The most widely used sperm sorting methods rely on the detection of quantifiable differences in the DNA content of X-chromosome bearing sperm and Y-chromosome bearing sperm. Various modifications to flow cytometers for this purpose are described in U.S. Pat. Nos. 5,135,759, 6,263,745, 7,371,517 and 7,758,811, each of which are incorporated herein by reference. In many species, the difference in DNA content can be small. In bovine, for example, Holstein bulls have about a 3.8% difference in DNA content, while Jersey bulls have about a 4.1% difference. The inexact nature of stoichiometric DNA staining makes these minor variations difficult to ascertain.
While the fluorescent dye Hoechst 33342 is suitable for distinguishing such variations in non-toxic concentrations, sperm must be incubated at elevated temperatures and at an elevated pH for Hoechst 33342 penetration to provide uniform staining Sperm are delicate cells in nature, as they lack the capacity to replicate and have a short life span. As such, injuries imposed by each of elevating sperm temperature and changing the sperm pH may result in a significant detriment to sperm health. Additionally, the pressure and sheering forces applied to sperm within a flow cytometer may further compromise sperm membranes. These factors accelerate the deterioration of sperm membranes further reducing the already limited shelf life of viable sperm for use in artificial insemination or other assisted reproductive procedures.
Accordingly, previous sperm sorting efforts focused on utilizing smaller insemination samples and producing the greatest amount of sorted sperm in the shortest amount of time. U.S. Pat. No. 6,149,867, incorporated herein by reference, describes methods and devices geared towards helping sperm better survive flow cytometric sorting in combination with reduced dosage inseminates. Subsequent advances in flow sorting focused on improvements in detection or throughput. However, as speeds and throughputs increased, larger quantities of sperm, including viable sperm of the desired sex, are discarded with waste. Additional tradeoffs between purity and recovery also exist. For example, where the desirable purity is greater than 95%, fewer sperm can be sex sorted with the requisite confidence level as compared to purities of 70%, 80% or even 90%. Meaning, fewer sperm are recovered at increasingly high purities and that more viable sperm are disposed with the waste stream.
Additional losses in efficiency exist as a consequence of discarding viable sperm due to the occurrence of coincident events. A coincident event occurs when two or more sperm are too close together to be separated. In such an event, all of coincident sperm may be discarded with waste, whereas some or all of those discarded cells may have been desirable to collect.
Previously, recovery problems were often overlooked, or moot, in view of raw flow sorting throughput. Bovine sperm, for example, is relatively easy to collect and process and high purities may be desirable in both the beef and dairy industries, even at the expense of discarding as much as about 90% of the sperm. However, this high throughput methodology is not acceptable for sperm in limited supply. For example, a specific animal could possess exceptionally desirable genetic qualities, but may produce poor sperm samples for sorting. A species could be rare, endangered, or difficult to collect, limiting the amount of sperm available for sorting. A previously collected sample may be preserved, but the animal or species may no longer be available for subsequent collections. Regardless of the circumstances, the wasteful sperm sorting process is undesirable for sperm in limited supply or sperm with high value. A need, therefore, exists for a method of sorting viable sperm with an improved efficiency in recovering sperm.
Additional limitations in convention sorting technology exist due to sperm damage produced during the staining and other processing stages. Even operational parameters of the flow cytometer instrument itself can introduce or exacerbate damage to relatively delicate sperm. Until a point of over staining is reached, generally higher pHs and longer staining times increase the uniformity with which dye associates with nuclear DNA allowing for a better distinction between X-chromosome bearing sperm and Y-chromosome bearing sperm. However, the overall health of sperm degenerates quickly with the elevated temperatures or an elevated pH. At a point, any additional resolution gained by extending staining time is lost due to the number of sperm that either die or become unviable.
In addition to the injuries caused by upfront handling or staining, the sorting process itself imposes pressures and stresses on sperm which have been reported as detrimental to sperm health. In particular, flow cytometer operating pressures of 50 psi (pounds per square inch), or approximately 3.45×105 Pascal, have been documented as damaging sperm in the sorting process. High pressure flow cytometric sorting damages sperm, Theriogenology 2005 Sep. 15:64(5) 1035-48. Accordingly, the industry standard has been to sort sperms at operating pressure less than 50 psi.