dilution rates (e.g., 10Â diluted), providing excellent longevity of
cold-stored poultry semen (Woelders, in preparation). We have
successfully applied the ASG poultry extender also as fresh extender
and as freezing medium base for semen of cranes, tragopan
pheasants, ducks, and geese, yielding better fresh longevity and
better post-thaw motility compared with commercial Avidiluent
extender (IMV Technologies, L’Aigle, France) (Woelders and
Zuidberg, unpublished). Therefore, the ASG extender (modified
L&R 7.1) is our preferred medium for avian semen.
1.4 Cryoprotective
Agents
Glycerol is seen as a good CPA for poultry semen, but it is contraceptive if present together with the semen in the hen. While the
mechanism of the contraceptive effect isn’t fully elucidated,
research with in vitro tissue cultures indicated that glycerol and
tissues of the hen genital tract have an interaction that negatively
affects sperm viability [38]. The contraceptive effect of glycerol is
not seen in mammalian species. Methods have been devised to
remove glycerol from poultry semen. Also, other CPAs that do
not appear to have the contraceptive effect have been tried as
alternatives to glycerol. DMA (0.6–0.7 M) and glycerol
(1.1–1.2 M) both appear effective CPAs, as high post-thaw fertility
rates of Æ90% have been reported for DMA [20, 39–41] and for
glycerol [19, 22, 42]. However, Blesbois and coworkers reported
glycerol to be the more effective than DMA with low fertility lines
[24]. Also with DMSO (0.6 M), fertility around 90% was
reported [43].
As pointed out by Donoghue and Wishart [44], there are many
confounding factors affecting comparisons of CPAs between or
within studies, such as the use of different CPA concentrations,
freezing rates, freezing methods, and “base” diluents. A rationale
for testing CPAs at different cooling rates may be that they may
have different optimal cooling rates when compared under otherwise identical conditions. For instance, in split sample comparison
of glycerol and DMA at equal concentrations and equal cooling
rates, we observed similar post-thaw sperm survival in poultry
semen frozen at cooling rates between 4 and 200
C/min, but
glycerol was not effective at cooling rates above 200
C/min, at
which DMA was more effective than glycerol [21]. Also, comparisons of CPAs may be made at different CPA concentrations, as the
apparent tolerated concentrations during freezing and thawing may
differ for different CPAs. Toxicity of the CPAs tested in semen
without freezing/thawing may differ from the toxicity
(or effectiveness) of these compounds during freezing and thawing.
For instance, Lake and Ravie [27] showed that fertility rate of fresh
semen with DMA or propanediol (PD) was close to control
(no CPA), whereas fertility of frozen semen was much lower
when using PD compared with either DMA or glycerol. Comparing fertility of semen frozen with glycerol or with alternative CPAs,
Cryopreservation of Avian Semen
383
cold-stored poultry semen (Woelders, in preparation). We have
successfully applied the ASG poultry extender also as fresh extender
and as freezing medium base for semen of cranes, tragopan
pheasants, ducks, and geese, yielding better fresh longevity and
better post-thaw motility compared with commercial Avidiluent
extender (IMV Technologies, L’Aigle, France) (Woelders and
Zuidberg, unpublished). Therefore, the ASG extender (modified
L&R 7.1) is our preferred medium for avian semen.
1.4 Cryoprotective
Agents
Glycerol is seen as a good CPA for poultry semen, but it is contraceptive if present together with the semen in the hen. While the
mechanism of the contraceptive effect isn’t fully elucidated,
research with in vitro tissue cultures indicated that glycerol and
tissues of the hen genital tract have an interaction that negatively
affects sperm viability [38]. The contraceptive effect of glycerol is
not seen in mammalian species. Methods have been devised to
remove glycerol from poultry semen. Also, other CPAs that do
not appear to have the contraceptive effect have been tried as
alternatives to glycerol. DMA (0.6–0.7 M) and glycerol
(1.1–1.2 M) both appear effective CPAs, as high post-thaw fertility
rates of Æ90% have been reported for DMA [20, 39–41] and for
glycerol [19, 22, 42]. However, Blesbois and coworkers reported
glycerol to be the more effective than DMA with low fertility lines
[24]. Also with DMSO (0.6 M), fertility around 90% was
reported [43].
As pointed out by Donoghue and Wishart [44], there are many
confounding factors affecting comparisons of CPAs between or
within studies, such as the use of different CPA concentrations,
freezing rates, freezing methods, and “base” diluents. A rationale
for testing CPAs at different cooling rates may be that they may
have different optimal cooling rates when compared under otherwise identical conditions. For instance, in split sample comparison
of glycerol and DMA at equal concentrations and equal cooling
rates, we observed similar post-thaw sperm survival in poultry
semen frozen at cooling rates between 4 and 200
C/min, but
glycerol was not effective at cooling rates above 200
C/min, at
which DMA was more effective than glycerol [21]. Also, comparisons of CPAs may be made at different CPA concentrations, as the
apparent tolerated concentrations during freezing and thawing may
differ for different CPAs. Toxicity of the CPAs tested in semen
without freezing/thawing may differ from the toxicity
(or effectiveness) of these compounds during freezing and thawing.
For instance, Lake and Ravie [27] showed that fertility rate of fresh
semen with DMA or propanediol (PD) was close to control
(no CPA), whereas fertility of frozen semen was much lower
when using PD compared with either DMA or glycerol. Comparing fertility of semen frozen with glycerol or with alternative CPAs,
Cryopreservation of Avian Semen
383