allow higher cooling rates [37]. In fact, this is how sugars were used
(but without real success) before the discovery of the cryoprotective properties of permeating CPAs [52] (cf. [2]). The higher
osmolalities are already a potential cause of cell stress and damage
even before freezing has started. Therefore, the term
non-permeating CPA may not be appropriate, notwithstanding
the fact that ionic strength and overall medium composition can
be an important factor for cryosurvival.
A similar discussion is valid with regard to macromolecules such
as polyvinylpyrrolidone [35], BSA, etc. Unless used at very high
concentrations, these compounds are not likely to affect cryobiological properties of the medium, but these compounds may have
specific interactions with the cell surface that may be beneficial
during or after freezing and thawing. For instance, the presence
of BSA after thawing can improve the apparent sperm motility in
poultry semen.
Another potential class of additives are the antioxidants. There
is much recent literature on the effect of antioxidants in the freezing
medium on post-thaw semen quality of avian semen. Additives with
antioxidant activity investigated in avian semen include catalase,
superoxide dismutase (reduced) glutathione, vitamins E and C,
selenium, carnitine, hyaluronic acid, lycopene, oleic acid, linoleic
acid, coenzyme Q10, resveratrol, and quercetin (see, for example,
[53–55] and references therein to earlier studies in both avian and
mammalian species).
Unlike bull semen extenders, poultry extenders described in
the literature often do not contain antibiotics, although antibiotics
may be added without negatively affecting semen fertility [56]. For
cold storage of “fresh” semen, the presence of antibiotics may
improve longevity.
1.6 Freezing
Methods
Avian semen is generally frozen in 0.25-mL straws or in “pellets.”
Frozen pellets can be obtained by dropping approximately 50 μL
droplets straight onto the surface of liquid nitrogen (LN 2 )
[39]. While this can yield very good results, the packaging in straws
is often preferred as it allows easier labelling of the semen. Another
disadvantage of pellet freezing is that a special device is needed for
rapid thawing of the pellets.
Cooling rates used may differ according to species and also
seem to be different for different types of CPA used. In addition,
for a given species and CPA, different authors may use or advise
different cooling rates. The pellet freezing method but also studies
on freezing in straws [21] indicate that poultry spermatozoa can
tolerate (and may perhaps prefer) quite high cooling rates. For
instance, the average cooling rate of 50-μL droplets in LN 2 is
approximately 600
C/min (it takes approximately 20 s to cool
and freeze from +5
C to À196
C).
Cryopreservation of Avian Semen
385
(but without real success) before the discovery of the cryoprotective properties of permeating CPAs [52] (cf. [2]). The higher
osmolalities are already a potential cause of cell stress and damage
even before freezing has started. Therefore, the term
non-permeating CPA may not be appropriate, notwithstanding
the fact that ionic strength and overall medium composition can
be an important factor for cryosurvival.
A similar discussion is valid with regard to macromolecules such
as polyvinylpyrrolidone [35], BSA, etc. Unless used at very high
concentrations, these compounds are not likely to affect cryobiological properties of the medium, but these compounds may have
specific interactions with the cell surface that may be beneficial
during or after freezing and thawing. For instance, the presence
of BSA after thawing can improve the apparent sperm motility in
poultry semen.
Another potential class of additives are the antioxidants. There
is much recent literature on the effect of antioxidants in the freezing
medium on post-thaw semen quality of avian semen. Additives with
antioxidant activity investigated in avian semen include catalase,
superoxide dismutase (reduced) glutathione, vitamins E and C,
selenium, carnitine, hyaluronic acid, lycopene, oleic acid, linoleic
acid, coenzyme Q10, resveratrol, and quercetin (see, for example,
[53–55] and references therein to earlier studies in both avian and
mammalian species).
Unlike bull semen extenders, poultry extenders described in
the literature often do not contain antibiotics, although antibiotics
may be added without negatively affecting semen fertility [56]. For
cold storage of “fresh” semen, the presence of antibiotics may
improve longevity.
1.6 Freezing
Methods
Avian semen is generally frozen in 0.25-mL straws or in “pellets.”
Frozen pellets can be obtained by dropping approximately 50 μL
droplets straight onto the surface of liquid nitrogen (LN 2 )
[39]. While this can yield very good results, the packaging in straws
is often preferred as it allows easier labelling of the semen. Another
disadvantage of pellet freezing is that a special device is needed for
rapid thawing of the pellets.
Cooling rates used may differ according to species and also
seem to be different for different types of CPA used. In addition,
for a given species and CPA, different authors may use or advise
different cooling rates. The pellet freezing method but also studies
on freezing in straws [21] indicate that poultry spermatozoa can
tolerate (and may perhaps prefer) quite high cooling rates. For
instance, the average cooling rate of 50-μL droplets in LN 2 is
approximately 600
C/min (it takes approximately 20 s to cool
and freeze from +5
C to À196
C).
Cryopreservation of Avian Semen
385