3.5 Semen Collection
and Dilution
with Primary Extender
Different methods and approaches can be employed for collecting
semen from domestic species.
1. Have all materials including the primary extender pre-warmed
at 37
C.
2. In case of collecting boar semen, collect the sperm-rich fraction
only. For stallion and bull, use the whole ejaculate. Directly
after semen collection, determine the ejaculate (gel-free) volume, appearance/consistency, and (approximate) sperm concentration (see Note 1).
3. Dilute raw semen by slowly adding a primary extender (i.e.,
INRA-82 for equine semen, TEY-1 for bovine semen, and BTS
for porcine semen). Add at least an equal volume of extender,
or dilute at ~100 Â 10
6 sperm/mL. This is referred to as
diluted semen.
4. Evaluate sperm motility and morphology using a phase contrast microscope with heated stage (see Note 10).
5. Let diluted semen sample cool down to room temperature (i.e.,
~22
C) for further handling. After attaining room temperature, diluted boar semen samples are typically cooled to 17
C.
3.6 Centrifugation
Processing
for Removal
of Seminal Plasma
and to Concentrate
the Sample
Stallion and boar sperm samples are generally subjected to centrifugation processing prior to cryopreservation, whereas bull sperm
samples are not. To increase the recovery rate while preventing
sperm packing in a dense pellet, a higher centrifugation speed can
be applied by adding cushion fluid below the sperm sample.
1. When performing ordinary centrifugation, for stallion or boar
sperm:
(a) Transfer diluted sperm sample in a tube (e.g., 50 mL
tube), and centrifuge. For stallion sperm, perform centrifugation for 10 min at 600 Â g. Perform centrifugation of
boar sperm after cooling to 17
C for 3 min at 2400 Â g.
(b) Carefully remove the supernatant using a Pasteur pipet
connected to a vacuum pump or a syringe, leaving a little
volume (~5 mL in case of 50 mL original volume) (see
Note 11). Gently resuspend the pellet, using a shaker.
2. When performing high-speed cushioned centrifugation, for
stallion sperm:
(a) Add diluted sperm in a centrifugation tube. Underlay a
small amount of cushion fluid in the bottom of the centrifugation tube, such that it covers the full diameter of the
tube (i.e., ~4 mL in case of a 50 mL tube). Centrifuge for
20 min at 1000 Â g at ambient temperature.
Cryopreservation of Sperm from Domestic Livestock
371
and Dilution
with Primary Extender
Different methods and approaches can be employed for collecting
semen from domestic species.
1. Have all materials including the primary extender pre-warmed
at 37
C.
2. In case of collecting boar semen, collect the sperm-rich fraction
only. For stallion and bull, use the whole ejaculate. Directly
after semen collection, determine the ejaculate (gel-free) volume, appearance/consistency, and (approximate) sperm concentration (see Note 1).
3. Dilute raw semen by slowly adding a primary extender (i.e.,
INRA-82 for equine semen, TEY-1 for bovine semen, and BTS
for porcine semen). Add at least an equal volume of extender,
or dilute at ~100 Â 10
6 sperm/mL. This is referred to as
diluted semen.
4. Evaluate sperm motility and morphology using a phase contrast microscope with heated stage (see Note 10).
5. Let diluted semen sample cool down to room temperature (i.e.,
~22
C) for further handling. After attaining room temperature, diluted boar semen samples are typically cooled to 17
C.
3.6 Centrifugation
Processing
for Removal
of Seminal Plasma
and to Concentrate
the Sample
Stallion and boar sperm samples are generally subjected to centrifugation processing prior to cryopreservation, whereas bull sperm
samples are not. To increase the recovery rate while preventing
sperm packing in a dense pellet, a higher centrifugation speed can
be applied by adding cushion fluid below the sperm sample.
1. When performing ordinary centrifugation, for stallion or boar
sperm:
(a) Transfer diluted sperm sample in a tube (e.g., 50 mL
tube), and centrifuge. For stallion sperm, perform centrifugation for 10 min at 600 Â g. Perform centrifugation of
boar sperm after cooling to 17
C for 3 min at 2400 Â g.
(b) Carefully remove the supernatant using a Pasteur pipet
connected to a vacuum pump or a syringe, leaving a little
volume (~5 mL in case of 50 mL original volume) (see
Note 11). Gently resuspend the pellet, using a shaker.
2. When performing high-speed cushioned centrifugation, for
stallion sperm:
(a) Add diluted sperm in a centrifugation tube. Underlay a
small amount of cushion fluid in the bottom of the centrifugation tube, such that it covers the full diameter of the
tube (i.e., ~4 mL in case of a 50 mL tube). Centrifuge for
20 min at 1000 Â g at ambient temperature.
Cryopreservation of Sperm from Domestic Livestock
371