Patent Publication Number: US-2020291351-A1

Title: Methods for sex-sorting sperm

Description:
REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Patent Application No. 62/817,135 filed Mar. 12, 2019. The entire disclosure of which is incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     Sperm sorting for sex selection (i.e., sex-sorting) via flow cytometry was scientifically established nearly 30 years ago (see Johnson, et. al.). Today, a portion of the global market for livestock artificial insemination (primarily dairy cattle) encompasses the use of sex-sorted sperm, typically having sex purities of 85% or higher. However, prior art sex-sorting methods typically only recover about 25% of the processed sperm, while discarding the remaining 75% of sperm. Those losses, when applied to sires with high genetic merit, represent a significant amount of “genetic potential” that is lost, i.e., the number of progeny born from such high value sires is reduced by the use of sex-sorted sperm. Additionally, for swine in particular, the need for large insemination doses has greatly limited the use of sex-sorted sperm due to the low yields associated with prior art sorting methods. 
     The main alternative to sex-sorted sperm is conventional (i.e., unsorted) sperm, which generally has a sex purity of about 50%. Conventional sperm still dominates the artificial insemination market in livestock, due in large part to the aforementioned inefficiencies of the sex-sorting process. Accordingly, there is an unmet need for a sperm sorting method that can provide sex purities higher than 50% (e.g., 55-65%) while utilizing high sort rates and simultaneously achieving high technical yields. 
     SUMMARY OF THE INVENTION 
     One embodiment of the invention comprises a method of producing a subpopulation of sperm enriched for a desired sex chromosome comprising: generating a stream containing a population of sperm; interrogating the population of sperm with a source of electromagnetic radiation; detecting signals produced in response to interrogation with the source of electromagnetic radiation; identifying sperm in the population of sperm that do not have the desired sex chromosome based on the detected signals; removing the identified sperm from the population of sperm to form a subpopulation of sperm enriched for the desired sex chromosome; and collecting the subpopulation of sperm. 
     Another embodiment of the invention comprises a method of producing a subpopulation of sperm enriched for a desired sex chromosome comprising: generating a stream containing a population of sperm; interrogating the population of sperm with a source of electromagnetic radiation; detecting signals above a threshold value at an event rate, wherein the signals are produced in response to interrogation with the source of electromagnetic radiation; identifying sperm in the population of sperm that do not have the desired sex chromosome based on the detected signals; perturbing the stream into regularly formed drops at a drop drive rate, wherein the event rate is between 85 percent and 150 percent of the drop drive rate; removing the identified sperm from the population of sperm to form a subpopulation of sperm enriched for the desired sex chromosome; and collecting the subpopulation of sperm. 
     Additional embodiments of the invention require the additional steps of: identifying dead, dying or damaged sperm based on the detected signals; and removing the identified dead, dying or damaged sperm from the population of sperm. 
     In a further embodiment of the invention, a collected subpopulation enriched for the desired sex chromosome is collected at a rate of: between about 20,000 sperm per second and 25,000 sperm per second; between about 25,000 sperm per second and about 30,000 sperm per second; between about 30,000 sperm per second and about 35,000 sperm per second; between about 35,000 sperm per second and about 40,000 sperm per second; between about 40,000 sperm per second and about 45,000 sperm per second; between about 45,000 sperm per second and about 50,000 sperm per second; between about 50,000 sperm per second and about 55,000 sperm per second; between about 55,000 sperm per second and about 60,000 sperm per second; between about 60,000 sperm per second and about 65,000 sperm per second; between about 65,000 sperm per second and about 70,000 sperm per second; between about 70,000 sperm per second and about 75,000 sperm per second; and between about 75,000 sperm per second and about 80,000 sperm per second. 
     In an even further embodiment, a step of removing identified sperm from a population of sperm comprises the charged deflection of drops formed from a stream in which the identified sperm are located, wherein said drops are regularly formed at a drop drive rate. 
     In a yet further embodiment of the invention, a step of removing identified sperm comprises deflecting all droplets expected to contain identified sperm, regardless of whether additional sperm is present in the droplet. In an alternative embodiment, a step of removing identified sperm comprises only deflecting droplets expected to contain identified sperm, and no other sperm, within the droplet. 
     In a specific embodiment of the invention, the purity of the subpopulation of sperm enriched for a desired sex chromosome is between about 55% and about 70%. In an even more specific embodiment, the purity of the subpopulation of sperm enriched for a desired sex chromosome comprises: between about 55% and about 60%; between about 60% and about 65%; or between about 65% and about 70%. 
     In an additional embodiment of the invention, a step of collecting a subpopulation of sperm enriched for a desired sex chromosome comprises collecting the subpopulation in a collector. 
     Another aspect of the invention comprises an insemination dosage comprising sperm from a subpopulation of sperm enriched for a desired sex chromosome by any of the methods of the invention. 
     In yet another embodiment of the invention, the desired sex chromosome comprises the X chromosome. In an alternative embodiment, the desired sex chromosome comprises the Y chromosome. 
     In a further embodiment, a collected subpopulation of sperm enriched for the desired sex chromosome comprises 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. 
     In yet another embodiment of the invention, a population of sperm is processed prior to a step of generating a stream by removing dead, dying or damaged sperm from the population. In a further embodiment, the population of sperm is treated with an antioxidant prior to the step of generating a stream. 
     An additional embodiment further comprises the step of establishing a gate for sperm that do not have the desired sex chromosome. In a further embodiment, the gate comprises between 35% and 55% of the population of sperm. 
     In another embodiment of the invention, detected signals above a threshold value are detected at an event rate. A further embodiment, comprises the step of perturbing a stream into regularly formed drops at a drop drive rate, wherein the event rate is between 85 percent and 150 percent of the drop drive rate. 
     In a specific embodiment of the invention, a subpopulation of sperm enriched for a desired sex chromosome is collected at a rate of at least 20,000 sperm per second, and wherein between 40 and 80 percent of the population of sperm are collected. 
     Another embodiment of the invention further comprises the step of collecting identified sperm removed from a population of sperm. 
     A yet further embodiment comprises the step of concentrating a population of sperm to a concentration of 200×10 6  to 400×10 6  sperm per ml prior to a step of generating a stream. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a depiction of a jet-in air flow cytometer used in connection with a method of the invention. 
         FIG. 2  is a graph showing sperm collection rates achieved with the invention when X is the desired sex chromosome and when Y is the desired sex chromosome. 
         FIG. 3  is a graph showing sperm collection rates achieved with the invention when using enrich and purify sort modes. 
         FIG. 4  is a graph showing sex chromosome purity achieved with the invention when X is the desired sex chromosome and when Y is the desired sex chromosome. 
         FIG. 5  is a graph showing sex chromosome purity achieved with the invention when using enrich and purify sort modes. 
         FIG. 6  is a graph showing technical yields achieved with the invention when X is the desired sex chromosome and when Y is the desired sex chromosome. 
         FIG. 7  is a graph showing technical yields achieved with the invention when using enrich and purify sort modes. 
         FIG. 8  is a graph showing motility of collected sperm achieved with the invention when X is the desired sex chromosome and when Y is the desired sex chromosome. 
         FIG. 9  is a graph showing motility of collected sperm achieved with the invention when using enrich and purify sort modes. 
         FIG. 10  is a graph showing post-thaw 0-hour motility, viability and percent intact acrosomes of collected sperm achieved with the invention with and without treatment with magnetic particles. 
         FIG. 11  is a graph showing post-thaw 3-hour motility, viability and percent intact acrosomes of collected sperm achieved with the invention with and without treatment with magnetic particles. 
         FIG. 12  is a graph showing post-thaw purity, 0-hour motility, 3-hour motility, viability and percent intact acrosomes of collected sperm achieved with the invention compared to sex-sorted sperm in which sperm of the desired sex were removed. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     One aspect of the invention encompasses the use of a “high event rate” sorting method having an event rate to drop drive rate ratio (“ER/DDR ratio”) greater than 0.85, and in some embodiments as high as 1.25, or higher. In flow cytometry, an “event” generally constitutes a detected signal that exceeds a threshold value. In the context of sex-sorting sperm specifically, the signals in question are produced in response to interrogation of sperm stained with a DNA-selective dye by a source of electromagnetic radiation, such as a laser. “Event rate” then, generally constitutes the number of detected signals that exceed the threshold value per second. In flow cytometers employing droplet sorting, “drop drive rate,” or drop drive frequency, is the number of drops formed per second. Typically, in droplet sorters, droplets are formed by subjecting a stream to vibrations produced by a piezoelectric or electromagnetic transducer. The wavelength (i.e., droplet spacing), λ, the frequency of the applied vibration, f, and the velocity of the stream are related to each other according to the following equation: v=f×λ. Thus, in order to maintain a specific droplet spacing, as droplet frequency is increased, stream velocity must also be increased. In accordance with Bernoulli&#39;s equation, in order to increase the velocity of a stream in a flow cytometer, all else remaining the same, fluid pressure must be increased. 
     In “selective” sorting, desired cells (i.e., target cells or cells of interest) are identified, removed from the population of cells and then collected for use, while the remaining population of cells is considered waste. In contrast to selective sorting, one aspect of the invention encompasses the use of “depletive” or “inverse” sorting. In inverse sorting, undesired cells are identified, removed from the population of cells, and then sent to waste, while the remaining population of cells is collected for use. In the context of the invention, instead of the selective sorting method in which subcategories of sperm are excluded and not collected (resulting in high purity samples produced at slow sort rates and modest technical yields), a depletive sorting method is applied in which the highly selective output (i.e., live sperm having the undesired sex chromosome) is not collected, but rather discarded, while the remaining sperm are collected rather than sent to waste as in the selective sorting method. 
     In the context of selective sorting, the ratio of sort rate to event rate (i.e., technical yield) is inversely proportional to event rate (if all other sort gate settings remain the same), due to the increase in aborted drops at higher event rates. This creates a tradeoff between high sort rates (i.e., the number of desired cells removed from the total population per hour) and high technical yields (the percentage of cells from the total population recovered for use). When utilizing the high event rate, inverse sorting method of the invention, however, the tradeoff between high sort rate (in the context of depletive sorting, this is equivalent to the number of cells collected per hour after removal of undesired cells) and high technical yield, as seen with the sorting methods of the prior art, is significantly reduced. 
     Another aspect of the invention encompasses the use of enrich (“ENR”) and purify (“PUR”) sort modes. As noted above, in selective sorting, desired cells are identified, removed from the population, and collected for use, while the remaining population of cells is sent to waste. When using selective sorting, the ENR mode is particularly useful when the desired cells are rare (e.g., less than 5% of the total cell population). This is because the ENR mode disables the abort function, which would otherwise cause a drop containing both a desired and an undesired cell to be sent to waste. By allowing drops containing both a desired and an undesired cell to be removed from the cell population and collected for use, the ENR mode results in a higher number of desired cells being collected, at the cost of purity (i.e., the percentage of desired cells in the collected cell population). However, in the context of inverse sorting, the ENR mode actually results in higher purity levels, because when the abort function is disabled, drops containing both a desired cell and an undesired cell are removed from the cell population and sent to waste. 
     When using the PUR mode in selective sorting, a drop with a desired and an undesired cell is aborted—i.e., it is not removed and collected for use, but is instead sent to waste. This results in a higher percentage of desired cells in the collected population (i.e., higher purity). In inverse sorting, however, the PUR mode leaves the aborted cells in the collected stream, allowing undesired cells to remain in the collected sample. Because of this, in the context of inverse sorting, the PUR mode is significantly faster (more sperm collected per hour at higher technical yield) than the ENR mode, all else equal, but at the cost of purity. 
     In certain embodiments of the invention, both the selected and unselected streams may be collected and processed for breeding. For example, the invention can be performed in an “unbalanced” skew at ER/DDR rates higher than 0.85 in which a higher purity female sample (e.g., 70-90%) is collected for use in the stream containing the selected sperm, while the stream containing unselected sperm is collected for use as a lower purity male sample (e.g., 55-65%). 
     Some advantages of the invention include a significant reduction, in the number of sorters and in the time needed, to process an entire ejaculate, since the invention can analyze sperm at rates that are about 150-250% of the rate of prior art methods. Moreover, since the technical yield achieved by the invention is in the range of 60-75% (compared to technical yields of 25% or less with prior art methods), the invention is able to produce about 3-5 times more processed sperm per hour, while being about 2-3 times more efficient in sperm usage, compared to prior art methods. Additionally, because the invention uses the same amount of sheath fluid per hour as prior art methods (i.e., about 350 ml per hour), the amount of sheath fluid utilized per artificial insemination dose is reduced with the invention. 
     An additional advantage of the invention is that the number of breeding units (e.g., cryopreserved straws) from an ejaculate may be increased compared to conventional sperm. For example, if conventional sperm is typically used at a dose of about 25 million total sperm per breeding unit/straw and the same sperm can be processed by the invention with overall yields of 60% and formulated at a level of 8 million total sperm per breeding unit/straw, then about double the number of breeding units/straws may be produced from the same amount of ejaculate. For high value bulls where the number of ejaculates available across a period of time is not enough to serve the demand, then a process that produces such an increase in breeding units is highly desirable. 
     Another advantage of the invention is reduction in drop surface charge effect. Specifically, in prior art sex-sorting methods, each drop that is collected has an applied voltage differential pulsed in a way to charge the drop when it is still attached to the fluid stream and then reverse the polarity of the stream when the drop has broken from the stream. The charge pulse energy distributes mainly along the surface of the drop. Since the size of the drops are empirically determined by the smallest drop not causing a side stream effect, it is common that a small portion of the sperm tail, or even the leading edge of the sperm head may be at the surface of the drop or protrude from the drop when the charge pulse is applied. This charge pulse has a small but detrimental effect on the sperm quality. In contrast, when using the invention, the stream that is collected may have a slight but constant charge on it in order to control the angle of deflection, but the droplets leaving in that stream are not experiencing charge pulses. 
     One example of the method of the invention can be described as follows. One or more freshly ejaculated sperm samples is secured from the same bull and pooled into one sample. The pH and sperm cell concentration are stabilized and a portion of the seminal fluid is washed out. The sperm are stained with Hoechst 33342 (or another DNA-selective dye or stain). In a particular embodiment, the entire sperm sample is stained at one time and the sample quality confirmed on one sorter before choosing the operational conditions (parameter settings) for a group of sorters to sort the sperm. Another embodiment includes using magnetic particles to remove dead, dying or damaged cells in the stained sample, prior to re-concentration and sorting. The stained sperm are concentrated to high concentrations, for example, at concentrations in the range of 200 million-400 million stained sperm per ml. Alignment of the sperm sample is established while using an ER/DDR ratio of &gt;0.85. Specific embodiments include the use of an ER/DDR ratio &gt;0.85, in the range of 1.00-1.10, or &gt;1.10. The stained sperm samples are sorted at high event rates (e.g., &gt;40,000 events per second) and in some embodiments, with an ER/DDR ratio of about 1.00 with a high percentage of live-oriented cells (e.g., 80% or more of the total sperm population), while the undesired live sex are removed from the cell population by establishing an appropriate gate, typically comprising about 42-48% of the live-oriented population. 
     Flow cytometry data analysis and sorting are based on the principles of gating. Typically, gates are created around populations of cells with common characteristics. In the context of the invention, these characteristics can include forward fluorescence and side fluorescence. Generally, the first step in gating when flow cytometrically analyzing sperm is distinguishing populations of sperm based on their forward and side fluorescence properties. Forward and side fluorescence provide an estimate of the DNA content of the cells and their orientation, respectively. Unoriented sperm will generate events having a lower level of side fluorescence, as noted above, and generally are not resolvable or are low resolution. In some embodiments of the invention, these events can be removed by gating oriented sperm only. In other embodiments of the invention, these events are not removed, i.e., no gates are established. One aspect of the invention also comprises providing a gate on a multivariate plot encompassing a subpopulation of sperm—in certain embodiments of the invention, this subpopulation of sperm comprises undesired sperm and in other embodiments of the invention, this subpopulation of sperm comprises desired sperm; in a further embodiment, the subpopulation of sperm can further comprise dead, dying or damaged sperm. 
     Some embodiments of the invention comprise the use of event rates of &gt;40,000 events per second (“eps”), 40,000-50,000 eps, 40,000-60,000 eps, 40,000-90,000 eps, &gt;50,000 eps, 50,000-70,000 eps, &gt;70,000 eps, or &gt;100,000 eps. Additionally, in order to establish an ER/DDR ratio at &gt;0.85 or higher, some embodiments of the invention comprise the use of a drop drive rate of &gt;40,000 drop per second (“dps”), 40,000-50,000 dps, 40,000-60,000 dps, 40,000-90,000 dps, &gt;50,000 dps, 50,000-70,000 dps, &gt;70,000 dps, or &gt;100,000 dps. 
     Certain embodiments include use of the ENR mode (which assures the most complete removal of the live cells having the undesired sex chromosome) and may include the gating of dead sperm for the purpose of removal. In one embodiment of the invention, the live-oriented sex gated sperm having the undesired sex chromosome are sorted into the selected stream location, while the unselected stream (i.e., all other cells) is collected for use into an appropriately sized fluid collection tube or container. 
     In certain embodiments of the invention sperm in the unselected stream are collected for use at a rate of &gt;50,000 sperm per second, 30,000-50,000 sperm per second, 40,000-60,0000 sperm per second, 50,000-80,000 sperm per second, &gt;60,000 sperm per second, &gt;70,000 sperm per second or &gt;80,000 sperm per second. Collected sperm may comprise both living and dead, dying or damaged sperm. 
     In one embodiment of the invention, diluted, collected sperm is concentrated using any appropriate method, such as centrifugation or membrane-based filtration, to provide a concentrated volume of sperm that may be subsequently processed for cryopreservation or used in appropriate fresh holding extenders for breeding within a short period. 
     The method of the invention requires the choice of an appropriate sheath fluid pressure. Certain embodiments of the invention comprise the use of 35-45 PSI sheath fluid pressure, but may include the use of pressures as high as 80 PSI, or higher. In specific embodiments, the invention encompasses, sheath fluid pressures of &gt;35 PSI, &gt;40 PSI, 35-40 PSI, 40-45 PSI, 40-50 PSI, 40-60 PSI, 40-80 PSI, 50-80 PSI, &gt;50 PSI or 45-50 PSI. 
     At high sheath fluid pressures, the drop drive rate may be as high as 120,000 drops per second. Based on the chosen pressure, a drop drive frequency is then established empirically such that the last attached drop is placed at a correct distance from the nozzle tip. For calibration purposes, a concentrated stained sperm sample is placed on the sorter and a sample pressure is applied that is sufficient to create an ER/DDR ratio of 0.95-1.05 and a side stream quality calibration is made to determine appropriate drop drive amplitude and deflection voltage. 
     The appropriate concentrations of sperm cells and a DNA-selective dye (typically Hoechst 33342) are incubated in a staining media, at appropriate temperature, for appropriate time. Optimal conditions result in sperm cell populations that are evenly stained and not overstained, since overstaining is a problem that often leads to a poor peak to valley ratio (PVR) at high event rates. 
     In one embodiment of the invention, the concentration of sperm for staining is in the range of 50-150×10 6  sperm cells per ml, while the concentration of sperm in the sample tube that provides cells to the sorter may be in the range of 250×10 6 -400×10 6  cells per ml. In general, this difference leads to a required step to concentrate stained cells. 
     In one aspect of the invention, manual or automated fluidic and optical alignment settings are empirically applied to create and sustain the highest possible differentiation between live X-chromosome bearing and live Y-chromosome bearing sperm populations, while using event rates that come within the range of ER/DDR ratio between about 0.85 and 1.15, or higher. Careful attention to the quality of this differentiation, along with appropriate setting of live-oriented gates and sort gates for cell removal, is then provided to allow the depletive removal of sperm that have two primary characteristics: the first characteristic is that they are live sperm; the second characteristic is that they have a high statistical chance (e.g., greater than 85%) of having the undesired sex chromosome. 
     In one embodiment of the invention, as droplets containing undesired cells are removed, the gating logic may be chosen to apply the PUR mode or the ENR mode to analyzed droplets. When the PUR mode is applied, droplets are not removed into the selected stream location if a second or even third sperm is present with the targeted live cell having the undesired sex chromosome. In the inverse sorting method of the invention, since droplets containing desired cells are not removed and remain in the unselected stream (i.e., the collection stream) and undesired cells remains in the selected stream (the waste stream), the sex purity of the collected sperm subpopulation will be lower (less sex selected or enriched) when PUR mode is used. Conversely, when the ENR mode is used in the invention, droplets are always removed to the selected stream location when they contain an undesired cell, and as a result, the sex purity of the collected sperm subpopulation will be higher. As noted above, in the inverse sorting, high event rate method of the invention, the ENR mode generates a higher sex purity at the cost of a slower collection rate of desired cells. 
     In certain embodiments of the invention, the dead sperm are also selectively removed by the sorter. In this case, the target cells to be removed comprise two populations which may be gated separately but for which the same sort logic may be applied. It should be noted that in standard sperm sorting methods of the prior art, dead sperm are removed inherently because only live sperm are selected for collection. A region placed around the dead sperm is typically only used to quantify them and is not used as a sorting gate. 
     In certain embodiments of the inverse sorting, high event rate method of the invention, unless dead cells are also gated for removal with the sort gate, the dead cells will remain in the stream that is collected for use. Since dead cells are much less fertile than live cells (if fertile at all), retaining the dead cells in the collected stream should be neutral to breeding outcomes. Additionally, since the purity of undesired, live cells in the selected stream is high, the purity of the desired, live cells that are collected will be high, which should have a positive effect on breeding outcomes in terms of the percentage of offspring being of the desired sex for a given sperm cell sex purity. 
     As mentioned above, the inverse sorting high event rate method of the invention will generate a much larger fluid stream, nearly 400-600% greater, compared to standard flow cytometric sorting. Standard sorting typically utilizes catch, or collection, tubes holding 50 ml. Accordingly, one embodiment of the invention comprises sorting into a 500 ml, or larger, catch tube. One of the positive outcomes in using the inverse sorting, high event rate method of the invention in commercial scale is that the same number of sorters process about 150-250% the amount of raw ejaculate and produce about 4-6 times as much sperm per hour per sorter compared to prior art methods. 
     Since the inverse sorting, high even rate method of the invention benefits when sperm are stained at a lower concentration and then concentrated to double or triple the standard concentration, and since methods like treatment with magnetic particles for removal of dead, dying or damaged sperm may be somewhat more selective at lower sperm concentrations, one aspect of the invention encompasses a method for staining in large volumes (e.g., 150 ml, or more) at one time. In an additional embodiment of the invention, a multi-head flow cytometer for use in the invention utilizes a single laser for each sorting head, which results in an increase in the stability of the signal due to shorter laser path lengths. 
     Once obtained from a non-human mammal, sperm to be used with the invention may be standardized to a predetermined concentration and/or towards a predetermined pH. Each of the predetermined concentrations and pH may be specific to different species, or even to different breeds of animals within a species. In one embodiment, the sperm may be combined with an initial buffer in the form of a high capacity buffer. Exemplary buffers may include TRIS citrate, sodium citrate, sodium bicarbonate, HEPES, TRIS, TEST, MOPS, KMT, TALP, and combinations thereof. Any buffer having a high capacity for buffering pH may also be employed, and may be used in combination with additional components which promote sperm viability such as egg yolk, and sources of citrates or citric acid. Additionally, antioxidants and antibiotics may be employed in the initial buffer to promote sperm viability. 
     The initial buffer may be set at a predetermined pH to normalize the pH of all the obtained sperm samples. In one embodiment, the buffer is adjusted to a pH of 7.2. Additionally, semen may become increasingly acidic over time, possibly due to proteins in the seminal fluid, or due to acidic byproducts of dying or dead cells. The initial buffer introduces enough free proton (i.e., H + ) binding sites to maintain pH near the predetermined target. Even in light of the natural tendency for sperm to become more acidic over time, the initial buffer provides a means for stabilizing pH throughout additional processing steps. 
     As one example, the sperm sample may be diluted in the high capacity buffer in ratios from about 1:1 to about 1:10. The resulting mixture will have a sperm concentration many times below natural sperm concentrations for a particular species. The extended sperm may be centrifuged in order to reconcentrate sperm. Centrifuging the sperm and removing supernatant allows the sperm to be reconcentrated into a predetermined concentration. The predetermined concentration may be selected based on additional sperm processing steps. For example, in the case of sex sorting bovine sperm, sperm may be reconcentrated at between about 240 million sperm per ml and about 900 million sperm per ml to simulate a natural range of concentrations. Other concentrations, such as between about 1400 million sperm per ml and about 2100 million sperm per ml, or between about 1700 million sperm per ml and about 2100 million sperm per ml may also be achieved for further processing. 
     Adjusting the sperm concentration and pH may provide a uniform starting point for further processing. For example, a relatively consistent pH and concentration may provide greater predictability in staining sperm, for example with a DNA selective dye. If each sample is adjusted to the same predetermined pH and concentration, fewer trials may be required on each new collection to ensure adequate staining for sex sorting. 
     The population of sperm will include X-chromosome bearing sperm and Y-chromosome bearing sperm. Additionally, each of the X-chromosome bearing sperm and the Y-chromosome bearing sperm will include viable sperm and nonviable sperm. Viable sperm can be considered sperm with intact membranes while nonviable sperm can be considered sperm with compromised membranes. The distinction between viable sperm and non-viable sperm in conventional sperm sorting is determined with the inclusion of a quenching dye that permeates membrane compromised sperm. Sperm which tends to be dead or dying absorbs the quenching dye and produces fluorescence signals distinct from the remaining sperm population, whereas sperm cells having intact membranes tend to be viable sperm cells that will prevent uptake of the quenching dye. Viable sperm, in the appropriate dosage, will generally be capable of achieving fertilization using artificial insemination, while nonviable sperm, or membrane compromised sperm, may be incapable of achieving fertilization using artificial insemination or will have a greatly reduced ability to do so. However, some sperm capable of fertilization may have compromised membranes, and some sperm with intact membranes may be incapable of fertilization. 
     Whether standardized or not, sperm may be stained with a staining buffer for introducing a DNA-selective dye. In the staining step, at least a portion of the population of sperm is incubated with a staining buffer and a DNA-selective fluorescent dye in order to stoichiometrically stain the DNA content of each cell in the sperm population. Hoechst 33342 tends to be less toxic than other DNA selective dyes. The vehicle for delivering this dye may be in the form of a modified TALP buffer adjusted to a pH of about 7.4. Hoechst 33342 is described in U.S. Pat. No. 5,135,759 and is commonly used for this purpose. However, other UV excitable dyes, as well as visible light excitable dyes, fluorescent polyamides, fluorescent nucleotide sequences, and sex specific antibodies could also be used. 
     Sperm in a natural state is often not readily permeable to such dyes. In order to produce a uniform staining, the first step of staining can include incubating at least a portion of the sperm population at an elevated temperature in a staining buffer at an elevated pH in addition to the dye. Examples of appropriate first staining buffers can be a TALP, TES-TRIS, TRIS citrate, sodium citrate, or a HEPES based medium, each described in WO2005/095960, incorporated herein by reference. As one example, the population of sperm, or a portion of the population of sperm, could be diluted with a first buffer to between 640×10 6  and 40×10 6  sperm/ml, to between about 320×10 6  and 80×10 6  sperm/ml, or to about 160×10 6  sperm/ml in the first buffer. The DNA selective fluorescent dye can be added to the sperm suspended in the first buffer in a concentration of between about 10 μM and 200 μM; between about 20 μM and 100 or between about 30 μM and 70 μM. The pH of the first buffer can be between about 6.8 and 7.9; about 7.1 and 7.6; or at about 7.4 in order to help ensure a uniform staining of nuclear DNA. Those of ordinary skill in the art will appreciate the pH can be elevated with the addition of NaOH and dropped with the addition of HCl. 
     While being stained, the population of sperm can be incubated between 30-39° C., between about 32-37° C., or at about 34° C. The period of incubation can range between about 20 minutes and about an hour and a half, between about 30 minutes and about 75 minutes, or for about 45 minutes to about 60 minutes. As one example, the population of sperm can be incubated for about 45 minutes at 34° C. In addition to the DNA-selective fluorescent dye, a quenching dye may be applied for the purpose of permeating membrane compromised sperm and quenching the signals they produce. A dead quenching dye can be understood to include dyes which differentially associate with membrane compromised sperm. It may be that these dyes enter membrane compromised sperm cells more easily because the membranes are breaking down or otherwise increasingly porous. It may also be that dead quenching dyes readily enter all sperm cells and that healthy sperm cells act to pump dead quenching dyes out faster than membrane compromised sperm. In either case, the sperm cells with which the dead quenching dyes associate includes a large portion of dead and dying sperm cells, although not necessarily all dead and dying sperm cells. The quenched signals produced from membrane compromised sperm having an association with quenching dye are distinct enough from the signals of healthy sperm that they may be removed from the further analysis and sorting applied to viable sperm. 
     In one embodiment, a second staining step is preformed which further reduces the concentration of sperm and introduces the dead quenching dye. The pH of the second staining solution may be targeted to achieve a target pH in the final sperm sample. Exemplary descriptions of two step staining processes are described in published PCT International Application WO 2011/123166 and International Application PCT/US12/58008, the entire disclosure of both are incorporated herein by reference. 
     In another embodiment, the quenching dye and the DNA selective dye are applied together in a single treatment. In this embodiment, the quenching dye is incubated along with the DNA selective dye at an elevated temperature in the modified TALP which may be at a pH of 7.4. In this embodiment, it is believed a synergy exists when the sperm is standardized at an elevated pH of about 7.2 before staining at 7.4. In this way, the pH to which the sperm is exposed remains in a constant range with minimal variations. Because both the staining buffer and the initial extender have high buffering capacities, it is believed the natural tendency of sperm to become more acidic over time will be avoided. Additionally, by minimizing the changes in pH seen by the sperm, it is believed the sperm are in a healthier condition to face the various pressures and stresses endured in the sex sorting process. 
     Sorting Stained Sperm 
     In one embodiment of the invention, sperm to be sorted are first processed by removing dead, dying or damaged sperm using magnetic particles, either before or after staining, as taught in WO 2014/035840 and WO 2016/090310, which are both incorporated by reference herein in their entirety. 
     In one aspect of the invention, it is contemplated that a sperm population is sorted by a flow cytometer. Referring to  FIG. 1 , a jet-in-air flow cytometer ( 10 ) is illustrated, although sorting may be performed with microfluidic chips or other types of flow cytometers, including flow cytometer having closed chambers and cytometers and cytometers incorporating ablating lasers. The flow cytometer ( 10 ) includes a cell source ( 12 ) for producing a flow of sperm sample, such as a flow of stained sperm sample, for sorting. The rate at which the sperm sample is delivered to the nozzle ( 14 ) may be considered the sample flow rate, and may be determined by a sample pressure applied at the cell source ( 12 ). The flow of stained sperm sample is deposited within a nozzle ( 14 ) and introduced into, or flowed into, a fluid stream ( 16 ) of sheath fluid ( 18 ). The sheath fluid ( 18 ) can be supplied by a sheath fluid source ( 20 ) so that as the cell source ( 12 ) supplies the sperm into the sheath fluid ( 18 ) they are concurrently fed through the nozzle ( 14 ). The sheath fluid ( 18 ) may be supplied at a sheath flow rate which is determined by a sheath pressure applied at the sheath fluid source ( 20 ). In this manner the sheath fluid ( 18 ) forms a fluid stream coaxially surrounding the sample having stained sperm which exits the nozzle ( 14 ) at the nozzle orifice ( 22 ). By providing an oscillator ( 24 ) which may be precisely controlled with an oscillator control ( 26 ), pressure waves may be established within the nozzle ( 14 ) and transmitted to the fluids exiting the nozzle ( 14 ) at nozzle orifice ( 22 ). In response to the pressure waves, the fluid stream ( 16 ) exiting the nozzle orifice ( 22 ) eventually forms regular droplets ( 28 ) at precise intervals. The frequency, and to some extent the shape of the formed droplets may be controlled by a drop drive rate (i.e., frequency) and a drop drive amplitude supplied to the oscillator ( 24 ) or the oscillator controller ( 26 ). 
     Each droplet, so formed, retains the sheath fluid and sperm sample that previously formed a portion of the fluid stream ( 16 ). Because the stained sperm are surrounded by the fluid stream ( 16 ) or sheath fluid environment, the droplets ( 28 ) ideally contain individually isolated sperm. However, the sample concentration, sample pressure, and other instrument parameters dictate the frequency with which multiple cells will regularly occupy a single droplet, as well as the percentage of droplets containing sperm cells. 
     The flow cytometer ( 10 ) acts to sort droplets based on the characteristics of sperm predicted to be contained within the droplets. This can be accomplished through a cell sensing system ( 30 ) in communication with an analyzer ( 36 ). The cell sensing system ( 30 ) includes at least one sensor ( 32 ) responsive to the cells contained within fluid stream ( 16 ). The cell sensing system ( 30 ) provides data to the analyzer ( 36 ), which may cause an action depending upon the relative presence or relative absence of a characteristic of cells in the fluid stream ( 16 ). Certain characteristics, such as the relative DNA content of sperm cells, can be detected through excitation with a source of electromagnetic radiation ( 34 ), such as a laser generating an irradiation beam to which the stained sperm are responsive. The source of electromagnetic radiation ( 34 ) can be a laser operated at UV wavelength, such as at about 355 nm. An example of such a laser can be a Vanguard 350 (available from Spectra-Physics), which operates at 350 mW. Various optics may be employed to shape the beam profile of the laser, split the beam to more than one stream, or reduce the beam power at a stream. Non-limiting examples of such optics can be found in WO/2004/104178 and WO/2001/85913, each being incorporated herein by reference. 
     The characteristics of individual sperm, particularly the presence of an X-chromosome or a Y-chromosome can be determined from the detected fluorescence produced in response to the electromagnetic radiation source ( 34 ). In particular, configurations of the cell sensing system ( 30 ) may be in communication with an analyzer for providing a variety of fluorescence information, such as the forward fluorescence of an event, the side fluorescence of an event, or the amount of scatter associated with an event. The analyzer ( 36 ) may include written instructions for analyzing the signals produced by the one or more sensors ( 32 ) in the cell sensing system ( 30 ). The DNA selective fluorescent dye binds stoichiometrically to sperm DNA. Because X-chromosome bearing sperm contain more DNA than Y-chromosome bearing sperm, the X-chromosome bearing sperm can bind a greater amount of DNA selective fluorescent dye than Y-chromosome bearing sperm. Thus, by measuring the fluorescence emitted by the bound dye upon excitation, it is possible to identify, or differentiate between, X-bearing spermatozoa and Y-bearing spermatozoa, or to identify spermatozoa that are not X-bearing or spermatozoa that are not Y-bearing. Distinctions, such as sperm which is viable or not viable, may be differentiated in addition to oriented and unoriented sperm by the analyzer ( 36 ) according to sorting logic incorporated gating regions. 
     In order to achieve separation and isolation based upon stained sperm characteristics, emitted light can be detected by the sensor ( 32 ) and the information fed to an analyzer ( 36 ) coupled to a droplet charger that charges droplets ( 28 ) based upon the characteristics of the stained sperm contained within that droplet ( 28 ). In this manner the analyzer ( 36 ) acts to permit the electrostatic deflection plate ( 38 ) to deflect droplets ( 28 ) based on whether or not they contain the appropriate particle or cell. In one embodiment of the high event rate, inverse sorting method of the invention, the flow cytometer ( 10 ) removes sperm having the undesired sex chromosome from the population of sperm by diverting droplets containing sperm having the undesired sex chromosome ( 44 ) (i.e., the selected stream) to waste ( 40 ). The remaining droplets (i.e., the unselected stream) ( 46 ) are collected for use in a collection container ( 47 ) and constitute a subpopulation of sperm enriched for the desired sex. 
     A controller ( 42 ) may form a portion of the analyzer ( 36 ) or may be a component external to the analyzer ( 36 ). The illustrated controller ( 42 ) may also represent a collection of individual controllers. The controller ( 42 ) may receive signals or instructions from the analyzer ( 36 ) and in response may modify one or more instrument parameters, such as the sample flow rate, sample pressure, sheath flow rate, sheath pressure, drop drive rate, or drop drive amplitude and the like. The controller ( 42 ) may also provide an interface for operator input to manually adjust the sample flow rate, sample pressure, sheath flow rate, sheath pressure, drop drive frequency, drop drive amplitude and the like. The analyzer ( 36 ) may include written instructions for modifying the instrument parameters in response to measured sorting parameters, or modifications to instrument parameters may be manually performed by an operator adjusting various settings. The modifications to instrument parameters may be carried out in the analyzer ( 36 ) such as for changing sorting logic, abort logic, sorting regions, or gate regions and other parameters specific to making sort decisions in the analyzer ( 36 ). Additional modifications to instrument parameters may be effected by a controller ( 42 ), for controlling various external components to the analyzer, such as for controlling the sample pressure, sample flow rate, sheath pressure, sheath flow rate, drop drive rate, and drop drive amplitude. 
     Once collected, the subpopulation of sperm enriched for the desired sex can be further processed by, for example, cryopreserving the sperm, or packaging the sperm into doses for use in artificial insemination. In one embodiment of the invention, the collected subpopulation is also processed by removing dead, dying or damaged sperm using magnetic particles, as taught in WO 2016/090310, which is incorporated by reference herein in its entirety. 
     One aspect of the invention encompasses its use to produce sex sorted sperm samples for use in parent gilt multiplication in swine. The output in terms of parent gilts produced per sow per year is directly proportional to the level of sperm cell sorting. For instance, with the use of 65% X-chromosome bearing sperm cell samples instead of conventional semen, the output per sow per year can increase from 7.50 to 9.75 gilts/sow per year or, at a fixed output, the number of multiplication sows per 100 gilts can be reduced from 13.3 to 10.3. 
     Example 1 
     In this example, semen from two bulls was inverse sorted at four event rates-40,000 events per second (“eps”), 50,000 eps, 60,000 eps and 70,000 eps—using two different Y chromosome sort gates having removal levels of 35% and 55%, respectively. Each semen sample was collected in an extender. Sperm concentration was normalized to 1200×10 6  sperm/ml. The normalized sample was then stained at a concentration of 120×10 6  sperm/ml with Hoechst 33342 and applied to a flow cytometer for alignment at an event rate of 40,000 eps and calibration of drop delay. A sort gate for removal of Y chromosome sperm (i.e., the undesired sex chromosome in this case) was established in a population consisting of live-oriented sperm. ENR mode was enabled. The drop drive rate was 60,000 drops per second (i.e., 60 kHz). In order to determine the empirical abort rate, just prior to sorting at each experimental condition, the sperm were sorted in PUR mode, which shows the sort rate (i.e., rate of removal of undesired cells) and abort rate. 
     A standard catch tube with 3.5 ml of TRIS-based media was sorted to a final volume of 30 ml. Sperm were cooled for a minimum of 30 minutes and then centrifuged at 850 G for 20 min, the supernatant decanted and replaced with 2.0 ml of cold TRIS-based freezing media. These tubes were left overnight in the refrigerator. The following day, the volumes were determined gravimetrically, the sperm concentrations were measured by NucleoCounter (DF 11 as 1.0 ml S-100 and 100 μl sperm) to determine the millions sorted. 2 hours after sperm samples were removed from the refrigerator, the visual motilities (by microscope) and the CASA motilities (motility and progressive motility) of 2004, samples incubated for 15-30 minutes on a warm block were determined. 
     Two samples from the first bull were also sorted as “70Keps/50% GateDead” method. This is a method in which a sort gate for removal of both Y chromosome sperm and dead/damaged sperm was established form a gated population consisting of both live-oriented and dead/damaged sperm. This method was then tested in both the ENR and PUR modes. Processed samples were resorted to determine purity levels. 
     Results for this experiment are shown in Table 1 below. All samples were sex enriched to ranges greater than 55% and in the case of the 50% Y chromosome gate treatments, typically over 60%. With respect to Table 1, “Live-Oriented” represents the gated live-oriented cells as a percentage of the cell population of the sample; “Dead” represents the gated dead cells as a percentage of the cell population of the sample; “Remove Gate” represents the gated undesired cells removed from the gated live-oriented cells as a percentage of the gated live-oriented cells; “Purity” represents the collected undesired cells (both living and dead, dying or damaged) as a percentage of the collected cells (both living and dead, dying or damaged). For the samples examined by microscope, the last two samples (in which dead cells were selectively removed) received a quality rating of “4” on a scale of 1-5, while the other 16 samples scored as “3”. 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                 METHOD 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 Event Rate 
               
               
                   
                 and Remove 
                   
                 Event 
                 Cell 
                 Cell 
                   
                   
                 Remove 
                 Resort 
                 CASA 
                 CASA 
               
               
                 Bull 
                 Gate Percent 
                 mode 
                 Rate 
                 Harvest Rate 
                 Remove Rate 
                 Live-Oriented 
                 Dead 
                 Gate 
                 Purity 
                 MOT 
                 PROG 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 40 KHz/35% 
                 ENR 
                 40,758 
                 31,430 
                 9,328 
                 77.4% 
                 9.5% 
                 35.9% 
                 58.0% 
                 89.2% 
                 73.1% 
               
               
                 1 
                 40 KHz/50% 
                 ENR 
                 39,745 
                 25,939 
                 13,806 
                 77.5% 
                 10.1% 
                 55.0% 
                 62.2% 
                 82.1% 
                 67.5% 
               
               
                 1 
                 50 KHz/35% 
                 ENR 
                 49,541 
                 38,576 
                 10,965 
                 75.3% 
                 10.7% 
                 37.1% 
                 58.8% 
                 86.3% 
                 70.5% 
               
               
                 1 
                 50 KHz/50% 
                 ENR 
                 49,212 
                 33,486 
                 15,726 
                 75.2% 
                 11.0% 
                 55.0% 
                 62.1% 
                 79.1% 
                 64.7% 
               
               
                 1 
                 60 KHz/35% 
                 ENR 
                 59,265 
                 48,173 
                 11,092 
                 72.0% 
                 11.3% 
                 34.9% 
                 58.5% 
                 85.2% 
                 69.1% 
               
               
                 1 
                 60 KHz/50% 
                 ENR 
                 59,756 
                 42,459 
                 17,297 
                 71.5% 
                 11.8% 
                 56.1% 
                 59.7% 
                 85.2% 
                 70.0% 
               
               
                 1 
                 70 KHz/35% 
                 ENR 
                 68,137 
                 58,250 
                 9,887 
                 66.5% 
                 14.6% 
                 35.5% 
                 55.0% 
                 66.1% 
                 47.4% 
               
               
                 1 
                 70 KHz/50% 
                 ENR 
                 60,553 
                 46,983 
                 13,570 
                 61.5% 
                 15.2% 
                 52.8% 
                 57.0% 
                 84.3% 
                 66.6% 
               
               
                 2 
                 40 KHz/35% 
                 ENR 
                 41,190 
                 31,832 
                 9,358 
                 78.1% 
                 8.9% 
                 35.3% 
                 58.5% 
                 87.6% 
                 55.0% 
               
               
                 2 
                 40 KHz/50% 
                 ENR 
                 39,631 
                 25,406 
                 14,225 
                 78.7% 
                 8.7% 
                 55.3% 
                 63.9% 
                 91.8% 
                 62.5% 
               
               
                 2 
                 50 KHz/35% 
                 ENR 
                 49,267 
                 39,230 
                 10,037 
                 76.0% 
                 8.7% 
                 36.5% 
                 57.3% 
                 90.5% 
                 51.4% 
               
               
                 2 
                 50 KHz/50% 
                 ENR 
                 49,780 
                 33,540 
                 16,240 
                 75.7% 
                 8.7% 
                 55.6% 
                 59.7% 
                 89.5% 
                 59.8% 
               
               
                 2 
                 60 KHz/35% 
                 ENR 
                 60,261 
                 48,109 
                 12,152 
                 72.8% 
                 8.9% 
                 37.0% 
                 57.0% 
                 62.2% 
                 44.1% 
               
               
                 2 
                 60 KHz/50% 
                 PUR 
                 59,531 
                 46,503 
                 13,028 
                 72.8% 
                 8.9% 
                 55.5% 
                 56.8% 
                 89.5% 
                 61.5% 
               
               
                 2 
                 70 KHz/35% 
                 ENR 
                 69,153 
                 56,926 
                 12,227 
                 70.7% 
                 9.3% 
                 38.0% 
                 55.4% 
                 87.2% 
                 52.8% 
               
               
                 2 
                 70 KHz/50% 
                 ENR 
                 66,354 
                 54,667 
                 11,687 
                 68.6% 
                 10.0% 
                 54.6% 
                 54.5% 
                 68.8% 
                 46.7% 
               
               
                 1 
                    50% Gate wit   
                 ENR 
                 71,440 
                 39,106 
                 32,334 
                 90.7% 
                 14.1% 
                 58.7% 
                 60.6% 
                 88.8% 
                 71.2% 
               
               
                 1 
                    50% Gate wit   
                 PUR 
                 69,980 
                 47,904 
                 22,076 
                 90.7% 
                 14.8% 
                 57.9% 
                 57.4% 
                 84.5% 
                 70.2% 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     Example 2 
     In this example, the range of event rates evaluated ranged from 50,000 eps to 90,000 eps, All samples, prior to being sorted, were processed as in Example 1. All samples were sorted with simultaneous dead removal (i.e., a sorting gate for both sperm having the undesired sex chromosome and dead/damaged sperm was established in a population consisting of live-oriented sperm and dead sperm), and both the ENR and PUR methods were compared. In total, almost 40 separate samples were sorted. Results are shown in Table 2 below and in  FIGS. 2-9 . 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
             
            
               
                   
                 Target 
                 Skew 
                   
                   
                   
                   
                 Cell 
               
               
                   
                 Event Rate 
                 Direction 
                   
                 ER/DDR 
                 Method Name 
                 Event Rate 
                 Harvest Rate 
               
               
                   
               
               
                 BULL 1 
                 40  
                 X 
                 ENR 
                 0.64 
                 40K ENR REMY + DEAD 
                 38,552 
                 14,297 
               
               
                   
                 50K 
                 X 
                 ENR 
                 0.84 
                 50K ENR REMY + DEAD 
                 50,557 
                 20,013 
               
               
                   
                   
                 X 
                 PUR 
                 0.82 
                 50K PUR REMY + DEAD 
                 49,498 
                 34,517 
               
               
                   
                   
                 Y 
                 PUR 
                 0.84 
                 50K PUR REMY + DEAD 
                 50,338 
                 36,773 
               
               
                   
                   
                 Y 
                 ENR 
                 0.88 
                 50K ENR REMY + DEAD 
                 52,704 
                 17,762 
               
               
                   
                 60K 
                 Y 
                 ENR 
                 1.02 
                 60K PUR REMY + DEAD 
                 61,322 
                 25,310 
               
               
                   
                   
                 Y 
                 PUR 
                 1.01 
                 60K PUR REMY + DEAD 
                 60,660 
                 50,330 
               
               
                   
                   
                 X 
                 PUR 
                 0.97 
                 60K PUR REMY + DEAD 
                 58,487 
                 41,392 
               
               
                   
                   
                 X 
                 ENR 
                 1.00 
                 60K ENR REMY + DEAD 
                 59,904 
                 17,577 
               
               
                   
                 70K 
                 X 
                 ENR 
                 1.20 
                 70K ENR REMY + DEAD 
                 71,721 
                 26,378 
               
               
                   
                   
                 X 
                 PUR 
                 1.18 
                 70K PUR REMY + DEAD 
                 70,618 
                 56,660 
               
               
                   
                   
                 Y 
                 PUR 
                 1.20 
                 70K PUR REMY + DEAD 
                 72,235 
                 62,796 
               
               
                   
                   
                 Y 
                 ENR 
                 1.19 
                 70K ENR REMY + DEAD 
                 71,178 
                 24,981 
               
               
                   
                 80K 
                 Y 
                 ENR 
                 1.37 
                 80K ENR REMY + DEAD 
                 82,259 
                 31,479 
               
               
                   
                   
                 Y 
                 PUR 
                 1.36 
                 80K PUR REMY + DEAD 
                 81,677 
                 70,319 
               
               
                   
                   
                 X 
                 PUR 
                 1.36 
                 80K PUR REMY + DEAD 
                 81,464 
                 63,333 
               
               
                   
                   
                 X 
                 ENR 
                 1.34 
                 80K ENR REMY + DEAD 
                 80,170 
                 21,954 
               
               
                   
                 90K 
                 Y 
                 ENR 
                 1.48 
                 90K ENR REMY + DEAD 
                 88,897 
                 32,644 
               
               
                   
                   
                 Y 
                 PUR 
                 1.50 
                 90K PUR REMY + DEAD 
                 90,122 
                 77,001 
               
               
                 BULL 2 
                 50K 
                 X 
                 PUR 
                 0.87 
                 50K PUR REMY + DEAD 
                 52,119 
                 35,905 
               
               
                   
                   
                 X 
                 ENR 
                 0.85 
                 50K ENR REMY + DEAD 
                 50,777 
                 16,427 
               
               
                   
                   
                 Y 
                 ENR 
                 0.84 
                 50K ENR REMX + DEAD 
                 50,356 
                 19,010 
               
               
                   
                   
                 Y 
                 PUR 
                 0.83 
                 50K PUR REMX + DEAD 
                 49,800 
                 38,872 
               
               
                   
                 60K 
                 Y 
                 PUR 
                 1.04 
                 60K PUR REMX + DEAD 
                 62,198 
                 46,011 
               
               
                   
                   
                 Y 
                 ENR 
                 1.04 
                 60K ENR REMX + DEAD 
                 62,553 
                 18,313 
               
               
                   
                   
                 X 
                 ENR 
                 1.03 
                 60K ENR REMY + DEAD 
                 61,865 
                 16,389 
               
               
                   
                   
                 X 
                 PUR 
                 1.04 
                 60K PUR REMY + DEAD 
                 62,284 
                 44,891 
               
               
                   
                 70K 
                 X 
                 PUR 
                 1.21 
                 70K PUR REMY + DEAD 
                 72,539 
                 53,911 
               
               
                   
                   
                 X 
                 ENR 
                 1.19 
                 70K ENR REMY + DEAD 
                 71,659 
                 16,508 
               
               
                   
                   
                 Y 
                 ENR 
                 1.17 
                 70K ENR REMX + DEAD 
                 70,319 
                 21,800 
               
               
                   
                   
                 Y 
                 PUR 
                 1.15 
                 70K PUR REMX + DEAD 
                 69,070 
                 53,371 
               
               
                   
                 80K 
                 Y 
                 PUR 
                 1.33 
                 80K PUR REMX + DEAD 
                 79,989 
                 60,909 
               
               
                   
                   
                 Y 
                 ENR 
                 1.32 
                 80K ENR REMX + DEAD 
                 79,296 
                 24,745 
               
               
                   
                   
                 X 
                 ENR 
                 1.33 
                 80K ENR REMY + DEAD 
                 79,862 
                 31,434 
               
               
                   
                   
                 X 
                 PUR 
                 1.36 
                 80K PUR REMY + DEAD 
                 81,414 
                 67,484 
               
               
                   
                 90K 
                 X 
                 PUR 
                 1.51 
                 90K PUR REMY + DEAD 
                 90,527 
                 75,629 
               
               
                   
                   
                 X 
                 ENR 
                 1.49 
                 90K ENR REMY + DEAD 
                 89,696 
                 27,636 
               
               
                   
                   
                 Y 
                 ENR 
                 1.54 
                 90K ENR REMX + DEAD 
                 92,143 
                 29,658 
               
               
                   
                   
                 Y 
                 PUR 
                 1.54 
                 90K PUR REMX + DEAD 
                 92,320 
                 73,438 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 Calculated 
                   
                   
                 Remove 
                 Live Sex 
                 Resort 
                 CASA 
                 CASA 
               
               
                   
                 Cell Remove Rate 
                 Live-Oriented 
                 Dead 
                 Gate 
                 Remove Gate 
                 Purity 
                 MOT 
                 PROG 
               
               
                   
               
               
                 BULL 1 
                 24,255 
                 93.0% 
                 8.8% 
                 58.1% 
                 49.3% 
                 68.6% 
                 88.3% 
                 68.3% 
               
               
                   
                 30,544 
                 91.8% 
                 8.9% 
                 52.7% 
                 43.8% 
                 64.7% 
                 74.9% 
                 44.6% 
               
               
                   
                 14,981 
                 91.8% 
                 9.1% 
                 54.0% 
                 44.9% 
                 58.5% 
                 88.5% 
                 52.6% 
               
               
                   
                 13,565 
                 91.8% 
                 9.2% 
                 50.7% 
                 41.5% 
                 60.3% 
                 93.8% 
                 54.7% 
               
               
                   
                 34,942 
                 91.6% 
                 9.2% 
                 49.8% 
                 40.7% 
                 65.6% 
                 91.2% 
                 72.0% 
               
               
                   
                 36,011 
                 90.8% 
                 9.0% 
                 48.0% 
                 39.0% 
                 62.1% 
                 89.5% 
                 64.9% 
               
               
                   
                 10,330 
                 90.9% 
                 8.9% 
                 47.8% 
                 38.9% 
                 55.4% 
                 69.5% 
                 36.8% 
               
               
                   
                 17,096 
                 91.0% 
                 9.0% 
                 56.8% 
                 47.7% 
                 57.9% 
                 82.5% 
                 51.1% 
               
               
                   
                 42,327 
                 90.9% 
                 9.0% 
                 55.1% 
                 46.1% 
                 62.3% 
                 75.4% 
                 49.2% 
               
               
                   
                 45,342 
                 89.4% 
                 8.6% 
                 56.2% 
                 47.6% 
                 61.1% 
                 72.1% 
                 36.8% 
               
               
                   
                 13,958 
                 89.6% 
                 8.6% 
                 55.0% 
                 46.4% 
                 52.7% 
                 83.4% 
                 53.4% 
               
               
                   
                 9,440 
                 88.4% 
                 8.8% 
                 51.8% 
                 42.9% 
                 60.2% 
                 71.1% 
                 28.3% 
               
               
                   
                 46,197 
                 89.5% 
                 9.0% 
                 45.8% 
                 36.8% 
                 63.0% 
                 78.4% 
                 55.8% 
               
               
                   
                 50,780 
                 89.1% 
                 8.7% 
                 48.6% 
                 39.9% 
                 60.1% 
                 75.4% 
                 53.8% 
               
               
                   
                 11,358 
                 89.0% 
                 8.7% 
                 50.1% 
                 41.4% 
                 55.8% 
                 66.4% 
                 41.5% 
               
               
                   
                 18,131 
                 89.0% 
                 8.8% 
                 54.2% 
                 45.4% 
                 52.8% 
                 76.4% 
                 38.7% 
               
               
                   
                 58,216 
                 89.1% 
                 8.7% 
                 54.8% 
                 46.0% 
                 56.0% 
                 69.5% 
                 45.6% 
               
               
                   
                 56,252 
                 88.4% 
                 8.7% 
                 50.6% 
                 41.9% 
                 60.5% 
                 67.6% 
                 31.6% 
               
               
                   
                 13,121 
                 88.3% 
                 8.7% 
                 52.3% 
                 43.6% 
                 55.5% 
                 62.3% 
                 38.7% 
               
               
                 BULL 2 
                 16,214 
                 91.7% 
                 12.3% 
                 56.3% 
                 44.0% 
                 58.5% 
                 85.7% 
                 66.4% 
               
               
                   
                 34,350 
                 91.7% 
                 12.2% 
                 57.5% 
                 45.2% 
                 63.9% 
                 91.1% 
                 74.3% 
               
               
                   
                 31,346 
                 91.6% 
                 12.1% 
                 53.6% 
                 41.5% 
                 66.9% 
                 87.1% 
                 66.4% 
               
               
                   
                 10,928 
                 91.7% 
                 12.1% 
                 55.9% 
                 43.8% 
                 61.7% 
                 75.4% 
                 59.9% 
               
               
                   
                 16,187 
                 90.4% 
                 11.9% 
                 55.1% 
                 43.3% 
                 61.2% 
                 67.3% 
                 45.1% 
               
               
                   
                 44,240 
                 90.2% 
                 11.7% 
                 53.1% 
                 41.4% 
                 63.9% 
                 82.5% 
                 46.9% 
               
               
                   
                 45,476 
                 90.3% 
                 11.6% 
                 57.0% 
                 45.4% 
                 59.2% 
                 91.2% 
                 62.3% 
               
               
                   
                 17,393 
                 90.3% 
                 11.6% 
                 57.8% 
                 46.2% 
                 58.9% 
                 84.8% 
                 44.2% 
               
               
                   
                 18,628 
                 89.5% 
                 11.5% 
                 57.0% 
                 45.6% 
                 56.4% 
                 64.1% 
                 37.5% 
               
               
                   
                 55,151 
                 89.4% 
                 11.6% 
                 60.9% 
                 49.3% 
                 61.1% 
                 92.1% 
                 71.5% 
               
               
                   
                 48,519 
                 89.7% 
                 11.7% 
                 50.7% 
                 39.0% 
                 62.8% 
                 78.2% 
                 56.7% 
               
               
                   
                 15,699 
                 89.7% 
                 11.9% 
                 52.4% 
                 40.5% 
                 58.8% 
                 80.0% 
                 63.0% 
               
               
                   
                 19,080 
                 86.8% 
                 11.3% 
                 57.4% 
                 46.2% 
                 58.3% 
                 59.2% 
                 32.6% 
               
               
                   
                 54,551 
                 87.0% 
                 11.3% 
                 57.0% 
                 45.7% 
                 58.9% 
                 88.1% 
                 67.2% 
               
               
                   
                 48,427 
                 87.0% 
                 11.3% 
                 53.4% 
                 42.1% 
                 54.9% 
                 89.9% 
                 71.0% 
               
               
                   
                 13,930 
                 86.9% 
                 11.2% 
                 56.0% 
                 44.7% 
                 54.0% 
                 71.1% 
                 46.2% 
               
               
                   
                 14,898 
                 87.8% 
                 11.5% 
                 49.6% 
                 38.1% 
                 53.9% 
                 91.7% 
                 69.9% 
               
               
                   
                 62,059 
                 87.8% 
                 11.5% 
                 50.8% 
                 39.3% 
                 57.1% 
                 75.8% 
                 54.9% 
               
               
                   
                 62,486 
                 87.7% 
                 11.5% 
                 54.2% 
                 42.7% 
                 56.6% 
                 85.5% 
                 73.6% 
               
               
                   
                 18,882 
                 87.5% 
                 11.2% 
                 55.6% 
                 44.4% 
                 54.1% 
                 87.4% 
                 71.9% 
               
               
                   
               
            
           
         
       
     
     Example 3 
     In this example, a higher sheath fluid pressure (60 PSI instead of the 40 PSI used in Examples 1 and 2) was used in order to increase the drop drive rate. In this way, event rates as high as 130,000 eps were evaluated. Prior to sorting, samples were processed as in Examples 1 and 2, above. Also in this example, inverse sorting was compared to selective, or standard, sorting as a control. Results are shown in Table 3 below. 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Pressure 
                 DDR 
                   
                   
                 Event 
                   
                   
                 Y + Dead 
                   
                 CALC Y 
                   
                 CASA 
               
               
                 (PSI) 
                 (KHz) 
                 Method 
                   
                 Rate 
                 ER/DDR 
                 Oriented 
                 Gate 
                 Dead 
                 Remove 
                 Purity 
                 MOT 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 60 
                 90 
                 Standard X 
                 ENR 
                 60 
                 0.67 
                 67.5% 
                 40.5% 
                 8.9% 
                   
                 85.7% 
                 59% 
               
               
                 60 
                 90 
                 Standard X 
                 PUR 
                 60 
                 0.67 
                 67.0% 
                 39.0% 
                 8.6% 
                   
                 87.9% 
                 76% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 PUR 
                 60 
                 0.67 
                 68.0% 
                 42.0% 
                 8.0% 
                   
                 59.8% 
                 58% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 PUR 
                 60 
                 0.67 
                 79.0% 
                 50.2% 
                 10.2% 
                 40.0% 
                 60.3% 
                 50% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 ENR 
                 60 
                 0.67 
                 79.0% 
                 51.2% 
                 10.2% 
                 41.0% 
                 61.5% 
                 47% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 ENR 
                 75 
                 0.83 
                 77.4% 
                 50.2% 
                 9.2% 
                 41.0% 
                 59.2% 
                 61% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 PUR 
                 75 
                 0.83 
                 77.6% 
                 49.8% 
                 8.9% 
                 40.9% 
                 58.1% 
                 50% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 PUR 
                 90 
                 1.00 
                 75.6% 
                 52.4% 
                 8.9% 
                 43.5% 
                 56.3% 
                 44% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 ENR 
                 90 
                 1.00 
                 75.2% 
                 51.3% 
                 9.1% 
                 42.2% 
                 57.3% 
                 74% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 ENR 
                 100 
                 1.11 
                 74.9% 
                 49.5% 
                 9.0% 
                 40.5% 
                 57.4% 
                 60% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 PUR 
                 100 
                 1.11 
                 75.2% 
                 49.2% 
                 9.0% 
                 40.2% 
                 55.3% 
                 39% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 PUR 
                 110 
                 1.22 
                 74.7% 
                 50.5% 
                 8.9% 
                 41.6% 
                 53.8% 
                 40% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 ENR 
                 110 
                 1.22 
                 74.6% 
                 51.0% 
                 9.0% 
                 42.0% 
                 56.5% 
                 52% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 ENR 
                 120 
                 1.33 
                 73.8% 
                 48.7% 
                 9.1% 
                 39.6% 
                 54.1% 
                 45% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 PUR 
                 120 
                 1.33 
                 73.6% 
                 49.1% 
                 9.1% 
                 40.0% 
                 55.0% 
                 35% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 PUR 
                 130 
                 1.44 
                 72.4% 
                 46.8% 
                 9.1% 
                 37.7% 
                 52.0% 
                 42% 
               
               
                 60 
                 90 
                 INV_REMY_DEAD 
                 ENR 
                 130 
                 1.44 
                 73.1% 
                 50.4% 
                 9.0% 
                 41.4% 
                 55.5% 
                 48% 
               
               
                   
               
            
           
         
       
     
     Example 4 
     In this Example, sperm from 5 bulls was processed as in Examples 1-3 above prior to sorting. Sorting was done at 40 PSI and an ER/DDR of 1.00 (60,000 eps/60,000 dps). Each bull&#39;s semen was sorted by two separate methods—by removing X-chromosome bearing sperm and by removing Y-chromosome bearing sperm—using the ENR mode and with removing dead cells with the sort gate, which gives the highest quality collected sperm at the cost of a lower collection rate. Collected sperm were cryopreserved and a post-thaw quality check was performed. The collection rate was determined empirically by using a NucleoCounter to determine the actual number of sperm that were collected (in the catch tube before centrifugation) and using the exact minutes and seconds that the sorting took (typically about 12-15 minutes to fill one catch tube). Results are shown in Tables 4A-9 below. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 4 A 
               
               
                   
               
             
            
               
                   
                   
                   
                 SORT 
                   
                 TIME 
                 TOTAL 
               
               
                   
                   
                 BULL 1 REMOVE X 
                 RATE 
                 TOTAL 
                 (SEC) 
                 EVENTS 
               
               
                   
               
               
                 Aug. 23, 2016 
                 12:49:17 
                 INV ENR REM_X_B1 
                 9,234 
                 33,348 
               
               
                 Aug. 23, 2016 
                 12:49:27 
                 INV ENR REM_X_B1 
                 9,095 
                 322,683 
                 10 
                 622,508 
               
               
                 Aug. 23, 2016 
                 12:49:58 
                 INV ENR REM_X_B1 
                 30,930 
                 1,304,503 
                 32 
                 2,051,086 
               
               
                 Aug. 23, 2016 
                 12:51:58 
                 INV ENR REM_X_B1 
                 30,941 
                 5,013,976 
                 120 
                 7,795,988 
               
               
                 Aug. 23, 2016 
                 12:53:59 
                 INV ENR REM_X_B1 
                 30,751 
                 8,699,179 
                 120 
                 7,796,751 
               
               
                 Aug. 23, 2016 
                 12:55:59 
                 INV ENR REM_X_B1 
                 30,183 
                 12,364,291 
                 120 
                 7,793,317 
               
               
                 Aug. 23, 2016 
                 12:57:59 
                 INV ENR REM_X_B1 
                 30,322 
                 16,018,085 
                 120 
                 7,818,392 
               
               
                 Aug. 23, 2016 
                 12:59:59 
                 INV ENR REM_X_B1 
                 30,319 
                 19,652,260 
                 120 
                 7,815,215 
               
               
                 Aug. 23, 2016 
                 13:01:59 
                 INV ENR REM_X_B1 
                 29,940 
                 23,256,030 
                 120 
                 7,802,708 
               
               
                 Aug. 23, 2016 
                 13:03:59 
                 INV ENR REM_X_B1 
                 29,869 
                 26,855,473 
                 120 
                 7,807,911 
               
               
                 Aug. 23, 2016 
                 13:05:46 
                 INV ENR REM_X_B1 
                 29,959 
                 30,077,357 
                 106 
                 6,922,602 
               
               
                 Aug. 23, 2016 
                 13:05:46 
                 INV ENR REM_X_B1 
                 29,957 
                 30,099,583 
                 1 
                 56,633 
               
               
                 Aug. 23, 2016 
                 13:05:58 
                 INV ENR REM_X_B1 
                 24,142 
                 30,232,956 
                 11 
                 732,498 
               
               
                   
                   
                   
                   
                 30,232,956 
                 1,001 
                 65,015,609 
               
            
           
           
               
               
               
            
               
                   
                 Y 
                 34,782,653 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 PURITY 
                 54.8% 
                   
                   
                 28,000,000 
               
               
                   
                 Straws Per Hour at 6Million 
                 17 
                   
                   
                 153,617,516 
               
            
           
           
               
               
            
               
                   
                 28,327 
               
               
                   
                 43% 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 NET LIVE 
               
               
                   
                   
                 TIME 
                 LIVE-ORIENTED 
                 DEAD 
                 GATE 
                 GATE 
               
               
                   
                   
               
               
                   
                 Aug. 23, 2016 
                   
                 86.4% 
                 10.6% 
                 66.17% 
                 55.59% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.4% 
                 10.6% 
                 66.48% 
                 55.85% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.3% 
                 10.7% 
                 67.89% 
                 57.20% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.3% 
                 10.6% 
                 66.89% 
                 56.25% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.4% 
                 10.8% 
                 66.87% 
                 56.06% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.4% 
                 10.8% 
                 66.29% 
                 55.53% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.4% 
                 10.8% 
                 65.88% 
                 55.13% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.4% 
                 10.8% 
                 65.68% 
                 54.84% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.5% 
                 10.9% 
                 65.49% 
                 54.58% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.4% 
                 10.9% 
                 65.45% 
                 54.55% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.4% 
                 10.9% 
                 65.28% 
                 54.36% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.3% 
                 10.9% 
                 65.26% 
                 54.35% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.4% 
                 10.9% 
                 65.00% 
                 54.09% 
               
               
                   
                   
                 0:16:28 
                 86.4% 
                 10.8% 
                 66.0% 
                 55.3% 
               
            
           
           
               
               
            
               
                   
                 BEFORE ABORT LOSSES 
               
               
                   
                 MEASURED RECOVERY/COLLECTED (NucleoCounter) 
               
               
                   
                 SPERM RECOVERED/COLLECTED PER HOUR 
               
               
                   
                 SPERM COLLECTED PER SECOND 
               
               
                   
                 TECHNICAL YIELD 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 4B 
               
               
                   
               
             
            
               
                   
                   
                   
                 SORT 
                   
                 TIME 
                 TOTAL 
               
               
                   
                   
                 BULL 1 REMOVE Y 
                 RATE 
                 TOTAL 
                 (SEC) 
                 EVENTS 
               
               
                   
               
               
                 Aug. 23, 2016 
                 13:32:13 
                 INV ENR REM_Y_B1 
                 1,414 
                 50,479 
               
               
                 Aug. 23, 2016 
                 13:32:24 
                 INV ENR REM_Y_B1 
                 13,585 
                 359,295 
                 11 
                 695,805 
               
               
                 Aug. 23, 2016 
                 13:32:24 
                 INV ENR REM_Y_B1 
                 14,165 
                 380,488 
                 1 
                 61,954 
               
               
                 Aug. 23, 2016 
                 13:34:05 
                 INV ENR REM_Y_B1 
                 29,336 
                 3,331,547 
                 100 
                 6,643,276 
               
               
                 Aug. 23, 2016 
                 13:36:05 
                 INV ENR REM_Y_B1 
                 29,739 
                 6,882,449 
                 120 
                 7,984,877 
               
               
                 Aug. 23, 2016 
                 13:38:05 
                 INV ENR REM_Y_B1 
                 29,753 
                 10,438,405 
                 120 
                 7,966,300 
               
               
                 Aug. 23, 2016 
                 13:40:05 
                 INV ENR REM_Y_B1 
                 29,256 
                 13,986,597 
                 120 
                 7,946,479 
               
               
                 Aug. 23, 2016 
                 13:42:05 
                 INV ENR REM_Y_B1 
                 30,231 
                 17,560,336 
                 120 
                 7,975,278 
               
               
                 Aug. 23, 2016 
                 13:44:05 
                 INV ENR REM_Y_B1 
                 29,356 
                 21,024,859 
                 120 
                 7,980,613 
               
               
                 Aug. 23, 2016 
                 13:46:05 
                 INV ENR REM_Y_B1 
                 29,306 
                 24,540,335 
                 120 
                 7,970,236 
               
               
                 Aug. 23, 2016 
                 13:46:40 
                 INV ENR REM_Y_B1 
                 29,763 
                 25,556,586 
                 34 
                 2,271,266 
               
               
                 Aug. 23, 2016 
                 13:48:05 
                 INV ENR REM_Y_B1 
                 29,179 
                 28,101,301 
                 86 
                 5,688,772 
               
               
                 Aug. 23, 2016 
                 13:50:06 
                 INV ENR REM_Y_B1 
                 29,176 
                 31,587,913 
                 120 
                 7,980,150 
               
               
                 Aug. 23, 2016 
                 13:50:42 
                 INV ENR REM_Y_B1 
                 28,672 
                 32,631,075 
                 37 
                 2,403,387 
               
               
                   
                   
                   
                   
                 32,631,075 
                 1,109 
                 73,568,394 
               
            
           
           
               
               
               
            
               
                   
                 X 
                 40,937,319 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 PURITY 
                 56.0% 
                   
                   
                 25,000,000 
               
               
                   
                 Stows Per Hour at 6M 
                 14 
                   
                   
                 81,127,346 
               
            
           
           
               
               
            
               
                   
                 22,535 
               
               
                   
                 34% 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 NET LIVE 
               
               
                   
                   
                 TIME 
                 LIVE-ORIENTED 
                 DEAD 
                 GATE 
                 GATE 
               
               
                   
                   
               
               
                   
                 Aug. 23, 2016 
                   
                 86.2% 
                 11.6% 
                 63.67% 
                 52.06% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.2% 
                 11.7% 
                 66.63% 
                 54.96% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.2% 
                 11.7% 
                 66.75% 
                 55.08% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.2% 
                 11.8% 
                 67.40% 
                 55.64% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.3% 
                 11.8% 
                 67.08% 
                 55.26% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.3% 
                 12.0% 
                 66.73% 
                 54.77% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.3% 
                 12.0% 
                 66.41% 
                 54.41% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.4% 
                 12.0% 
                 66.73% 
                 54.77% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.4% 
                 12.1% 
                 66.26% 
                 54.19% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.5% 
                 12.2% 
                 65.62% 
                 53.44% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.5% 
                 12.2% 
                 65.76% 
                 53.56% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.5% 
                 12.2% 
                 65.83% 
                 53.61% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.7% 
                 12.5% 
                 64.93% 
                 52.43% 
               
               
                   
                 Aug. 23, 2016 
                   
                 86.6% 
                 12.4% 
                 64.96% 
                 52.54% 
               
               
                   
                   
                 0:18:29 
                 86.4% 
                 12.0% 
                 66.1% 
                 54.1% 
               
            
           
           
               
               
            
               
                   
                 BEFORE ABORT LOSSES 
               
               
                   
                 MEASURED RECOVERY (NucleoCounter) 
               
               
                   
                 SPERM RECOVERED PER HOUR 
               
               
                   
                 EFFECTIVE SORT RATE BASED ON YIELD TO TRIS A   
               
               
                   
                 TECHNICAL YIELD 
               
               
                   
                   
               
               
                   
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 5A 
               
               
                   
               
             
            
               
                   
                   
                   
                 SORT 
                   
                 TIME 
                 TOTAL 
               
               
                   
                   
                 BULL 2 REMOVE X 
                 RATE 
                 TOTAL 
                 (SEC) 
                 EVENTS 
               
               
                   
               
               
                 Aug. 23, 2016 
                 14:16:08 
                 INV ENR REM_X_B2 
                 0 
                 13,113 
               
               
                 Aug. 23, 2016 
                 14:17:13 
                 INV ENR REM_X_B2 
                 4,748 
                 136,648 
                 65 
                 4,133,714 
               
               
                 Aug. 23, 2016 
                 14:17:14 
                 INV ENR REM_X_B2 
                 6,008 
                 173,384 
                 1 
                 90,255 
               
               
                 Aug. 23, 2016 
                 14:18:08 
                 INV ENR REM_X_B2 
                 23,786 
                 1,477,625 
                 54 
                 3,448,938 
               
               
                 Aug. 23, 2016 
                 14:20:08 
                 INV ENR REM_X_B2 
                 24,636 
                 4,408,074 
                 120 
                 7,661,980 
               
               
                 Aug. 23, 2016 
                 14:22:06 
                 INV ENR REM_X_B2 
                 24,553 
                 7,279,458 
                 118 
                 7,526,447 
               
               
                 Aug. 23, 2016 
                 14:22:09 
                 INV ENR REM_X_B2 
                 24,550 
                 7,332,938 
                 3 
                 165,218 
               
               
                 Aug. 23, 2016 
                 14:24:09 
                 INV ENR REM_X_B2 
                 25,422 
                 10,272,795 
                 120 
                 7,667,785 
               
               
                 Aug. 23, 2016 
                 14:26:09 
                 INV ENR REM_X_B2 
                 24,264 
                 13,182,808 
                 120 
                 7,669,411 
               
               
                 Aug. 23, 2016 
                 14:27:24 
                 INV ENR REM_X_B2 
                 24,504 
                 14,998,815 
                 75 
                 4,791,927 
               
               
                 Aug. 23, 2016 
                 14:27:24 
                 INV ENR REM_X_B2 
                 24,514 
                 15,017,010 
                 0 
                 28,490 
               
               
                 Aug. 23, 2016 
                 14:27:28 
                 INV ENR REM_X_B2 
                 23,729 
                 15,071,575 
                 4 
                 222,235 
               
               
                   
                   
                   
                   
                 15,071,575 
                 680 
                 43,406,401 
               
            
           
           
               
               
               
            
               
                   
                 Y 
                 28,334,826 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 PURITY 
                 58.5% 
                   
                   
                 29,000,000 
               
               
                   
                 Straws Per Hour at 6M 
                 26 
                   
                   
                 153,617,516 
               
            
           
           
               
               
            
               
                   
                 42,672 
               
               
                   
                 67% 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 NET LIVE 
               
               
                   
                   
                 TIME 
                 LIVE-ORIENTED 
                 DEAD 
                 GATE 
                 GATE 
               
               
                   
                   
               
               
                   
                 Aug. 23, 2016 
                   
                 84.2% 
                 7.9% 
                 59.63% 
                 51.74% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.2% 
                 7.9% 
                 60.75% 
                 52.87% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.2% 
                 7.9% 
                 61.32% 
                 53.45% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.2% 
                 7.8% 
                 60.71% 
                 52.91% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.2% 
                 7.8% 
                 60.81% 
                 52.99% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.1% 
                 7.9% 
                 61.08% 
                 53.22% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.1% 
                 7.9% 
                 61.17% 
                 53.31% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.1% 
                 7.8% 
                 61.40% 
                 53.58% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.1% 
                 7.8% 
                 61.40% 
                 53.59% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.1% 
                 7.8% 
                 60.89% 
                 53.11% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.1% 
                 7.8% 
                 60.90% 
                 53.12% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.1% 
                 7.8% 
                 60.85% 
                 53.09% 
               
               
                   
                   
                 0:11:20 
                 84.1% 
                 7.8% 
                 60.9% 
                 53.1% 
               
            
           
           
               
               
            
               
                   
                 BEFORE ABORT LOSSES 
               
               
                   
                 MEASURED RECOVERY (NucleoCounter) 
               
               
                   
                 SPERM RECOVERED PER HOUR 
               
               
                   
                 EFFECTIVE SORT RATE BASED ON YIELD TO TRIS A   
               
               
                   
                 TECHNICAL YIELD 
               
               
                   
                   
               
               
                   
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 5B 
               
               
                   
               
             
            
               
                   
                   
                   
                 SORT 
                   
                 TIME 
                 TOTAL 
               
               
                   
                   
                 BULL 2 REMOVE Y 
                 RATE 
                 TOTAL 
                 (SEC) 
                 EVENTS 
               
               
                   
               
               
                 Aug. 23, 2016 
                 13:55:46 
                 INV ENR REM_Y_B2 
                 31,905 
                 1,248,222 
               
               
                 Aug. 23, 2016 
                 13:56:07 
                 INV ENR REM_Y_B2 
                 28,763 
                 1,826,864 
                 21 
                 1,689,701 
               
               
                 Aug. 23, 2016 
                 13:58:07 
                 INV ENR REM_Y_B2 
                 25,440 
                 4,853,557 
                 120 
                 7,723,781 
               
               
                 Aug. 23, 2016 
                 14:00:07 
                 INV ENR REM_Y_B2 
                 25,748 
                 7,906,513 
                 120 
                 7,683,234 
               
               
                 Aug. 23, 2016 
                 14:02:07 
                 INV ENR REM_Y_B2 
                 24,189 
                 10,891,344 
                 120 
                 7,653,956 
               
               
                 Aug. 23, 2016 
                 14:04:07 
                 INV ENR REM_Y_B2 
                 24,723 
                 13,844,619 
                 120 
                 7,666,836 
               
               
                 Aug. 23, 2016 
                 14:06:07 
                 INV ENR REM_Y_B2 
                 24,517 
                 16,816,796 
                 120 
                 7,653,310 
               
               
                 Aug. 23, 2016 
                 14:07:01 
                 INV ENR REM_Y_B2 
                 24,324 
                 18,097,324 
                 53 
                 3,405,430 
               
               
                 Aug. 23, 2016 
                 14:08:08 
                 INV ENR REM_Y_B2 
                 25,251 
                 19,779,849 
                 67 
                 4,268,095 
               
               
                 Aug. 23, 2016 
                 14:10:08 
                 INV ENR REM_Y_B2 
                 24,055 
                 22,640,202 
                 120 
                 7,658,602 
               
               
                 Aug. 23, 2016 
                 14:10:50 
                 INV ENR REM_Y_B2 
                 24,954 
                 23,674,031 
                 42 
                 2,672,546 
               
               
                 Aug. 23, 2016 
                 14:11:42 
                 INV ENR REM_Y_B2 
                 21,917 
                 24,906,898 
                 52 
                 3,296,199 
               
               
                   
                   
                   
                   
                 24,906,898 
                 956 
                 61,371,690 
               
            
           
           
               
               
               
            
               
                   
                 X 
                 36,464,792 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 PURITY 
                 55.7% 
                   
                   
                 28,000,000 
               
               
                   
                 Straws Per Hour at 6M 
                 18 
                   
                   
                 105,422,348 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 29,284 
               
               
                 8/23/2016 
                   
                   
                   
                   
                   
                 46% 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 NET LIVE 
               
               
                   
                   
                 TIME 
                 LIVE-ORIENTED 
                 DEAD 
                 GATE 
                 GATE 
               
               
                   
                   
               
               
                   
                 Aug. 23, 2016 
                   
                 78.2% 
                 7.3% 
                 69.00% 
                 61.68% 
               
               
                   
                 Aug. 23, 2016 
                   
                 81.0% 
                 7.6% 
                 65.54% 
                 57.94% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.9% 
                 7.8% 
                 62.79% 
                 54.99% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.9% 
                 7.8% 
                 62.92% 
                 55.14% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.7% 
                 7.8% 
                 62.61% 
                 54.77% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.6% 
                 7.9% 
                 62.03% 
                 54.10% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.5% 
                 7.9% 
                 61.99% 
                 54.05% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.5% 
                 7.9% 
                 62.23% 
                 54.33% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.5% 
                 8.0% 
                 61.81% 
                 53.85% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.4% 
                 7.9% 
                 61.73% 
                 53.79% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.3% 
                 7.9% 
                 61.79% 
                 53.86% 
               
               
                   
                 Aug. 23, 2016 
                   
                 84.3% 
                 7.9% 
                 61.69% 
                 53.75% 
               
               
                   
                   
                 0:15:56 
                 83.7% 
                 7.8% 
                 63.0% 
                 55.2% 
               
            
           
           
               
               
               
            
               
                   
                   
                 BEFORE ABORT LOSSES 
               
               
                   
                   
                 MEASURED RECOVERY (NucleoCounter) 
               
               
                   
                   
                 SPERM RECOVERED PER HOUR 
               
               
                   
                   
                 EFFECTIVE SORT RATE BASED ON YIELD TO TRIS A   
               
               
                   
                 8/23/2016 
                 TECHNICAL YIELD 
               
               
                   
                   
               
               
                   
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 6A 
               
               
                   
               
             
            
               
                   
                   
                   
                 SORT 
                   
                 TIME 
                 TOTAL 
               
               
                   
                   
                 BULL 3 REMOVE X 
                 RATE 
                 TOTAL 
                 (SEC) 
                 EVENTS 
               
               
                   
               
               
                 Aug. 24, 2016 
                 16:39:22 
                 INV ENR REM_X_B3 
                 0 
                 0 
               
               
                 Aug. 24, 2016 
                 16:40:13 
                 INV ENR REM_X_B3 
                 2,781 
                 83,376 
                 51 
                 3,131,266 
               
               
                 Aug. 24, 2016 
                 16:40:15 
                 INV ENR REM_X_B3 
                 4,883 
                 134,829 
                 2 
                 144,587 
               
               
                 Aug. 24, 2016 
                 16:41:23 
                 INV ENR REM_X_B3 
                 21,993 
                 1,645,372 
                 67 
                 4,206,886 
               
               
                 Aug. 24, 2016 
                 16:43:23 
                 INV ENR REM_X_B3 
                 22,656 
                 4,373,575 
                 120 
                 7,424,917 
               
               
                 Aug. 24, 2016 
                 16:45:23 
                 INV ENR REM_X_B3 
                 25,949 
                 7,424,528 
                 120 
                 7,447,735 
               
               
                 Aug. 24, 2016 
                 16:47:23 
                 INV ENR REM_X_B3 
                 25,132 
                 10,510,062 
                 120 
                 7,436,269 
               
               
                 Aug. 24, 2016 
                 16:49:09 
                 INV ENR REM_X_B3 
                 25,230 
                 13,232,647 
                 106 
                 6,546,857 
               
               
                 Aug. 24, 2016 
                 16:49:23 
                 INV ENR REM_X_B3 
                 25,502 
                 13,590,487 
                 14 
                 880,703 
               
               
                 Aug. 24, 2016 
                 16:51:23 
                 INV ENR REM_X_B3 
                 25,986 
                 16,688,099 
                 120 
                 7,438,345 
               
               
                 Aug. 24, 2016 
                 16:53:23 
                 INV ENR REM_X_B3 
                 26,809 
                 19,853,695 
                 120 
                 7,413,728 
               
               
                 Aug. 24, 2016 
                 16:55:23 
                 INV ENR REM_X_B3 
                 25,885 
                 22,976,366 
                 120 
                 7,432,509 
               
               
                 Aug. 24, 2016 
                 16:57:07 
                 INV ENR REM_X_B3 
                 25,642 
                 25,678,232 
                 103 
                 6,338,233 
               
               
                   
                   
                   
                   
                 25,678,232 
                 1,064 
                 65,842,035 
               
            
           
           
               
               
               
            
               
                   
                 Y 
                 40,163,803 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 PURITY 
                 59.3% 
                   
                   
                 42,000,000 
               
               
                   
                 Straws Per Hour at 6M 
                 24 
                   
                   
                 142,058,133 
               
            
           
           
               
               
            
               
                   
                 39,461 
               
               
                   
                 64% 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 NET LIVE 
               
               
                   
                   
                 TIME 
                 LIVE-ORIENTED 
                 DEAD 
                 GATE 
                 GATE 
               
               
                   
                   
               
               
                   
                 Aug. 24, 2016 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.9% 
                 12.4% 
                 48.6% 
                 36.2% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.9% 
                 12.4% 
                 49.1% 
                 36.7% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 11.7% 
                 47.4% 
                 35.7% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 11.7% 
                 48.6% 
                 37.0% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 11.8% 
                 54.5% 
                 42.7% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 11.8% 
                 54.6% 
                 42.8% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 11.8% 
                 54.9% 
                 43.1% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 11.8% 
                 54.7% 
                 42.9% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 11.9% 
                 55.6% 
                 43.7% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 56.8% 
                 44.6% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 56.6% 
                 44.5% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 56.6% 
                 44.5% 
               
               
                   
                   
                 0:17:44 
                 92.7% 
                 12.0% 
                 53.2% 
                 41.2% 
               
            
           
           
               
               
            
               
                   
                 BEFORE ABORT LOSSES 
               
               
                   
                 MEASURED RECOVERY (NucleoCounter) 
               
               
                   
                 SPERM RECOVERED PER HOUR 
               
               
                   
                 EFFECTIVE SORT RATE BASED ON YIELD TO TRIS A   
               
               
                   
                 TECHNICAL YIELD 
               
               
                   
                   
               
               
                   
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 6B 
               
               
                   
               
             
            
               
                   
                   
                   
                 SORT 
                   
                 TIME 
                 TOTAL 
               
               
                   
                   
                 BULL 3 REMOVE Y 
                 RATE 
                 TOTAL 
                 (SEC) 
                 EVENTS 
               
               
                   
               
               
                 Aug. 24, 2016 
                 16:57:10 
                 INV ENR REM_Y_B3 
                 22,824 
                 0 
               
               
                 Aug. 24, 2016 
                 16:57:13 
                 INV ENR REM_Y_B3 
                 19,465 
                 0 
               
               
                 Aug. 24, 2016 
                 16:57:23 
                 INV ENR REM_Y_B3 
                 8,321 
                 0 
               
               
                 Aug. 24, 2016 
                 16:59:19 
                 INV ENR REM_Y_B3 
                 27,311 
                 1,894,150 
                 115 
                 7,119,484 
               
               
                 Aug. 24, 2016 
                 16:59:19 
                 INV ENR REM_Y_B3 
                 27,303 
                 1,914,992 
                 1 
                 44,427 
               
               
                 Aug. 24, 2016 
                 16:59:24 
                 INV ENR REM_Y_B3 
                 27,362 
                 2,038,416 
                 4 
                 264,836 
               
               
                 Aug. 24, 2016 
                 17:01:24 
                 INV ENR REM_Y_B3 
                 26,996 
                 5,294,608 
                 120 
                 7,448,722 
               
               
                 Aug. 24, 2016 
                 17:03:24 
                 INV ENR REM_Y_B3 
                 26,752 
                 8,515,192 
                 120 
                 7,415,335 
               
               
                 Aug. 24, 2016 
                 17:05:24 
                 INV ENR REM_Y_B3 
                 26,986 
                 11,750,987 
                 120 
                 7,434,223 
               
               
                 Aug. 24, 2016 
                 17:06:27 
                 INV ENR REM_Y_B3 
                 27,068 
                 13,453,882 
                 63 
                 3,899,936 
               
               
                 Aug. 24, 2016 
                 17:06:29 
                 INV ENR REM_Y_B3 
                 27,071 
                 13,501,755 
                 2 
                 117,187 
               
               
                 Aug. 24, 2016 
                 17:07:24 
                 INV ENR REM_Y_B3 
                 27,077 
                 14,995,123 
                 55 
                 3,419,487 
               
               
                 Aug. 24, 2016 
                 17:09:25 
                 INV ENR REM_Y_B3 
                 27,332 
                 18,251,960 
                 120 
                 7,440,723 
               
               
                 Aug. 24, 2016 
                 17:11:25 
                 INV ENR REM_Y_B3 
                 26,841 
                 21,477,082 
                 120 
                 7,432,032 
               
               
                 Aug. 24, 2016 
                 17:13:25 
                 INV ENR REM_Y_B3 
                 27,041 
                 24,707,380 
                 120 
                 7,413,152 
               
               
                 Aug. 24, 2016 
                 17:15:25 
                 INV ENR REM_Y_B3 
                 0 
                 27,080,060 
                 120 
                 7,433,550 
               
               
                   
                   
                   
                   
                 27,080,060 
                 1,082 
                 59,763,610 
               
            
           
           
               
               
               
            
               
                   
                 X 
                 32,683,550 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 PURITY 
                 52.2% 
                   
                   
                 42,000,000 
               
               
                   
                 Stows Per Hour at 6M 
                 23 
                   
                   
                 139,789,022 
               
            
           
           
               
               
            
               
                   
                 38,830 
               
               
                   
                 70% 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 NET LIVE 
               
               
                   
                   
                 TIME 
                 LIVE-ORIENTED 
                 DEAD 
                 GATE 
                 GATE 
               
               
                   
                   
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 56.5% 
                 44.4% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 56.5% 
                 44.4% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 12.1% 
                 56.2% 
                 44.2% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 57.9% 
                 45.8% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 57.9% 
                 45.8% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 57.7% 
                 45.7% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.2% 
                 55.8% 
                 43.7% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 55.9% 
                 43.8% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 56.3% 
                 44.2% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 56.3% 
                 44.2% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 56.3% 
                 44.2% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.5% 
                 12.1% 
                 56.4% 
                 44.4% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.5% 
                 12.1% 
                 56.5% 
                 44.4% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 56.2% 
                 44.1% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 55.9% 
                 43.9% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 12.1% 
                 57.3% 
                 45.3% 
               
               
                   
                   
                 0:18:02 
                 92.6% 
                 12.1% 
                 56.6% 
                 44.6% 
               
            
           
           
               
               
            
               
                   
                 BEFORE ABORT LOSSES 
               
               
                   
                 MEASURED RECOVERY (NucleoCounter) 
               
               
                   
                 SPERM RECOVERED PER HOUR 
               
               
                   
                 EFFECTIVE SORT RATE BASED ON YIELD TO TRIS A   
               
               
                   
                 TECHNICAL YIELD 
               
               
                   
                   
               
               
                   
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 7A 
               
               
                   
               
             
            
               
                   
                   
                   
                 SORT 
                   
                 TIME 
                 TOTAL 
               
               
                   
                   
                 BULL 4 REMOVE X 
                 RATE 
                 TOTAL 
                 (SEC) 
                 EVENTS 
               
               
                   
               
               
                 Aug. 24, 2016 
                 15:25:59 
                 INV ENR REM_X_B4 
                 0 
                 0 
               
               
                 Aug. 24, 2016 
                 15:26:11 
                 INV ENR REM_X_B4 
                 4,340 
                 121,136 
                 12 
                 697,168 
               
               
                 Aug. 24, 2016 
                 15:27:17 
                 INV ENR REM_X_B4 
                 26,328 
                 1,855,021 
                 67 
                 3,961,499 
               
               
                 Aug. 24, 2016 
                 15:29:17 
                 INV ENR REM_X_B4 
                 24,757 
                 4,819,574 
                 120 
                 7,087,139 
               
               
                 Aug. 24, 2016 
                 15:31:18 
                 INV ENR REM_X_B4 
                 24,417 
                 7,846,790 
                 120 
                 7,135,810 
               
               
                 Aug. 24, 2016 
                 15:33:18 
                 INV ENR REM_X_B4 
                 25,341 
                 10,848,011 
                 120 
                 7,155,376 
               
               
                 Aug. 24, 2016 
                 15:35:18 
                 INV ENR REM_X_B4 
                 24,517 
                 13,807,468 
                 120 
                 7,186,757 
               
               
                 Aug. 24, 2016 
                 15:36:29 
                 INV ENR REM_X_B4 
                 24,628 
                 15,524,507 
                 71 
                 4,205,028 
               
               
                 Aug. 24, 2016 
                 15:37:18 
                 INV ENR REM_X_B4 
                 24,781 
                 16,741,755 
                 49 
                 2,926,996 
               
               
                 Aug. 24, 2016 
                 15:39:18 
                 INV ENR REM_X_B4 
                 23,640 
                 18,899,870 
                 120 
                 7,149,588 
               
               
                 Aug. 24, 2016 
                 15:41:18 
                 INV ENR REM_X_B4 
                 23,962 
                 21,737,803 
                 120 
                 7,133,491 
               
               
                 Aug. 24, 2016 
                 15:42:16 
                 INV ENR REM_X_B4 
                 23,199 
                 23,076,144 
                 57 
                 3,395,105 
               
               
                 Aug. 24, 2016 
                 15:42:18 
                 INV ENR REM_X_B4 
                 22,424 
                 23,111,417 
                 2 
                 143,680 
               
               
                   
                   
                   
                   
                 23,111,417 
                 979 
                 58,177,639 
               
            
           
           
               
               
               
            
               
                   
                 Y 
                 35,066,222 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 PURITY 
                 59.0% 
                   
                   
                 38,000,000 
               
               
                   
                 Stows Per Hour at 6M 
                 23 
                   
                   
                 139,703,457 
               
            
           
           
               
               
            
               
                   
                 38,807 
               
               
                   
                 65% 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 NET LIVE 
               
               
                   
                   
                 TIME 
                 LIVE-ORIENTED 
                 DEAD 
                 GATE 
                 GATE 
               
               
                   
                   
               
               
                   
                 Aug. 24, 2016 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 16.6% 
                 58.1% 
                 41.5% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 16.6% 
                 59.5% 
                 42.9% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 16.6% 
                 53.5% 
                 36.9% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 16.5% 
                 52.8% 
                 36.2% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 16.6% 
                 52.9% 
                 36.3% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 16.6% 
                 52.8% 
                 36.2% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 16.6% 
                 52.8% 
                 36.2% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 16.7% 
                 53.2% 
                 36.5% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 16.7% 
                 50.9% 
                 34.2% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 16.7% 
                 50.8% 
                 34.1% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 16.7% 
                 50.8% 
                 34.1% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 16.6% 
                 50.7% 
                 34.1% 
               
               
                   
                   
                 0:16:19 
                 92.7% 
                 16.6% 
                 53.6% 
                 37.0% 
               
            
           
           
               
               
            
               
                   
                 BEFORE ABORT LOSSES 
               
               
                   
                 MEASURED RECOVERY (NucleoCounter) 
               
               
                   
                 SPERM RECOVERED PER HOUR 
               
               
                   
                 EFFECTIVE SORT RATE BASED ON YIELD TO TRIS A   
               
               
                   
                 TECHNICAL YIELD 
               
               
                   
                   
               
               
                   
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 7B 
               
               
                   
               
             
            
               
                   
                   
                   
                 SORT 
                   
                 TIME 
                 TOTAL 
               
               
                   
                   
                 BULL 4 REMOVE Y 
                 RATE 
                 TOTAL 
                 (SEC) 
                 EVENTS 
               
               
                   
               
               
                 Aug. 24, 2016 
                 15:43:19 
                 INV ENR REM_Y_B4 
                 0 
                 0 
               
               
                 Aug. 24, 2016 
                 15:44:30 
                 INV ENR REM_Y_B4 
                 2,699 
                 79,162 
                 71.52 
                 4,201,390 
               
               
                 Aug. 24, 2016 
                 15:45:19 
                 INV ENR REM_Y_B4 
                 26,550 
                 1,376,872 
                 49 
                 3,007,303 
               
               
                 Aug. 24, 2016 
                 15:47:19 
                 INV ENR REM_Y_B4 
                 27,840 
                 4,666,947 
                 120 
                 7,375,455 
               
               
                 Aug. 24, 2016 
                 15:49:19 
                 INV ENR REM_Y_B4 
                 25,853 
                 7,873,046 
                 120 
                 7,372,456 
               
               
                 Aug. 24, 2016 
                 15:49:20 
                 INV ENR REM_Y_B4 
                 25,881 
                 7,911,820 
                 1 
                 89,901 
               
               
                 Aug. 24, 2016 
                 15:51:19 
                 INV ENR REM_Y_B4 
                 27,698 
                 11,057,298 
                 119 
                 7,317,938 
               
               
                 Aug. 24, 2016 
                 15:53:19 
                 INV ENR REM_Y_B4 
                 27,236 
                 14,273,385 
                 120 
                 7,400,047 
               
               
                 Aug. 24, 2016 
                 15:55:19 
                 INV ENR REM_Y_B4 
                 26,495 
                 17,477,322 
                 120 
                 7,383,017 
               
               
                 Aug. 24, 2016 
                 15:57:19 
                 INV ENR REM_Y_B4 
                 26,696 
                 20,730,544 
                 120 
                 7,380,124 
               
               
                 Aug. 24, 2016 
                 15:59:20 
                 INV ENR REM_Y_B4 
                 27,104 
                 24,014,725 
                 120 
                 7,389,357 
               
               
                 Aug. 24, 2016 
                 16:01:09 
                 INV ENR REM_Y_B4 
                 26,607 
                 26,954,167 
                 109 
                 6,697,674 
               
               
                 Aug. 24, 2016 
                 16:01:12 
                 INV ENR REM_Y_B4 
                 25,633 
                 27,012,489 
                 3 
                 202,694 
               
               
                   
                   
                   
                   
                 27,012,489 
                 1,073 
                 65,817,356 
               
            
           
           
               
               
               
            
               
                   
                 X 
                 38,804,867 
               
            
           
           
               
               
               
               
               
            
               
                   
                 PURITY 
                 56.2% 
                   
                 42,000,000 
               
               
                   
                 Stews Per Hour at 6M 
                 23 
                   
                 140,881,291 
               
            
           
           
               
               
            
               
                   
                 39,134 
               
               
                   
                 64% 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 NET LIVE 
               
               
                   
                   
                 TIME 
                 LIVE-ORIENTED 
                 DEAD 
                 GATE 
                 GATE 
               
               
                   
                   
               
               
                   
                 Aug. 24, 2016 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.8% 
                 16.6% 
                 59.0% 
                 42.4% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 16.5% 
                 57.1% 
                 40.5% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 16.6% 
                 56.7% 
                 40.1% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 16.6% 
                 56.9% 
                 40.3% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 16.6% 
                 57.0% 
                 40.4% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 16.6% 
                 57.2% 
                 40.6% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.7% 
                 16.6% 
                 57.1% 
                 40.5% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 16.7% 
                 56.7% 
                 40.1% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 16.6% 
                 57.2% 
                 40.6% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 16.6% 
                 57.0% 
                 40.3% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 16.6% 
                 57.3% 
                 40.7% 
               
               
                   
                 Aug. 24, 2016 
                   
                 92.6% 
                 16.6% 
                 57.3% 
                 40.7% 
               
               
                   
                   
                 0:17:53 
                 92.6% 
                 16.6% 
                 57.0% 
                 40.4% 
               
            
           
           
               
               
            
               
                   
                 BEFORE ABORT LOSSES 
               
               
                   
                 MEASURED RECOVERY (NucleoCounter) 
               
               
                   
                 SPERM RECOVERED PER HOUR 
               
               
                   
                 EFFECTIVE SORT RATE BASED ON YIELD TO TRIS A   
               
               
                   
                 TECHNICAL YIELD 
               
               
                   
                   
               
               
                   
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 8A 
               
               
                   
               
             
            
               
                   
                   
                   
                 SORT 
                   
                 TIME 
                 TOTAL 
               
               
                   
                   
                 BULL 5 REMOVE X 
                 RATE 
                 TOTAL 
                 (SEC) 
                 EVENTS 
               
               
                   
               
               
                 Aug. 24, 2016 
                 16:23:21 
                 INV ENR REM_X_B5 
                 10,412 
                 274,257 
                 120 
                 7,139,663 
               
               
                 Aug. 24, 2016 
                 16:23:24 
                 INV ENR REM_X_B5 
                 12,991 
                 337,062 
                 3 
                 170,379 
               
               
                 Aug. 24, 2016 
                 16:23:26 
                 INV ENR REM_X_B5 
                 14,939 
                 392,820 
                 2 
                 142,943 
               
               
                 Aug. 24, 2016 
                 16:25:21 
                 INV ENR REM_X_B5 
                 20,136 
                 2,766,174 
                 115 
                 6,951,908 
               
               
                 Aug. 24, 2016 
                 16:27:22 
                 INV ENR REM_X_B5 
                 20,330 
                 5,211,250 
                 120 
                 7,227,246 
               
               
                 Aug. 24, 2016 
                 16:29:22 
                 INV ENR REM_X_B5 
                 20,763 
                 7,701,630 
                 120 
                 7,220,736 
               
               
                 Aug. 24, 2016 
                 16:31:22 
                 INV ENR REM_X_B5 
                 21,170 
                 10,189,443 
                 120 
                 7,233,042 
               
               
                 Aug. 24, 2016 
                 16:33:22 
                 INV ENR REM_X_B5 
                 20,969 
                 12,716,527 
                 120 
                 7,228,917 
               
               
                 Aug. 24, 2016 
                 16:35:22 
                 INV ENR REM_X_B5 
                 20,806 
                 15,202,349 
                 120 
                 7,230,294 
               
               
                 Aug. 24, 2016 
                 16:37:22 
                 INV ENR REM_X_B5 
                 20,795 
                 17,697,093 
                 120 
                 7,221,184 
               
               
                 Aug. 24, 2016 
                 16:38:05 
                 INV ENR REM_X_B5 
                 21,145 
                 18,623,045 
                 43 
                 2,603,267 
               
               
                 Aug. 24, 2016 
                 16:38:09 
                 INV ENR REM_X_B5 
                 19,967 
                 18,662,040 
                 3 
                 200,665 
               
               
                 Aug. 24, 2016 
                 16:38:10 
                 INV ENR REM_X_B5 
                 19,543 
                 18,662,040 
                 1 
                 58,268 
               
               
                   
                   
                   
                   
                 18,662,040 
                 1,009 
                 53,318,469 
               
            
           
           
               
               
               
            
               
                   
                 Y 
                 34,656,429 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 PURITY 
                 58.8% 
                   
                   
                 43,000,000 
               
               
                   
                 Stews Per Hour at 6M 
                 26 
                   
                   
                 153,449,643 
               
            
           
           
               
               
            
               
                   
                 42,625 
               
               
                   
                 81% 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 NET LIVE 
               
               
                   
                   
                 TIME 
                 LIVE-ORIENTED 
                 DEAD 
                 GATE 
                 GATE 
               
               
                   
                   
               
               
                   
                 Aug. 24, 2016 
                   
                 90.8% 
                 5.7% 
                 46.5% 
                 40.7% 
               
               
                   
                 Aug. 24, 2016 
                   
                 90.8% 
                 5.7% 
                 46.9% 
                 41.2% 
               
               
                   
                 Aug. 24, 2016 
                   
                 90.8% 
                 5.7% 
                 47.2% 
                 41.5% 
               
               
                   
                 Aug. 24, 2016 
                   
                 90.6% 
                 5.6% 
                 47.2% 
                 41.6% 
               
               
                   
                 Aug. 24, 2016 
                   
                 90.7% 
                 5.6% 
                 47.3% 
                 41.7% 
               
               
                   
                 Aug. 24, 2016 
                   
                 90.8% 
                 5.6% 
                 47.4% 
                 41.8% 
               
               
                   
                 Aug. 24, 2016 
                   
                 90.8% 
                 5.6% 
                 47.6% 
                 42.0% 
               
               
                   
                 Aug. 24, 2016 
                   
                 90.8% 
                 5.6% 
                 47.5% 
                 41.9% 
               
               
                   
                 Aug. 24, 2016 
                   
                 90.9% 
                 5.7% 
                 48.0% 
                 42.3% 
               
               
                   
                 Aug. 24, 2016 
                   
                 90.9% 
                 5.6% 
                 48.0% 
                 42.4% 
               
               
                   
                 Aug. 24, 2016 
                   
                 90.9% 
                 5.6% 
                 47.7% 
                 42.1% 
               
               
                   
                 Aug. 24, 2016 
                   
                 90.9% 
                 5.6% 
                 47.7% 
                 42.1% 
               
               
                   
                 Aug. 24, 2016 
                   
                 90.9% 
                 5.6% 
                 47.7% 
                 42.0% 
               
               
                   
                   
                 0:16:49 
                 90.8% 
                 5.6% 
                 47.4% 
                 41.8% 
               
            
           
           
               
               
            
               
                   
                 BEFORE ABORT LOSSES 
               
               
                   
                 MEASURED RECOVERY (NucleoCounter) 
               
               
                   
                 SPERM RECOVERED PER HOUR 
               
               
                   
                 EFFECTIVE SORT RATE BASED ON YIELD TO TRIS A   
               
               
                   
                 TECHNICAL YIELD 
               
               
                   
                   
               
               
                   
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 8B 
               
               
                   
               
             
            
               
                   
                   
                   
                 SORT 
                   
                 TIME 
                 TOTAL 
               
               
                   
                   
                 BULL 5 REMOVE Y 
                 RATE 
                 TOTAL 
                 (SEC) 
                 EVENTS 
               
               
                   
               
               
                 Aug. 24, 2016 
                 16:03:20 
                 INV ENR REM_Y_B5 
                 0 
                 0 
               
               
                 Aug. 24, 2016 
                 16:05:00 
                 INV ENR REM_Y_B5 
                 1,521 
                 45,322 
                 101 
                 6,158,478 
               
               
                 Aug. 24, 2016 
                 16:05:04 
                 INV ENR REM_Y_B5 
                 4,987 
                 145,509 
                 4 
                 233,083 
               
               
                 Aug. 24, 2016 
                 16:05:20 
                 INV ENR REM_Y_B5 
                 18,544 
                 525,158 
                 16 
                 950,835 
               
               
                 Aug. 24, 2016 
                 16:07:20 
                 INV ENR REM_Y_B5 
                 19,699 
                 3,378,417 
                 120 
                 7,270,439 
               
               
                 Aug. 24, 2016 
                 16:09:20 
                 INV ENR REM_Y_B5 
                 22,254 
                 5,472,451 
                 120 
                 7,217,618 
               
               
                 Aug. 24, 2016 
                 16:11:20 
                 INV ENR REM_Y_B5 
                 22,449 
                 8,164,448 
                 120 
                 7,199,972 
               
               
                 Aug. 24, 2016 
                 16:13:20 
                 INV ENR REM_Y_B5 
                 22,074 
                 10,837,026 
                 120 
                 7,192,848 
               
               
                 Aug. 24, 2016 
                 16:15:21 
                 INV ENR REM_Y_B5 
                 21,593 
                 13,478,622 
                 120 
                 7,229,656 
               
               
                 Aug. 24, 2016 
                 16:17:21 
                 INV ENR REM_Y_B5 
                 21,845 
                 16,171,610 
                 120 
                 7,226,619 
               
               
                 Aug. 24, 2016 
                 16:19:21 
                 INV ENR REM_Y_B5 
                 22,452 
                 18,840,137 
                 120 
                 7,222,437 
               
               
                 Aug. 24, 2016 
                 16:20:53 
                 INV ENR REM_Y_B5 
                 20,858 
                 20,820,523 
                 92 
                 5,470,771 
               
               
                   
                   
                   
                   
                 20,820,523 
                 1,053 
                 63,372,757 
               
            
           
           
               
               
               
            
               
                   
                 X 
                 42,552,234 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 PURITY 
                 59.0% 
                   
                   
                 45,000,000 
               
               
                   
                 Straws Per Hour at 6M 
                 26 
                   
                   
                 153,798,539 
               
            
           
           
               
               
            
               
                   
                 42,722 
               
               
                   
                 71% 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 NET LIVE 
               
               
                   
                   
                 TIME 
                 LIVE-ORIENTED 
                 DEAD 
                 GATE 
                 GATE 
               
               
                   
                   
               
               
                   
                 Aug. 24, 2016 
               
               
                   
                 Aug. 24, 2016 
                   
                 91.3% 
                 5.5% 
                 57.6% 
                 52.1% 
               
               
                   
                 Aug. 24, 2016 
                   
                 91.2% 
                 5.4% 
                 56.7% 
                 51.3% 
               
               
                   
                 Aug. 24, 2016 
                   
                 91.3% 
                 5.5% 
                 54.8% 
                 49.3% 
               
               
                   
                 Aug. 24, 2016 
                   
                 91.4% 
                 5.5% 
                 53.6% 
                 48.1% 
               
               
                   
                 Aug. 24, 2016 
                   
                 91.4% 
                 5.6% 
                 49.7% 
                 44.1% 
               
               
                   
                 Aug. 24, 2016 
                   
                 91.3% 
                 5.6% 
                 49.2% 
                 43.6% 
               
               
                   
                 Aug. 24, 2016 
                   
                 91.3% 
                 5.6% 
                 49.0% 
                 43.3% 
               
               
                   
                 Aug. 24, 2016 
                   
                 91.2% 
                 5.7% 
                 48.8% 
                 43.1% 
               
               
                   
                 Aug. 24, 2016 
                   
                 91.2% 
                 5.7% 
                 49.1% 
                 43.5% 
               
               
                   
                 Aug. 24, 2016 
                   
                 91.0% 
                 5.6% 
                 49.4% 
                 43.8% 
               
               
                   
                 Aug. 24, 2016 
                   
                 91.0% 
                 5.7% 
                 49.1% 
                 43.5% 
               
               
                   
                   
                 0:17:33 
                 91.2% 
                 5.6% 
                 51.5% 
                 46.0% 
               
            
           
           
               
               
            
               
                   
                 BEFORE ABORT LOSSES 
               
               
                   
                 MEASURED RECOVERY (NucleoCounter) 
               
               
                   
                 SPERM RECOVERED PER HOUR 
               
               
                   
                 EFFECTIVE SORT RATE BASED ON YIELD TO TRIS A   
               
               
                   
                 TECHNICAL YIELD 
               
               
                   
                   
               
               
                   
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 9 
               
               
                   
                   
               
               
                   
                   
                 0-HOUR 
                 3-HOUR 
                   
               
               
                   
                 PUR- 
                 VISUAL 
                 VISUAL 
                 RATIO 
               
               
                   
                 ITY 
                 MOTILITY 
                 MOTILITY 
                 3 H/0 H 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 BULL 1 
                 Remove X 
                 54.8% 
                 57.0% 
                 45.0% 
                 78.9% 
               
               
                   
                 Remove Y 
                 56.0% 
                 58.0% 
                 57.0% 
                 98.3% 
               
               
                 BULL 2 
                 Remove X 
                 58.5% 
                 58.0% 
                 36.0% 
                 62.1% 
               
               
                   
                 Remove Y 
                 55.7% 
                 55.0% 
                 48.0% 
                 87.3% 
               
               
                 BULL 3 
                 Remove X 
                 59.3% 
                 54.0% 
                 39.0% 
                 72.2% 
               
               
                   
                 Remove Y 
                 52.2% 
                 55.0% 
                 34.0% 
                 61.8% 
               
               
                 BULL 4 
                 Remove X 
                 59.0% 
                 48.0% 
                 42.0% 
                 87.5% 
               
               
                   
                 Remove Y 
                 56.2% 
                 49.0% 
                 50.0% 
                 102.0% 
               
               
                 BULL 5 
                 Remove X 
                 58.8% 
                 68.0% 
                 41.0% 
                 60.3% 
               
               
                   
                 Remove Y 
                 59.0% 
                 60.0% 
                 48.0% 
                 80.0% 
               
               
                 AVERAGE 
                 Remove X 
                 58.1% 
                 57.0% 
                 40.6% 
                 71.2% 
               
               
                   
                 Remove Y 
                 55.8% 
                 55.4% 
                 47.4% 
                 85.6% 
               
               
                   
               
            
           
         
       
     
     Example 5 
     1. Get three fresh ejaculates each from a different bull.
 
2. Check volume, concentration, motility, morphology and pH. Add antibiotics.
 
3. Standardize with an extender, concentrate to 1800 mill/ml.
 
4. Stain 20 mL sperm per tube at 160 million sperm per mL with Hoechst 33342.
 
5. Once the staining mix is created, split the volume in two aliquots (10 mL each):
         Add magnetic particles (ULTRAsep) to ALIQUOT A.   No ULTRAsep will be added to ALIQUOT B.
 
6. Incubate for 60 minutes at 34° C. and apply the separation system for 10 minutes to the ULTRAsep treated sample.
 
7. Add ⅓ vol of 8% egg yolk TALP-based media to each aliquot.
 
8. Transfer to 15 mL Falcon tubes, centrifuge and remove an amount of supernatant to target a new concentration of 320 sperm/ml.
 
9. Confirm the final concentrations and place the fluid volumes into 6 mL tubes for use on Saratoga Sorters.
 
10. Align samples at 65 KHz. Establish INV_ENR_YREM method gate (Y removal gate with ENR mode enabled) at 65 KHz Event Rate (ER).
 
11. Prior to sorting measure the PVR of each sample at 80 KHz and 50 KHz like standard sex sorting.
 
12. Bulk sort (standard) sperm into 7.0 mL of Tris based media to a final of 40 mL with sheath fluid (6 pre-weighted tubes total).
 
13. Using the Y gate, remove a percentage of sperm that is close to the dead percentage plus 45%.
 
14. Inverse sort sperm into 7.0 mL of Tris based media to a final of 40 ml with sheath fluid (6 pre-weighted tubes total).
 
15. Place all catch tubes in a CoolingCastle at the same time and for a minimum of 30 minutes.
 
16. Centrifuge all tubes and decant following production procedures. Calculate recovery (using tare and end weight of tubes and final concentration of pellets).
 
17. Add freezing media for a final of 6 million sperm per ¼ cc straw.
 
18. Hold sperm over-night in freezing media and freeze (3 straws per treatment).
 
19. Perform quality control check for 0 and 3 h motility, viability (PI) and PIA (PNA). Perform purity analysis.
       

     Results of the experiment are found in Table 10 below and in  FIGS. 10 and 11 . 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                   
                   
                   
                   
                   
                   
                 ORI- 
                   
                   
                   
                   
                 MILLION 
                 MILLION 
                   
               
               
                   
                   
                 Sorted 
                 EVENT 
                 SORT 
                 TOTAL 
                 ENTED 
                 DEAD 
                 X 
                 PVR 
                 NET 
                 RECOVERED 
                 SORTED/ 
                 PURI- 
               
               
                 Bull 
                 Method 
                 Time 
                 AVG 
                 AVG 
                 SORT 
                 AVG 
                 AVG 
                 AVG 
                 AVG 
                 Gate 
                 (1TUBE) 
                 MIN 
                 TY 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 B1 
                 PVR Check 
                   
                 50,404 
                   
                   
                   
                   
                   
                 61.7% 
                   
                   
                   
                   
               
               
                   
                 Standard 
                 0:29:02 
                 50,224 
                 20,698 
                 37,010,494 
                 64.8% 
                 11.0% 
                 99.3% 
                 58.6% 
                   
                 41.9 
                 1.4 
                 50.0% 
               
               
                   
                   
                 0:27:10 
                 49,909 
                 22,579 
                 38,038,888 
                 69.1% 
                 9.7% 
                 99.3% 
                 55.8% 
                   
                 44.7 
                 1.7 
                 50.0% 
               
               
                   
                 Inverse 
                 0:12:17 
                 65,093 
                 38,626 
                 28,448,852 
                 88.4% 
                 11.4% 
                 60.2% 
                 22.4% 
                 48.8% 
                 32.5 
                 2.7 
                 60.4% 
               
               
                   
                   
                 0:11:18 
                 64,825 
                 40,006 
                 27,134,067 
                 87.3% 
                 9.5% 
                 57.5% 
                 14.6% 
                 48.0% 
                 36.6 
                 3.3 
                 60.5% 
               
               
                   
                 PVR Check 
                   
                 79,838 
                   
                   
                   
                   
                   
                 52.6% 
               
               
                 B2 
                 PVR Check 
                   
                 51,818 
                   
                   
                   
                   
                   
                 64.9% 
               
               
                   
                 Standard 
                 0:24:27 
                 50,836 
                 25,239 
                 38,355,238 
                 74.2% 
                 7.6% 
                 99.1% 
                 65.3% 
                   
                 45.1 
                 1.9 
                 50.0% 
               
               
                   
                   
                 0:24:34 
                 50,429 
                 25,105 
                 37,994,720 
                 73.8% 
                 7.9% 
                 99.2% 
                 63.6% 
                   
                 42.6 
                 1.8 
                 50.0% 
               
               
                   
                 Inverse 
                 0:11:47 
                 65,193 
                 41,791 
                 29,526,163 
                 87.8% 
                 7.5% 
                 57.3% 
                 45.1% 
                 49.9% 
                 33.5 
                 2.8 
                 58.5% 
               
               
                   
                   
                 0:11:57 
                 65,218 
                 39,117 
                 28,050,183 
                 87.4% 
                 7.4% 
                 60.9% 
                 43.4% 
                 53.5% 
                 33.6 
                 2.8 
                 60.9% 
               
               
                   
                 PVR Check 
                   
                 87,310 
                   
                   
                   
                   
                   
                 55.8% 
               
               
                 B3 
                 PVR Check 
                   
                 51,448 
                   
                   
                   
                   
                   
                 54.7% 
               
               
                   
                 Standard 
                 0:25:25 
                 50,788 
                 23,998 
                 37,693,153 
                 70.6% 
                 10.4% 
                 99.1% 
                 53.0% 
                   
                 45.6 
                 1.8 
                 50.0% 
               
               
                   
                   
                 0:26:03 
                 50,537 
                 23,008 
                 36,993,115 
                 69.8% 
                 11.0% 
                 98.0% 
                 52.6% 
                   
                 39.7 
                 1.5 
                 50.0% 
               
               
                   
                 Inverse 
                 0:10:31 
                 65,100 
                 47,101 
                 29,729,475 
                 88.7% 
                 9.7% 
                 61.2% 
                 1.8% 
                 51.4% 
                 37.6 
                 3.8 
                 41.2% 
               
               
                   
                   
                 0:10:42 
                 65,590 
                 49,550 
                 31,817,520 
                 88.4% 
                 10.9% 
                 57.4% 
                 1.3% 
                 46.4% 
                 36.1 
                 3.6 
                 42.9% 
               
               
                   
                 PVR Check 
                   
                 80,560 
                   
                   
                   
                   
                   
                 45.7% 
               
               
                   
               
            
           
         
       
     
     Example 6 
     1. Get three fresh ejaculates each from a different bull.
 
2. Check volume, concentration, motility, morphology and pH. Add antibiotics.
 
3. Remove 1 ml of ejaculate and process as with conventional semen (unsorted) procedures (25 Million/straw).
 
4. Standardize with extender, concentrate to 1800 million/ml.
 
5. Stain with Hoechst 33342 as follows:
         TUBE 0 (CONTROL): 10 ml of sperm at 160 million sperm per ml with magnetic particles (ULTRAsep).   TUBE 1 (INV): 10 mL of sperm at 160 million sperm per ml with magnetic particles.   TUBE 2 (INV): 10 mL of sperm at 160 million sperm per ml with magnetic particles.   TUBE 3 (INV): 10 mL of sperm at 160 million sperm per ml with magnetic particles.
 
6, Incubate for 60 minutes at 34° C. Add ⅓ vol of 8% egg yolk TALP based media to each aliquot.
 
7. Apply the magnetic separation system for 10 minutes.
 
8. Transfer TUBE 0 to 4 mL sample tubes.
 
9. Align samples at 40 KHz. Prior to sorting measure the PVR of each sample at 40 KHz.
 
10. Sex sort (40 KHz, 65% X purity) sperm into 7.0 ml of Tris based media to a final of 40 ml using sheath fluid (1 catch tube per bull=3 pre-weighted tubes total) on flow cytometer.
 
11. Transfer TUBES 1-3 to 15 mL Falcon tubes, centrifuge and remove an amount of supernatant to target a new concentration of 300 sperm/ml.
 
12. Confirm the final concentrations and place the fluid volumes into 6 mL tubes for use on flow cytometers.
 
13. Align samples at 61 KHz. Establish INV_ENR_YREM_DEADREM gates (Y removal and dead removal gate with ENR mode enabled) at 61 KHz ER for optimal Y removal. Regions will be established based on resolution and not to a specific percent.
 
14. Prior to sorting, measure the PVR of each sample at 80 KHz and 50 KHz with zoom like standard sorting.
 
15. Inverse sort (61 KHz) sperm into 7.0 ml of Tris based media to a final of 40 mL using sheath fluid (3 catch tubes per bull=9 pre-weighted tubes) on flow cytometer.
 
16. Place all catch tubes in the CoolingCastle. Cool for 30 minutes.
 
17. Centrifuge all tubes and decant. Calculate recovery.
 
18. Add freeze media for a final of 4 million (CONTROL) or 6 million (INV) sperm per ¼ cc straw.
 
19. Hold over-night and freeze (3 straws per treatment).
 
20. Perform quality control checks—0 and 3 h motility, viability (PI) and PIA (PNA). Perform purity analysis.
       

     Results are found in Tables 11 and 12 below and in  FIG. 12 . 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 11 
               
             
            
               
                   
                   
               
               
                   
                 0 H 
                 3 H 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 VISUAL 
                 IVOS II 
                 A. SORTER 
                 VISUAL 
                 IVOS II 
                 A. SORTER 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 BULL 
                 METHOD 
                 PURITY 
                 MOTILE 
                 MOTILE 
                 PROG 
                 VIABLE 
                 PIA 
                 MOTILE 
                 MOTILE 
                 PROG 
                 VIABLE 
                 PIA 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 CONTROL (65% X) 
                 66.9 
                 85 
                 91.3 
                 85.2 
                 55.75 
                 *3.05 
                 68 
                 73.5 
                 41.2 
                 47.89 
                 64.36 
               
               
                   
                 INV (Rem Y) 
                 58.4 
                 80 
                 74.7 
                 69.6 
                 44.24 
                 73.21 
                 41 
                 47.8 
                 24.4 
                 37.3 
                 56.64 
               
               
                 2 
                 CONTROL (65% X) 
                 68.1 
                 72 
                 77.1 
                 59.6 
                 65.23 
                 85.65 
                 55 
                 59.9 
                 30.1 
                 48.06 
                 64.76 
               
               
                   
                 INV (Rem Y) 
                 59.6 
                 71 
                 64.2 
                 58.2 
                 53.12 
                 77.77 
                 47 
                 35 
                 18.8 
                 39.95 
                 56.36 
               
               
                 3 
                 CONTROL (65% X) 
                 69.1 
                 76 
                 73.7 
                 63 
                 50.1 
                 86.71 
                 55 
                 59.1 
                 40.7 
                 44.47 
                 63.11 
               
               
                   
                 lNV (Rem Y) 
                 60.5 
                 70 
                 66.5 
                 56.6 
                 54 
                 82.23 
                 50 
                 46 
                 39.4 
                 46.24 
                 64.18 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 12 
               
               
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 X Viable 
                 X Viable 
               
               
                   
                   
                   
                   
                   
                   
                 Sort 
                 sperm sorted 
                 sperm sorted 
               
               
                   
                   
                   
                 0 h 
                 3 h 
                 Recovery 
                 Time 
                 per minute 
                 per minute 
               
               
                 BULL 
                 METHOD 
                 PURITY 
                 Viable 
                 Viable 
                 Million/Tube 
                 Minutes 
                 (0 h Viable) 
                 (3 h Viable) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 BULK SORT 
                 67% 
                 56% 
                 48% 
                 25.8 
                 37 
                 0.26 
                 0.22 
               
               
                   
                 INVERSE SORT 
                 58% 
                 44% 
                 37% 
                 24.0 
                 12 
                 0.52 
                 0.44 
               
               
                 2 
                 BULK SORT 
                 68% 
                 66% 
                 48% 
                 26.7 
                 40 
                 0.30 
                 0.22 
               
               
                   
                 INVERSE SORT 
                 60% 
                 53% 
                 40% 
                 25.4 
                 12 
                 0.67 
                 0.50 
               
               
                 3 
                 BULK SORT 
                 69% 
                 60% 
                 44% 
                 26.2 
                 35 
                 0.31 
                 0.23 
               
               
                   
                 INVERSE SORT 
                 61% 
                 54% 
                 46% 
                 24.4 
                 12 
                 0.67 
                 0.57