Systems, methods, and apparatus for sexing sperm by electrical charge interaction and rheotaxis

Abstract

Disclosed herein is process and method for sexing non-human animal sperm in using a microfluidic chip utilizing the electrical charge interaction between the sperm cells and the chip and rheotaxis. In some examples, a non-human animal semen sample is collected and loaded into a two chamber microfluidic chip, wherein the two chambers are connected by a plurality of microchannels. In some examples, the non-human animal semen sample can be sexed for one or both of X chromosome or Y chromosome-bearing sperm cells from a single sample. In some examples, magnetic beads are used to improve differentiation between X and Y chromosome-bearing sperm cells.

Description

IN THE UNITED STATES PATENT AND TRADEMARK OFFICE UNITED STATES PATENT APPLICATIONFORSYSTEMS, METHODS, AND APPARATUS FOR SEXING SPERM BY ELECTRICAL CHARGE INTERACTION AND RHEOTAXISBYPURTELL, LUKE WINTERS, REBECCAXIA, ZHENG ZHAO, WUJUNROTI ROTI. ELONBOTTS, MICHAELGenus Docket No.: GS-58-2024-WO1SYSTEMS, METHODS, AND APPARATUS FOR SEXING SPERM BY ELECTRICAL CHARGE INTERACTION AND RHEOTAXISFIELD OF THE INVENTION

[0001] Disclosed are methods, devices, and systems for sexing non-human sperm cells using a microfluidic chip and fluid flows. Microfluidic chips for sexing non-human semen samples are disclosed. Microfluidic chips may be used in conjunction with magnetic beads to improve differentiation between sample subpopulations. The approach can provide, for example and without limitation, systems and methods for separating bovine sperm cells to produce sexed bovine sperm samples.BACKGROUND

[0002] In the beef production industry, mating is typically done through natural service, whereby male cattle (bulls) are present on a farm and roaming freely with a herd of cows during breeding season. This practice requires minimal intervention but comes with certain notable disadvantages. For example, breeders have ven,' little control when selecting the traits of the resulting offspring. As a result, there is no guarantee that the desired traits (e g., a specific sex and / or the like) will ultimately be selected. And while artificial insemination techniques exist to assist in increasing the control breeders have over such selection, these techniques have varying success rates.

[0003] In the dairy industry, farmers have historically used conventional semen and have fertilized their herds via artificial insemination. However, with the development of sexed semen technology, farmers are using an increasing proportion of sexed semen in their herds. Specifically, farmers are using sexed semen from high quality genetics sources (e.g., high Net Merit bulls) to breed their highest genomic value cows to produce replacement animals for their herds. As a result, the demand for high quality sexed semen continues to increase and outpaces producers’ ability to meet that demand. In other contexts, demand is higher for any sexed semen, regardless of quality. In these other contexts, it may be more desirable to produce large volumes of sexed semen at the lowest possible cost. The type of product desired will necessitate different approaches to sexing the semen, including using either a slower, more expensive approach the produces a higher quality product, or using a faster, less expensive approach that produces a lower quality product. In these contexts, “lower quality” generally refers to a product having a lower skew, or ratio of sperm cells bearing the desired sex chromosome to those having the undesired sex chromosome, and having a lower ratio of collected cells to total input cells, the “eligibility”, while “higher quality” generally refers to a product having a higher skew, and having a higher ratio of collected cells to total input cells.

[0004] Microfluidics chips have recently been developed to enable the manipulation of fluids on microscopic scales, and are being applied to address the above-noted problems in semen selection. Conventional techniques for focusing fluids in a microfluidic chip include shaping the microchannels to focus the fluids. However, existing microfluidic chips, while capable of tightly focusing cells to provide for accurate differentiation, may be relatively slow and expensive to operate.

[0005] Other methods of sexing semen without using microfluidic chips or flow cytometry have recently been introduced. These methods include using magnetic beads having a surface coating to selective bind to X or Y chromosome bearing sperm cells and then removing the undesired fraction of particles. These methods also include using a combination of layered medias and / or centrifugation to enable either the X or Y chromosome bearing sperm cells to selectively “swim up” to be removed from a sample. However, these existing methods for sexing semen which do not use microfluidic chips suffer from many drawbacks including being difficult to automate, requiring highly skilled technicians, being slow, producing low quality products with low skews and yields, having low repeatability’, and being difficult to scale commercially.

[0006] What is needed are systems, methods, and apparatuses for sexing large quantities of non-human animal sperm that can provide higher quality’ products at a lower price or cost. Additionally, what is needed are systems, methods, and apparatuses for sexing non-human animal sperm that address the deficiencies of existing methods such as those discussed hereinabove.BRIEF SUMMARY

[0007] In an embodiment, what is provided is a device for sexing non-human animal sperm, the device comprising a substrate, the device further comprising: a first inlet for introducing a non-human animal sperm sample into the device; a second inlet for introducing a media into the device; a sorting chamber fluidically coupled to the first inlet; a collection chamber fluidically coupled to the second inlet; a first outlet fluidically coupled to the sorting chamber for flowing a waste fluid out of the sorting chamber; a second outlet fluidically coupled to the collection chamber for flowing a processed sample out of the collection chamber; and a plurality of microchannels disposed between the sorting chamber and the collection chamber; wherein when the non-human animal sperm is flowed into the sorting chamber via the first inlet and the media is flowed into the collection chamber via the second inlet, sperm cells in the non-human animal sperm are able to swim against a cunent flow provided by’ the media and move to the collection chamber.

[0008] In various embodiments, the interior of the device is coated with a hydrophilic coating.

[0009] In various embodiments, the coating comprises a polyethylene glycol surfactant modification.

[0010] In various embodiments, the coating is selected from the group consisting of Pluronic F-127, Pluronic F-68, Poloxamer 407, Synperonic F-108, or PLL-g-PEG.

[0011] In various embodiments, the device further comprises: a sample inlet channel; a medium inlet channel; a waste outlet channel; and a cell outlet channel.

[0012] In various embodiments, the device further comprises a set of valves.

[0013] In various embodiments, the set of valves comprises a first inlet valve, a second inlet valve, a first outlet valve, and a second outlet valve.

[0014] In various embodiments, each valve in the set of valves is operate to permit fluid flow into or out of a corresponding inlet and into or out of one of the sorting chamber or collection chamber.

[0015] In various embodiments, a height for each microchannel in the plurality of microchannels is smaller than a height of both the sorting chamber and the collection chamber.

[0016] In various embodiments, each of the sorting chamber and the collection chamber comprise a shallow cylindrical shape.

[0017] In various embodiments, each microchannel in the plurality of microchannels comprises a width of 20-200 microns, a depth of 20-200 microns, and a pitch of 50-1000 microns between each channel in the plurality of microchannels.

[0018] In various embodiments, each microchannel in the plurality of microchannels comprises a width of 100 microns, a depth of 100 microns, and a pitch of 400 microns between each microchannel in the plurality of microchannels.

[0019] In various embodiments, each of the first inlet, the second, the sorting chamber, the collection, the first outlet, the second outlet, and the plurality of microchannels are disposed in a first layer of the substrate.

[0020] In various embodiments, the first layer of the substrate is covered by a second layer of the substrate.

[0021] In various embodiments, the substrate comprises one of polydimethylsiloxane (“PDMS”), boro-silicate glass, or polycarbonate (“PC”).

[0022] In various embodiments, the substrate comprises one of polymethyl methacrylate (“PMMA”), cyclic olefin copolymer (“COC”), polystyrene, or cyclic olefin polymer (“COP”).

[0023] In an embodiment, what is provided is a method for sexing non-human animal sperm in a microlluidic chip using electrical charge interactions and rheotaxis, the method comprising: collecting an ejaculate sample; extending the ejaculate sample with an extender to create an extended ejaculate sample; reconcentrating the extended ejaculate sample to create a reconcentrated sample; resuspending the reconcentrated sample to create a prepared sample; loading the prepared sample into a sample inlet of the microfluidic chip; processing the sample on the microfluidic chip to create a processed sample; and collecting the processed sample.

[0024] In various embodiments, the non-human animal sperm is bovine sperm, porcine sperm, equine sperm, ovine sperm, or avian sperm.

[0025] In various embodiments, the ejaculate sample comprises a porcine ejaculate sample comprising a concentration of 100-500 million cells per mL and a volume of 200-300 mL.

[0026] In various embodiments, the ejaculate sample comprises a bovine ejaculate sample comprising a concentration of 80-4000 million cells per mL and a volume of 2-10 mL.

[0027] In various embodiments, the extender is a citrate extender.

[0028] In various embodiments, the extended ejaculate sample comprises a concentration of 40-200 million cells per mL in a 70 mL volume.

[0029] In various embodiments, the extended ejaculate sample is extended to a ratio of 1:0.5, 1: 1, or 1 :2 ejaculate to extender.

[0030] In various embodiments, the reconcentration comprises centrifuging the extended ejaculate sample.

[0031] In various embodiments, the method further comprises resuspending the concentrated sample comprises washing the concentrated sample in an extender, Tyrode's albumin lactate pyruvate (“TALP”), Tris, a sheath fluid, or Hanks’ balanced salt solution (“HBSS”).

[0032] In various embodiments, the resuspended sample comprises a concentration of 40- 1500 million cells per mL.

[0033] In various embodiments, the method further comprises loading a collection chamber and sorting chamber of the microfluidic chip with 1-1000 microliters of media.

[0034] In various embodiments, the media is an extender, TALP, Tris, a sheath fluid, or HBSS.

[0035] In various embodiments, the loading the prepared sample into the microfluidic chip comprises loading 10-1000 microliters of the prepared sample into the microfluidic chip.

[0036] In various embodiments, the 10-1000 microliters of the prepared sample comprises 10-400 million sperm cells.

[0037] In various embodiments, the processing the sample on the microfluidic chip to create processed sample further comprises: wherein the inlet of the microfluidic chip is fluidically coupled to a sorting chamber of the microfluidic chip, and wherein the prepared sample is permitted to flow into the sorting chamber; waiting 5-90 minutes while holding the microfluidic chip at a temperature of 19-37C; removing 100-1000 microliters of volume from a collection chamber of the microfluidic chip to collect a Y chromosome-bearing sperm cell fraction; waiting 5-90 minutes while holding the microfluidic chip at a temperature of 19- 7C; removing 100-1000 microliters of volume from a collection chamber of the microfluidic chip to collect an X chromosome-bearing sperm cell fraction; and repeating the loading the prepared sample into the sample inlet of the microfluidic chip and processing the sample on the microfluidic chip steps up to five times to maximize processing efficiency.

[0038] In various embodiments, the method further comprises removing the remaining volume of the prepared sample from the sorting chamber.

[0039] In various embodiments, the method further comprises adding 100-1000 microliters of the removed prepared sample back into the sorting chamber.

[0040] In various embodiments, the method further comprises removing the remaining volume of the prepared sample from the sorting chamber and adding 100-1000 microliters of the removed prepared sample back into the sorting chamber.

[0041] In various embodiments, the method further comprises waiting 5-30 minutes while holding the microfluidic chip at a temperature of 19-37C after flowing the sample into the sorting chamber.

[0042] In various embodiments, the method further comprises waiting 5-30 minutes while holding the microfluidic chip at a temperature of 19-37C after removing the 100-1000 microliters of volume from the collection chamber of the microfluidic chip to collect the Y chromosome-bearing sperm cell fraction.

[0043] In various embodiments, the method further comprises performing quality control measurements on the processed sample.

[0044] In various embodiments, the processed sample is diluted for use in artificial insemination, in vitro fertilization, or intracytoplasmic sperm injection.

[0045] In various embodiments, the processed sample is not further diluted for use in artificial insemination, in vitro fertilization, or intracytoplasmic sperm injection.

[0046] In various embodiments, the method further comprises cryopreserving the processed sample.

[0047] In various embodiments, the processed sample is used for trans -cervical artificial insemination, post-cervical artificial insemination, deep-intrauterine artificial insemination, laparoscopic artificial insemination, in vitro fertilization, or intracytoplasmic sperm injection.

[0048] In an embodiment, what is provided is a sex-sorted sperm sample prepared according to any of the above methods.

[0049] In various embodiments, the sex-sorted sperm sample comprises a majority of motile sperm cells.

[0050] In an embodiment, what is provided is a method for sex-sorting non-human animal sperm cells using a microfluidic chip utilizing electrical charge interactions and rheotaxis, the method comprising: preparing the microfluidic chip, the microfluidic chip comprising: a first inlet for introducing a non-human animal sperm sample; a second inlet for introducing a media; a sorting chamber fluidically coupled to the first inlet; a collection chamber fluidi cally coupled to the second inlet; a first outlet fluidically coupled to the sorting chamber for flowing a waste fluid out of the sorting chamber; a second outlet fluidically coupled to the collection chamber for flowing a processed sample out of the collection chamber; and a plurality of microchannels disposed between the sorting chamber and the collection chamber; wherein preparing the microfluidic chip comprises setting: a first valve for controlling flow into the first inlet to an closed position; a second valve for controlling flow7into the second inlet to an open position; a third valve for controlling flow out of the first outlet to an open position; and a fourth valve for controlling flow out of the second outlet to a closed position; loading the media into the collection chamber and the sorting chamber via the second inlet; closing second valve and opening the first valve; loading the non-human animal sperm sample into the sorting chamber via the first inlet; closing the first valve and opening the second valve; flowing the media from the collection chamber into the sorting chamber and waiting for sperm cells in the non-human animal sperm sample to migrate or swim from the sorting chamber to the collection chamber via the plurality of microchannels, wherein a population ofY chromosome-bearing sperm cells will migrate or swim from the sorting chamber through the plurality of microchannels to the collection chamber at a different rate than a population of X chromosome-bearing sperm cells based on an electrical charge interaction a rheotaxic counterflow effect; opening the fourth valve and closing the third valve; collecting the population of Y chromosome-bearing sperm cells from the collection chamber by flowing fluid from the second inlet into the collection chamber and out of the second outlet.

[0051] In various embodiments, the method further comprises collecting the population of X chromosome-bearing sperm cells.

[0052] In various embodiments, the method further comprises closing the fourth valve and opening the third valve; and flushing the chip with the media.

[0053] In various embodiments, the method further comprises repeating the steps of the method to further process remaining cells in the non-human animal sperm sample.

[0054] In various embodiments, a sexed non-human animal sperm sample is prepared using any of the above methods, devices, apparatuses, or systems.

[0055] In various embodiments, a sexed non-human animal sperm sample is prepared using any of the above methods, devices, apparatuses, or systems, wherein the sexed non-human animal sperm sample comprises predominantly X chromosome-bearing sperm cells or Y chromosome-bearing sperm cells.

[0056] In various embodiments, a sexed non-human animal sperm sample is prepared using any of the above methods, devices, apparatuses, or systems, wherein the sexed non-human animal sperm sample comprises predominantly motile or progressively motile sperm cells.

[0057] In various embodiments, what is provided is a method for improving non-human mammalian semen sample quality by introducing and subsequently removing a discriminating agent into a non-human mammalian semen sample which selectively binds to a subset of sperm cells in the non-human mammalian semen sample. In some embodiments, the discriminating agent is a plurality7of magnetic beads coated with, or on which is disposed, a binding agent. In some embodiments, the binding agent is an acrosome associated molecule. In some embodiments, the discriminating agent is used with a microfluidic chip. In some embodiments, the discriminating agent is removed from the sample by a magnet or magnetically receptive material. In some embodiments, the discriminating agent and bound particles are removed from the sample by a magnet or magnetically receptive material and a microfluidic chip.

[0058] In one embodiment, the magnetic beads comprise Lens culinaris agglutinin (LCA) conjugated to HiSur magnetic beads from Ocean NanoTech (HiSur, Ocean NanoTech, San Diego, CA). LCA is a plant-derived lectin protein that binds sugars on proteins enriched in the sperm acrosome.

[0059] In some embodiments, the discriminating agent comprises beads having a magnetic substrate and coated with a silicon containing compound.

[0060] In some embodiments, the discriminating further comprises: contacting the sample with a binding agent that binds to the acrosome associated molecule to form a mixture of the semen and the binding agent; and separating sperm cells bound to the binding agent from sperm cells not bound to the binding agent.

[0061] In some embodiments, the discriminating further comprises: collecting the sperm cells not bound to the binding agent.

[0062] In some embodiments, the binding agent is a polysaccharide binding agent.

[0063] In some embodiments, the binding agent is an agglutinin.

[0064] In some embodiments, the binding agent is a lectin.

[0065] In some embodiments, the agglutinin is derived from an organism selected from the group consisting of Lens culinaris, Ricinus communis, Arachis hypogaea, Glycine max, Bandeiraea simplicifolia (Griffonia simplicifolia), Dolichos biflorus, Erythrina cristagalli, Helix pomatia, Lycopersicon esculentum, Phaseolus vulgaris, Pisum sativum, Sambucus nigra, Triticum vulgaris, Ulex europaeus, and Wisteria floribunda.

[0066] In some embodiments, the agglutinin is derived from an organism selected from the group consisting of Lens culinaris and Pisum sativum.

[0067] In some embodiments, the binding agent is bound to a solid support.

[0068] In some embodiments, the solid support is selected from the group consisting of a bead, a resin, and agarose.

[0069] In some embodiments, the solid support is a magnetic bead.

[0070] In some embodiments, the solid support is coated in the binding agent.

[0071] In some embodiments, the solid support is a magnetic bead coated in an agglutinin.

[0072] In some embodiments, the solid support is a magnetic bead coated in Lens culinaris agglutinin (LCA).

[0073] In some embodiments, the binding agent comprises a binding moiety.

[0074] In some embodiments, the binding moiety is biotin.

[0075] In some embodiments, the binding moiety is an affinity tag is selected from the group consisting of an ALFA-tag, an AViTag. a C-tag, a Calmodulin-tag. a poly glutamate tag, a polyarginine tag, an E-tag, a FLAG-tag, an HA-tag, a His tag, a Myc tag, an NE-tag, a RholD4 tag, an S-tag, an SBP-tag, a Softag 1, a Softag 3, a Spot-tag, a Strep-tag, a T7-tag, a TC tag, a Ty tag, a V5 tag, a VSV-tag, and an Xpress tag.

[0076] In some embodiments, the separating comprises contacting the mixture of semen and binding agent with a molecule that binds to the binding moiety, wherein the molecule that binds to the binding moiety is immobilized to a solid support.

[0077] In some embodiments, the molecule that binds to the binding moiety7is selected from the group consisting of an antibody, avidin, streptavidin, and nickel.

[0078] In some embodiments, the discriminating comprises: contacting the semen with a binding agent immobilized on beads wherein the binding agent binds to the acrosome associated molecule; and separating the beads from the rest of the mixture.

[0079] In some embodiments, the beads magnetic beads, microbeads, or affinity beads.

[0080] In some embodiments, the beads are coated in the binding agent.

[0081] In some embodiments, the beads are magnetic beads and the separating comprises exposing the mixture to a magnet.

[0082] In some embodiments, the beads are heterogenous in size.

[0083] In some embodiments, the separating comprises passing the mixture through an opening smaller than the beads, but larger than the unbound sperm.

[0084] In some embodiments, the separating the sperm cells bound to the agglutinin comprises: contacting the sample with agglutinin-conjugated magnetic beads to form an agglutinin-semen mixture; exposing the beads to a magnetic field to form a pellet comprising the beads and a supernatant comprising unbound sperm cells; and collecting the supernatant.

[0085] In some embodiments, the semen is bovine semen.

[0086] In some embodiments, the semen is Bos taurus semen.

[0087] In some embodiments, the semen is porcine semen.

[0088] In some embodiments, the semen is Sus scrofa semen.

[0089] In some embodiments, the semen is sexed semen.

[0090] In some embodiments, the method further comprises sex selecting the semen.

[0091] In another embodiment, what is provided is a method of sexing non-human animal semen comprising: contacting a mammalian semen sample comprising sperm cells with coated magnetic beads that are conjugated to a lectin to form a semen-bead mixture; exposing the semen-bead mixture to a magnetic field to separate cells bound to antibody from cells not bound to the lectin; separating bound cells from unbound cells to form an X fraction and a Y fraction; and collecting the X fraction and / or the Y fraction to form sex selected semen.

[0092] In another embodiment, what is provided is a method of producing a sexed semen sample comprising: producing a sample according to any of the preceding claims and sexing said sample to produce a sexed semen product.

[0093] In another embodiment, what is provided is a method for sexing non-human animal sperm in a microfluidic chip using electrical charge interactions and rheotaxis, the method comprising: collecting an ejaculate sample; extending the ejaculate sample with an extender to create an extended ejaculate sample; reconcentrating the extended ejaculate sample to create a reconcentrated sample; resuspending the reconcentrated sample to create a prepared sample;loading the prepared sample into a sample inlet of the microfluidic chip; processing the sample on the microfluidic chip by flowing the sample through a channel structure in the microfluidic chip to create a processed sample; and collecting the processed sample.

[0094] In some embodiments, the method further comprises adding magnetic beads to the extended ejaculate sample, wherein the magnetic beads differentially bind to Y chromosome bearing sperm cells forming a bead-bound cell population.

[0095] In some embodiments, the method further comprises retaining the bead-bound cell population in the microfluidic chip using a magnetic substrate layer.

[0096] In another embodiment, what is provided is a device for sexing non-human animal sperm, the device comprising a substrate, the device further comprising; a first inlet for introducing a non-human animal sperm sample into the device; a second inlet for introducing a media into the device; a sorting chamber disposed in the substrate and fluidically coupled to the first inlet; a collection chamber disposed in the substrate and fluidically coupled to the second inlet; a first outlet fluidically coupled to the sorting chamber for flowing a waste fluid out of the sorting chamber; a second outlet fluidically coupled to the collection chamber for flowing a processed sample out of the collection chamber; a plurality of microchannels disposed between the sorting chamber and the collection chamber in the substrate; and a magnetic substrate layer disposed on an exterior surface of the device; wherein when the non- human animal sperm is flowed into the sorting chamber via the first inlet and the media is flowed into the collection chamber via the second inlet, sperm cells in the non-human animal sperm are able to swim against a current flow provided by the media and move to the collection chamber; and wherein a set of sperm cells bound to magnetic beads in the non-human animal sperm are retained in the sorting chamber by the magnetic substrate layer.BRIEF DESCRIPTION OF THE DRAWINGS

[0097] FIG. 1 provides a block diagram flowchart of a process for sexing non-human animal sperm according to one embodiment.

[0098] FIG. 2 provides a diagram of a device for sexing non-human animal sperm according to one embodiment.

[0099] FIG. 3 provides a plan view of a microfluidic chip for sexing non-human animal sperm according to one embodiment.

[0100] FIG. 4 provides a top isometric perspective view of a microfluidic chip for sexing non-human animal sperm according to one embodiment.

[0101] FIG. 5 provides a magnified top isometric perspective view of a portion of a microfluidic chip for sexing non-human animal sperm according to one embodiment.

[0102] FIG. 6 provides a diagram view of a microfluidic chip channel design for sexing non-human animal sperm according to one embodiment.

[0103] FIG. 7 provides a diagram view of an inertial microfluidic chip channel design for sexing non-human animal sperm according to one embodiment.DETAILED DESCRIPTION

[0104] Embodiments of the present disclosure are described with reference to the drawings. Like numerals may designate identical or corresponding elements in each of the several views.

[0105] As used herein, “particle” refers to any thing which may be present in a flow stream. For example, a particle may be a polystyrene bead, a magnetic bead, a protein, cellular debris, small inorganic matter, or a cell, such as a sperm cell.

[0106] As used herein, “sample fluid” refers to a fluid comprising an analyte to be measured. A sample fluid may be, for example, a fluid mixture comprising a buffer fluid media and a plurality of polystyrene beads, or may be a fluid mixture of sperm cells in seminal plasma, or may be a fluid mixture of sperm cells, with or without seminal plasma, in an other media.

[0107] As used herein, “sheath fluid” or “buffer fluid” refers to a fluid which provides sheath flow and / or hydrodynamic focusing of a sample fluid in a microfluidic environment in laminar flow conditions or which may be flowed through a microfluidic chip to act as a biologically compatible buffer media.

[0108] As used herein, “structure” is an element or collection of elements of a physical thing. For example, a sheath fluid structure may comprise one or more sheath fluid channels and all elements or components of the sheath fluid channels. For example, a microfluidic channel structure may comprise all elements therein, such as sample fluid channels, flow channels, sheath fluid channels, and outlet channels. A structure may comprise one or more other structures.

[0109] Throughout this description, the term “upstream” will refer to portions of the device or component thereof that are closer to portions of the device configured to receive fluids therein (such as inlets and / or microwells), and the term “downstream” will refer to portions of the device or component that are farther from portions of the device that are configured to receive the fluids therein.

[0110] In the drawings and the description that follows, terms such as front, rear, upper, lower, top, bottom, and similar directional terms are used simply for convenience of description and are not intended to limit the disclosure unless expressly stated otherwise. Well-known functions and / or constructions are not described in detail to avoid obscuring the present disclosure in unnecessary detail.[OHl] Described herein are systems, methods and apparatus for sexing non-human animal sperm cells using a microfluidic chip without using interrogation, discrimination, or direct action on individual cells by a device to sex the non-human animal sperm cells. Described herein are systems, methods, and apparatus for sexing non-human animal sperm cells using a microfluidic chip by using an electrical charge interaction force, which affects a first subpopulation of cells to a greater degree than a second subpopulation of cells, and rheotaxis to provide for sexing of the non-human animal sperm cells.

[0112] Described herein is a method of improving semen sample quality comprising: providing a mammalian semen sample comprising sperm cells and discriminating between sperm cells based on the exposure or availability' of an acrosome associated molecule. An acrosome associated molecule can include a polysaccharide or a protein capable of binding to a ligand. Without being limited by theory, it is believed that these molecules either become exposed on the cell surface, change conformation, or associate or disassociate with certain molecules as a result of capacitation or sperm activation. These molecules are therefore useful for discriminating between X and Y chromosome bearing sperm cells.

[0113] The present teachings provide for and include discriminating between X and Y chromosome bearing sperm cells on the basis of differential binding of a binding agent. As discussed above, an acrosome associated molecule is able to differentially bind to the binding agent for X and Y chromosome bearing sperm cells. Thus the ability of the binding agent to bind to a sperm is dependent on the sperm being X or Y chromosome bearing. The present teachings utilize this difference in binding in order to separate X and Y chromosome bearing sperm cells. The separation can comprise collecting the cells bound to the binding agent or the cells not bound to the binding agent.

[0114] In various configurations, the binding agent can be a protein, a polysaccharide, a lipid, a nucleic acid, an ion, or an inorganic compound. In some configurations, the protein can be an antibody, a natural ligand, an enzy me, an agglutinin or a lectin. In some configurations, the binding agent is a biomimetic.

[0115] In some configurations, the protein can be an agglutinin or a lectin. The agglutinin or lectin may be isolated from any species, such as, but without limitation, Lens culmaris.Ricinus communis, Arachis hypogaea, Glycine max, Bandeiraea simplicifolia (Griffonia simplicifolia), Dolichos biflorus, Erythrina cristagalli, Helix pomatia, Lycopersicon esculentum, Phaseolus vulgaris, Pisum sativum, Sambucus nigra, Triticum vulgaris, Ulex europaeus, and Wisteria floribunda. Many lectins are commercially available from vendors such as Millipore Sigma (formerly Sigma Aldrich).

[0116] Skilled artisans will recognize that there are many methods of separating the bound sperm cells from the unbound sperm cells. Such methods may include: size exclusion columns, sucrose gradients, or using other molecules or substances that bind to or are conjugated to the binding agent.

[0117] In one embodiment, a magnetic or magnetically receptive plate or location may be used in combination with a microfluidic chip as described herein to separate the differentially bound X and Y chromosome bearing sperm cells

[0118] In some configurations, the binding agent may be bound to a solid support. As used herein, “bound to a solid support” may mean that the binding agent is chemically bound to the solid support, ionically bound to the solid support, conjugated to the solid support, coating the solid support, reversibly attached to. ionically bound to. or non-ionically bound to the solid support. Further, a binding agent bound to a solid support may mean that the solid support is coated in the binding agent. The binding may be specific binding, such as an antibody that recognizes a specific epitope, or non-specific binding, such as an ionic bond. The binding may also be through an intermediary, such as a secondary antibody conjugated to a solid support, a his-tagged binding agent bound to a nickel column, or an antibody to a particular tag conjugated to a bead or resin. Exemplary solid supports can include a plate, a bead, a resin, agarose, or a gel. In some configurations, the resin can be an ionic exchange resin, an immunoresin, or a size exclusion resin. In some configurations, the bead can be a magnetic bead, an immunobead, an affinity bead, or an ionic bead. In some configurations, the solid support can be a plurality of beads. In some configurations, the plurality of beads can include beads of heterogenous sizes. In various configurations, the beads can comprise, be coated in, or be conjugated to the binding agent. In some configurations, the bead can comprise, be coated in, or be conjugated a molecule that recognizes a binding moiety on the binding agent. As used herein, a binding moiety can be a small molecule, an epitope, a binding motif, an epitope tag, or an affinity tag. Small molecule binding molecules can include biotin or hapten. Affinity tags can include ALFA-tag, an AViTag, a C-tag, a Calmodulin-tag, a poly glutamate tag, a polyarginine tag, an E-tag, a FLAG-tag, an HA-tag, a His tag, a Myc tag, an NE-tag, a RholD4 tag, an S-tag, an SBP-tag, a Softag I, a Softag 3, a Spot-tag, a Strep-tag, a T7-tag, a TC tag, a Ty tag, a V5 tag, a VSV-tag, or an Xpress tag. Other affinity tags are know n in the art. Binding moieties can be recognized by a binding partner that can be bound to a solid support. For example, but without limitation, biotinylated agglutinin can bind to both an acrosome associated molecule and to streptavidin or avidin coated beads. Alternatively, a his-tagged lectin can bind to both an acrosome associatedmolecule and to nickel beads. Various affinity columns are commercially available and well known in the art.

[0119] Semen suitable for use in the present teachings can be semen from any type of mammalian livestock, including, such as but without limitation, bovine semen, porcine semen, ovine semen, or equine semen. Semen may also be from, for example, a ruminant animal, an even-toed ungulate animal, or an odd-toed ungulate animal. Bovine semen, such as Bos taurus semen or Bos indicus semen and porcine semen, such as Sus scrofa semen, are especially preferred. Semen suitable for use in the present teachings can be semen from a collected ejaculate or epidi dymal semen. Methods of collecting both types of semen are known in the art.

[0120] 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

[0121] 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.

[0122] By virtue of the implementation of the systems, methods, and apparatus described herein, animal protein production, such as dairy and / or meat production, can be improved. More specifically, in one embodiment in a dairy application semen from bulls (e g., the top 20- 50% of the most genetically desirable bulls) can be selected by flowing the semen through the microfluidic chips and using the methods described herein. Once inseminated, the resulting cows may be used to replace the existing herd, thereby continually improving the herd's overall milk production. Alternatively, the systems, method, and apparatus described herein may be used to sex bovine, porcine, equine, ovine, or avian sperm cells to achieve a similar effect. For example, the systems, method, and apparatus described herein may be used to sex porcine semen to produce porcine sows having desired genetic characteristics, or may be used to produce primarily male bovine animals for meat production.

[0123] Referring now to FIG. 1 , what is provided is a block diagram flowchart of a process 100 for sexing non-human animal sperm according to one embodiment. The process 100 depicted in FIG. 1 provides an embodiment of steps involved in using a microfluidic chip device to sex non-human animal semen samples; specifically, in one embodiment to sex non- human animal sperm cells using a combination of an electrical charge interaction betw een thesperm cells and the interior surface of the microfluidic chip and rheotaxis to sex the population of non-human animal sperm cells into an X chromosome-bearing sperm cell population and a Y chromosome-bearing sperm cell population. Additionally, the process 100 will separate non- motile sperm cells from motile sperm cells, providing, in one embodiment, for the collection of sexed, motile X and Y chromosome-bearing sperm cell subpopulations.

[0124] The process 100 may vary’ from embodiment to embodiment based on the non- human animal sperm sample being processed, but will generally follow the steps outlined in the process 100. In an embodiment, the process 100 begins with the collection of raw ejaculate in step 110. Raw ejaculate, or unprocessed ejaculate, is collected from a male animal at a bam or farm. It is important to process the raw ejaculate as close in time to its collection as possible, as the differentiable difference between X and Y chromosome bearing sperm cells of varying non-human animals species is greatest at the time of collection, TO, and decreases over time, at Tl, T2, etc. For bovine raw ejaculate, the raw ejaculate may comprise a volume of 2-10 mL at a concentration of 80-4000 million cells per mL ("M / mL"). For porcine raw ejaculate, the raw ejaculate may comprise a volume of 200-300 mL at a concentration of 100-500 M / mL.

[0125] After collection in step 110, the raw ejaculate is extended with a semen extender product in step 120. The semen extender product may be a commercially available porcine or bovine extender, or may be a custom formulation. In a preferred embodiment, the extender is a commercially available extender suitable for use with the particular species from which the non-human animal semen sample was collected. In one embodiment, raw ejaculate is collected from one or more bulls at a collection site in step 1 10. Then, the raw ejaculate collected from the bulls is extended at step 120 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). In this embodiment, the ejaculate is extended with the extender in an approximately 2: 1 ratio. In another embodiment, the ejaculate is extended in a concentration range of 40 - 25 00 M motile sperm / mL with the extender described in US PGPUB 2020 / 0347347 (Roti-Roti et al.) or using any of the described extenders with an amount of citrate. In one embodiment, sheath or buffer fluids may be used as an extender or media and may comprise nutrients to maintain the viability of the sperm cells in the extended ejaculate mixture. Commercially available Tris, as sold by Chata Biosystems, is one example, and a sheath or buffer fluid may be formulated to include the following: Water — 0.9712 L; Tris — 23.88 gg; citric acid monohydrate — 11.63 g; D-fructose — 8.55 g. The pH is adjusted to 6.80 ±0.05 with hydrochloric acid, and osmolarity is adjusted, if necessary, to 270-276 mOsm with fructose high purity. The mixture is filtered using a 0.22 -micron filter.In another embodiment, the extender may be Tyrode's albumin lactate pyruvate (“TALP”) or Hanks’ balanced salt solution (“HBSS”). In various embodiments, the ratio of raw ejaculate to extender may be 1 :0.5, 1 : 1, or 1:2.

[0001] In one embodiment, magnetic beads are used to further increase the differential separation of X and Y chromosome bearing sperm cells.

[0002] In one embodiment, magnetic beads are used to selectively bind to Y chromosome bearing sperm cells. The needed volume of beads is aliquoted, and placed on a magnetic rack for 15 seconds. The liquid is removed and replaced with an equal volume of Staining TALP. The beads are resuspended and replaced on the rack, resuspended, and the wash repeated. The beads are resuspended in Staining TALP.

[0003] The concentration of semen is determined by first removing an aliquot of a raw ejaculate sample and combining 10 pL of the raw ejaculate with a lysis solution and homogenizing the sample and lysis solution. The homogenized solution is loaded into a cartridge and then placed into a concentration counter, such as for example a spectrophotometer or imaging-based cell counting system. Propidium iodide is added and the total DNA content is determined. Concentration and progressive motility of the sample may be further evaluated using an IVOS.

[0126] Cell suspensions with an incoming concentration of 1000 million cells / mL (M / mL) or more are diluted to 200 M / mL. Cells with lower incoming concentrations, for example 800- 1000 M / mL or 600-1000 M / mL. are diluted to 125 M / mL. The total volume for the cell dilutions depends on the desired throughput on the sex selection machines, which may be, for example 3 mL for 17500 cells / second or 4 mL for 25000 cells / second. 10 pL of beads are added for each mL of sample. Additional buffer is added to the final volume needed. The mixture is incubated at 34° C for 45 minutes. Further volumes of buffer may then added to each tube to achieve a desired concentration or volume.

[0127] Depending on the desired result and the type of non-human animal sperm cells being processed, one or more of the media solutions described above may be used in later steps, such as in the processing of the sample in a microfluidic chip.

[0128] The extended ejaculate, which may have been combined with magnetic beads as described above, is then reconcentrated using centrifugation in step 130, for example at 1000 g for 10 minutes at room temperature. In vary ing embodiments, the centrifugation may be between lOO-lOOOg for 1-10 minutes at 19-37C depending on ejaculate condition and the desired result. The reconcentrated cells are washed or resuspended in step 140 using a mediasuch as a commercially available extender, TALP, or HBSS to a concentration of 40-1500 m / ML.

[0129] Before the reconcentrated cells can be loaded into a microfluidic chip for processing - sexing the sperm cells into subpopulations based on the chromosome present in the cells - in step 150 the microfluidic chip is pre-loaded with media. In one embodiment, the microfluidic chip is loaded with 1-1000 microliters (“uL”) of media based on the internal volume of the chambers and channels in the microfluidic chip. The media used to pre-load the microfluidic chip in step 150 may be a commercially available extender, sheath fluid, TALP, HBSS, or Tris. The pre-loaded chip may then be loaded with the reconcentrated sample fluid for processing.

[0130] In step 160, the reconcentrated sample fluid is processed in the microfluidic chip. Depending on the volume of reconcentrated sample to be processed, the entire volume may not be able to be processed in a single step and may need to be processed in repeated steps until the entire volume has been used. For example, in one embodiment 10-400 million sperm cells may be flowed, injected, or loaded into the microfluidic chip at a time. The sample in the chip is permitted to dwell for 5-90 minutes at a temperature of 19-37C, during which time Y chromosome-bearing sperm cells swim, migrate, or flow from the sorting chamber of the microfluidic chip into the collection chamber of the microfluidic chip. In another embodiment, the sample in the chip is permitted to dwell for 5-30 minutes at a temperature of 19-37C. After this first period, 100-1000 uL volume of fluid, comprising predominantly Y chromosomebearing sperm cells, is collected from the collection chamber of the chip. The collected Y chromosome-bearing sperm cell volumes will be pooled for later use and processing. After the Y chromosome-bearing sperm cell volume is collected from the collection chamber, the remaining volume in the sorting chamber is flushed from the microfluidic chip and separately collected. In various embodiments, Y chromosome bearing sperm cells are bound to magnetic beads and the bound magnetic beads are attracted to a magnetic or magnetically receptive plate or area in the microfluidic chip to further improve the separation of X and Y chromosome bearing sperm cells.

[0131] After removing the first fraction from the collection chamber, the sample in the chip is permitted to dwell for 5-90 minutes at a temperature of 19-37C, during which time X chromosome-bearing sperm cells swim, migrate, or flow from the sorting chamber of the microfluidic chip into the collection chamber of the microfluidic chip. In one embodiment, a magnetic plate, magnet, magnetic or magnetically receptive layer, may be used in or with the microfluidic chip to attract sperm cells bound to a magnetic bead. In this embodiment, the magnetic bead separation may further improve the separation efficiency of X and Ychromosome bearing sperm cells. After this first period, 100-1000 uL volume of fluid, comprising predominantly X chromosome-bearing sperm cells, is collected from the collection chamber of the chip.

[0132] In another embodiment, prior to collecting the X chromosome-bearing sperm cell fraction from the collection chamber but after collecting the Y chromosome-bearing sperm cell fraction, the chip may be re-primed with 100-1000 uL of media as done before initially loading the chip. Then, the collected waste fraction is then re-flowed, injected, or inserted into the sorting chamber of the microfluidic chip.

[0133] In another embodiment, the sample in the chip is permitted to dwell for 5-30 minutes at a temperature of 19-37C.

[0134] The processing step 160 as described above may be performed multiple times on a single sample to improve collection efficiency and skew to provide for a higher quality product.

[0135] In step 170 the sample processed in the step 160 is collected as an X chromosomebearing sperm cell fraction or subpopulation and a Y chromosome-bearing sperm cell fraction or subpopulation. These two subpopulations may then be subjected to quality control steps including measuring for motility and progressive motility, skew, and number of collected cells relative to the number of cells processed.

[0136] After collection, the processed subpopulations may either be cryopreserved or immediately used in step 180 in either a diluted or an undiluted form for end uses such as transcervical artificial insemination, post-cervical artificial insemination, deep-intrauterine artificial insemination, laparoscopic artificial insemination, in vitro fertilization, or intracytoplasmic sperm injection. When cryopreserved, 2-50 million cells may be packaged in individual straws and frozen in liquid nitrogen.

[0137] With reference now to FIG. 2. what is provided is a diagram of a device 200 for sexing non-human animal sperm according to one embodiment. In one embodiment, the device 200 shown in the diagram of FIG. 2 may be used to process non-human animal sperm according to the method 100 shown in FIG. 1. The device 200 is a microfluidic chip. In some embodiments, the microfluidic chip is comprised of one or more materials. For example, the microfluidic chip may be comprised of one or more of polydimethylsiloxane (PDMS), thermoplastics, glass, silicon, metals, and composites. In various embodiments, the substrate comprises one of poly dimethylsiloxane (“PDMS’’), boro-silicate glass, polycarbonate (“PC’’), polymethyl methacrylate (“PMMA’j, cyclic olefin copolymer (“COC”), polysty rene, or cyclic olefin polymer (‘ COP’ ).

[0138] As described below, the microfluidic chip includes one or more structures therein. The microfluidic chip may be manufactured using one or more manufacturing techniques. These techniques can include photolithography, soft lithography, hot embossing, micro machining, injection molding, and / or 3D printing as appropriate for the corresponding material type.

[0139] The interior surface, or internal structure, of the microfluidic chip is coated with a suitable hydrophilic material coating to enhance the sexing effect of the electrostatic and rheotaxic forces on the sperm cells being processed. In various embodiments, the coating may be a polyethylene glycol surfactant modification. In various embodiments, the coating may be ThermoFisher Pluronic F-127, Pluronic F-68, or Poloxamer 407. In various embodiments, the coating may be Synperonic F-108. In various embodiments, the coating may be PLL-g-PEG.

[0140] The structures of the device 200 comprise a sorting side 210, a collection side 250, and a connecting plurality or array of microchannels 240 including at least a first microchannel 242. The sorting side 210 comprises a sorting chamber 212, a cell or first inlet 220, and a waste or first outlet 230. An inlet or first valve 222 controls the flowing or ingress of fluids into the sorting chamber 212 via the sorting inlet 220, and a waste or third valve 232 for controls the outflow of fluids from the sorting chamber 212.

[0141] The collection side 250 comprises a collection chamber 252, a media or second inlet 260, and a cell, collection, or second outlet 270. A media or second valve 262 controls the flowing or ingress of media into the collection chamber 252 via the media inlet 260, and a cell, collection, or fourth valve 272 controls the outflow of fluids from the collection chamber 252. Each of the valves may be a solenoidal valve or other suitable valve type. The flow of fluids into the device may be configured at range of pressures or flow rates as controlled by a flow controller.

[0142] In one embodiment, in operation of the device 200, a prepared non-human animal sperm sample, such as either a fresh, unprocessed sample or a sample that has been extended, reconcentrated, and resuspended, is processed in the device 200. First, media is loaded into the device via the media inlet 260 into both the collection chamber 252 and the sorting chamber 212 with the first valve 222 in a closed position, the second valve 262 in an open position, the third valve 232 in an open position, and the fourth valve 272 in a closed position. A flow rate of 0. 1-0.5 mL / min may be used to fill the collection 252 and sorting 212 chambers.

[0143] The first valve 222 is then opened and the second valve 262 is closed. The sample, which in some embodiments comprises a sample conjugated with magnetic beads, is then flowed or loaded into the sorting chamber 212 via the cell inlet 220. In one embodiment, a flowrate of 0-5 mm / minute (sample velocity) is used to flow the cells into the sorting chamber 212. Once the cells have been loaded, the first valve 222 is closed and the second valve 262 is opened. A counter flow of media is established by flowing media into the media inlet 260. Cells migrate or swim from the sorting chamber to the collection chamber via the plurality of microchannels 240. The counterflow velocity7should be no greater than the sample (particle or sperm cell) velocity of 0-5 mm / min.

[0144] The sperm cells in the sample being processed migrate or swim through the sorting chamber 212 and through the plurality of microchannels 240 and different rates, with X chromosome-bearing sperm cells migrating at a slower rate than Y chromosome-bearing sperm cells due to a combination of electrical charge interactions and rheotaxis. The Y chromosomebearing sperm cells are thereby able to move into the collection chamber 252 at a greater rate then X chromosome-bearing sperm cells.

[0145] The sorted cells, initially Y chromosome-bearing sperm cells, are then collected from the collection chamber 252 by opening the fourth valve 272 and closing the third valve 232 and flowing media through the device 200. After collecting the sexed fraction of cells, the remaining sample is flushed from the chip by opening the third valve 232 and closing the fourth valve 272. The device 200 is then ready for re-priming and for processing the same sample batch additional times, or for processing another non-human animal semen sample batch.

[0146] With reference now to FIGs. 3-5, various views of a microfluidic chip 300 for sexing non-human animal sperm according to one embodiment are provided. The microfluidic chip 300 may be used as the device 200 via the process 100 as described hereinabove. The microfluidic chip 300 is a microfluidic chip comprising an internal channel structure. In some embodiments, the microfluidic chip 300 is comprised of one or more materials. For example, the microfluidic chip 300 may be comprised of one or more of poly dimethylsiloxane (PDMS), thermoplastics, glass, silicon, metals, and composites. As described below, the microfluidic chip 300 includes one or more structures therein. The microfluidic chip 300 may be manufactured using one or more manufacturing techniques. These techniques can include photolithography, soft lithography, hot embossing, micro machining, injection molding, and / or 3D printing as appropriate for the corresponding material type.

[0147] The interior surface, or internal structure, of the microfluidic chip 300 is coated with a suitable hydrophilic material coating to enhance the sexing effect of the electrostatic and rheotaxic forces on the sperm cells being processed. In various embodiments, the coating may be a polyethylene glycol surfactant modification. In various embodiments, the coating may beThermoFisher Pluronic F-127, Pluronic F-68, or Poloxamer 407. In various embodiments, the coating may be Synperonic F-108. In various embodiments, the coating may be PLL-g-PEG.

[0148] The structures of the microfluidic chip 300 comprise a sorting side 310, a collection side 350, and a connecting plurality or array of microchannels 340 including at least a first microchannel 342. The sorting side 310 comprises a sorting chamber 312, a cell or first inlet 320, an inlet channel 323, a waste or first outlet 330, and a waste channel 333.

[0149] In one embodiment, as shown in FIG. 4, the microfluidic chip 300 may be used with, bound to, coupled with, or affixed to a magnet 399. Magnet 399 may be any suitable magnet such as a ferromagnet, ferromagnetic material, neodymium magnet, or suitable treated or prepared magnetically receptive material. In operation, magnet 399 may attract magnetic beads bound to particles flowed through the microfluidic chip 399. In one embodiment, the microchannels 340 may comprise openings which are smaller than any magnetic beads bound to particles, such as sperm cells, but which are larger than the particles to not permit the passing of magnetic bead-bound particles through the microchannels 340.

[0150] The collection side 350 comprises a collection chamber 352, a media or second inlet 360, a media channel 363, a cell, collection, or second outlet 370, and a collection channel 373.

[0151] As shown in detail in FIG. 5, the plurality of microchannels 340, comprising at least microchannel 342, is disposed between and fluidically connects the sorting chamber 312 and the collection chamber 352. In various embodiments, each microchannel in the plurality of microchannels comprises a width of 20-200 microns, a depth of 20-200 microns, and a pitch of 50-1000 microns between each channel in the plurality of microchannels. In one embodiment, each of the microchannels in the plurality of microchannels 342 has a width of 100 microns, a height of 100 microns, and the pitch betw een the microchannels is 400 microns.

[0152] The operation of the microfluidic chip 300 and the purpose and function of the structures of the microfluidic chip 300 correspond to the associated structures shown and described with respect to the device 200 shown in FIG. 2.

[0153] With reference now to FIG. 6 and FIG. 7, what are provided are diagrams of continuous flow microchannel structures which may be disposed in a microfluidic chip and used for sexing of non-human animal sperm cells as described herein. Generally, the microfluidic channel structures shown in FIG. 6 and FIG. 7 function similarly to the device 200 and microfluidic chip 300, but utilize a continuous How instead of alternating flow's controlled by a set or series of valves.

[0154] In FIG. 6, what is provided a diagram view of a microfluidic chip channel design 600 for sexing non-human animal sperm according to one embodiment.

[0155] The microfluidic chip channel design 600 is disposed in a microfluidic chip. In some embodiments, the microfluidic chip is comprised of one or more materials as described hereinabove.

[0156] The interior surface, or internal structure, of the microfluidic chip channel design 600 is coated with a suitable hydrophilic material coating to enhance the sexing effect of the electrostatic and rheotaxic forces on the sperm cells being processed.

[0157] The microfluidic chip channel design 600 comprises a sorting side 610, a collection side 650, and a connecting plurality or array of microchannels 640 including at least a first microchannel 642 and a final microchannel 644. The sorting side 610 comprises a sorting channel 612, a cell or first inlet 620 and a waste or first outlet 630.

[0158] The collection side 650 comprises a collection channel 652. a media or second inlet 660 and a cell, collection, or second outlet 670. The operation of the microfluidic chip channel design 600 and is similar to the microfluidic chip 300 and device 200, however, the microfluidic chip channel design 600 instead utilizes a constant flow of sample into the sample inlet 620 and media into the media inlet 660 instead of alternating flows. While the sample flows through the sample channel 612, sperm cells in the sample swim against the flow of media from the media channel 660 via the plurality of microchannels 640 into the collection channel 652 and out of the second outlet 670.

[0159] In FIG. 7, what is provided a diagram view of an inertial microfluidic chip channel design 700 for sexing non-human animal sperm according to one embodiment.

[0160] The inertial microfluidic chip channel design 700 is disposed in a microfluidic chip. In some embodiments, the microfluidic chip is comprised of one or more materials as described hereinabove.

[0161] The interior surface, or internal structure, of the inertial microfluidic chip channel design 700 is coated with a suitable hydrophilic material coating to enhance the sexing effect of the electrostatic and rheotaxic forces on the sperm cells being processed.

[0162] The inertial microfluidic chip channel design 700 comprises a sorting side 710, a collection side 750, and a connecting plurality or array of microchannels 740 including at least a first microchannel 742 and a final microchannel 744. The sorting side 710 comprises a sorting channel 712, a cell or first inlet 720 and a waste or first outlet 730.

[0163] The collection side 750 comprises a collection channel 752, a media or second inlet 760 and a cell, collection, or second outlet 770. The operation of the inertial microfluidic chip channel design 700 and is similar to the microfluidic chip channel design 600, the microfluidic chip 300 and device 200, however, the inertial microfluidic chip channel design 700 furtherutilizes inertial forces on the particles in the channels due to the curved design of the sorting channel 712 and the collection channel 752. While the sample flows through the sample channel 712, sperm cells in the sample swim against the flow of media from the media channel 760 via the plurality of microchannels 740 into the collection channel 752 and out of the second outlet 770. Additionally, the inertial forces on the particles work the push the sperm cells from the sorting channel 712 into the collection channel 752.

[0164] As illustrated, the spiral of the inertial microfluidic chip channel design 700 is an Archimedean spiral. The width of the spiral along points of the spiral may be between 50 pm and 250 pm and the height of the spiral may be between 40 pm and 50 pm.

[0165] The foregoing method descriptions and the process flow diagrams are provided merely as illustrative examples and are not intended to require or imply that the steps of the various embodiments must be performed in the order presented. The steps in the foregoing embodiments may be performed in any order. Words such as '‘then,” “next,” etc., are not intended to limit the order of the steps; these words are simply used to guide the reader through the description of the methods. Although process flow diagrams may describe the operations as a sequential process, many of the operations can be performed in parallel or concurrently. In addition, the order of the operations may be re-arranged. A process may correspond to a method, a function, a procedure, a subroutine, a subprogram, and the like. When a process corresponds to a function, the process termination may correspond to a return of the function to a calling function or a main function.

[0166] Some non-limiting embodiments of the present disclosure may be described herein in connection with a threshold. As described herein, satisfying a threshold may refer to a value being greater than the threshold, more than the threshold, higher than the threshold, greater than or equal to the threshold, less than the threshold, fewer than the threshold, lower than the threshold, less than or equal to the threshold, equal to the threshold, and / or the like.

[0167] No aspect, component, element, structure, act, step, function, instruction, and / or the like used herein should be construed as critical or essential unless explicitly described as such. In addition, as used herein, the articles "a" and "an" are intended to include one or more items and may be used interchangeably with "one or more" and "at least one." Furthermore, as used herein, the term "set" is intended to include one or more items (e.g., related items, unrelated items, a combination of related and unrelated items, etc.) and may be used interchangeably with "one or more" or "at least one." Where only one item is intended, the term "one" or similar language is used. Also, as used herein, the terms "has," "have," "having." or the like are

Claims

intended to be open ended terms. Further, the phrase "based on" is intended to mean "based at least partially on" unless explicitly stated otherwise.[0168] The preceding description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the disclosure. Thus, the present disclosure is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the following claims and the principles and novel features disclosed herein. [0169] While various aspects and embodiments have been disclosed, other aspects and embodiments are contemplated. The various aspects and embodiments disclosed are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.CLAIMSWhat is claimed is:

1. A device for sexing non-human animal sperm, the device comprising a substrate, the device further comprising: a first inlet for introducing a non-human animal sperm sample into the device; a second inlet for introducing a media into the device; a sorting chamber disposed in the substrate and fluidically coupled to the first inlet; a collection chamber disposed in the substrate and fluidically coupled to the second inlet; a first outlet fluidically coupled to the sorting chamber for flowing a waste fluid out of the sorting chamber; a second outlet fluidically coupled to the collection chamber for flowing a processed sample out of the collection chamber; and a plurality of microchannels disposed between the sorting chamber and the collection chamber in the substrate; wherein when the non-human animal sperm is flowed into the sorting chamber via the first inlet and the media is flowed into the collection chamber via the second inlet, sperm cells in the non-human animal sperm are able to swim against a current flow provided by the media and move to the collection chamber.

2. The device of claim 1, wherein the interior of the device is coated with a hydrophilic coating.

3. The device of claim 2, wherein the coating comprises a polyethylene glycol surfactant modification.

4. The device of claim 2, wherein the coating is selected from the group consisting of Pluronic F-127, Pluronic F-68, Poloxamer 407, Synperonic F-108, or PLL-g- PEG.

5. The device of claim 1, wherein the device further comprises: a sample inlet channel; a medium inlet channel; a waste outlet channel; and a cell outlet channel.

6. The device of claim 1, wherein the device further comprises a set of valves, the set of valves comprising a first inlet valve, a second inlet valve, a first outlet valve, and a second outlet valve.

7. The device of claim 6, wherein each valve in the set of valves is operate to permit fluid flow into or out of a corresponding inlet and into or out of one of the sorting chamber or collection chamber.

8. The device of claim 1, the device further comprising a magnetic substrate layer disposed on an exterior surface of the device.

9. The device of claim 1, wherein a height for each microchannel in the plurality of microchannels is less than a height of both the sorting chamber and the collection chamber.

10. The device of claim 1, wherein each of the sorting chamber and the collection chamber comprise a shallow cylindrical shape.

11. The device of claim 1, wherein each microchannel in the plurality of microchannels comprises a width of 100 microns, a depth of 100 microns, and a pitch of 400 microns between each microchannel in the plurality of microchannels.

12. The device of claim 1, wherein each of the first inlet, the second, the sorting chamber, the collection, the first outlet, the second outlet, and the plurality of microchannels are disposed in a first layer of the substrate.

13. The device of claim 12, wherein the first layer of the substrate is covered by a second layer of the substrate.

14. The device of claim 1, wherein the substrate comprises one of polydimethylsiloxane (“PDMS”), boro-silicate glass, polycarbonate (“PC”), polymethyl methacrylate (“PMMA”), cyclic olefin copolymer (“COC”), polystyrene, or cyclic olefin polymer (“COP”).

15. A method for sexing non-human animal sperm in a microfluidic chip using electrical charge interactions and rheotaxis, the method comprising: collecting an ejaculate sample; extending the ejaculate sample with an extender to create an extended ejaculate sample; reconcentrating the extended ejaculate sample to create a reconcentrated sample; resuspending the reconcentrated sample to create a prepared sample; loading the prepared sample into a sample inlet of the microfluidic chip;processing the sample on the microfluidic chip by flowing the sample through a channel structure in the microfluidic chip to create a processed sample; and collecting the processed sample.

16. The method of claim 15, further comprising adding magnetic beads to the extended ejaculate sample, wherein the magnetic beads differentially bind to Y chromosome bearing sperm cells forming a bead-bound cell population.

17. The method of claim 16, further comprising retaining the bead-bound cell population in the microfluidic chip using a magnetic substrate layer.

18. The method of claim 15, wherein the ejaculate sample comprises a porcine ejaculate sample comprising a concentration of 100-500 million cells per rnL and a volume of 200-300 mL.

19. The method of claim 15, wherein the ejaculate sample comprises a bovine ejaculate sample comprising a concentration of 80-4000 million cells per mL and a volume of 2-10 mL.

20. The method of claim 15, wherein the extended ejaculate sample comprises a concentration of 40-200 million cells per mL in a 70 mL volume.

21. The method of claim 15, wherein the extended ejaculate sample is extended to a ratio of 1 :0.5, 1: 1, or 1:2 ejaculate to extender.

22. The method of claim 15, wherein the reconcentration comprises centrifuging the extended ejaculate sample.

23. The method of claim 15, wherein resuspending the concentrated sample comprises washing the concentrated sample in an extender, Tyrode's albumin lactate pyruvate (“TALP”), Tris, a sheath fluid, or Hanks’ balanced salt solution (“HBSS”).

24. The method of claim 15, wherein the resuspended sample comprises a concentration of 40-1500 million cells per rnL.

25. The method of claim 15, further comprising loading a collection chamber and sorting chamber of the microfluidic chip with 1-1000 microliters of media.

26. The method of claim 25, wherein the media is an extender, TALP, Tris, a sheath fluid, or HBSS.

27. The method of claim 15, wherein the loading the prepared sample into the microfluidic chip comprises loading 10-1000 microliters of the prepared sample into the microfluidic chip.

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