System to separate 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 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.
1. A method of using stained sperm cells collected from mammals, comprising the steps of:
collecting semen from a male of a non-human mammal;
freezing said semen;
thawing said semen;
incubating sperm cells contained within said semen in a concentration of Hoechst 33342 stain of greater than 40 micro-molar;
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;
adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain;
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;
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 collected element;
establishing a sample from said sperm cells isolated in said collection element;
successfully fertilizing at least one egg of a female non-human mammal with said sample at success levels of at least about 70% of success with spermatozoa that have not been separated and/or frozen; and
successfully producing offspring non-human mammal of the desired sex.
2. A method of using stained sperm cells collected from mammals as described in claim 1 , wherein said male of a non-human mammal is selected from the group of mammals consisting of non-human primates, swine, ovids, bovids, equids, canids, felids, and dolphins.
3. A method of using stained sperm cells collected from mammals as described in claim 2 , wherein said male of a non-human 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 using stained sperm cells collected from mammals as described in claim 2 , wherein said male of a non-human mammal comprises said bovid and said concentration of Hoechst 33342 stain is 224 micro-molar.
5. A method of using stained sperm cells collected from mammals as described in claim 2 , wherein said male of a non-human mammal comprises said bovid and said concentration of Hoechst 3342 stain is 2240 micro-molar.
6. A method of using stained sperm cells collected from mammals as described in claim 1 , wherein said step of adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain comprises adjusting said duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain between about 50 minutes and about 200 minutes.
7. A method of using stained sperm cells collected from mammals as described in claim 1 , wherein said step of adjusting a duration of time said sperm cells are incubated with said concentration of Hoechst 33342 stain 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 using stained 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 comprises differentiating said Y-chromosome bearing sperm cells from said X-chromosome bearing sperm cells based upon the magnitude of fluorescence at a purity rate selected from the group consisting of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, and 95%.
9. A method of using stained 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 using stained sperm cells collected from mammals as described in claim 1 , wherein said step of isolating sperm cells separated according to the determination of their sex in said collected 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.
11. A method of using stained sperm cells collected from mammals as described in claim 1 , wherein said step of isolating sperm cells separated according to the determination of their sex in said collected 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.
12. A method of using stained 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 comprises differentiating said magnitude of fluorescence with a flow cytometer.
13. A method of using stained sperm cells collected from mammals as described in claim 1 , wherein said step of successfully fertilizing at least one egg of said female non-human mammal comprises the step of successfully fertilizing at least one egg of said female non-human mammal by in vitro fertilization.
14. A method of using stained sperm cells collected from mammals as described in claim 13 , wherein said in vitro fertilization success levels are selected from a group consisting of at least about 17%, at least about 23%, at least about 16%, between about 16% and about 23%, about 17%, about 16%, about 23%; and of success levels with spermatozoa that have not been separated and/or frozen of at least about 90%, and between about 70% and about 90%.
15. A method of using stained sperm cells collected from mammals as described in claim 1 , wherein said step of successfully fertilizing at least one egg of said female non-human mammal comprises the step of successfully fertilizing at least one egg of said female non-human mammal by artificial insemination.
16. A method of using stained sperm cells collected from mammals as described in claim 15 , wherein said success levels with spermatozoa that have not been separated and/or frozen are selected from a group consisting of at least 41% and at least 50%.
17. A method of using stained sperm cells collected form mammals as described in claim 13 , wherein said success levels with spermatozoa that have not been separated and/or frozen are selected from a group consisting of at least 41% and at least 50%.
18. A method of using stained sperm cells collected form mammals as described in claim 1 , wherein said success levels with spermatozoa that have not been separated and/or frozen are selected from a group consisting of at least 41% and at least 50%.