Methods for sex-sorting sperm

A method of producing a subpopulation of sperm cells enriched for a sex chromosome using depletive sorting with a flow cytometer comprising:

• setting the flow cytometer to an enrich (ENR) sort mode;
• generating a stream containing a population of sperm cells;
• interrogating the population with a source of electromagnetic radiation;
• detecting signals produced in response to interrogation with the source of electromagnetic radiation at an event rate greater than 40,000 events per second;
• identifying i) dead sperm cells and ii) live sperm cells in the population that do not have the sex chromosome based on the detected signals;
• forming droplets from the stream at a drop drive rate, wherein the ratio of the event rate to the drop drive rate is greater than 0.85;
• charging droplets comprising the identified live sperm cells in the population that do not have the sex chromosome with a charge pulse;
• deflecting the charged droplets away from droplets to which a charge pulse is not applied;
• collecting the droplets to which a charge pulse is not applied, wherein the collected droplets to which a charge pulse is not applied comprise i) a subpopulation of sperm cells enriched for the sex chromosome wherein the purity of the subpopulation is between about 55% and about 70% and ii) the identified dead sperm cells;
• and
• producing an insemination dosage comprising sperm cells from the collected droplets.