Separation Systems of Frozen-Thawed Spermatozoa Into X-Chromosome Bearing and Y-Chromosome Bearing Populations

US 20070042342A1
Filed: 09/28/2006
Published: 02/22/2007
Est. Priority Date: 11/29/2000
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

Devices, compositions, and methods for handling, separating, packaging, and utilization of spermatozoa (1) that can be derived from previously frozen sperm samples collected from a male mammal. Specifically, techniques to uniformly stain (2) spermatozoal DNA even when derived from previously frozen sperm and separation techniques to separate and isolate spermatozoa even when derived from previously frozen sperm samples into X-chromosome bearing and Y-chromosome bearing populations having high purity.

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54 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.

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Current Assignee
XY, LLC (Inguran LLC)
Original Assignee
Colorado State University (Colorado State University System), XY Incorporated (Inguran LLC)
Inventors
Seidel, George, Lu, Kehaun, Cran, David, Suh, Tae

Granted Patent

US 7,771,921 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 1/0226   Physiologically active agen...

G01N 1/30   Staining; Impregnating ; Fi...

Separation Systems of Frozen-Thawed Spermatozoa Into X-Chromosome Bearing and Y-Chromosome Bearing Populations

First Claim

9 Assignments

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Abstract

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54 Claims

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Separation Systems of Frozen-Thawed Spermatozoa Into X-Chromosome Bearing and Y-Chromosome Bearing Populations

First Claim

9 Assignments

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0 Petitions

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Accused Products

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Abstract

Citations

54 Claims

Specification

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Solutions

Use Cases

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