Methods and apparatus for reducing protein content in sperm cell extenders

• US 7,618,770 B2
• Filed: 09/02/2005
• Issued: 11/17/2009
• Est. Priority Date: 07/29/2005
• Status: Active Grant

• Current Assignee: XY, LLC (Inguran LLC)
• Original Assignee: XY Incorporated (Inguran LLC)
• Inventors: Schenk, John L.
• Primary Examiner(s): Davis; Ruth A
• Application Number:
  US11/219,607
• Publication Number:
  US 20070026378A1
• Time in Patent Office: 1,537 Days
• Field of Search: 435/2, 435/325, 424/93.7
• US Class Current: 435/2
• 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

The inventive technology relates to methods and apparatus for reducing protein content in sperm cell extenders and may include one or more of the following features: techniques for reducing protein content in a sperm cell extender; techniques for reducing protein content in a cryoprotectant-containing B fraction of a sperm cell extender; techniques for preparing sperm cell extenders that do not require clarification; techniques for preparing low density gradient sperm cell extenders suitable for centrifugation; techniques for reducing protein content between individual steps in preparing a sperm cell extender, and techniques for establishing novel values of reduced protein content in sperm cell extenders.

Claims

1. A method for freezing sorted sperm cells compromised by a sorting event comprising the steps of:

obtaining a plurality of sperm cells;

subjecting said plurality of sperm cells to sorting stresses;

selecting said plurality of sperm cells for a desired characteristic;

adding a protein-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture;

cooling said first extended sperm cell mixture to create a first cooled extended sperm cell mixture;

adding a protein-free cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture;

freezing said second cooled extended sperm cell mixture.

2. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of obtaining a plurality of sperm cells comprises the step of obtaining a plurality of mammalian sperm cells.

3. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 2, wherein said step of obtaining a plurality of mammalian sperm cells comprises the step of obtaining a plurality of mammalian sperm cells selected from the group consisting of bovine sperm cells, equine sperm cells, porcine sperm cells, ovine sperm cells, cervid sperm cells, canine sperm cells, and delphinidae sperm cells.

4. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of selecting said plurality of sperm cells to sorting stresses comprises the step of separating said selected sperm cells having a desired characteristic.

5. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 4, wherein said step of separating said selected sperm cells having a desired characteristic comprises the step of separating said selected sperm cells having a desired characteristic by flow cytometery.

6. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 5, wherein said step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to a pre-freeze sperm cell concentration comprises the step establishing a pre-freeze egg yolk content of said second extended sperm cell mixture of about 16.5 percent.

7. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of selecting said plurality of sperm cells for a desired characteristic comprises the step of selecting said plurality of sperm cells for a sex characteristic.

8. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 7, wherein said step of selecting said plurality of sperm cells for a sex characteristic comprises the step of selecting said plurality of sperm cells for a sex characteristic selected from the group consisting of a X-chromosome-bearing characteristic and a Y-chromosome-bearing characteristic.

9. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of adding a protein-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture comprises the step of adding an animal-based protein-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture.

10. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 9, wherein said step of adding an animal-based protein-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture comprises the step of adding a lipoprotein-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture.

11. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 10, wherein said step of adding a lipoprotein-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture comprises the step of adding an egg-yolk-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture.

12. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 11, wherein said step of adding an egg-yolk-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture comprises the step of adding an egg-yolk-containing sperm cell extender having a percentage of egg yolk selected from the group consisting of less than about 50 percent egg yolk, less than about 45 percent egg yolk, less than about 40 percent egg yolk, less than about 35 percent egg yolk, less than about 30 percent egg yolk, less than about 25 percent egg yolk, less than about 20 percent egg yolk, less than about 15 percent egg yolk, less than about 10 percent egg yolk, and less than 5 percent egg yolk.

13. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 12, wherein said step of adding an egg-yolk-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture comprises the step of adding an egg-yolk-containing sperm cell extender having about 20 percent egg yolk.

14. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 13, wherein said step of adding an egg-yolk-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture comprises the step of adding an egg-yolk-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture having a percentage of egg yolk selected from the group consisting of more than about 0.8 percent egg yolk, more than about 1.6 percent egg yolk, more than about 3.2 percent egg yolk, and more than about 6.4 percent egg yolk.

15. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 11, wherein said step of adding an egg-yolk-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture comprises the step of adding an egg-yolk-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture having about 3.2 percent egg yolk.

16. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of adding a protein-containing sperm cell extender to said plurality of selected sperm cells to form a first extended sperm cell mixture comprises the step of adding a protein-containing sperm cell extender selected from the group consisting of an egg yolk based extender, a milk based extender, a citrate extender, a tris-hydroxymethyl-aminomethane (Tris) extender, and a N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid plus Tris (TEST) extender.

17. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of cooling said first extended sperm cell mixture to create a first cooled extended sperm cell mixture comprises the step of cooling said first extended sperm cell mixture to a temperature selected from the group consisting of less than about 10 degrees Celsius, less than about 9 degrees Celsius, less than about 8 degrees Celsius, less than about 7 degrees Celsius, less than about 6 degrees Celsius, less than about 5 degrees Celsius, less than about 4 degrees Celsius, less than about 3 degrees Celsius, less than about 2 degrees Celsius, and less than about 1 degree Celsius.

18. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of cooling said first extended sperm cell mixture to create a first cooled extended sperm cell mixture comprises the step of cooling said first extended sperm cell mixture to a temperature of about 5 degrees Celsius.

19. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of adding a protein-free cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a glycerol-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture.

20. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 19, wherein said step of adding a glycerol-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a glycerol-containing sperm cell extender having a percentage of glycerol selected from the group consisting of more than about 3 percent glycerol, more than about 6 percent glycerol, more than about 12 percent glycerol, and more than about 24 percent glycerol.

21. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 19, wherein said step of adding a glycerol-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a glycerol-containing sperm cell extender having about 6 percent glycerol.

22. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of adding a protein-free cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a protein-free cryoprotectant-containing sperm cell extender having a volume equal to a volume of said first cooled extended sperm cell mixture.

23. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 22, further comprising the step of adding said protein-free cryoprotectant-containing sperm cell extender having a volume equal to a volume of said first cooled extended sperm cell mixture in two steps.

24. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of adding a protein-free cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a sterile cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture.

25. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of adding a protein-free cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a low density gradient cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture.

26. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 25, wherein said step of adding a low density gradient cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a substantially liquid cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture.

27. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 25, wherein said step of adding a low density gradient cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a centrifugation-efficient cryoprotectant-containing sperm cell extender.

28. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 25, wherein said step of adding a low density gradient cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a substantially uniform density cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture.

29. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 25, wherein said step of adding a low density gradient cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a cryoprotectant-containing sperm cell extender with substantially no sperm cell compaction particles.

30. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 25, wherein said step of adding a low density gradient cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a low viscosity cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture.

31. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of adding a protein-free cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a protein-free cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture having a percentage of egg yolk selected from the group consisting of more than about 0.4 percent egg yolk, more than about 0.8 percent egg yolk, more than about 1.6 percent egg yolk, and more than about 3.2 percent egg yolk.

32. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, wherein said step of adding a protein-free cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture comprises the step of adding a protein-free cryoprotectant-containing sperm cell extender to said first cooled extended sperm cell mixture to form a second cooled extended sperm cell mixture having about 1.6 percent egg yolk.

33. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, further comprising the step of maintaining said second cooled extended sperm cell mixture in an unclarified state.

34. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 33, further comprising the step of subjecting said unclarified second cooled extended sperm cell mixture to centrifugation.

35. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 34, further comprising the step of decanting a portion of said centrifuged unclarified second cooled extended sperm cell mixture.

36. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 1, further comprising the step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to a pre-freeze sperm cell concentration.

37. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 36, wherein said step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to a pre-freeze sperm cell concentration comprises the step of adding a protein-containing sperm cell extender to said second extended sperm cell mixture.

38. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 37, wherein said step of adding a protein-containing sperm cell extender to said second extended sperm cell mixture comprises the step of adding an animal-based protein-containing sperm cell extender to said second extended sperm cell mixture.

39. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 38, wherein said step of adding an animal-based protein-containing sperm cell extender to said second extended sperm cell mixture comprises the step of adding a lipoprotein-containing sperm cell extender to said second extended sperm cell mixture.

40. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 39, wherein said step of adding a lipoprotein-containing sperm cell extender to said second extended sperm cell mixture comprises the step of adding an egg-yolk-containing sperm cell extender to said second extended sperm cell mixture.

41. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 40, wherein said step of adding an egg-yolk-containing sperm cell extender to said second extended sperm cell mixture comprises the step of adding an egg-yolk-containing sperm cell extender having a percentage of egg yolk selected from the group consisting of less than about 50 percent egg yolk, less than about 45 percent egg yolk, less than about 40 percent egg yolk, less than about 35 percent egg yolk, less than about 30 percent egg yolk, less than about 25 percent egg yolk, less than about 20 percent egg yolk, less than about 15 percent egg yolk, less than about 10 percent egg yolk, and less than 5 percent egg yolk.

42. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 40, wherein said step of adding an egg-yolk-containing sperm cell extender to said second extended sperm cell mixture comprises the step of adding an egg-yolk-containing sperm cell extender having about 20 percent egg yolk.

43. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 37, wherein said step of adding a protein-containing sperm cell extender to said second extended sperm cell mixture comprises the step of adding a protein-containing sperm cell extender having a percentage of glycerol selected from the group consisting of more than about 3 percent glycerol, more than about 6 percent glycerol, more than about 12 percent glycerol, and more than about 24 percent glycerol.

44. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 37, wherein said step of adding a protein-containing sperm cell extender to said second extended sperm cell mixture comprises the step of adding a protein-containing sperm cell extender to said second extended sperm cell mixture having about 6 percent glycerol.

45. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 36, wherein said step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to a pre-freeze sperm cell concentration comprises the step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to a species-appropriate pre-freeze sperm cell concentration.

46. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 45, wherein said step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to a species-appropriate pre-freeze sperm cell concentration comprises the step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to a pre-freeze sperm cell concentration selected from the group consisting of a bovine pre-freeze sperm cell concentration, an equine pre-freeze sperm cell concentration, and a porcine pre-freeze sperm cell concentration, an ovine pre-freeze sperm cell concentration, a cervid pre-freeze sperm cell concentration, a canine pre-freeze sperm cell concentration, and a delphinidae pre-freeze sperm cell concentration.

47. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 45, wherein said step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to a species-appropriate pre-freeze sperm cell concentration comprises the step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to a sperm cell concentration selected from the group consisting of less than about 100 million sperm cells per milliliter, less than about 50 million sperm cells per milliliter, less than about 40 million sperm cells per milliliter, less than about 30 million sperm cells per milliliter, less than about 20 million sperm cells per milliliter, less than about 15 million sperm cells per milliliter, less than about 10 million sperm cells per milliliter, less than about 5 million sperm cells per milliliter, and less than about 2 million sperm cells per milliliter.

48. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 45, wherein said step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to a species-appropriate pre-freeze sperm cell concentration comprises the step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to about 10 million sperm cells per milliliter.

49. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 36, wherein said step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to a pre-freeze sperm cell concentration comprises the step of establishing a pre-freeze egg yolk content of said second extended sperm cell mixture selected from the group consisting of within about 50 percent of the pre-freeze egg yolk content of a typical method for freezing sorted sperm cells, within about 25 percent of the pre-freeze egg yolk content of a typical method for freezing sorted sperm cells, within about 20 percent of the pre-freeze egg yolk content of a typical method for freezing sorted sperm cells, within about 15 percent of the pre-freeze egg yolk content of a typical method for freezing sorted sperm cells, within about 10 percent of the pre-freeze egg yolk content of a typical method for freezing sorted sperm cells, within about 5 percent of the pre-freeze egg yolk content of a typical method for freezing sorted sperm cells, and within about 1 percent of the pre-freeze egg yolk content of a typical method for freezing sorted sperm cells.

50. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 36, wherein said step of adjusting a sperm cell concentration of said second cooled extended sperm cell mixture to a pre-freeze sperm cell concentration comprises the step establishing a pre-freeze egg yolk content of said second extended sperm cell mixture selected from the group consisting of less than about 50 percent egg yolk, less than about 45 percent egg yolk, less than about 40 percent egg yolk, less than about 35 percent egg yolk, less than about 30 percent egg yolk, less than about 25 percent egg yolk, less than about 20 percent egg yolk, less than about 15 percent egg yolk, and less than about 10 percent egg yolk.