Methods and apparatus for reducing protein content in sperm cell extenders

• US 20070026378A1
• Filed: 09/02/2005
• Published: 02/01/2007
• Est. Priority Date: 07/29/2005
• Status: Active Grant

• Current Assignee: XY, LLC (Inguran LLC)
• Original Assignee: XY Incorporated (Inguran LLC)
• Inventors: Schenk, John
• Granted Patent: US 7,618,770 B2
• Time in Patent Office: Days
• 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, and porcine 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 for a desired characteristic comprises the step of sorting said plurality of sperm cells.

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

6. 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.

7. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 6, 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.

8. 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.

9. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 8, 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.

10. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 9, 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.

11. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 10, 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.

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 about 20 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 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.

14. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 10, 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.

15. 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 extender, and a TEST extender.

16. 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.

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 of about 5 degrees Celsius.

18. 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.

19. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 18, 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.

20. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 18, 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.

21. 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.

22. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 21, 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.

23. 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.

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 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.

25. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 24, 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.

26. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 24, 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.

27. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 24, 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.

28. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 24, 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.

29. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 24, 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.

30. 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.

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 about 1.6 percent egg yolk.

32. 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.

33. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 32, further comprising the step of subjecting said unclarified second cooled extended sperm cell mixture to centrifugation.

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

35. 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.

36. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 35, 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.

37. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 36, 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.

38. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 37, 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.

39. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 38, 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.

40. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 39, 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.

41. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 39, 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.

42. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 36, 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.

43. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 36, 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.

44. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 35, 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.

45. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 44, 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.

46. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 44, 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.

47. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 44, 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.

48. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 35, 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.

49. A method for freezing sorted sperm cells compromised by a sorting event as described in claim 35, 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.

50. 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.

51. An incipient compromised sorted sperm cell admixture comprising:

a nascent plurality of sperm cells selected for a desired characteristic in incipient admixture relation;

a nascent protein-free sperm cell extender component in incipient admixture relation;

a nascent protein-free cryoprotectant-containing sperm cell extender component in incipient admixture relation; and

a nascent protein-containing sperm cell extender component in incipient admixture relation.

52. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent plurality of sperm cells selected for a desired characteristic comprises a plurality of sperm cells selected for a desired characteristic proximately located in a substantially uncombined state to at least one component of said incipient admixture.

53. An incipient compromised sorted sperm cell admixture as described in claim 52, further comprising a barrier-free zone between said plurality of sperm cells selected for a desired characteristic and said at least one component of said incipient admixture.

54. An incipient compromised sorted sperm cell admixture as described in claim 53, further comprising an induced combination force to which said plurality of sperm cells selected for a desired characteristic and said at least one component of said incipient admixture are responsive.

55. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent plurality of sperm cells comprises a nascent plurality of mammalian sperm cells.

56. An incipient compromised sorted sperm cell admixture as described in claim 55, wherein said nascent plurality of mammalian sperm cells comprises a nascent plurality of mammalian sperm cells selected from the group consisting of bovine sperm cells, equine sperm cells, and porcine sperm cells.

57. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent plurality of sperm cells selected for a desired characteristic comprises a nascent plurality of sorted sperm cells.

58. An incipient compromised sorted sperm cell admixture as described in claim 57, wherein said nascent plurality of sorted sperm cells comprises a nascent plurality of sorted sperm cells sorted by flow cytometery.

59. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent plurality of sperm cells selected for a desired characteristic comprises a nascent plurality of sperm cells selected for a sex characteristic.

60. An incipient compromised sorted sperm cell admixture as described in claim 59, wherein said nascent plurality of sperm cells selected for a sex characteristic comprises a nascent plurality of sperm cells selected for a sex characteristic selected from the group consisting of an X-chromosome-bearing characteristic and a Y-chromosome-bearing characteristic.

61. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent protein-free sperm cell extender component comprises a protein-free sperm cell extender component proximately located in a substantially uncombined state to at least one component of said incipient admixture.

62. An incipient compromised sorted sperm cell admixture as described in claim 61, further comprising a barrier-free zone between said protein-free sperm cell extender component and said at least one component of said incipient admixture.

63. An incipient compromised sorted sperm cell admixture as described in claim 62, further comprising an induced combination force to which said protein-free sperm cell extender component and said at least one component of said incipient admixture are responsive.

64. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent protein-free sperm cell extender component comprises a sterile nascent protein-free sperm cell extender component.

65. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent protein-free cryoprotectant-containing sperm cell extender component comprises a protein-free cryoprotectant-containing sperm cell extender component proximately located in a substantially uncombined state to at least one component of said incipient admixture.

66. An incipient compromised sorted sperm cell admixture as described in claim 65, further comprising a barrier-free zone between said protein-free cryoprotectant-containing sperm cell extender component and said at least one component of said incipient admixture.

67. An incipient compromised sorted sperm cell admixture as described in claim 66, further comprising an induced combination force to which said protein-free cryoprotectant-containing sperm cell extender component and said at least one component of said incipient admixture are responsive.

68. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent protein-free cryoprotectant-containing sperm cell extender component comprises a glycerol component.

69. An incipient compromised sorted sperm cell admixture as described in claim 68, wherein said glycerol component comprises 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.

70. An incipient compromised sorted sperm cell admixture as described in claim 68, wherein said glycerol component comprises about 6% glycerol.

71. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent protein-free cryoprotectant-containing sperm cell extender component comprises a sterile nascent protein-free cryoprotectant-containing sperm cell extender component.

72. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent protein-free cryoprotectant-containing sperm cell extender component comprises a nascent low density gradient cryoprotectant-containing sperm cell extender component.

73. An incipient compromised sorted sperm cell admixture as described in claim 72, wherein said nascent low density gradient cryoprotectant-containing sperm cell extender component comprises a nascent substantially liquid cryoprotectant-containing sperm cell extender component.

74. An incipient compromised sorted sperm cell admixture as described in claim 72, wherein said nascent low density gradient cryoprotectant-containing sperm cell extender component comprises a nascent centrifugation-efficient cryoprotectant-containing sperm cell extender component.

75. An incipient compromised sorted sperm cell admixture as described in claim 72, wherein said nascent low density gradient cryoprotectant-containing sperm cell extender component comprises a nascent substantially uniform density cryoprotectant-containing sperm cell extender component.

76. An incipient compromised sorted sperm cell admixture as described in claim 72, wherein said nascent low density gradient cryoprotectant-containing sperm cell extender component comprises a cryoprotectant-containing sperm cell extender component having substantially no sperm cell compaction particles.

77. An incipient compromised sorted sperm cell admixture as described in claim 72, wherein said nascent low density gradient cryoprotectant-containing sperm cell extender component comprises a nascent low viscosity cryoprotectant-containing sperm cell extender component.

78. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent protein-containing sperm cell extender component comprises a protein-containing sperm cell extender component proximately located in a substantially uncombined state to at least one component of said incipient admixture.

79. An incipient compromised sorted sperm cell admixture as described in claim 78, further comprising a barrier-free zone between said protein-containing sperm cell extender component and said at least one component of said incipient admixture.

80. An incipient compromised sorted sperm cell admixture as described in claim 79, further comprising an induced combination force to which said protein-containing sperm cell extender component and said at least one component of said incipient admixture are responsive.

81. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent protein-containing sperm cell extender component comprises an animal-based protein-containing sperm cell extender component.

82. An incipient compromised sorted sperm cell admixture as described in claim 81, wherein said animal-based protein-containing sperm cell extender component comprises a lipoprotein-containing sperm cell extender component.

83. An incipient compromised sorted sperm cell admixture as described in claim 82, wherein said lipoprotein-containing sperm cell extender component comprises an egg-yolk-containing sperm cell extender component.

84. An incipient compromised sorted sperm cell admixture as described in claim 83, wherein said egg-yolk-containing sperm cell extender component comprises an egg yolk percentage 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.

85. An incipient compromised sorted sperm cell admixture as described in claim 83, wherein said egg-yolk-containing sperm cell extender component comprises about 20 percent egg yolk.

86. An incipient compromised sorted sperm cell admixture as described in claim 83, further comprising an egg yolk content of said incipient admixture 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.

87. An incipient compromised sorted sperm cell admixture as described in claim 83, further comprising an egg yolk content of said incipient admixture of about 1.6 percent.

88. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said incipient admixture further comprises an unclarified incipient admixture.

89. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said incipient admixture further comprises a cool incipient admixture.

90. An incipient compromised sorted sperm cell admixture as described in claim 89, wherein said cool incipient admixture comprises an incipient admixture at 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.

91. An incipient compromised sorted sperm cell admixture as described in claim 89, wherein said cool incipient admixture comprises an incipient admixture at a temperature of about 5 degrees Celsius.

92. An incipient compromised sorted sperm cell admixture as described in claim 51, wherein said nascent protein-containing sperm cell extender component comprises a protein-containing sperm cell extender component selected from the group consisting of an egg yolk based extender, a milk based extender, a citrate extender, a Tris extender, and a TEST extender.

93-235. -235. (canceled)