Multiple sexed embryo production system for mammals

• US 6,372,422 B1
• Filed: 11/24/1999
• Issued: 04/16/2002
• Est. Priority Date: 12/31/1997
• Status: Expired due to Term

• Current Assignee: XY, LLC (Inguran LLC)
• Original Assignee: Colorado State University Research Foundation
• Inventors: Schenk, John, Seidel, George, Herickhoff, Lisa
• Primary Examiner(s): Myers, Carla J.
• Application Number:
  US09/448,643
• Time in Patent Office: 874 Days
• Field of Search: 435/6, 435/7.1, 435/7.21, 435/4, 435/2, 800/2, 800/8, 514/12, 514/573, 422/73, 422/81, 422/99, 422/100, 356/23, 356/246, 356/336, 356/337
• US Class Current: 435/2
• CPC Class Codes: A01K 67/027 New or modified breeds of vertebrates
• CPC Class Codes: A61D 19/02 for artificial insemination
• 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: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties

Abstract

Improved insemination systems particularly adapted to use for sex-selected sperm sorting include systems which achieve superovulation and then multiple embryo production with sexed embryos. These systems combine with other techniques, including techniques for enhanced sheath fluid and other strategies which minimize stress on the sperm cells, and, potentially, 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 application of sperms samples as well as the resulting animals may be achieved.

Claims

1. A method of producing multiple, sexed embryos from a non-human female mammal comprising;

a. creating superovulation in said female mammal to create at least two eggs comprising the step of using an ovulatory pharmaceutical to cause multiple eggs to be produced;

b. determining a sex of a sperm cell of a male mammal;

c. sorting according to said sex of said sperm cells;

d. inserting at least a portion of said sorted sperm cells into a uterus of said female mammal after an onset of estrus; and

e. fertilizing a plurality of said eggs to produce at least two sexed embryos of the desired sex from said female mammal.

2. A method of producing multiple, sexed embryos according to claim 1 wherein said creating superovulation is encouraged during the estrus cycle.

3. A method of producing multiple, sexed embryos according to claim 2 wherein said step of using an ovulatory pharmaceutical comprises the step of injecting said ovulatory pharmaceutical in half days increments between any of days 2 and 18 of said estrus cycle.

4. A method of producing multiple, sexed embryos as described in claim 3 wherein injecting said ovulatory pharmaceutical in half day increments comprises injecting at least seven injections and further comprising the step of incorporating an estrus manipulation system at least on about the sixth and seventh injections.

5. A method of producing multiple, sexed embryos as described in claim 4 wherein inserting at least a portion of said sorted sperm cells into said uterus comprises inserting said sperm cells into both uterine horns of said uterus.

6. A method of producing multiple, sexed embryos as described in claim 5 wherein inserting into both uterine horns comprises inserting said sperm cells approximately between 20 to 24 hours inclusive after said onset of said estrus.

7. A method of producing multiple, sexed embryos as described in claim 1 wherein said step of using an ovulatory pharmaceutical to cause multiple eggs to be produced comprises the step of injecting a dosage of follicle stimulating hormone a plurality of times a day.

8. A method of producing multiple, sexed embryos as described in claim 7 wherein said step of creating superovulation in said mammal to create at least two eggs further comprises the step of incorporating an estrus manipulation system comprising the step of supplementing said dosage of follicle stimulant hormone with prostaglandin F-2-alpha.

9. A method of producing multiple, sexed embryos as described in claim 8 wherein injecting said dosage of follicle stimulating hormone a plurality of times a day comprises injecting said follicle stimulating hormone in approximately half day increments at a dosage level of 6, 6, 4, 4, 2, 2, 2, and 2 mg between days 9 and 12 inclusive of the estrus cycle and wherein administering said dosage of follicle stimulant hormone with prostaglandin F-2-alpha comprises supplementing 25 and 12.5 mg of prostaglandin F-2-alpha on the sixth and seventh dosages, respectively, of said follicle stimulating hormone.

10. A method of producing multiple, sexed embryos as described in claim 1 and further comprising the steps of:

a. staining sperm cells of a male mammal;

b. sorting according to said sex of said sperm cells at sperm cell sorting rates selected from the group consisting of;

above 500 sorts per second, above 1000 sorts per second, and above 1200 sorts per second; and

c. concentrating said sorted sperm cells.

11. A method of producing multiple, sexed embryos as described in claim 1 wherein inserting at least a portion of said sorted sperm cells comprises using a low dose of said sperm cells.

12. A method of producing multiple, sexed embryos as described in claim 10 wherein inserting at least a portion of said sorted sperm cells comprises using a low dose of said sperm cells.

13. A method of producing a mammal of a desired sex using the processes as described in claim 1.

14. A method of producing a mammal of a desired sex as described in claim 13 wherein said step of sorting comprises the steps of:

a. sorting said sperm cells at sperm cell sorting rates selected from the group consisting of;

above 500 sorts per second, above 1000 sorts per second, and above 1200 sorts per second; and

b. producing a sexed sperm cell specimen.

15. A method of producing a mammal of a desired sex as described in claim 14, wherein said sexed sperm cell specimen comprises a low dose of said sperm cells.

16. A method of producing a mammal of a desired sex as described in claim 13 further comprising chemically coordinating a sheath fluid to create a sheath fluid environment for said cells which is coordinated with both a pre-sort and a post-sort cell fluid environment comprising establishing a sheath fluid which contains about 2.9% sodium citrate.

17. A method of producing a mammal of a desired sex as described in claim 16 wherein chemically coordinating a sheath fluid to create a sheath fluid environment for said cells which is coordinated with both a pre-sort and a post-sort cell fluid environment comprises establishing a sheath fluid which contains a HEPES buffered medium.

18. A method of producing a mammal of a desired sex as described in claim 17 further comprising collecting sperm cells of said desired sex and cushioning said cells from impact with a collection container through use of a collection container which has a wide opening.