System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations
First Claim
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1. A method of staining sperm cells collected from mammals, comprising the steps of:
- a. collecting semen from a male mammal;
b. incubating sperm cells contained within said semen in a concentration of Hoechst 33342 stain of greater than 40 micro-molar;
c. 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;
d. 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; and
e. 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 rate of greater than about 85%.
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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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Citations
75 Claims
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1. A method of staining sperm cells collected from mammals, comprising the steps of:
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a. collecting semen from a male mammal;
b. incubating sperm cells contained within said semen in a concentration of Hoechst 33342 stain of greater than 40 micro-molar;
c. 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;
d. 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; and
e. 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 rate of greater than about 85%. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of staining DNA within frozen-thawed sperm cells, comprising the steps of:
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a. collecting semen containing sperm cells from a male mammal;
b. freezing said semen containing said sperm cells;
c. thawing said semen containing said sperm cells;
d. combining frozen-thawed semen with Hoechst 33342 stain;
e. establishing a concentration of said Hoechst 33342 stain between about 200 micro-molar and about 2500 micro-molar;
f. adjusting the temperature of said frozen-thawed semen in said concentration of Hoechst 33342 stain to between about 30 degrees Centigrade and about 40 degrees Centigrade; and
g. adjusting the duration of time said frozen-thawed semen in said concentration of Hoechst 33342 stain incubates to between about 50 minutes and 200 minutes, whereby DNA within sperm cells contained in said frozen-thawed are stained with sufficient uniformity to differentiate between X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells on the basis of magnitude of fluorescence. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A method of generating mammalian embryos, comprising the steps of:
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a. collecting semen from a male mammal;
b. combining said semen from said male mammal with an amount of Hoechst 33342 stain;
c. establishing a concentration of Hoechst 33342 stain combined with said semen to a concentration between about 40 micro-molar and about 2500 micro-molar;
d. adjusting the temperature at which said sperm cells are incubated with said Hoechst 33342 stain between about 30 degrees centigrade and about 40 degrees centigrade;
e. adjusting the duration of time said sperm cells are incubated in said concentration of Hoechst 33342 between about 60 minutes and about 200 minutes;
f. staining DNA within sperm cells contained in said semen with said Hoechst 33342 stain; and
g. fertilizing oocytes with stained sperm cells, whereby increasing the concentration of Hoechst 33342 stain and decreasing the duration of time said sperm cells are incubated in said concentration of Hoechst 33342 stain increases the percentage of said mammalian embryos produced. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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40. A flow cytometer system for isolating desired sperm cells, comprising:
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a. sperm cells obtained by thawing previously frozen semen, wherein said sperm cells are incubated with a concentration of Hoechst 33342 stain between about 200 micro-molar and about 2500 micro-molar until DNA within said sperm cells are stained 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 rate of greater than about 85%;
b. a sperm cell source that supplies said sperm cells to a flow cytometer;
c. a sheath fluid source that creates a sheath fluid environment within said flow cytometer in which said sperm cells are entrained;
d. a nozzle through which said sperm cells pass while entrained in said sheath fluid environment;
e. an oscillator that acts upon said sheath fluid as it passes through said nozzle;
f. a sperm cell sensing system responsive to said sperm cells;
g. a separation discrimination system that acts to separate said sperms cells having a desired characteristic; and
h. a containment element into which said sperm cells having said desired characteristic are collected. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56)
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57. A method of producing a mammal having a predetermined sex comprising the steps of:
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a. collecting semen from a male mammal;
b. freezing said semen;
c. thawing said semen;
b. determining the sex characteristic of a plurality of sperm cells contained within said frozen-thawed semen;
c. separating said sperm cells according to the determination of their sex characteristic;
d. isolating sperm cells separated according to the determination of their sex in a collection element;
d. establishing an artificial insemination sample from said sperm cells isolated in said collection element;
e. inserting said artificial insemination sample into a female mammal of the same species from which said semen was collected;
f. fertilizing at least one egg within said female mammal; and
g. producing an offspring mammal of the desired sex. - View Dependent Claims (58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75)
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Specification