Chalah and coworkers [40] obtained fertility with semen frozen
with DMA similar to that obtained with fresh semen, while fertility
with glycerol and dimethylformamide (DMF) was a bit lower. Also,
Tselutin and coworkers [20] reported higher fertility with DMA
than with glycerol, although this depended on the method (cooling
rate) used. Semen frozen by plunging droplets of semen with DMA
in LN 2 (pellet method) resulted in very good fertility, but not
semen frozen conventionally in straws at much lower cooling
rates [20, 22]. Woelders and coworkers [21] showed that very
good fertility can be equally obtained using DMA as CPA with
either pellet and straw freezing, provided that also the straws are
frozen at a very high cooling rate (200
C/min). The ´lie and coworkers [22] found better fertility with glycerol than with DMA but
using DMA in a straw method (i.e., different from the pellet
method in [20, 40]) and using a lower cooling rate than that used
in [21].
All in all, good fertility of Æ90% can apparently be obtained
with frozen-thawed poultry semen using either DMA or glycerol as
cryoprotectant. However, when using glycerol, the semen cannot
be inseminated right from the straw. Instead, extra steps are needed
after thawing to remove glycerol prior to insemination, which
implies extra labor and costs. Methods to remove glycerol include
dialysis [2, 45], stepwise dilution, followed by centrifugation
[22, 42], and discontinuous density gradient centrifugation [46].
1.5 Other Stabilizing
Additives
For semen of mammalian species, the presence in the freezing
medium of milk components or egg yolk is beneficial, if not essential, for post-thaw sperm survival and fertility. However, it seems
there is little if any evidence for positive effects of yolk or milk
components for avian semen. Instead, chicken egg yolk reduces
fertility of chicken semen, but not of turkey and red jungle fowl
[47–49]. Low-density lipoprotein extracted from egg yolk [50],
egg yolk plasma, and soybean lecithin [51] were found beneficial
for freezing poultry semen.
The use of sugars and other non-permeating neutral (nonelectrolyte) solutes in (cryo)preservation media results in a lower ionic
strength, which may be beneficial [37, 41], especially as salts and
other solutes become strongly concentrated in the “unfrozen fraction” during freezing. Very high ionic strengths affect lipid membranes and (membrane) proteins and may lead to “salt loading” of
the cells. Sugars and other neutral solutes are sometimes referred to
as “non-permeating cryoprotectants.” However, in media with
normal (physiological) osmotic strength, the ratio of electrolytes/
nonelectrolytes will not influence freezing point depression, or the
degree of shrinking of the unfrozen fraction and of the cells therein
during ice formation, nor can it affect intracellular cryobiological
properties. High osmolalities (be it by sugars or any other solutes)
can work to reduce the chance of intracellular ice formation and
384
Henri Woelders
with DMA similar to that obtained with fresh semen, while fertility
with glycerol and dimethylformamide (DMF) was a bit lower. Also,
Tselutin and coworkers [20] reported higher fertility with DMA
than with glycerol, although this depended on the method (cooling
rate) used. Semen frozen by plunging droplets of semen with DMA
in LN 2 (pellet method) resulted in very good fertility, but not
semen frozen conventionally in straws at much lower cooling
rates [20, 22]. Woelders and coworkers [21] showed that very
good fertility can be equally obtained using DMA as CPA with
either pellet and straw freezing, provided that also the straws are
frozen at a very high cooling rate (200
C/min). The ´lie and coworkers [22] found better fertility with glycerol than with DMA but
using DMA in a straw method (i.e., different from the pellet
method in [20, 40]) and using a lower cooling rate than that used
in [21].
All in all, good fertility of Æ90% can apparently be obtained
with frozen-thawed poultry semen using either DMA or glycerol as
cryoprotectant. However, when using glycerol, the semen cannot
be inseminated right from the straw. Instead, extra steps are needed
after thawing to remove glycerol prior to insemination, which
implies extra labor and costs. Methods to remove glycerol include
dialysis [2, 45], stepwise dilution, followed by centrifugation
[22, 42], and discontinuous density gradient centrifugation [46].
1.5 Other Stabilizing
Additives
For semen of mammalian species, the presence in the freezing
medium of milk components or egg yolk is beneficial, if not essential, for post-thaw sperm survival and fertility. However, it seems
there is little if any evidence for positive effects of yolk or milk
components for avian semen. Instead, chicken egg yolk reduces
fertility of chicken semen, but not of turkey and red jungle fowl
[47–49]. Low-density lipoprotein extracted from egg yolk [50],
egg yolk plasma, and soybean lecithin [51] were found beneficial
for freezing poultry semen.
The use of sugars and other non-permeating neutral (nonelectrolyte) solutes in (cryo)preservation media results in a lower ionic
strength, which may be beneficial [37, 41], especially as salts and
other solutes become strongly concentrated in the “unfrozen fraction” during freezing. Very high ionic strengths affect lipid membranes and (membrane) proteins and may lead to “salt loading” of
the cells. Sugars and other neutral solutes are sometimes referred to
as “non-permeating cryoprotectants.” However, in media with
normal (physiological) osmotic strength, the ratio of electrolytes/
nonelectrolytes will not influence freezing point depression, or the
degree of shrinking of the unfrozen fraction and of the cells therein
during ice formation, nor can it affect intracellular cryobiological
properties. High osmolalities (be it by sugars or any other solutes)
can work to reduce the chance of intracellular ice formation and
384
Henri Woelders