System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations

• US 20040049801A1
• Filed: 05/29/2003
• Published: 03/11/2004
• Est. Priority Date: 11/29/2000
• Status: Active Grant

• Current Assignee: XY, LLC (Inguran LLC)
• Original Assignee: XY, LLC (Inguran LLC)
• Inventors: Lu, Kehuan, Seidel, George E., Suh, Tae Kwang
• Granted Patent: US 7,713,687 B2
• Time in Patent Office: Days
• US Class Current: 800/21
• 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: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

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 uniformity 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.

Claims

1. A method of staining sperm cells collected from mammals, comprising the steps of:

a. collecting semen from a male mammal;

b. incubating sperm cells contained within said semen in a concentration of Hoechst 33342 stain of greater than 40 micro-molar;

c. establishing the temperature at which said sperm cells in said concentration of Hoechst 33342 stain are incubated between about 30 degrees centigrade and about 40 degrees centigrade;

d. adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain between about 50 minutes and about 200 minutes; and

e. staining DNA within said sperm cells with sufficient uniformity to allow X-chromosome bearing sperm cells to be differentiated from Y-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate of greater than about 85%.

2. A method of staining sperm cells collected from mammals as described in claim 1, wherein said male mammal is selected from the group of mammals consisting of primates, humans, swine, ovids, bovids, equids, canids, felids, and dolphins.

3. A method of staining sperm cells collected from mammals as described in claim 2, wherein said male mammal comprises said bovid and said concentration of Hoechst 33342 stain is between about 200 micro-molar and about 2500 micro-molar.

4. A method of staining sperm cells collected from mammals as described in claim 2, wherein said male mammal comprises said bovid and said concentration of Hoechst 33342 stain is 224 micro-molar.

5. A method of staining sperm cells collected from mammals as described in claim 2, wherein said male mammal comprises said bovid and said concentration of Hoechst 3342 stain is 2240 micro-molar.

6. A method of staining sperm cells collected from mammals as described in claim 4, wherein said step of adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain between about 50 minutes and about 200 minutes comprises adjusting said duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain to about 190 minutes.

7. A method of staining sperm cells collected from mammals as described in claim 5, wherein said step of adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain between about 50 minutes and about 200 minutes comprises adjusting said duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain to about 60 minutes.

8. A method of staining sperm cells collected from mammals as described in claim 1, wherein said step of staining DNA within said sperm cells with sufficient uniformity to allow X-chromosome bearing sperm cells to be differentiated from Y-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate of greater than about 85% comprises differentiating said Y-chromosome bearing sperm cells from said X-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate selected from the group consisting of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

9. A method of staining sperm cells collected from mammals as described in claim 8, wherein said X-chromosome bearing sperm cells differentiated from said Y-chromosome bearing sperm cells comprise viable sperm cells.

10. A method of staining sperm cells collected from mammals as described in claims 1, 2, 6, 7, 8, or 9, further comprising the step of freezing said semen.

11. A method of staining sperm cells collected from mammals as described in claim 10, further comprising the step of thawing said semen.

12. A method of staining sperm cells collected from mammals as described in claim 11, wherein said step of staining DNA within said sperm cells with sufficient uniformity to allow X-chromosome bearing sperm cells to be differentiated from Y-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate of greater than about 85% comprises differentiating said magnitude of fluorescence with a flow cytometer.

13. A method of staining DNA within frozen-thawed sperm cells, comprising the steps of:

a. collecting semen containing sperm cells from a male mammal;

b. freezing said semen containing said sperm cells;

c. thawing said semen containing said sperm cells;

d. combining frozen-thawed semen with Hoechst 33342 stain;

e. establishing a concentration of said Hoechst 33342 stain between about 200 micro-molar and about 2500 micro-molar;

f. adjusting the temperature of said frozen-thawed semen in said concentration of Hoechst 33342 stain to between about 30 degrees Centigrade and about 40 degrees Centigrade; and

g. adjusting the duration of time said frozen-thawed semen in said concentration of Hoechst 33342 stain incubates to between about 50 minutes and 200 minutes, whereby DNA within sperm cells contained in said frozen-thawed are stained with sufficient uniformity to differentiate between X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells on the basis of magnitude of fluorescence.

14. A method of staining sperm cells collected from mammals as described in claim 13, wherein said male mammal is selected from the group of mammals consisting of primates, humans, swine, ovids, bovids, equids, canids, felids, and dolphins.

15. A method of staining sperm cells collected from mammals as described in claim 14, wherein said male mammal comprises said bovid and said concentration of Hoechst 33342 stain is between about 200 micro-molar and about 2500 micro-molar.

16. A method of staining sperm cells collected from mammals as described in claim 14, wherein said male mammal comprises said bovid and said concentration of Hoechst 33342 stain is 224 micro-molar.

17. A method of staining sperm cells collected from mammals as described in claim 14, wherein said male mammal comprises said bovid and said concentration of Hoechst 3342 stain is 2240 micro-molar.

18. A method of staining sperm cells collected from mammals as described in claim 14, wherein said step of adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain between about 50 minutes and about 200 minutes comprises adjusting said duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain to about 190 minutes.

19. A method of staining sperm cells collected from mammals as described in claim 17, wherein said step of adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain between about 50 minutes and about 200 minutes comprises adjusting said duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain to about 60 minutes.

20. A method of staining sperm cells collected from mammals as described in claims 13, 15, 16, 17, 18, 19, or 20, wherein said step of staining DNA within said sperm cells with sufficient uniformity to allow X-chromosome bearing sperm cells to be differentiated from Y-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate of greater than about 85% comprises differentiating said Y-chromosome bearing sperm cells from said X-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate selected from the group consisting of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

21. A method of staining sperm cells collected from mammals as described in claim 20, wherein said X-chromosome bearing sperm cells differentiated from said Y-chromosome bearing sperm cells comprise viable sperm cells.

22. A method of staining sperm cells collected from mammals as described in claim 20, wherein said step of staining DNA within said sperm cells with sufficient uniformity to allow X-chromosome bearing sperm cells to be differentiated from Y-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate of greater than about 85% comprises differentiating said magnitude of fluorescence with a flow cytometer.

23. A method of generating mammalian embryos, comprising the steps of:

a. collecting semen from a male mammal;

b. combining said semen from said male mammal with an amount of Hoechst 33342 stain;

c. establishing a concentration of Hoechst 33342 stain combined with said semen to a concentration between about 40 micro-molar and about 2500 micro-molar;

d. adjusting the temperature at which said sperm cells are incubated with said Hoechst 33342 stain between about 30 degrees centigrade and about 40 degrees centigrade;

e. adjusting the duration of time said sperm cells are incubated in said concentration of Hoechst 33342 between about 60 minutes and about 200 minutes;

f. staining DNA within sperm cells contained in said semen with said Hoechst 33342 stain; and

g. fertilizing oocytes with stained sperm cells,

whereby increasing the concentration of Hoechst 33342 stain and decreasing the duration of time said sperm cells are incubated in said concentration of Hoechst 33342 stain increases the percentage of said mammalian embryos produced.

24. A method of staining sperm cells collected from mammals as described in claim 23, wherein said male mammal is selected from the group of mammals consisting of primates, humans, swine, ovids, bovids, equids, canids, felids, and dolphins.

25. A method of staining sperm cells collected from mammals as described in claim 24, wherein said male mammal comprises said bovid and said concentration of Hoechst 33342 stain is between about 200 micro-molar and about 2500 micro-molar.

26. A method of staining sperm cells collected from mammals as described in claim 24, wherein said male mammal comprises said bovid and said concentration of Hoechst 33342 stain is 224 micro-molar.

27. A method of staining sperm cells collected from mammals as described in claim 24, wherein said male mammal comprises said bovid and said concentration of Hoechst 3342 stain is 2240 micro-molar.

28. A method of staining sperm cells collected from mammals as described in claim 26, wherein said step of adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain between about 50 minutes and about 200 minutes comprises adjusting said duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain to about 190 minutes.

29. A method of staining sperm cells collected from mammals as described in claim 27, wherein said step of adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain between about 50 minutes and about 200 minutes comprises adjusting said duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain to about 60 minutes.

30. A method of staining sperm cells collected from mammals as described in claims 23, 25, 26, 27, 28, 29, or 30, wherein said step of staining DNA within said sperm cells with sufficient uniformity to allow X-chromosome bearing sperm cells to be differentiated from Y-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate of greater than about 85% comprises differentiating said Y-chromosome bearing sperm cells from said X-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate selected from the group consisting of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

31. A method of staining sperm cells collected from mammals as described in claim 30, wherein said X-chromosome bearing sperm cells differentiated from said Y-chromosome bearing sperm cells comprise viable sperm cells.

32. A method of staining sperm cells collected from mammals as described in claim 30, wherein said step of staining DNA within said sperm cells with sufficient uniformity to allow X-chromosome bearing sperm cells to be differentiated from Y-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate of greater than about 85% comprises differentiating said magnitude of fluorescence with a flow cytometer.

33. A method of staining sperm cells collected from mammals as described in claim 32, further comprising the step of isolating differentiated X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells into separate collection elements.

34. A method of staining sperm cells collected from mammals as described in claim 33, wherein said step of isolating differentiated X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells into separate collection elements comprises isolating Y-chromosome bearing sperm cells into a separate collection element at a rate of about 1000 per second.

35. A method of staining sperm cells collected from mammals as described in claim 33, wherein said step of isolating differentiated X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells into separate collection elements comprises isolating X-chromosome bearing sperm cells into a separate collection element at a rate of about 1000 per second.

36. A method of generating mammalian embryos as described in claims 23, further comprising the step of freezing said semen.

37. A method of generating mammalian embryos as described in claims 30, further comprising the step of freezing said semen.

38. A method of generating mammalian embryos as described in claim 36, further comprising the step of thawing said semen.

39. A method of generating mammalian embryos as described in claim 37, further comprising the step of thawing said semen.

40. A flow cytometer system for isolating desired sperm cells, comprising:

a. sperm cells obtained by thawing previously frozen semen, wherein said sperm cells are incubated with a concentration of Hoechst 33342 stain between about 200 micro-molar and about 2500 micro-molar until DNA within said sperm cells are stained with sufficient uniformity to allow X-chromosome bearing sperm cells to be differentiated from Y-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate of greater than about 85%;

b. a sperm cell source that supplies said sperm cells to a flow cytometer;

c. a sheath fluid source that creates a sheath fluid environment within said flow cytometer in which said sperm cells are entrained;

d. a nozzle through which said sperm cells pass while entrained in said sheath fluid environment;

e. an oscillator that acts upon said sheath fluid as it passes through said nozzle;

f. a sperm cell sensing system responsive to said sperm cells;

g. a separation discrimination system that acts to separate said sperms cells having a desired characteristic; and

h. a containment element into which said sperm cells having said desired characteristic are collected.

41. A flow cytometer system for isolating desired sperm cells as described in claim 40, wherein said sperm cells obtained by thawing previously frozen semen are obtained from male mammals selected from the group consisting of primates, humans, swine, ovids, bovids, equids, canids, felids, and dolphins.

42. A flow cytometer system for isolating desired sperm cells as described in claim 41, wherein said male mammal comprises said bovid, and wherein said concentration of Hoechst 33342 stain is between about 200 micro-molar and about 2500 micro-molar.

43. A flow cytometer system for isolating desired sperm cells as described in claim 41, wherein said male mammal comprises said bovid, and wherein said concentration of Hoechst 33342 stain is about 224 micro-molar.

44. A flow cytometer system for isolating desired sperm cells as described in claim 41, wherein said male mammal comprises said bovid, and wherein said concentration of Hoechst 3342 stain is 2240 micro-molar.

45. A flow cytometer system for isolating desired sperm cells as described in claim 42, further comprising a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain between about 50 minutes and about 200 minutes.

46. A flow cytometer system for isolating desired sperm cells as described in claim 43, wherein said duration of time about 190 minutes.

47. A flow cytometer system for isolating desired sperm cells as described in claim 44, wherein said duration of time about 60 minutes.

48. A flow cytometer system for isolating desired sperm cells as described in claims 40, 42, 43, 45, or 48, wherein said X-chromosome bearing sperm cells to be differentiated from Y-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate of greater than about 85% is a rate selected from the group consisting of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

49. A flow cytometer system for isolating desired sperm cells as described in claim 48, further comprising a collection element into which differentiated X-chromosome bearing sperm cells are isolated.

50. A flow cytometer system for isolating desired sperm cells as described in claim 48, further comprising a collection element into which differentiated Y-chromosome bearing sperm are isolated.

51. A flow cytometer system for isolating desired sperm cells as described in claim 49, further comprising a rate at which X-chromosome bearing sperm cells are isolated greater than about 1000 per second.

52. A flow cytometer system for isolating desired sperm cells as described in claim 50, further comprising a rate at which Y-chromosome bearing sperm cells are isolated greater than about 1000 per second.

53. A method of generating mammalian embryos as described in claim 40, further comprising the step of freezing said semen.

54. A method of generating mammalian embryos as described in claims 51, further comprising the step of freezing said semen.

55. A method of generating mammalian embryos as described in claim 53, further comprising the step of thawing said semen.

56. A method of generating mammalian embryos as described in claim 54, further comprising the step of thawing said semen.

57. A method of producing a mammal having a predetermined sex comprising the steps of:

a. collecting semen from a male mammal;

b. freezing said semen;

c. thawing said semen;

b. determining the sex characteristic of a plurality of sperm cells contained within said frozen-thawed semen;

c. separating said sperm cells according to the determination of their sex characteristic;

d. isolating sperm cells separated according to the determination of their sex in a collection element;

d. establishing an artificial insemination sample from said sperm cells isolated in said collection element;

e. inserting said artificial insemination sample into a female mammal of the same species from which said semen was collected;

f. fertilizing at least one egg within said female mammal; and

g. producing an offspring mammal of the desired sex.

58. A method of producing a mammal having a predetermined sex as described in claim 57, wherein said male mammal is selected from the group of mammals consisting of primates, humans, swine, ovids, bovids, equids, canids, felids, and dolphins.

59. A method of producing a mammal having a predetermined sex as described in claim 58, further comprising the step of staining DNA within said sperm cells with a concentration of Hoechst 33342 greater than 40 micro-molar.

60. A method of producing a mammal having a predetermined sex as described in claim 59, wherein said step of staining DNA within said sperm cells with a concentration of Hoechst 33342 greater than 40 micro-molar comprises staining of sufficient uniformity to allow X-chromosome bearing sperm cells to be differentiated from Y-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate of greater than about 85%.

61. A method of producing a mammal having a predetermined sex as described in claim 59, wherein said male mammal comprises said bovid, and wherein said concentration of Hoechst 33342 stain is between about 200 micro-molar and about 2500 micro-molar.

62. A method of producing a mammal having a predetermined sex as described in claim 61, wherein said male mammal comprises said bovid, and wherein said concentration of Hoechst 33342 stain is 224 micro-molar.

63. A method of producing a mammal having a predetermined sex as described in claim 61, wherein said male mammal comprises said bovid and wherein said concentration of Hoechst 3342 stain is 2240 micro-molar.

64. A method of producing a mammal having a predetermined sex as described in claim 61, further comprising the step of adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain between about 50 minutes and about 200 minutes.

65. A method of producing a mammal having a predetermined sex as described in claim 62, wherein said step of adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain between about 50 minutes and about 200 minutes comprises adjusting said duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain to about 190 minutes.

66. A method of producing a mammal having a predetermined sex as described in claim 63, wherein said step of adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain between about 50 minutes and about 200 minutes comprises adjusting said duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain to about 60 minutes.

67. A method of producing a mammal having a predetermined sex as described in claims 57, 58, 61, 62, 63, 64, 65, or 66, wherein said step of staining DNA within said sperm cells with a concentration of Hoechst 33342 greater than 40 micro-molar comprises staining of sufficient uniformity to allow X-chromosome bearing sperm cells to be differentiated from Y-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate of greater than about 85% comprises a rate selected from the group consisting of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

68. A method of producing a mammal having a predetermined sex as described in claim 67, wherein said step of staining DNA within said sperm cells with sufficient uniformity to allow X-chromosome bearing sperm cells to be differentiated from Y-chromosome bearing sperm cells based upon the magnitude of fluorescence at a rate of greater than about 85% comprises differentiating said magnitude of fluorescence with a flow cytometer.

69. A method of staining sperm cells collected from mammals as described in claim 68, wherein said step of isolating sperm cells separated according to the determination of their sex in a collection element comprises isolating Y-chromosome bearing sperm cells into a separate collection element at a rate of about 1000 per second.

70. A method of staining sperm cells collected from mammals as described in claim 68, wherein said step of isolating sperm cells separated according to the determination of their sex in a collection element comprises isolating X-chromosome bearing sperm cells into a separate collection element at a rate of about 1000 per second.

71. A method of producing a mammal having a predetermined sex as described in claim 57, further comprising the step of limiting the number of isolated sperm cells in said artificial insemination sample to about 10% to about 50% of the number of said sperm cells relative to a typical unseparated artificial insemination sample.

72. A method of producing a mammal having a predetermined sex as described in claim 58, wherein said mammal comprises said bovid and wherein said artificial insemination sample has the number of isolated sperm cells limited to about one million to three million.

73. A method of producing a mammal having a predetermined sex as described in claim 58, wherein said mammal is a bovid and wherein said artificial insemination sample has the number of isolated sperm cells limited to between about one-hundred and fifty thousand and about one million.

74. A method of producing a mammal having a predetermined sex as described in claim 57, wherein said mammal comprises said equid and wherein said artificial insemination sample has the number of isolated sperm cells limited to between about forty million and about one hundred million.

75. A method of producing a mammal having a predetermined sex as described in claims 71, 72, 73 or 74, further comprising the step of 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, and wherein said ovulatory pharmaceutical is injected in half day increments between any of days 2 and 18 of the estrus cycle.