Sperm Suspensions For Use in Insemination

US 20070092860A1
Filed: 12/07/2006
Published: 04/26/2007
Est. Priority Date: 11/24/1999
Status: Active Grant

First Claim

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1. A process for storing unsorted spermatozoa, the process comprising a. forming a sperm dispersion, the sperm dispersion comprising spermatozoa, a composition that induces sperm immotility, and an antibiotic, and b. storing the sperm dispersion.

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Abstract

The present invention provides a method of cryopreserving sperm that have been selected for a specific characteristic. In a preferred embodiment, the method is employed to freeze sex-selected sperm. Although the cryopreservation method of the invention can be used to freeze sperm selected by any number of selection methods, selection using flow cytometry is preferred. The present invention also provides a frozen sperm sample that has been selected for a particular characteristic, such as sex-type. In preferred embodiments, the frozen sperm sample includes mammalian sperm, such as, for example, human, bovine, equine, porcine, ovine, elk, or bison sperm. The frozen selected sperm sample can be used in a variety of applications. In particular, the sample can be thawed and used for fertilization. Accordingly, the invention also includes a method of using the frozen selected sperm sample for artificial insemination or in vitro fertilization.

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81 Claims

1. A process for storing unsorted spermatozoa, the process comprising a. forming a sperm dispersion, the sperm dispersion comprising spermatozoa, a composition that induces sperm immotility, and an antibiotic, and b. storing the sperm dispersion.
- View Dependent Claims (12, 13, 14, 15, 24, 27, 30, 31, 32)
- - 12. The process of claim 1, wherein the concentration of spermatozoa in the sperm dispersion is at least about 0.04×
    - 10⁶sperm/ml.
  - 13. The process of claim 1, wherein the concentration of spermatozoa in the sperm dispersion is at least about 1.5×
    - 10⁶sperm/ml.
  - 14. The process of claim 1, wherein the concentration of spermatozoa in the sperm dispersion is at least about 1×
    - 10⁷sperm/ml.
  - 15. The process of claim 1, wherein the concentration of spermatozoa in the sperm dispersion is at least about 12×
    - 10⁷sperm/ml.
  - 24. The process of claim 1, wherein the composition that induces sperm immotility comprises potassium and optionally sodium, the molar ratio of potassium to sodium being greater than 1:
    - 1, respectively.
  - 27. The process of claim 1, wherein the composition that induces sperm immotility comprises potassium and optionally sodium, the molar ratio of potassium to sodium being greater than 1.75:
    - 1.
  - 30. The process of claim 1, wherein the sperm dispersion comprises a source of carbonate.
  - 31. The process of claim 1, wherein the dispersion comprises NaHCO₃, KHCO₃, and C₆H₈O₇.H₂O.
  - 32. The process of claim 1, wherein the dispersion comprises a buffer comprising 0.097 moles/L of NaHCO₃, 0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈O₇.H₂O in water.

2. A process for storing sorted spermatozoa, the process comprising a. forming a sperm dispersion, the sperm dispersion comprising spermatozoa and a composition that induces sperm immotility, b. sorting the sperm dispersion into separate populations, wherein the spermatozoa of one of the populations comprises at least about 65% X chromosome bearing sperm cells or at least about 65% Y chromosome bearing sperm cells, and c. storing the one population at a temperature of about −
- 4°
  
  C. to about 30°
  
  C.
- View Dependent Claims (9, 10, 11, 16, 17, 18, 19, 25, 28, 33, 34, 35, 39, 40, 41, 42)
- - 9. The process of claim 2, wherein the sperm dispersion is stored for at least about 24 hours.
  - 10. The process of claim 2, wherein the sperm dispersion is stored for at least about 72 hours.
  - 11. The process of claim 2, wherein the sperm dispersion is stored for at least about 1 week.
  - 16. The process of claim 2, wherein the concentration of spermatozoa in the sperm dispersion is at least about 0.04×
    - 10⁶sperm/ml.
  - 17. The process of claim 2, wherein the concentration of spermatozoa in the sperm dispersion is at least about 1.5×
    - 10⁶sperm/ml.
  - 18. The process of claim 2, wherein the concentration of spermatozoa in the sperm dispersion is at least about 1×
    - 10⁷sperm/ml.
  - 19. The process of claim 2, wherein the concentration of spermatozoa in the sperm dispersion is at least about 12×
    - 10⁷sperm/ml.
  - 25. The process of claim 2, wherein the composition that induces sperm immotility comprises potassium and optionally sodium, the molar ratio of potassium to sodium being greater than 1:
    - 1, respectively.
  - 28. The process of claim 2, wherein the composition that induces sperm immotility comprises potassium and optionally sodium, the molar ratio of potassium to sodium being greater than 1.75:
    - 1.
  - 33. The process of claim 2, wherein the sperm dispersion comprises a source of carbonate.
  - 34. The process of claim 2, wherein the dispersion comprises NaHCO₃, KHCO₃, and C₆H₈O₇.H₂O.
  - 35. The process of claim 2, wherein the dispersion comprises a buffer comprising 0.097 moles/L of NaHCO₃, 0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈O₇.H₂O in water.
  - 39. The process of claim 2, wherein the spermatozoa of one of the populations comprises at least about 70% X chromosome bearing or at least about 70% Y chromosome bearing sperm cells.
  - 40. The process of claim 2, wherein the spermatozoa of one of the populations comprises at least about 80% X chromosome bearing or at least about 80% Y chromosome bearing sperm cells.
  - 41. The process of claim 2, wherein the spermatozoa of one of the populations comprises at least about 90% X chromosome bearing or at least about 90% Y chromosome bearing sperm cells.
  - 42. The process of claim 2, wherein the spermatozoa of one of the populations comprises at least about 95% X chromosome bearing or at least about 95% Y chromosome bearing sperm cells.

3. A process for inseminating a female mammal, the process comprising inseminating a female mammal with a sperm dispersion, the sperm dispersion comprising immotile spermatozoa and a composition that induces sperm immotility.
- View Dependent Claims (43, 44, 45, 46, 47)
- - 43. The process of claim 3, wherein the sperm dispersion comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 65% X chromosome bearing sperm cells or at least about 65% Y chromosome bearing sperm cells.
  - 44. The process of claim 3, wherein the sperm dispersion comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 75% X chromosome bearing sperm cells or at least about 75% Y chromosome bearing sperm cells.
  - 45. The process of claim 3, wherein the sperm dispersion comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 85% X chromosome bearing sperm cells or at least about 85% Y chromosome bearing sperm cells.
  - 46. The process of claim 3, wherein the sperm dispersion comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 90% X chromosome bearing sperm cells or at least about 90% Y chromosome bearing sperm cells.
  - 47. The process of claim 3, wherein the sperm dispersion comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 95% X chromosome bearing sperm cells or at least about 95% Y chromosome bearing sperm cells.

4. A process for providing a fresh sperm dispersion for inseminating a female mammal, the process comprising:
- a. forming a sperm dispersion, the sperm dispersion comprising spermatozoa and a composition that induces sperm immotility, b. placing the sperm dispersion in a container for shipment to a remote location, and c. shipping the sperm dispersion in the container to a remote location within about 24 hours after forming the sperm dispersion.
- View Dependent Claims (6, 7, 8, 48, 49, 50, 51, 52, 53, 54)
- - 6. The process of claim 4, wherein the sperm dispersion is stored at about −
    - 4°
      
      C. to about 30°
      
      C.
  - 7. The process of claim 4, wherein the sperm dispersion is stored at about 0°
    - C. to about 5°
      
      C.
  - 8. The process of claim 4, wherein the sperm dispersion is stored at about 5°
    - C. to about 30°
      
      C.
  - 48. The process of claim 4, wherein the sperm dispersion comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 65% X chromosome bearing sperm cells or at least about 65% Y chromosome bearing sperm cells.
  - 49. The process of claim 4, wherein the sperm dispersion comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 75% X chromosome bearing sperm cells or at least about 75% Y chromosome bearing sperm cells.
  - 50. The process of claim 4, wherein the sperm dispersion comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 85% X chromosome bearing sperm cells or at least about 85% Y chromosome bearing sperm cells.
  - 51. The process of claim 4, wherein the sperm dispersion comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 90% X chromosome bearing sperm cells or at least about 90% Y chromosome bearing sperm cells.
  - 52. The process of claim 4, wherein the sperm dispersion comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 95% X chromosome bearing sperm cells or at least about 95% Y chromosome bearing sperm cells.
  - 53. The process of claim 4, wherein the sperm dispersion is shipped within about 24 hours after the formation of the sperm dispersion.
  - 54. The process of claim 4, wherein the sperm dispersion is shipped within about 48 hours after the formation of the sperm dispersion.

5. A combination comprising:
- a. an elongated container for use in the insemination of a female mammal, and b. a sperm dispersion, the sperm dispersion comprising immotile spermatozoa and a composition that induces sperm immotility, and wherein the sperm dispersion is contained in the elongated container.
- View Dependent Claims (20, 21, 22, 23, 26, 29, 36, 37, 38)
- - 20. The combination of claim 5, wherein the concentration of spermatozoa in the sperm dispersion is at least about 0.04×
    - 10⁶sperm/ml.
  - 21. The combination of claim 5, wherein the concentration of spermatozoa in the sperm dispersion is at least about 1.5×
    - 10⁶sperm/ml.
  - 22. The combination of claim 5, wherein the concentration of spermatozoa in the sperm dispersion is at least about 1×
    - 10⁷sperm/ml.
  - 23. The combination of claim 5, wherein the concentration of spermatozoa in the sperm dispersion is at least about 12×
    - 10⁷sperm/ml.
  - 26. The combination of claim 5, wherein the composition that induces sperm immotility comprises potassium and optionally sodium, the molar ratio of potassium to sodium being greater than 1:
    - 1, respectively.
  - 29. The combination of claim 5, wherein the composition that induces sperm immotility comprises potassium and optionally sodium, the molar ratio of potassium to sodium being greater than 1.75:
    - 1.
  - 36. The combination of claim 5, wherein the sperm dispersion comprises a source of carbonate.
  - 37. The combination of claim 5, wherein the dispersion comprises NaHCO₃, KHCO₃, and C₆H₈O₇.H₂O.
  - 38. The combination of claim 5, wherein the dispersion comprises a buffer comprising 0.097 moles/L of NaHCO₃, 0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈O₇.H₂O in water.

55. A process for storing unsorted spermatozoa, the process comprising a. forming a sperm suspension, the sperm suspension comprising spermatozoa, a composition that reduces sperm motility, and an antibiotic, and b. storing the sperm suspension.

56. A staining mixture comprising viable spermatozoa, a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly, and a DNA selective dye, the concentration of the composition in the staining mixture being greater than 50 μ
- M when the composition is pyruvate.
- View Dependent Claims (57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 76, 77, 78, 79, 80, 81)
- - 57. The staining mixture of claim 56, wherein the composition is selected from the group consisting of pyruvate, vitamin K, lipoic acid, glutathione, flavins, quinones, superoxide dismutase, superoxide dismutase mimics, and any combinations thereof.
  - 58. The staining mixture of claim 57, wherein the composition is selected from the group consisting of pyruvate, vitamin K, lipoic acid, and combinations thereof.
  - 59. The staining mixture of claim 56, wherein the composition comprises pyruvate at a concentration selected from the group consisting of about 2.5 mM, about 10 mM, about 15 mM, about 25 mM, and about 50 mM.
  - 60. The staining mixture of claim 56, wherein the composition comprises vitamin K at a concentration selected from the group consisting of about 10 μ
    - M, about 50 μ
      
      M, about 75 μ
      
      M, and about 100 μ
      
      M.
  - 61. The staining mixture of claim 56, wherein the composition comprises lipoic acid at a concentration selected from the group consisting of about 0.1 mM, about 0.5 mM, about 0.75 mM, about 1.0 mM, and about 1.5 mM.
  - 62. The staining mixture of claim 56, wherein the DNA selective dye is a DNA selective fluorescent dye.
  - 63. The staining mixture of claim 57, wherein the DNA selective dye is a DNA selective fluorescent dye.
  - 64. The staining mixture of claim 56, wherein the dye is a UV excitable or a visible light excitable dye.
  - 65. The staining mixture of claim 57, wherein the dye is a UV excitable or a visible light excitable dye.
  - 66. The staining mixture claim 56, wherein the dye is selected from the group consisting of Hoechst 33342, Hoechst 33258, SYBR-14, and bisbenzimide-BODIPY®
    - conjugate 6-{[3-((2Z)-2-{[1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2-yl]methylene}-2H-pyrrol-5-yl)propanoyl]amino}-N-[3-(methyl {3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3′
      
      H-2,5′
      
      -bibenzimidazol-2′
      
      -yl-]phenoxy)acetyl)amino]propyl}amino)propyl]hexanamide.
  - 67. The staining mixture claim 57, wherein the dye is selected from the group consisting of Hoechst 33342, Hoechst 33258, SYBR-14, and bisbenzimide-BODIPY®
    - conjugate 6-{[3-((2Z)-2-{[1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2-yl]methylene}-2H-pyrrol-5-yl)propanoyl]amino}-N-[3-(methyl {3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3′
      
      H-2,5′
      
      -bibenzimidazol-2′
      
      -yl-]phenoxy)acetyl)amino]propyl}amino)propyl]hexanamide.
  - 76. The staining mixture of claim 56, wherein the staining mixture further comprises a buffer that inhibits sperm motility.
  - 77. The staining mixture of claim 57, wherein the staining mixture further comprises a buffer that inhibits sperm motility.
  - 78. The staining mixture of claim 76, wherein the buffer is a carbonate-based buffer.
  - 79. The staining mixture of claim 77, wherein the buffer is a carbonate-based buffer.
  - 80. The staining mixture of claim 76, wherein the buffer comprises 0.097 moles/L of NaHCO₃, 0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈O₇.H₂O in water.
  - 81. The staining mixture of claim 77, wherein the buffer comprises 0.097 moles/L of NaHCO₃, 0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈O₇.H₂O in water.

68. A process for staining sperm cells, the process comprising forming a staining mixture containing intact viable sperm cells, a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly, and a DNA selective dye, the concentration of the composition in the staining mixture being greater than 50 μ
- M when the composition is pyruvate.
- View Dependent Claims (69, 70, 71, 72, 73, 74, 75)
- - 69. The process of claim 68, wherein the DNA-selective dye is selected from the group consisting of Hoechst 33342, Hoechst 33258, SYBR-14, and bisbenzimide-BODIPY®
    - conjugate 6-{[3-((2Z)-2-{[1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2-yl]methylene}-2H-pyrrol-5-yl)propanoyl]amino}-N-[3-(methyl{3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3′
      
      H-2,5′
      
      -bibenzimidazol-2′
      
      -yl-]phenoxy)acetyl)amino]propyl}amino)propyl]hexanamide.
  - 70. The process of claim 68, wherein the staining mixture is subjected to a temperature of about 4°
    - C. to about 30°
      
      C.
  - 71. The process of claim 69, wherein the staining mixture is subjected to a temperature of about 30°
    - C. to about 39°
      
      C.
  - 72. The process of claim 69, wherein the staining mixture is subjected to a temperature of about 40°
    - C. to about 50°
      
      C.
  - 73. The process of claim 68, wherein the composition comprises pyruvate at a concentration selected from the group consisting of about 2.5 mM, about 10 mM, about 15 mM, about 25 mM, and about 50 mM.
  - 74. The process of claim 68, wherein the composition comprises vitamin K at a concentration selected from the group consisting of about 10 μ
    - M, about 50 μ
      
      M, about 75 μ
      
      M, and about 100 μ
      
      M.
  - 75. The process of claim 68, wherein the composition comprises lipoic acid at a concentration selected from the group consisting of about 0.1 mM, about 0.5 mM, about 0.75 mM, about 1.0 mM, and about 1.5 mM.

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Current Assignee
XY, LLC (Inguran LLC)
Original Assignee
XY Incorporated (Inguran LLC)
Inventors
Schenk, John

Granted Patent

US 7,820,425 B2
Time in Patent Office

Days
Field of Search
US Class Current

435/2
CPC Class Codes

A01N 1/02   Preservation of living parts

A01N 1/021   Preservation or perfusion m...

A01N 1/0215   Disinfecting agents, e.g. a...

A01N 1/0221   Freeze-process protecting a...

A01N 1/0226   Physiologically active agen...

Sperm Suspensions For Use in Insemination

First Claim

6 Assignments

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Abstract

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81 Claims

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Sperm Suspensions For Use in Insemination

First Claim

6 Assignments

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Accused Products

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Abstract

Citations

81 Claims

Specification

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Solutions

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