Use of a Composition which Regulates Oxidation/Reduction Reactions Intracellularly and/or Extracellularly in a Staining or Sorting Process

• US 20070099260A1
• Filed: 12/20/2006
• Published: 05/03/2007
• Est. Priority Date: 12/31/1997
• Status: Abandoned Application

• Current Assignee: XY Incorporated (Inguran LLC)
• Original Assignee: XY Incorporated (Inguran LLC)
• Inventors: Schenk, John, Seidel, George, Herickhoff, Lisa
• Application Number:
  US11/613,605
• Publication Number:
  US 20070099260A1
• Time in Patent Office: Days
• US Class Current: 435/40.5
• CPC Class Codes: A01K 67/027 New or modified breeds of vertebrates
• CPC Class Codes: A01N 1/02 Preservation of living parts
• 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
• CPC Class Codes: A61D 19/02 for artificial insemination
• CPC Class Codes: A61D 19/04 for embryo transplantation
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 5/0612 sorting of gametes, e.g. according to sex or motility
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/149 Sorting the particles
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent

Abstract

Improved flow cytometer system particularly adapted to use for sex-selected sperm sorting include enhanced sheath fluid and other strategies which minimize stress on the sperm cells, including a 2.9 percent sodium citrate sheath solution for bovine species and a hepes bovine gamete media for equine species. Improved collection systems and techniques for the process are described so that commercial applications of sperms samples as well as the resulting animals may be achieved.

Claims

1. 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.

2. The staining mixture of claim 1, 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.

3. The staining mixture of claim 2, wherein the composition is selected from the group consisting of pyruvate, vitamin K, lipoic acid, and combinations thereof.

4. The staining mixture of claim 1, 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.

5. The staining mixture of claim 1, 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.

6. The staining mixture of claim 1, 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.

7. The staining mixture of claim 1, wherein the DNA selective dye is a DNA selective fluorescent dye.

8. The staining mixture of claim 2, wherein the DNA selective dye is a DNA selective fluorescent dye.

9. The staining mixture of claim 1, wherein the dye is a UV excitable or a visible light excitable dye.

10. The staining mixture of claim 2, wherein the dye is a UV excitable or a visible light excitable dye.

11. The staining mixture claim 1, 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.

12. The staining mixture claim 2, 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.

21. The staining mixture of claim 1, wherein the staining mixture further comprises a buffer that inhibits sperm motility.

22. The staining mixture of claim 2, wherein the staining mixture further comprises a buffer that inhibits sperm motility.

23. The staining mixture of claim 21, wherein the buffer is a carbonate-based buffer.

24. The staining mixture of claim 22, wherein the buffer is a carbonate-based buffer.

25. The staining mixture of claim 21, wherein the buffer comprises 0.097 moles/L of NaHCO₃, 0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈O₇H₂O in water.

26. The staining mixture of claim 22, wherein the buffer comprises 0.097 moles/L of NaHCO₃, 0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈O₇H₂O in water.

13. 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.

14. The process of claim 13, 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.

15. The process of claim 13, wherein the staining mixture is subjected to a temperature of about 4°

C. to about 30°

C.

16. The process of claim 14, wherein the staining mixture is subjected to a temperature of about 30°

C. to about 39°

C.

17. The process of claim 14, wherein the staining mixture is subjected to a temperature of about 40°

C. to about 50°

C.

18. The process of claim 13, 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.

19. The process of claim 13, 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.

20. The process of claim 13, 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.