Methods for seperating frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations
AssigneeInguran, LLC
InventorsGeorge E. Seidel, Tae Kwang Suh, Kehuan Lu
Life of this patent
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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 uniformily 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.
Claims21
-75. (canceled)
A method of producing a mammal having a predetermined sex comprising the steps of:
- a. collecting sperm from a mammal;
- b. freezing said sperm;
- c. thawing said sperm;
- d. determining a sex characteristic of a plurality of sperm cells contained within said frozen thawed sperm;
- e. separating said sperm according to the determination of their sex characteristic;
- f. isolating sperm cells separated according to the determination of their sex in a collection element;
- g. establishing an artificial insemination sample from said sperm cells isolated in said collection element.
- h. fertilizing at least one egg with the insemination sample.
The method according to claim 1 further comprising the steps of:
- a. staining DNA within the frozen-thawed sperm cells with a concentration of a flourochrome dye greater than 40 micromolar;
- and
- b. adjusting the staining temperature to between about 30° C. and about 40° C.
The method according to claim 1 wherein the step of fertilizing at least one egg is accomplished at success levels of at least about 70% of the success levels with sperm that have not been separated and/or frozen.
The method according to claim 1 further comprising the step of fertilizing at least one egg within a female.
The method according to claim 1 further comprising the step of fertilizing at least one egg in vitro.
The method according to claim 1 wherein the sperm cells isolated in said collection element comprise spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 85% Y chromosome bearing sperm cells.
The method according to claim 1 wherein the sperm cells isolated in said collection element comprise spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 85% X chromosome bearing sperm cells.
The method according to claim 1 wherein the mammal comprises a bovine and wherein said artificial insemination sample has a number of isolated sperm cells between about one million to about three million.
The method according to claim 1 wherein the mammal comprises a bovine and wherein said artificial insemination sample has a number of isolated sperm cells between about one hundred and fifty thousand to about one million.
The method according to claim 1 wherein the mammal comprises an equine and wherein said artificial insemination sample has a number of isolated sperm cells between about forty million and one hundred million.
The method according to claim 1 wherein the step of isolating the sperm cells separated according to the determination of their sex in a collection element.
The method according to claim 1 wherein the step of isolating sperm cells separated according to the determination of their sex in a collection element comprises isolating Y-chromosome bearing sperm into a separate collection element at a rate of at least about 1000 per second.
The method according to claim 1 wherein the step of isolating sperm cells separated according to the determination of their sex in a collection element comprises isolating X-chromosome bearing sperm into a separate collection element at a rate of at least about 1000 per second.
A method of producing an artificial insemination sample comprising the steps of:
- a. obtaining frozen sperm;
- b. thawing said frozen sperm;
- c. staining DNA within the frozen-thawed sperm cells with a concentration of a flourochrome dye greater than 40 micromolar;
- d. adjusting the staining temperature to between about 30° C. and about 40° C.;
- e. determining a sex characteristic of a plurality of sperm cells contained within said frozen thawed sperm;
- f. separating said sperm according to the determination of their sex characteristic;
- g. isolating sperm cells separated according to the determination of their sex in a collection element;
- h. establishing an artificial insemination sample from said sperm cells isolated in said collection element.
The method according to claim 12 wherein the artificial insemination sample is capable of fertilizing at least one egg at success levels of at least about 70% of the success levels with sperm that have not been separated and/or frozen.
The method according to claim 1 wherein the sperm cells isolated in said collection element comprise spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 85% Y chromosome bearing sperm cells.
The method according to claim 1 wherein the sperm cells isolated in said collection element comprise spermatozoa sorted into separate populations, wherein the spermatozoa of one of the populations comprises at least about 85% X chromosome bearing sperm cells.
The method according to claim 1 wherein the mammal comprises a bovine and wherein said artificial insemination sample has a number of isolated sperm cells between about one million to about three million.
The method according to claim 1 wherein the mammal comprises a bovine and wherein said artificial insemination sample has a number of isolated sperm cells between about one hundred and fifty thousand to about one million.
The method according to claim 1 wherein the mammal comprises an equine and wherein said artificial insemination sample has a number of isolated sperm cells between about forty million and one hundred million.
Dependencies are inferred from the claim text.