4. If available, one may employ computer-assisted sperm analysis
(CASA) for motility and other variables, or flow cytometry,
using fluorescent dyes for plasma membrane integrity, acrosome intactness, or mitochondrial function.
3.3 Fresh Storage
1. The semen is best stored at a constant temperature of approximately 5
C. In routine practice, the dilution rate for fresh
poultry semen is usually very low, but a higher dilution rate
(with an extender of the appropriate osmolality) would be
beneficial for longevity during cold storage. A tenfold dilution
of the semen with ASG-PE would still be compatible with a
sufficient sperm number for insemination with fresh semen (see
Note 12).
3.4 Semen Freezing
A relatively high sperm dosage is required when using frozenthawed semen [19]. In chicken hens, an inseminate volume of
0.2 mL is possible without having too much backflow. A sperm
concentration of 1200 Â 106 sperm cells/mL would thus give
240 million total sperm per dose. This final sperm concentration
is usually feasible in chicken, where initial ejaculate sperm cell
concentrations of 4000 million per mL or more can be expected,
but this may be different in special cases. If glycerol is used as CPA,
one may actually freeze at lower sperm concentrations, as the semen
is reconcentrated by centrifugation in the course of post-thaw
glycerol removal. In order to compare freezing methods and to
validate a chosen freezing method as to the actual cooling rate
obtained inside the straws, it is advised to measure cooling rate
using thin “Type K” copper constantan thermocouples mounted
inside “dummy” straws. A tiny hole can be bored in the side of an
empty straw, and a thermocouple wire can be placed with the
sensing “tip” inside the straw and fixed with a bit of glue. After
the glue is cured, the straw can be filled with freezing medium and
sealed in the normal fashion. The thermocouple is connected to the
computer interface, and the dummy straw is placed between straws
to be frozen.
3.4.1 Method DMA
in Straws
With this method, the semen can be used directly for insemination
after thawing. No need to remove the cryoprotectant prior to
insemination. Use ASG-PE as the extender. The sperm concentration of the prediluted semen is determined with a spectrophotometer or hemocytometer. Handling is performed at 5
C (cold room
or open top cooler cabinet).
1. The semen can then be further diluted with ASG-PE to a
chosen sperm concentration (e.g., 1.8 Â 109 sperm/mL).
0.25-mL straws are printed with a straw printer.
2. Then, half a volume of ASG-PE with three times the final
concentration of DMA, e.g., 1.8 mol/L, is added to 1 volume
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Henri Woelders
(CASA) for motility and other variables, or flow cytometry,
using fluorescent dyes for plasma membrane integrity, acrosome intactness, or mitochondrial function.
3.3 Fresh Storage
1. The semen is best stored at a constant temperature of approximately 5
C. In routine practice, the dilution rate for fresh
poultry semen is usually very low, but a higher dilution rate
(with an extender of the appropriate osmolality) would be
beneficial for longevity during cold storage. A tenfold dilution
of the semen with ASG-PE would still be compatible with a
sufficient sperm number for insemination with fresh semen (see
Note 12).
3.4 Semen Freezing
A relatively high sperm dosage is required when using frozenthawed semen [19]. In chicken hens, an inseminate volume of
0.2 mL is possible without having too much backflow. A sperm
concentration of 1200 Â 106 sperm cells/mL would thus give
240 million total sperm per dose. This final sperm concentration
is usually feasible in chicken, where initial ejaculate sperm cell
concentrations of 4000 million per mL or more can be expected,
but this may be different in special cases. If glycerol is used as CPA,
one may actually freeze at lower sperm concentrations, as the semen
is reconcentrated by centrifugation in the course of post-thaw
glycerol removal. In order to compare freezing methods and to
validate a chosen freezing method as to the actual cooling rate
obtained inside the straws, it is advised to measure cooling rate
using thin “Type K” copper constantan thermocouples mounted
inside “dummy” straws. A tiny hole can be bored in the side of an
empty straw, and a thermocouple wire can be placed with the
sensing “tip” inside the straw and fixed with a bit of glue. After
the glue is cured, the straw can be filled with freezing medium and
sealed in the normal fashion. The thermocouple is connected to the
computer interface, and the dummy straw is placed between straws
to be frozen.
3.4.1 Method DMA
in Straws
With this method, the semen can be used directly for insemination
after thawing. No need to remove the cryoprotectant prior to
insemination. Use ASG-PE as the extender. The sperm concentration of the prediluted semen is determined with a spectrophotometer or hemocytometer. Handling is performed at 5
C (cold room
or open top cooler cabinet).
1. The semen can then be further diluted with ASG-PE to a
chosen sperm concentration (e.g., 1.8 Â 109 sperm/mL).
0.25-mL straws are printed with a straw printer.
2. Then, half a volume of ASG-PE with three times the final
concentration of DMA, e.g., 1.8 mol/L, is added to 1 volume
392
Henri Woelders