13. Aluminum foil should be placed on LN as shortly as possible before placing microdrops onto them to avoid white
frost formation on metal surface at the time of vitrification.
When a divided cooling surface is used, LN tends to appear
on the cooling surface. LN on the surface does not reduce
the efficacy of vitrification. On the contrary, it is beneficial
because it prevents sticking of droplets to the metal surface
caused by ice formation. Therefore placing a few (1À2) mL
of LN on the cooling surface is indeed beneficial. Nevertheless, this approach cannot be considered as a solid
surface.
14. This movement takes a lot of practice. Microdrops are formed
by blowing the mouthpiece and flicking the dropping pipette
simultaneously. Avoid keeping pipettes (oocytes) in the cool
gas above the cooling surface for an extended time. Before the
actual experiments, it is advised to practice dropping vitrification medium without oocytes until transparent 1–2 μL vitrified
droplets can be achieved.
15. To avoid/reduce sticking oocytes to the dropping pipette,
approximately the first 10 mm of the dropping pipette tip
(from the opening) should contain only vitrification medium
without oocytes.
16. White (“snowball”) microdrops contain ice crystals and therefore should be omitted.
17. The use of sterile LN should be considered for a pathogen-free
approach.
18. Warming at the 42
C is crucial for immature porcine
oocytes. However, for zygotes, the warming dish can be
kept at 38
C.
19. The cooling surface should be kept as close to the warming
dish as possible. Before grabbing microdrops, the tip of the
forceps must always be cooled in LN for a few seconds.
20. This staining procedure did not exert harmful effects on the
in vitro developmental competence of matured porcine oocytes
[17]. However, to our knowledge, its toxicity has not been
tested on immature porcine oocytes.
21. New FDA working solution must be prepared as soon as it
starts to change color to green. Ideally, a new working solution
should be prepared for each group. The FDA base solution can
be kept in sealed vessels at À20
C for at least 2 days. However,
the exact shelf life of the FDA base solution has not been
determined.
Porcine Oocyte Vitrification
467
frost formation on metal surface at the time of vitrification.
When a divided cooling surface is used, LN tends to appear
on the cooling surface. LN on the surface does not reduce
the efficacy of vitrification. On the contrary, it is beneficial
because it prevents sticking of droplets to the metal surface
caused by ice formation. Therefore placing a few (1À2) mL
of LN on the cooling surface is indeed beneficial. Nevertheless, this approach cannot be considered as a solid
surface.
14. This movement takes a lot of practice. Microdrops are formed
by blowing the mouthpiece and flicking the dropping pipette
simultaneously. Avoid keeping pipettes (oocytes) in the cool
gas above the cooling surface for an extended time. Before the
actual experiments, it is advised to practice dropping vitrification medium without oocytes until transparent 1–2 μL vitrified
droplets can be achieved.
15. To avoid/reduce sticking oocytes to the dropping pipette,
approximately the first 10 mm of the dropping pipette tip
(from the opening) should contain only vitrification medium
without oocytes.
16. White (“snowball”) microdrops contain ice crystals and therefore should be omitted.
17. The use of sterile LN should be considered for a pathogen-free
approach.
18. Warming at the 42
C is crucial for immature porcine
oocytes. However, for zygotes, the warming dish can be
kept at 38
C.
19. The cooling surface should be kept as close to the warming
dish as possible. Before grabbing microdrops, the tip of the
forceps must always be cooled in LN for a few seconds.
20. This staining procedure did not exert harmful effects on the
in vitro developmental competence of matured porcine oocytes
[17]. However, to our knowledge, its toxicity has not been
tested on immature porcine oocytes.
21. New FDA working solution must be prepared as soon as it
starts to change color to green. Ideally, a new working solution
should be prepared for each group. The FDA base solution can
be kept in sealed vessels at À20
C for at least 2 days. However,
the exact shelf life of the FDA base solution has not been
determined.
Porcine Oocyte Vitrification
467