2. Remove the straw containing the embryo from the liquid
nitrogen tank, and place it in a styrofoam box containing liquid
nitrogen, for handling in the lab.
3. While keeping the straw with the embryo under liquid nitrogen, remove the label and, using a pair of fine tweezers, remove
the hemistraw from the 0.5 mL straw. Take care in performing
the whole procedure while maintaining all materials in liquid
nitrogen.
4. Take the hemistraw from the liquid nitrogen, and quickly
submerge the tip containing the embryo in well 1 of the
warming dish which is kept at 37
C (see Note 8). Start the
timer set for 5 min. After 1 min, transfer the embryo to well
2, also containing WS1.
5. With the minimum volume possible, take the embryo and
transfer to well 3. Incubate in WS2 for 5 min.
6. With the minimum volume possible, take the embryo and
transfer to well 4. Incubate in HS for 5 min.
7. Hereafter the embryo can be used for transfer into a mare or
for in vitro culture.
4 Notes
1. Here we describe the use of a hemistraw as a carrier for vitrification and storage of the embryos. Other carriers such as cryotops or cryoleafs can be used as well.
2. Use protective measures when working with liquid nitrogen.
3. When using ampoules containing DMSO, use within 3 weeks
after opening.
4. During the aspiration of the blastocoel fluid of the blastocyst,
the micropipette should be moved inside the embryo to avoid
the interruption of the aspiration by blockage of the micropipette opening with embryonic cells. The embryo will fold and
collapse during aspiration, and the micropipette should be
moved within the blastocoel capsule to ensure continuous
flow of the fluid.
5. Immediately after collapsing, the embryos will start accumulating fluid inside the blastocoel cavity in order to recover their
size. This should be avoided by starting the vitrification process
immediately after micromanipulation for collapse. The accumulation of fluid in the blastocoel cavity may result in ice
formation during vitrification and the death of the embryo.
Therefore, before starting the micromanipulation for collapsing the embryo, the operator should have the vitrification
materials ready to avoid any delays.
Vitrification of Equine Embryos After Blastocoel Aspiration
521
nitrogen tank, and place it in a styrofoam box containing liquid
nitrogen, for handling in the lab.
3. While keeping the straw with the embryo under liquid nitrogen, remove the label and, using a pair of fine tweezers, remove
the hemistraw from the 0.5 mL straw. Take care in performing
the whole procedure while maintaining all materials in liquid
nitrogen.
4. Take the hemistraw from the liquid nitrogen, and quickly
submerge the tip containing the embryo in well 1 of the
warming dish which is kept at 37
C (see Note 8). Start the
timer set for 5 min. After 1 min, transfer the embryo to well
2, also containing WS1.
5. With the minimum volume possible, take the embryo and
transfer to well 3. Incubate in WS2 for 5 min.
6. With the minimum volume possible, take the embryo and
transfer to well 4. Incubate in HS for 5 min.
7. Hereafter the embryo can be used for transfer into a mare or
for in vitro culture.
4 Notes
1. Here we describe the use of a hemistraw as a carrier for vitrification and storage of the embryos. Other carriers such as cryotops or cryoleafs can be used as well.
2. Use protective measures when working with liquid nitrogen.
3. When using ampoules containing DMSO, use within 3 weeks
after opening.
4. During the aspiration of the blastocoel fluid of the blastocyst,
the micropipette should be moved inside the embryo to avoid
the interruption of the aspiration by blockage of the micropipette opening with embryonic cells. The embryo will fold and
collapse during aspiration, and the micropipette should be
moved within the blastocoel capsule to ensure continuous
flow of the fluid.
5. Immediately after collapsing, the embryos will start accumulating fluid inside the blastocoel cavity in order to recover their
size. This should be avoided by starting the vitrification process
immediately after micromanipulation for collapse. The accumulation of fluid in the blastocoel cavity may result in ice
formation during vitrification and the death of the embryo.
Therefore, before starting the micromanipulation for collapsing the embryo, the operator should have the vitrification
materials ready to avoid any delays.
Vitrification of Equine Embryos After Blastocoel Aspiration
521