10. Sperm motility can be observed using a 10 Â 20–40 magnification; morphological abnormalities can be more easily evaluated using immobilized/fixed specimens and a 10 Â 100
magnification with an oil immersion objective. Specimens can
be fixed by adding formal saline solution, or sperm smears on
slides can be prepared after staining with eosin-nigrosin. When
available, computer-assisted sperm analysis can be used to
assess sperm motility characteristics. Furthermore, specific
fluorescent dyes can be used with fluorescence microscopy or
flow cytometric analysis, e.g., for determining plasma and
acrosomal membrane integrity.
11. Presence of large contents of seminal plasma in preserved
semen is described to negatively affect sperm quality. Seminal
plasma contents of ~5%, however, have been reported to result
in higher percentages of motile sperm post-thaw.
12. Iodixanol (commercially available as OptiPrep) has been
described to have cryoprotective properties. For cryopreservation, 1–2% final iodixanol concentrations can remain in sperm
samples.
13. For commercial purposes, take into account regulations on
minimum values for numbers and/or percentages of motile
and morphological normal sperm which need to be present in
a dose for use for artificial insemination. Generally, the total
number of sperm per straw or insemination dose is calculated.
For bulls, one 0.25-mL straw containing ~20 Â 10
6 sperm
typically results in ~10 Â 10
6 progressively motile sperm postthaw, when taking into account a 50% survival after cryopreservation. For stallions, 4–8 straws of 0.5 mL containing
~50–100 Â 10
6 sperm typically result in ~250 Â 10
6 progressively motile sperm post-thaw.
14. For bull and equine sperm, filling of straws can be done at
ambient temperature, after which cooling down to 5
C can be
done using a controlled rate freezer, prior to freezing to subzero temperatures.
15. Straws should be labeled with at least information concerning
the date of semen collection, donor, and manufacturer.
16. If desired, one can use a protocol with a hold at a particular
subzero temperature at which ice nucleation can be induced
(mechanically), as well as different cooling rates in different
temperature ranges.
17. A thermocouple can be used for actually measuring the cooling
rates. Allow time for building up the vapor phase by using a
large enough box and keeping the box closed before us. Note
that cooling rates dramatically decrease with placing straws
further away from the liquid nitrogen level, and if stapling
racks with straws, these will be frozen at different rates.
376
Harrie ¨ tte Oldenhof et al.
magnification with an oil immersion objective. Specimens can
be fixed by adding formal saline solution, or sperm smears on
slides can be prepared after staining with eosin-nigrosin. When
available, computer-assisted sperm analysis can be used to
assess sperm motility characteristics. Furthermore, specific
fluorescent dyes can be used with fluorescence microscopy or
flow cytometric analysis, e.g., for determining plasma and
acrosomal membrane integrity.
11. Presence of large contents of seminal plasma in preserved
semen is described to negatively affect sperm quality. Seminal
plasma contents of ~5%, however, have been reported to result
in higher percentages of motile sperm post-thaw.
12. Iodixanol (commercially available as OptiPrep) has been
described to have cryoprotective properties. For cryopreservation, 1–2% final iodixanol concentrations can remain in sperm
samples.
13. For commercial purposes, take into account regulations on
minimum values for numbers and/or percentages of motile
and morphological normal sperm which need to be present in
a dose for use for artificial insemination. Generally, the total
number of sperm per straw or insemination dose is calculated.
For bulls, one 0.25-mL straw containing ~20 Â 10
6 sperm
typically results in ~10 Â 10
6 progressively motile sperm postthaw, when taking into account a 50% survival after cryopreservation. For stallions, 4–8 straws of 0.5 mL containing
~50–100 Â 10
6 sperm typically result in ~250 Â 10
6 progressively motile sperm post-thaw.
14. For bull and equine sperm, filling of straws can be done at
ambient temperature, after which cooling down to 5
C can be
done using a controlled rate freezer, prior to freezing to subzero temperatures.
15. Straws should be labeled with at least information concerning
the date of semen collection, donor, and manufacturer.
16. If desired, one can use a protocol with a hold at a particular
subzero temperature at which ice nucleation can be induced
(mechanically), as well as different cooling rates in different
temperature ranges.
17. A thermocouple can be used for actually measuring the cooling
rates. Allow time for building up the vapor phase by using a
large enough box and keeping the box closed before us. Note
that cooling rates dramatically decrease with placing straws
further away from the liquid nitrogen level, and if stapling
racks with straws, these will be frozen at different rates.
376
Harrie ¨ tte Oldenhof et al.