3 Methods
3.1 Freeze-Drying
Complete species-specific steps of sperm collection. The following
includes preparation of murine sperm for freeze-drying (see Note 3
for other species).
1. Aseptically dissect two cauda epididymides, and place them in
an empty sterile culture dish.
2. Gently puncture the cauda epididymides with a 30-G needle
under a dissecting microscope, and transfer them to the bottom of a 1.5 mL microcentrifuge tube containing 1 mL freezedrying solution (see Note 4).
3. Incubate the microcentrifuge tube at 37
C for 10 min to
disperse the sperm.
4. Carefully aspirate the top 0.8 mL sperm suspension, and distribute into sterile glass ampules as 100 μL aliquots.
5. Turn on your freeze-dryer, and make it ready for the freezedrying process depending on specifications of your freezedryer.
6. Partially immerse glass ampules containing 100 μL of sperm
suspension into liquid nitrogen for 30–60 s to freeze the sperm
samples.
7. Quickly connect each ampule to your manifold freeze-dryer.
8. Freeze-dry the sperm samples for 4 h under vacuum pressure at
around 0.03 mBar (0.003 kPa).
9. Flame seal each ampule using a gas torch under vacuum
(0.03 mBar) after freeze-drying.
10. Store the sealed ampules at 4
C in the dark to avoid any
potential light-induced damage (see Note 5).
3.2 Rehydration
and Preparation
of Freeze-Dried Sperm
for ICSI
1. Open an ampule, preferentially under a laminar flow hood, and
rehydrate the sperm by adding 100 μL of sterile ultrapure
water.
2. Aspirate 5 μL sperm suspension, and mix it with a small drop
(20–30 μL) of a HEPES-buffered medium (e.g., HEPESCZB) containing 12% (w/v) polyvinylpyrrolidone (PVP; M r
360,000).
3. For ICSI, select only sperm displaying normal morphology (see
Note 6).
Freeze-Drying of Mammalian Sperm
727
3.1 Freeze-Drying
Complete species-specific steps of sperm collection. The following
includes preparation of murine sperm for freeze-drying (see Note 3
for other species).
1. Aseptically dissect two cauda epididymides, and place them in
an empty sterile culture dish.
2. Gently puncture the cauda epididymides with a 30-G needle
under a dissecting microscope, and transfer them to the bottom of a 1.5 mL microcentrifuge tube containing 1 mL freezedrying solution (see Note 4).
3. Incubate the microcentrifuge tube at 37
C for 10 min to
disperse the sperm.
4. Carefully aspirate the top 0.8 mL sperm suspension, and distribute into sterile glass ampules as 100 μL aliquots.
5. Turn on your freeze-dryer, and make it ready for the freezedrying process depending on specifications of your freezedryer.
6. Partially immerse glass ampules containing 100 μL of sperm
suspension into liquid nitrogen for 30–60 s to freeze the sperm
samples.
7. Quickly connect each ampule to your manifold freeze-dryer.
8. Freeze-dry the sperm samples for 4 h under vacuum pressure at
around 0.03 mBar (0.003 kPa).
9. Flame seal each ampule using a gas torch under vacuum
(0.03 mBar) after freeze-drying.
10. Store the sealed ampules at 4
C in the dark to avoid any
potential light-induced damage (see Note 5).
3.2 Rehydration
and Preparation
of Freeze-Dried Sperm
for ICSI
1. Open an ampule, preferentially under a laminar flow hood, and
rehydrate the sperm by adding 100 μL of sterile ultrapure
water.
2. Aspirate 5 μL sperm suspension, and mix it with a small drop
(20–30 μL) of a HEPES-buffered medium (e.g., HEPESCZB) containing 12% (w/v) polyvinylpyrrolidone (PVP; M r
360,000).
3. For ICSI, select only sperm displaying normal morphology (see
Note 6).
Freeze-Drying of Mammalian Sperm
727