well, and add it to the third well, and mix to achieve a sucrose
concentration of 0.05 M. Then remove 500 μL of medium
from the third well. The fourth well contains BM. Keep this
dish on the 38.0
C stage of the stereo microscope.
6. Prepare the Styrofoam box and LN.
3.4 Warming
1. Fill the Styrofoam box can with LN leaving at least 1 cm rim on
the box.
2. Take out the tube-holding cane with the cryo-vial containing
the desired lot of oocytes to be warmed, and place it in the
Styrofoam box submerged at least partially in LN.
3. Place the aluminum foil cooling surface on LN, and fix it with
needles (see Fig. 4).
4. Remove the cryo-vial from the cane using long forceps. Then
keep it partially submerged in LN, remove the lid with another
forceps, and unload (pour) the content (both LN and microdrops) on the cooling surface. Then fill the cryo-vial with LN,
and repeat the procedure to make sure all droplets are
unloaded.
Fig. 4 Steps of warming and re-hydration of porcine oocytes/zygotes vitrified in microdrops. The cryo-vial
containing a vitrified lot of oocytes/zygotes is taken out from the LN tank, and its content is unloaded (poured)
onto an aluminum cooling surface prepared as described earlier. First, the microdrops are transferred
consecutively with pre-cooled forceps into 2.5 mL of WM kept on a warm plate either at 42
C (for immature
oocytes) or 38
C (for zygotes) and mixed. After incubation for 2–3 min, the oocytes/zygotes are gathered and
transferred as a group for periods of 1 min (each) to 500 μL droplets of BM supplemented with 0.2, 0.1, or
0.05 M sucrose at 38.0
C. Finally, the group of oocytes/zygotes is incubated in BM for 3 min
Porcine Oocyte Vitrification
463
concentration of 0.05 M. Then remove 500 μL of medium
from the third well. The fourth well contains BM. Keep this
dish on the 38.0
C stage of the stereo microscope.
6. Prepare the Styrofoam box and LN.
3.4 Warming
1. Fill the Styrofoam box can with LN leaving at least 1 cm rim on
the box.
2. Take out the tube-holding cane with the cryo-vial containing
the desired lot of oocytes to be warmed, and place it in the
Styrofoam box submerged at least partially in LN.
3. Place the aluminum foil cooling surface on LN, and fix it with
needles (see Fig. 4).
4. Remove the cryo-vial from the cane using long forceps. Then
keep it partially submerged in LN, remove the lid with another
forceps, and unload (pour) the content (both LN and microdrops) on the cooling surface. Then fill the cryo-vial with LN,
and repeat the procedure to make sure all droplets are
unloaded.
Fig. 4 Steps of warming and re-hydration of porcine oocytes/zygotes vitrified in microdrops. The cryo-vial
containing a vitrified lot of oocytes/zygotes is taken out from the LN tank, and its content is unloaded (poured)
onto an aluminum cooling surface prepared as described earlier. First, the microdrops are transferred
consecutively with pre-cooled forceps into 2.5 mL of WM kept on a warm plate either at 42
C (for immature
oocytes) or 38
C (for zygotes) and mixed. After incubation for 2–3 min, the oocytes/zygotes are gathered and
transferred as a group for periods of 1 min (each) to 500 μL droplets of BM supplemented with 0.2, 0.1, or
0.05 M sucrose at 38.0
C. Finally, the group of oocytes/zygotes is incubated in BM for 3 min
Porcine Oocyte Vitrification
463