Composition and method for preparation, storage and activation of large populations of immotile sperm

• US 6,309,815 B1
• Filed: 08/06/1999
• Issued: 10/30/2001
• Est. Priority Date: 08/07/1998
• Status: Expired due to Fees

• Current Assignee: University of Kansas Medical Center
• Original Assignee: University of Kansas Medical Center
• Inventors: Tash, Joseph S., Bracho, Gerácimo E.
• Primary Examiner(s): Saucier, Sandra E.
• Application Number:
  US09/369,559
• Time in Patent Office: 816 Days
• Field of Search: 435/2
• US Class Current: 435/2
• 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

Abstract

A method and composition for extending the viability of immotile sperm is disclosed. The method comprises the steps of (a) preparing an animal for semen collection; (b) collecting semen from the animal; (c) treating the collected semen with a storage buffer solution to substantially inhibit sperm motility; (d) storing the semen for a length of time and at a certain temperature; and (e) reactivating the sperm to normal motility by mixing the inhibited sperm with an activation buffer.

Claims

1. A method for extending the viability of sperm which has the capacity to become motile comprising the steps of:

(a) preparing an animal for semen collection by washing the animal with a preparation solution;

(b) collecting semen from said animal;

(c) treating the collected semen with a storage buffer solution to inhibit motility by at least 90 percent;

(d) storing the semen for a length of time and at a certain temperature in said storage buffer solution; and

(e) reactivating the sperm to normal motility by mixing the inhibited sperm with an activation buffer.

2. The method of claim 1 wherein said preparing comprises washing the animal by immersion into a buffer solution containing 2-[N-morpholino]-ethanesulfonic acid.

3. The method of claim 1 wherein the storage buffer solution comprises a solution containing 2-[N-morpholino]-ethanesulfonic acid.

4. The method of claim 1 wherein sperm storage buffer solution comprises a formulation of 50 mM potassium chloride, 5 mM 2-[N-morpholino]ethanesulfonic acid, 5 mM 3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid, 425 mM sodium chloride, 27 mM magnesium chloride, 29 mM magnesium sulfate, 10 mM calcium chloride, and 2.4 mM sodium bicarbonate.

5. The method of claim 1 wherein the stored sperm is reactivated to normal motility by diluting the stored sperm with an activation buffer at a ratio of four parts sperm to seventeen parts activation buffer.

6. The method of claim 5 wherein the activation buffer is hardware sea water.

7. The method of claim 1 wherein said storage is performed in a sperm storage buffer solution having a pH of about 6.0.

8. The method of claim 1 wherein said storage is performed at a temperature of about 4-5°

C.

9. The method of claim 8 wherein the semen is incubated at room temperature prior to said storage at about 4-5°

C.

10. The method of claim 1 wherein said storage is performed in a storage solution containing NaCl.

11. The method of claim 1 wherein said washing the animal with a preparation solution comprises washing the exterior of the animal with the preparation solution.

12. The method of claim 1 wherein said sperm is stored for 4 days.

13. A method for extending the viability of sperm which has the capacity to become motile comprising the steps of:

(a) preparing an animal for semen collection;

(b) collecting semen from said animal;

(c) treating the collected semen with a storage buffer solution comprising 2-[N-morpholino]-ethanesulfonic acid to inhibit motility by at least 90 percent;

(d) storing the semen for a length of time and at a certain temperature in said storage buffer solution; and

(e) reactivating the sperm to normal motility by mixing the inhibited sperm with an activation buffer.

14. The method of claim 13 wherein said storage is performed in a sperm storage buffer solution having a pH of about 6.0.

15. The method of claim 13 wherein said storage is performed at a temperature of 4-5°

C.

16. The method of claim 13 wherein said storage is performed in a storage solution containing NaCl.

17. The method of claim 13 wherein said sperm is stored for 4 days.

18. A composition for preserving and extending the viability of immotile sperm having a pH of about 6.0, comprising about:

(a) 50 mM potassium chloride;

(b) 5 mM 2-[N-morpholino]ethanesulfonic acid;

(c) 5 mM 3-[tris(hydroxymethyl)methylamino]-1 -propanesulfonic acid;

(d) 425 mM sodium chloride;

(e) 27 mM magnesium chloride;

(f) 29 mM magnesium sulfate;

(g) 10 mM calcium chloride; and

(h) 2.4 mM sodium bicarbonate.