WO2023212529A1 - Compositions and methods for enhancing sperm cell quality - Google Patents

Compositions and methods for enhancing sperm cell quality Download PDF

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Publication number
WO2023212529A1
WO2023212529A1 PCT/US2023/066131 US2023066131W WO2023212529A1 WO 2023212529 A1 WO2023212529 A1 WO 2023212529A1 US 2023066131 W US2023066131 W US 2023066131W WO 2023212529 A1 WO2023212529 A1 WO 2023212529A1
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Prior art keywords
enhancing compound
fertilization
fertility
inhibitor
cells
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PCT/US2023/066131
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French (fr)
Inventor
Elon ROTI-ROTI
Xiao Peng
Rebecca WINTERS
Ashley SCHROEDER
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Abs Global, Inc.
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Publication of WO2023212529A1 publication Critical patent/WO2023212529A1/en

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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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C12N2501/392—Sexual steroids
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)

Definitions

  • the present technology relates to the field of animal husbandry and breeding.
  • the present disclosure includes improved compositions and methods for use with reproductive cell samples, and in fertilization processes. Such compositions and methods provide improved viability, enhanced protection and positive functional improvement of reproductive cells.
  • reproductive cells e.g., sperm cells (spermatozoa), oocytes, zygotes, embryos, embryonic stem cells and spemiatogonial stem cells.
  • sperm cells are collected m 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.
  • the present teachings include a composition comprising: a non-human mammalian reproductive cell selected from the group consisting of a sperm cell, an oocyte, an embryo, an embryonic stem cell, and a spermatogonial stem cell; and an effective amount of a fertility enhancing compound for enhancing cell viability during or after one or more of: storage, staining, freezing, thawing, cell selection, packaging, or in vitro fertilization; wherein the fertility enhancing compound can be a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5- HT 2A receptor antagonist, aNrf2 (nuclear
  • the fertility enhancing compound can be DS2, SKA 31, SR 3677 hydrochloride, BAY 41-2272, BI 78D3, SQ 22536, BINA, spiperone hydrochloride, ML 385, Ro 3306, Bax, (R)-DPN, resveratrol, Tempol, Capsazepine, Nifedipine, or Quercetin.
  • the fertility enhancing compound can be DS2.
  • the fertility enhancing compound can be SKA 31.
  • the fertility enhancing compound can be SR 3677 hydrochloride.
  • the fertility enhancing compound can be BAY 41-2272.
  • the fertility enhancing compound can be BINA. In various configurations, the fertility enhancing compound can be spiperone hydrochloride . In various configurations, the fertility enhancing compound can be ML 385. In various configurations, the fertility enhancing compound can be Ro 3306. In various configurations, the fertility enhancing compound can be Bax. In various configurations, the fertility enhancing compound can be (R)-DPN. In various configurations, the fertility enhancing compound can be resveratrol. In various configurations, the fertility enhancing compound can be Tempol. In various configurations, the fertility enhancing compound can be Capsazepine. In various configurations, the fertility enhancing compound can be Nifedipine. In various configurations, the fertility enhancing compound can be DS2.
  • the fertility enhancing compound can be Quercetin. In various configurations, the fertility enhancing compound can be SQ 22536. In various configurations, the fertility enhancing compound can be Bl 78D3. In various configurations, the fertility enhancing compound can be present at a concentration ranging from 1 nM to 100 mM.
  • the non-human mammalian reproductive cell can comprise bovine, porcine, equine, ovine, elk, or bison sperm cells. In various configurations, the non-human mammalian reproductive cell can be bovine or porcine sperm cells. In various configurations, the non-human mammalian reproductive cells can be bovine sperm cells.
  • the non-human mammalian reproductive cells can be porcine sperm cells.
  • the bovine, porcine, equine, ovine, elk, or bison sperm cells can be sex selected sperm cells.
  • the bovine, porcine, equine, ovine, elk, or bison sex selected sperm cells can be bovine or porcine sex selected sperm cells.
  • the bovine, porcine, equine, ovine, elk, or bison sex selected sperm cells can be bovine sperm cells.
  • the bovine, porcine, equine, ovine, elk, or bison sex selected sperm cells can be porcine sperm cells.
  • the composition can further comprise a medium.
  • the present teachings can include a method for enhancing non-human mammalian sperm cell viability during or after a sexing process at one or more of staining, freezing, thawing, cell selection, or packaging during the sexing process or in vitro fertilization following the sexing process, wherein the method can comprise: adding to the non-human mammalian sperm cells an effective amount of a fertility enhancing compound or a composition thereof; and wherein the fertility enhancing compound is a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear
  • the fertility enhancing compound can be added during or after the sexing process.
  • adding the fertility compound to the non-human mammalian sperm cells at a pre-freeze stage can provide a positive functional improvement in sperm cells at a post-thaw stage.
  • the fertility compound can be a c-Jun Kinase inhibitor or an adenylyl cyclase inhibitor.
  • the fertility' enhancing compound can be DS2, SKA 31, SR 3677 hydrochloride, BAY 41-2272, BI 78D3, SQ 22536, BINA, spiperone hydrochloride, ML 385, Ro 3306, Bax channel blocker, (R)-DPN, resveratrol, Tempol, Capsazepine, Nifedipine, or Quercetin.
  • the fertility enhancing compound can be DS2.
  • the fertility enhancing compound can be SKA 31.
  • the fertility enhancing compound can be, SR 3677 hydrochloride.
  • the fertility enhancing compound can be BAY 41-2272.
  • the fertility enhancing compound can be BINA. In various configurations, the fertility enhancing compound can be spiperone hydrochloride . In various configurations, the fertility enhancing compound can be ML 385. Tn various configurations, the fertility enhancing compound can be Ro 3306. In various configurations, the fertility enhancing compound can be Bax. In various configurations, the fertility enhancing compound can be (R)-DPN. In various configurations, the fertility enhancing compound can be resveratrol. In various configurations, the fertility enhancing compound can be Tempol. In various configurations, the fertility enhancing compound can be Capsazepine. In various configurations, the fertility enhancing compound can be Nifedipine. In various configurations, the fertility enhancing compound can be DS2.
  • the fertility enhancing compound can be Quercetin. In various configurations, the fertility enhancing compound can be SQ 22536. In various configurations, the fertility enhancing compound can be BI 78D3.
  • the non-human mammalian sperm cells can comprise bovine, porcine, equine, ovine, elk, or bison sperm cells. In various configurations, the non-human mammalian sperm cells can comprise bovine or porcine sperm cells. In various configurations, the non-human mammalian sperm cells can comprise bovine sperm cells. In various configurations, the non-human mammalian semen sample can comprise porcine sperm cells.
  • the method can further comprise adding the fertility enhancing compound to the sperm cells at the staining step. In various configurations, the method can further comprise adding the fertility enhancing compound to the sperm cells at the centrifugation step. In various configurations, the method can further comprise adding the fertility enhancing compound to the sperm cells prior to the cry opreservation step.
  • the present teachings can include a method of preserving viability of non-human mammalian semen during sex selection and cryopreservation comprising: obtaining an ejaculate comprising a semen sample; contacting the semen sample with a fertility enhancing compound selected from a c-Jun Kinase inhibitor and an adenylyl cyclase inhibitor; sex selecting the semen sample while maintaining a concentration of the fertility enhancing compound; and cry opreserving the sex selected semen sample.
  • the fertility enhancing compound can be SQ 22536.
  • the fertility enhancing compound can be BI 78D3.
  • the non-human mammalian semen sample can comprise a bovine, porcine, equine, ovine, elk, or bison semen sample.
  • the non- human mammalian semen sample can comprise a bovine or porcine semen sample.
  • the non-human mammalian semen sample can comprise a bovine semen sample.
  • the non-human mammalian semen sample can comprise a porcine semen sample.
  • compositions comprising: a reproductive cell selected from the group consisting of a sperm cell, an oocyte, an embryo, an embryonic stem cell and a spermatogonial stem cell; and an effective amount of a fertility enhancing compound for enhancing cell viability during and/or after one or more of staining, freezing, thawing, cell sorting, packaging, or in vitro fertilization; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D 2 -like dopamine receptor antagonist, a 5-HT 2A receptor antagonist, a Nrf2 (nuclear factor eryth)
  • compositions comprising: a reproductive cell selected from the group consisting of a sperm cell, an oocyte, an embryo, an embryonic stem cell and a spermatogonial stem cell; an effective amount of a fertility enhancing compound for enhancing cell viability during and/or after one or more of staining, freezing, thawing, cell sorting, packaging, or in vitro fertilization; and wherein the fertility enhancing compound is an agonist and/or an antagonist of upstream or downstream regulatory proteins of GABA pathway , potassium channel pathway, Rho-kinase pathway, soluble guanylyl cyclase (sGC) pathway, c-Jun Kinase pathway, adenylyl cyclase pathway, mGluR2 pathway, Dz-like receptor pathway, 5-HT2A pathway, Nrf2 (nuclear factor erythroid 2-related factor 2) pathway, cyclin dependent kinase 1 pathway, Bax channel pathway, estrogen receptor 0 pathway
  • a media composition comprising an amount of a fertility enhancing compound effective to improve the function of sperm, oocyte, embryo, embryonic stem cell or spermatogonial stem cell, wherein the improvement in function comprises improvement in sexed semen production, improvement in efficiency of the sexing process, improvement in fertility/viability/physiological function of sexed semen, improvement in in vitro fertilization, improvement in rates of embryo production and/or increased implantation, and live births; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A
  • Another aspect of the disclosure relates to a composition
  • a composition comprising semen, an extender composition, and an effective amount of a fertility enhancing compound
  • the semen provides a concentration of motile sperm in the composition ranging from 0.01 M motile sperm/ml to 2000 M motile sperm/ml
  • the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) Inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker
  • Another aspect of the disclosure relates to a method for enhancing reproductive cell viability during and/or after one or more of staining, freezing, thawing, cell sorting, packaging, or in vitro fertilization, the method comprising, adding to the reproductive cells, an effective amount of a fertility enhancing compound or a composition thereof, wherein the fertility enhancing compound is added at a concentration ranging from 1 nM to 100 mM; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor
  • Another aspect of the disclosure relates to a method of protecting sperm cells throughout the sexing process comprising adding to the sperm cells an effective amount of a fertility enhancing compound or a composition thereof, wherein the fertility enhancing compound is added before, during and/or after the sexing process at a concentration ranging from 1 nM to 100 mM; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-hke dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) Inhibitor, a cyclin dependent kina
  • Another aspect of the disclosure relates to a method of eliciting a positive functional improvement in sperm cells at the post-thaw stage, the method comprising, adding to the sperm cells at a pre-freeze, an effective amount of a fertility enhancing compound or a composition thereof, wherein the fertility enhancing compound is added at a concentration ranging from 1 nM to 100 mM; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) Inhibitor,
  • Another aspect of the disclosure relates to a method of improving quality of a semen sample comprising contacting the semen sample with an effective amount of a fertility enhancing compound or a composition thereof, wherein the fertility enhancing compound is added at a concentration ranging from 1 nM to 100 mM; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) Inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker, an estrogen receptor 0
  • FIG. 1 is a flowchart showing the addition of a fertilization enhancing compound at various stages during the sexing process, each media that could contain a fertilization enhancing compound is designated by a star.
  • FIG. 2A shows a comparison in normalized Motile (M/mL) after 2 hr storage of post-thaw straw material treated with BI 78D3 between 0.1 pM and 30 pM BI 78D3. Both TO and T2 timepoint raw’ values were normalized to the TO DMSO control. Dashed lines represent the DMSO control at TO and T2.
  • FIG. 2B show's a comparison in curvilinear velocity (VCL) after 2 hr storage of post-thaw straw material treated with BI 78D3 between 0.1 pM and 30 pM BI 78D3, with the DMSO control. Both TO and T2 timepoint raw values were normalized to the TO DMSO control.
  • VCL curvilinear velocity
  • FIG. 3A shows the time it takes to reach 80% saturation (EC80) for Hoechst dye uptake obtained using compounds SQ22536, R-DPN, BI 78D3, Bax, and Ro3306, compared to a DMSO control.
  • FIG. 3B shows CellTiter Gio assay results obtained using compounds SQ22536, BI 78D3, R-DPN, Ro3306, and Bax, compared to DMSO and SOP control.
  • FIGs. 4A-4B show a comparison in Motile % 2 hours after stain between BI 78D3 (0.03 pM, 0.1 pM, and 30 pM, respectively), DMSO, and SOP (control) in JE4264 media (FIG. 4A) and HO0916 media (FIG. 4B).
  • FIG. 5 shows a comparison in normalized progressive Motile (M/straw) at 3 hours post thaw between compounds Bax, BI 78D3, SQ22536, Ro3306, and R-DPN, at various concentrations.
  • FIG. 6 shows normalized progressive Motile (M/straw) for compounds R-DPN (FIG. 6A), BI 78D3 (FIG. 6B), and SQ22536 (FIG. 6C), at 0, 1, 2, and 3 hours post thaw compared to the control.
  • FIG. 7A shows initial dead (%) of the cells treated with BI 78D3 and SQ22536, compared to the control.
  • FIG. 7B shows initial eligibility (%) of the cells treated with BI 78D3 and SQ22536, compared to the control.
  • FIG. 8A shows straws/catch tube for samples treated with BI 78D3, SQ22536, and (R)-DPN, compared to SOP control.
  • FIG. SB shows progressive motile post-thaw (M/straw) of the cells treated with BI 78D3, SQ22536, and R-DPN, compared to the control.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present technology. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • the term “about” in reference to a number is generally taken to include numbers that fall within a range of 10% (including, e.g., 1%, 5% or 10%) in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
  • compositions and methods include the recited elements, but do not exclude others.
  • Consisting essentially of shall mean excluding other elements of any essential significance to the combination for the stated purpose Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characterIstic(s) of the claimed invention. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention. It will be understood that use of any of these expressions — “comprising,” “consisting essentially thereof,” or “consisting of’ — also contemplate and provides disclosure for use of any of the other terms
  • the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, or a mammal. In some embodiments, the individual, patient or subject is cattle, pig, or sheep.
  • the term “effective amount” refers to an amount sufficient to produce a biological effect.
  • “fertility” includes one or more of the following: a level or degree of ability of an animal to conceive and bear young; a level or degree of ability of an animal to become pregnant; a level or degree of ability of an animal to reproduce; a level or degree of ability of a spermatozoa to fertilize an oocyte; a level or degree of ability of an oocyte to be fertilized by a spermatozoa; and a level or degree of ability of an oocyte fertilized by a spermatozoa to develop into a zygote capable of progressing through embryonic and fetal development.
  • Various methods of evaluating fertility, such as in gametes, are described in U.S. Patent Pub. 2020/0347347 by Roti-Roti, which is incorporated herein by reference and for all purposes.
  • fertilization enhancing compound refers to compounds that can provide beneficial effect in fertilization, including but not limited to compounds that can improve the fertility competency of individual sperm cells, compounds that can improve the amount of motile, live cells in the straw; and/or compounds that can increase the recovery of motile cells.
  • exing 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 or sperm cells.
  • the sexing process can be accomplished using a number of different techniques, including droplet sorting, mechanical sorting, and laser ablation
  • reproductive cell as referred herein is defined as sperm, eggs, and the formation of embryo/blastocyst, also gametes; haploid cells; germ cells; sex cells; sperm cells and egg cells.
  • medium refers to an essentially liquid composition that may contain nutrients, salts, and other substances or constituents.
  • ejaculate refers to the combination of semen and spermatozoa, which may comprise any amount of seminal plasma, produced by a male mammal, as released by ejaculation.
  • seminal fluid components as referred herein is the substances that make up and/or are commonly found in mammalian semen.
  • Seminal fluid components include, but are not limited to, amino acids, prostate specific antigen, proteolytic enzymes, citric acid, citrate, sialic acid, vitamin C, acid phosphatase, fibrinolysin, lipids, fructose, prostaglandins, phosphorylcholine, glycerophosphocholine, flavins, basic amines such as putrescine, spermine, spermidine and cadaverine, zinc, galactose, mucus and other organic and inorganic constituents.
  • sperm cells are collected in the form of raw ejaculate from male animals and must be stored before further use. Storage can comprise hours or even days.
  • cell samples are usually manipulated in one or more ways before use. Thus, it is important to maintain the viability and function of the cells throughout the process.
  • Sexing procedures disclosed can be implemented for use with fresh, un-extended ejaculate.
  • ejaculate that is not supplemented with an extender, as an ejaculate declines in quality continually after collection.
  • Sperm that undergoes sexing is exposed to numerous insults including temperature swings, high dilution, and pH changes during cell processing. Insults during sexing include shear stress, high fluid pressure, and the high force caused by the sexing process on cytometers (Gamer, D.L. and Seidel, G.E., Canadian Journal of Animal Science, 2003, 83, 375-384; Gamer, D.L., Theriogenology, 2006, 65, 943-957). These insults lead to a decrease in the number of cells recovered after processing.
  • That loss of cells during processing, while detrimental, is not the main source of projected product loss.
  • the major loss is due to fresh ejaculates having a steady increase in dead cell population over time after collection.
  • a major factor of this is that the fresh ejaculates must be stored at close to room temperature, as the sexing process happens at room temperature. Keeping the ejaculate at room temperature, rather than at a cooler temperature that better preserves cell survivability prevents time spent on equilibrations to the cooler temperature and allows sexing of the ejaculates continuously. This also increases the rapidity in which the viable cells are lost before the sexing process begins. This continual loss of viability when paired with the time required to perform the sexing and the packaging steps led to the standard policy of freezing a sexed ejaculate no later than 12 hours after collection.
  • An extender or other medium formulated for use in a sperm sexing facility could help to mitigate losses.
  • the medium can slow the decline of sperm cells held at room temperature before sexing, allowing a larger number of ejaculates to be collected at a single time point, and decreasing the number of times per day ejaculates are collected.
  • instruments would only be shut down to change to a new 7 ejaculate as needed on a bull by bull basis, rather than the entire production floor at once. This would decrease the time it takes to change to a new bull, as it increases the available staff per instrument.
  • Maintaining cell viability 7 also means ejaculates could be run until exhaustion, maximizing the number of sexed sperm obtained per ejaculate, and decreasing the total number of times per day a bull change would be performed per instrument. By mitigating these causes of cell loss, the number of insemination doses produced would increase making the superior product more available to farmers globally.
  • a fertility enhancing compound would also be useful in reducing or mitigating cell stress involved in a staining step or process, such as a staining process with Hoechst 33342 prior to enriching or sexing a cell population on an instrument.
  • a staining process a semen sample may be mixed with staining media and other compounds which alter the concentration and pH of the semen sample, and the semen sample may be subjected to elevated temperatures, such as temperatures above 30 oC.
  • the changes in sample chemistry, concentration, and temperature are stressors on the cells that impact cell health. Cell viability is improved by reducing the stressors, or by reducing or mitigating the impact of the sources of stress on the cells.
  • a fertility enhancing compound is used to reduce or mitigate the impact of the stressors on the cells thereby maintaining cell viability, reducing cell loss, and maximizing the number of sexed sperm that may be obtained per ejaculate.
  • the present disclosure relates to certain fertilization enhancing compounds, and compositions thereof, and methods that improve reproductive cell viability and activity.
  • the fertilization enhancing compound is an element of a pre-made or pre-formulated media (or a composition). In some embodiments, the fertilization enhancing compound is added (e g., to the reproductive cells) on its own as a discrete element or step in a process described herein.
  • the fertilization enhancing compounds, compositions and/or methods described herein may be used in a conventional process (e g., conventional (i.e. non-sexed) semen).
  • a conventional process e g., conventional (i.e. non-sexed) semen.
  • the fertilization enhancing compounds (and/or compositions) described herein may be added to conventional semen after it has been collected.
  • the fertilization enhancing compounds (and/or compositions described) herein are added to conventional semen immediately prior to packaging in straws.
  • the fertilization enhancing compounds (and/or compositions described) herein are added to conventional semen immediately before usage in an Al or IVF process.
  • the fertilization enhancing compounds, compositions and/or methods described herein may be used in a sexing process (e g., sexed semen). Tn some embodiments, the fertilization enhancing compounds (and/or compositions) described herein may be added to semen that is to be sexed.
  • the fertilization enhancing compounds (and/or compositions described) herein are added to semen that is to be sexed at one or more of the following stages, including 1) immediately after collection; 2) during incubation or pre-instrument QC (quality control); 3) immediately prior to, during, or after staining; 3) pre-sexing instrument; 4) postinstrument in the catch tube; 5) prior to centrifugation; 6) after centrifugation; 7) prepackaging, either prior or post-cryopreservation addition; and 8) post-thaw, such as before an Al or IVF event.
  • the present specification provides and includes fertilization enhancing compounds and/or compositions that impart increased activity and viability to reproductive cells, in particular, sperm cells.
  • the present specification also provides and includes methods for processing reproductive cell samples, wherein the methods and processes produce samples in which the sperm cells have increased viability and activity, and improve production efficiency.
  • the present specification also provides and includes the reproductive cell samples produced by these methods, wherein reproductive cells in the samples have increased viability and activity.
  • the present specification still further provides and includes methods using these reproductive cell samples with increased viability and activity, including sexing (selecting X-chromosome bearing or Y- chromosome bearing cells), sorting, separating, freezing, artificial insemination, in vitro fertilization, cooling and transport, and related processes.
  • the present disclosure relates to new compositions, new media formulations, new processes for addition of a fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) or a salt thereof, to spemi cells (before/, during, ot after sexing, or in a conventional process without sexing) and/or to embryos (before, during, or after IVF).
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compounds described herein are included in a composition or a media before adding to sperm cells and/or to embry os.
  • the fertilization enhancing compounds described herein are added directly (e.g, not included in a composition or a media before use) to sperm cells and/or to embryos.
  • the present disclosure relates to improvements in conventional (i.e. non-sexed) semen production.
  • the present disclosure relates to improvements in sexed semen production. Exemplary' improvements include but are not limited to, improved efficiency of the sexing process and improved fertility/viability/physiological function of sexed semen; improvements in in vitro fertilization, including improved rates of embryo production and/or increased implantation, live births.
  • compositions which include a reproductive cell selected from the group consisting of a sperm cell, oocyte, embryo, embryonic stem cell and a spermatogonial stem cell; an effective amount of a fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) for enhancing cell viability during and/or after one or more of staining, freezing, thawing, cell sorting, packaging, or in vitro fertilization; and optionally, a medium.
  • the medium is an extender medium.
  • the medium may be a maturation medium or other medium used in an IVF process, or could be a staining solution in a sexing process, or a collection medium (e.g., comprising a cryoprotectant) in the sexing and/or the conventional process.
  • a maturation medium or other medium used in an IVF process or could be a staining solution in a sexing process, or a collection medium (e.g., comprising a cryoprotectant) in the sexing and/or the conventional process.
  • Suitable fertilization enhancing compounds include, but are not limited to a GABA positive allosteric modulator (e.g, DS2), a potassium channel activator (e.g., an activator of KCa3.1 and KCa2 channels such as SKA 31), a Rho-kinase inhibitor (e.g., SR 3677 or SR 3677 hydrochloride), a soluble guanylyl cyclase (sGC) activator (e.g., BAY 41-2272), a c-Jun Kinase inhibitor (BI 78D3), an adenylyl cyclase inhibitor (e.g., SQ 22536), a mGluR2 positive allosteric modulator (e.g., BINA), a D2-like dopamine receptor antagonist (e.g., spiperone or spiperone hydrochloride), a 5-HT2A receptor antagonist (e.g., spiperone or spiperone hydrochloride),
  • the fertilization enhancing compound is a c-Jun Kinase inhibitor. In any embodiments, the fertilization enhancing compound is BI 78D3 or a salt thereof (collectively referred to as “BI 78D3”). In any embodiments, the fertilization enhancing compound is a an adenylyl cyclase inhibitor. In any embodiments, the fertilization enhancing compound is SQ 22536 or a salt thereof (collectively referred to as “SQ 22536”).
  • the fertilization enhancing compound e.g. , a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound can be provided between about 0.
  • 1 nM and about 100 mM including, for example, about 0.001 pM to about 50 mM, about 0.01 pM to about 500 pM, about 0.1 pM to about 250 pM, about I pM to about 100 pM, about 2 pM to about 50 pM, about 3 pM to about 30 pM, about 4 pM to about 20 pM, about 5 pM to about 10 pM, about 0.1 pM to about 5 pM, about 0.1 pM to about 10 pM, or about 0.1 pM.
  • the fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • a concentration ranging from about 0.1 pM to about 5 pM is present at a concentration ranging from about 0.1 pM to about 5 pM.
  • fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound is present at a concentration of less than about 500 pM, or alternatively less than about 100 pM, or alternatively about less than 50 pM, or alternatively less than about 30 pM, or alternatively less than about 20 pM, or alternatively less than about 10 pM, or alternatively less than about 5pM, or alternatively less than about 3 pM, or alternatively less than about 2 pM.
  • the fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound is present at a concentration of greater than about 0.01 pM or alternatively greater than about 0.1 pM, or alternatively about greater than 1 pM, or alternatively greater than about 2 pM, or alternatively greater than about 5 pM, or alternatively greater than about 10 pM, or alternatively greater than about 15 pM, or alternatively greater than about 20 pM, or alternatively greater than about 50 pM.
  • the fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound is present at a concentration ranging from about 10 nM to about 100 pM.
  • the fertilization enhancing compound e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound e g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound is present at a concentration ranging from about 5 pM to about 50 pM.
  • the fertilization enhancing compound is a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536, and it is present at a concentration of about 10 nM to about 100 pM.
  • the fertilization enhancing compound is a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536, and it is present at a concentration of less than about 5 pM. In some embodiments, the fertilization enhancing compound is a c-Jun Kinase inhibitor such as BI 78D3, and it is present at a concentration of about 0.1 pM. In some embodiments, the fertilization enhancing compound is an adenylyl cyclase inhibitor such as SQ 22536, and it is present at a concentration of about 0.3 pM.
  • the fertilization enhancing compounds described herein are included in a composition or a media before adding to sperm cells and/or to embryos, and the concentrations described herein are the concentration of the fertilization enhancing compounds in the composition or media.
  • the fertilization enhancing compounds described herein are added directly (e.g., not included in a composition or a media before use) to sperm cells and/or to embryos, and the concentrations described herein are the concentration of the fertilization enhancing compounds in the resulting mixture (resulting composition) after addition.
  • a fertilization enhancing compound e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • a fertilization enhancing compound is provided in an amount effective to enhance cell viability during and/or after one or more of staining, freezing, thawing, cell sorting, packaging, or in vitro fertilization.
  • one or more fertilization enhancing compounds described herein are provided in an amount effective to enhance cell viability during and/or after one or more of staining, freezing, thawing, cell sorting, packaging, or in vitro fertilization. Effective amounts for these compounds may range from about 0.01 uM to about 30 uM.
  • the compositions may also include one or more types of reproductive cells. Suitable reproductive cells, include, without limitation, sperm cells, oocytes, embryos, embryonic stem cells and spermatogonial stem cells.
  • the composition may include a single sperm cell or a plurality of sperm cells.
  • the composition may include a single oocyte or a plurality of oocytes.
  • the composition may include a single embryo or a plurality of embryos.
  • the composition may include a single embryonic stem cell or a plurality' of embryonic stem cells. In some embodiments, the composition may include a single spermatogonia! stem cell or a plurality of spermatogonia! stem cells. In some embodiments, the composition may include a plurality' of sperm cells. In some embodiments, the sperm cells include mammalian sperm cells. Suitable mammalian sperm cells may include, without limitation, human, bovine, porcine, equine, ovine, elk, or bison sperm cells. The concentration of reproductive cells in the composition can be adapted based on the type of cells and the process stage.
  • the initial cell concentration can be higher (e.g., 1000 M/mL or less), which can then diluted in a medium or buffer to a concentration of 66 M/mL for sexing
  • 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.
  • composition of the present technology may further include a medium.
  • Suitable mediums include without limitation, an extender medium, a cryoprotectant, a buffer, a diluent, an energy source, an extender medium, an antibiotic, and a bolus.
  • the potassium channel blocker may be added to the composition separately or included in the medium.
  • the fertilization enhancing compound e g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536, or a salt thereof, is included in the extender medium.
  • the fertilization enhancing compound can be added altogether in a single dose or as a bolus, slowly titrating in, and combinations of the two.
  • the media composition may contain the fertilization enhancing compound, and can be combined with cells in a variety of ways, for example by using a set volume of media, a set ratio of media to cell sample, or media provided at a set volume in relation to a measured aspect of the sample (i.e., sperm cell concentration).
  • media can be added to the sample, in other embodiments, the sample can be added to the media. In other embodiments, both sample and media can be added to a third container/receptacle.
  • the fertilization enhancing compound can include a c-Jun Kinase inhibitor (e.g., BI 78D3 or a salt thereof) or an adenylyl cyclase inhibitor (e.g. , SQ 22536 or a salt thereof).
  • the c-Jun Kinase inhibitor or adenylyl cyclase inhibitor is present, e.g, in the composition and/or medium in an amount effective to improve the function of sperm, oocyte, embryo, embryonic stem cell or spermatogonia!
  • the improvement in function comprises improvement in semen production (sexed semen production and/or non-sexed semen production), improvement in efficiency of the sexing process, improvement in fertility/viability /physiological function of semen (sexed semen and/or non-sexed semen), improvement in in vitro fertilization, improvement in rates of embryo production and/or increased implantation, and live births.
  • the present technology preserves the viable, motile sperm population eligible for sexing and significantly reduces the cost of sexed semen to farmers by increasing yield, such as by increasing the number or percentage of successful or viable pregnancies from artificial insemination events using said semen.
  • the ideal extender formulation must maintain a high motile sperm population and must not interfere with the ability to separate the X and Y populations using a fluorescent DNA stain, which is required to separate the two cell populations on the sexing cytometry instruments.
  • additives may be included in a composition (and/or a medium) comprising the fertilization enhancing compounds to bring about the desired properties and create the inventive media.
  • the compositions comprise a medium which includes at least one additive selected from the group consisting of antioxidants, phosphatidylserine (PS), coumarin compounds or pyranocoumarin compounds, zinc chloride, coenzyme Q10, a nonsteroidal antiinflammatory drug (NSAID), linolenic acid, fatty acids, D-aspartic acid, and combinations thereof.
  • the medium contains sodium fluoride.
  • the additives comprise one or more of phosphatidylserine (PS), decursin, zinc chloride, coenzyme Q10, acetylsalicylic acid (aspirin), linolenic acid, fatty acids, D-aspartic acid, sodium fluoride, and combinations thereof. In some embodiments, all of the additives are included in the formulation. In other embodiments, one or more of decursin, zinc chloride, coenzyme Q10, acetylsalicylic acid (aspirin), linolenic acid, fattyacids, D-aspartic acid, or sodium fluoride are omitted.
  • PS phosphatidylserine
  • decursin zinc chloride
  • coenzyme Q10 acetylsalicylic acid
  • linolenic acid fatty acids
  • D-aspartic acid sodium fluoride
  • the medium further includes a buffer.
  • the buffer may be TRIS or HEPES.
  • TRIS may be used due to its longer shelf life in the formulation as measured by pH stability.
  • the medium may be supplemented with a salt such as sodium chloride (NaCl), potassium chloride (KC1), calcium chloride dehydrate (CaC 12 (H 2 O) 2 ), magnesium chloride hexahydrate (MgC 12 (H 2 O) 6 ), sodium bicarbonate (NaHCO 3 ), sodium phosphate dihydrate (NaH 2 PO 4 (H 2 O) 2 ), potassium phosphate ( KH 2 PO 4 ), and sodium fluoride (NaF).
  • a salt such as sodium chloride (NaCl), potassium chloride (KC1), calcium chloride dehydrate (CaC 12 (H 2 O) 2 ), magnesium chloride hexahydrate (MgC 12 (H 2 O) 6 ), sodium bicarbonate (NaHCO 3 ), sodium phosphate dihydrate (NaH 2 PO 4 (H 2 O) 2 ), potassium phosphate ( KH 2 PO 4 ), and sodium fluoride (NaF).
  • the medium may further include antioxidants, which are added to decrease the amount of stress the cells are subjected to during the sexing process. All sperm are exposed to LV light during the sexing process, which typically causes oxidative damage to DNA (rather than direct strand breaks), and sperm is likely subject to elevated reactive oxygen species (ROS) during the cryoprotectant step (Aitken, R.J., et al., Asian J Androl., 2015, 17, 633-639; Farber, J.L., Environmental Health Perspectives, 102 (Suppl 10), 1994, 17-24).
  • ROS reactive oxygen species
  • ROS can also cause DNA damage such as single and double strand breaks, and base pair modification (Richter, C , et al., Proc. Natl. Acad. Sci., 1988, 85, 6465-6467).
  • Fatehi et al. (Fatehi, A.N. et al., J. Andrology, 2006, 27, 176-188) reported oocytes fertilized with DNA damaged bovine spermatozoa exhibited cleavage rates similar to controls, but further development halted in the damaged experimental group.
  • the compositions of the present technology which include sperm cells treated with potassium channel blocker compound may exhibit less DNA damage than non-extended controls.
  • compositions comprise Nonsteroidal Antiinflammatory- Drugs (NSAIDs), which are a class of drugs and compounds capable of reducing inflammation, primarily through inhibition of cyclooxygenase enzymes (COX-1 and/or COX-2).
  • NSAIDs Nonsteroidal Antiinflammatory- Drugs
  • COX-1 and/or COX-2 cyclooxygenase enzymes
  • compositions can include one or more NSAID including , but not limited to: salicylates, including aspirin (acetylsalicylic acid), diflunisal (Dolobid); salicylic acid and other salicylates, and salsalate (Disalcid); Propionic acid derivatives, including Ibuprofen, Dexibuprofen, Naproxen, F enoprofen, Ketoprofen, Dexketoprofen, Flurbiprofen, Oxaprozin, and Loxoprofen; acetic acid derivatives, including indomethacin, Tolmetin, Sulindac, Etodolac, Ketorolac, Diclofenac, Aceclofenac, and Nabumetone: enolic acid (Oxicam) derivatives, including Piroxicam, Meloxicam, tenoxicam, Droxicam, Lomoxicam, Isoxicam, and phenylbutazone (Bute); anthranilic acid derivative
  • compositions comprise coumarin compounds or pyranocoumarin compounds.
  • the coumarin compound or pyranocoumarin compound comprises decursin.
  • the medium may further include one or more of antioxidants, phosphatidylserine (PS), phosphatidylcholine, coumarin compounds, pyranocoumarin compounds, zinc chloride, coenzyme Q10), a nonsteroidal anti-inflammatory' drug (NSAID), linolenic acid, fatty' acids, D-aspartic acid, sodium fluoride, decursin, acetylsalicylic acid (aspirin), a salt (e.g., sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium bicarbonate, sodium phosphate dihydrate, potassium phosphate, sodium fluoride), a buffer (HEPES, Tris, etc.), a sugar source (glucose, fructose, etc.), citric acid, pyruvate, ascorbic acid, glycerol or other cry ⁇ protective agents, natural or synthetic ice blockers, BSA or other protein source,
  • antioxidants phosphati
  • the medium may' also include beads, such as magnetic (or other) beads coated with lectin, DNA-binding moieties or ubiquitin-targeted moieties.
  • beads such as magnetic (or other) beads coated with lectin, DNA-binding moieties or ubiquitin-targeted moieties.
  • lectin-beads can be used to remove the dead cells during the staining process.
  • reproductive cell activity' of stored and/or manipulated samples can be maintained or even increased, by the measure of either motility', fertilization, or both. Fertilization can be measured by blastocyst formation. Concentration and progressive motility of the sample may be further evaluated using an integrated visual optical system (“IVOS”).
  • IVOS integrated visual optical system
  • a composition with semen, an extender composition, and an effective amount of a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the semen provides a concentration of motile sperm in the composition ranging from about 0.01 M motile sperm/ml to about 2000 M motile sperm/ml.
  • concentration of motile sperm in the composition may range from about 75-200 M motile sperm cells per mL in the stain reaction; about 25-100 M motile sperm cells per mL as the samples are on -instrument; about 0.25-4 M motile sperm cells/ mL at the postinstrument stage; about 10-600 M motile sperm cells/mL after centrifugation; about 5-300 M motile sperm cells / mL at cooling/pre-freeze stage after addition of Part 2 of a two- part extender (e.g., Tris B); and about 0.25-15 M motile/ mL at freeze.
  • a two- part extender e.g., Tris B
  • the fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the present technology can provide a number of important benefits, including but not limited to: extends cell viability, with a reduced loss of progressively motile cells; does not interfere with Hoechst 33342 (or an alternative) staining and red dye viability counterstaining of the cells which is necessary for proper sexing on the cytometers; and does not negatively interfere with fertilization capacity or embryonic development.
  • the present technology relates to a fertilization enhancing compound, e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536, or a salt thereof, as well as a composition or a medium thereof, which maximizes recovery and packaging of functional, fertilization competent sperm.
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536, or a salt thereof
  • This can improve the flexibility and efficiency of production by exhausting ej aculates, optimizing bull changes, and decreasing the need for backup ejaculates.
  • An additional advantage is an increase in motile cells recovered post-processing (e.g., sexing). When semen samples are processed using the inventive compositions and methods, increased activity is seen.
  • Increased activity can be increased viability, increased motility' or both.
  • use of the present technology allows for greater yields of semen samples during and after processing.
  • inclusion of a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • semen e.g., sexed semen
  • semen samples e.g., sexed semen samples
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • Hoechst dye can reduce the time required to achieve staining saturation, and increase the number of cells eligible for sexing, increase resolution (separation of X- and Y- chromosome cell peaks in a histogram derived from interrogation of spemi cells by an instrument), and sex skew, with the potential to positively impact batch yields.
  • the present technology may exhibit enhanced sperm cell survivability and motility compared to commercially available extenders: ANDROMED® (Minitube, Delavan, WI USA) and OptiXell (IMV technologies. Maple Grove, MN USA), and a previously described media formulation CEP2 (Verberckmoes, S., et al., Reproduction in Domestic Animals, 2004, 39, 410-416).
  • the present technology' may extend the window of cell survivability before sexing, while still maintaining cells measured as live and motile after the sexing process.
  • the present technology may result in an increase of the percent of blastocysts per oocyte.
  • the compositions may result in less cleavage.
  • the technology may successfully maintain the motile, viable sperm population for an enhanced period of time, e.g., 24 hours, before sexing, and may result in frozen-thawed sexed semen that meets quality control standards with no increased risk for batch failure compared to current standard operating procedures.
  • fertilization enhancing compounds as well as the compositions thereof, therefore, has the potential to increase utilization of the total ejaculate volume and concurrently increase the number of insemination doses produced per ejaculate, increasing the availability of sexed semen for farmers.
  • the present technology may maintain sperm in a fertilization competent state. Fertilization competence includes, but is not limited to, the capability of sperm cells exposed to compositions according to the present technology for producing pregnancies via artificial insemination, and fertilization, cleavage, and blastocyst conversion both in vitro and in vivo.
  • compositions of the present technology which include sperm cells treated with a fertilization enhancing compound (e.g , a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536), may produce more blastocysts per oocyte compared to non-extended, paired controls.
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • Sources of reproductive cell samples are typically from ejaculate, obtained by methods commonly known in the art.
  • the ejaculate samples can be a single source or pooled .
  • in vitro produced or expanded sperm cell populations may be used.
  • Samples are obtained from animals, preferably mammalian animals; more preferably livestock; samples are most preferably porcine or bovine.
  • the media composition containing the fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • a “hold media” to store raw ejaculate and minimize loss of reproductive cell components.
  • the composition is utilized as a “hold media” to store isolated sperm cells after processing and before use in breeding procedures. This can also be referred to as an “extender media” since samples remain viable for longer when the inventive media is used.
  • the composition functions as a medium to use for processing of reproductive cell samples that are used for further processing (such as, e.g., sexing).
  • the composition may be utilized as a staining media, a sexing processing media, and/or a freezing media.
  • compositions comprising reproductive cells and the inventive “hold media” can maintain an acceptable viability and/or motility for hours (e.g., 24 hours).
  • the inventive compositions can maintain an acceptable level of live cells throughout cell processing. For example, the percentage of dead cells may be ⁇ 25% throughout sexing duration of processing.
  • Samples can be combined with the fertilization enhancing compounds and/or the improved media in a variety of ways.
  • the fertilization enhancing compounds and/or the media can be added directly after collecting the raw ejaculate sample, within a set amount of time after collecting the raw ejaculate; or the raw' ejaculate can be collected directly into the media.
  • Sperm cells that are exposed to the fertilization enhancing compounds (or the inventive compositions thereof) can exhibit enhanced motility, viability , and functionality (including the ability to fertilize ova) over time, compared to sperm exposed to existing commonly -used media.
  • these fertilization enhancing compounds (or the compositions thereof) can enhance yields in both conventional and sexed semen production.
  • the fertilization enhancing compounds (or the inventive compositions thereof) can maximize recovery and packaging of functional, fertilization competent sperm.
  • the present technology' may provide beneficial effects for IVF outcomes, measured as cleavage and blastocyst conversion rates, which may be due, at least in part, to mitigation of DNA damage, increased ROS production, and/or capacitati on-like changes caused by sexing.
  • the fertilization enhancing compounds (or the compositions thereof) of the present technology may also allow for increased run time for each ejaculate and thereby increase frozen semen (e.g., sexed semen) product per volume of ejaculate collected and decreased the cost of each insemination dose.
  • semen e.g., sexed semen
  • compositions are applicable not only to frozen sexed bovine semen, but could also have applications in extending the life of a fresh ejaculate in a setting where extended transport times are required or specifically for preservation of ejaculates of impaired quality.
  • One aspect of the disclosure relates to methods of processing mammalian reproductive cells comprising the steps of providing a mammalian reproductive cells sample, processing the mammalian reproductive cells sample, and adding a fertilization enhancing compound (or a media composition thereof) of the present technology.
  • a fertilization enhancing compound e.g. , a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound e.g. , a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound can be added to the mammalian reproductive cells sample by itself.
  • the fertilization enhancing compound can be a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536, or a salt thereof.
  • the media can include an additive.
  • the processing can comprise at least one of the following steps: collecting a semen sample, sexing, selecting, separating, freezing, artificial insemination, in vitro fertilization, cooling, transport, or related processes.
  • the sexing can be accomplished via droplet sorting, mechanical sorting, micro fluidic processing, microchip processing, jet and air processing, flow cytometry processing, or laser ablation.
  • the mammalian reproductive cells can be obtained from a male mammal. In yet other embodiments, the male mammal can be a bull or boar. In any embodiments, the processed mammalian reproductive cells can be gathered in a container, tube, or straw.
  • the mammalian reproductive cells can be selected from the group consisting of gametes, haploid cells, germ cells, sex cells, sperm cells, and egg cells.
  • a sperm cell composition can be produced by this processing method.
  • Processing of raw ejaculate can include many downstream applications.
  • processing of raw ejaculate can include one or more of the applications including, but not limited to sexing (selecting X-chromosome bearing or Y-chromosome bearing cells), freezing, artificial insemination, or IVF (with and without sexing).
  • this can include cooling and transport of samples, concentrating sperm cells and suspending before staining/sexing.
  • One aspect of the disclosure relates to methods of protecting sperm cells throughout the process (e.g., the sexing process or the conventional process), wherein the method can include the step of adding to the sperm cells an effective amount of a fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) or a composition thereof.
  • a fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the composition can further include a medium.
  • the fertilization enhancing compound e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the medium can be an extender medium.
  • the medium can be a staining buffer, a cry opreservation buffer, and/or a semen processing buffer.
  • the fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound can be added to the sperm cells and the fertilization enhancing compound can have a concentration ranging from 1 nM to 100 mM in the resulting mixture, including, without limitation, about 0.
  • Another aspect of the disclosure relates to methods of eliciting a positive functional improvement in sperm cells at the post-thaw stage.
  • the method can include adding to the sperm cells at a pre-freeze stage an effective amount of a fertilization enhancing compound (e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) or a composition thereof.
  • a fertilization enhancing compound e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the composition can further include a medium.
  • the fertilization enhancing compound e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the medium can be an extender medium.
  • the medium can be a staining buffer, a cry opreservation buffer, and/or a semen processing buffer.
  • the fertilization enhancing compound e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the process e.g., the sexing process or the conventional process.
  • the fertilization enhancing compound e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536
  • the fertilization enhancing compound has a concentration ranging from 1 nM to 100 mM in the resulting mixture, including, without limitation, about 0.
  • 1 pM to about 500 pM about 0.5 pM to about 250 pM, about 1 pM to about 100 pM, about 2 pM to about 50 pM, about 3 pM to about 30 pM, about 4 pM to about 20 pM, about 5 pM to about 10 pM, or about 0.1 pM to about 5 pM.
  • the compositions herein are sperm cell compositions that comprise sperm cells, a fertilization enhancing compound (e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) and a medium.
  • the compositions may include seminal fluid components.
  • the media may include one or more of extender media, staining media (stain TALP), collection media (Part 1 or TRIS A), and cry opreservation media (packaging extender).
  • the medium may include one or more of NaCl, KC1, Na 2 HPO 4 , NaHCO 3 .
  • the medium may include one or more of Tyrode’ s, Sodium lactate syrup, Glucose, HEPES, Sodium Pyruvate, BSA (Stain TALP). In some embodiments, the medium may include one or more of Stain TALP, Egg Yolk, Quenching Dye (Red TALP). In some embodiments, the medium may include one or more of Sterile Milli-Q H 2 O, TRIS, Egg Yolk, GTLS (Part 1 or TRIS A).
  • the medium may include one or more of Sterile Milli-Q H 2 O, TRIS, Egg Yolk, Glycerol, Green Food Color, GTLS (Part 2 or TRIS B).
  • the medium may include one or more of TRIS A (Part 1 ) and TRIS B (Part 2) (Packaging Extender9.
  • the medium may include one or more of Gentamicin Sulfate (powdered, Amresco # 0304), Sterile Milli-Q H 2 O (Gentamycin Sulfate Solution).
  • the medium may include one or more of Tylosin (Tylosin Tartrate; Midwest Vet Supply), Sterile Milli-Q H 2 O (Tylosin Solution).
  • the medium may include one or more of Gentamicin Sulfate Solution, Tylosin Solution, Linco-Spectin Stock (50 mg Lincomycin/lOOmg Spectinomycin; Midwest Vet Supply) (GTLS)
  • Other components which may be included in the medium may include, without limitation, one or more of sodium chloride, potassium chloride, disodium phosphate, sodium bicarbonate, magnesium chloride hexahydrate, D-(+)-Glucose, sodium pyruvate, sodium lactate, HEPES, BSA (Fraction V), red food dye, tris(hydroxymethyl)aminomethane (Trizma), citric acid monohydrate, D-Fructose, egg yolk, glycerol, green food dye, CaCl 2 (H 2 O) 2 .
  • MgCl 2 (H 2 O) 6 NaHCO 3 , NaH 2 PO 4 dihydrate, KH 2 PO 4 , fructose, sorbitol, phosphatidylserine, decursin, zinc chloride, coenzyme Q10, aspirin, linolenic acid, fatty acid supplement, and D-aspartic acid.
  • the present technology can include a container of sperm cells comprising a plurality of sperm cells, a medium, one or more of fertilization enhancing compounds, or combinations thereof.
  • the container may further comprise seminal fluid components.
  • Such containers can be used for storage, or for further procedures such as IVF or Al.
  • Tyrode NaCl, KC1, Na 2 HPO 4 , NaHCO 3 , MgCl 2 .6H 2 O.
  • Stain TALP Tyrode's, Sodium lactate syrup, Glucose, HEPES, Sodium Pyruvate, BSA.
  • Red TALP Stain TALP, Egg Yolk, Quenching Dye.
  • TRIS A Sterile Milli-Q H 2 O, TRIS, Egg Yolk, GTLS.
  • TRIS B Sterile Milli-Q H 2 O, TRIS, Egg Yolk, Glycerol, Green Food Color, GTLS.
  • TRIS A Part 1
  • TRIS B Part 2
  • Gentamycin Sulfate Solution Gentamicin Sulfate (powdered, Amresco # 0304), Sterile Milli-Q H 2 O.
  • Tylosin Solution Tylosin (Tylosin Tartrate; Midwest Vet Supply), Sterile Milli-Q H 2 O.
  • IVF Testable Unit Generation Design This study utilizes sires from one breed, Holstein. The study design includes one ejaculate collection from 2 unique sires to generate sexed semen units in a split batch design with the fertilization enhancing compounds and a paired control.
  • the sexed semen is packaged and frozen during a regularly scheduled production freeze following standard operating procedures (SOPs).
  • SOPs standard operating procedures
  • the outgoing quality control measurements are performed by a trained research technician. Post-thaw motile concentration and the presence and/or absence of bacterial contamination are completed. Insemination doses have to pass standard production outgoing quality control parameters to be utilized for the IVF trial.
  • the IVF facility receives the four treatment groups for each individual ejaculate.
  • the four treatments from each split ejaculate are used concurrently to fertilize oocytes from the same pooled batch of slaughterhouse oocytes.
  • Trained IVF technicians performs the outlined fertilizations. Day 2 cleavage rate, Day 7 and 8 blastocyst rates will be recorded, as well as polyspermy post-fertilization.
  • Ejaculate Collections All ejaculate collections were performed on-site by experienced technicians following standard collection procedures.
  • Ejaculate extension and Incoming Quality Assessment Ejaculates were transported in an insulated cooler to prevent temperature fluctuation during transport to the second facility. The volume of the ejaculate was determined by mass. Immediately, within 15 minutes, Citrate buffer was added in a 0.5: 1 ratio to the ejaculates. GTLS antibiotic solution was added at a 2% v/v of ejaculate. [0102] Within 45 minutes of initial ejaculate collection, the cell concentration and incoming motility parameters were collected. Concentration was determined using a Nucleocounter SP-100TM with Reagent 5100 and SP100 cassettes (ChemoMetec Allerod, Denmark).
  • Motility characteristics were calculated by diluting 10 pL sample in 990 pL motility diluent and reading 7 frames each in 2 chambers of Leja 4 chamber capillary slides with a known chamber depth on a Hamilton Thome IVOS II using HTCasa II software at 60 Hz frame capture speed in a 37° C enclosed stage with a Zeiss lOx objective. An ejaculate was utilized if the cell concentration was greater than 500 million/mL, and the percent of progressive motile cells in the sample was greater than or equal to 65%.
  • Sample Preparation for Sperm Sexing A stained sample was prepared at room temperature that contained 200 M/mL sperm cells in 0.06 rng/mL Hoechst 33342 diluted to a final volume in Stain TALP with Magnetic Beads with (+ fertilization enhancing compound such as BI 78D3, SQ 22536) or without (control) such that the fertilization enhancing compound would be at a suitable concentration (e.g., 0.1 pM) in the final stained sample volume.
  • a suitable concentration e.g., 0.1 pM
  • Sexing Cytometer Metrics The stained, filtered sample was then run on proprietary sexing cytometers. The sample throughput w 7 as adjusted to 23,000 cells/sec and the detection and kill lasers were focused. To confirm proper laser focus, kill count assessments were performed before collecting sex skewed sample. A successful kill count has a population that is greater than or equal to 75% dead and greater than or equal to 95% sliced with at least 200 cells being counted. If an instrument could not achieve the above metrics, the instrument w r as not used to collect sex skewed semen. After a successful kill count, a gate w ?
  • 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 were 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 %.
  • Sex Skewed Sample Collection and Processing The sample was run to collect between 300 and 400 mLs of sex skewed sample, the composition of which is approximately 17% TRIS A (Part 1) buffer, 80% sheath fluid, and 2% cell sample. The sample was collected in 50 mL conical tubes containing 5 mLs of TRIS A (Part 1), and each tube was filled to a maximum volume of 30 mLs before being replaced. After the requisite total volume was collected, sexed sperm was centrifuged at room temperature at 2400 x g for 10 minutes. The supernatant was aspirated and discarded to reach a 1 mL pellet volume.
  • the cryoprotected sample was diluted to final live, motile cell concentration, 2.6 M/mL, in Packaging Extender containing 0. 1-0.3 pM fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3) or no fertilization enhancing compound (control) and placed in Mini Straws which hold 0.25 mL volume (IMV technologies, Maple Grove, MN USA) using an MX4 straw filling and sealing machine (IMV technologies, Maple Grove, MN USA). Filled straws were rapidly cooled using a freeze tunnel before storage in liquid nitrogen
  • fertilization enhancing compounds can be first added in a mixture with magnetic beads in the staining step with Stain TALP and Hoechst 33342, added a second time in the Tris A (Part 1 of a two part extender)/GTLS addition after the centrifugation that occurs post-instrument, and added for a third time in the packaging extender pre- freeze.
  • the fertilization enhancing compound is maintained at a steady concentration, (e.g., 0.1 ⁇ M) throughout the process.
  • a citrate-based extender is used pre-instrument and magnetic beads are used to remove dead cells pre-instrument.
  • an IVF trial quantifies cleavage and blastocyst conversion rates, providing insight into mechanisms underlying apparent changes in the number of embryos produced (Bermejo- Alvarez, P., et al., Reprod. Fertil. Dev., 2010, 22, 426-436; Blondin, P., et al., Theriogenology, 2009, 71, 30-38; Greve, T. and Madison, V., Reprod. Nutr. Dev., 1991, 31, 147-157). Quantified outcomes in this IVF trial will include percent fertilization, cleavage conversion, and blastocyst conversion rates day 7 and day 8.
  • EXAMPLE 2 An illustrative flowchart showing the addition of fertilization enhancing compounds at various stages during the sexing process, is shown in FIG. 1 .
  • Each media containing fertilization enhancing compounds is designated by a star.
  • an ejaculate sample is obtained and a volume of Stain TALP and Hoechst 33362 are added such that a fertilization enhancing compound is introduced to the sample at a certain concentration.
  • the staining step is earned out by incubating the treated ejaculate sample for about 45 minutes at 34° C. Red TALP is added to the stained sample, and the sample is then sorted on a suitable instrument to provide a sex-skewed sample.
  • TrisA Part I of a two-part extender/GTLS, which may include additional fertilization enhancing compounds.
  • Tris B Part 2 is added to the sample, followed by a packaging extender medium, which may again include fertilization enhancing compounds, and the sample packaged in straws or other packaging for freezing and cry opreservation.
  • a fertilization enhancing compound e.g., BI 78D3, SQ22536, or (R)-DPN
  • a fertilization enhancing compound is BI 78D3.
  • the Stain TALP medium includes 0. 1 uM BI 78D3 or BI 78D3 is added to achieve or maintain 0. 1 uM concentration;
  • TRIS A (Part 1)/GTLS includes 0.1 uM of BI 78D3 or BI 78D3 is added to achieve or maintain 0.
  • cryopreservation media also includes 0. 1 uM of BI 78D3 or BI 78D3 is added to achieve or maintain 0. 1 uM concentration.
  • a fertilization enhancing compound is SQ22536.
  • the Stain TALP medium includes 0.3 uM of SQ22536 or SQ22536 is added to achieve or maintain 0.3 uM concentration;
  • TRIS A (Part 1)/GTLS includes 0.3 uM of SQ22536 or SQ22536 is added to achieve or maintain 0.3 uM concentration; and cry opreservation media also includes 0.3 uM of SQ22536 or SQ22536 is added to achieve or maintain 0.3 uM concentration.
  • a fertilization enhancing compound is (R)-DPN.
  • the Stain TALP medium includes 0.3 uM of (R)-DPN or (R)-DPN is added to achieve or maintain 0.3 uM concentration;
  • TRIS A (Part 1)/GTLS may include 0.3 uM of (R)-DPN or (R)-DPN is added to achieve/maintain 0.3 uM concentration; and cry opreservation media also includes 0.3 uM of (R)-DPN or (R)-DPN is added to achieve or maintain 0.3 uM concentration.
  • the frozen sample may then be thawed and used for IVF embryo production or artificial insemination.
  • the sample may be checked or evaluated through quality control to determine that fertility/motility levels (see other FIGs) have been achieved.
  • Oocyte prep Four well fertilization plates are prepared by filling all 4 wells with 400 pL of BO-IVF (MOFA Verona, WI) and equilibrated in a 37° C 5% CO2 for at least 1 hour. At this same time, four well embryo culture plates filled with 450 pL of BO-1VC (MOFA Verona, WI) are made and equilibrated at 37° C 5% CO2, 5% O2. A sample of each lot of BO-IVC used during these fertilizations is aliquoted and stored at -80oC as control media for assessing conditioned embryo media. All handling of oocytes and zygotes is done with heat pulled glass pipettes.
  • BO-IVF MOFA Verona, WI
  • COCs Cumulus oocyte complexes
  • Semen prep Three insemination straws per treatment group are thawed at 37oC for 45 seconds. They are then layered over 80% BOVIPURETM density gradient (Nidacon international AB. Sweden). The samples are centrifuged at 500 x g for 15 minutes, aspirated close to the pellet, and then resuspended in warm TL HEPES (MOFA Verona, WI). They are centrifuged at 300 x g for 5 minutes, aspirated to 100 pLs, and the pellet is resuspended in that low volume. A 5pL sample aliquot is added to 95 pLs 4% NaCl to immobilize the cells, and cell concentration is quantified using a hemocytometer.
  • the presumptive zygotes are washed in TL HEPES plates and then placed in the embryo culture plates containing maturation media that are equilibrated for 24 hours prior to use (oocyte prep above).
  • the presumptive zygote containing plates are then placed at 37° C 5% CO 2 , 5% O 2 for the rest of the IVF trial.
  • Development assessments Developmental assessments are performed three times during the 8-day post-fertilization incubation. Cleavage events are quantified 48 hours after initial fertilization. Blastocysts are scored on a binary scale of yes/no blastocyst based on its developmental stage. If the embryo had reached al least the early blastocyst stage it is scored as a blastocyst. The differences between early, expanding, and hatched blastocysts are not recorded, nor are the blastocysts scored, but blastocysts are fixed to facilitate future characterization. Blastocyst conversion per oocyte is visually determined on both day 7 and day 8 after initial fertilization. All determinations of developmental stages are done by trained IVF technicians using a dissecting scope on a heated stage set to 37oC.
  • FIG. 2A shows a comparison in normalized Motile (M/mL) after 2 hr storage of post-thaw straw material treated with BI 78D3 bet ween 0.1 pM and 30 pM BI 78D3. Both TO and T2 timepoint raw values were normalized to the TO DMSO control. Dashed lines represent the DMSO control at TO and T2.
  • FIG. 2B shows a comparison in curvilinear velocity (VCL) after 2 hr storage of post-thaw straw material treated with BI 78D3 between 0. 1 pM and 30 pM BI 78D3, with the DMSO control. Both TO and T2 timepoint raw values were normalized to the TO DMSO control. The screening was performed to find any toxicities (test TO and T2 post thaw). The loss of more than 10% Motile M/mL compared to time matched DMSO control at either/or both time points was considered to be indicative of toxic effect on cells.
  • FIG. 3A shows EC80 time for Hoechst uptake obtained using compounds SQ22536, R- DPN, BI 78D3, Bax, and Ro3306, compared to DMSO control. These results indicate that the tested compounds have no risk for reducing Hoechst uptake and are therefore compatible with the staining step required for sexing.
  • FIG. 3B shows CELLTITER- GLO® assay results obtained using compounds SQ22536, BI 78D3, R-DPN, Ro3306, and Bax, compared to DMSO and SOP control. These results indicate that the tested compounds have no risk for increasing cell death during the time frame assessed. Indeed some compound doses increase signal from the CELLTITER-GLO® assay, illustrating an increase in viable cells following the stain step.
  • FIGs. 4A-4B show a comparison in Motile % 2 hours after stain between BI 78D3 (0.03 pM, 0.1 pM, and 30 pM, respectively), DMSO, and SOP (control
  • FIG. 5 shows a comparison in normalized progressive Motile (M/straw) at 3 hours post thaw between compounds Bax, BI 78D3, SQ22536, Ro3306, and R-DPN, at various concentrations. Data demonstrate specific compound concentrations improve the 3 hour survival of motile cells as indicated as a mean > 0 in FIG. 5.
  • FIG. 6 shows normalized progressive Motile (M/straw) for compounds R-DPN (FIG. 6A), BI 78D3 (FIG. 6B), and SQ22536 (FIG. 6C), at 0, 1 , 2, and 3 hours post thaw, compared to the control.
  • FIGs. 7A-7B show on-instrument performance of the cells.
  • FIG. 7A shows initial dead (%) of the cells treated with BI 78D3 and SQ22536, compared to the control, demonstrating that the compounds have the ability to lower the percentage of dead cells following staining.
  • FIG. 7B show s initial eligibility (%) of the cells treated with BI 78D3 and SQ22536, compared to the control.
  • FIG. 8A shows straws/catch tube for samples treated with BI 78D3, SQ22536, and (R)-DPN, compared to SOP control.
  • FIG. 8B shows progressive motile post-thaw (M/straw) of the cells treated with BI 78D3, SQ22536, and R-DPN, compared to the control.
  • M/straw motile post-thaw

Abstract

The present disclosure relates generally to fertilization enhancing compounds (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536, or a salt thereof) and compositions thereof; and methods for improving reproductive cell viability and quality, during and/or after one or more of staining, freezing, thawing, cell selecting, packaging, or in vitro fertilization using the fertilization enhancing compounds. These compounds are especially useful for preserving sperm cell viability during a sex selection or sexing process.

Description

COMPOSITIONSAND METHODS FOR
ENHANCING SPERM CELL QUALITY
PRIORITY CLAIM
[0001 ] This application claims benefit of and priority to US Provisional application 63/334,636 filed April 25, 2022 and US Provisional application 63/357,589, filed June 30, 2022. Each application is hereby incorporated by reference, each in its entirety.
TECHNICAL FIELD
[0002] The present technology relates to the field of animal husbandry and breeding. In particular, the present disclosure includes improved compositions and methods for use with reproductive cell samples, and in fertilization processes. Such compositions and methods provide improved viability, enhanced protection and positive functional improvement of reproductive cells.
BACKGROUND
[0003] An important aspect of animal husbandry, particularly in agriculture, is the collection and use of reproductive cells, e.g., sperm cells (spermatozoa), oocytes, zygotes, embryos, embryonic stem cells and spemiatogonial stem cells. Generally, sperm cells are collected m 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.
[0004] Artificial insemination (Al) and in vitro fertilization (IVF) are common techniques used in cattle and swine farming. Generally, semen samples need to be sorted and/or preserved prior to long-term storage. The sexing process subjects the sperm to cellular insults (Alvarz, J.G. and Storey, B.T., J. Andrology, 13, 1992, 232-241). These stresses decrease the viable cell population, and rapid losses are expected during at least three steps: incubation (at about 19ºC) before staining and sexing; the staining step; and during freezing for long-term storage. For example, stress during sexing can cause premature capacitati on-like changes that minimize the sperm cell’s ability to undergo timely capacitation in the reproductive tract, limiting fertilization potential (Leahy, T. et al., Reproduction 201 1 , 142, 759-778; de Graaf, S. P. et al., Bioscientifica Proceedings 2014, DOI: 10. 1530/biosciprocs.8.035). Induced oxidative DNA damage in sperm decreases fertilization rates and high levels of damage cause developmental arrest after embryonic transcript activation (Aitken, R.J., et al.. International J. Andrology, 2009, 32, 46-56; Fatehi, A.N. et al., J. Andrology, 2006, 27, 176-188). Various types of mediums, extender solutions and compounds have been developed to reduce the metabolic activity of sperm and allow for extended preservation. However, commercially available media do not provide the necessary performance characteristics, and may not allow for adequate maintenance of viability and/or activity of sperm. Additionally, commercially available media also can cause interference with downstream use of the sperm, such as during sexsorting or in IVF or AI Therefore, new and improved compounds, media and/or extender solutions are needed to improve Al, IVF and embryo culture outcomes.
SUMMARY
[0005] Certain aspects and embodiments of the claimed invention are summarized below. These embodiments are not intended to limit the scope of the claimed invention, but rather serve as brief descriptions of possible forms of the claimed invention. The claimed invention may encompass a variety' of forms which differ from these summaries.
[0006] In various embodiments, the present teachings include a composition comprising: a non-human mammalian reproductive cell selected from the group consisting of a sperm cell, an oocyte, an embryo, an embryonic stem cell, and a spermatogonial stem cell; and an effective amount of a fertility enhancing compound for enhancing cell viability during or after one or more of: storage, staining, freezing, thawing, cell selection, packaging, or in vitro fertilization; wherein the fertility enhancing compound can be a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5- HT2A receptor antagonist, aNrf2 (nuclear factor erythroid 2-related factor 2) inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker, an estrogen receptor β agonist, an anti-inflammatory compound, an antioxidant, a TRPV 1 antagonist, or a calcium channel blocker. In some configurations, the fertility enhancing compound can be a c-Jun Kinase inhibitor or an adenylyl cyclase inhibitor.
[0007] In various configurations, the fertility enhancing compound can be DS2, SKA 31, SR 3677 hydrochloride, BAY 41-2272, BI 78D3, SQ 22536, BINA, spiperone hydrochloride, ML 385, Ro 3306, Bax, (R)-DPN, resveratrol, Tempol, Capsazepine, Nifedipine, or Quercetin. In various configurations, the fertility enhancing compound can be DS2. In various configurations, the fertility enhancing compound can be SKA 31. In various configurations, the fertility enhancing compound can be SR 3677 hydrochloride. In van ous configurations, the fertility enhancing compound can be BAY 41-2272. In various configurations, the fertility enhancing compound can be BINA. In various configurations, the fertility enhancing compound can be spiperone hydrochloride . In various configurations, the fertility enhancing compound can be ML 385. In various configurations, the fertility enhancing compound can be Ro 3306. In various configurations, the fertility enhancing compound can be Bax. In various configurations, the fertility enhancing compound can be (R)-DPN. In various configurations, the fertility enhancing compound can be resveratrol. In various configurations, the fertility enhancing compound can be Tempol. In various configurations, the fertility enhancing compound can be Capsazepine. In various configurations, the fertility enhancing compound can be Nifedipine. In various configurations, the fertility enhancing compound can be DS2. In various configurations, the fertility enhancing compound can be Quercetin. In various configurations, the fertility enhancing compound can be SQ 22536. In various configurations, the fertility enhancing compound can be Bl 78D3. In various configurations, the fertility enhancing compound can be present at a concentration ranging from 1 nM to 100 mM. In various configurations, the non-human mammalian reproductive cell can comprise bovine, porcine, equine, ovine, elk, or bison sperm cells. In various configurations, the non-human mammalian reproductive cell can be bovine or porcine sperm cells. In various configurations, the non-human mammalian reproductive cells can be bovine sperm cells. In various configurations, the non-human mammalian reproductive cells can be porcine sperm cells. In various configurations, the bovine, porcine, equine, ovine, elk, or bison sperm cells can be sex selected sperm cells. In vanous configurations, the bovine, porcine, equine, ovine, elk, or bison sex selected sperm cells can be bovine or porcine sex selected sperm cells. In various configurations, the the bovine, porcine, equine, ovine, elk, or bison sex selected sperm cells can be bovine sperm cells. In various configurations, the bovine, porcine, equine, ovine, elk, or bison sex selected sperm cells can be porcine sperm cells. In various configurations, the composition can further comprise a medium.
[0008] In various embodiments, the present teachings can include a method for enhancing non-human mammalian sperm cell viability during or after a sexing process at one or more of staining, freezing, thawing, cell selection, or packaging during the sexing process or in vitro fertilization following the sexing process, wherein the method can comprise: adding to the non-human mammalian sperm cells an effective amount of a fertility enhancing compound or a composition thereof; and wherein the fertility enhancing compound is a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) Inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker, an estrogen receptor β agonist, anti-inflammatory, an antioxidant, a TRPV1 antagonist, or a calcium channel blocker. In some configurations, the fertility enhancing compound can be added during or after the sexing process. In various configurations, adding the fertility compound to the non-human mammalian sperm cells at a pre-freeze stage can provide a positive functional improvement in sperm cells at a post-thaw stage. In various configurations, the fertility compound can be a c-Jun Kinase inhibitor or an adenylyl cyclase inhibitor. In various configurations, the fertility' enhancing compound can be DS2, SKA 31, SR 3677 hydrochloride, BAY 41-2272, BI 78D3, SQ 22536, BINA, spiperone hydrochloride, ML 385, Ro 3306, Bax channel blocker, (R)-DPN, resveratrol, Tempol, Capsazepine, Nifedipine, or Quercetin. In various configurations, the fertility enhancing compound can be DS2. In various configurations, the fertility enhancing compound can be SKA 31. In various configurations, the fertility enhancing compound can be, SR 3677 hydrochloride. In various configurations, the fertility enhancing compound can be BAY 41-2272. In various configurations, the fertility enhancing compound can be BINA. In various configurations, the fertility enhancing compound can be spiperone hydrochloride . In various configurations, the fertility enhancing compound can be ML 385. Tn various configurations, the fertility enhancing compound can be Ro 3306. In various configurations, the fertility enhancing compound can be Bax. In various configurations, the fertility enhancing compound can be (R)-DPN. In various configurations, the fertility enhancing compound can be resveratrol. In various configurations, the fertility enhancing compound can be Tempol. In various configurations, the fertility enhancing compound can be Capsazepine. In various configurations, the fertility enhancing compound can be Nifedipine. In various configurations, the fertility enhancing compound can be DS2. In various configurations, the fertility enhancing compound can be Quercetin. In various configurations, the fertility enhancing compound can be SQ 22536. In various configurations, the fertility enhancing compound can be BI 78D3. In various configurations, the non-human mammalian sperm cells can comprise bovine, porcine, equine, ovine, elk, or bison sperm cells. In various configurations, the non-human mammalian sperm cells can comprise bovine or porcine sperm cells. In various configurations, the non-human mammalian sperm cells can comprise bovine sperm cells. In various configurations, the non-human mammalian semen sample can comprise porcine sperm cells.
[0009] In various configurations, the method can further comprise adding the fertility enhancing compound to the sperm cells at the staining step. In various configurations, the method can further comprise adding the fertility enhancing compound to the sperm cells at the centrifugation step. In various configurations, the method can further comprise adding the fertility enhancing compound to the sperm cells prior to the cry opreservation step.
[0010] In various embodiments, the present teachings can include a method of preserving viability of non-human mammalian semen during sex selection and cryopreservation comprising: obtaining an ejaculate comprising a semen sample; contacting the semen sample with a fertility enhancing compound selected from a c-Jun Kinase inhibitor and an adenylyl cyclase inhibitor; sex selecting the semen sample while maintaining a concentration of the fertility enhancing compound; and cry opreserving the sex selected semen sample. In various configurations, the fertility enhancing compound can be SQ 22536. In various configurations, the fertility enhancing compound can be BI 78D3. In various configurations, the non-human mammalian semen sample can comprise a bovine, porcine, equine, ovine, elk, or bison semen sample. In various configurations, the non- human mammalian semen sample can comprise a bovine or porcine semen sample. In various configurations, the non-human mammalian semen sample can comprise a bovine semen sample. In various configurations, the non-human mammalian semen sample can comprise a porcine semen sample.
[0011] One aspect of the disclosure relates to compositions comprising: a reproductive cell selected from the group consisting of a sperm cell, an oocyte, an embryo, an embryonic stem cell and a spermatogonial stem cell; and an effective amount of a fertility enhancing compound for enhancing cell viability during and/or after one or more of staining, freezing, thawing, cell sorting, packaging, or in vitro fertilization; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker, an estrogen receptor β agonist, an anti-inflammatory compound, an antioxidant, a TRPV 1 antagonist, and a calcium channel blocker.
[0012] Another aspect of the disclosure relates to compositions comprising: a reproductive cell selected from the group consisting of a sperm cell, an oocyte, an embryo, an embryonic stem cell and a spermatogonial stem cell; an effective amount of a fertility enhancing compound for enhancing cell viability during and/or after one or more of staining, freezing, thawing, cell sorting, packaging, or in vitro fertilization; and wherein the fertility enhancing compound is an agonist and/or an antagonist of upstream or downstream regulatory proteins of GABA pathway , potassium channel pathway, Rho-kinase pathway, soluble guanylyl cyclase (sGC) pathway, c-Jun Kinase pathway, adenylyl cyclase pathway, mGluR2 pathway, Dz-like receptor pathway, 5-HT2A pathway, Nrf2 (nuclear factor erythroid 2-related factor 2) pathway, cyclin dependent kinase 1 pathway, Bax channel pathway, estrogen receptor 0 pathway, TRPV1 pathway, or calcium channel pathway.
[0013] Another aspect of the disclosure relates to a media composition comprising an amount of a fertility enhancing compound effective to improve the function of sperm, oocyte, embryo, embryonic stem cell or spermatogonial stem cell, wherein the improvement in function comprises improvement in sexed semen production, improvement in efficiency of the sexing process, improvement in fertility/viability/physiological function of sexed semen, improvement in in vitro fertilization, improvement in rates of embryo production and/or increased implantation, and live births; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) Inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker, an estrogen receptor 0 agonist, anti-inflammatory, an antioxidant, a TRPV 1 antagonist, and a calcium channel blocker.
[0014| Another aspect of the disclosure relates to a composition comprising semen, an extender composition, and an effective amount of a fertility enhancing compound, wherein the semen provides a concentration of motile sperm in the composition ranging from 0.01 M motile sperm/ml to 2000 M motile sperm/ml; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) Inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker, an estrogen receptor 0 agonist, antiinflammatory, an antioxidant, a TRPV1 antagonist, and a calcium channel blocker. [0015] Another aspect of the disclosure relates to a method for enhancing reproductive cell viability during and/or after one or more of staining, freezing, thawing, cell sorting, packaging, or in vitro fertilization, the method comprising, adding to the reproductive cells, an effective amount of a fertility enhancing compound or a composition thereof, wherein the fertility enhancing compound is added at a concentration ranging from 1 nM to 100 mM; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) Inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker, an estrogen receptor P agonist, anti-inflammatory, an antioxidant, a TRPV 1 antagonist, and a calcium channel blocker.
[0016] Another aspect of the disclosure relates to a method of protecting sperm cells throughout the sexing process comprising adding to the sperm cells an effective amount of a fertility enhancing compound or a composition thereof, wherein the fertility enhancing compound is added before, during and/or after the sexing process at a concentration ranging from 1 nM to 100 mM; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-hke dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) Inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker, an estrogen receptor P agonist, antiinflammatory, an antioxidant, a TRPV1 antagonist, and a calcium channel blocker.
[0017] Another aspect of the disclosure relates to a method of eliciting a positive functional improvement in sperm cells at the post-thaw stage, the method comprising, adding to the sperm cells at a pre-freeze, an effective amount of a fertility enhancing compound or a composition thereof, wherein the fertility enhancing compound is added at a concentration ranging from 1 nM to 100 mM; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) Inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker, an estrogen receptor 0 agonist, antiinflammatory, an antioxidant, a TRPV1 antagonist, and a calcium channel blocker.
[0018] Another aspect of the disclosure relates to a method of improving quality of a semen sample comprising contacting the semen sample with an effective amount of a fertility enhancing compound or a composition thereof, wherein the fertility enhancing compound is added at a concentration ranging from 1 nM to 100 mM; and wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) Inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker, an estrogen receptor 0 agonist, antiinflammatory, an antioxidant, a TRPV1 antagonist, and a calcium channel blocker.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] To illustrate the disclosure, depicted in the drawings are certain features of the aspects and embodiments of the disclosure. However, the disclosure is not limited to the preci se arrangements and instrumentalities of the aspects depicted in the drawings.
[0020] FIG. 1 is a flowchart showing the addition of a fertilization enhancing compound at various stages during the sexing process, each media that could contain a fertilization enhancing compound is designated by a star.
[0021] FIG. 2A shows a comparison in normalized Motile (M/mL) after 2 hr storage of post-thaw straw material treated with BI 78D3 between 0.1 pM and 30 pM BI 78D3. Both TO and T2 timepoint raw’ values were normalized to the TO DMSO control. Dashed lines represent the DMSO control at TO and T2. FIG. 2B show's a comparison in curvilinear velocity (VCL) after 2 hr storage of post-thaw straw material treated with BI 78D3 between 0.1 pM and 30 pM BI 78D3, with the DMSO control. Both TO and T2 timepoint raw values were normalized to the TO DMSO control.
[0022] FIG. 3A shows the time it takes to reach 80% saturation (EC80) for Hoechst dye uptake obtained using compounds SQ22536, R-DPN, BI 78D3, Bax, and Ro3306, compared to a DMSO control. FIG. 3B shows CellTiter Gio assay results obtained using compounds SQ22536, BI 78D3, R-DPN, Ro3306, and Bax, compared to DMSO and SOP control.
[0023] FIGs. 4A-4B show a comparison in Motile % 2 hours after stain between BI 78D3 (0.03 pM, 0.1 pM, and 30 pM, respectively), DMSO, and SOP (control) in JE4264 media (FIG. 4A) and HO0916 media (FIG. 4B).
[0024] FIG. 5 shows a comparison in normalized progressive Motile (M/straw) at 3 hours post thaw between compounds Bax, BI 78D3, SQ22536, Ro3306, and R-DPN, at various concentrations.
[0025] FIG. 6 shows normalized progressive Motile (M/straw) for compounds R-DPN (FIG. 6A), BI 78D3 (FIG. 6B), and SQ22536 (FIG. 6C), at 0, 1, 2, and 3 hours post thaw compared to the control.
[0026] FIG. 7A shows initial dead (%) of the cells treated with BI 78D3 and SQ22536, compared to the control. FIG. 7B shows initial eligibility (%) of the cells treated with BI 78D3 and SQ22536, compared to the control.
[0027] FIG. 8A shows straws/catch tube for samples treated with BI 78D3, SQ22536, and (R)-DPN, compared to SOP control. FIG. SB shows progressive motile post-thaw (M/straw) of the cells treated with BI 78D3, SQ22536, and R-DPN, compared to the control. DETAILED DESCRIPTION
[0028] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.
Definitions
[0029] Throughout this application, various embodiments of the present technology may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present technology. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
[0030] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ ranges between" a first indicate number and a second indicate number and "ranging/ ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.
[0031 ] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory' procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.
[0032} As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 10% (including, e.g., 1%, 5% or 10%) in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
[0033] The expression “comprising” means “including, but not limited to.” For example, compositions and methods include the recited elements, but do not exclude others.
“Consisting essentially of” shall mean excluding other elements of any essential significance to the combination for the stated purpose Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characterIstic(s) of the claimed invention. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention. It will be understood that use of any of these expressions — “comprising,” “consisting essentially thereof,” or “consisting of’ — also contemplate and provides disclosure for use of any of the other terms
[0034] As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, or a mammal. In some embodiments, the individual, patient or subject is cattle, pig, or sheep.
[0035 ] As used herein, the term “effective amount” refers to an amount sufficient to produce a biological effect.
[0036] As used herein, “fertility” includes one or more of the following: a level or degree of ability of an animal to conceive and bear young; a level or degree of ability of an animal to become pregnant; a level or degree of ability of an animal to reproduce; a level or degree of ability of a spermatozoa to fertilize an oocyte; a level or degree of ability of an oocyte to be fertilized by a spermatozoa; and a level or degree of ability of an oocyte fertilized by a spermatozoa to develop into a zygote capable of progressing through embryonic and fetal development. Various methods of evaluating fertility, such as in gametes, are described in U.S. Patent Pub. 2020/0347347 by Roti-Roti, which is incorporated herein by reference and for all purposes.
[0037] As used herein, “fertilization enhancing compound” refers to compounds that can provide beneficial effect in fertilization, including but not limited to compounds that can improve the fertility competency of individual sperm cells, compounds that can improve the amount of motile, live cells in the straw; and/or compounds that can increase the recovery of motile cells.
[0038] 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 or sperm cells. The sexing process can be accomplished using a number of different techniques, including droplet sorting, mechanical sorting, and laser ablation
[0039] The term “reproductive cell” as referred herein is defined as sperm, eggs, and the formation of embryo/blastocyst, also gametes; haploid cells; germ cells; sex cells; sperm cells and egg cells.
[ 0040 ] The term “medium” or “media” as used herein refers to an essentially liquid composition that may contain nutrients, salts, and other substances or constituents.
[0041] The term “ejaculate” as used herein refers to the combination of semen and spermatozoa, which may comprise any amount of seminal plasma, produced by a male mammal, as released by ejaculation.
[0042 ] The term “seminal fluid components” as referred herein is the substances that make up and/or are commonly found in mammalian semen. Seminal fluid components include, but are not limited to, amino acids, prostate specific antigen, proteolytic enzymes, citric acid, citrate, sialic acid, vitamin C, acid phosphatase, fibrinolysin, lipids, fructose, prostaglandins, phosphorylcholine, glycerophosphocholine, flavins, basic amines such as putrescine, spermine, spermidine and cadaverine, zinc, galactose, mucus and other organic and inorganic constituents.
[0043] The collection and use of sperm cells is a central part of animal husbandry and breeding. Sperm cells are collected in the form of raw ejaculate from male animals and must be stored before further use. Storage can comprise hours or even days.
Additionally, cell samples are usually manipulated in one or more ways before use. Thus, it is important to maintain the viability and function of the cells throughout the process.
[0044] Sexing procedures disclosed can be implemented for use with fresh, un-extended ejaculate. However, there are inherent issues to using fresh ejaculate that is not supplemented with an extender, as an ejaculate declines in quality continually after collection. Sperm that undergoes sexing is exposed to numerous insults including temperature swings, high dilution, and pH changes during cell processing. Insults during sexing include shear stress, high fluid pressure, and the high force caused by the sexing process on cytometers (Gamer, D.L. and Seidel, G.E., Canadian Journal of Animal Science, 2003, 83, 375-384; Gamer, D.L., Theriogenology, 2006, 65, 943-957). These insults lead to a decrease in the number of cells recovered after processing.
[0045] That loss of cells during processing, while detrimental, is not the main source of projected product loss. The major loss is due to fresh ejaculates having a steady increase in dead cell population over time after collection. A major factor of this is that the fresh ejaculates must be stored at close to room temperature, as the sexing process happens at room temperature. Keeping the ejaculate at room temperature, rather than at a cooler temperature that better preserves cell survivability prevents time spent on equilibrations to the cooler temperature and allows sexing of the ejaculates continuously. This also increases the rapidity in which the viable cells are lost before the sexing process begins. This continual loss of viability when paired with the time required to perform the sexing and the packaging steps led to the standard policy of freezing a sexed ejaculate no later than 12 hours after collection.
[0046] This policy of having to package an ejaculate no later than 12 hours after collection leaves a lot of ejaculate volume behind due to surplus ejaculate volume. These excess volumes cannot be utilized because fresher ejaculates are being collected before they can be used entirely. Ejaculates are collected every 6 hours, 24 hours a day, to prevent a lapse in instrument running time due to lack of sample. This means that ejaculates from an early morning collection frequently still have viable volume left to run when the second daily collection arrives but are still removed and replaced in favor of the fresher ejaculate because the older sample is not likely to run an additional 6 hours on the instrument. Favoring fresh ejaculates leads to a second major source of loss: downtime on sexing instruments. All ejaculates from a given time point are removed and replaced at the same times each day, meaning instruments are shut down, cleaned, and restarted four times daily. This means that a minimum of 40 minutes of run time is lost, four times daily, for each instrument. These numbers calculated in a number of cells is 46.9 x 108 skewed cells uncollected per instrument per day; which translates to approximately one thousand insemination doses lost per day.
[0047] An extender or other medium formulated for use in a sperm sexing facility could help to mitigate losses. For example, the medium can slow the decline of sperm cells held at room temperature before sexing, allowing a larger number of ejaculates to be collected at a single time point, and decreasing the number of times per day ejaculates are collected. In this model, instruments would only be shut down to change to a new7 ejaculate as needed on a bull by bull basis, rather than the entire production floor at once. This would decrease the time it takes to change to a new bull, as it increases the available staff per instrument. Maintaining cell viability7 also means ejaculates could be run until exhaustion, maximizing the number of sexed sperm obtained per ejaculate, and decreasing the total number of times per day a bull change would be performed per instrument. By mitigating these causes of cell loss, the number of insemination doses produced would increase making the superior product more available to farmers globally.
[0048] A fertility enhancing compound would also be useful in reducing or mitigating cell stress involved in a staining step or process, such as a staining process with Hoechst 33342 prior to enriching or sexing a cell population on an instrument. For example, in a staining process, a semen sample may be mixed with staining media and other compounds which alter the concentration and pH of the semen sample, and the semen sample may be subjected to elevated temperatures, such as temperatures above 30 ºC. The changes in sample chemistry, concentration, and temperature are stressors on the cells that impact cell health. Cell viability is improved by reducing the stressors, or by reducing or mitigating the impact of the sources of stress on the cells. A fertility enhancing compound is used to reduce or mitigate the impact of the stressors on the cells thereby maintaining cell viability, reducing cell loss, and maximizing the number of sexed sperm that may be obtained per ejaculate.
[0049] In addition, a large amount of cell loss is observed during the freeze-thaw process. Both conventional (i.e. non-sexed) and sexed semen are typically frozen in straws for storage and distribution. The straws must be subsequently thawed pnor to use for insemination or fertilization. This process of freezing and thawing results in the death of a large proportion of the cells in the straw. The compositions and processes disclosed herein can be used to alleviate this cell loss due to the freeze-thaw process.
Compositions
[0050] The present disclosure relates to certain fertilization enhancing compounds, and compositions thereof, and methods that improve reproductive cell viability and activity.
|0051 ] In some embodiments, the fertilization enhancing compound is an element of a pre-made or pre-formulated media (or a composition). In some embodiments, the fertilization enhancing compound is added (e g., to the reproductive cells) on its own as a discrete element or step in a process described herein.
[0052] In some embodiments, the fertilization enhancing compounds, compositions and/or methods described herein may be used in a conventional process (e g., conventional (i.e. non-sexed) semen). For example, the fertilization enhancing compounds (and/or compositions) described herein may be added to conventional semen after it has been collected. In some embodiments, the fertilization enhancing compounds (and/or compositions described) herein are added to conventional semen immediately prior to packaging in straws. In some embodiments, the fertilization enhancing compounds (and/or compositions described) herein are added to conventional semen immediately before usage in an Al or IVF process. [ 0053] In some embodiments, the fertilization enhancing compounds, compositions and/or methods described herein may be used in a sexing process (e g., sexed semen). Tn some embodiments, the fertilization enhancing compounds (and/or compositions) described herein may be added to semen that is to be sexed. In some embodiments, the fertilization enhancing compounds (and/or compositions described) herein are added to semen that is to be sexed at one or more of the following stages, including 1) immediately after collection; 2) during incubation or pre-instrument QC (quality control); 3) immediately prior to, during, or after staining; 3) pre-sexing instrument; 4) postinstrument in the catch tube; 5) prior to centrifugation; 6) after centrifugation; 7) prepackaging, either prior or post-cryopreservation addition; and 8) post-thaw, such as before an Al or IVF event.
[0054] In some embodiments, the present specification provides and includes fertilization enhancing compounds and/or compositions that impart increased activity and viability to reproductive cells, in particular, sperm cells. The present specification also provides and includes methods for processing reproductive cell samples, wherein the methods and processes produce samples in which the sperm cells have increased viability and activity, and improve production efficiency. The present specification, also provides and includes the reproductive cell samples produced by these methods, wherein reproductive cells in the samples have increased viability and activity. The present specification still further provides and includes methods using these reproductive cell samples with increased viability and activity, including sexing (selecting X-chromosome bearing or Y- chromosome bearing cells), sorting, separating, freezing, artificial insemination, in vitro fertilization, cooling and transport, and related processes.
[0055] In various aspects and embodiments, the present disclosure relates to new compositions, new media formulations, new processes for addition of a fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) or a salt thereof, to spemi cells (before/, during, ot after sexing, or in a conventional process without sexing) and/or to embryos (before, during, or after IVF). In some embodiments, the fertilization enhancing compounds described herein are included in a composition or a media before adding to sperm cells and/or to embry os. In some embodiments, the fertilization enhancing compounds described herein are added directly (e.g, not included in a composition or a media before use) to sperm cells and/or to embryos. In embodiments, the present disclosure relates to improvements in conventional (i.e. non-sexed) semen production. In embodiments, the present disclosure relates to improvements in sexed semen production. Exemplary' improvements include but are not limited to, improved efficiency of the sexing process and improved fertility/viability/physiological function of sexed semen; improvements in in vitro fertilization, including improved rates of embryo production and/or increased implantation, live births.
[0056] In some embodiments, provided herein are compositions which include a reproductive cell selected from the group consisting of a sperm cell, oocyte, embryo, embryonic stem cell and a spermatogonial stem cell; an effective amount of a fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) for enhancing cell viability during and/or after one or more of staining, freezing, thawing, cell sorting, packaging, or in vitro fertilization; and optionally, a medium. In some embodiments, the medium is an extender medium. In other embodiments, the medium may be a maturation medium or other medium used in an IVF process, or could be a staining solution in a sexing process, or a collection medium (e.g., comprising a cryoprotectant) in the sexing and/or the conventional process.
[0057] Suitable fertilization enhancing compounds include, but are not limited to a GABA positive allosteric modulator (e.g, DS2), a potassium channel activator (e.g., an activator of KCa3.1 and KCa2 channels such as SKA 31), a Rho-kinase inhibitor (e.g., SR 3677 or SR 3677 hydrochloride), a soluble guanylyl cyclase (sGC) activator (e.g., BAY 41-2272), a c-Jun Kinase inhibitor (BI 78D3), an adenylyl cyclase inhibitor (e.g., SQ 22536), a mGluR2 positive allosteric modulator (e.g., BINA), a D2-like dopamine receptor antagonist (e.g., spiperone or spiperone hydrochloride), a 5-HT2A receptor antagonist (e.g., spiperone or spiperone hydrochloride), a Nrf2 (nuclear factor erythroid 2-related factor 2) inhibitor (e.g., ML 385), a cyclin dependent kinase 1 inhibitor (e.g., Ro 3306), a Bax channel blocker (e.g, Bax), an estrogen receptor P agonist (e.g., (R)-DPN), an antiinflammatory compound (e.g, Tempol or Quercetin), an antioxidant (e.g., resveratrol, Tempol, or Quercetin), a TRPVl antagonist (e.g., Capsazepine), and a calcium channel blocker (e.g., Nifedipine), or a salt thereof Tn any embodiments, the fertilization enhancing compound is BI 78D3, SQ 22536, Ro 3306, Bax, (R)-DPN, or a salt thereof. In any embodiments, the fertilization enhancing compound is a c-Jun Kinase inhibitor. In any embodiments, the fertilization enhancing compound is BI 78D3 or a salt thereof (collectively referred to as “BI 78D3”). In any embodiments, the fertilization enhancing compound is a an adenylyl cyclase inhibitor. In any embodiments, the fertilization enhancing compound is SQ 22536 or a salt thereof (collectively referred to as “SQ 22536”).
[0058] The fertilization enhancing compound (e.g. , a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) can be provided in an amount effective to enhance cell viability during and/or after one or more of staining, freezing, thawing, cell selection, packaging, or in vitro fertilization. Depending on the type, the fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) can be provided between about 0. 1 nM and about 100 mM, including, for example, about 0.001 pM to about 50 mM, about 0.01 pM to about 500 pM, about 0.1 pM to about 250 pM, about I pM to about 100 pM, about 2 pM to about 50 pM, about 3 pM to about 30 pM, about 4 pM to about 20 pM, about 5 pM to about 10 pM, about 0.1 pM to about 5 pM, about 0.1 pM to about 10 pM, or about 0.1 pM. In some embodiments, the fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) is present at a concentration ranging from about 0.1 pM to about 5 pM. In some embodiments, fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) is present at a concentration ranging from about 5 pM to about 50 pM. In some embodiments, the fertilization enhancing compound (e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) is present at a concentration of less than about 500 pM, or alternatively less than about 100 pM, or alternatively about less than 50 pM, or alternatively less than about 30 pM, or alternatively less than about 20 pM, or alternatively less than about 10 pM, or alternatively less than about 5pM, or alternatively less than about 3 pM, or alternatively less than about 2 pM. In some embodiments, the fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) is present at a concentration of greater than about 0.01 pM or alternatively greater than about 0.1 pM, or alternatively about greater than 1 pM, or alternatively greater than about 2 pM, or alternatively greater than about 5 pM, or alternatively greater than about 10 pM, or alternatively greater than about 15 pM, or alternatively greater than about 20 pM, or alternatively greater than about 50 pM. In some embodiments, the fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) is present at a concentration ranging from about 10 nM to about 100 pM. In some embodiments, the fertilization enhancing compound (e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) is present at a concentration ranging from about 1 mM to about 50 mM. In some embodiments, the fertilization enhancing compound (e g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) is present at a concentration ranging from about 5 pM to about 50 pM. In some embodiments, the fertilization enhancing compound is a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536, and it is present at a concentration of about 10 nM to about 100 pM. In some embodiments, the fertilization enhancing compound is a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536, and it is present at a concentration of less than about 5 pM. In some embodiments, the fertilization enhancing compound is a c-Jun Kinase inhibitor such as BI 78D3, and it is present at a concentration of about 0.1 pM. In some embodiments, the fertilization enhancing compound is an adenylyl cyclase inhibitor such as SQ 22536, and it is present at a concentration of about 0.3 pM. In some embodiments, the fertilization enhancing compounds described herein are included in a composition or a media before adding to sperm cells and/or to embryos, and the concentrations described herein are the concentration of the fertilization enhancing compounds in the composition or media. In some embodiments, the fertilization enhancing compounds described herein are added directly (e.g., not included in a composition or a media before use) to sperm cells and/or to embryos, and the concentrations described herein are the concentration of the fertilization enhancing compounds in the resulting mixture (resulting composition) after addition. [0059] In some embodiments, a fertilization enhancing compound (e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) is provided in an amount effective to enhance cell viability during and/or after one or more of staining, freezing, thawing, cell sorting, packaging, or in vitro fertilization. In some embodiments, one or more fertilization enhancing compounds described herein are provided in an amount effective to enhance cell viability during and/or after one or more of staining, freezing, thawing, cell sorting, packaging, or in vitro fertilization. Effective amounts for these compounds may range from about 0.01 uM to about 30 uM.
[0060] In addition to the fertilization enhancing compound (e.g. , a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536), the compositions may also include one or more types of reproductive cells. Suitable reproductive cells, include, without limitation, sperm cells, oocytes, embryos, embryonic stem cells and spermatogonial stem cells. In some embodiments, the composition may include a single sperm cell or a plurality of sperm cells. In some embodiments, the composition may include a single oocyte or a plurality of oocytes. In some embodiments, the composition may include a single embryo or a plurality of embryos. In some embodiments, the composition may include a single embryonic stem cell or a plurality' of embryonic stem cells. In some embodiments, the composition may include a single spermatogonia! stem cell or a plurality of spermatogonia! stem cells. In some embodiments, the composition may include a plurality' of sperm cells. In some embodiments, the sperm cells include mammalian sperm cells. Suitable mammalian sperm cells may include, without limitation, human, bovine, porcine, equine, ovine, elk, or bison sperm cells. The concentration of reproductive cells in the composition can be adapted based on the type of cells and the process stage. For example, traditionally, sex selection is performed at 66 million cells per mL (M/mL). Accordingly, the initial cell concentration can be higher (e.g., 1000 M/mL or less), which can then diluted in a medium or buffer to a concentration of 66 M/mL for sexing
[0061 ] Different sexes of livestock are preferred depending on the application. For example, only female dairy cattle produce milk, and male cattle have greater muscle mass for beef production. 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. 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.
[0062] The composition of the present technology may further include a medium.
Suitable mediums, include without limitation, an extender medium, a cryoprotectant, a buffer, a diluent, an energy source, an extender medium, an antibiotic, and a bolus. The potassium channel blocker may be added to the composition separately or included in the medium. In some embodiments, the fertilization enhancing compound, e g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536, or a salt thereof, is included in the extender medium. In some embodiments, the fertilization enhancing compound can be added altogether in a single dose or as a bolus, slowly titrating in, and combinations of the two. In any embodiment, the media composition may contain the fertilization enhancing compound, and can be combined with cells in a variety of ways, for example by using a set volume of media, a set ratio of media to cell sample, or media provided at a set volume in relation to a measured aspect of the sample (i.e., sperm cell concentration). In certain embodiments, media can be added to the sample, in other embodiments, the sample can be added to the media. In other embodiments, both sample and media can be added to a third container/receptacle.
[0063} The present technology provides fertilization enhancing compounds and compositions thereof. In some embodiments, the fertilization enhancing compound can include a c-Jun Kinase inhibitor (e.g., BI 78D3 or a salt thereof) or an adenylyl cyclase inhibitor (e.g. , SQ 22536 or a salt thereof). The c-Jun Kinase inhibitor or adenylyl cyclase inhibitor is present, e.g, in the composition and/or medium in an amount effective to improve the function of sperm, oocyte, embryo, embryonic stem cell or spermatogonia! stem cell, wherein the improvement in function comprises improvement in semen production (sexed semen production and/or non-sexed semen production), improvement in efficiency of the sexing process, improvement in fertility/viability /physiological function of semen (sexed semen and/or non-sexed semen), improvement in in vitro fertilization, improvement in rates of embryo production and/or increased implantation, and live births.
10064] The present technology preserves the viable, motile sperm population eligible for sexing and significantly reduces the cost of sexed semen to farmers by increasing yield, such as by increasing the number or percentage of successful or viable pregnancies from artificial insemination events using said semen. The ideal extender formulation must maintain a high motile sperm population and must not interfere with the ability to separate the X and Y populations using a fluorescent DNA stain, which is required to separate the two cell populations on the sexing cytometry instruments.
[0065] In some embodiments, additives may be included in a composition (and/or a medium) comprising the fertilization enhancing compounds to bring about the desired properties and create the inventive media. In any embodiment, the compositions comprise a medium which includes at least one additive selected from the group consisting of antioxidants, phosphatidylserine (PS), coumarin compounds or pyranocoumarin compounds, zinc chloride, coenzyme Q10, a nonsteroidal antiinflammatory drug (NSAID), linolenic acid, fatty acids, D-aspartic acid, and combinations thereof. In some embodiments, the medium contains sodium fluoride. In any embodiment, the additives comprise one or more of phosphatidylserine (PS), decursin, zinc chloride, coenzyme Q10, acetylsalicylic acid (aspirin), linolenic acid, fatty acids, D-aspartic acid, sodium fluoride, and combinations thereof. In some embodiments, all of the additives are included in the formulation. In other embodiments, one or more of decursin, zinc chloride, coenzyme Q10, acetylsalicylic acid (aspirin), linolenic acid, fattyacids, D-aspartic acid, or sodium fluoride are omitted. The additives, when present, maybe included in a concentration range of from about 0 to about 100 mM or 0 to 1000 pg/mL. [0066] In any embodiment, the medium further includes a buffer. For applications where cell survivability and/or shelf life is of preeminent concern, the buffer may be TRIS or HEPES. In certain embodiments, TRIS may be used due to its longer shelf life in the formulation as measured by pH stability.
[0067] In other embodiments, the medium may be supplemented with a salt such as sodium chloride (NaCl), potassium chloride (KC1), calcium chloride dehydrate (CaC12(H2O)2), magnesium chloride hexahydrate (MgC12(H2O)6), sodium bicarbonate (NaHCO3), sodium phosphate dihydrate (NaH2PO4(H2O)2), potassium phosphate ( KH2PO4), and sodium fluoride (NaF).
[0068] In one embodiment, the medium may further include antioxidants, which are added to decrease the amount of stress the cells are subjected to during the sexing process. All sperm are exposed to LV light during the sexing process, which typically causes oxidative damage to DNA (rather than direct strand breaks), and sperm is likely subject to elevated reactive oxygen species (ROS) during the cryoprotectant step (Aitken, R.J., et al., Asian J Androl., 2015, 17, 633-639; Farber, J.L., Environmental Health Perspectives, 102 (Suppl 10), 1994, 17-24). ROS can also cause DNA damage such as single and double strand breaks, and base pair modification (Richter, C , et al., Proc. Natl. Acad. Sci., 1988, 85, 6465-6467). Fatehi et al. (Fatehi, A.N. et al., J. Andrology, 2006, 27, 176-188) reported oocytes fertilized with DNA damaged bovine spermatozoa exhibited cleavage rates similar to controls, but further development halted in the damaged experimental group. The compositions of the present technology which include sperm cells treated with potassium channel blocker compound may exhibit less DNA damage than non-extended controls.
[ 0069] In some embodiments, the compositions comprise Nonsteroidal Antiinflammatory- Drugs (NSAIDs), which are a class of drugs and compounds capable of reducing inflammation, primarily through inhibition of cyclooxygenase enzymes (COX-1 and/or COX-2). The compositions can include one or more NSAID including , but not limited to: salicylates, including aspirin (acetylsalicylic acid), diflunisal (Dolobid); salicylic acid and other salicylates, and salsalate (Disalcid); Propionic acid derivatives, including Ibuprofen, Dexibuprofen, Naproxen, F enoprofen, Ketoprofen, Dexketoprofen, Flurbiprofen, Oxaprozin, and Loxoprofen; acetic acid derivatives, including indomethacin, Tolmetin, Sulindac, Etodolac, Ketorolac, Diclofenac, Aceclofenac, and Nabumetone: enolic acid (Oxicam) derivatives, including Piroxicam, Meloxicam, tenoxicam, Droxicam, Lomoxicam, Isoxicam, and phenylbutazone (Bute); anthranilic acid derivatives (Fenamates), including mefenamic acid, meclofenamic acid, flufenamic acid, and tolfenamic acid; selective COX-2 inhibitors (Coxibs), including Celecoxib, Rofecoxib, Vai decoxib, Parecoxib, Lumiracoxib, Etoricoxib, and Firocoxib;
Sulfonanilides, including Nimesulide; and other NSAIDs, including Clonixin, Licofelone, and H-harpagide (in Figwort or Devil’s Claw). In some embodiments, the compositions comprise coumarin compounds or pyranocoumarin compounds. In certain embodiments, the coumarin compound or pyranocoumarin compound comprises decursin.
[0070] In one embodiment, the medium may further include one or more of antioxidants, phosphatidylserine (PS), phosphatidylcholine, coumarin compounds, pyranocoumarin compounds, zinc chloride, coenzyme Q10), a nonsteroidal anti-inflammatory' drug (NSAID), linolenic acid, fatty' acids, D-aspartic acid, sodium fluoride, decursin, acetylsalicylic acid (aspirin), a salt (e.g., sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium bicarbonate, sodium phosphate dihydrate, potassium phosphate, sodium fluoride), a buffer (HEPES, Tris, etc.), a sugar source (glucose, fructose, etc.), citric acid, pyruvate, ascorbic acid, glycerol or other cry ©protective agents, natural or synthetic ice blockers, BSA or other protein source, egg yolk, polyvinylalcohol, polyvinylpyrrolidone, and other polymers. In one embodiment, the medium may' also include beads, such as magnetic (or other) beads coated with lectin, DNA-binding moieties or ubiquitin-targeted moieties. For example, lectin-beads can be used to remove the dead cells during the staining process.
[0071] In any embodiment, by using the present technology, reproductive cell activity' of stored and/or manipulated samples can be maintained or even increased, by the measure of either motility', fertilization, or both. Fertilization can be measured by blastocyst formation. Concentration and progressive motility of the sample may be further evaluated using an integrated visual optical system (“IVOS”). [0072] In any embodiment, provided is a composition with semen, an extender composition, and an effective amount of a fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) wherein the semen provides a concentration of motile sperm in the composition ranging from about 0.01 M motile sperm/ml to about 2000 M motile sperm/ml. For example, concentration of motile sperm in the composition may range from about 75-200 M motile sperm cells per mL in the stain reaction; about 25-100 M motile sperm cells per mL as the samples are on -instrument; about 0.25-4 M motile sperm cells/ mL at the postinstrument stage; about 10-600 M motile sperm cells/mL after centrifugation; about 5-300 M motile sperm cells / mL at cooling/pre-freeze stage after addition of Part 2 of a two- part extender (e.g., Tris B); and about 0.25-15 M motile/ mL at freeze. In some embodiments, the fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) is added to a composition having semen in a concentration range of 400 -- 2,000 M motile sperm I mL for the extender described in US PGPUB 2020/0347347 (Roti-Roti et al.) and the same extender with an amount of citrate.
[0073] The present technology can provide a number of important benefits, including but not limited to: extends cell viability, with a reduced loss of progressively motile cells; does not interfere with Hoechst 33342 (or an alternative) staining and red dye viability counterstaining of the cells which is necessary for proper sexing on the cytometers; and does not negatively interfere with fertilization capacity or embryonic development.
[0074] The present technology relates to a fertilization enhancing compound, e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536, or a salt thereof, as well as a composition or a medium thereof, which maximizes recovery and packaging of functional, fertilization competent sperm. This can improve the flexibility and efficiency of production by exhausting ej aculates, optimizing bull changes, and decreasing the need for backup ejaculates. An additional advantage is an increase in motile cells recovered post-processing (e.g., sexing). When semen samples are processed using the inventive compositions and methods, increased activity is seen. Increased activity can be increased viability, increased motility' or both. Moreover, use of the present technology allows for greater yields of semen samples during and after processing. Specifically, inclusion of a fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) at various stages (including various stages of sexing) can improve fertility7 and processing. For example, including a fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) in the process (including the sexing process) can improve fertility of semen (e.g., sexed semen), which can be assessed by embryo cleavage and blastocyst formation post-IVF. In particular, semen samples (e.g., sexed semen samples) treated with a fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) can exhibit increased cellular function, which can be measured by mitochondrial membrane potential, motility, ATP production, plasma membrane integrity, and other cell physiological readouts. In addition, the presence of a fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) during the process of staining sperm cells with Hoechst dye can reduce the time required to achieve staining saturation, and increase the number of cells eligible for sexing, increase resolution (separation of X- and Y- chromosome cell peaks in a histogram derived from interrogation of spemi cells by an instrument), and sex skew, with the potential to positively impact batch yields.
[0075] The present technology may exhibit enhanced sperm cell survivability and motility compared to commercially available extenders: ANDROMED® (Minitube, Delavan, WI USA) and OptiXell (IMV technologies. Maple Grove, MN USA), and a previously described media formulation CEP2 (Verberckmoes, S., et al., Reproduction in Domestic Animals, 2004, 39, 410-416).
[0076] The present technology' may extend the window of cell survivability before sexing, while still maintaining cells measured as live and motile after the sexing process. The present technology may result in an increase of the percent of blastocysts per oocyte. In some embodiments, the compositions may result in less cleavage. In some embodiments, the technology may successfully maintain the motile, viable sperm population for an enhanced period of time, e.g., 24 hours, before sexing, and may result in frozen-thawed sexed semen that meets quality control standards with no increased risk for batch failure compared to current standard operating procedures. Use of the fertilization enhancing compounds, as well as the compositions thereof, therefore, has the potential to increase utilization of the total ejaculate volume and concurrently increase the number of insemination doses produced per ejaculate, increasing the availability of sexed semen for farmers. In any embodiment the present technology may maintain sperm in a fertilization competent state. Fertilization competence includes, but is not limited to, the capability of sperm cells exposed to compositions according to the present technology for producing pregnancies via artificial insemination, and fertilization, cleavage, and blastocyst conversion both in vitro and in vivo. The compositions of the present technology which include sperm cells treated with a fertilization enhancing compound (e.g , a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536), may produce more blastocysts per oocyte compared to non-extended, paired controls. In some embodiments, the fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) may produce enhanced zygote/blastocyst formation from germ cells (i.e., increased fertilization or increased activity of reproductive cells).
[0077| Other advantages realized by use of the present technology are a decreased need for multiple collections of ejaculates or moving of animals to the process site. Additionally, the present technology, due to its maintenance of viability and motility of reproductive cells, can allow for shipping of ejaculates for further processing (e.g., sexing). This eliminates the need to move and quarantine animals.
[0078] Sources of reproductive cell samples are typically from ejaculate, obtained by methods commonly known in the art. The ejaculate samples can be a single source or pooled . In some embodiments, in vitro produced or expanded sperm cell populations may be used. Samples are obtained from animals, preferably mammalian animals; more preferably livestock; samples are most preferably porcine or bovine.
[0079| In some embodiments, the media composition containing the fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) is utilized as a “hold media” to store raw ejaculate and minimize loss of reproductive cell components. In further embodiments, the composition is utilized as a “hold media” to store isolated sperm cells after processing and before use in breeding procedures. This can also be referred to as an “extender media” since samples remain viable for longer when the inventive media is used. In other embodiments, the composition functions as a medium to use for processing of reproductive cell samples that are used for further processing (such as, e.g., sexing). For example, the composition may be utilized as a staining media, a sexing processing media, and/or a freezing media.
[0080] In certain embodiments, compositions comprising reproductive cells and the inventive “hold media” can maintain an acceptable viability and/or motility for hours (e.g., 24 hours). In other embodiments, the inventive compositions can maintain an acceptable level of live cells throughout cell processing. For example, the percentage of dead cells may be < 25% throughout sexing duration of processing.
[0081 ] Samples can be combined with the fertilization enhancing compounds and/or the improved media in a variety of ways. The fertilization enhancing compounds and/or the media can be added directly after collecting the raw ejaculate sample, within a set amount of time after collecting the raw ejaculate; or the raw' ejaculate can be collected directly into the media.
[0082] Sperm cells that are exposed to the fertilization enhancing compounds (or the inventive compositions thereof) can exhibit enhanced motility, viability , and functionality (including the ability to fertilize ova) over time, compared to sperm exposed to existing commonly -used media. Thus, these fertilization enhancing compounds (or the compositions thereof) can enhance yields in both conventional and sexed semen production. The fertilization enhancing compounds (or the inventive compositions thereof) can maximize recovery and packaging of functional, fertilization competent sperm.
[0083} The present technology' may provide beneficial effects for IVF outcomes, measured as cleavage and blastocyst conversion rates, which may be due, at least in part, to mitigation of DNA damage, increased ROS production, and/or capacitati on-like changes caused by sexing. The fertilization enhancing compounds (or the compositions thereof) of the present technology may also allow for increased run time for each ejaculate and thereby increase frozen semen (e.g., sexed semen) product per volume of ejaculate collected and decreased the cost of each insemination dose. This allows semen (e.g., sexed semen) products to be more widely available to farmers who would profit from the use of semen (e.g., sexed semen) on their cattle farms. Further the compositions are applicable not only to frozen sexed bovine semen, but could also have applications in extending the life of a fresh ejaculate in a setting where extended transport times are required or specifically for preservation of ejaculates of impaired quality.
Methods
[0084] One aspect of the disclosure relates to methods of processing mammalian reproductive cells comprising the steps of providing a mammalian reproductive cells sample, processing the mammalian reproductive cells sample, and adding a fertilization enhancing compound (or a media composition thereof) of the present technology. In any embodiments the fertilization enhancing compound (e.g. , a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) can be included in a media composition before adding to the mammalian reproductive cells sample. In any embodiments the fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) can be added to the mammalian reproductive cells sample by itself. In any embodiments, the fertilization enhancing compound can be a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536, or a salt thereof. In any embodiment, the media can include an additive. In any embodiment, the processing can comprise at least one of the following steps: collecting a semen sample, sexing, selecting, separating, freezing, artificial insemination, in vitro fertilization, cooling, transport, or related processes. In any aspect or embodiment, the sexing can be accomplished via droplet sorting, mechanical sorting, micro fluidic processing, microchip processing, jet and air processing, flow cytometry processing, or laser ablation. In any aspect or embodiment, the mammalian reproductive cells can be obtained from a male mammal. In yet other embodiments, the male mammal can be a bull or boar. In any embodiments, the processed mammalian reproductive cells can be gathered in a container, tube, or straw. In any embodiments, the mammalian reproductive cells can be selected from the group consisting of gametes, haploid cells, germ cells, sex cells, sperm cells, and egg cells. In any embodiments, a sperm cell composition can be produced by this processing method.
[0085] Processing of raw ejaculate can include many downstream applications. For example, processing of raw ejaculate can include one or more of the applications including, but not limited to sexing (selecting X-chromosome bearing or Y-chromosome bearing cells), freezing, artificial insemination, or IVF (with and without sexing). In some embodiments, this can include cooling and transport of samples, concentrating sperm cells and suspending before staining/sexing.
[0086} One aspect of the disclosure relates to methods of protecting sperm cells throughout the process (e.g., the sexing process or the conventional process), wherein the method can include the step of adding to the sperm cells an effective amount of a fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) or a composition thereof. In any embodiments the composition can further include a medium. In any embodiments, the fertilization enhancing compound (e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) can be included in the medium. In any embodiments, the medium can be an extender medium. In any embodiments, the medium can be a staining buffer, a cry opreservation buffer, and/or a semen processing buffer. In any embodiments, the fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) can be added before, during, and/or after the sexing process. In any embodiments, the fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) can be added to the sperm cells and the fertilization enhancing compound can have a concentration ranging from 1 nM to 100 mM in the resulting mixture, including, without limitation, about 0. 1 pM to about 500 pM, about 0.5 pM to about 250 pM, about 1 pM to about 100 pM, about 2 pM to about 50 pM, about 3 pM to about 30 pM, about 4 pM to about 20 pM, about 5 pM to about 10 pM, or about 0.1 pM to about 5 pM. [0087] Another aspect of the disclosure relates to methods of eliciting a positive functional improvement in sperm cells at the post-thaw stage. Tn any embodiment, the method can include adding to the sperm cells at a pre-freeze stage an effective amount of a fertilization enhancing compound (e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) or a composition thereof. In any embodiments the composition can further include a medium. In any embodiments, the fertilization enhancing compound (e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) can be included in the medium. In any embodiments, the medium can be an extender medium. In any embodiments, the medium can be a staining buffer, a cry opreservation buffer, and/or a semen processing buffer. In any embodiments, the fertilization enhancing compound (e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) can be added before, during and/or after the process (e.g., the sexing process or the conventional process). In any embodiments, the fertilization enhancing compound (e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) can be added to the sperm cells and the fertilization enhancing compound has a concentration ranging from 1 nM to 100 mM in the resulting mixture, including, without limitation, about 0. 1 pM to about 500 pM, about 0.5 pM to about 250 pM, about 1 pM to about 100 pM, about 2 pM to about 50 pM, about 3 pM to about 30 pM, about 4 pM to about 20 pM, about 5 pM to about 10 pM, or about 0.1 pM to about 5 pM.
[0088] In any aspects and embodiments, the compositions herein are sperm cell compositions that comprise sperm cells, a fertilization enhancing compound (e.g, a c-Jun Kinase inhibitor such as BI 78D3 or an adenylyl cyclase inhibitor such as SQ 22536) and a medium. The compositions may include seminal fluid components. In some embodiments, the media may include one or more of extender media, staining media (stain TALP), collection media (Part 1 or TRIS A), and cry opreservation media (packaging extender). In some embodiments, the medium may include one or more of NaCl, KC1, Na2HPO4 , NaHCO3. MgCl26H2O (Tyrode ’s). In some embodiments, the medium may include one or more of Tyrode’ s, Sodium lactate syrup, Glucose, HEPES, Sodium Pyruvate, BSA (Stain TALP). In some embodiments, the medium may include one or more of Stain TALP, Egg Yolk, Quenching Dye (Red TALP). In some embodiments, the medium may include one or more of Sterile Milli-Q H2O, TRIS, Egg Yolk, GTLS (Part 1 or TRIS A). Tn some embodiments, the medium may include one or more of Sterile Milli-Q H2O, TRIS, Egg Yolk, Glycerol, Green Food Color, GTLS (Part 2 or TRIS B). In some embodiments, the medium may include one or more of TRIS A (Part 1 ) and TRIS B (Part 2) (Packaging Extender9. In some embodiments, the medium may include one or more of Gentamicin Sulfate (powdered, Amresco # 0304), Sterile Milli-Q H2O (Gentamycin Sulfate Solution). In some embodiments, the medium may include one or more of Tylosin (Tylosin Tartrate; Midwest Vet Supply), Sterile Milli-Q H2O (Tylosin Solution). In some embodiments, the medium may include one or more of Gentamicin Sulfate Solution, Tylosin Solution, Linco-Spectin Stock (50 mg Lincomycin/lOOmg Spectinomycin; Midwest Vet Supply) (GTLS)
[0089] Other components which may be included in the medium, may include, without limitation, one or more of sodium chloride, potassium chloride, disodium phosphate, sodium bicarbonate, magnesium chloride hexahydrate, D-(+)-Glucose, sodium pyruvate, sodium lactate, HEPES, BSA (Fraction V), red food dye, tris(hydroxymethyl)aminomethane (Trizma), citric acid monohydrate, D-Fructose, egg yolk, glycerol, green food dye, CaCl2 (H2O)2. MgCl2(H2O)6, NaHCO3, NaH2PO4 dihydrate, KH2PO4, fructose, sorbitol, phosphatidylserine, decursin, zinc chloride, coenzyme Q10, aspirin, linolenic acid, fatty acid supplement, and D-aspartic acid.
[0090] In other aspects and embodiments, the present technology can include a container of sperm cells comprising a plurality of sperm cells, a medium, one or more of fertilization enhancing compounds, or combinations thereof. The container may further comprise seminal fluid components. Such containers can be used for storage, or for further procedures such as IVF or Al.
[0091] In vitro fertilization can be carried out by methods and procedures known in the art. Many factors can affect successful IVF, including, but not limited to sources of eggs, sperm samples, additional processing and/or manipulation, fertilization, presence and/or concentration of media components and/or sperm cells during fertilization step, and presence and/or concentration of media components during blastocyst formation and/or embryo development. [0092] Some aspects and embodiments of the disclosure are illustrated by the following examples. These examples are provided to describe specific embodiments of the technology and do not limit the scope of the disclosure. It will be understood by those skilled in the art that the full scope of the disclosure is defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.
EXAMPLES
[0093] Materials and Methods:
[0094] Examples of media formulations wherein the fertilization enhancing compounds are added are outlined below:
• Tyrode’s: NaCl, KC1, Na2HPO4, NaHCO3, MgCl2.6H2O.
• Stain TALP: Tyrode's, Sodium lactate syrup, Glucose, HEPES, Sodium Pyruvate, BSA.
• Red TALP: Stain TALP, Egg Yolk, Quenching Dye.
• TRIS A (Part 1): Sterile Milli-Q H2 O, TRIS, Egg Yolk, GTLS.
• TRIS B (Part 2): Sterile Milli-Q H2O, TRIS, Egg Yolk, Glycerol, Green Food Color, GTLS.
• Packaging Extender: TRIS A (Part 1) and TRIS B (Part 2).
• Gentamycin Sulfate Solution: Gentamicin Sulfate (powdered, Amresco # 0304), Sterile Milli-Q H2O.
• Tylosin Solution: Tylosin (Tylosin Tartrate; Midwest Vet Supply), Sterile Milli-Q H2O.
GTLS: Gentamicin Sulfate Solution, Tylosin Solution, Linco-Spectin Stock (50 mg Lincomycin/lOOmg Spectinomycin; Midwest Vet Supply). [0095] IVF Testable Unit Generation Design: This study utilizes sires from one breed, Holstein. The study design includes one ejaculate collection from 2 unique sires to generate sexed semen units in a split batch design with the fertilization enhancing compounds and a paired control.
[0096] Final breed tally of the collections are 2 Holsteins. Ejaculates selected for use in this trial are processed following standard production procedures outlined in detail below. Insemination doses, freeze canes, and all associated documentation are blind labeled during the incoming quality check. This is to prevent technician bias on quality control assessments after freeze-thaw as well as during the IVF outcome quantitation assessments.
[0097] After processing, the sexed semen is packaged and frozen during a regularly scheduled production freeze following standard operating procedures (SOPs). Within one week of unit generation, the outgoing quality control measurements are performed by a trained research technician. Post-thaw motile concentration and the presence and/or absence of bacterial contamination are completed. Insemination doses have to pass standard production outgoing quality control parameters to be utilized for the IVF trial.
[0098] The IVF facility receives the four treatment groups for each individual ejaculate. The four treatments from each split ejaculate are used concurrently to fertilize oocytes from the same pooled batch of slaughterhouse oocytes.
[0099] Trained IVF technicians performs the outlined fertilizations. Day 2 cleavage rate, Day 7 and 8 blastocyst rates will be recorded, as well as polyspermy post-fertilization.
[0100] Ejaculate Collections: All ejaculate collections were performed on-site by experienced technicians following standard collection procedures.
[0101 ] Ejaculate extension and Incoming Quality Assessment: Ejaculates were transported in an insulated cooler to prevent temperature fluctuation during transport to the second facility. The volume of the ejaculate was determined by mass. Immediately, within 15 minutes, Citrate buffer was added in a 0.5: 1 ratio to the ejaculates. GTLS antibiotic solution was added at a 2% v/v of ejaculate. [0102] Within 45 minutes of initial ejaculate collection, the cell concentration and incoming motility parameters were collected. Concentration was determined using a Nucleocounter SP-100™ with Reagent 5100 and SP100 cassettes (ChemoMetec Allerod, Denmark). Motility characteristics were calculated by diluting 10 pL sample in 990 pL motility diluent and reading 7 frames each in 2 chambers of Leja 4 chamber capillary slides with a known chamber depth on a Hamilton Thome IVOS II using HTCasa II software at 60 Hz frame capture speed in a 37° C enclosed stage with a Zeiss lOx objective. An ejaculate was utilized if the cell concentration was greater than 500 million/mL, and the percent of progressive motile cells in the sample was greater than or equal to 65%.
[0103] Sample Preparation for Sperm Sexing: A stained sample was prepared at room temperature that contained 200 M/mL sperm cells in 0.06 rng/mL Hoechst 33342 diluted to a final volume in Stain TALP with Magnetic Beads with (+ fertilization enhancing compound such as BI 78D3, SQ 22536) or without (control) such that the fertilization enhancing compound would be at a suitable concentration (e.g., 0.1 pM) in the final stained sample volume. (R-DPN was added at end of sexing process in any of Part 1 or Part 2 of two-part extender or in a Packaging Exten der.) The sample w as then incubated in a 37° C w7ater bath for 45 minutes. After 45 minutes Red Stain TALP was added to the stained sample in a 2: 1 v/v ratio. The sample plus Red TALP was then mixed using inversion, filtered using tube top 20 pm Partec filters (Partec# 04-0042-2315), and aliquoted into round bottom 5 mL tubes.
[0104] Sexing Cytometer Metrics: The stained, filtered sample was then run on proprietary sexing cytometers. The sample throughput w7as adjusted to 23,000 cells/sec and the detection and kill lasers were focused. To confirm proper laser focus, kill count assessments were performed before collecting sex skewed sample. A successful kill count has a population that is greater than or equal to 75% dead and greater than or equal to 95% sliced with at least 200 cells being counted. If an instrument could not achieve the above metrics, the instrument wras not used to collect sex skewed semen. After a successful kill count, a gate w?as 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 were 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 %.
[0105] Sex Skewed Sample Collection and Processing: The sample was run to collect between 300 and 400 mLs of sex skewed sample, the composition of which is approximately 17% TRIS A (Part 1) buffer, 80% sheath fluid, and 2% cell sample. The sample was collected in 50 mL conical tubes containing 5 mLs of TRIS A (Part 1), and each tube was filled to a maximum volume of 30 mLs before being replaced. After the requisite total volume was collected, sexed sperm was centrifuged at room temperature at 2400 x g for 10 minutes. The supernatant was aspirated and discarded to reach a 1 mL pellet volume. 144 pL of TRIS A/Part 1)/GTLS mixture containing 40 pM fertilization enhancing compounds or no fertilization enhancing compound (control) was added to each pellet after resuspension. At this stage, compounds BI 78D3 and SQ 22536 were added separately to their respective samples to maintain effective concentration or R- DPN was added for the first time at an effective concentration. The tubes were then placed in beakers filled with 150 mL of room temperature water, to prevent cold shock, and were then transferred to a 4ºC cold room.
[0106] After 90 minutes of equilibration at 4ºC the samples were cryoprotected by adding TRIS B (Part 2, which contains glycerol) to a final concentration of 20% v/v of sample in 3 separate additions, 15 minutes apart. At this stage, compounds BI 78D3, SQ 22536, and R-DPN were added separately to their respective samples to maintain effective concentration. The concentration of progressively motile cells was determined using the Hamilton Thorne IVOS II with HTCasa Animal Breeders II software set to the same capture settings listed above, but with a Xenon light source and an Olympus lOx UplanSApo objective. The cryoprotected sample was diluted to final live, motile cell concentration, 2.6 M/mL, in Packaging Extender containing 0. 1-0.3 pM fertilization enhancing compound (e.g., a c-Jun Kinase inhibitor such as BI 78D3) or no fertilization enhancing compound (control) and placed in Mini Straws which hold 0.25 mL volume (IMV technologies, Maple Grove, MN USA) using an MX4 straw filling and sealing machine (IMV technologies, Maple Grove, MN USA). Filled straws were rapidly cooled using a freeze tunnel before storage in liquid nitrogen
[0107] Outgoing Quality Control Assessment: To assess the number of motile cells that survived the freezing process a single straw is thawed in a 37ºC water bath for 45 seconds. The straw is then plunged into a pre-warmed Eppendorf tube. The sample is then gently vortexed 10 seconds to homogenize the sample. After which 20 pLs of sample is added to 20 pLs of QC diluent, gently vortexed for 5 seconds, and read on the CASA fluorescent settings described above. The remainder of the straw volume is spread on a blood agar plate and left at 37ºC for 24 hours before bacterial colonies are counted.
[0108] During the course of development of a robust medium for use during various stages of sexing, the inventors tested the effect of fertilization enhancing compounds on the sexing process by adding the fertilization enhancing compounds to each media beginning with the staining media, throughout the sexing process, and in the collection and cooling steps and assessed the effects on cell viability and fertility, and impact on production efficiency. Additionally, the addition of fertilization enhancing compound samples only after sexing was also tested in order to assess the effects independent of the impacts on processing. Multiple concentrations of the fertilization enhancing compounds were tested in various compositions for different applications as well as the effect of timing of addition of fertilization enhancing compounds was studied. The addition of the fertilization enhancing compounds was studied at various stages as shown in Fig. 1. For example, fertilization enhancing compounds can be first added in a mixture with magnetic beads in the staining step with Stain TALP and Hoechst 33342, added a second time in the Tris A (Part 1 of a two part extender)/GTLS addition after the centrifugation that occurs post-instrument, and added for a third time in the packaging extender pre- freeze. The fertilization enhancing compound is maintained at a steady concentration, (e.g., 0.1 μM) throughout the process. A citrate-based extender is used pre-instrument and magnetic beads are used to remove dead cells pre-instrument. EXAMPLE 1
[0109] Sexing procedures require that spermatozoa survive a multitude of insults, and preliminary data demonstrated that the medium containing certain fertilization enhancing compounds facilitates successful sexing up to about 24 hours post-collection, depending on the background extender formulation. While scientists have attempted to correlate in vitro sperm characteristics with fertilization outcomes, no such assay has become a gold standard. Cellular assessments of the plasma membrane, intracellular structures, as well as tests of velocity parameters with a CASA system or with the bovine cervical mucus penetration test (BCMPT) often have conflicting results in how they relate to IVF or Al fertility. For example, PI staining of plasma membrane integrity correlating with field fertility for Januskauskas, et al., (Januskauskas, A., et al., Theriogenology, 2003, 60, 743- 758) and no correlation for Oliveira et al. (Oliveira, L.Z., et al., Andrologia, 2012, 44, 9- 15). Also, distance traveled in BCMPT appeared to correlate to NRR (Tas, M., et al., Theriogenology 2007, 68, 981-987; Bacinoglu, S., et al., Anim. Reprod. Sci., 2008, 104, 38-46) but that this assay did not relate well to IVF outcomes (Keel, B.A. and Schalue, T.K., Arch. Androl., 2000, 44, 109- 115). These conflicting results prevent relating sperm viability or motility measurements to performance in IVF or Al. Accurately assessing spermatozoa fertility, therefore, requires performing an IVF or Al.
[0110] While an Al trial is required to apply the compositions described herein to commercial products, an IVF trial quantifies cleavage and blastocyst conversion rates, providing insight into mechanisms underlying apparent changes in the number of embryos produced (Bermejo- Alvarez, P., et al., Reprod. Fertil. Dev., 2010, 22, 426-436; Blondin, P., et al., Theriogenology, 2009, 71, 30-38; Greve, T. and Madison, V., Reprod. Nutr. Dev., 1991, 31, 147-157). Quantified outcomes in this IVF trial will include percent fertilization, cleavage conversion, and blastocyst conversion rates day 7 and day 8. These two time points to quantify blastocyst development are based on literature showing a developmental delay in IVF using sexed semen (Lu, K.H., et al., Theriogenology, 52, 1999, 1393-1405), and will indicate whether the compositions described herein change the rate of embryonic development.
EXAMPLE 2 [0111] An illustrative flowchart showing the addition of fertilization enhancing compounds at various stages during the sexing process, is shown in FIG. 1 . Each media containing fertilization enhancing compounds is designated by a star. For example, an ejaculate sample is obtained and a volume of Stain TALP and Hoechst 33362 are added such that a fertilization enhancing compound is introduced to the sample at a certain concentration. The staining step is earned out by incubating the treated ejaculate sample for about 45 minutes at 34° C. Red TALP is added to the stained sample, and the sample is then sorted on a suitable instrument to provide a sex-skewed sample. The sex-skewed sample is subjected to room temperature centrifugation and is then treated with TrisA Part I (of a two-part extender)/GTLS, which may include additional fertilization enhancing compounds. Tris B (Part 2) is added to the sample, followed by a packaging extender medium, which may again include fertilization enhancing compounds, and the sample packaged in straws or other packaging for freezing and cry opreservation.
[0112] As noted in the foregoing procedure, a fertilization enhancing compound (e.g., BI 78D3, SQ22536, or (R)-DPN) can be added at multiple stages (e.g., at the staining step, centrifugation step and prior to cry opreservation step) to maintain the desired concentration. For example, a fertilization enhancing compound is BI 78D3. The Stain TALP medium includes 0. 1 uM BI 78D3 or BI 78D3 is added to achieve or maintain 0. 1 uM concentration; TRIS A (Part 1)/GTLS includes 0.1 uM of BI 78D3 or BI 78D3 is added to achieve or maintain 0. 1 uM concentration; and cryopreservation media also includes 0. 1 uM of BI 78D3 or BI 78D3 is added to achieve or maintain 0. 1 uM concentration. In another example, a fertilization enhancing compound is SQ22536. The Stain TALP medium includes 0.3 uM of SQ22536 or SQ22536 is added to achieve or maintain 0.3 uM concentration; TRIS A (Part 1)/GTLS includes 0.3 uM of SQ22536 or SQ22536 is added to achieve or maintain 0.3 uM concentration; and cry opreservation media also includes 0.3 uM of SQ22536 or SQ22536 is added to achieve or maintain 0.3 uM concentration. In another example, a fertilization enhancing compound is (R)-DPN. The Stain TALP medium includes 0.3 uM of (R)-DPN or (R)-DPN is added to achieve or maintain 0.3 uM concentration; TRIS A (Part 1)/GTLS may include 0.3 uM of (R)-DPN or (R)-DPN is added to achieve/maintain 0.3 uM concentration; and cry opreservation media also includes 0.3 uM of (R)-DPN or (R)-DPN is added to achieve or maintain 0.3 uM concentration.
[0113] The frozen sample may then be thawed and used for IVF embryo production or artificial insemination. The sample may be checked or evaluated through quality control to determine that fertility/motility levels (see other FIGs) have been achieved.
EXAMPLE 3
[0114| In Vitro Fertilization and Assessment:
[0115| Oocyte prep: Four well fertilization plates are prepared by filling all 4 wells with 400 pL of BO-IVF (MOFA Verona, WI) and equilibrated in a 37° C 5% CO2 for at least 1 hour. At this same time, four well embryo culture plates filled with 450 pL of BO-1VC (MOFA Verona, WI) are made and equilibrated at 37° C 5% CO2, 5% O2. A sample of each lot of BO-IVC used during these fertilizations is aliquoted and stored at -80ºC as control media for assessing conditioned embryo media. All handling of oocytes and zygotes is done with heat pulled glass pipettes.
[0116| Cumulus oocyte complexes (COCs) are collected from slaughterhouse ovaries by aspiration. The COCs are kept warm in oocyte maturation media and handled on 37° C heated stages. COCs are grouped and separated into 3 wells of 60 oocytes each per treatment group in a 4 well plate.
[0117] Semen prep: Three insemination straws per treatment group are thawed at 37ºC for 45 seconds. They are then layered over 80% BOVIPURE™ density gradient (Nidacon international AB. Sweden). The samples are centrifuged at 500 x g for 15 minutes, aspirated close to the pellet, and then resuspended in warm TL HEPES (MOFA Verona, WI). They are centrifuged at 300 x g for 5 minutes, aspirated to 100 pLs, and the pellet is resuspended in that low volume. A 5pL sample aliquot is added to 95 pLs 4% NaCl to immobilize the cells, and cell concentration is quantified using a hemocytometer. Cells with visible membrane damage are not counted towards cell density' calculations. Sperm suspension is added to the COC containing wells at 1.2 million sperm per w ell (20,000 sperm/oocyte) [0118] Cumulus oocyte complex removal: 24 hours after sperm addition. COCs from the same treatment group are pooled in a 15-mL conical tube containing 0.5 mL TL HEPES with 1 mg/mL hyaluronidase. COCs are vortexed for 1 minute, put back into the 37° C heating block for 1 minute, and vortexed again for 1 minute. The presumptive zygotes are washed in TL HEPES plates and then placed in the embryo culture plates containing maturation media that are equilibrated for 24 hours prior to use (oocyte prep above). The presumptive zygote containing plates are then placed at 37° C 5% CO2, 5% O2 for the rest of the IVF trial.
[0119] Development assessments: Developmental assessments are performed three times during the 8-day post-fertilization incubation. Cleavage events are quantified 48 hours after initial fertilization. Blastocysts are scored on a binary scale of yes/no blastocyst based on its developmental stage. If the embryo had reached al least the early blastocyst stage it is scored as a blastocyst. The differences between early, expanding, and hatched blastocysts are not recorded, nor are the blastocysts scored, but blastocysts are fixed to facilitate future characterization. Blastocyst conversion per oocyte is visually determined on both day 7 and day 8 after initial fertilization. All determinations of developmental stages are done by trained IVF technicians using a dissecting scope on a heated stage set to 37ºC.
[0120] Assessment of early fertilization events: 24 hours after initial fertilization and after the presumptive zygotes are stripped of their COCs, a subset of 20-30 zygotes are fixed and stained using a proprietary kit created to assess for monospermic/polyspermic events. The DNA stain is Hoechst 33342. All presumptive zygotes are scored in 1 of 4 categories: monospermic fertilization, polyspemnc fertilization, unfertilized, or other. The other category encompasses zygotes that are present, but un-scorable due to either obscuring fluorescence from COC not fully removed or because the zygote is fragmented. Those zygotes presenting with two pronuclei are considered monospermic, and those presenting with 3 or more are scored as polyspermic (Yang et al., 1993).
[0121 ] Statistical Analysis: All statistical analysis is performed using OriginPro 2021 64- bit software. Threshold for significance is set at a = 0.05. Results:
[0122] Initial screening (compounds applied post-thaw).
[0123] Certain compounds were applied post-thaw in POD to determine their toxic effect on cells. Results obtained from the initial screening are summarized in Table A.
Specifically, for compound Bl 78D3, FIG. 2A shows a comparison in normalized Motile (M/mL) after 2 hr storage of post-thaw straw material treated with BI 78D3 bet ween 0.1 pM and 30 pM BI 78D3. Both TO and T2 timepoint raw values were normalized to the TO DMSO control. Dashed lines represent the DMSO control at TO and T2. FIG. 2B shows a comparison in curvilinear velocity (VCL) after 2 hr storage of post-thaw straw material treated with BI 78D3 between 0. 1 pM and 30 pM BI 78D3, with the DMSO control. Both TO and T2 timepoint raw values were normalized to the TO DMSO control. The screening was performed to find any toxicities (test TO and T2 post thaw). The loss of more than 10% Motile M/mL compared to time matched DMSO control at either/or both time points was considered to be indicative of toxic effect on cells.
Table A.
Figure imgf000045_0001
Figure imgf000046_0001
[0124] Pre-stain application
[0125] Certain compounds were applied to citrate-buffered ejaculates during stain, and the Hoechst uptake EC80 (FIG. 3A), as well as the corresponding post-thaw motile cells at 0 and 2 hours post-stain (FIG. 4) were accessed. After the Hoechst uptake assay was done, cells were transferred to a black opaque plate, CELLTITER-GLO® (Promega Corporation, Madison, WI) was then added and read. The toxicity of the tested compounds were quantified using CELLTITER-GLO® assay (FIG. 3B) Specifically, FIG. 3A shows EC80 time for Hoechst uptake obtained using compounds SQ22536, R- DPN, BI 78D3, Bax, and Ro3306, compared to DMSO control. These results indicate that the tested compounds have no risk for reducing Hoechst uptake and are therefore compatible with the staining step required for sexing. FIG. 3B shows CELLTITER- GLO® assay results obtained using compounds SQ22536, BI 78D3, R-DPN, Ro3306, and Bax, compared to DMSO and SOP control. These results indicate that the tested compounds have no risk for increasing cell death during the time frame assessed. Indeed some compound doses increase signal from the CELLTITER-GLO® assay, illustrating an increase in viable cells following the stain step. FIGs. 4A-4B show a comparison in Motile % 2 hours after stain between BI 78D3 (0.03 pM, 0.1 pM, and 30 pM, respectively), DMSO, and SOP (control).
[0126] Extender pre-freeze application.
[0127| Certain compounds were added to the extender, and the test results are summarized in FIGs. 5-6. Briefly, FIG. 5 shows a comparison in normalized progressive Motile (M/straw) at 3 hours post thaw between compounds Bax, BI 78D3, SQ22536, Ro3306, and R-DPN, at various concentrations. Data demonstrate specific compound concentrations improve the 3 hour survival of motile cells as indicated as a mean > 0 in FIG. 5. FIG. 6 shows normalized progressive Motile (M/straw) for compounds R-DPN (FIG. 6A), BI 78D3 (FIG. 6B), and SQ22536 (FIG. 6C), at 0, 1 , 2, and 3 hours post thaw, compared to the control.
[0128 ] Sustained compound addition during, sexed unit collection.
[0129] Certain compounds (DPN, BI 78D3 or SQ22536) were added at multiple steps throughout the process (i.e., at the beginning/ staining step, after centrifuging, and before packaging), and test results are summarized in FIGs. 7-8. Specifically, FIGs. 7A-7B show on-instrument performance of the cells. FIG. 7A shows initial dead (%) of the cells treated with BI 78D3 and SQ22536, compared to the control, demonstrating that the compounds have the ability to lower the percentage of dead cells following staining. FIG. 7B show s initial eligibility (%) of the cells treated with BI 78D3 and SQ22536, compared to the control. The increase in the percentage of cells eligible for sexing demonstrates that compounds have the ability to improve the yield of viable cells through the sexing process, thereby increasing the efficiency with which ejaculates are sexed. FIG. 8A shows straws/catch tube for samples treated with BI 78D3, SQ22536, and (R)-DPN, compared to SOP control. FIG. 8B shows progressive motile post-thaw (M/straw) of the cells treated with BI 78D3, SQ22536, and R-DPN, compared to the control. The increase in straws/catch tube demonstrates compounds have the potential to increase output efficiency of the sexing process, yielding more motile sperm cells and resulting in more straws of sexed semen per batch.
[0130J All publications are hereby incorporated by reference, each in their entirety.

Claims

WHAT IS CLAIMED IS:
1. A composition comprising: a non-human mammalian reproductive cell selected from the group consisting of a sperm cell, an oocyte, an embryo, an embryonic stem cell, and a spermatogonial stem cell; and an effective amount of a fertility enhancing compound for enhancing cell viability during or after one or more of: storage, staining, freezing, thawing, cell selection, packaging, or in vitro fertilization; wherein the fertility enhancing compound is selected from the group consisting of a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker, an estrogen receptor agonist, an anti-inflammatory compound, an antioxidant, a TRPV 1 antagonist, and a calcium channel blocker.
2. The composition of claim 1, wherein the fertility enhancing compound is selected from the group consisting of DS2, SKA 31, SR 3677 hydrochloride, BAY 41-2272, BI 78D3, SQ 22536, BINA, spiperone hydrochloride, ML 385, Ro 3306, Bax, (R)-DPN, resveratrol, Tempol, Capsazepine, Nifedipine, and Quercetin.
3. The composition of claim 1, wherein the fertility enhancing compound is SQ 22536.
4. The composition of claim 1, wherein the fertility enhancing compound is a c-Jun
Kinase inhibitor or an adenylyl cyclase inhibitor.
5. The composition of claim 1, wherein the fertility enhancing compound is BI 78D3.
6. The composition of any one of claims 1-5, wherein the fertility enhancing compound is present at a concentration ranging from 1 nM to 100 mM.
7. The composition of claim 1, wherein the non-human mammalian reproductive cell comprises bovine, porcine, equine, ovine, elk, or bison sperm cells.
8. The composition of claim 7, wherein the bovine, porcine, equine, ovine, elk, or bison sperm cells are sex selected sperm cells.
9. The composition of any one of claims 1-8, further comprising a medium.
10. A method for enhancing non-human mammalian sperm cell viability during or after a sexing process at one or more of staining, freezing, thawing, cell selection, or packaging during the sexing process or in vitro fertilization following the sexing process, the method comprising: adding to the non-human mammalian sperm cells an effective amount of a fertility enhancing compound or a composition thereof; and wherein the fertility enhancing compound is a GABA positive allosteric modulator, a potassium channel activator, a Rho-kinase inhibitor, a soluble guanylyl cyclase (sGC) activator, a c-Jun Kinase inhibitor, an adenylyl cyclase inhibitor, a mGluR2 positive allosteric modulator, a D2-like dopamine receptor antagonist, a 5-HT2A receptor antagonist, a Nrf2 (nuclear factor erythroid 2-related factor 2) Inhibitor, a cyclin dependent kinase 1 inhibitor, a Bax channel blocker, an estrogen receptor β agonist, anti-inflammatory, an antioxidant, a TRPV 1 antagonist, or a calcium channel blocker.
1 1 . The method of claim 10, wherein the fertility enhancing compound is added during or after the sexing process.
12. The method of claim 10, wherein adding the fertility compound to the non-human mammalian sperm cells at a pre-freeze stage provides positive functional improvement in sperm cells at a post-thaw stage.
13. The method of any one of claims 10-12, wherein the fertility enhancing compound is DS2, SKA 31, SR 3677 hydrochloride, BAY 41-2272, BI 78D3, SQ 22536, BINA, spiperone hydrochloride, ML 385, Ro 3306, Bax channel blocker, (R)-DPN, resveratrol, Tempol, Capsazepine, Nifedipine, or Quercetin.
14. The method of any one of claims 10-12, wherein the fertility compound is a c-Jun Kinase inhibitor or an adenylyl cyclase inhibitor.
15. The method of any one of claims 10-12, wherein the fertility enhancing compound is SQ 22536.
16. The method of any one of claims 10-12, wherein the fertility enhancing compound is BI 78D3.
17. The method of any one of claims 10-16, wherein the non-human mammalian sperm cells comprise bovine, porcine, equine, ovine, elk, or bison sperm cells.
18. The method of any one of claims 10-16, further comprising adding the fertility enhancing compound to the sperm cells at the staining step.
19. The method of any one of claims 10-16, further comprising adding the fertility enhancing compound to the sperm cells at the centrifugation step.
20. The method of any one of claims 10-16, further comprising adding the fertility enhancing compound to the sperm cells prior to the cryopreservation step.
21. A method of preserving viability of non-human mammalian semen during sex selection and cryopreservation comprising: obtaining an ejaculate comprising a semen sample; contacting the semen sample with a fertility enhancing compound selected from a c- Jun Kinase inhibitor and an adenylyl cyclase inhibitor; sex selecting the semen sample while maintaining a concentration of the fertility enhancing compound; and cry opreserving the sex selected semen sample.
22. The method of claim 21, wherein the fertility enhancing compound is SQ 22536.
23. The method of claim 21, wherein the fertility enhancing compound is BI 78D3.
24. The method of claim 21, wherein the non-human mammalian semen sample comprises a bovine, porcine, equine, ovine, elk, or bison semen sample.
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