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Sperm freezing protocol using gametes harvested
from epididymides after transportation at
refrigerated temperatures (4-8°C)
This protocol is similar with the mouse sperm cryopreservation protocol presented on
Infrafrontier website. The only major change is the equilibration time of sperm in gCPA during
cryopreservation, which is extended from 3min to 15min. This ensures more sperm is collected
from the epididymides prior to freezing. This protocol is based on the work published in Takeo
et al., (2012 & 2014)
1.
Media and solutions
1.1. L-glutamine supplemented cryoprotective agent (gCPA) made up using 18% D (+)
raffinose and 3% skim milk and 100mM L-glutamine
1.2. D (+) raffinose (Sigma: R7630)
1.3. Skim milk (BD Diagnostics: 232100)
1.4. L-Glutamine (Sigma: G8540)
1.5. Ultrapure H2O (e.g., Sigma embryo tested water)
1.6. Modified HTF medium (300-310mOsm) (see reagents and solutions)
1.7. TYH+MBCD sperm pre-incubation medium (285-295mOsm) (see reagents and
solutions).
1.8. 70% ethanol
1.9. Mineral oil (Sigma: M8410)
2. Animals
Male
Male mice over 12 weeks of age
3. Equipment
3.1. Osmometer (e.g., Research instruments)
3.2. Hotplate (37°C)
3.3. Analytical balance
3.4. A supply of LN2
3.5.
Small LN2 dewar
3.6. Large LN2 dewar (e.g., Planer: MVE-XC 47-11-6)
3.7. Oxygen monitors (e.g., Drager Pac 3500
3.8. Water bath (60°C)
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3.9. Paper tissue
3.10. 1.0ml and 10ml syringes
3.11. 0.22µl syringe end filters (e.g., Millipore)
3.12. 16g metal rod
3.13. Indelible marker pen or cryo-fast labels (e.g., Brady labels)
3.14. 0.25ml plastic semen straws (Planer: FZA201)
3.15. 35mm culture dishes (Falcon: 351008)
3.16. 200µl and 1.0ml pipette, plus tips (e.g., Gilson)
3.17. Wedge-shaped 10µl pipette tip (e.g., Thistle Scientific: AX-T-400-L-R-S)
3.18. Dissecting microscope (10x-100x magnification)
3.19. Dissecting instruments e.g. standard dissecting forceps, fine watchmakers forceps and
scissors
3.20. Laboratory timers
3.21. Floating sperm freezing device (See Fig. 2b)
4. Sperm collection and freezing
4.1. For each male to be frozen label ten 250μl plastic semen straws, 1 x Kleenex tissue and 1
x 35mm Falcon 351008 petri dish with the appropriate sample code. Using a permanent
marker pen, mark each straw at a distance approximately 4.5cm and 2.3cm (Figure 1)
from the end furthest from the cotton/PVA plug. Label the straws with the appropriate
sample code near the end of the cotton/PVA plug.
4.5cm
cotton plug
2.3cm
Figure 1
4.2. Prepare the cooling chamber e.g. a 47 litre LN2 Dewar (Figure 2a) filled with LN2 to a
depth of 25cm. Also, prepare the floating sperm freezing apparatus. This can be made
from a 50ml syringe attached to a perspex rod. It is important to seal the needle hub
(Figure 2b)
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Figure 2a Cooling chamber. Depth of LN2 (H2) = 25 cm.
Acrylic bar
50cm
50ml syringe
Styrofoam
(30mm thick)
Heat sealed
Figure 2b Floating sperm freezing apparatus
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4.3. Place a 60µl aliquot of the sperm cryoprotective agent (gCPA containing 100mM Lglutamine - described in Appendix A) into the base of a 35 mm culture dish and cover
with mineral oil.
4.4. A second 60µl drop of gCPA should be added into the drop (final volume: 120µl) to make
a tall, semi-spherical drop (Figure 3). Prepare a separate dish for each male to be sperm
frozen.
NOTE: The optimal volume of gCPA is 120µl per two epididymides. If you need to freeze
down one epididymis, use a 60µl drop.
gCPA
Figure 3
4.5. On arrival the recipient should remove the aluminium lined box from the polystyrene
container, open the aluminium box and find the biotube. Then retrieved the microfuge
tube, which containing the epididymides from the biotube.
4.6. The cauda epididymides should be wiped free of any Lifor solution using paper tissue.
4.7. Wash each cauda epididymis through 4 x drops of gCPA (Figure 4). Steps 4.6 & 4.7 are
very important, if you skip these two steps sperm motility may be compromised.
NOTE: To make a wash dish, put 4 drops (80ul) IVF media into a 35mm petri dish
(Falcon 351008). There is no need to overly with mineral oil.
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Figure 4
4.8. Clean off all adipose and vascular tissue. This is best achieved by placing the organs on a
paper tissue and examining them under a dissecting microscope lit from above.
4.9. Transfer the cauda epididymides into a 120µl drop of gCPA.
4.10. Make six to seven cuts across the cauda epididymides using a pair of fine scissors or a
similar sharp bladed tool and place the dish on a 37°C hot plate (Figure 5). Keep the
dishes on 37°C hot plate for 15min avoiding the light from microscope. During the last 3
minutes, gently swirls the dish every minute to help disperse the sperm.
Cauda epididymides
120µl gCPA
Mineral oil
Figure 5
4.11. While the sperm is equilibrating, prepare the straws (250μl plastic semen straws,
Planer; FZA201) for freezing, as follows:
1. Attach a 1ml syringe to the labelled end of the straw. Using the syringe, aspirate HTF
solution until the meniscus reaches 4.5cm marker.
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2. Then aspirate 2.3cm air and lay the syringe and straw assembly on the bench until
required.
4.12. After 15 minutes, the sperm suspension should be divided into 10 x 10µl aliquots on a
culture dish (Figure 6). Then aspirate one drop into each of the 10 straws.
10 x 10µl aliquots of sperm
Figure 6
4.13. Aspirate air until the HTF meniscus reaches the polyvinyl alcohol section, half way along
the cotton plug. This will seal the labelled end of the straw.
4.14. Seal both ends of the straw (Figure 7) using a double impulse heat sealer or similar
device.
heat sealed
aliquots of sperm
HTF
cotton plug
10ul
air
heat sealed
air
Figure 7
4.15. Load the sealed straws into a floating sperm freezing device and then rest the device on
the surface of the LN2 in the pre-prepared cooling chamber (Figure 8) for 10 minutes.
After 10 minutes plunge the straws directly into liquid nitrogen.
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H1= 28cm
LN2
H2 = 25cm
Figure 8
4.16. Whilst minimising their exposure to air, transfer the straws into their long term storage
locations.
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