Sperm CryoProtec II Instructions for use
Recommendations
Although it is possible to freeze unprocessed semen, we recommend that the ejaculate be prepared on a PureSperm® density gradient and washed with PureSperm®Wash before adding Sperm CryoProtec™ II. This method removes seminal plasma as well as ROS and their sources, thereby ensuring optimal recovery of motile sperm on thawing.
1a) Gradient-prepared sperm
1. Resuspend sperm pellet in a small volume of PureSperm®Wash to obtain desired con
centration of sperm
2. Add 1/4 of Sperm CryoProtec™II dropwise, ensuring thorough mixing after adding each
drop (e.g 0.100mL SCP II to 0.300mL sperm sample)
3. Fill straws with sperm suspension or aliquot into vials
4. Equilibrate straws in refrigerator for 1 hr
5. Place the straws horizontally in nitrogen vapour (1cm above the liquid nitrogen surface
(on a piece of Styrofoam)). Leave for 30 minutes.
6. Transfer the straws quickly into the liquid nitrogen and store in liquid nitrogen
1b) Unprocessed ejaculate
2. Add 1/4 of Sperm CryoProtec™II dropwise, ensuring thorough mixing after adding each
drop (e.g 0.100mL SCP II to 0.300mL semen)
3-6. Continue as for gradient-prepared sperm
2) Thawing of sperm
1. Remove straw from liquid nitrogen tank
2. Place straw in water at 37°C for 30 secs
3. Dry surface of straw
4. Cut one end of straw
5. Resuspend contents in 5mL PureSperm®Wash by cutting the upper end of straw. Any
sperm suspension remaining in straw can be expelled using a pipette
6. Centrifuge at 500 x g for 10 minutes. Do not use the brake
7. Aspirate PureSperm®Wash supernatant leaving as much liquid as required for desired
concentration. If no pellet is seen, leave the bottom 0.10mL fluid
Sperm CryoProtec II Procedure
|
Sperm CryoProtec™II dilution table
| Sperm Volume (mL) |
Medium (mL) |
|
Sperm Volume (mL) |
Medium (mL) |
|
Sperm Volume (mL) |
Medium (mL) |
|
Sperm Volume (mL) |
Medium (mL) |
| 0,1 |
0,03 |
|
1,6 |
0,53 |
|
3,1 |
1,03 |
|
4,6 |
1,53 |
| 0,2 |
0,07 |
|
1,7 |
0,57 |
|
3,2 |
1,07 |
|
4,7 |
1,57 |
| 0,3 |
0,10 |
|
1,8 |
0,60 |
|
3,3 |
1,10 |
|
4,8 |
1,60 |
| 0,4 |
0,13 |
|
1,9 |
0,63 |
|
3,4 |
1,13 |
|
4,9 |
1,63 |
| 0,5 |
0,17 |
|
2,0 |
0,67 |
|
3,5 |
1,17 |
|
5,0 |
1,67 |
| 0,6 |
0,20 |
|
2,1 |
0,70 |
|
3,6 |
1,20 |
|
5,1 |
1,70 |
| 0,7 |
0,23 |
|
2,2 |
0,73 |
|
3,7 |
1,23 |
|
5,2 |
1,73 |
| 0,8 |
0,27 |
|
2,3 |
0,77 |
|
3,8 |
1,27 |
|
5,3 |
1,77 |
| 0,9 |
0,30 |
|
2,4 |
0,80 |
|
3,9 |
1,30 |
|
5,4 |
1,80 |
| 1,0 |
0,33 |
|
2,5 |
0,83 |
|
4,0 |
1,33 |
|
5,5 |
1,83 |
| 1,1 |
0,37 |
|
2,6 |
0,87 |
|
4,1 |
1,37 |
|
5,6 |
1,87 |
| 1,2 |
0,40 |
|
2,7 |
0,90 |
|
4,2 |
1,40 |
|
5,7 |
1,90 |
| 1,3 |
0,43 |
|
2,8 |
0,93 |
|
4,3 |
1,43 |
|
5,8 |
1,93 |
| 1,4 |
0,47 |
|
2,9 |
0,97 |
|
4,4 |
1,47 |
|
5,9 |
1,97 |
| 1,5 |
0,50 |
|
3,0 |
1,00 |
|
4,5 |
1,50 |
|
6,0 |
2,00 |
|
