High pressure sperm sorting and flow cytometer methods

1. A method of sorting sperm comprising:

staining a sperm sample with a DNA selective dye and a quenching dye;

establishing a calibrated drop drive frequency as the highest drop drive frequency in a flow cytometer with live stained sperm at which there is no spraying in a calibration side stream;

establishing a calibrated drop delay at the calibrated drop drive frequency by forming puddles at different drop delays and counting the number of live sperm in the formed puddles;

determining whether the puddles formed during the step of establishing the calibrated drop delay comprise a round shape or a split puddle shape; and

sorting stained sperm in the sperm sample with the flow cytometer at the calibrated drop drive frequency and the calibrated drop delay at an elevated sheath fluid pressure between about 45 psi and about 65 psi if the shape of the puddles formed during the step of calibrating the drop delay comprise round puddles.

2. The method of claim 1 further comprising the step of: adjusting a concentration of the sperm sample and adjusting a pH of the sperm sample towards a predetermined pH by extending the sperm sample in an initial extender at a sperm sample to initial extender ratio to form an extended sperm sample, centrifuging the extended sperm sample, and removing supernatant to reach a predetermined concentration.

3. The method of claim 2 , wherein the step of staining the sperm sample with a DNA selective dye and a quenching dye further comprises staining sperm sample with the DNA selective dye and the quenching dye in a single dilution.

4. The method of claim 3 , wherein adjusting the concentration of the sperm sample and adjusting the pH of the sperm sample prior to staining and the step of staining the sperm sample in a single dilution reduce damage imposed on the sperm by the elevated pressure.

5. The method of claim 2 , wherein the initial extender comprises a pH buffering extender.

6. The method of claim 2 , wherein the sperm sample to initial extender ratio comprises a ratio between about 1:1 and 1:10.

7. The method of claim 2 , wherein the initial extender comprises one or more selected from the group of: sodium bicarbonate, TRIS citrate, sodium citrate, HEPES, TRIS, TEST, MOPS, KMT, TALP, and combinations thereof.

8. The method of claim 7 , wherein the initial extender further comprises egg yolk, milk, lipoproteins, lecithin, or combinations thereof.

9. The method of claim 7 , wherein the initial extender has a pH of between about 6.8 and about 7.4.

10. The method of claim 2 , wherein the steps of centrifuging the extended sperm sample and removing supernatant to reach a predetermined concentration further comprises adjusting the concentration to between about 1400 million sperm per ml and about 2100 million sperm per ml.

11. The method of claim 7 , wherein the initial extender further comprises an antioxidant.

12. The method of claim 11 , wherein the antioxidant comprises: catalase, SOD, an SOD mimic, glutathione, glutathione reductase, glutathione peroxidase, pyruvate, caproic acid, mercaptoethanol, BHT, lipoic acid, flavins, quinines, vitamin K (and related vitamers), vitamin B12, vitamin B12 vitamers, vitamin E (and related vitamers), tocopherols, tocotrienols, α-tocopheryl, alpha ketoglutarate (AKG), malondialdehyde (MDA), asymmetric dimethylarginine (ADMA) and biologically active derivatives thereof, or combinations thereof.

13. The method of claim 11 , wherein the antioxidant concentration is in the range of 0.01 mg/ml to 5.0 mg/ml; 0.01 mg/ml to 0.25 mg/ml; 0.01 mg/ml to 0.5 mg/ml; 0.01 mg/ml to 1 mg/ml; 0.01 mg/ml to 2.5 mg/ml; 0.01 mg/ml to 5 mg/ml; 0.05 mg/ml to 0.1 mg/ml; 0.05 mg/ml to 1.0 mg/ml; 0.05 mg/ml to 2.5 mg/ml; 0.1 mg/ml to 0.25 mg/ml; 0.1 mg/ml to 0.5 mg/ml; 0.1 mg/ml to 1 mg/ml; 0.1 mg/ml to 2.5 mg/ml; 0.1 mg/ml to 5 mg/ml; 0.15 mg/ml to 0.45 mg/ml; 0.15 mg/ml to 0.5 mg/ml; 0.25 mg/ml to 0.35 mg/ml; 0.25 mg/ml to 0.5 mg/ml; 0.25 mg/ml to 1 mg/ml; 0.25 mg/ml to 2.5 mg/ml; 0.25 mg/ml to 5 mg/ml; 0.35 mg/ml to 0.5 mg/ml; 0.35 mg/ml to 1 mg/ml; 0.35 mg/ml to 2.5 mg/ml; 0.35 mg/ml to 5 mg/ml; 0.5 mg/ml to 1 mg/ml; 0.5 mg/ml to 2.5 mg/ml; 0.5 mg/ml to 5 mg/ml; 1 mg/ml to 2.5 mg/ml; or 1 mg/ml to 5 mg/ml.

14. The method of claim 1 , wherein the step of calibrating the flow cytometer further comprises providing each droplet in the calibration side stream which is determined to contain live sperm with a uniform trajectory.

15. The method of claim 1 , wherein the step of calibrating the flow cytometer further comprises adjusting instrument parameters to place live sperm in a leading edge of forming droplets.

16. The method of claim 1 , wherein the elevated pressure comprises sheath fluid pressure in a range: about 50 psi to about 55 psi; about 55 psi to about 60 psi; or about 60 psi to about 65 psi.

17. The method of claim 3 , wherein the steps of adjusting the concentration of the sperm sample and adjusting the pH of the sperm sample prior to staining and the step of staining the sperm sample in a single dilution reduces the additional sperm damage imposed by pressures greater than 40 psi by about 50%, about 60%, about 70%, about 80%, about 90%, or by nearly 100%.

18. The method of claim 1 , wherein the step of sorting the stained sperm sample further comprises the step of sex sorting sperm into a viable X chromosome bearing population and/or a viable Y chromosome bearing.

19. The method of claim 1 , wherein the elevated pressure further comprises a sheath fluid pressure between about 50 psi and about 60 psi, or at about 60 psi.

20. The method of claim 19 , further comprising the step of adjusting the sheath fluid pressure selected for sorting in order to change a distance between a catch fluid level and flow cytometer nozzle.

21. The method of claim 1 wherein the split puddle shape comprises an elongated puddle.