of semen. Final concentrations will thus be 1200 million sperm
cells/mL and 0.6 mol/L DMA.
3. The semen may be held in the DMA medium at 5
C, if
wanted, for any time up to 1 h. Straws are filled and sealed.
Make sure that the straw contains a 10-mm-long air pocket.
This is especially important at the relatively high cooling rates
employed.
4. Sealing can be done by pressing the (wet) open end of the straw
on polyvinyl alcohol (PVA) powder, which then forms a plug.
But it is to be preferred to make a heat seal instead, e.g., with a
straw filling machine.
5. The straws are then frozen. Use a freezing rate of 200
C/min.
This is the rate as measured inside the straw after the dissipation
of heat of fusion, e.g., between À10 and À60
C. Programmable freezers are not intended for such high cooling rates. One
may use a programmable freezer, but set at a constant temperature of, for example, À160
C (see Note 13). Straws are placed
on a steel rack for freezing 0.25-mL straws (IMV, L’Aigle,
France). Depending on the ventilation rate and the strength
of the jets of injected LN 2 , it may be necessary in these programmable freezers to restrain the straws, which can be conveniently done with a rubber band stretching along the length of
the rack. The rack with straws is then placed inside the precooled freeing cabinet. After 2 min, the straws can be removed
from the cabinet and be plunged in LN 2 .
6. CGN is using custom-built ventilated nitrogen vapor freezers,
set at a constant vapor temperature of À140
C (Fig. 3). This
works similarly as the freezing cabinet described above, except
that these machines allow very easy access to place the racks and
for removing the straws and do not have the risk of blowing
away the straws from the racks. Figure 3 shows two images of
this apparatus.
7. A simple low-cost way is to place the straws on a Styrofoam
“floating rack,” as described above, with a thickness of
12.5 mm. A large (approximately 30 Â 30 cm) Styrofoam
box filled with a layer of LN 2 is prepared. The rack with straws
is then placed floating on the surface of the LN 2 . This will
provide the desired cooling rate. After 1 min, the straws can
be plunged in LN 2 .
8. A last alternative is to use a programmable freezer and freeze
with a constant rate of 50
C/min (¼ maximum rate of most
programmable freezers).
9. Straws are thawed in a 5
C water bath, and the thawed semen
is best maintained at 5
C until use for semen assessment or
insemination as it will deteriorate faster at elevated temperatures (see Note 14). Straws are taken from liquid nitrogen and
Cryopreservation of Avian Semen
393
cells/mL and 0.6 mol/L DMA.
3. The semen may be held in the DMA medium at 5
C, if
wanted, for any time up to 1 h. Straws are filled and sealed.
Make sure that the straw contains a 10-mm-long air pocket.
This is especially important at the relatively high cooling rates
employed.
4. Sealing can be done by pressing the (wet) open end of the straw
on polyvinyl alcohol (PVA) powder, which then forms a plug.
But it is to be preferred to make a heat seal instead, e.g., with a
straw filling machine.
5. The straws are then frozen. Use a freezing rate of 200
C/min.
This is the rate as measured inside the straw after the dissipation
of heat of fusion, e.g., between À10 and À60
C. Programmable freezers are not intended for such high cooling rates. One
may use a programmable freezer, but set at a constant temperature of, for example, À160
C (see Note 13). Straws are placed
on a steel rack for freezing 0.25-mL straws (IMV, L’Aigle,
France). Depending on the ventilation rate and the strength
of the jets of injected LN 2 , it may be necessary in these programmable freezers to restrain the straws, which can be conveniently done with a rubber band stretching along the length of
the rack. The rack with straws is then placed inside the precooled freeing cabinet. After 2 min, the straws can be removed
from the cabinet and be plunged in LN 2 .
6. CGN is using custom-built ventilated nitrogen vapor freezers,
set at a constant vapor temperature of À140
C (Fig. 3). This
works similarly as the freezing cabinet described above, except
that these machines allow very easy access to place the racks and
for removing the straws and do not have the risk of blowing
away the straws from the racks. Figure 3 shows two images of
this apparatus.
7. A simple low-cost way is to place the straws on a Styrofoam
“floating rack,” as described above, with a thickness of
12.5 mm. A large (approximately 30 Â 30 cm) Styrofoam
box filled with a layer of LN 2 is prepared. The rack with straws
is then placed floating on the surface of the LN 2 . This will
provide the desired cooling rate. After 1 min, the straws can
be plunged in LN 2 .
8. A last alternative is to use a programmable freezer and freeze
with a constant rate of 50
C/min (¼ maximum rate of most
programmable freezers).
9. Straws are thawed in a 5
C water bath, and the thawed semen
is best maintained at 5
C until use for semen assessment or
insemination as it will deteriorate faster at elevated temperatures (see Note 14). Straws are taken from liquid nitrogen and
Cryopreservation of Avian Semen
393