seen in chicken (unpublished) and have reported for turkey semen
[32] that freezing at a fixed concentration is better than at a fixed
dilution rate.
A diluent that is a good extender for cold storage of “fresh”
semen may also likely be a good base medium for freezing semen,
assuming that “happy sperm are freezable sperm”. Obviously, a
diluent that causes cellular stress by, for example, inappropriate
pH, osmolality, electrolyte composition and concentration, etc.
provides a wrong starting point for cells to cope with the additional
stresses of freezing and thawing. Electrolyte concentration seems
also important, as solutes become strongly concentrated during
freezing. Thus, a freezing medium may consist of a good fresh
extender supplemented with cryoprotective agents (CPAs) such as
glycerol, etc. and possibly other components that are deemed
beneficial. Original extenders for poultry semen were “inspired”
by the composition of seminal plasma [33, 34] in that they contain
a high concentration glutamate, serving as an organic anion. A
freezing semen that is often used is Lake’s “Solution 1” [35] (see,
for example, [32] and references therein). This medium as well as
the fresh extender “A” [34] contained very little pH buffering
capacity. Addition of buffering substances (phosphate and sulfonic
acid-based, zwitterionic “Good’s” buffers like MES, BES, TES,
and Hepes) was then introduced [28, 29, 36]. Lake and Ravie
[28] reported best fertility with 24-h cold-stored semen when
using an MES (pH 6.8) or BES (pH 7.1) buffered diluent, compared with similar diluents but with other buffers and other pH
values.
In our own experience, we also obtained very good results
when using the BES-containing Lake and Ravie [28] diluent of
pH 7.1 (“L&R 7.1”) as fresh extender or as “base” for a freezing
medium, compared with a large number of other possible poultry
semen extenders, culture media, or mammalian semen extenders
we tried for poultry semen. However, the osmolality of the L&R
7.1 medium (411 mOsm/kg of water) is outside the physiological
range. At low dilution rates (e.g., 1:1 [28]), this poses no problem,
as the final osmolality remains at an acceptable value between that
of seminal plasma and that of the diluent. At higher dilution rates,
the resulting dehydration of the sperm cells inhibits sperm motility
(cf. [37]). We therefore modified the L&R 7.1 medium to have an
osmolality of 325 mOsm/kg of water, similar to that of poultry
semen (referred to as “ASG poultry extender” in [21, 32]). We
have used this medium since 2004 as a fresh extender and as base
freezing medium, combined with glycerol, dimethylacetamide
(DMA), or other cryoprotectants. We obtained better fertility
with frozen/thawed poultry [21] and turkey [32] semen frozen
in “ASG”-based freezing medium compared with Lake‘s freezing
medium (“Solution 1
00 [35]). Thanks to the physiological osmolality, the ASG extender can be used as fresh extender at higher
382
Henri Woelders
[32] that freezing at a fixed concentration is better than at a fixed
dilution rate.
A diluent that is a good extender for cold storage of “fresh”
semen may also likely be a good base medium for freezing semen,
assuming that “happy sperm are freezable sperm”. Obviously, a
diluent that causes cellular stress by, for example, inappropriate
pH, osmolality, electrolyte composition and concentration, etc.
provides a wrong starting point for cells to cope with the additional
stresses of freezing and thawing. Electrolyte concentration seems
also important, as solutes become strongly concentrated during
freezing. Thus, a freezing medium may consist of a good fresh
extender supplemented with cryoprotective agents (CPAs) such as
glycerol, etc. and possibly other components that are deemed
beneficial. Original extenders for poultry semen were “inspired”
by the composition of seminal plasma [33, 34] in that they contain
a high concentration glutamate, serving as an organic anion. A
freezing semen that is often used is Lake’s “Solution 1” [35] (see,
for example, [32] and references therein). This medium as well as
the fresh extender “A” [34] contained very little pH buffering
capacity. Addition of buffering substances (phosphate and sulfonic
acid-based, zwitterionic “Good’s” buffers like MES, BES, TES,
and Hepes) was then introduced [28, 29, 36]. Lake and Ravie
[28] reported best fertility with 24-h cold-stored semen when
using an MES (pH 6.8) or BES (pH 7.1) buffered diluent, compared with similar diluents but with other buffers and other pH
values.
In our own experience, we also obtained very good results
when using the BES-containing Lake and Ravie [28] diluent of
pH 7.1 (“L&R 7.1”) as fresh extender or as “base” for a freezing
medium, compared with a large number of other possible poultry
semen extenders, culture media, or mammalian semen extenders
we tried for poultry semen. However, the osmolality of the L&R
7.1 medium (411 mOsm/kg of water) is outside the physiological
range. At low dilution rates (e.g., 1:1 [28]), this poses no problem,
as the final osmolality remains at an acceptable value between that
of seminal plasma and that of the diluent. At higher dilution rates,
the resulting dehydration of the sperm cells inhibits sperm motility
(cf. [37]). We therefore modified the L&R 7.1 medium to have an
osmolality of 325 mOsm/kg of water, similar to that of poultry
semen (referred to as “ASG poultry extender” in [21, 32]). We
have used this medium since 2004 as a fresh extender and as base
freezing medium, combined with glycerol, dimethylacetamide
(DMA), or other cryoprotectants. We obtained better fertility
with frozen/thawed poultry [21] and turkey [32] semen frozen
in “ASG”-based freezing medium compared with Lake‘s freezing
medium (“Solution 1
00 [35]). Thanks to the physiological osmolality, the ASG extender can be used as fresh extender at higher
382
Henri Woelders