Methods for sex-sorting sperm
The invention comprises depletive methods of sex-sorting sperm at high event rates, while simultaneously achieving high technical yields.
1 . 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.
2 . The method of claim 1 , wherein the step of collecting comprises collecting the subpopulation in a collector.
3 . The method of claim 1 , wherein the sex chromosome comprises the X chromosome.
4 . The method of claim 1 , wherein the sex chromosome comprises the Y chromosome.
5 . The method of claim 1 , wherein the collected droplets comprise between about 40% and about 45%, about 45% and about 50%, about 50% and about 55%, about 55% and about 60%, about 60% and about 65%, about 65% and about 70%, about 70% and about 75%, or about 75% and about 80%, of the population of sperm cells.
6 . The method of claim 1 , wherein the population of sperm cells is processed prior to the step of generating a stream by removing dead, dying or damaged sperm from the population.
7 . The method of claim 1 , wherein the population of sperm cells is treated with an antioxidant prior to the step of generating a stream.
8 . The method of claim 1 , further comprising the step of establishing a gate for sperm cells that do not have the sex chromosome.
9 . The method of claim 8 , wherein the gate comprises between 35% and 55% of the population of sperm cells.
10 . The method of claim 1 , wherein the subpopulation is collected at a rate of at least 20,000 sperm cells per second, and wherein between 40 and 80 percent of the population of sperm cells are collected.
11 . The method of claim 1 , further comprising the step of concentrating the population of sperm cells to a concentration of 200×10 6 to 400×10 6 sperm cells per ml prior to the step of generating a stream.