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Xenopus Tropicalis Sperm Freezing

This protocol is modified from the protocol provided by Sargent et al. Many thanks to Mike Sargent and Lyle Zimmerman for providing us with the protocol months before publication.

 

Sperm Preparation

1. Isolate testis.

2. Macerate both testis in a single 1.5 ml Eppendorf tube with 500µl L15/CS (L-15 Medium [Leibovitz-15] supplemented to 10% calf serum)

3. Add 500µl cryoprotectant.

4. Divide evenly into 4 separate tubes.

 

NOTE: We test the fresh sperm to ensure the sperm is initially viable and fertile. One tube is used fresh, while the remaining three are frozen. If significantly low rates are obtained using the fresh sperm, the frozen tubes are discarded.

NOTE: The night before harvesting testis we boost the males with 100 u hCG. In our experience, this appears to improve fertilization rates.

Sperm Freezing

1. Place tubes into Styrofoam box. Wrap lid with foil.

NOTE: We have tested three different freezing techniques (slow, medium, and fast) and found the slow freeze with a Styrofoam box to work the best. This is in contrast to the original published protocol of Sargent et al. which uses a slow freeze using either a dry ice/ethanol bath or thermoregulator for freezing.

 2. Place the box with the tubes of sperm in -80ºC for at least 24 hours. Once completely frozen, we transfer the tubes of sperm from the Styrofoam box and into a regular tube rack or other holder for long term storage.

 

Using Frozen Sperm for In Vitro Fertilization

1. Harvest eggs from females into a dry Petri dish. Check the quality of the eggs under a microscope before thawing sperm. There is no point wasting sperm on poor quality eggs.

 2. Thaw frozen sperm in 25ºC water bath. Remove immediately when thawed (<30s).

 NOTE: We’ve tested thawing methods and found the water bath to work best. The key is to thaw quickly.

 3. Dilute sperm with 2000µl distilled water. Add the diluted sperm suspension to the eggs. Remember, the eggs are not in any liquid prior to adding the sperm suspension to them. This is important because the sperm should not be too dilute.

 NOTE: We’ve tested diluting the sperm with 1/9xMR or L15/CS, but find that distilled water works much better.

 4. Gently mix the sperm and eggs well to ensure exposure of sperm and eggs. A pipet tip works well.

 5. After 2 minutes, cover the eggs completely with distilled water. Fertilization should be readily seen as sperm entry points within 40 minutes and the first cell division should occur within 90 minutes.

 

Sperm entry point

Using the above protocol we’ve obtained confident fertilization rates of 10-15% resulting in 100-150 embryos.

 

Cryoprotectant

 

1. Disperse one egg yolk (about 15ml) in an equal volume of distilled water. We only want the egg yolk, not the egg white. Here is a video demonstration for an easy method of isolating the yolk.

Double Click to start Movie

 

2. Dilute the yolk to 20% with a solution containing 0.4 M sucrose, 10mM sodium bicarbonate, and 2mM pentoxyfylline.

3. Centrifuge the mixture for 20min at 13,000 rpm.

4. 1000µl aliquots of the supernatant are stored in Eppendorf tubes at -20ºC. We do not recommend using cryoprotectant that is more than a month or two old.

 

Freezing Methods
 
Three methods of freezing sperm were tested: Slow (Styrofoam box), Medium (dry ice/ethanol bath), and Fast (liquid nitrogen). The Styrofoam box is a common box used for shipping. The dry ice/ethanol bath is adopted from Sargent and Mohun. A small container with ethanol and a magnetic stir bar is placed inside a larger container filled with a dry ice/ethanol freezing mixture (see below). Tubes are placed on a rack inside the smaller container. The fast method is simply submerging the tubes of sperm into liquid nitrogen.

Styrofoam box Side View
Styrofoam box Side View
Styrofoam box inner view
Dry ice ethanol bath

We find that the slow method consistently works the best.

 

Note: Each letter corresponds to an individual frog. Fertilizations were done with >1000 eggs.

 

 

Thawing Methods
 
We compared thawing sperm in a 25ºC incubator versus a 25ºC water bath. We used the slow freeze (Styrofoam box) method and diluted with distilled water. Thawing in the incubator took over 3min, while the water bath was less than 30s. The incubator gave more variable times for thawing, while the water bath was more consistent.
 
With the incubator, we had fertilization rates of 3-5%, but had fertilization rates of 10-15%* with the water bath. Thawing with the water bath is clearly the method of choice.
 
* Note: Each letter corresponds to an individual frog. Fertilizations were done with >1000 eggs.

 

Dilution Methods
 
We tested distilled water, 1/9xMR, and L15/CS for diluting the sperm before fertilization. L15/CS consistently had 0% fertilization rates. While distilled water and 1/9xMR were similar, distilled water consistently had higher rates comparatively.
 
Note: Each letter corresponds to an individual frog. Fertilizations were done with >1000 eggs.

 

 

Contributed by Kendon Kuo, Angie M. Sera, and Timothy C. Grammer