(b) Carefully remove the supernatant. Cushion fluid can be
aspirated from below the layer with sperm (see Note 12).
Transfer the sperm to a new tube when needed.
3. Determine the sperm concentration in the resuspended pellet
or recovered layer using a hemocytometer (see Note 1).
4. After centrifugation, either a “one-” or “two-step” dilution can
be used to transfer into freezing extender.
3.7 One-Step
Approach for Diluting
Sperm with Freezing
Extender
When performing a one-step dilution, freezing extender containing
the desired final concentrations of cryoprotective agents is directly
added to a concentrated sperm sample. This approach is followed at
many equine breeding stations and uses INRA-82 supplemented
with 2–10% clarified egg yolk (freeze-thawed aliquots) to which
glycerol is added just prior to use. Final concentrations are
~400 Â 10
6 sperm/mL in INRA-82 supplemented with egg yolk
and 2.5% glycerol (see Note 13).
1. After initial dilution (in INRA-82) and centrifugation (10 min,
600 Â g, at 22
C), resuspend the obtained sperm pellet in a
little volume (i.e., remaining after removal of the supernatant),
and determine the sperm concentration.
2. Determine the amount of freezing extender (i.e., volume)
needed for diluting to the desired final sperm concentration
(e.g., 400 Â 10
6 sperm/mL).
3. Supplement diluent without penetrating protectants (INRA82 containing egg yolk) with glycerol such that the final glycerol concentration is 2.5% (e.g., 0.25 mL 100% glycerol to
9.75 mL diluent).
4. Slowly add freezing extender containing the final concentrations of cryoprotective agents. Do so dropwise on the side of
the tube with the sperm sample while gently turning the tube
to ensure mixing.
3.8 Two-Step
Approach for Diluting
Sperm with Freezing
Extender
In case of a two-step dilution, a sperm sample is first diluted with a
primary extender which does not contain glycerol; this is done to a
concentration 2-fold (stallion and bull) or (2/3)-fold (boar) the
desired final sperm concentration. Then, for cryopreservation
freezing, extender is added which contains 2-fold (stallion and
bull) or (1/3)-fold (boar) the desired final concentrations of cryoprotective agents (see Note 13).
1. Stallion sperm:
(a) Use INRA-82 for initial dilution and centrifugation
(10 min, 600 Â g, at 22
C).
(b) Use INRA-82 for diluting to 200–800 Â 10
6 sperm/mL.
(c) Add an equal volume (e.g., 10 mL to 10 mL) of INRA-82
freezing extender (i.e., INRA-82 supplemented with 4%
372
Harrie ¨ tte Oldenhof et al.
aspirated from below the layer with sperm (see Note 12).
Transfer the sperm to a new tube when needed.
3. Determine the sperm concentration in the resuspended pellet
or recovered layer using a hemocytometer (see Note 1).
4. After centrifugation, either a “one-” or “two-step” dilution can
be used to transfer into freezing extender.
3.7 One-Step
Approach for Diluting
Sperm with Freezing
Extender
When performing a one-step dilution, freezing extender containing
the desired final concentrations of cryoprotective agents is directly
added to a concentrated sperm sample. This approach is followed at
many equine breeding stations and uses INRA-82 supplemented
with 2–10% clarified egg yolk (freeze-thawed aliquots) to which
glycerol is added just prior to use. Final concentrations are
~400 Â 10
6 sperm/mL in INRA-82 supplemented with egg yolk
and 2.5% glycerol (see Note 13).
1. After initial dilution (in INRA-82) and centrifugation (10 min,
600 Â g, at 22
C), resuspend the obtained sperm pellet in a
little volume (i.e., remaining after removal of the supernatant),
and determine the sperm concentration.
2. Determine the amount of freezing extender (i.e., volume)
needed for diluting to the desired final sperm concentration
(e.g., 400 Â 10
6 sperm/mL).
3. Supplement diluent without penetrating protectants (INRA82 containing egg yolk) with glycerol such that the final glycerol concentration is 2.5% (e.g., 0.25 mL 100% glycerol to
9.75 mL diluent).
4. Slowly add freezing extender containing the final concentrations of cryoprotective agents. Do so dropwise on the side of
the tube with the sperm sample while gently turning the tube
to ensure mixing.
3.8 Two-Step
Approach for Diluting
Sperm with Freezing
Extender
In case of a two-step dilution, a sperm sample is first diluted with a
primary extender which does not contain glycerol; this is done to a
concentration 2-fold (stallion and bull) or (2/3)-fold (boar) the
desired final sperm concentration. Then, for cryopreservation
freezing, extender is added which contains 2-fold (stallion and
bull) or (1/3)-fold (boar) the desired final concentrations of cryoprotective agents (see Note 13).
1. Stallion sperm:
(a) Use INRA-82 for initial dilution and centrifugation
(10 min, 600 Â g, at 22
C).
(b) Use INRA-82 for diluting to 200–800 Â 10
6 sperm/mL.
(c) Add an equal volume (e.g., 10 mL to 10 mL) of INRA-82
freezing extender (i.e., INRA-82 supplemented with 4%
372
Harrie ¨ tte Oldenhof et al.