WO2023010101A1 - Procédés d'amélioration de la qualité d'un échantillon de cellule reproductrice - Google Patents

Procédés d'amélioration de la qualité d'un échantillon de cellule reproductrice Download PDF

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WO2023010101A1
WO2023010101A1 PCT/US2022/074287 US2022074287W WO2023010101A1 WO 2023010101 A1 WO2023010101 A1 WO 2023010101A1 US 2022074287 W US2022074287 W US 2022074287W WO 2023010101 A1 WO2023010101 A1 WO 2023010101A1
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semen
cells
sample
sperm
tag
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PCT/US2022/074287
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English (en)
Inventor
Elon Roti Roti
Xiao Peng
Michael BOTTS
Rebecca WINTERS
Ashley SCHROEDER
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Abs Global, Inc.
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Publication of WO2023010101A1 publication Critical patent/WO2023010101A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0612—Germ cells sorting of gametes, e.g. according to sex or motility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

Definitions

  • the present technology relates to methods of improving reproductive cell sample quality. More particularly, the present technology relates to methods of improving the quality of mammalian semen cell samples by discriminating between live and dead sperm cells in a semen sample obtained from livestock, such as cattle or pigs.
  • sperm cells spermatozoa
  • sperm cells are collected in the form of raw ejaculate from male animals. Subsequent use and manipulation of the sperm cells requires that the viability and function of the cells be maintained for hours or even days.
  • a substantial problem with the manipulation of reproductive cells in vitro can be a significant loss of reproductive cell characteristics such as alteration of the lipid bilayer, alteration of cellular organelles, cell apoptosis, or cell necrosis, and decreased motility of sperm cells. The loss of reproductive cell characteristics can result in decreased fertility or decreased viability of the reproductive cells, or both.
  • incoming or collected semen samples comprise a varying quality including the percentage of dead cells in the sample.
  • Dead cells represent wasted instrument processing time for a semen sexing instrument as the dead cells do not contribute to the number of sexed cells in a processed straw.
  • What is needed is a bulk method to remove dead cells from a sample to improve the percentage of eligible cells in the sample.
  • a method for the bulk removal of dead cells would provide for faster processing of ejaculates with less waste and less leftover ejaculate thereby improving efficiency, increasing yield, and improving sample to sample production predictability.
  • 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.
  • the discriminating agent is a plurality of magnetic beads coated with, or on which is disposed, a binding agent.
  • the binding agent is an acrosome associated molecule.
  • magnetic beads are verified through a quality control process. Beads from verified bead stock are aliquoted and washed to remove potentially harmful or toxic storage fluid. Washed beads are added to a sample, such as anon-human mammalian semen sample, and incubated. Magnetic beads are then removed from the sample by a magnet prior to processing the sample on a sexing instrument, such as a droplet or microfluidic chip- based flow cytometer.
  • a sexing instrument such as a droplet or microfluidic chip- based flow cytometer.
  • magnetic beads are used to remove dead cells before a sample is processed on a sexing instrument.
  • Running dead cells, acrosome reacted cells, or other damaged, malformed, or otherwise undesirable cells through the sexing instrument constitutes a wasted effort as dead cells do not contribute to a final, salable product (i.e., a straw of sexed non-human mammalian semen) and do not contribute to the number of straws produced by a sexing run.
  • Removing dead cells both improves the number of cells that are produced from the same amount of instrument time and labor using a set amount of a semen sample, and normalize bulls of differing quality (e.g., number of dead cells in a semen sample) to provide a more consistent and predictable sexing process.
  • 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. The acrosome is typically not accessible to lectins, but in dead cells, cells with malformed heads/acrosomes, and spontaneously capacitated cells, lectin binding is high.
  • LCA Lens culinaris agglutinin 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.
  • the acrosome is typically not accessible to lectins, but in dead cells, cells with malformed heads/acrosomes, and spontaneously capacitated cells, lectin binding is high.
  • a magnet is
  • a 5 percentage point reduction in dead cells in a sample equates to roughly a 1 percentage point increase in the number of eligible cells in the sample which provides an approximately 3 percent increase in instrument output for a sexing instrument.
  • This improvement improves efficiency and reduces the cost of goods for a sexing operation. This improvement further reduces instrument downtime as a result of samples comprising too many dead cells to be effectively processed.
  • a method of improving semen sample quality comprising: discriminating between live and dead sperm cells in a mammalian semen sample comprising sperm cells based on the exposure or availability of an acrosome associated molecule.
  • 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. [0015] In some embodiments, the discriminating further comprises: collecting the sperm cells not bound to the binding agent.
  • the discriminating further comprises: collecting the sperm cells bound to the binding agent thereby removing the dead sperm cells from the sample.
  • the binding agent is a polysaccharide binding agent.
  • the binding agent is an agglutinin.
  • the binding agent is a lectin.
  • 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.
  • the agglutinin is derived from an organism selected from the group consisting of Lens culinaris and Pisum sativum.
  • the binding agent is bound to a solid support.
  • the solid support is selected from the group consisting of a bead, a resin, and agarose.
  • the solid support is a magnetic bead.
  • the solid support is coated in the binding agent.
  • the solid support is a magnetic bead coated in an agglutinin.
  • the solid support is a magnetic bead coated in Lens culinaris agglutinin (LCA).
  • the binding agent comprises a binding moiety.
  • the binding moiety is biotin.
  • 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.
  • 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 mo
  • the molecule that binds to the binding moiety is selected from the group consisting of an antibody, avidin, streptavidin, and nickel.
  • 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.
  • the beads magnetic beads, microbeads, or affinity beads.
  • the beads are coated in the binding agent.
  • the beads are magnetic beads and the separating comprises exposing the mixture to a magnet.
  • the beads are heterogenous in size.
  • the beads are immunomagnetic beads and the separating comprises running the mixture over a ferromagnetic resin column. [0039] In some embodiments, the separating comprises passing the mixture through an opening smaller than the beads, but larger than the unbound sperm.
  • the discriminating comprises: contacting the sample with an agglutinin to form a semen-agglutinin mixture; and separating sperm cells bound to the agglutinin from sperm cells not bound to the agglutinin.
  • the method further comprises collecting the sperm cells not bound to the agglutinin.
  • the method further comprises removing the cells bound to the agglutinin from the mixture.
  • 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.
  • the semen is bovine semen.
  • the semen is Bos taurus semen.
  • the semen is porcine semen.
  • the semen is Sus scrofa semen.
  • the semen is sexed semen.
  • the method further comprises sex selecting the semen.
  • the method further comprises: measuring the sperm cell concentration of the semen; staining the cells at 200 M/mL if the sperm cell concentration is greater than 1000 M/mL, or at 125M/mL - 150 M/mL if the concentration is less than 1000 M/mL; and sex selecting the semen.
  • the method further comprises: measuring the sperm cell concentration of the semen; staining the cells at 200 M/mL if the sperm cell concentration is greater than 1000 M/mL, or at 150 M/mL if the sperm cell concentration is less than 1000 M/mL; and sex selecting the semen.
  • the method further comprises: measuring the sperm cell concentration of the semen; staining the cells at 200 M/mL if the sperm cell concentration is greater than 1000 M/mL, or at 125 M/mL if the sperm cell concentration is less than 1000 M/mL; and sex selecting the semen.
  • the method further comprises: measuring the sperm cell concentration of the semen; staining the cells at 200 M/mL if the sperm cell concentration is greater than 600 M/mL, or at 125M/mL - 150 M/mL if the concentration is less than 600 M/mL; and sex selecting the semen.
  • the method further comprises: measuring the sperm cell concentration of the semen; staining the cells at 200 M/mL if the sperm cell concentration is greater than 600 M/mL, or at 150 M/mL if the sperm cell concentration is less than 600 M/mL; and sex selecting the semen.
  • the method further comprises: measuring the sperm cell concentration of the semen; staining the cells at 200 M/mL if the sperm cell concentration is greater than 600 M/mL, or at 125 M/mL if the sperm cell concentration is less than 600 M/mL; and sex selecting the semen.
  • the sex selecting the semen comprises discriminating sperm cells within the semen based on the expression or activity of a Toll-like receptor (TLR).
  • TLR Toll-like receptor
  • the discriminating comprises contacting the semen with an anti-TLR7 antibody. [0058] In some embodiments, the discriminating comprises contacting the semen with a
  • the discriminating comprises contacting the semen with a compound selected from the group consisting of Resiquimod, Imiquimod, and SM360320. [0060] In some embodiments, the discriminating comprises contacting the semen with a TLR agonist.
  • the TLR agonist is a TLR7 agonist.
  • the TLR agonist is a TLR8 agonist.
  • the TLR agonist a TLR7 agonist and a TLR8 agonist.
  • a method of improving sexed semen sample quality 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; collecting the supernatant; contacting the supernatant with a TLR binding molecule; 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.
  • 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.
  • the sexing comprises droplet sorting, mechanical sorting, acoustic, electrophoretic, diversion, radiation pressure, optical tweezers, or laser ablation.
  • a method of processing a semen sample to prepare the sample for a sexing operation the method comprising: contacting a mammalian semen sample with a staining media and a Slo3 antagonist.
  • the method further comprises sexing the semen sample.
  • the Slo3 antagonist is clofilium tosylate.
  • the contacting the semen sample with the Slo3 antagonist improves the uptake of a dye in the staining media by sperm cells in the semen sample.
  • the improvement in the uptake of the dye is a reduction in time required to stain the sperm cells in the semen sample.
  • the improvement in the uptake of the dye is an increase in an amount of the dye bound to DNA in the sperm cells in the semen sample.
  • the improvement in the uptake of the dye is an increase in the amount of dye by the sperm cells in the semen sample per unit time.
  • the method further comprises 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.
  • FIG. 1 provides a histogram representing instrument output of a sexing instrument evaluating a semen sample processed using magnetic beads according to one embodiment.
  • FIG. 2 provides a box-plot graph representing a percentage of eligible cells in a semen sample for a sample processed using magnetic beads as compared to control according to one embodiment.
  • FIG. 3 provides a box-plot graph representing a percentage of dead cells in a semen sample for a sample processed using magnetic beads as compared to control according to one embodiment.
  • the present teachings describe 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 sperm cells that have prematurely undergone capacitation or are otherwise unable to fertilize an ovum.
  • the present teachings provide for and include discriminating between active and acrosome reacted cells on the basis of differential binding of a binding agent.
  • an acrosome associated molecule is able to bind to the binding agent following capacitation, sperm activation, sperm cell membrane compromise, or sperm cell death, but is not accessible or available to be bound to an active, healthy sperm.
  • the ability of the binding agent to bind to a sperm is dependent on the sperm being incapable of fertilizing an ovum.
  • the present teachings utilize this difference in binding in order to separate fertilization competent sperm cells from incompetent sperm cells.
  • the separation can comprise collecting the cells bound to the binding agent or the cells not bound to the binding agent.
  • the binding agent can be a protein, a polysaccharide, a lipid, a nucleic acid, an ion, or an inorganic compound.
  • the protein can be an antibody, a natural ligand, an enzyme, an agglutinin or a lectin.
  • the binding agent is a biomimetic.
  • 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 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. Many lectins are commercially available from vendors such as Milbpore Sigma (formerly Sigma Aldrich).
  • the binding agent may be bound to a solid support.
  • 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.
  • 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.
  • the resin can be an ionic exchange resin, an immunoresin, or a size exclusion resin.
  • the bead can be a magnetic bead, an immunobead, an affinity bead, or an ionic bead.
  • the solid support can be a plurality of beads.
  • the plurality of beads can include beads of heterogenous sizes.
  • the beads can comprise, be coated in, or be conjugated to the binding agent.
  • the bead can comprise, be coated in, or be conjugated a molecule that recognizes a binding moiety on the binding agent.
  • 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 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, or an Xpress tag.
  • Other affinity tags are known in the art.
  • Binding moieties can be recognized by a binding partner that can be bound to a solid support.
  • a binding partner that can be bound to a solid support.
  • biotinylated agglutinin can bind to both an acrosome associated molecule and to streptavidin or avidin coated beads.
  • a his-tagged lectin can bind to both an acrosome associated molecule and to nickel beads.
  • affinity columns are commercially available and well known in the art.
  • Skilled artisans will recognize that there are several avenues available to remove the beads bound to dead sperm from the live sperm, such as, but without limitation, centrifugation, immunoprecipitation, a membrane or porous filter, magnetism, column separation, or allowing the beads to settle gravitationally.
  • Magnetic beads can be separated from solution by applying a magnetic field or running the mixture over a ferromagnetic column. Affinity beads are usually placed on a column with a filter of pore size larger than the sperm cells but smaller than the beads, so that unbound sperm cells flow through and while sperm cells bound to beads cannot.
  • Other methods of separating bound and unbound beads can include filters or size exclusion columns.
  • 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 epididymal semen. Methods of collecting both types of semen are known in the art.
  • 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.
  • 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 sexed using a DNA dye, the dye is typically the fluorescent dye Hoechst 33342, although other non toxic, biocompatible dyes are known in the art and may be used.
  • 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 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.
  • the increase in eligibility due to the clean-up process above leads to an increase in the efficiency of production of sex selected semen.
  • sex selection is performed at 66 million cells per mL (M/mL).
  • cells may be stained at a concentration of 200 M/mL and then diluted in a buffer or extender to a concentration of 66 M/mL for sexing.
  • the present inventors have determined that this concentration is efficient for non-extended sperm cell samples having initial cell concentrations greater than or equal to 1000 M/mL.
  • non-extended sperm cells samples having initial concentrations lower than about 1000 M/mL, for example between 800-1000 M/mL the eligibility of samples after staining is reduced.
  • non-extended samples having a cell concentration of 800-1000 M/mL having a cell concentration of 800-1000 M/mL, higher eligibility can be achieved by staining and sex selecting at a concentration of 125-150 M/mL, or at a concentration of 140 M/mL.
  • the concentration may be at least 125 M/mL, at least 130 M/mL, at least 135 M/mL, at least 140 M/mL, at least 145 M/mL, or at least 150 M/mL.
  • the present inventors have further determined that while this concentration is efficient for extended sperm cell samples having initial cell concentrations greater than or equal to 600 M/mL.
  • the eligibility of samples after staining is reduced.
  • higher eligibility can be achieved by staining and sex selecting at a concentration of 125-150 M/mL, or at a concentration of 140 M/mL.
  • the concentration may be at least 125 M/mL, at least 130 M/mL, at least 135 M/mL, at least 140 M/mL, at least 145 M/mL, or at least 150 M/mL.
  • Propidium iodide is added and the total DNA content is determined.
  • Cell concentration may also be determined by standard microscopic or optical density techniques. Concentration and progressive motility of the sample may be further evaluated using a computer assisted semen analysis (“CASA”) such as an integrated visual optical system (“IVOS”).
  • CASA computer assisted semen analysis
  • IVOS integrated visual optical system
  • the present invention provides compositions and methods for enhancing the staining of sperm cells, for example with Hoechst 33342.
  • sperm cells may be exposed to one or more stain-enhancing compounds (“SECs”).
  • SECs according to the present invention include, but are not limited to, Slo3 inhibitors, Slo3 activators, multiple drug resistance (“MDR”) protein inhibitors, and MDR activators.
  • Slo3 activators include, but are not limited to, compounds that cause or promote a decrease in the concentration of protons within the sperm (re. an increase of the pH inside the cell), or change the levels of calcium ions.
  • Slo3 inhibitors include compounds that decrease or inhibit the function of the Slo3 potassium channel, in particular barium, mibefradil, clofilium (e.g., clofilium tosylate), profolol, 4-aminopyridine, progesterone, Charybdotoxin, Penitrem A, and/or quinidine.
  • Cells may be exposed to one or more SECs before staining or during staining.
  • procedures are provided for improving the staining of sperm cells; these procedures may comprise exposing sperm cells to a Slo3 inhibitor during a staining or dying process to improve the uptake of a dye in a staining media or solution.
  • the improvement in the uptake of the dye may be an improvement in the quantity of the dye bound to DNA within the sperm cells, or may be a reduction in the amount of time required to achieve a desired level of staining of the sperm cells, or may be an increase in the quantity of the dye bound to the sperm cells per unit time.
  • Other improvements from contacting a semen sample with a Slo3 inhibitor during a dying or staining process include an observable left shift Hoechst intracellular accumulation curve when interrogated cells are examined in a flow cytometry instrument or fluorescent plate reader, a suppressed Resazurin signal, and an observable increase in the percentage of progressively motile cells.
  • the present teachings also provide for and include a method of producing sex selected mammalian livestock comprising: discriminating sperm cells within the semen based on the expression or activity of a Toll-like receptor (TLR).
  • TLR Toll-like receptors are pattern recognition receptors expressed on the surface of immune cells, the activation of which can provoke an immune response (Chi, H., et al., Frontiers in Pharmacology, 2017, 8, article 304).
  • TLR7 and TLR8 are X- linked receptors that have been recently demonstrated to be expressed on the cell surface of X-chromosome bearing sperm (Umehara, T. et al., PLoS Biol, 2019, 17, e3000398).
  • a TLR binding molecule can include a TLR agonist, a TLR ligand, an anti-TLR antibody, or another molecule that binds to TLR. This binding can be reversable or irreversible.
  • a TLR agonist is a drug or other small molecule that binds to and activates a TLR receptor.
  • TLR agonists can include, for example and without limitation, an Imidazoquinoline compound, an adenine analog, a nucleotide base analog, a guanosine analog, a thymidine homopolymer, or a benzyoazepine analog.
  • TLR agonists include, for example and without limitation, Imiquimod, Resiquimod, gardiquimod, SM360320, SM- 27600, 852A, CL075, CL097, CL264, CL307, 2-amin-5-bromo-6-phenyl-4(3)-pyrimidinone, 8-(3-(pyrrolidin-l -ylmethyl) benzyl)-4-amino-2-butoxy-7,8-dihydropteridin-6(5H)-one], and 3M-052.
  • Imiquimod, Resiquimod, and SM360320 can be fluorescently labeled (Russo, C., et al., Blood, 117, 2011, 5683-5691).
  • TLR binding molecules include anti-TLR antibodies.
  • anti-TLR antibodies are commercially available (for example from ThermoFisher Scientific), and methods of producing TLR antibodies are known in the art.
  • TLR binding molecules including TLR agonists and anti-TLR antibodies can be labelled to comprise a tag.
  • fluorophores such as and without limitation, [5(6)]- carboxytetramethylrhodamine and ALEXA FLUORTM 488 (ThermoFisher Scientific, Waltham, MA), are available commercially and suitable for use with the TLR methods of the present teachings.
  • a third possibility for discriminating between X- and Y-chromosome bearing sperm is performing a modified version of the classic swim-up procedure.
  • motile Y-chromosome bearing sperm can be separated from X-chromosome bearing sperm bound to a TLR binding molecule, referred to herein as ligand-impaired X- chromosome bearing sperm.
  • Swim-up procedures make use of the natural tendency of sperm to swim against gravity and are known in the art as a method of separating live sperm cells from dead sperm cells.
  • semen in a diluent comprising a TLR binding molecule forms a bottom layer.
  • a middle layer is comprised by the diluent and the TLR binding molecule.
  • a top layer is comprised by the TLR binding molecule and culture media.
  • the TLR binding molecule binds to the TLR on the surface of an X-chromosome bearing sperm, impeding its movement. Therefore, the majority of sperm that make it to the top layer will be Y-chromosome bearing sperm, and this layer will have Y-skew. In contrast, the bottom layer will be largely X-chromosome bearing sperm and therefore have X-skew.
  • Other motility-based assays can also be used to separate sperm cells based on differential effects on motility due to binding of TLR receptors, including, for example, rheotaxis-based separation.
  • An additional approach of the present invention is Y-skewing semen by contacting the semen with a TLR ligand that irreversibly binds to TLR, thus permanently binding the ligand to the X-chromosome bearing sperm cells, preventing them from fertilizing an ovum.
  • the semen comprising this irreversibly bound ligand would therefore be Y-skewed with no further processing.
  • TLR sex selection can be performed in conjunction with other sex selection processes to boost overall skew.
  • the TLR process can be run before or after the other process.
  • a person of ordinary skill in the art would understand that various procedures could be combined to further improve semen quality. Overall, improved semen quality input leads to higher yield of sexed semen. Therefore, multiple steps to improve semen quality can lead to improved yield of the overall process.
  • a semen processing method of improving sexed semen quality may comprise contacting a semen sample with magnetic beads coated in Lens culinaris agglutinin (LCA) to form a semen-bead mixture, exposing the semen-bead mixture to a magnetic field to separate cells bound to the LCA from cells not bound to the LCA, collecting the supernatant, contacting the supernatant with a TLR binding molecule, separating the bound cells from the unbound cells to form an X fraction and a Y fraction, and, finally, subjecting either fraction to cytometric sex selection. Alternately, cytometric sex selection could be performed prior to TLR sex selection.
  • LCA Lens culinaris agglutinin
  • magnetic beads coated with a binding agent are used to bind to proteins associated with dead, damaged, malformed, deformed, or otherwise undesirable sperm cells in a sample of sperm cells such that the cells to which the magnetic beads are bound may be removed from the remainder of the sample by a magnet.
  • lectin conjugated magnetic beads are introduced to or mixed with a sperm sample. The lectin on the magnetic beads targets, directs, or binds the magnetic beads to dead or acrosome reacted sperm cells in the sperm sample.
  • a magnet such as a rare earth magnet, is used to remove or sequester dead or acrosome reacted bound cells such that live, healthy, or viable cells can be aliquoted, transferred, or otherwise removed or separated from the undesired, dead, or acrosome reacted cells bound to the magnetic beads.
  • Bead preparation 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.
  • concentration of Cells 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.
  • 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.
  • Hoechst 33342 stain is added to 60 pg/mL (10 pL of 5 mg/mL stock per 1 mL of sample volume). 10 pL of beads are added for each mL of sample.
  • Staining TALP is added to the final volume needed. The mixture is incubated at 34 ° C for 45 minutes. Two volumes of Red TALP is then added to each tube.
  • the tubes are then placed on a magnetic rack for 15 minutes. The supernatant is collected, passed through a 50 pm filter in aliquots, and then sex selected as described below. [0099] 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, 25,0000 cells/sec, 30,000 cells/sec, 35,000 cells/sec,
  • 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.
  • a gate is placed to collect the X chromosome cells, which is 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 %.
  • 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.
  • the sample is run to collect between 300 and 400 mL 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.
  • 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.
  • 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.
  • TLR Sexing Media The following are media recipes for use in TLR sex selection protocols of the present teachings:
  • the ingredients are mixed and the medium is passed through a 0.45-pm filter and then incubated in a CCh incubator at 37° C for at least 12 h.
  • TLR Sex Selection Sample Prep. All supplies and media are prewarmed to between 34-37 ° C. Frozen semen straws from the same batch are thawed and pooled. Samples are pelleted at 700 x g for 5 minutes. Sample are washed 2 times with mHTF. Motility and concentration are determined using the standard CASA methods. [0110] TLRXY or Y Enrichment. Samples are resuspended in 3 mL mHTF + 500 mM
  • Creatine + 0.03 mM Resiquimod (R848; Novus Biologicals) and then incubated for 60 minutes at 37 ° C. The top 1 mL is collected, spun at 400 x g for 5 minute at 37 ° C, and then washed in mHTF and the spin is repeated. The washed sperm are then resuspended at lxlO 7 sperm/mL in mHTF.
  • An IVOS is used to evaluate motility, such as described herein, and skew is determined using ddPCR (see, e.g., U.S. Pat. No. 10961577, issued 30 MAR 2021, which is incorporated by reference herein in its entirety).
  • TLR XX Enrichment Samples are resuspended in 3 mL mHTF + 0.03 pM R848 and then incubated for 30 minutes at 37 ° C with 5% CCh. The bottom 1 mL is collected and layered onto a fresh 2 mL mHTF + 0.03 pM R848. This mixture is then incubated 10 minutes at 37 ° C with 5% CCh. The bottom 1 mL is collected, spun at 400 x g for 5 minutes at 37 ° C, then washed in mHTF. After respinning, the sperm is resuspended at 1 x 10 7 sperm/mL in mHTF. An IVOS is used to evaluate motility, such as described herein, and skew is determined using ethidium monoazide (EMA) and ddPCR (see. e.g., U.S. Pat. No.
  • EMA ethidium monoazide
  • This example illustrates a dead cell clean-up process of the present teachings.
  • Raw semen samples from four bulls were split. Half of each sample was stained as raw ejaculate (control). The other half was mixed with LCA-Biotin conjugate and incubated for 10 minutes at 19° C. Stain-TALP buffer bearing streptavidin coated magnetic beads was added, and the mixture was incubated for 45 minutes at 34 ° C. The mixture was then incubated in a magnetic rack for 5 minutes to pellet the magnetic beads and any bound sperm cells. The supernatant was then transferred to Red TALP to create a stained sample.
  • Table 1 Mean percentage dead cells and eligible cells in control and clean-up samples. [0116] The number of the dead cells in the cleaned-up samples was significantly reduced.
  • Eligibility and dead percent were determined for the samples processed in Example 2. The results are shown in Table 2. Table 2. Percent eligible and dead semen cells from samples processed according to Example 2.
  • This example illustrates TLR sexing for enrichment of Y-chromosome bearing sperm.
  • Example 7 This example illustrates TLR sexing for enrichment of X-chromosome bearing sperm.
  • the mixture will be incubated at 34 ° C for 45 minutes. 8 mL of Red TALP will be added, and then the tubes will be placed on a magnetic rack for 15 minutes to pellet the beads. The supernatant will be collected, passed through a 50 pm filter into 2.1 mL aliquots. The sample will then be sex selected as described above at a throughput of 17500 cells/sec. The 2% increase in eligibility expected from the use of this process would lead to a 10% increase in straw production for the batch.
  • This example illustrates an alternative commercial method of sex selection using a bead clean-up step.
  • Raw ejaculate of a bull will be collected according to methods known in the art. Concentration will be measured as above, and the concentration will be 500 M/mL. 750 pL of raw ejaculate (for a final concentration of 125 M/mL in 3 mL) will be mixed with 36 pL of 5 mg/mL Hoechst 33342 stain and 30 pL of Pisum sativum lectin-conjugated magnetic beads will be added. The total volume will be brought up to 3 mL using 2.22 mL of staining TALP. The mixture will be incubated at 34 ° C for 30 minutes.
  • This example illustrates bead-clean up in conjunction with multiple modes of XX sex selection.
  • Raw ejaculate of a bull will be collected, spun down at 700 x g for 5 minutes, washed once in mHTF, and resuspended in 3 mL of mHTF. 30 pL of LCA beads will be added to the sample, which will then be incubated at 34 ° C for 30 minutes. The tube will then be placed on a magnetic rack to remove the beads. The supernatant will be collected, spun at 700 x g for 5 minutes, and then resuspended in the treatment for TLR XX sex enrichment as described above. Cell concentration will then be determined to be 750 M/mL.
  • Cells will then be diluted to 200 M/mL in staining TALP containing 60 pg/mL Hoest stain in a total volume of 4 mL. Cells will be stained at 37 ° C for 45 minutes. 8 mL of red TALP will be added, and then passed through a 50 pm filter and subjected to cytometric sex selection.
  • This example illustrates bead clean-up in conjunction with multiple modes of XX sex selection.
  • Raw ejaculate of a bull will be collected according to methods known in the art. Concentration will be measured as above, and the concentration will be 500 M/mL. 750 pL of raw ejaculate (for a final concentration of 125 M/mL in 3 mL) will be mixed with 36 pL of 5 mg/mL Hoechst 33342 stain and 30 pL of LCA-conjugated magnetic beads will be added. The total volume will be brought up to 3 mL using 2.22 mL of staining TALP. The mixture will be incubated at 34 ° C for 30 minutes. 6 mL of Red TALP will be added, and then the tubes will be placed on a magnetic rack for 15 minutes to pellet the beads.
  • the supernatant will be collected and passed through a 50 pm filter into 2.9 mL aliquots.
  • the sample will then be sex selected at 23,000 cells/second as described above.
  • the cells will be pelleted at 700 x g for 5 minutes, washed twice with mHTF, and then TLR XX enrichment is performed as described above.
  • This example illustrates the use of a Slo3 channel inhibitor in a dyeing or staining process in conjunction with methods for sex selection.
  • Raw ejaculate of a bull will be collected according to methods known in the art. Hoechst 33342 stain will be added to achieve a final stain concentration of 12.5 pL/mL and a pH constant buffer will be added to achieve a sperm concentration of 150-200 M/mL. An effective amount of a Slo3 channel inhibitor, such as clofilium tosylate, will be added to the mixture to improve the dye uptake and other characteristics of the stained semen sample, wherein the effective amount is 0.2-250 micromolar. The sample will be incubated at 34-37 °
  • a volume of red TALP equal to twice the volume of the sample and staining media will be added, and 3 mL volumes of the mixture will be aliquoted into separate sample tubes. The aliquoted samples will be used in a sex selection process.
  • the administration of the Slo3 channel inhibitor is expected to improve the dye uptake of the semen sample.
  • This example illustrates the preparation and use of LCA conjugated magnetic beads to improve the quality of a sperm cell sample.
  • All staining TALP is aspirated from each sample tube in a single aliquot, and the washing step is repeated. After the second washing, each tube is filled with staining TALP and vortexed to homogenize the magnetic beads in the staining TALP.
  • the washed beads in staining TALP are aliquoted into 1.5 mL microcentrifuge tubes and stored at 4 ° C.
  • the beads are ideally fully resuspended after each vortexing step. This is important to achieve a consistent bead concentration.
  • the magnetic beads may cluster on the side of the tube especially after being attracted by the magnet. After vortexing beads vigorously for 6 seconds, the tube is visually inspected to identify any beads clustered on the side of the tube. If clustered beads are identified, vortexing is repeated for another 6 seconds. If vortexing splashes some beads to the cap of the tube, a pipette is used to transfer the volume from the cap back to the tube.
  • a semen sample is prepared by first removing one or more aliquots of washed beads from storage and allowing the aliquots to equilibrate to room temperature for at least 30 minutes. A volume of 10 microlitres of washed beads is added to each 1 mL of semen sample. If more beads than are required are mistakenly added into the staining reaction, no issue will occur unless an amount in excess of five times the required amount has been added. An amount of washed beads greater than five times the required amount will have an adverse effect on instrument performance when sexing the semen sample. If fewer than the required amount of beads is added to the sample, the sample is not adversely impacted, but an insufficient amount of dead cells will be removed from the sample to improve instrument performance when sexing the semen sample.
  • sample tube In a 50 mL sample tube, raw ejaculate (semen sample) is first added to the tube, followed by a volume of staining TALP, the volume of washed magnetic beads, and a volume of DNA intercalating dye, which is Hoechst 33342. The sample is then incubated at 34 ° C for 45 minutes. After incubation, the sample tube is placed in a magnetic tube rack with the cap loosely covering the tube. After 15 minutes on the magnetic tube rack, a serological pipette is used to remove the incubated, stained sample from the tube and the sample is aliquoted into 5 mL sample tubes. The sample tube is ideally not rotated or moved as the sample is being aspirated from the tube, or else the sample in the tube must be vortexed and placed back in the magnetic tube rack for an additional 15 minutes.
  • the sample is ideally removed at once in a single aliquot by the serological pipette. Multiple draws may not be performed as multiple draws of the sample will re-distribute the magnetic beads into the sample. A volume of up to 500 microlitres is left in the bottom of the 50 mL tube to prevent beads from being disturbed or being introduced into the aliquoted sample. If beads are aliquoted with the sample, the sample is placed back into the tube and placed on the magnetic rack for 15 minutes.
  • This example illustrates the validation of LCA conjugated magnetic beads to improve the quality of a sperm cell sample.
  • First a new lot of magnetic beads is prepared by washing the magnetic beads at least twice in a staining solution.
  • a sample of quality controlled (previously evaluated for characteristics such as motility, morphology, and concentration) non-human mammalian semen is provided and prepared for staining.
  • the sample is stained with a staining solution and magnetic beads, and the magnetic beads are removed.
  • the percentage of eligible cells (the number of live, processable cells out of a total number of cells in the sample), a percentage of dead cells, and the peak to valley ratio for interrogated cells are compared to baseline number (minimum acceptable values for those measurements) to determine if the magnetic beads have met quality control standards.
  • a split-batch comparison on a single instrument may be used to compare magnetic bead processed semen sample characteristics to samples not processed by magnetic beads.
  • This example illustrates the preparation and use of LCA conjugated magnetic beads to improve the quality of a sperm cell sample.
  • a sample of LCA conjugated magnetic beads was prepared and used in a semen sample staining process to produce improved results (instrument and sample metrics) compared to a semen sample that was not processed with magnetic beads.
  • Magnetic beads were washed and prepared for use in processing the sample.
  • Materials required were 1000 microliter filtered pipette tips, 1.7 mL microcentrifuge tubes, 15 mL tubes, 50 mL tubes, tube labels, a P1000 pipette, a 50 mL conical magnetic rack, and a vortex.
  • Reagents required were staining TALP and LCA conjugated magnetic beads in a storage buffer.
  • Tubes were first labeled as appropriate indicating stock and lot numbers, dates, and associated volumes for the containers.
  • a stock bottle 100 mL bottle of beads in a shipment buffer was removed from storage at 4 ° C and then vortexed at 3000 rpm for 6 seconds to fully resuspend the beads heterogeneously in the buffer.
  • the stock bottle was inverted 5 times and revortexed at 3000 rpm for 6 seconds.
  • a bead volume to be washed was calculated using the formula (([number of instruments]* [volume of semen sample per tube]* [number of sample preparations per day]*[0.010 mL of washed beads])* ([number of days until staining media expiration])).
  • a volume of magnetic beads in the shipment buffer was aliquoted into 50 mL tubes, with a maximum of 35 mL in each tube.
  • Staining TALP was removed from storage at 4 ° C.
  • the volume of staining TALP used was three times (3x) the volume of magnetic beads aliquoted into the 50 mL tubes.
  • Each 50 mL tube of magnetic beads in shipment buffer was vortexed at 3000 rpm for 6 seconds and then placed in a magnetic rack for 15 minutes with the tube cap loosely secured or placed on the tube. After 15 minutes, the liquid volume in each tube was removed in a single aliquot by serological pipette.
  • each tube was removed from the magnetic rack and a volume of staining TALP equal to the initial wash volume was added to each tube. The process of vortexing, aspirating the liquid volume, and adding staining TALP was repeated a second time.
  • the washed magnetic beads in staining TALP were then ready to be aliquoted into 1.7 mL microcentrifuge tubes for storage. Each 50 mL tube was vortexed at 3000 rpm for 6 seconds. 1 mL of the washed beads in staining TALP was aliquoted into each 1.7 mL microcentrifuge tube. The 50 mL tube was revortexed after every 10 aliquots removed from the tube.
  • the washed beads in the 1.7 mL microcentrifuge tubes were stored at 2-4 ° C until ready to be used in the staining process.
  • washed beads were used in the processing of non-human mammalian semen (bovine semen).
  • Materials required were 5 mL tubes, 50 mL tubes, 200 microliter filtered pipette tips, 1000 microliter pipette tips, 5 mL serological pipette, 10 mL serological pipette, 25 mL serological pipette, 50 micron filters, microcentrifuge, water bath at 34 ° C, 200 microliter pipette, 1 mL pipette, heat block at 34 ° C, vortex mixer, wipes, disinfecting wipes, and an incubator at 19 ° C.
  • Reagents required were Hoechst 33342 stock, staining TALP (biocompatible salt buffer media), isopropyl alcohol, and washed magnetic beads.
  • a sample of collected semen was obtained and placed in the incubator at 19 ° C in a 50 mL tube.
  • Staining TALP was warmed on the heat block for at least 30 minutes.
  • Staining TALP was drawn into a 10 mL pipette tip and dispensed into the 50 mL tube with the semen sample by dispensing the staining TALP down the side of the tube.
  • a volume of washed beads (10 microliters per 1 mL of semen sample) was aliquoted by pipette and dispensed into the 50 mL tube of semen and staining TALP.
  • the pipette was washed by drawing the contents of the 50 mL tube into the pipette and dispensing the contents into the tube.
  • Room temperature Hoechst 33342 was added to the 50 mL tube.
  • the tube was vortexed at 1350 rpm for 10 seconds and then placed in the water bath to incubate at 34 ° C for 45 minutes.
  • the tube was placed into a magnetic rack after incubation. The top of the tube was loosened but remained on the tube. The tube remained in the magnetic rack for 15 minutes. The entire usable volume of the 50 mL tube was removed via serological pipette with special care taken to not disturb the magnetic beads, and the volume was aliquoted into 5 mL tubes through the filter.
  • the percentage of dead cells in the sample is reduced (i.e., with respect to control).
  • processing the sample with magnetic beads improved the percentage of eligible cells (percentage of live cells out of total cells in the sample that may be sexed by the instrument) by approximately one (1) percentage point, or approximately 3 percent over control.
  • processing the sample with magnetic beads reduced the percentage of dead cells in the sample by approximately 5 percentage points.
  • the reduction in the percentage of dead cells and the improvement in cell eligibility improved the number of straws of sexed semen that were produced per instrument, per hour of instrument run time, and therefore also improved the number of straws produced per instrument, per day of instrument run time.
  • a method of improving semen sample quality comprising: discriminating between live and dead sperm cells in a mammalian semen sample comprising sperm cells based on the exposure or availability of an acrosome associated molecule.
  • the method 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.
  • the method further comprises collecting the sperm cells bound to the binding agent thereby removing the dead sperm cells from the sample.
  • the binding agent is selected from the group consisting of an agglutinin, a lectin, and a polysaccharide binding agent.
  • the agglutinin is derived from an organism selected from the group consisting of Lens culinaris, Ricinus communis, Arachis hypogaea, Glycine max, Bandeiraea simplicifolia ( Griffonia simplici folia), Dolichos biflorus, Erythrina cristagalli, Helix pomatia, Lycopersicon esculentum, Phaseolus vulgaris, Pisum sativum, Sambucus nigra, Triticum vulgaris, Ulex europaeus, and Wisteria floribunda.
  • the agglutinin is derived from an organism selected from the group consisting of Lens culinaris and Pisum sativum.
  • binding agent is bound to a solid support.
  • the binding agent is bound to a solid support selected from the group consisting of a bead, a resin, and agarose.
  • the solid support is a magnetic bead.
  • the solid support is coated in the binding agent.
  • the solid support is a magnetic bead coated in Lens culinaris agglutinin (LCA).
  • the binding agent comprises a binding moiety.
  • the binding moiety is biotin.
  • 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.
  • 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.
  • molecule that binds to the binding moiety is selected from the group consisting of an antibody, avidin, streptavidin, and nickel.
  • 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.
  • the beads are selected from the group consisting of magnetic beads, microbeads, and affinity beads.
  • the beads are magnetic beads and the separating comprises exposing the mixture to a magnet.
  • the beads are immunomagnetic beads and the separating comprises running the mixture over a ferromagnetic resin column.
  • the discriminating comprises: contacting the sample with an agglutinin to form a semen-agglutinin mixture; and separating sperm cells bound to the agglutinin from sperm cells not bound to the agglutinin. [0186] In various embodiments, the method further comprises collecting the sperm cells not bound to the agglutinin.
  • the method further comprises removing the cells bound to the agglutinin from the mixture.
  • 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.
  • the semen is non-human mammalian semen selected from the group consisting of bovine semen, and porcine semen.
  • the method further comprises sex selecting the semen.
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

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Abstract

La présente invention concerne de manière générale des procédés d'amélioration de la qualité d'un échantillon de cellules reproductrices. Les procédés comprennent l'amélioration de la qualité d'échantillons de cellules de sperme de mammifère par la discrimination effectuée entre des cellules de sperme vivant et mort dans un échantillon de sperme obtenu à partir de bétail, tel que des bovins ou des animaux porcins. Les procédés comprennent l'amélioration de la qualité des échantillons de cellules reproductrices sur la base de l'exposition ou de la disponibilité d'une molécule associée à l'acrosome par mise en contact de l'échantillon avec un agent de liaison qui se lie à la molécule associée à l'acrosome pour former un mélange de la semence et de l'agent de liaison et la séparation des spermatozoïdes liés à l'agent de liaison à partir de cellules spermatiques non liées à l'agent de liaison.
PCT/US2022/074287 2021-07-30 2022-07-29 Procédés d'amélioration de la qualité d'un échantillon de cellule reproductrice WO2023010101A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995016916A1 (fr) * 1993-12-17 1995-06-22 The University Of Melbourne Procedes et reactifs pour l'evaluation de sperme
US5665582A (en) * 1990-10-29 1997-09-09 Dekalb Genetics Corp. Isolation of biological materials
US5897988A (en) * 1998-01-21 1999-04-27 Yale University Process and system for selection of mature sperm by surface membrane determinants for assisted reproduction
US20060199231A1 (en) * 2004-11-04 2006-09-07 Moore Sean C Detection of activation of endothelial cells as surrogate marker for angiogenesis
US20170137773A1 (en) * 2015-11-13 2017-05-18 Hams-Werner Heinrich Cell strainer with functionalized mesh to separate biological components

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5665582A (en) * 1990-10-29 1997-09-09 Dekalb Genetics Corp. Isolation of biological materials
WO1995016916A1 (fr) * 1993-12-17 1995-06-22 The University Of Melbourne Procedes et reactifs pour l'evaluation de sperme
US5897988A (en) * 1998-01-21 1999-04-27 Yale University Process and system for selection of mature sperm by surface membrane determinants for assisted reproduction
US20060199231A1 (en) * 2004-11-04 2006-09-07 Moore Sean C Detection of activation of endothelial cells as surrogate marker for angiogenesis
US20170137773A1 (en) * 2015-11-13 2017-05-18 Hams-Werner Heinrich Cell strainer with functionalized mesh to separate biological components

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MOUTAFFIAN ET AL.: "Selection and characterization of human acrosome reacted spermatozoa", HUMAN REPRODUCTION, vol. 10, no. 11, 1 November 1995 (1995-11-01), pages 2948 - 2951, XP009172029 *
UMEHARA ET AL.: "A simple sperm-sexing method that activates TLR7l8 on X sperm for the efficient production of sexed mouse or cattle embryos", NATURE PROTOCOLS, vol. 15, August 2020 (2020-08-01), pages 2645 - 2667, XP037207392, DOI: 10.1038/s41596-020-0348-y *

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