then collect semen of a number of roosters consecutively in that
collection tube. Alternatively, one may collect semen from
roosters consecutively in a collection tube and after each
rooster wash down the ejaculate with 0.5 mL of extender.
8. If small tubes and/or small volumes are used, one may place
the tubes surrounded with tissue paper in a beaker or tube rack
to prevent a perhaps too abrupt cooling.
9. In frozen semen, the apparent sperm motility may be better if
the final dilution contains approximately 10 mg of bovine
serum albumin per mL.
10. Poultry semen motility can best be estimated at room temperature rather than at 40
C, as the sperm cells may be too agile
on a heated stage.
11. The fixative (cross-linking agent) glutaraldehyde can render
cell membranes permeable for salts. However, it does not
make the spermatozoa permeable to DAPI (see, for example,
[61]).
12. During prolonged storage, e.g., several days, bacterial growth
may play a role especially if no antibiotics are used in the
extender (but, see [56]).
13. The actual cooling rate will depend on the freezing cabinet
temperature and the ventilation rate (windchill) in the freezing
cabinet.
14. One may use a thermostatic cooled bath. An easy low-cost
alternative is to have a Styrofoam box filled with water and
another one containing ice flakes. Simply check water temperature regularly, and add a handful of ice flakes if temperature
rises above 7
C.
Acknowledgments
Thanks are due to AAC de Wit for expert technical assistance.
Funding from the Dutch Ministry of Agriculture, Nature and
Food Quality (project KB-Dier, WOT-03-436-077) and from
European Union’s Horizon 2020 Research and Innovation
Programme (project IMAGE, grant agreement n
677353) are
gratefully acknowledged.
References
1. Polge C, Smith AU, Parkes AS (1949) Revival
of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666–666
2. Polge C (1951) Functional survival of fowl
spermatozoa after freezing at À79
C. Nature
167:949–950
396
Henri Woelders
collection tube. Alternatively, one may collect semen from
roosters consecutively in a collection tube and after each
rooster wash down the ejaculate with 0.5 mL of extender.
8. If small tubes and/or small volumes are used, one may place
the tubes surrounded with tissue paper in a beaker or tube rack
to prevent a perhaps too abrupt cooling.
9. In frozen semen, the apparent sperm motility may be better if
the final dilution contains approximately 10 mg of bovine
serum albumin per mL.
10. Poultry semen motility can best be estimated at room temperature rather than at 40
C, as the sperm cells may be too agile
on a heated stage.
11. The fixative (cross-linking agent) glutaraldehyde can render
cell membranes permeable for salts. However, it does not
make the spermatozoa permeable to DAPI (see, for example,
[61]).
12. During prolonged storage, e.g., several days, bacterial growth
may play a role especially if no antibiotics are used in the
extender (but, see [56]).
13. The actual cooling rate will depend on the freezing cabinet
temperature and the ventilation rate (windchill) in the freezing
cabinet.
14. One may use a thermostatic cooled bath. An easy low-cost
alternative is to have a Styrofoam box filled with water and
another one containing ice flakes. Simply check water temperature regularly, and add a handful of ice flakes if temperature
rises above 7
C.
Acknowledgments
Thanks are due to AAC de Wit for expert technical assistance.
Funding from the Dutch Ministry of Agriculture, Nature and
Food Quality (project KB-Dier, WOT-03-436-077) and from
European Union’s Horizon 2020 Research and Innovation
Programme (project IMAGE, grant agreement n
677353) are
gratefully acknowledged.
References
1. Polge C, Smith AU, Parkes AS (1949) Revival
of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666–666
2. Polge C (1951) Functional survival of fowl
spermatozoa after freezing at À79
C. Nature
167:949–950
396
Henri Woelders