1. Prepare TRIS stock solution by dissolving the following chemicals in 1 L distilled water: 36.05 g TRIS, 20.24 g citric acid
monohydrate, 14.88 g fructose, and antibiotics (see Note 6).
The pH should be adjusted to 7.0, after which the solution
should be filter-sterilized by passing through a 0.2 μm filter. If
needed, the solution can be stored at 4
C for use within
1–2 weeks.
2. For 1 L TEY-1, add together 672 mL TRIS stock solution,
128 mL distilled water, and 200 mL egg yolk (~100%
solution).
3. For 1 L TEY-2 add together 672 mL TRIS stock solution,
200 mL egg yolk (~100% solution), and 128 mL glycerol
(~100% solution).
4. In case of performing a “one-step” dilution with freezing
extender, prepare TEY extender with the desired final concentration of cryoprotective agents. Therefore, add together
672 mL TRIS stock solution, 64 mL water, 200 mL egg yolk
(~100% solution), and 64 mL glycerol (~100% solution). TEY
is also prepared by mixing equal volumes of TEY-1 and TEY-2.
3.4 Preparation
of Diluents (BTS, LEY)
With(out)
Cryoprotective Agents
for Porcine Semen
For porcine sperm, for example, Beltsville Thawing Solution (BTS)
can be used for primary dilution [11] and lactose-egg yolk (LEY)
cooling and freezing extenders for cryopreservation [12, 13]. Final
concentrations for cryopreservation are typically 20% (v/v) egg
yolk, 0.5% Equex paste (see Note 9), and 2% (v/v) glycerol.
1. Prepare BTS by dissolving the following chemicals in 1 L distilled water: 6.0 g trisodium citrate dihydrate, 1.25 g sodium
hydrogen carbonate, 0.75 g potassium chloride, 40.5 g glucose
monohydrate, 1.25 g ethylenediaminetetraacetic acid disodium salt dihydrate, and antibiotics (see Note 6). The pH
should be adjusted to 7.2 and osmolality should be
330 mOsm/kg. Filter-sterilize via passing the solution through
a 0.2 μm filter. If needed, the solution can be stored at 4
C for
use within 1–2 weeks.
2. Prepare lactose solution via dissolving 11 g lactose in 100 mL
distilled water.
3. Prepare LEY cooling extender by combining 80 mL lactose
solution and 20 mL egg yolk (~100% solution). Filter the
solution through a folded/fluted paper filter.
4. Prepare LEY freezing extender (also referred to as LEYGO:
LEY-glycerol-Orvus-ES-paste), by adding 1.5 mL Equex paste
and 6 mL glycerol (~100% solution) to 92.5 mL LEY cooling
extender.
5. The final cryopreservation solution is prepared by adding 1 volume of LEY freezing extender to 2 volumes of LEY cooling
extender.
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Harrie ¨ tte Oldenhof et al.
monohydrate, 14.88 g fructose, and antibiotics (see Note 6).
The pH should be adjusted to 7.0, after which the solution
should be filter-sterilized by passing through a 0.2 μm filter. If
needed, the solution can be stored at 4
C for use within
1–2 weeks.
2. For 1 L TEY-1, add together 672 mL TRIS stock solution,
128 mL distilled water, and 200 mL egg yolk (~100%
solution).
3. For 1 L TEY-2 add together 672 mL TRIS stock solution,
200 mL egg yolk (~100% solution), and 128 mL glycerol
(~100% solution).
4. In case of performing a “one-step” dilution with freezing
extender, prepare TEY extender with the desired final concentration of cryoprotective agents. Therefore, add together
672 mL TRIS stock solution, 64 mL water, 200 mL egg yolk
(~100% solution), and 64 mL glycerol (~100% solution). TEY
is also prepared by mixing equal volumes of TEY-1 and TEY-2.
3.4 Preparation
of Diluents (BTS, LEY)
With(out)
Cryoprotective Agents
for Porcine Semen
For porcine sperm, for example, Beltsville Thawing Solution (BTS)
can be used for primary dilution [11] and lactose-egg yolk (LEY)
cooling and freezing extenders for cryopreservation [12, 13]. Final
concentrations for cryopreservation are typically 20% (v/v) egg
yolk, 0.5% Equex paste (see Note 9), and 2% (v/v) glycerol.
1. Prepare BTS by dissolving the following chemicals in 1 L distilled water: 6.0 g trisodium citrate dihydrate, 1.25 g sodium
hydrogen carbonate, 0.75 g potassium chloride, 40.5 g glucose
monohydrate, 1.25 g ethylenediaminetetraacetic acid disodium salt dihydrate, and antibiotics (see Note 6). The pH
should be adjusted to 7.2 and osmolality should be
330 mOsm/kg. Filter-sterilize via passing the solution through
a 0.2 μm filter. If needed, the solution can be stored at 4
C for
use within 1–2 weeks.
2. Prepare lactose solution via dissolving 11 g lactose in 100 mL
distilled water.
3. Prepare LEY cooling extender by combining 80 mL lactose
solution and 20 mL egg yolk (~100% solution). Filter the
solution through a folded/fluted paper filter.
4. Prepare LEY freezing extender (also referred to as LEYGO:
LEY-glycerol-Orvus-ES-paste), by adding 1.5 mL Equex paste
and 6 mL glycerol (~100% solution) to 92.5 mL LEY cooling
extender.
5. The final cryopreservation solution is prepared by adding 1 volume of LEY freezing extender to 2 volumes of LEY cooling
extender.
370
Harrie ¨ tte Oldenhof et al.