Separation systems of frozen-thawed spermatozoa into X-chromosome bearing and Y-chromosome bearing populations
View Patent ↗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 uniformly 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.
1. A process for preparing sperm cell samples, the process comprising:
collecting semen from a male mammal;
freezing said semen to form frozen semen;
thawing said frozen semen to form frozen-thawed semen;
incubating sperm cells contained within said frozen-thawed semen in a concentration of Hoechst 33342 stain of greater than 40 micro-molar;
establishing the temperature at which said sperm cells are incubated between about 30 degrees centigrade and about 40 degrees centigrade;
adjusting a duration of time said sperm cells are incubated in said concentration of Hoechst 33342 stain;
staining 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;
determining the sex characteristic of a plurality of sperm cells contained within said frozen-thawed semen;
separating said sperm cells according to the determination of their sex characteristic;
isolating sperm cells separated according to the determination of their sex in a collection element;
establishing a sample from said sperm cells isolated in said collection element;
wherein fertilizing at least one egg of a female mammal with said sample at success levels of at least about 70% of success with spermatozoa that have not been separated and/or frozen is achieved.
2. The process of claim 1 , wherein the sperm cells isolated in said collection element comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 85% X chromosome bearing sperm cells or at least about 85% Y chromosome bearing sperm cells.
3. The process of claim 1 , wherein the sperm cells isolated in said collection element comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 90% X chromosome bearing sperm cells or at least about 90% Y chromosome bearing sperm cells.
4. The process of claim 1 , wherein the sperm cells isolated in said collection element comprises spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 95% X chromosome bearing sperm cells or at least about 95% Y chromosome bearing sperm cells.
5. The process of 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.
6. The process of claim 1 , 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 up to about 95%.
7. The process of claim 6 , 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 purity of up to about 95% comprises differentiating said magnitude of fluorescence with a flow cytometer.
8. The process of claim 7 , 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 selected from a group consisting of about 1000 per second, and about 2000 per second.
9. The process of claim 7 , 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 selected from a group consisting of about 1000 per second, and about 2000 per second.
10. The process of claim 1 , wherein said male mammal is a bovid, and wherein said concentration of Hoechst 33342 stain is between about 200 micro-molar and about 2500 micro-molar.
11. The process of claim 10 , wherein said male mammal is a bovid, and wherein said concentration of Hoechst 33342 stain is 224 micro-molar.
12. The process of claim 10 , wherein said male mammal is a bovid and wherein said concentration of Hoechst 3342 stain is 2240 micro-molar.
13. The process of claim 10 , 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.
14. The process of claim 1 , further comprising the step of limiting the number of isolated sperm cells in said sample to about 10% to about 50% of the number of said sperm cells relative to a typical unseparated artificial insemination sample.
15. The process of claim 1 , wherein said sample has the number of isolated sperm cells limited to about one million to three million.
16. The process of claim 1 , wherein said sample has the of number isolated sperm cells limited to between about one-hundred and fifty thousand and about one million.
17. The process of claim 1 , wherein said sample has the number of isolated sperm cells limited to between about forty million and about one hundred million.
18. The process of claim 1 , wherein said step of establishing said sample from said sperm cells isolated in said collection element comprises the step of establishing an in-vitro fertilization sample from said sperm cells isolated in said collection element.
19. The process of claim 1 , wherein said step of establishing said sample from said sperm cells isolated in said collection element comprises the step of establishing an artificial insemination sample from said sperm cells isolated in said collection element.
20. A process for preparing sperm cell samples, the process comprising:
collecting semen from a male mammal;
freezing said semen to form frozen semen;
thawing said frozen semen to form frozen-thawed semen;
incubating sperm cells contained within said frozen-thawed semen in a concentration of Hoechst 33342 stain of greater than 40 micro-molar;
establishing the temperature at which said sperm cells are incubated between about 30 degrees centigrade and about 40 degrees centigrade;
adjusting a duration of time said sperm cells are incubated in said concentration of Hoechst 33342 stain;
staining 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;
determining the sex characteristic of a plurality of sperm cells contained within said frozen-thawed semen;
separating said sperm cells according to the determination of their sex characteristic;
isolating sperm cells separated according to the determination of their sex in a collection element, wherein the sperms cells are stained with sufficient uniformity to produce a population of either X-chromosome bearing sperm cells or Y-chromosome bearing sperm cells with a rate of between about 85% and about 95%;
establishing a sample from said sperm cells isolated in said collection element; wherein fertilizing at least one egg of a female mammal with said sample at success levels of at least about 70% of success with spermatozoa that have not been separated and/or frozen is achieved.