submersed in the 5
C water while being moved vigorously
through the water during 30 s. This is to maximize warming
rate and prevent growth of an insulating sheath of ice outside
the straw. Despite the low temperature of the water bath, the
thawing rate is still high enough (average thawing rate between
Æ À190 and + 5
C is 500–600
C/min). Do not thaw bundles
of straws as this will reduce the thawing rate.
10. For insemination, the semen can be used as is. The semen does
not need to be warmed first. For post-thaw semen assessment,
the thawed semen must be extended with ASG-PE of 5
C to
the appropriate low concentrations needed for the chosen
sperm assessment method. It is advised to do the initial postthaw dilutions in a stepwise fashion.
3.4.2 Method DMA
in Pellets
Semen is prepared exactly as described in the “Method DMA in
Straws” above. Tselutin [39] describes that the semen is frozen
1 min after adding DMA, although longer exposure to DMA
prior to freezing does not appear to be harmful (Woelders et al.,
in preparation).
1. Prepare a dewar with LN 2 .
2. A “blue” tip of a 1-mL pipette is cut off just a bit such that
droplets formed are approximately 0.5 mL.
3. Then the semen is pipetted with this tip, and droplets are
dropped onto the surface of the LN 2 . It is best to wait for the
droplet to be cooled (20 s) and then sinks, or else droplets will
collate together which will affect cooling and thawing rates.
4. A sort of “tea sieve”-like insert may be convenient for easy
collection of the pellets for storage.
5. The pellets can be conveniently thawed using a custom-built
apparatus as shown in [39]. The pellets are thawed by contact
to a warm (60
C) surface without the risk of overheating the
semen, as the semen drips down as soon as the pellet melts.
6. The collected semen can be maintained at 5
C.
3.4.3 Method Glycerol
in Straws
1. Semen is prepared as described in the “Method DMA in
Straws” above, using either ASG-PE or Lake’s “Solution 1”
and using glycerol as CPA. Glycerol is not suitable for very high
cooling rates as obtained in the pellet method. Also, the relatively high CPA concentration of 1.2 M allows freezing at lower
cooling rates. Cooling rates between 4 and 50
C/min seem
appropriate. The ´lie and coworkers [22] used 7
C/min with
good post-thaw fertility results. That means that straws can be
frozen in a controlled rate freezer. Alternatively, straws can be
frozen in static nitrogen vapor, as described above in “Method
DMA in Straws,” but using Styrofoam floating racks of 5-cm
thickness.
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Henri Woelders
C water while being moved vigorously
through the water during 30 s. This is to maximize warming
rate and prevent growth of an insulating sheath of ice outside
the straw. Despite the low temperature of the water bath, the
thawing rate is still high enough (average thawing rate between
Æ À190 and + 5
C is 500–600
C/min). Do not thaw bundles
of straws as this will reduce the thawing rate.
10. For insemination, the semen can be used as is. The semen does
not need to be warmed first. For post-thaw semen assessment,
the thawed semen must be extended with ASG-PE of 5
C to
the appropriate low concentrations needed for the chosen
sperm assessment method. It is advised to do the initial postthaw dilutions in a stepwise fashion.
3.4.2 Method DMA
in Pellets
Semen is prepared exactly as described in the “Method DMA in
Straws” above. Tselutin [39] describes that the semen is frozen
1 min after adding DMA, although longer exposure to DMA
prior to freezing does not appear to be harmful (Woelders et al.,
in preparation).
1. Prepare a dewar with LN 2 .
2. A “blue” tip of a 1-mL pipette is cut off just a bit such that
droplets formed are approximately 0.5 mL.
3. Then the semen is pipetted with this tip, and droplets are
dropped onto the surface of the LN 2 . It is best to wait for the
droplet to be cooled (20 s) and then sinks, or else droplets will
collate together which will affect cooling and thawing rates.
4. A sort of “tea sieve”-like insert may be convenient for easy
collection of the pellets for storage.
5. The pellets can be conveniently thawed using a custom-built
apparatus as shown in [39]. The pellets are thawed by contact
to a warm (60
C) surface without the risk of overheating the
semen, as the semen drips down as soon as the pellet melts.
6. The collected semen can be maintained at 5
C.
3.4.3 Method Glycerol
in Straws
1. Semen is prepared as described in the “Method DMA in
Straws” above, using either ASG-PE or Lake’s “Solution 1”
and using glycerol as CPA. Glycerol is not suitable for very high
cooling rates as obtained in the pellet method. Also, the relatively high CPA concentration of 1.2 M allows freezing at lower
cooling rates. Cooling rates between 4 and 50
C/min seem
appropriate. The ´lie and coworkers [22] used 7
C/min with
good post-thaw fertility results. That means that straws can be
frozen in a controlled rate freezer. Alternatively, straws can be
frozen in static nitrogen vapor, as described above in “Method
DMA in Straws,” but using Styrofoam floating racks of 5-cm
thickness.
394
Henri Woelders