Separation systems of frozen-thawed spermatozoa into X-chromosome bearing and Y-chromosome bearing populations
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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.
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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 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.
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20 Claims
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1. A process for preparing sperm cell samples, the process comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A process for preparing sperm cell samples, the process comprising:
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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.
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Specification