2. When the freezing program is complete (e.g., reaching
À100
C or lower), plunge straws into LN 2 and store.
3. Straws can be thawed exactly as described above in “Method
DMA in Straws.” If glycerol is used as CPA, the glycerol must
be removed, or its concentration substantially lowered before
insemination. A method that is practical and simple, and can
yield apparently good results [22, 59], is dilution in extender
and reconcentration of the sperm cells by centrifugation.
Semen is progressively diluted in ASG-PE in a stepwise fashion
[42] with gentle agitation to a final dilution of minimally ten
times. The equilibration of glycerol (at 5
C) is probably very
fast, but one may use 2 min intervals between dilution steps.
The diluted semen is then centrifuged at 500 Â g at 4
C
[22]. The sperm pellet is then gently resuspended in the appropriate volume of ASG-PE.
4 Notes
1. Glycerol must be removed prior to insemination, for example,
by stepwise dilution, followed by centrifugation [22, 42].
2. This procedure (Table 3) of accommodating the CPA as well as
all solutes as used in the medium without CPA into the same
final volume as used in the medium without CPA is different
from adding CPA to a ready isotonic medium. If the latter
would be done, the molar concentration of non-permeating
solutes would be lowered by the addition of CPA, causing
swelling of the cells above their isotonic volume after equilibrating with the CPA.
3. The massage technique [60] is also applied (in adapted form)
to nondomestic species, but see refs. 15, 16 for species specific
recommendations.
4. Semen may be collected twice or three times per week. Training the males for collection and regular collection make
subsequent collections easier. More importantly, regular collection results in better semen quality.
5. Ejaculate volume and sperm concentration depend on breed
and animal, and different values may apply to other species than
chicken.
6. The ejaculate is quite viscous. Directly after collection, the
volume is roughly estimated, and an equal volume of ASG-PE
is added to wash down the semen and supply an initial predilution. Semen with inadequate volume or too watery or contaminated with blood or feces must be discarded.
7. For some purposes, pooled ejaculates may be collected from a
series of cocks. One method often employed for practical reasons is to place a few mL of extender in a collection tube and
Cryopreservation of Avian Semen
395
À100
C or lower), plunge straws into LN 2 and store.
3. Straws can be thawed exactly as described above in “Method
DMA in Straws.” If glycerol is used as CPA, the glycerol must
be removed, or its concentration substantially lowered before
insemination. A method that is practical and simple, and can
yield apparently good results [22, 59], is dilution in extender
and reconcentration of the sperm cells by centrifugation.
Semen is progressively diluted in ASG-PE in a stepwise fashion
[42] with gentle agitation to a final dilution of minimally ten
times. The equilibration of glycerol (at 5
C) is probably very
fast, but one may use 2 min intervals between dilution steps.
The diluted semen is then centrifuged at 500 Â g at 4
C
[22]. The sperm pellet is then gently resuspended in the appropriate volume of ASG-PE.
4 Notes
1. Glycerol must be removed prior to insemination, for example,
by stepwise dilution, followed by centrifugation [22, 42].
2. This procedure (Table 3) of accommodating the CPA as well as
all solutes as used in the medium without CPA into the same
final volume as used in the medium without CPA is different
from adding CPA to a ready isotonic medium. If the latter
would be done, the molar concentration of non-permeating
solutes would be lowered by the addition of CPA, causing
swelling of the cells above their isotonic volume after equilibrating with the CPA.
3. The massage technique [60] is also applied (in adapted form)
to nondomestic species, but see refs. 15, 16 for species specific
recommendations.
4. Semen may be collected twice or three times per week. Training the males for collection and regular collection make
subsequent collections easier. More importantly, regular collection results in better semen quality.
5. Ejaculate volume and sperm concentration depend on breed
and animal, and different values may apply to other species than
chicken.
6. The ejaculate is quite viscous. Directly after collection, the
volume is roughly estimated, and an equal volume of ASG-PE
is added to wash down the semen and supply an initial predilution. Semen with inadequate volume or too watery or contaminated with blood or feces must be discarded.
7. For some purposes, pooled ejaculates may be collected from a
series of cocks. One method often employed for practical reasons is to place a few mL of extender in a collection tube and
Cryopreservation of Avian Semen
395