egg yolk and 5% glycerol). Add slowly, dropwise on the
side of the tube while gently turning to ensure mixing.
(d) This results in 100–400 Â 10
6 sperm/mL, 2% egg yolk,
and 2.5% glycerol.
2. Bull sperm:
(a) Use TEY-1 (contains 20% egg yolk) for diluting raw
semen to 120 Â 10
6 sperm/mL.
(b) Add an equal volume of TEY-2 (containing 20% egg yolk
and 12.8% glycerol). Add slowly, dropwise on the side of
the tube while gently turning to ensure mixing.
(c) This results in 60 Â 10
6 sperm/mL, 20% egg yolk, and
6.4% glycerol.
3. Boar sperm:
(a) Use BTS for initial dilution, cooling sperm to 17
C, and
centrifugation (3 min, 2400 Â g, at 17
C).
(b) Use LEY cooling extender (contains 20% egg yolk) for
diluting to 1500–750 Â 10
6 sperm/mL.
(c) Slowly cool sperm to 5
C.
(d) Dilute with LEY freezing extender (containing 20% egg
yolk, 1.5% Equex paste, and 6% glycerol) for diluting to
1000–500 Â 10
6 sperm/mL. This is done by adding one
volume per two volumes: e.g., 10 mL LEY freezing
extender to 20 mL sperm in LEY cooling extender. Add
slowly, dropwise on the side of the tube while gently
turning.
(e) This results in 1000–500 Â 10
6 sperm/mL, 20% egg yolk,
0.5% Equex paste, and 2% glycerol.
3.9 Slow Cooling
and Freezing of Sperm
Packaged in Straws
Stallion and bull sperm are cooled to 5
C after being diluted in
freezing extender at room temperature. In contrast, boar sperm is
cooled from 17 down to 5
C in cooling extender, after which
freezing extender is added at 5
C. In all cases, a slow cooling
procedure needs to be performed for cooling to 5
C.
1. Slowly cool sperm down to 5
C at ~0.1
C/min. Such a slow
cooling rate is reached by placing a tube with sperm sample of
ambient temperature (e.g., 50 mL) in a beaker with room
temperature water (e.g., 250 mL) at 5
C during 2–4 h (see
Note 14). For bull sperm, equilibration periods at 5
C up to
18 h can be used to allow sufficient time for exchange of
protective extender components and to slow down sperm
metabolism.
2. Load sperm samples in freezing extender into 0.25 mL (bull)
or 0.5 mL (stallion, boar) labeled straws while maintaining
samples at 5
C (in a cooling cabinet/cold room) (see Note
Cryopreservation of Sperm from Domestic Livestock
373
side of the tube while gently turning to ensure mixing.
(d) This results in 100–400 Â 10
6 sperm/mL, 2% egg yolk,
and 2.5% glycerol.
2. Bull sperm:
(a) Use TEY-1 (contains 20% egg yolk) for diluting raw
semen to 120 Â 10
6 sperm/mL.
(b) Add an equal volume of TEY-2 (containing 20% egg yolk
and 12.8% glycerol). Add slowly, dropwise on the side of
the tube while gently turning to ensure mixing.
(c) This results in 60 Â 10
6 sperm/mL, 20% egg yolk, and
6.4% glycerol.
3. Boar sperm:
(a) Use BTS for initial dilution, cooling sperm to 17
C, and
centrifugation (3 min, 2400 Â g, at 17
C).
(b) Use LEY cooling extender (contains 20% egg yolk) for
diluting to 1500–750 Â 10
6 sperm/mL.
(c) Slowly cool sperm to 5
C.
(d) Dilute with LEY freezing extender (containing 20% egg
yolk, 1.5% Equex paste, and 6% glycerol) for diluting to
1000–500 Â 10
6 sperm/mL. This is done by adding one
volume per two volumes: e.g., 10 mL LEY freezing
extender to 20 mL sperm in LEY cooling extender. Add
slowly, dropwise on the side of the tube while gently
turning.
(e) This results in 1000–500 Â 10
6 sperm/mL, 20% egg yolk,
0.5% Equex paste, and 2% glycerol.
3.9 Slow Cooling
and Freezing of Sperm
Packaged in Straws
Stallion and bull sperm are cooled to 5
C after being diluted in
freezing extender at room temperature. In contrast, boar sperm is
cooled from 17 down to 5
C in cooling extender, after which
freezing extender is added at 5
C. In all cases, a slow cooling
procedure needs to be performed for cooling to 5
C.
1. Slowly cool sperm down to 5
C at ~0.1
C/min. Such a slow
cooling rate is reached by placing a tube with sperm sample of
ambient temperature (e.g., 50 mL) in a beaker with room
temperature water (e.g., 250 mL) at 5
C during 2–4 h (see
Note 14). For bull sperm, equilibration periods at 5
C up to
18 h can be used to allow sufficient time for exchange of
protective extender components and to slow down sperm
metabolism.
2. Load sperm samples in freezing extender into 0.25 mL (bull)
or 0.5 mL (stallion, boar) labeled straws while maintaining
samples at 5
C (in a cooling cabinet/cold room) (see Note
Cryopreservation of Sperm from Domestic Livestock
373