Modifying allocation of sample processing procedures for elite animals to maximize throughput

Abstract

Disclosed are methods of increasing production of sex selected semen for elite animals without a loss of plant efficiency. Assignment of elite animal ejaculate to a number of instruments so that a set number of cells is applied to each instrument is disclosed. Also disclosed is allowing the instrument to run until the ejaculate sample is exhausted, the instrument reports a dead percent of 25, or the sample has been processing for eight hours. Methods of pairing elite bulls with less valuable animals to preserve plant efficiency are also disclosed.

Description

PCT Application Docket No. GS-30-2023-W01MODIFYING ALLOCATION OF SAMPLE PROCESSING PROCEDURES FOR ELITE ANIMALS TO MAXIMIZE THROUGHPUTCross-Reference and Claim of Priority

[0001] This patent application claims the benefit of and priority to United States Provisional Patent Application No. 63 / 504,094, filed May 24. 2023, which is incorporated herein by reference in its entirety.Background

[0002] In the separation of various particles or cellular materials - for example, the processing of sperm into subpopulations of viable and motile sperm and non-viable or non- motile sperm, or the sexing of semen samples based on the sex chromosome (e.g., Y- chromosome bearing or X-chromosome bearing sperm cells) - the process is often a timeconsuming task, with severe volume restrictions. These restrictions can be complicated by variance in sample quality typical of biological samples, such as low cell concentration ejaculate samples from animals known to produce samples with low' concentration or other individual factors that may make a given ejaculate easier or harder to sex select.

[0003] Further, the ejaculate of different animals can have different economic values, depending on factors such as the genetic characteristics of the animal — for example, prices for sex selected Holstein bovine semen can range from $15 to $60 per straw, yet the processing time and yield per processing instrument is fixed. Conventional methods of allocating instrument time to a single bull for an approximately 8 hour collection allocates the same number of instruments to each animal at a set volume. This leads to wasted semen. However, if more instruments are allocated, there may not be enough instruments to process ejaculate of another animal — leading to a situation which results in either larger amounts of waste or idle instruments. Instrument runs are generally limited to a single 8 hour shift both for logistics of personnel shift changes and to allow sufficient time for other processing steps while still freezing the sample before it is 12 hours old. (Sample quality will degrade over time at room temperature if left for too long.)

[0004] Methods of allocating processing instruments that allow for less wasted semen of elite bulls while maintaining plant efficiency are needed.Summary

[0005] In one embodiment, what is provided is a method of optimizing efficiency of sex selection for a given semen sample comprising: obtaining an ejaculate from a non-human mammal; measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; dividing the number of cells by a set number of cells per instrument to determine how many sample processing instruments are needed to process the ejaculate; preparing the ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments calculated in a prior step; dividing leftover ejaculate between the samples prepared in a prior step so that all ejaculate is spread evenly between the processing instruments; and processing the sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced.

[0006] In various embodiments, the method further comprises taking unprocessed sample from an instrument that was stopped because it read a dead percent of 25 or higher and consolidating it with a sample from a still running instrument.

[0007] In various embodiments, the set number of cells per instrument is 1.0 billion cells to 3.0 billion cells.

[0008] In various embodiments, the set number of cells per instrument is 1.75 billion cells.

[0009] In various embodiments, the non-human mammal is an elite non-human mammal.

[0010] In various embodiments, the method further comprises sacrificing instrument time for ejaculate from a non-elite non-human mammal in order to continue processing ejaculate from the elite non-human mammal.

[0011] In various embodiments, the method further comprises pairing the elite non-human mammal with a non-elite non-human mammal for prioritizing the elite non-human mammal’s ejaculate over the non-elite non-human mammal’s ejaculate.

[0012] In various embodiments, the method further comprises freezing the ejaculate within 16 hours of a prior step

[0013] In various embodiments, what is provided is a straw of processed semen processed according to a method recited hereinabove.

[0014] In another embodiment, what is provided is a method of processing an ejaculate from an elite bull comprising: obtaining an ejaculate from a mammal elite bull: measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; dividing the number of cells by a set number of cells per instrument to determinehow many sample processing instruments are needed to process the ejaculate; preparing ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments calculated in a prior step; dividing leftover ejaculate between the samples prepared in a prior step so that all ejaculate is spread evenly between the processing instruments; and processing the sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced.

[0015] In various embodiments, the method further comprises taking unprocessed sample from an instrument that was stopped because it read a dead percent of 25 or higher and consolidating it with a sample from a still running instrument.

[0016] In various embodiments, the set number of cells per instrument is 1.25 billion cells to 2.0 billion cells.

[0017] In various embodiments, the set number of cells per instrument is 1.75 billion cells.

[0018] In various embodiments, the method further comprises sacrificing instrument time for ejaculate from a non-elite bull in order to continue processing ejaculate from the elite bull.

[0019] In various embodiments, the method further comprises pairing the elite bull with a non-elite bull for prioritizing the elite bull’s ejaculate over the non-elite bull’s ejaculate.

[0020] In various embodiments, the method further comprises freezing the ejaculate within 16 hours of a prior step.

[0021] In various embodiments, what is provided is a straw of semen processed according to a method recited hereinabove.

[0022] In another embodiment, what is provided is a method of operating a sex selected semen production plant comprising: obtaining an ejaculate from a non-human mammal; measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; dividing the number of cells by a set number of cells per instrument to determine how many sample processing instruments are needed to process the ejaculate; preparing ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments calculated in a prior step; dividing leftover ejaculate between the samples prepared in a prior step so that all ejaculate is spread evenly between the processing instruments; and processing the sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced.

[0023] In various embodiments, the method further comprises taking unprocessed sample from an instrument that was stopped because it read a dead percent of 25 or higher and consolidating it with a sample from a still running instrument.

[0024] In various embodiments, the set number of cells per instrument is 1.25 billion cells to 2.0 billion cells.

[0025] In various embodiments, the set number of cells per instrument is 1.75 billion cells.

[0026] In various embodiments, the method further comprises wherein the non-human mammal is an elite non-human mammal and further comprising sacrificing instrument time for ejaculate from a non-elite non-human mammal in order to continue processing ejaculate from the elite animal.

[0027] In various embodiments, the method further comprises pairing the elite non-human mammal with the non-elite non-human mammal for prioritizing the elite non-human mammal’s ejaculate over the non-elite non-human mammal’s ejaculate.

[0028] In various embodiments, the method further comprises freezing the ejaculate within 16 hours a prior step.

[0029] In various embodiments, what is provided is a straw of semen processed according to a method recited hereinabove.

[0030] In another embodiment, what is provided is a method of automatically increasing sex selected semen production for an elite animal comprising: obtaining an ejaculate from an elite animal, placing the ejaculate in a container, and placing a tag that indicates animal’s identity on the container; reading the tag; using information from the tag to determine identity and value of the animal; allocating system processing time based on the value of the animal; if the animal is an elite animal: assigning enough processing instruments to the ejaculate to process all of the ejaculate based on a constant number of cells per instrument; pairing the ejaculate with ejaculate from a low value animal, wherein the ejaculate from the low value animal is assigned to any open processing instruments that remain; and running each of the processing instruments until the ejaculate has been exhausted, the individual processing instrument reads a dead percent of 25, or until 8 hours have elapsed; or if the animal is a low value animal, assigning instruments to the ejaculate that are open while an elite animal is run on most of the instruments and sex selecting the semen.

[0031] In another embodiment, what is provided is a system for increasing sex selected semen production for an elite animal comprising: a bank of one or more sex selection instruments, the instruments configured to run a sample to completion, until a reading of deadpercent of 25 or higher is obtained, or until 8 hours have elapsed after system activation; and a computer processor configured to read data from the instruments.

[0032] In another embodiment, what is provided is a method of optimizing efficiency of sex selection for a given semen sample comprising: obtaining an ejaculate from an elite nonhuman mammal; measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; dividing the number of cells by a set number of cells per instrument to determine how many sample processing instruments are needed to process the ejaculate; preparing the ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments calculated in a prior step; dividing leftover ejaculate between the samples prepared in a prior step so that all ejaculate is spread evenly between the processing instruments; processing the sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced; and sacrificing instrument time for ejaculate from a non-elite non-human mammal in order to continue processing ejaculate from the elite non-human mammal.

[0033] In various embodiments, the method further comprises taking unprocessed sample from an instrument that was stopped because it read a dead percent of 25 or higher and consolidating it with a sample from a still running instrument.

[0034] In various embodiments, the set number of cells per instrument is 1.0 billion cells to 3.0 billion cells.

[0035] In various embodiments, the set number of cells per instrument is 1 .75 billion cells.

[0036] In various embodiments, the method further comprises pairing the elite non-human mammal with a non-elite non-human mammal for prioritizing the elite non-human mammal's ejaculate over the non-elite non-human mammal’s ejaculate.

[0037] In various embodiments, the method further comprises freezing the ejaculate within 16 hours of a prior step.

[0038] In various embodiments, what is provided is a straw of semen processed according to a method recited hereinabove.

[0039] In another embodiment, what is provided is a method of processing an ejaculate from an elite bull comprising: obtaining an ejaculate from an elite bull; measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; dividing the number of cells by a set number of cells per instrument to determine how many sample processing instruments are needed to process the ejaculate; preparing ejaculate to form at least one sample to be run on the processing instruments and dividing the at least onesample into collection tubes equal to the number of processing instruments calculated in a prior step; dividing leftover ejaculate between the samples prepared in a prior step so that all ejaculate is spread evenly between the processing instruments; processing the sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced; and sacrificing instrument time for ejaculate from a non-elite bull in order to continue processing ejaculate from the elite bull.

[0040] In various embodiments, the method further comprises taking unprocessed sample from an instrument that was stopped because it read a dead percent of 25 or higher and consolidating it with a sample from a still running instrument.

[0041] In various embodiments, the set number of cells per instrument is 1.25 billion cells to 2.0 billion cells.

[0042] In various embodiments, the set number of cells per instrument is 1.75 billion cells.

[0043] In various embodiments, the method further comprises pairing the elite bull with a non-elite bull for prioritizing the elite animal's ejaculate over the non-elite animal's ejaculate.

[0044] In various embodiments, the method further comprises freezing the ejaculate within 16 hours of a prior step.

[0045] In various embodiments, what is provided is a straw of semen processed according to a method recited hereinabove.

[0046] In various embodiments, what is provided is method of inseminating a female nonhuman animal with sex selected sperm from an elite non-human animal, the method comprising: obtaining an ejaculate from the elite non-human mammal; measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; dividing the number of cells by a set number of cells per instrument to determine a number of processing instruments needed to process the ejaculate; preparing the ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments; dividing leftover ejaculate so that all ejaculate is spread evenly between the processing instruments; processing the at least one sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced to generate processed sample; producing at least one straw of semen from the processed sample; and inseminating the female non-human animal with the at least one straw of semen.

[0047] In various embodiments, what is provided is a method of fertilizing an oocyte of a female non-human animal with sex selected sperm from an elite non-human animal in an in- vitro fertilization process, the method comprising: obtaining an ejaculate from the elite non- human mammal; measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; dividing the number of cells by a set number of cells per instrument to determine a number of processing instruments needed to process the ejaculate; preparing the ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments; dividing leftover ejaculate so that all ejaculate is spread evenly between the processing instruments; processing the at least one sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced to generate processed sample; producing at least one straw of semen from the processed sample; and fertilizing the oocyte of the female non- human animal with the at least one straw of semen in an in-vitro fertilization process.

[0048] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.Brief Description of the Drawings

[0049] In order to facilitate a full understanding of the present invention, reference is now made to the accompanying drawings, in which like elements are referenced with like numerals. These drawings should not be construed as limiting the present invention but are intended to be exemplary and for reference.

[0050] FIG. 1 depicts a flow chart of internal quality control for ejaculate samples

[0051] FIG. 2 depicts a flow chart for general sample preparation

[0052] FIG. 3 depicts a system for use in the semen allocation process of the present teachings.Detailed Description

[0053] The systems and methods herein will now' be described in more detail with reference to exemplary embodiments as shown in the accompanying drawings. While the present invention is described herein with reference to the exemplary embodiments, it shouldbe understood that the systems and methods herein are not limited to such exemplary embodiments. Those possessing ordinary skill in the art and having access to the teachings herein will recognize additional implementations, modifications, and embodiments, as well as other applications for use of the invention, which are fully contemplated herein as within the scope of the systems and methods as disclosed and claimed herein, and with respect to which the systems and methods herein could be of significant utility.

[0054] In general, a method of the present teachings comprises allocating a sufficient number of instruments to run an ejaculate from a non-human mammal such that a constant number of cells is run on each instrument and then running each instrument until either the ejaculate sample runs out, the instrument reads a dead percent of 25, or 8 hours have elapsed since sample processing commenced. As discussed supra, the 8 hour time frame is important in order to contain a collection within a single shift and to ensure that samples are not kept at room temperature longer than 12 hours, as sample quality begins to degrade after that time as cells begin to die off. Typically, the industry' previously allowed only 6 hours of instrument run time in order to allow sufficient time for sample collection and pre- and post-instrument processing while still keeping the semen at room temperature no more than 12 hours. This time limit would frequently result in wastage of unprocessed sample. In the instant teachings, extra time is allowed for collection processing in order to not waste ejaculate from elite bulls, even if this means needing to rush post-sample processing in order to still freeze the sex selected semen within 12 hours of collection.

[0055] Further, typical standard procedures call for stopping the instrument well before the sample tube has been emptied to over 1 ml, because when the sample volume in the sample tube reaches a volume of 1 ml or less it allows for the possible introduction of air into the system. In the method of the present teachings, recovery’ of as much sample as possible is prioritized over possible instrument downtime, and the instrument is run until the collection tube contains as close to 1 ml as possible (without allowing the volume to go under 1 ml). Instruments suitable for use with the present teachings include sex selection instruments that make use of electromagnetic radiation and a photodetector, electrical detection and flow sorting (e.g. bubble / piezo sorting), laser ablation, holographic and optical trapping, or any other method of sex selection known in the art. A dead percent is a percentage of evaluated cells that are determined to be dead, non-motile, or otherwise non-viable based upon analysis by a sexing cytometer. In various configurations, unprocessed sample that remains once the collection tubes have been prepared is divided among the collection tubes so that the entire sample is processed.

[0056] An exemplary procedure for sample processing includes the collected semen being subjected to internal quality control, sample preparation, loading onto the instruments, room temperature centrifugation, and then cry opreservation of the skewed semen. Internal Quality Control (FIG. 1) can comprise checking in the ejaculate, cryovial sampling (obtaining a sample for freezing), determination of the raw volume of the ejaculate, GTLS addition (antibiotics gentamycin, tylosin, lincomycin and spectinomycin), measurement of the sperm concentration of the ejaculate, measurement of the sperm mobility and sperm morphology, and pooling of the raw ejaculate. A preferred method of measuring sperm mobility can be Computer Assisted Sperm Analysis (CASA); various computer analysis software is commercially available and known in the art. Sample preparation can comprise stain calculation, aliquoting of pooled raw ejaculate — a traditional sample prep calls for the refrigeration of remaining ejaculate, a step that the present teachings seeks to eliminate, addition of stain TALP and then Hoechest stain, mixing the stain in, incubation, addition of Red TALP, mixing, and then aliquot and filtering of the incubated raw ejaculate. The ejaculate can then be applied to the instruments. See FIG. 2. Filtering the sample can comprise the use of glass wool, separating live cells via a PERCOLL® (Cytiva, Marlborough, MA)) gradient, passing the cells through a sufficiently fine filter (e.g., a 10-25 micron filter), or by removing dead cells in the sample via a process utilizing magnetic beads. The sample is then sex selected on an instrument or pod of instruments, the samples are then centrifuged at room temperature, placed in extender or other cry opreservation medium, and then frozen until ready for use.

[0057] In various embodiments, the non-human mammal can be a bovine, a swine, an ovid, or an equine. In some configurations, the non-human mammal can be a bull, a pig, or a horse. In some configurations, the bull can be a Bos taurus, a Bos indicus. or aBubcihis bubalis. In various configurations, the bull can be a Holstein, a Jersey, a Brown Swiss, an Ayrshire, a Grassland, a Norw egian Red, an Angus, a Red Angus, a Simmental, a Brangus, a Charolais, a Gebvieh, a Hereford, or a Waygyu. In various configurations, the bull can be a Zeebu.

[0058] In various embodiments, unprocessed sample from an instrument that is stopped or idle because it reads a dead percent over 25 can be consolidated with a sample or several samples that are on still running instruments so that the entire sample can still be processed.

[0059] The number of cells in an ejaculate sample can be from 1.0 billion to 3.0 billion. The number of cells can be about 1.0 billion, about 1.25 billion, about 1.5 billion, about 1.75 billion, about 2.0 billion, about 2.25 billion, about 2.5 billion, about 2.75 billion, or about 3.0billion cells. In various configurations, 1.75 billion cells can be applied to or processed by each instrument in an 8 hour processing run.

[0060] In various configurations, the non-human mammal can be an elite animal. As used herein, elite animals are defined as animals that have estimated breeding values in the top 10% of animals for their age and breed. The estimated breeding value can be based on genomic traits or traits of the animal’s offspring, such as, but without limitation, daughter proofs with dairy cattle. Alternatively, or in addition, elite animals may command a price in the top 10% of price ranges prepared by the processing plant.

[0061] In various configurations, a non-elite animal can be paired with an elite animal so that the non-elite animal can be run on the instruments not being used by the elite animal. Production of sexed semen from the non-elite animal may therefore be sacrificed in favor of the elite animal. Instruments may be changed over to the non-elite animal as they finish processing sample from the elite animal (or read at least 25 dead percent) in order to maintain plant efficiency. The same number of straws can be made whether the processing instruments are divided evenly between the animals or are divided according to the value of the animal: by dividing the instruments such that the entirety of the elite animal’s ejaculate is processed, more valuable product is produced and profit is maximized. The ejaculate of lower value animals (either in terms of estimated breeding value or other valuable traits or merely in terms of price) can be sacrificed (or sold as conventional semen instead of sex selected semen) in order to produce more sex selected semen from the elite animal.

[0062] In various embodiments, after the semen has been sex selected, it can be packaged into straws; semen straws are commonly used in artificial insemination for both livestock and humans.

[0063] In various embodiments, the semen can be frozen after sex selection. Semen is tj pically cryopreserved and stored in liquid nitrogen until ready for use. There are various methods of freezing known in the art, including flash freezing, slow freezing, and vitrification. Such cry opreservation preserves the motility and viability of the sperm cells and maintains their ability to fertilize an ovum.

[0064] It is expected that assigning resources according to the present teachings can increase the yield of straws by 10-50% for an elite bull.

[0065] There are several methods of sex selecting semen know n in the art. Commercial sexed semen can be produced by staining sperm with Hoechst 33342. a dye that penetrates live cells, binds stoichiometrically to DNA, and releases a fluorescent signal which quantitatively reflects total cellular DNA content when binding has been driven to completion(See, Lalande, M.E., et al., The Journal of Experimental Medicine, 1980, 151,12-19; Lalande, M.E., et al., The Journal of Histochemistry and Cytochemistry, 1979. 27. 394-397; Loken, M.R., The Journal of Histochemistry and Cytochemistry, 1980, 28, 36-39; and Loken, M.R., Cytometry, 1980, 1, 136-142). Custom, high-throughput sexing cytometers can discriminate the roughly 4% difference in total DNA content between X and Y chromosome containing sperm (due to the relative size of the sex chromosomes) by quantifying Hoechst 33342 emission fluorescence (Moruzzi. J.F., J. Reprod. Fertil., 1979, 57319-323; van Munster. E.B.. et al.. Cytometry, 1979, 35, 125-128; and Johnson, L. A., Reprod. Fertil. Dev., 1995, 7, 893- 903). Once the sex of the sperm is determined, sperm can either be segregated into separate containers based on sex chromosome (Johnson, LA. and Welch, G.R., Theriogenology, 1999, 52, 1323-1341), or the undesired cell population can be eliminated by laser-ablation (Faust, M.A., et al.. Journal of Animal Science, 2016, 94, 544-544). The maximum achievable sex skew (enrichment for either the X or Y population) can depend upon how well the cells are stained, alignment of the pancake shaped sperm head in the optical detection plane, and speed at which sperm flow through the cytometers (Johnson, L.A. and Welch, G.R., Theriogenology . 1999. 52. 1323-1341). Custom cytometers are described in. for example and without limitation, US Patent 10,928,298.

[0066] Differentiation Technologies

[0067] The term “sexing"’ or “sex selection” as used herein refers to any process that selects X-chromosome bearing or Y-chromosome bearing sperm cells from a population that comprises a mixture of both X-chromosome and Y-chromosome bearing sperm cells. The sperm cell population can be raw ejaculate, or any other mixture of sperm cells. Sex selection can be performed by any method known in the art, such as, without limitation, droplet sorting, mechanical sorting, acoustic, electrophoretic, diversion, radiation pressure, optical tweezers, laser ablation, etc. Sex selection or sexing may also include bulk sexing methods such as the instant teachings described hereinbelow^. In general, sexing of sperm involves differentiating the sperm cells in a population to select or selectively enrich for either live X- chromosome bearing sperm cells or live Y-chromosome bearing sperm cells. Typically, this is accomplished by staining the sperm cells with a dye that binds the DNA of the cells, such as a DNA intercalating dye, permitting discrimination on the basis of DNA content: X- chromosome bearing sperms cells have roughly 3-4% more DNA than Y-chromosome bearing sperm cells. Other methods for differentiating sperm cells are known in the art, including through the detection or binding of surface markers or through detecting differences in volume or mass of the sperm cells. In applications where sperm cells are sexedusing a DNA dye, the dye is typically the fluorescent dye Hoechst 33342, although other nontoxic, biocompatible dyes are known in the art and may be used.

[0068] Different sexes of livestock are preferred depending on the application — for example, only female dairy cattle produce milk; however, for beef production, male cattle have greater muscle mass. Therefore, it is desirable to select sperm cells based on their chromosomal content: X-chromosome bearing sperm to produce female offspring and Y- chromosome bearing sperm to produce male offspring. Clean-up of the semen prior to sex selection increases the efficiency of the sexing process because the dead cells have already been removed, so that more live, functional sperm are run through the machine and processed. Eligibility, as used herein, is the percentage of sperm that are capable of being effectively discriminated - essentially the proportion of all cells from a raw ejaculate that (1) are alive, (2) took up the stain, (3) are not clumped, and (4) are oriented properly to allow effective detection; in essence, the cells that fluoresce well enough to be able to resolve whether they have an X chromosome or a Y chromosome. In other words, eligibility is the percentage of live, motile cells that are collected after the discrimination process as a percentage of cells that were processed.

[0069] A number of techniques, directly or indirectly based on differences in size, mass, or densi have been disclosed for use in discriminating X-chromosome-bearing from Y- chromosome-bearing spermatozoa. The most commonly used methods utilize flow cytometer techniques for the sex-skewing of spermatozoa and generally involve staining spermatozoa with a fluorochrome; the stained spermatozoa are made to flow in a narrow7stream or band passing by an excitation or irradiation source such as a laser beam. As stained particles or cells pass through the excitation or irradiation source, the fluorochrome emits fluorescent light. An optical lens assembly collects the fluorescent light, focused on a detector — typically a photomultiplier tube or avalanche photodiode — that generates and multiplies an electronic signal, which may then be analyzed by an analyzer. The data can then be displayed as multiple or single parameter chromatograms or histograms. The number of cells and fluorescence per cell may be used as coordinates. Detection of the two populations provides the opportunity to skew the population towards one population or the other, including by sorting X- and Y-chromosome bearing cells into separate populations, enriching the population for either X- or Y-chromosome bearing cells, or selectively removing, destroying, or otherwise inactivating either X- or Y-chromosome bearing cells in a population. However, with respect to this type of technology a variety of problems remain unresolved, and ensuringthat chromosomal differentiation techniques yield highly purified populations (e.g. X- chromosome bearing or Y-chromosome bearing sperm cells) can be difficult.

[0070] Cytometric Sex Selection. The stained, filtered sample is then run on proprietary sexing cytometers. The sample throughput is adjusted to 17,500 cells / sec-25,000 cells / sec, or may be adjusted to 10,000 cells / sec, 13,000 cells / sec, 13,500 cells / sec, 15,000 cells / sec, 17,500 cells / sec, 20,000 cells / sec, 23,500 cells / sec, 25,0000 cells / sec, 28.000 cells / sec. 30.000 cells / sec. 35,000 cells / sec, 40,000 cells / sec, 45,000 cells / sec, or 50.000 cells / sec or about or between any of these throughput values, and the detection and kill lasers are focused. To confirm proper laser focus, kill count assessments are performed before collecting sex skewed sample. A successful kill count has a population that is > 75% dead and > 95% sliced with at least 200 cells being counted. If an instrument cannot achieve the above metrics, the instrument is not used to collect sex selected semen and / or the sample may be discarded. After a successful kill count, a gate is placed to collect the desired cells, which may be the Y chromosome bearing cells or the X chromosome bearings cells. In the instance of collection for X chromosome bearing cells, these cells are in the cell population with the brighter Hoechst 33342 fluorescence as measured with a 355 nm wavelength excitation laser. Cytometer performance metrics are collected 15 minutes after instrument set up, and 15 minutes after the placement of the last sample collection tube, including the height of the Y - peak, the height of the X-peak, the height of the trough from the histogram of events per emitted fluorescent intensity, gated %. and dead %.

[0071] Peak to trough ratio is a measure of the distance from the peak to the trough of a histogram for unselected semen where X is the height of the of the X peak, Y is the height of the Y peak, and trough is the height of the trough, the ratio is calculated as [(X + Y) / 2]] / trough. Higher Peak:Trough ratios lead to better discrimination between X- and Y- bearing sperm.

[0072] The sample is run to collect between 300 and 400 rnL of sex skewed sample, the composition of which is approximately 17% TRIS A buffer, 80% sheath fluid, and 2% cell sample. The sample is collected in 50 mL conical tubes containing 1-5 mL of TRIS A, and each tube is filled to a max volume of 30 mL before being replaced. For conical tubes containing less than 5 mL of TRIS A, a remaining volume of TRIS A may be added to the conical tube after centrifugation to reach a total volume of TRIS A of about 5 mL. After the requisite total volume is collected, sexed sperm is centrifuged at room temperature at 2400 x g for 10 minutes. The supernatant is aspirated and discarded to reach a 1 mL pellet volume. 17 pL of GTLS antibiotic solution is added to each pellet after resuspension. The tubes arethen placed in beakers filled with 150 mL of room temperature water, to prevent cold shock, and are then transferred to a 4° C cold room.

[0073] Typically, cytometric instruments are organized into pods of two to four instruments. A sample can be run over 2-3 pods of 6 to 12 instruments.

[0074] Sample Analysis. Analysis of samples is performed on the instrument during sex selection. Both eligibility’ and dead percent are measured. Dead percent is calculated from the peak of dead cells found in the cytometry of the instrument and reflects a measure of dead cells relative to total cells in the sample. Additionally, an aliquot of a processed sample may be removed and evaluated under a microscope to confirm the dead cell percentage.

[0075] With reference now to FIG. 3, what is provided is a block diagram of a system 100 for processing an elite non-human mammal semen 102. Sample is collected from a nonhuman mammal in a bam (300), and then the sample is brought into a facility (100) for processing. The sample is processed (110), and then loaded onto a pod (120) of two tables (122 / 124) of instruments such as 123. Data from the instruments are gathered by a computer (140) and stored on the computer, on a local server (150). or on a cloud drive (400). After the sample has been processed to exhaustion, it is frozen and stored in liquid nitrogen (130).

[0076] Examples

[0077] The present teachings including descriptions provided in the Examples that are not intended to limit the scope of any claim or embodiment. The following non-limiting examples are provided to further illustrate the present teachings. Those of skill in the art. in light of the present disclosure, will appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.

[0078] Example 1

[0079] This example illustrates a method of allocating resources for the processing of an elite bull according to the present teachings.

[0080] Ejaculate will be collected from an elite bull named Venture, who is highly ranked in his age group. The ejaculate will be extended with Xdextender, run through quality control checks, and then the concentration and volume will be measured. The extended semen may have a volume of 10 ml and a concentration of 1600 million cells per milliliter (M / ml). The extended ejaculate will be split into ten samples of approximately 1.75 billion sperm cells. Semen leftover after these samples have been created will be distributed evenly between the samples. The samples will be assigned to selection instruments 1-10. After 4 hours, instrument 1 may read a dead percent of 27. The instrument will then be stopped, andremaining sample will be redistributed over instruments 2-10. Instrument 3 might stop after 5 hours, having exhausted the sample. Instrument 7 might read a dead percent of 30 after 6 hours, and the remaining sample would be distributed between instruments 2, 4, 5, 6, and 8- 10. Instruments 2, 4, and 5 might stop between hours 6 and 7, having also exhausted the sample. Instruments 6 and 8-10 might stopped after 8 hours of processing. The total yield of straws may be 25% higher than if the instruments were run according to traditional methods.

[0081] Example 2

[0082] This example illustrates a method of allocating resources for the processing of an elite bull according to the present teachings.

[0083] Raw ejaculate is collected from one or more bulls at a collection site. The raw ejaculate collected from the bulls is extended in an extender solution such as one comprising water. Tris buffer, a protein source (e.g., egg yolk), glycerol, and an antibiotic cocktail such as GTLS (gentamycin, tylosin. lincomycin, and spectinomycin). The ejaculate is extended with the extender in an approximately 2: 1 ratio. After collection, the extended ejaculate is transferred to a facility for processing, such as sexing for sex skew.

[0084] At the processing facility, the extended ejaculate is analyzed for motility, is pooled and concentrated, and evaluated for morphology. Samples are then prepared for processing on a sexing instrument. The samples are prepared by first using a magnetic bead solution to remove dead cells from the samples. The sample processed by the magnetic bead solution is then aliquoted in to an equal number of 4 mL sample tubes, with no more than 4 mL per tube. If excess sample remains after aliquoting, the sample is evenly distributed among the already prepared tubes.

[0085] Instruments at the processing facility are scheduled to process the prepared samples, and any other samples received by the facility at the same time. Instruments are scheduled to reduce the number of “bull changes” or sample changes on an instrument during its typical 8-hour runtime. Instruments may refer to a single instrument or to a cluster or pod of 2-8 instruments that are managed by a single operator. A single operator may also manage several instruments or pods of instruments.

[0086] For example, if samples are collected from 6 bulls and there are 6 instruments, the instruments and samples are scheduled in a way to maximize the collection from the highest value samples. Additionally, the samples are scheduled on the instruments to maximize the number of straws of semen that can be produced per billion cells collected. The number of straws per billion cells collected may range from 13 straws / billion to 55 straws / billion. with the target being the higher end of the range. Reducing the number of bull changes on aninstrument and running instruments at a lower processing rate for higher value samples can improve the number of straws collected per billion cells.

[0087] In this example, assume that bull 1 is the highest value, bulls 2-5 are relatively equal value, and that bull 6 is the lowest value. Also assume that the volume of material or volume of sample is not equal for all 6 bulls. In this example, bull 1 is scheduled on instrument such that the entire volume of sample can be processed. However, this cannot be done on a single instrument in an 8-hour processing period. Therefore, the sample for bull 1 is scheduled on instrument 1 for the entirety of its processing period, and for instrument 2 for the first portion of its processing period. The second portion of the processing period for instrument 2 is used by either bull 6 or a reserve sample. Bull 2 is assigned to instrument 3, but does not have enough sample to use the entirety of the processing period, and therefore, as with instrument 2, bull 6 or a reserve bull are used for the remaining time for instrument 3. Bulls 3 and 4 are assigned to instruments 4 and 5 respectively for the entire processing period duration for those instruments. Bull 5 is assigned to instrument 6, but only has enough sample for a portion of the processing period. Therefore, as with instruments 2 and 3, either bull 6 or a reserve sample is processed using the remaining instrument time for instrument 6 for the processing period.

[0088] In this manner of scheduling, for 6 instruments there are a maximum of 3 bull changes and the entire volume of the highest value samples is able to be processed within the 12-hour processing window without sample waste.

[0089] After processing on instrument, the samples are prepared for cryogenic freezing and are then frozen in straws for storage and distribution. The frozen samples are then thawed and used for artificial insemination or for in-vitro fertilization.

Claims

Listing of the ClaimsWhat is claimed is:

1. A method of optimizing efficiency of sex selection for a given semen sample comprising: i) obtaining an ejaculate from a non-human mammal; ii) measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; iii) dividing the number of cells by a set number of cells per instrument to determine how many sample processing instruments are needed to process the ejaculate; iv) preparing the ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments calculated in step iii); v) dividing leftover ejaculate between the samples prepared in step iv) so that all ejaculate is spread evenly between the processing instruments; and vi) processing the sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced.

2. The method according to claim 1, further comprising: vii) taking unprocessed sample from an instrument that was stopped because it read a dead percent of 25 or higher and consolidating it with a sample from a still running instrument.

3. The method according to claim 1 wherein the set number of cells per instrument is 1.0 billion cells to 3.0 billion cells.

4. The method according to claim 1 wherein the set number of cells per instrument is 1.75 billion cells.

5. The method according to claim 1, wherein the non-human mammal is an elite non-human mammal.

6. The method according to claim 5, further comprising sacrificing instrument time for ejaculate from a non-elite non-human mammal in order to continue processing ejaculate from the elite non-human mammal.

7. The method according to claim 5, further comprising pairing the elite non-human mammal with a non-elite non-human mammal for prioritizing the elite non-human mammal’s ejaculate over the non-elite non-human mammal’ s ejaculate.

8. The method according to claim 1, further comprising freezing the ejaculate within 16 hours of step i).

9. A straw of semen processed according to claims 1-8.

10. A method of processing an ejaculate from an elite bull comprising: i) obtaining an ejaculate from a mammal elite bull; ii) measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; iii) dividing the number of cells by a set number of cells per instrument to determine how many sample processing instruments are needed to process the ejaculate; iv) preparing ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments calculated in step iii); v) dividing leftover ejaculate between the samples prepared in step iv) so that all ejaculate is spread evenly between the processing instruments; and vi) processing the sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced.

11. The method according to claim 10, further comprising: vii) taking unprocessed sample from an instrument that was stopped because it read a dead percent of 25 or higher and consolidating it with a sample from a still running instrument.

12. The method according to claim 10, wherein the set number of cells per instrument is 1.25 billion cells to 2.0 billion cells.

13. The method according to claim 10, wherein the set number of cells per instrument is 1.75 billion cells.

14. The method according to claim 10, further comprising sacrificing instrument time for ejaculate from a non-elite bull in order to continue processing ejaculate from the elite bull.

15. The method according to claim 10, further comprising pairing the elite bull with a nonelite bull for prioritizing the elite bull’s ejaculate over the non-elite bull’s ejaculate.

16. The method according to claim 10, further comprising freezing the ejaculate within 16 hours of step i).

17. A straw of semen processed according to claims 10-16.

18. A method of operating a sex selected semen production plant comprising: i) obtaining an ejaculate from a non-human mammal; ii) measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; iii) dividing the number of cells by a set number of cells per instrument to determine how many sample processing instruments are needed to process the ejaculate; iv) preparing ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments calculated in step iii); v) dividing leftover ejaculate between the samples prepared in step iv) so that all ejaculate is spread evenly between the processing instruments; and vi) processing the sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced.

19. The method according to claim 18, further comprising: vii) taking unprocessed sample from an instrument that was stopped because it read adead percent of 25 or higher and consolidating it with a sample from a still running instrument.

20. The method according to claim 19, wherein the set number of cells per instrument is 1.25 billion cells to 2.0 billion cells.

21. The method according to claim 19, wherein the set number of cells per instrument is 1.75 billion cells.

22. The method according to claim 19, wherein the non-human mammal is an elite nonhuman mammal and further comprising sacrificing instrument time for ejaculate from a nonelite non-human mammal in order to continue processing ejaculate from the elite animal.

23. The method according to claim 22, further comprising pairing the elite non-human mammal with the non-elite non-human mammal for prioritizing the elite non-human mammal’s ejaculate over the non-elite non-human mammal’s ejaculate.

24. The method according to claim 18, further comprising freezing the ejaculate within 16 hours of step i).

25. A straw of semen processed according to claims 18-24.

26. A method of automatically increasing sex selected semen production for an elite animal comprising: obtaining an ejaculate from an elite animal, placing the ejaculate in a container, and placing a tag that indicates animal’s identity on the container; reading the tag; using information from the tag to determine identity and value of the animal; allocating system processing time based on the value of the animal; if the animal is an elite animal: assigning enough processing instruments to the ejaculate to process all of the ejaculate based on a constant number of cells per instrument; pairing the ejaculate with ejaculate from a low value animal, wherein the ejaculate from the low value animal is assigned to any open processing instruments thatremain; and running each of the processing instruments until the ejaculate has been exhausted, the individual processing instrument reads a dead percent of 25, or until 8 hours have elapsed; or if the animal is a low value animal, assigning instruments to the ejaculate that are open while an elite animal is run on most of the instruments and sex selecting the semen.

27. A system for increasing sex selected semen production for an elite animal comprising: a bank of one or more sex selection instruments, the instruments configured to run a sample to completion, until a reading of dead percent of 25 or higher is obtained, or until 8 hours have elapsed after system activation; and a computer processor configured to read data from the instruments.

28. A method of optimizing efficiency of sex selection for a given semen sample comprising: i) obtaining an ejaculate from an elite non-human mammal; ii) measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; iii) dividing the number of cells by a set number of cells per instrument to determine how many sample processing instruments are needed to process the ejaculate; iv) preparing the ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments calculated in step iii); v) dividing leftover ejaculate between the samples prepared in step iv) so that all ejaculate is spread evenly between the processing instruments; vi) processing the sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced; and vii) sacrificing instrument time for ejaculate from a non-elite non-human mammal in order to continue processing ejaculate from the elite non-human mammal.

29. The method according to claim 28, further comprising: viii) taking unprocessed sample from an instrument that w as stopped because it read a dead percent of 25 or higher and consolidating it with a sample from a still running instrument.

30. The method according to claim 28 wherein the set number of cells per instrument is 1.0 billion cells to 3.0 billion cells.

31. The method according to claim 28 wherein the set number of cells per instrument is 1.75 billion cells.

32. The method according to claim 28, further comprising pairing the elite non-human mammal with a non-elite non-human mammal for prioritizing the elite non-human mammal’s ejaculate over the non-elite non-human mammal’s ejaculate.

33. The method according to claim 28, further comprising freezing the ejaculate within 16 hours of step i).

34. A straw of semen processed according to claims 28-33.

35. A method of processing an ejaculate from an elite bull comprising: i) obtaining an ejaculate from an elite bull; ii) measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; iii) dividing the number of cells by a set number of cells per instrument to determine how many sample processing instruments are needed to process the ejaculate; iv) preparing ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments calculated in step iii); v) dividing leftover ejaculate between the samples prepared in step iv) so that all ejaculate is spread evenly between the processing instruments; vi) processing the sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out. or 8 hours have elapsed since processing commenced; and vii) sacrificing instrument time for ejaculate from a non-elite bull in order to continue processing ejaculate from the elite bull.

36. The method according to claim 35, further comprising: viii) taking unprocessed sample from an instrument that was stopped because it read a dead percent of 25 or higher and consolidating it with a sample from a still running instrument.

37. The method according to claim 35, wherein the set number of cells per instrument is 1.25 billion cells to 2.0 billion cells.

38. The method according to claim 35, wherein the set number of cells per instrument is 1.75 billion cells.

39. The method according to claim 35, further comprising pairing the elite bull with a nonelite bull for prioritizing the elite animal’s ejaculate over the non-elite animal’s ejaculate.

40. The method according to claim 35, further comprising freezing the ejaculate within 16 hours of step i).

41. A straw of semen processed according to claims 35-40.

42. A method of inseminating a female non-human animal with sex selected sperm from an elite non-human animal, the method comprising: obtaining an ejaculate from the elite non-human mammal; measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; dividing the number of cells by a set number of cells per instrument to determine a number of processing instruments needed to process the ejaculate; preparing the ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments; dividing leftover ejaculate so that all ejaculate is spread evenly between the processing instruments; processing the at least one sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced to generate processed sample;producing at least one straw of semen from the processed sample; and inseminating the female non-human animal with the at least one straw of semen.

43. A method of fertilizing an oocyte of a female non-human animal with sex selected sperm from an elite non-human animal in an in-vitro fertilization process, the method comprising: obtaining an ejaculate from the elite non-human mammal: measuring volume and cell concentration of the ejaculate to calculate total number of cells in the ejaculate; dividing the number of cells by a set number of cells per instrument to determine a number of processing instruments needed to process the ejaculate; preparing the ejaculate to form at least one sample to be run on the processing instruments and dividing the at least one sample into collection tubes equal to the number of processing instruments; dividing leftover ejaculate so that all ejaculate is spread evenly between the processing instruments; processing the at least one sample on the processing instruments until each instrument reads a dead percent of 25 or higher, the sample runs out, or 8 hours have elapsed since processing commenced to generate processed sample; producing at least one straw of semen from the processed sample: and fertilizing the oocyte of the female non-human animal with the at least one straw of semen in an in-vitro fertilization process.

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