4. Measure the osmolality (270–290 mOsm/kg) and pH (~7.2)
and record, and filter into an appropriately sized sterile bottle
using 0.22 μm pore size filters.
5. Aliquot into desired volume, and label the tubes with make and
expiration date (3 months from make date), initials, and batch
number.
6. Store in À20
C freezer for up to 3 months.
3.3 Preparation
of FERTIUP Medium
TYH + MBCD (FERTIUP) is a bicarbonate-buffered medium
which was formulated to perform post-thaw sperm preincubation
for capacitation before in vitro fertilization in mice. It can only be
used in incubators having 5% CO 2 and 95% humidified air at 37
C.
1. Bring an appropriate size Griffin beaker, pour embryo transfer
water (~80% of the total volume), and then add the following
components in gram as depicted in Table 3. All the recipes are
calculated based on 1 L final volume, so adjust the volume of
the media for your needs (see Note 1).
2. Solubilize the CaCl 2 .2H 2 O and polyvinyl alcohol separately in
a small volume of water, and add it at the end. After all components are completely dissolved in Griffin beaker, transfer the
contents to the appropriate volumetric flask, and bring to 1 L
with embryo transfer water.
3. Gas the media with 5% CO 2 /nitrogen balance for ~30 min.
4. Measure the osmolality (280–295 mOsm/kg), pH (7.2–7.4)
and record.
Table 3
Composition of sperm preincubation medium (TYH + MBCD)
Components
Company
Add per L (g)
Concentration (mM)
CaCl 2 .2H 2 O
Sigma C7902
0.251
1.71
KCl
Sigma P5405
0.356
4.78
NaCl
Sigma S5886
6.976
119.37
NaHCO 3
Sigma S5761
2.106
25.07
D-glucose
Sigma G6152
1.00
5.56
Sodium pyruvate
Sigma P4562
0.055
0.5
Penicillin G potassium salt
Sigma P7794
0.075
N/a
Streptomycin sulfate
Sigma S1277
0.05
N/a
KH 2 PO 4
Sigma P5655
0.162
1.19
MgSO 4 .7H 2 O
Sigma M1880
0.293
1.19
Methyl-β-cyclodextrin
Sigma C-4555
0.983
0.75
Polyvinyl alcohol (30–70,000 mw)
Sigma P8136
1.00
N/a
406
Yuksel Agca and Cansu Agca
and record, and filter into an appropriately sized sterile bottle
using 0.22 μm pore size filters.
5. Aliquot into desired volume, and label the tubes with make and
expiration date (3 months from make date), initials, and batch
number.
6. Store in À20
C freezer for up to 3 months.
3.3 Preparation
of FERTIUP Medium
TYH + MBCD (FERTIUP) is a bicarbonate-buffered medium
which was formulated to perform post-thaw sperm preincubation
for capacitation before in vitro fertilization in mice. It can only be
used in incubators having 5% CO 2 and 95% humidified air at 37
C.
1. Bring an appropriate size Griffin beaker, pour embryo transfer
water (~80% of the total volume), and then add the following
components in gram as depicted in Table 3. All the recipes are
calculated based on 1 L final volume, so adjust the volume of
the media for your needs (see Note 1).
2. Solubilize the CaCl 2 .2H 2 O and polyvinyl alcohol separately in
a small volume of water, and add it at the end. After all components are completely dissolved in Griffin beaker, transfer the
contents to the appropriate volumetric flask, and bring to 1 L
with embryo transfer water.
3. Gas the media with 5% CO 2 /nitrogen balance for ~30 min.
4. Measure the osmolality (280–295 mOsm/kg), pH (7.2–7.4)
and record.
Table 3
Composition of sperm preincubation medium (TYH + MBCD)
Components
Company
Add per L (g)
Concentration (mM)
CaCl 2 .2H 2 O
Sigma C7902
0.251
1.71
KCl
Sigma P5405
0.356
4.78
NaCl
Sigma S5886
6.976
119.37
NaHCO 3
Sigma S5761
2.106
25.07
D-glucose
Sigma G6152
1.00
5.56
Sodium pyruvate
Sigma P4562
0.055
0.5
Penicillin G potassium salt
Sigma P7794
0.075
N/a
Streptomycin sulfate
Sigma S1277
0.05
N/a
KH 2 PO 4
Sigma P5655
0.162
1.19
MgSO 4 .7H 2 O
Sigma M1880
0.293
1.19
Methyl-β-cyclodextrin
Sigma C-4555
0.983
0.75
Polyvinyl alcohol (30–70,000 mw)
Sigma P8136
1.00
N/a
406
Yuksel Agca and Cansu Agca