Sperm Suspensions for Sorting Into X or Y Chromosome-bearing Enriched Populations

• US 20070099171A1
• Filed: 12/07/2006
• Published: 05/03/2007
• Est. Priority Date: 11/24/1999
• Status: Abandoned Application

• Current Assignee: XY Incorporated (Inguran LLC)
• Original Assignee: XY Incorporated (Inguran LLC)
• Inventors: Schenk, John
• Application Number:
  US11/608,039
• Publication Number:
  US 20070099171A1
• Time in Patent Office: Days
• US Class Current: 435/2
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/021 Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
• CPC Class Codes: A01N 1/0215 Disinfecting agents, e.g. antimicrobials for preserving 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
• CPC Class Codes: A01N 1/0226 Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

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.

Claims

1. A sperm cell suspension comprising viable spermatozoa, the spermatozoa having a motility more characteristic of epididymal sperm then endogenous ejaculated spermatozoa of the same species, the concentration of spermatozoa in the suspension being less than about 1×

10⁶ or at least about 1×

10⁸ spermatozoa per ml.

2. A sperm cell suspension comprising viable, immotile sperm, the concentration of spermatozoa in the suspension being less than about 1×

10⁶ or at least about 1×

10⁸ spermatozoa per ml.

12. The sperm suspension of claim 2, wherein the suspension comprises a source of carbonate.

13. The sperm suspension of claim 2, wherein the suspension comprises NaHCO₃, KHCO₃, and C₆H₈O₇.H₂O.

14. The sperm suspension of claim 2, wherein the suspension is formed from a buffer comprising 0.097 moles/L of NaHCO₃, 0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈O₇H₂O in water.

15. The sperm suspension of claim 2, wherein the concentration of spermatozoa in the suspension is at least 1.25×

10⁸ spermatozoa per ml.

16. The sperm suspension of claim 2, wherein the concentration of spermatozoa in the suspension is at least 1.5×

10⁸ spermatozoa per ml.

17. The sperm suspension of claim 2, wherein the concentration of spermatozoa in the suspension is at least 1.75×

10⁸ spermatozoa per ml.

18. The suspension of claim 2, wherein the concentration of spermatozoa in the suspension is less than about 9.0.×

10⁵ spermatozoa per ml.

19. The suspension of claim 2, wherein the concentration of spermatozoa in the suspension is less than about 7×

10⁵ spermatozoa per ml.

20. The suspension of claim 2, wherein the concentration of spermatozoa in the suspension is less than about 5×

10⁵ spermatozoa per ml.

21. The suspension of claim 2, wherein the concentration of spermatozoa in the suspension is less than about 2×

10⁵ spermatozoa per ml.

22. The suspension of claim 2, wherein the concentration of spermatozoa in the suspension is less than about 1×

10⁵ spermatozoa per ml.

27. The suspension of claim 13, further comprising a DNA-selective dye.

3. A sperm cell suspension comprising viable sperm, a motility inhibiting amount of potassium, and sodium, the concentration of spermatozoa in the suspension being less than about 1×

10⁶ or at least about 1×

10⁸ spermatozoa per ml and the molar ratio of potassium to sodium being greater than 1;

1, respectively.

4. The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 1.25:

1, respectively.

5. The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 1.5:

1, respectively.

6. The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 1.75:

1, respectively.

7. The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 2:

1, respectively.

8. A sperm cell suspension comprising viable, immotile sperm and a DNA-selective dye.

9. The suspension of claim 8, wherein the dye is a DNA selective fluorescent dye.

10. The suspension of claim 8, wherein the dye is a UV excitable or a visible light excitable dye.

11. The suspension of claim 8, 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.

23. A process for staining sperm cells, the process comprising forming a staining mixture containing intact viable sperm cells, a motility inhibiting amount of potassium, and a DNA selective dye.

24. The process of claim 23 wherein the staining mixture is under an atmosphere having an enriched partial pressure of CO₂ relative to air.

25. A process of forming a sperm cell suspension for use in cell sorting, the process comprising combining a sperm cell source with a composition which inhibits the motility of sperm cells to form a sperm cell suspension, the concentration of sperm cells in the suspension being less than about 1×

10⁶ or at least 1×

10⁸ sperm cells per milliliter.

26. The process of claim 25, wherein the cell suspension comprises a DNA-selective dye.

28. A sperm cell suspension comprising viable, immotile spermatozoa, the spermatozoa having a DNA-selective dye associated with their DNA.

29. The suspension of claim 28, wherein the dye is a UV excitable or a visible light excitable dye.

30. The sperm suspension of claim 28, wherein the suspension comprises NaHCO₃, KHCO₃, and C₆H₈O₇.H₂O.

31. The sperm suspension of claim 28, wherein the suspension is formed from a buffer comprising 0.097 moles/L of NaHCO₃, 0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈O₇.H₂O in water.

32. A sperm cell suspension comprising viable spermatozoa and a composition which down-regulates carbohydrate uptake by the spermatozoa, the concentration of spermatozoa in the suspension being less than about 1×

10⁶ or at least about 1×

10³ spermatozoa per ml.

33. A sperm cell suspension comprising viable sperm, potassium and optionally sodium, the concentration of spermatozoa in the suspension being less than about 1×

10⁶ or at least about 1×

10⁸ spermatozoa per ml and the molar ratio of potassium to sodium being greater than 1;

1, respectively.