Cryopreservation of swine embryos

• US 6,503,698 B1
• Filed: 06/16/2000
• Issued: 01/07/2003
• Est. Priority Date: 06/16/2000
• Status: Expired due to Fees

• Current Assignee: United States Department Of Agriculture
• Original Assignee: United States Department Of Agriculture
• Inventors: Nagashima, Hiroshi, Dobrinsky, John R.
• Primary Examiner(s): Tate, Christopher R.
• Assistant Examiner(s): Flood, Michele C.
• Application Number:
  US09/594,704
• Time in Patent Office: 935 Days
• Field of Search: 435/1.3, 435/374, 435/378, 435/404, 530/853, 600/34, 604/55, 424/582
• US Class Current: 435/1.3
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0221 Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents

Abstract

A method for the cryopreservation of zona pellucida-intact, early-stage swine embryos, morulae and blastocysts includes centrifuging the embryos prior to cryopreservation in order to remove intracytoplasmic lipids. The lipids are localized external to the embryo proper inside the zona pellucida, thus neutralizing harmful effects which prevent subsequent embryological development. The embryos may be cryopreserved by vitrification or conventional freezing. After recovery and rehydration, zona pellucidae are removed, and intact, viable embryos are transferred to recipient females.

Claims

1. A process for the cryopreservation and recovery of swine embryos having intact zona pellucidae comprising providing swine embryos, culturing said embryos, centrifuging the cultured embryos, equilibrating the centrifuged embryos, cryopreserving the centrifuged and equilibrated embryos by vitrification or freezing, storing the cryopreserved embryos, and recovering the cryopreserved embryos having intact zona pellucidae, wherein said recovery process comprises warming vitrified embryos or thawing frozen embryos, rehydrating the embryos and removing the zona pellucidae of rehydrated embryos.

2. The process of claim 1, where said culturing, centrifuging, equilibrating, cryopreserving and storing the embryos is carried out in the presence of at least one cryoprotectant agent and/or cytoskeletal and cellular stabilizing agent, and wherein the concentration of said agent is diluted in warmed or thawed embryos during the recovery process.

3. The process of claim 2, wherein said cytoskeletal and cellular stabilizing agent is cytochalasin.

4. The process of claim 3, wherein said cytochalasin is cytochalasin-b.

5. The vitrification process of claim 1, wherein said centrifugation and equilibration occur simultaneously.

6. The process of claim 5, wherein centrifugation and equilibration are carried out for an amount of time effective for a sufficient amount of lipids present in embryos to localize outside an embryo within the zona pellucida such that toxic effects of the lipids are neutralized during cryopreservation and storage, and for dehydration of embryos.

7. The freezing process of claim 1, wherein said centrifugation is carried out for an amount of time effective for a sufficient amount of lipids present in embryos to localize outside an embryo within the zona pellucida such that toxic effects of the lipids are neutralized during cryopreservation and storage, followed by equilibration effective for dehydration of embryos.

8. The process of claim 1, wherein said warming or thawing is carried out in a water or alcohol bath.

9. The process of claim 8, wherein said warming or thawing in said water or alcohol bath is carried out at room temperature.

10. The process of claim 2, wherein said diluting and rehydrating is carried out sufficient for replacing intracellular fluid lost during dehydration.

11. The process of claim 1, wherein zona pellucidae are removed by treatment with a proteolytic enzyme or acidified culture medium or by mechanical removal.

12. The process of claim 1, wherein the zona pellucidae are removed by treatment with a proteolytic enzyme.

13. The process of claim 12, wherein the proteolytic enzyme is protease or pronase.

14. The process of claim 1, wherein, after removal of the zona pellucidae, embryos are cultured for a time sufficient to allow cytoskeletal repolymerization and cellular stabilization to occur.

15. A cryopreserved swine embryo effective for embryo transfer prepared according to the process of any of claims 1, 2-7, or 8-14.