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

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.