Sperm staining and sorting methods
View Patent ↗A method of sex sorting sperm is disclosed. The sperm may be stained with a DNA selective fluorescent dye, which fluoresces when excited, a dead quenching dye, which selectively quenches fluorescence emitted by the DNA selective fluorescent dye within the membrane of compromised sperm, and a split enhancing dye. The stained sperm may then sorted into one or more gender enriched subpopulations of viable sperm.
1. A sperm suspension comprising: viable, sperm having a DNA selective fluorescent dye associated with nuclear DNA;
a medium supporting sperm viability;
a second dye that permeates the membranes of compromised sperm and quenches fluorescence emitted from the DNA selective fluorescent dye, the second dye having a first local absorption maximum wavelength; and
a third dye that quenches fluorescence noise, the third dye having a second local absorption maximum wavelength that is greater than the first local absorption wavelength of the second dye.
2. The sperm suspension of claim 1 further comprising an intermediate product collected from a flow cytometer.
3. The sperm suspension of claim 1 further comprising an inseminate having trace amounts of the second dye and the third dye.
4. The sperm suspension of claim 1 wherein the sperm suspension is frozen.
5. The sperm suspension of claim 1 , wherein the second dye and the third dye are different color dyes.
6. The sperm suspension of claim 1 , wherein the third dye is impedance matched to non-specific noise.
7. The sperm suspension of claim 1 , wherein the third dye is split enhancing dye.
8. The sperm suspension of claim 1 , wherein the third dye absorbs fluorescence emissions having a wavelength within the range of about 520 nm to about 600 nm.
9. The sperm suspension of claim 1 , wherein local absorption maximum wavelength of the third dye is about 520 nm.
10. The sperm suspension of claim 1 , wherein the viable sperm comprises a gender enriched population of sperm.
11. The sperm suspension of claim 1 , wherein the viable sperm comprises a sex sorted population of sperm.
12. The sperm suspension of claim 1 , wherein the second dye is a dead quenching dye.
13. The sperm suspension of claim 1 , wherein the sperm suspension comprises a flow sorted sperm suspension.
14. The sperm suspension of claim 1 , wherein the sperm suspension comprises an artificial inseminate.