3.2 Preparation
of Diluents (INRA-82)
With(out)
Cryoprotective Agents
for Equine Semen
For stallion sperm, for example, INRA-82 skim milk extender can
be used as a primary extender [9], while this can be supplemented
with clarified egg yolk and glycerol for use as freezing extender.
Final concentrations for cryopreservation are typically 2% (v/v)
clarified egg yolk and 2.5% (v/v) glycerol (see Note 5).
1. Prepare buffered glucose saline solution by dissolving the following chemicals in 500 mL distilled water: 25 g glucose
(anhydrous), 1.5 g lactose monohydrate, 1.5 g raffinose pentahydrate, 0.25 g sodium citrate dihydrate, 0.41 g potassium
citrate monohydrate, 4.76 g HEPES, and antibiotics (see Note
6). Mix well, via using a magnetic stirrer. The pH should be
adjusted to 7.2 using KOH and osmolality to ~330 mOsm/kg
by adding water or glucose if needed (see Note 7). Filtersterilize the solution by passing through a 0.2 μm filter, and
store at 4
C for use within 1–2 weeks. This solution can be
frozen as aliquots (e.g., of 50 mL) for later use.
2. INRA-82, for primary dilution and cooled storage, is prepared
by mixing equal volumes (typically 50 or 500 mL each) of the
above described buffered glucose saline solution and commercial 0.3% ultra-heat-treated skim milk (see Note 8). This should
give a pH of 6.8–7.0 and osmolality of 300–330 mOsm/kg.
Prepare fresh, prior to use. If needed, the solution can be
stored at 4
C for use within 3 days.
3. For preparing INRA-82 freezing extender, first prepare modified buffered saline solution by adding the abovementioned
amounts of chemicals in 370 mL (instead of 500 mL as
described above). Then, prepare 1 L INRA-82 freezing
extender by also adding 500 mL commercial 0.3% ultra-heattreated skim milk, 80 mL clarified egg yolk (~50% solution,
prepared as described above), and 50 mL glycerol (~100%
solution). Mix well, and if needed freeze as aliquots for
later use.
4. In case of performing a “one-step” dilution with freezing
extender (i.e., direct addition to a sperm pellet resuspended
in a little volume as remaining after centrifugation), prepare
INRA-82 containing 2% (v/v) egg yolk and 2.5% (v/v) glycerol. This can be done by mixing equal volumes of INRA-82
and INRA-82 freezing extender.
3.3 Preparation
of Diluents (TEY)
With(out)
Cryoprotective Agents
for Bovine Semen
For bull sperm, for example, TRIS-egg yolk (TEY) extenders can be
used [10, and references therein]. Both TEY-1 and TEY-2 contain
egg yolk, whereas TEY-2 added for cryopreservation also includes
glycerol. Solutions are typically prepared fresh/prior to use. Final
concentrations for cryopreservation are 20% (v/v) egg yolk and
6.4% (v/v) glycerol.
Cryopreservation of Sperm from Domestic Livestock
369
of Diluents (INRA-82)
With(out)
Cryoprotective Agents
for Equine Semen
For stallion sperm, for example, INRA-82 skim milk extender can
be used as a primary extender [9], while this can be supplemented
with clarified egg yolk and glycerol for use as freezing extender.
Final concentrations for cryopreservation are typically 2% (v/v)
clarified egg yolk and 2.5% (v/v) glycerol (see Note 5).
1. Prepare buffered glucose saline solution by dissolving the following chemicals in 500 mL distilled water: 25 g glucose
(anhydrous), 1.5 g lactose monohydrate, 1.5 g raffinose pentahydrate, 0.25 g sodium citrate dihydrate, 0.41 g potassium
citrate monohydrate, 4.76 g HEPES, and antibiotics (see Note
6). Mix well, via using a magnetic stirrer. The pH should be
adjusted to 7.2 using KOH and osmolality to ~330 mOsm/kg
by adding water or glucose if needed (see Note 7). Filtersterilize the solution by passing through a 0.2 μm filter, and
store at 4
C for use within 1–2 weeks. This solution can be
frozen as aliquots (e.g., of 50 mL) for later use.
2. INRA-82, for primary dilution and cooled storage, is prepared
by mixing equal volumes (typically 50 or 500 mL each) of the
above described buffered glucose saline solution and commercial 0.3% ultra-heat-treated skim milk (see Note 8). This should
give a pH of 6.8–7.0 and osmolality of 300–330 mOsm/kg.
Prepare fresh, prior to use. If needed, the solution can be
stored at 4
C for use within 3 days.
3. For preparing INRA-82 freezing extender, first prepare modified buffered saline solution by adding the abovementioned
amounts of chemicals in 370 mL (instead of 500 mL as
described above). Then, prepare 1 L INRA-82 freezing
extender by also adding 500 mL commercial 0.3% ultra-heattreated skim milk, 80 mL clarified egg yolk (~50% solution,
prepared as described above), and 50 mL glycerol (~100%
solution). Mix well, and if needed freeze as aliquots for
later use.
4. In case of performing a “one-step” dilution with freezing
extender (i.e., direct addition to a sperm pellet resuspended
in a little volume as remaining after centrifugation), prepare
INRA-82 containing 2% (v/v) egg yolk and 2.5% (v/v) glycerol. This can be done by mixing equal volumes of INRA-82
and INRA-82 freezing extender.
3.3 Preparation
of Diluents (TEY)
With(out)
Cryoprotective Agents
for Bovine Semen
For bull sperm, for example, TRIS-egg yolk (TEY) extenders can be
used [10, and references therein]. Both TEY-1 and TEY-2 contain
egg yolk, whereas TEY-2 added for cryopreservation also includes
glycerol. Solutions are typically prepared fresh/prior to use. Final
concentrations for cryopreservation are 20% (v/v) egg yolk and
6.4% (v/v) glycerol.
Cryopreservation of Sperm from Domestic Livestock
369