System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations
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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.
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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 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.
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18 Claims
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1. A method of using stained sperm cells collected from mammals, comprising the steps of:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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Specification