BJournal%5D)">Sperm-egg association and its relationship to activation of the egg in salmonid fishes.</a> J. Embryol. Exp. Morphol. 11, 13-33 (1963).</li>
<li>Harvey, B., Kelley, R. N., Ashwood-Smith, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryopreservation+of+zebra+fish+spermatozoa+using+methanol%5BTitle%5D)+AND+%22Can.+J.+Zoology%22%5BJournal%5D)">Cryopreservation of zebra fish spermatozoa using methanol.</a> Can. J. Zoology. 60, 1867-1870 (1982).</li>
</ol>

A number of other investigators are now using this sperm freezing protocol in their labs. Some have reported similar fertility and the same sample-to-sample variability that we have observed. Others have reportedly failed to make this protocol work in their hands. We believe that likely sources of problems are: 
<ol>
<li>Failure to use the exact cryovials or conical tubes we have suggested in the protocol, leading to a different rate of cooling when in dry ice;</li>
<li>Failure to use powdered dry ice, leading to a sub-optimal cooling rate;</li>
<li>Using male fish with poor fertility: you should get at least 1 &mu;l of sperm from a single male. You can also test male fertility by doing an in vitro fertilization with fresh sperm, however this can be misleading because it takes very little fresh sperm to get near 100% fertility. Larger, more robustly colored males typically give more higher quality sperm.</li>
<li>Using sub-standard quality eggs in the in vitro fertilization. Using anything but perfect clutches always leads to near 0% fertility, even from sperm samples that actually have good fertility (based on the fertility of the duplicate vial). </li>
</ol>

<table><tbody><tr><td>10 &amp;mu;l disposable pipettes</td><td>Fisher Scientific</td><td>22-358697</td><td></td></tr><tr><td>Watch glasses</td><td>Pyrex</td><td>9985-75</td><td></td></tr><tr><td>2 ml cryogenic vials</td><td>Corning</td><td>430488</td><td></td></tr><tr><td>10 x 10 cryoboxes</td><td>Nalge Nunc international</td><td>03-337-7AA</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Falcon BD</td><td>352099</td><td></td></tr><tr><td>Tongs</td><td>Fisher Scientific</td><td>10-316B</td><td></td></tr><tr><td>Cryofreezer</td><td>Taylor-Wharton</td><td>K-series</td><td>For example</td></tr></tbody></table>

a high-throughput method for zebrafish sperm cryopreservation and in vitro fertilization

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.

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 ...

Hi, I'm Bruce Draper from the University of California Davis, department of Molecular and Cellular Biology, And I'm Cecilia Moans from the Division of Basic Science at the Fred Hutchinson Cancer Research Center in Seattle. Today, Cecilia and I are going to demonstrate the zebrafish sperm cry preservation protocol, which is a modification of the original Brian Harvey protocol. We will show you that working in teams of two, it is possible to cryopreserve sperm from 100 individual males in under two hours.While there is a wide range of fertility upon thawing, on average, we receive 25%fertility, and this is from a library that's been cryopreserved for over seven years. So let's get started. Before performing this procedure, first, make the sperm freezing solutions.The Ginsburg Fish ringer solution is made fresh every three days, and the freezing medium is made fresh daily with and without methanol. The composition of these solutions can be found in the online supplemental information accompanying this protocol. The materials used in this procedure can affect the rate of freezing and thawing, which can in turn affect the fertility of frozen sperm.For this reason, a list of recommended materials is also provided as supplemental online information. It is best to freeze the sperm on finely crushed dry ice as opposed to dry ice pellets. One simple way to crush dry ice is to wrap it in a towel and pulverize it with a hammer.A more efficient method is to use a dry ice grinder such as a claws and model RE two to freeze zebrafish sperm. First mark 10 microliter capillary tubes with a lab pen at the level of 3.33 microliters or about 16.7 millimeters from the bottom to anesthetize the males place two to four males in a beaker containing trica diluted and fish water. The recipe can be found in the zebrafish book.Once anesthetized, lift the fish out of the anesthetic using a plastic spoon and gently blot them dry on a paper towel. Pay special attention to drawing the ventral side. Water activates sperm so it is critical to thoroughly dry around the ika.Position the fish on the sponge holder ventral side up. If the region around the pelvic fins is still moist, gently dry it with a Kim wipe. Place marked capillary tube in a rubber mouth pipette adapter that is included with the capillary tubes.The adapter is a rubber hose that has a mouthpiece on one end and a capillary holder on the other. Alternatively, the capillary tube can be connected to a 50 microliter Hamilton syringe. Using a short piece of tigon tubing of the appropriate diameter, place the male under the scope and expose the urogenital opening by carefully spreading the pelvic fins apart.Using the end of the capillary tube, express the sperm by gently stroking the sides of the fish with smooth forceps or by gently squeezing the sides of the fish with an index finger and thumb, collect the sperm in a capillary tube as it is expelled using gentle suction. Avoid feces that may be expelled with the sperm. The target volume of sperm is 3.3 microliters.The minimum amount of sperm that is acceptable varies depending on the quality of the sperm. Good sperm is white and opaque while pore sperm looks watery. In general, the minimal acceptable amount of sperm is one microliter of good sperm or one third of the target, and two microliters of pore sperm or two thirds of the target.If sperm volume does not reach the target volume of 3.3 microliters, then it is necessary to normalize the volume to the pen mark using freeze medium without methanol. If the volume of sperm already reaches or exceeds the pen mark on the capillary tube, then proceed to the next step. Now add the cryoprotectant to the sperm.Suck up freezing medium with methanol to the orange mark. The approximate total volume is now 20 microliters. Expel the sperm and cryoprotectant mixture onto a clean area of a watch glass gently mixed by pipetting up and down about four times and avoid introducing bubbles.Aliquot the sperm into two cryo vials by pipetting 10 microliters of the sperm cryoprotectant solution into the bottom of each labeled vial. Moving quickly cap the vials and drop each cryo vial into a separate 15 milliliter falcon tube. At room temperature, cap the falcon tubes and insert them into finely powdered dry ice.The tubes should be inserted deep enough so only the caps show to keep track of the falcon tubes. Number them and record the time that each tube goes into the dry ice. Allow the sperm to freeze in the dry ice for 20 minutes.After the sperm is frozen, transfer the two cryo vials into a liquid nitrogen containing doer flask. For temporary storage. Place the freezer box in a bath of liquid nitrogen so the samples do not heat up.Use long metal tongs to recover the vials from the doer flask and handle them with cryo gloves. Alternatively, cryo vials can be transferred directly from the dry ice into the freezer. If the freezer box is kept immersed in liquid nitrogen in a styrofoam container for long-term storage, place the cryo vials in a cryogenic liquid nitrogen freezer.To maintain sperm viability, it is crucial that the vials are stored immersed in liquid nitrogen vials stored in the vapor phase may lose viability over time. Speed is also critical in maintaining sperm viability. The time between adding the cryoprotectant and placing the sperm vials on dry ice should be no more than 30 seconds In vitro fertilization.Using cryo-preserved sperm works best with two people. One person squeezes eggs from the females while the other person thaws the sperm. Set a water bath to 33 degrees Celsius.Remove a cryo vial from the liquid nitrogen freezer and transfer it to a liquid nitrogen containing D or flask until it is ready to thaw. Squeeze eggs from anesthetized females into a 35 millimeter plastic Petri dish. This is described in detail in the JoVE protocol entitled Making Gyno Genetic deployed zebrafish by early pressure, which provides a detailed description of zebrafish.In vitro fertilization, a good clutch contains more than 150 eggs that are uniform and yellowish. Any white debris is indicative of degradation. If possible, try to obtain three clutches of eggs and combined into one dish.If three good clutches are not obtained within one minute of the first clutch, then proceed to the next step. Keep the dish covered while the sperm is thawed. Meanwhile, remove the vial from the liquid nitrogen and remove the cap.Quickly immerse the vial halfway in a 33 degree water bath for eight to 10 seconds. Quickly add 70 microliters of room temperature, Hank's buffered salt solution to the vial and mix by pipetting up and down immediately add to the eggs in the Petri dish and mix gently with the pipette tip. Then immediately activate the sperm and eggs by adding 750 microliters of fish water swirl to mix and incubate for five minutes at room temperature.After five minutes, fill the dish with fish, water, and incubate at 28 degrees Celsius. After two to three hours, count and transfer viable embryos to 100 millimeter dishes at 50 embryos per dish. Also count on fertilized eggs so that the fertility of the sperm sample can be determined.Sperm fertility is the ratio of viable embryos to the total number of eggs that was used for the invitro fertilization. Eggs can be sorted anytime from two to six hours after in vitro fertilization. When infertile eggs are easily distinguished from viable embryos, infertile eggs are clear and undivided.When viewed with trans illumination on a stereo microscope, the average fertility of cryopreserved sperm varies widely. However, it should be possible to get an average of 25%fertility that is stable for multiple years. This graph shows the average percent fertility of sperm frozen between 2001 and 2003 and thought as recently as 2008.We've just demonstrated a high throughput method for cry preserving zero fish sperm. In doing this protocol, it's important to use all the recommended plastic wear and equipment as well as to use pulverized crushed dry ice rather than dry ice pellets. It's critical to use really high quality sperm and eggs, and in the case of the eggs when doing the in vitro fertilization, upon hawing the sperm, it's important to use as large a number of eggs as possible to improve the number of fertile embryos you get at the other end, Which could take three to four Females, right?Another key component of the protocol is speed. It should never take more than about a minute to go from expressing the sperm from the male to putting it into the dry ice. Well, I think that's it.Good luck with your experiments.

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Learn a high-throughput zebrafish sperm cryopreservation protocol achieving 25% fertility in vitro. This method enables long-term genetic storage for reproductive biology research.

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

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