5. Pick up the microdrops with small forceps, and drop them into
the warming dish kept on the warm plate (see Notes 18 and
19). Several (up to 6) microdrops can be transferred consecutively in the warming dish at once [6]. After transferring each
microdrop into the warming dish, mix its content by a gentle
shake (still, on the warm plate). Then move the warming dish
to the stereo microscope. Gather oocytes/zygotes in one
group in the center of the warming dish with a glass transfer
pipette under the stereo microscope. The oocytes/zygotes are
kept in the warming dish for 2 min.
6. Move all oocytes/zygotes from the warming dish into the first
well of the re-hydration dish (0.2 M sucrose), and mix it by
gentle shaking. Gather oocytes/zygotes in one group in the
center of the well. The oocytes/zygotes are kept in this well for
1 min.
7. Move all oocytes/zygotes into the second well of the
re-hydration dish (0.1 M sucrose), and mix it by gentle shaking. Gather oocytes/zygotes in one group in the center of the
well. The oocytes/zygotes are kept in this well for 1 min.
8. Move all oocytes/zygotes into the third well of the
re-hydration dish (0.05 M sucrose), and mix it by gentle shaking. Gather oocytes/zygotes in one group in the center of the
well. The oocytes/zygotes are kept in this well for 1 min.
9. Move all oocytes/zygotes into the fourth well of the
re-hydration dish (BM) and mix it by gentle shaking. Gather
oocytes/zygotes in one group in the center of the well. The
oocytes/zygotes are kept in this well for 3 min.
3.5 Subsequent
Culture and Evaluation
of Live/Dead Status
To generate embryos, vitrified/warmed oocytes can be subjected to
IVM and IVF as reported previously [6, 14, 15]. Vitrified/warmed
zygotes are subjected to in vitro embryo culture as reported previously [7–9]. Live/dead status of the oocytes and zygotes can be
assayed by morphological evaluation under a stereo microscope or
by supravital staining. When evaluated by morphological appearance, oocytes with an intact oolemma showing a normal spherical
shape, a definite smooth surface, and a dark and evenly granular
cytoplasm are considered alive (see Fig. 5). Oocytes with membrane
damage and brownish faded cytoplasm are considered dead (see
Fig. 5). However, when CEOs are vitrified, such evaluation is
reliable only after IVM and IVF, when the cumulus cells are
removed. Live/dead status of CEOs can be assayed by viability
staining using fluorescein diacetate (FDA), following the method
of Mohr and Trounson [16] (see Note 20). The steps of the FDA
staining are as follows:
464
Tamas Somfai and Kazuhiro Kikuchi
the warming dish kept on the warm plate (see Notes 18 and
19). Several (up to 6) microdrops can be transferred consecutively in the warming dish at once [6]. After transferring each
microdrop into the warming dish, mix its content by a gentle
shake (still, on the warm plate). Then move the warming dish
to the stereo microscope. Gather oocytes/zygotes in one
group in the center of the warming dish with a glass transfer
pipette under the stereo microscope. The oocytes/zygotes are
kept in the warming dish for 2 min.
6. Move all oocytes/zygotes from the warming dish into the first
well of the re-hydration dish (0.2 M sucrose), and mix it by
gentle shaking. Gather oocytes/zygotes in one group in the
center of the well. The oocytes/zygotes are kept in this well for
1 min.
7. Move all oocytes/zygotes into the second well of the
re-hydration dish (0.1 M sucrose), and mix it by gentle shaking. Gather oocytes/zygotes in one group in the center of the
well. The oocytes/zygotes are kept in this well for 1 min.
8. Move all oocytes/zygotes into the third well of the
re-hydration dish (0.05 M sucrose), and mix it by gentle shaking. Gather oocytes/zygotes in one group in the center of the
well. The oocytes/zygotes are kept in this well for 1 min.
9. Move all oocytes/zygotes into the fourth well of the
re-hydration dish (BM) and mix it by gentle shaking. Gather
oocytes/zygotes in one group in the center of the well. The
oocytes/zygotes are kept in this well for 3 min.
3.5 Subsequent
Culture and Evaluation
of Live/Dead Status
To generate embryos, vitrified/warmed oocytes can be subjected to
IVM and IVF as reported previously [6, 14, 15]. Vitrified/warmed
zygotes are subjected to in vitro embryo culture as reported previously [7–9]. Live/dead status of the oocytes and zygotes can be
assayed by morphological evaluation under a stereo microscope or
by supravital staining. When evaluated by morphological appearance, oocytes with an intact oolemma showing a normal spherical
shape, a definite smooth surface, and a dark and evenly granular
cytoplasm are considered alive (see Fig. 5). Oocytes with membrane
damage and brownish faded cytoplasm are considered dead (see
Fig. 5). However, when CEOs are vitrified, such evaluation is
reliable only after IVM and IVF, when the cumulus cells are
removed. Live/dead status of CEOs can be assayed by viability
staining using fluorescein diacetate (FDA), following the method
of Mohr and Trounson [16] (see Note 20). The steps of the FDA
staining are as follows:
464
Tamas Somfai and Kazuhiro Kikuchi