4. The chicken ejaculate may have a volume of approximately
0.5 mL and may contain 2–6 Â 10
9 spermatozoa per mL (see
Notes 5 and 6).
5. The tube is closed with a stopper to prevent evaporation
(dehydration) and is placed in a thermostat-controlled cool
box at 5
C (see Note 7).
6. The semen is preferably cooled relatively rapidly (but not
abruptly), starting cooling directly after collection and predilution with extender. Cooling can be done, for example, by
placing the tubes of pre-extended semen in a 5
C cool box.
The heat capacity of the tube and the volume of semen will
result a slow enough cooling (see Note 8).
7. When ejaculates from all cocks are collected, the semen is
transported to the laboratory for further processing and freezing. All further handling is performed at 5
C (cold room or
open top cooler cabinet).
3.2 Quality
Assessment
1. The sperm concentration can be determined using a photometer, which measures transmission decrease resulting from light
scattering by the sperm cells. This requires a very small volume
of the ejaculate which is diluted with a saline. Each species
would require a dedicated calibration, preferably made at various sperm concentrations as the extinction is not exactly proportional to sperm concentration. Calibrations can be done by
using a hemocytometer with immobilized sperm cells.
2. Motility can be estimated in extended semen, e.g., using a
tenfold or stronger dilution (with an appropriate diluent such
as ASG-PE). Use a final sperm concentration that would allow
to see individual freely swimming sperm cells with a limited
(10–100) number of cells per field of view (see Note 9). Four
μL of the diluted semen preparation can be placed on a microscope slide and covered with a coverslip. The percentage of
motile sperm is estimated using phase contrast microscopy in
five different fields of view at different places of the coverslip,
and the estimates are then averaged (see Note 10).
3. Plasma membrane integrity can be estimated in (strongly)
diluted semen. Add a volume of poultry extender containing
DAPI (4
0 ,6-Diamidine-2
0 -phenylindole dihydrochloride), having the same temperature as the semen, to reach a final DAPI
concentration of 5 μM. Add just a smidge of 0.5% glutaraldehyde solution (e.g., 10 μL added to 200 μL diluted DAPIstained semen) to immobilize the live sperm cells and place the
tube at RT for 4 min (see Note 11). Then place 3 μL on a slide
covered with an 18 Â 18 mm coverslip, and assess a minimum
of 200 cells in different fields of view in a microscope combining phase contrast and fluorescence microscopy.
Cryopreservation of Avian Semen
391
0.5 mL and may contain 2–6 Â 10
9 spermatozoa per mL (see
Notes 5 and 6).
5. The tube is closed with a stopper to prevent evaporation
(dehydration) and is placed in a thermostat-controlled cool
box at 5
C (see Note 7).
6. The semen is preferably cooled relatively rapidly (but not
abruptly), starting cooling directly after collection and predilution with extender. Cooling can be done, for example, by
placing the tubes of pre-extended semen in a 5
C cool box.
The heat capacity of the tube and the volume of semen will
result a slow enough cooling (see Note 8).
7. When ejaculates from all cocks are collected, the semen is
transported to the laboratory for further processing and freezing. All further handling is performed at 5
C (cold room or
open top cooler cabinet).
3.2 Quality
Assessment
1. The sperm concentration can be determined using a photometer, which measures transmission decrease resulting from light
scattering by the sperm cells. This requires a very small volume
of the ejaculate which is diluted with a saline. Each species
would require a dedicated calibration, preferably made at various sperm concentrations as the extinction is not exactly proportional to sperm concentration. Calibrations can be done by
using a hemocytometer with immobilized sperm cells.
2. Motility can be estimated in extended semen, e.g., using a
tenfold or stronger dilution (with an appropriate diluent such
as ASG-PE). Use a final sperm concentration that would allow
to see individual freely swimming sperm cells with a limited
(10–100) number of cells per field of view (see Note 9). Four
μL of the diluted semen preparation can be placed on a microscope slide and covered with a coverslip. The percentage of
motile sperm is estimated using phase contrast microscopy in
five different fields of view at different places of the coverslip,
and the estimates are then averaged (see Note 10).
3. Plasma membrane integrity can be estimated in (strongly)
diluted semen. Add a volume of poultry extender containing
DAPI (4
0 ,6-Diamidine-2
0 -phenylindole dihydrochloride), having the same temperature as the semen, to reach a final DAPI
concentration of 5 μM. Add just a smidge of 0.5% glutaraldehyde solution (e.g., 10 μL added to 200 μL diluted DAPIstained semen) to immobilize the live sperm cells and place the
tube at RT for 4 min (see Note 11). Then place 3 μL on a slide
covered with an 18 Â 18 mm coverslip, and assess a minimum
of 200 cells in different fields of view in a microscope combining phase contrast and fluorescence microscopy.
Cryopreservation of Avian Semen
391