15). Use an automatic filling machine or a syringe with little
piece of tubing for attaching straws on their plugged sites.
Make sure that the sealing powder on the plugged site is
moistened upon filling to close the straw on that site.
3. Seal the straws further using a sealing machine, sealing powder,
or balls. Place straws on the racks used for freezing.
4. For cooling down to subzero temperatures (i.e., freezing), use
a controlled rate freezer connected to a liquid nitrogen tank, or
place straws in the vapor phase of liquid nitrogen built up in a
Styrofoam box.
(a) When using a controlled rate freezer, place racks with
straws in the freezing chamber of the device held at
5
C, and cool down to À120
C at 10–60
C/min (see
Note 16).
(b) Alternatively, straws can be frozen in the vapor phase
above liquid nitrogen in a Styrofoam box (see Fig. 1),
e.g., of 60 Â 40 Â 20 cm, filled with liquid nitrogen up
to 3 cm height. In this case, a cooling rate of ~30
C/min
is reached by placing straws in horizontal position ~3 cm
above the surface of liquid nitrogen. Temperatures below
À80
C are then attained within 15 min (see Note 17).
5. Plunge the straws in liquid nitrogen (i.e., after attaining À80 to
À120
C), after which they can be transferred into goblets and
stored in liquid nitrogen containers. Store for a minimum of
24 h, after which post-thaw sperm motility can be assessed.
3.10 Thawing
of Cryopreserved
Samples
1. Thaw straws by incubating in a water bath for 30 s at 37
C (see
Note 18).
4 Notes
1. The sperm concentration can be determined using a Neubauer
improved counting chamber or hemocytometer. Alternatively,
a photometer can be employed; however, note that when measuring the optical density, one cannot discriminate between
cells and debris. Moreover, milk and egg yolk particles will
interfere. Determining cell concentrations and percentages of
membrane intact sperm by using a NucleoCounter eliminates
the latter issue, since a DNA-intercalating fluorescent dye is
used. This approach is based on comparing fluorescence intensities with a calibration curve, both before and after full membrane permeabilization.
2. In addition to “home-made” extenders, many species-specific
commercial extenders are available. Manufacturers for
374
Harrie ¨ tte Oldenhof et al.
piece of tubing for attaching straws on their plugged sites.
Make sure that the sealing powder on the plugged site is
moistened upon filling to close the straw on that site.
3. Seal the straws further using a sealing machine, sealing powder,
or balls. Place straws on the racks used for freezing.
4. For cooling down to subzero temperatures (i.e., freezing), use
a controlled rate freezer connected to a liquid nitrogen tank, or
place straws in the vapor phase of liquid nitrogen built up in a
Styrofoam box.
(a) When using a controlled rate freezer, place racks with
straws in the freezing chamber of the device held at
5
C, and cool down to À120
C at 10–60
C/min (see
Note 16).
(b) Alternatively, straws can be frozen in the vapor phase
above liquid nitrogen in a Styrofoam box (see Fig. 1),
e.g., of 60 Â 40 Â 20 cm, filled with liquid nitrogen up
to 3 cm height. In this case, a cooling rate of ~30
C/min
is reached by placing straws in horizontal position ~3 cm
above the surface of liquid nitrogen. Temperatures below
À80
C are then attained within 15 min (see Note 17).
5. Plunge the straws in liquid nitrogen (i.e., after attaining À80 to
À120
C), after which they can be transferred into goblets and
stored in liquid nitrogen containers. Store for a minimum of
24 h, after which post-thaw sperm motility can be assessed.
3.10 Thawing
of Cryopreserved
Samples
1. Thaw straws by incubating in a water bath for 30 s at 37
C (see
Note 18).
4 Notes
1. The sperm concentration can be determined using a Neubauer
improved counting chamber or hemocytometer. Alternatively,
a photometer can be employed; however, note that when measuring the optical density, one cannot discriminate between
cells and debris. Moreover, milk and egg yolk particles will
interfere. Determining cell concentrations and percentages of
membrane intact sperm by using a NucleoCounter eliminates
the latter issue, since a DNA-intercalating fluorescent dye is
used. This approach is based on comparing fluorescence intensities with a calibration curve, both before and after full membrane permeabilization.
2. In addition to “home-made” extenders, many species-specific
commercial extenders are available. Manufacturers for
374
Harrie ¨ tte Oldenhof et al.