2. Gently wipe off the straw ends to get rid of any excess liquid.
3. Seal the end of the straw using the heat sealer.
4. Open the Styrofoam box containing the tube rack and LN 2 ,
place the straws on the metal tube rack, and close the Styrofoam box lid (see Fig. 1).
5. Cool sperm for 5 min in LN 2 vapor (~160
C), and following
cooling, plunge the French straws directly into the LN 2 in the
Styrofoam box.
6. Place the cryo-sleeves in 2 L capacity LN 2 dewar for at least
1 min before placing the canes with cryo-goblets containing
the French straws containing the frozen sperm.
7. Place the cryo-canes into a designated LN 2 dewar for longterm storage.
3.7 Thawing
Procedure
1. To measure the post-thaw motility characteristics, remove the
designated straw from LN 2 , place the straw in a 37
C water
bath, and hold until the ice crystals disappear (~10 s). Dry the
outside of the straw using a Kimwipe.
2. Expel the French straw contents into 1 mL of FHM/BSA in a
microcentrifuge tube by first cutting the end opposite the label
first, angling the straw over the tube, then cutting right below
the filter, and expelling the remaining contents of the straw
using a syringe (see Fig. 5a).
3. Centrifuge for 5 min at 300 Â g, gently discard the supernatant
(see Fig. 5b), and resuspend the pellet by gently adding 90 μL
FHM/BSA at about 45
angle.
4. Close the cap, and allow the sperm to swim in a bath for 30 min
before measuring the post-thaw motility characteristics (see
Fig. 5c) (see Note 7).
4 Notes
1. If any precipitates form in the solution, discard the solution and
start over. Use glassware that has been rinsed at least ten times
with Milli-Q water, which has been properly dried.
2. Solubilize the CaCl 2 .2H 2 O separately from the other reagents
before combining the solutions.
Fig. 4 Pre-labeled 0.25 cc French straw connected to 1 cc syringes to load the
sperm suspension in freezing solution
410
Yuksel Agca and Cansu Agca
3. Seal the end of the straw using the heat sealer.
4. Open the Styrofoam box containing the tube rack and LN 2 ,
place the straws on the metal tube rack, and close the Styrofoam box lid (see Fig. 1).
5. Cool sperm for 5 min in LN 2 vapor (~160
C), and following
cooling, plunge the French straws directly into the LN 2 in the
Styrofoam box.
6. Place the cryo-sleeves in 2 L capacity LN 2 dewar for at least
1 min before placing the canes with cryo-goblets containing
the French straws containing the frozen sperm.
7. Place the cryo-canes into a designated LN 2 dewar for longterm storage.
3.7 Thawing
Procedure
1. To measure the post-thaw motility characteristics, remove the
designated straw from LN 2 , place the straw in a 37
C water
bath, and hold until the ice crystals disappear (~10 s). Dry the
outside of the straw using a Kimwipe.
2. Expel the French straw contents into 1 mL of FHM/BSA in a
microcentrifuge tube by first cutting the end opposite the label
first, angling the straw over the tube, then cutting right below
the filter, and expelling the remaining contents of the straw
using a syringe (see Fig. 5a).
3. Centrifuge for 5 min at 300 Â g, gently discard the supernatant
(see Fig. 5b), and resuspend the pellet by gently adding 90 μL
FHM/BSA at about 45
angle.
4. Close the cap, and allow the sperm to swim in a bath for 30 min
before measuring the post-thaw motility characteristics (see
Fig. 5c) (see Note 7).
4 Notes
1. If any precipitates form in the solution, discard the solution and
start over. Use glassware that has been rinsed at least ten times
with Milli-Q water, which has been properly dried.
2. Solubilize the CaCl 2 .2H 2 O separately from the other reagents
before combining the solutions.
Fig. 4 Pre-labeled 0.25 cc French straw connected to 1 cc syringes to load the
sperm suspension in freezing solution
410
Yuksel Agca and Cansu Agca