High-throughput and non-invasive method to vitrify porcine embryos
The present invention provides a practical, non-invasive, and efficient method for cryopreservation of an in-vitro-produced porcine embryo. The inventive method treats the NP (such as IVF- or NT-derived) embryo at the one-cell or cleavage stage prior to compaction with high osmolality followed by high speed centrifugation. The high osmolality treatment enlarges the periviteline space, and with centrifugation, enables the separation of the lipids from the cytoplasm. The lipid-separated embryos after high osmolality treatment have been successfully cryopreserved and later recovered and transferred to produce live offspring.
1. A method for delipation of an in vitro produced porcine embryo comprising:
incubating an in vitro produced porcine embryo in a medium having an osmolality of 350 mOsm to 600 mOsm to produce a condensed embryo; and
centrifuging the condensed embryo to separate lipids from cytoplasm to produce a lipid-separated embryo.
2. The method of claim 1 , further comprising cryopreserving the in vitro produced porcine embryo, wherein said preserving comprises:
culturing the lipid-separated embryo to the blastocyst stage to produce a lipid-separated blastocyst; and
cryopreserving the blastocyst by vitrification or freezing to produce a cryopreserved embryo.
3. The method of claim 2 further comprising recovery and transfer of the cryopreserved embryo into a recipient, wherein said recovery and transfer comprise:
warming and rehydrating the cryopreserved embryo;
removing the zona pellucida from the embryo; and
transferring the embryo into a recipient.
4. The method of claim 1 , wherein the medium has an osmolality of 350 mOsm to 500 mOsm, or 350 mOsm to 450 mOsm.
5. The method of claim 1 , wherein the medium has an osmolality of 350 mOsm, 400 mOsm, or 500 mOsm.
6. The method of claim 1 , wherein the osmolality of the medium is adjusted by the addition of a salt, a sugar, or an organic reagent to produce the medium having an osmolality.
7. The method of claim 6 , wherein the salt is sodium chloride; wherein the sugar is sucrose, raffinose, fructose, mannitol, or trehalose; or wherein the organic reagent is dimethyl sulfoxide (DMSO) or ethylene glycol.
8. The method of claim 7 wherein the osmolality of the medium is adjusted by the addition of sodium chloride or sucrose to produce the medium having an osmolality, the osmolality being 350 mOsm, 400 mOsm, or 500 mOsm.
9. The method of claim 8 , wherein the medium is adjusted by the addition of sodium chloride or sucrose to produce the medium having an osmolality, the osmolality being 400 mOsm.
10. The method of claim 1 , wherein the embryo is produced by in vitro fertilization or nuclear transfer.
11. The method of claim 1 , wherein the embryo to be incubated in the medium is at the one cell or cleavage stage, prior to compaction.
12. The method of claim 1 wherein the embryo is incubated in the medium for a period of 5 to 10 minutes and wherein the embryo is centrifuged at 13,400×g for 6 to 20 minutes.
13. The method claim 1 wherein multiple embryos are delipated at the same time.
14. A method for delipation, cryopreservation, and recovery of an in vitro produced porcine embryo comprising:
incubating an in vitro produced porcine embryo in a medium having an osmolality of 350 mOsm to 600 mOsm to produce a condensed embryo;
centrifuging the condensed embryo to separate lipids from cytoplasm to produce a lipid-separated embryo;
culturing the lipid-separated embryo to the blastocyst stage to produce a lipid-separated blastocyst;
cryopreserving the blastocyst by vitrification or freezing to produce a cryopreserved embryo;
warming and rehydrating the cryopreserved embryo;
removing the zona pellucida from the embryo; and
transferring the embryo into a recipient.
15. The method of claim 14 , wherein the medium has an osmolality of 350 mOsm to 500 mOsm, or 350 mOsm to 450 mOsm.
16. The method of claim 14 , wherein the medium has an osmolality of 350 mOsm, 400 mOsm, or 500 mOsm.
17. The method of claim 14 , wherein the osmolality of the medium is adjusted by the addition of a salt, a sugar, or an organic reagent.
18. The method of claim 17 , wherein the salt is sodium chloride; wherein the sugar is sucrose, raffinose, fructose, mannitol, or trehalose; or wherein the organic reagent is dimethyl sulfoxide (DMSO) or ethylene glycol.
19. The method of claim 14 , wherein the embryo to be incubated in the medium is at the one cell or cleavage stage, prior to compaction.
20. The method claim 14 wherein multiple embryos are delipated at the same time.