5. Two long forcipes to handle cryo-vials in LN.
6. A fine tweezer (10–11 cm) to handle vitrified microdrops. For
the firm grip, consider the use of a cross-action tweezer
(as described in Subheading 2.2).
7. A Styrofoam box (parameters as above).
8. A single cooling surface prepared from aluminum foil as
described above (see Fig. 1a).
9. Injection needles or drawing pins (3 pieces, any type) to fix the
cooling surface.
10. Block tube heater (such as Corning LSE Dipike Dry Bath).
11. LN.
12. LN tank.
13. Timer.
2.4 Tools
for Subsequent Culture
After Warming
and Evaluation of Live/
Dead Status
of Oocytes/Zygotes
1. A stereo microscope for the evaluation of live/dead status of
oocytes/zygotes by morphology and visualization during
handling (such as Olympus SZX16).
2. A portable warm plate for handling oocytes/zygotes under the
stereo microscope (as described earlier).
3. Media for the subsequent culture of vitrified immature oocytes
after warming prepared according to Somfai et al. [14]. For the
subsequent culture of vitrified zygotes prepare media and culture dishes as according to Yoshioka et al. [15].
4. A water-jacketed CO 2 incubator for oocyte/zygote culture.
5. Fluorescein diacetate (FDA) for viability staining (optional).
6. Acetone for viability staining (optional).
7. A cryo-vial and a 4-well dish for viability staining (see Note 8).
8. A phenol red-free oocyte handling medium (recommended:
PXM-Hepes [15]) for viability staining (optional).
9. A fluorescence stereo microscope (such as MZ16FA; Leica,
Tokyo, Japan) or inverted microscope equipped with fluorescent device and filter to detect green fluorescence (see Note 9)
for viability staining (optional).
3 Methods
3.1 Setting Up
for Vitrification
1. Dehumidify the air of the room (see Note 10).
2. Pre-warm 1 mL aliquots of BM, EM, and VM to 25
C for each
oocyte/zygote lot to be vitrified together as a group.
3. Prepare glass pipettes and aluminum foil cooling device.
Porcine Oocyte Vitrification
459
6. A fine tweezer (10–11 cm) to handle vitrified microdrops. For
the firm grip, consider the use of a cross-action tweezer
(as described in Subheading 2.2).
7. A Styrofoam box (parameters as above).
8. A single cooling surface prepared from aluminum foil as
described above (see Fig. 1a).
9. Injection needles or drawing pins (3 pieces, any type) to fix the
cooling surface.
10. Block tube heater (such as Corning LSE Dipike Dry Bath).
11. LN.
12. LN tank.
13. Timer.
2.4 Tools
for Subsequent Culture
After Warming
and Evaluation of Live/
Dead Status
of Oocytes/Zygotes
1. A stereo microscope for the evaluation of live/dead status of
oocytes/zygotes by morphology and visualization during
handling (such as Olympus SZX16).
2. A portable warm plate for handling oocytes/zygotes under the
stereo microscope (as described earlier).
3. Media for the subsequent culture of vitrified immature oocytes
after warming prepared according to Somfai et al. [14]. For the
subsequent culture of vitrified zygotes prepare media and culture dishes as according to Yoshioka et al. [15].
4. A water-jacketed CO 2 incubator for oocyte/zygote culture.
5. Fluorescein diacetate (FDA) for viability staining (optional).
6. Acetone for viability staining (optional).
7. A cryo-vial and a 4-well dish for viability staining (see Note 8).
8. A phenol red-free oocyte handling medium (recommended:
PXM-Hepes [15]) for viability staining (optional).
9. A fluorescence stereo microscope (such as MZ16FA; Leica,
Tokyo, Japan) or inverted microscope equipped with fluorescent device and filter to detect green fluorescence (see Note 9)
for viability staining (optional).
3 Methods
3.1 Setting Up
for Vitrification
1. Dehumidify the air of the room (see Note 10).
2. Pre-warm 1 mL aliquots of BM, EM, and VM to 25
C for each
oocyte/zygote lot to be vitrified together as a group.
3. Prepare glass pipettes and aluminum foil cooling device.
Porcine Oocyte Vitrification
459