Methods and apparatus for producing gender enriched sperm
First Claim
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1. A method for sorting semen containing predominantly living, viable sperm into GES (gender enriched sperm) comprising:
- a) staining DNA in the sperm for an incubation period effective for staining nuclei of living sperm cells sufficient to distinguish chromosomal determinants of sex in individual sperm based on resulting fluorescence under a fluorescence stimulating light source using a quantitative DNA vital stain (QDVS) under conditions selected from the following and combinations thereof;
(a) temperature in the range from about 18°
C. to less than about 30°
C., (b) pH in the range of about 7.1 to about 7.6 (c) incubation for an extended period at a lower temperature followed by a shorter period at higher temperature to enhance staining; and
b) separating the thus-incubated sperm into GES using FACS (fluorescence-activated cell sorting and producing GES consisting predominantly of living sperm of which greater than 90% are of one sex.
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Abstract
Sperm in semen are sorted by fluorescence-activated cell sorting into gender-enriched populations enriched in X-chromosome or Y-chromosome bearing sperm by use of a fluorescent quantitative DNA-binding vital stain.
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Citations
29 Claims
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1. A method for sorting semen containing predominantly living, viable sperm into GES (gender enriched sperm) comprising:
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a) staining DNA in the sperm for an incubation period effective for staining nuclei of living sperm cells sufficient to distinguish chromosomal determinants of sex in individual sperm based on resulting fluorescence under a fluorescence stimulating light source using a quantitative DNA vital stain (QDVS) under conditions selected from the following and combinations thereof;
(a) temperature in the range from about 18°
C. to less than about 30°
C., (b) pH in the range of about 7.1 to about 7.6 (c) incubation for an extended period at a lower temperature followed by a shorter period at higher temperature to enhance staining; and
b) separating the thus-incubated sperm into GES using FACS (fluorescence-activated cell sorting and producing GES consisting predominantly of living sperm of which greater than 90% are of one sex. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A process for producing GES (gender enriched semen) comprising:
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a) providing a suspension of viable sperm produced from collected semen ejaculate that is extended and transported to a sorting facility;
b) staining the sperm using a QDVS (quantitative DNA vital stain) in the presence of a medium comprising a buffer system and further optionally including other components, the medium effective for maintaining viability of at least a portion of the semen;
c) producing at least one of X-enriched and Y-enriched GES based on the extent of QDVS staining;
d) collecting the resulting GES;
e) wherein steps c) and d) occur in the presence of media comprising at least said buffer system, and f) partitioning the collected GES into dosage quantities for use or shipment. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A process for producing GES (gender enriched semen) comprising:
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a) providing a suspension of viable sperm produced from collected semen ejaculate that is extended and transported to a sorting facility;
b) staining the sperm using a QDVS (quantitative DNA vital stain);
c) based on the extent of QDVS staining producing at least one of X-enriched and Y-enriched GES;
d) collecting the resulting GES; and
e) partitioning the collected GES into dosage quantities for use or shipment;
f) wherein from step a) starting after collection of the semen ejaculate until completion of step e) all steps occur at a temperature in a range from above the thermotropic phase transition temperature Tm of the membranes of the sperm being sorted up to less than about 30°
C. and in the presence of a buffer system which is used at least for steps b), c) and d) and at an effective pH between about 6.8 and about 7.6. - View Dependent Claims (23, 24, 25, 27, 28, 29)
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- 22. The method of claim 21 wherein step c) is conducted using a FACS (fluorescence-activated cell sorter) and the FACS is operated at ambient temperature.
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22-1. A process for producing GES (gender enriched semen) comprising:
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a) providing a suspension of viable sperm produced from collected semen ejaculate that is extended and transported to a sorting facility;
b) staining the sperm using a QDVS (quantitative DNA vital stain) that fluoresces in response to visible light irradiation;
c) based on the extent of QDVS staining producing at least one of X-enriched and Y-enriched GES;
d) collecting the resulting GES; and
e) partitioning the collected GES into dosage quantities for use or shipment;
f) wherein from step a) starting after collection of the semen ejaculate until completion of step e) all steps occur (i) at a temperature in a range from above the lower semen viability temperature to less than the upper semen viability temperature and (ii) in the presence of sperm maintenance media effective for maintaining viability of at least a portion of the semen throughout the process.
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Specification