CA2561661A1 - Sperm suspensions for sorting into x or y chromosome-bearing enriched populations - Google Patents
Sperm suspensions for sorting into x or y chromosome-bearing enriched populations Download PDFInfo
- Publication number
- CA2561661A1 CA2561661A1 CA002561661A CA2561661A CA2561661A1 CA 2561661 A1 CA2561661 A1 CA 2561661A1 CA 002561661 A CA002561661 A CA 002561661A CA 2561661 A CA2561661 A CA 2561661A CA 2561661 A1 CA2561661 A1 CA 2561661A1
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- Prior art keywords
- suspension
- sperm
- spermatozoa
- concentration
- motility
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 229960003343 ouabain Drugs 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- 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
- C12N5/061—Sperm cells, spermatogonia
-
- 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
- C12N5/0611—Primordial germ cells, e.g. embryonic germ cells [EG]
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
Abstract
Sperm cell suspensions comprising a motility inhibitor are disclosed. The cells contained in such suspensions tend to have a greater capacity for enduring the various process steps typically associated with the sorting of sperm cells into gender enriched populations, thereby resulting in post-sort compositions with an increased number of viable or motile sperm. Processes for forming such cell suspensions, as well as processes for staining sperm cells, are also disclosed.
Description
SPERM SUSPENSIONS FOR SORTING INTO X OR Y CHROMOSOME
BEARING ENRICHED POPULATIONS
FIELD OF THE INVENTION
[0001] The present invention generally relates to a process of sorting sperm cells. More specifically, the present invention relates to the preparation of suspensions of sperm cells having reduced motif ity, and more particularly a temporarily reduced motility, relative to endogenous ejaculated sperm, the suspensions having utility, for example, in a process for sorting sperm cells into an enriched population of X or Y chromosome-bearing sperm cells.
BACKGROUND
BEARING ENRICHED POPULATIONS
FIELD OF THE INVENTION
[0001] The present invention generally relates to a process of sorting sperm cells. More specifically, the present invention relates to the preparation of suspensions of sperm cells having reduced motif ity, and more particularly a temporarily reduced motility, relative to endogenous ejaculated sperm, the suspensions having utility, for example, in a process for sorting sperm cells into an enriched population of X or Y chromosome-bearing sperm cells.
BACKGROUND
[0002] The fertilization of animals by artificial insemination (AI) and embryo transplant following in vitro fertilization is an established practice. In the livestock production industry, the ability to influence the reproductive outcome toward offspring having one or more desired characteristics has obvious advantages. By way of example, there would be an economic benefit in the dairy industry to preselect offspring in favor of the female sex to ensure the production of dairy cows. The separation of sperm into enriched populations of X and Y chromosome-bearing cells, known as gender enri shed semen or gender enriched sperm, is one method of achieving preselected offspring.
[0003] In order to obtain gender enriched semen, sperm cells must be stained with a dye and subsequently sorted into X and Y chromosome-bearing cells. Each of staining and sorting processes places a stress on the sperm cells that decreases sperm cell viability or motility, particularly progressive motility.
[0004] Salisbury et al. describe a technique for the collection of ejaculated bovine semen directly into a diluent which inhibits cell motility and prevents the absorption of carbohydrates from the surrounding seminal plasma. When the ejaculate is collected into the diluent and the air phase above the liquid is replaced by gassing with 100% C02, the cells in the ejaculate became immotile.
As long as the cells remained in the diluent and air was excluded, the cells remained immotile for several hours at room temperature and for at least 8 days at 5°C.
SUMMARY OF THE INVENTION
As long as the cells remained in the diluent and air was excluded, the cells remained immotile for several hours at room temperature and for at least 8 days at 5°C.
SUMMARY OF THE INVENTION
[0005) Among the various aspects of the present invention are sperm suspensions having utility, for example, in processes used to sort sperm into enriched populations of X or Y-chromosome bearing sperm.
[0006) Briefly, therefore, the present invention is directed to a sperm cell suspension comprising viable spermatozoa and a composition which down-regulates carbohydrate uptake by the spermatozoa, the concentration of spermatozoa in the suspension being less than about 1 X 106 or at least 1 x 108 spermatozoa per ml.
[0007] The present invention is further directed to a sperm cell suspension comprising viable, immotile sperm, the concentration of spermatozoa in the suspension being less than about 1 X 10~ or at least 1 x 10$ spermatozoa per ml.
[0008] The present invention is further directed to a sperm cell suspension comprising viable spermatozoa, the spermatozoa having a motility more characteristic of epididymal spermatozoa than endogenous ejaculated spermatozoa of the same species, the concentration of spermatozoa in the suspension being less than about 1 X 106 or at least 1 x 108 spermatozoa per ml.
[0009] The present invention is further directed to a sperm cell suspension comprising viable sperm, potassium and optionally sodium, the concentration of spermatozoa in the suspension being at least 1 x 108 spermatozoa per ml and the molar ratio of potassium to sodium being greater than 1:1, respectively.
[0010] The present invention is further directed to a sperm cell suspension comprising viable spermatozoa, a composition which down-regulates carbohydrate uptake by the spermatozoa, and a DNA-selective dye.
[0011] The present invention is further directed to a sperm cell suspension comprising viable, immotile sperm and a DNA-selective dye.
[0012] The present invention is further directed to a sperm cell suspension comprising viable spermatozoa and a DNA-selective dye, the spermatozoa having a metabolic rate and motility more characteristic of epididymal spermatozoa than endogenous ejaculated spermatozoa of the same species.
[0013] The present invention is further directed to a sperm cell suspension comprising viable, immotile spermatozoa, the spermatozoa having a DNA-selective dye associated with their DNA.
[0014] The present invention is further directed to a sperm cell suspension comprising viable spermatozoa, the spermatozoa having a metabolic rate and motility more characteristic of epididymal spermatozoa than endogenous ejaculated spermatozoa of the same species, the spermatozoa also having a DNA-selective dye associated with their DNA.
[0015] The present invention is further directed to a process for staining sperm oells, the process comprising forming a staining mixture containing intact viable sperm cells, a motility inhibiting amount of potassium, and a DNA
selective dye.
selective dye.
[0016] The present invention is further directed to a process of forming a sperm cell suspension for use in a flow cytometry process, the process comprising combining a sperm cell source with a composition which inhibits the motility of sperm cells to form a sperm cell suspension, the concentration of sperm cells in the suspension being less than about 1 X 106 or at least 1 x 108 sperm cells per milliliter.
[0017] The present invention is further directed to a process of forming a sperm cell suspension for use in a flow cytometry process, the process comprising collecting the ejaculate of a mammal in a buffer containing an inhibitory amount of a motility inhibitor to form a sperm cell suspension, the suspension comprising less than about 1 X 106 or at least 1 x 10B sperm cells per milliliter.
[0018] Other aspects and features of the invention will be in part apparent and in part pointed out hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIGURE 1 graphically depicts the results of the study carried out in Example 1 wherein percent progressive motility of sperm cells is measured for sperm cells stained with 600~,M Hoechst 33342 dye at 28°C in TCA containing lOmM
pyruvate or in carbon dioxide-blanketed TCA containing lOmM
pyruvate.
pyruvate or in carbon dioxide-blanketed TCA containing lOmM
pyruvate.
[0020] FIGURE 2 graphically depicts the results of the study carried out in Example 1 wherein percent progressive motility of sperm cells is measured for sperm cells stained with 600~M Hoechst 33342 dye at 28°C in TCA containing lOmM
pyruvate or a carbonate-based inhibitor at pH 7.3.
pyruvate or a carbonate-based inhibitor at pH 7.3.
[0021] FIGURE 3 graphically depicts the results of the study carried out in Example 1 wherein percent progressive motility of sperm cells is measured for sperm cells stained with 600,uM Hoechst 33342 dye at 28°C in TCA containing lOmM
5 pyruvate or a carbonate-based inhibitor at pH 6.2.
5 pyruvate or a carbonate-based inhibitor at pH 6.2.
[0022] FIGURE 4 graphically depicts the results of the study carried out in Example 2 wherein percent progressive motility of sperm cells is measured for sperm cells stained with 1000~,M Hoechst 33342 dye at 28°C in TCA containing lOmM pyruvate and then diluted 1 to 3 with either TCA
containing lOmM pyruvate or a carbonate-based inhibitor at pH 6.2.
containing lOmM pyruvate or a carbonate-based inhibitor at pH 6.2.
[0023] FIGURE 5 graphically depicts the results of the study carried out in Example 2 wherein percent progressive motility of sperm cells is measured for sperm cells stained with 1000~,M Hoechst 33342 dye at 28°C in (1) TCA containing lOmM pyruvate and diluted 1 to 3 with the same or (2) a carbonate-based buffer at pH 7.3 and diluted 1 to 3 with carbonate-based inhibitor at pH 6.2.
[0024] FIGURE 6 graphically depicts the results of the study carried out in Example 2 wherein percent progressive motility of sperm cells is measured for sperm cells stained with 1000~.M Hoechst 33342 dye at 28°C in TCA containing lOmM pyruvate or a carbonate-based inhibitor at pH 6.2.
L0025] FIGURE 7 graphically depicts the results of the study carried out in Example 3 wherein percent progressive motility of sperm cells is measured for sperm cells stained with 300~,M Hoechst 33342 dye at 41°C in TCA containing lOmM
pyruvate and then diluted 1 to 3 with either TCA containing lOmM pyruvate or a carbonate-based inhibitor at pH 6.2.
L0026] FIGURE 8 graphically depicts the results of the study carried out in Example 3 wherein percent progressive motility of sperm cells is measured for sperm cells stained with 300~,M Hoechst 33342 dye at 41°C in (1) TCA containing lOmM pyruvate and diluted 1 to 3 with the same or (2) a carbonate-based buffer at pH 7.3 and diluted 1 to 3 with carbonate-based inhibitor at pH 6.2.
[0027] FIGURE 9 graphically depicts the results of the study carried out in Example 3 wherein percent progressive motility of sperm cells is measured for sperm cells stained with 300~,M Hoechst 33342 dye at 41°C in TCA containing lOmM
pyruvate or a carbonate-based inhibitor at pH 6.2.
[0028] FIGURE 10 graphically depicts the results of the study carried out in Example 4 wherein percent progressive motility of sperm is measured for sperm stained with 400~tM Hoechst 33342 dye at 41°C in either a TCA buffer or a TCA buffer containing lOmM pyruvate.
[0029] FIGURE 11 graphically depicts the results of the study carried out in Example 5 wherein percent progressive motility of sperm is measured for sperm stained with 400~,M Hoechst 33342 dye at 41°C in either a TCA buffer or a TCA buffer containing 10~,M vitamin K.
[0030] FIGURE 12 graphically depicts the results of the study carried out in Example 6 wherein percent progressive motility of sperm is measured for sperm stained with 400~,M Hoechst 33342 dye at 41°C in either a TCA buffer or a TCA buffer containing 100~M vitamin K.
[0031] FIGURE 13 graphically depicts the results of the study carried out in Example 7 wherein percent progressive motility of sperm is measured for sperm stained with 400~CM Hoechst 33342 dye at 41°C in either a TCA buffer or a TCA buffer containing 1mM lipoic acid.
[0032] FIGURE 14 graphically depicts the results of the study carried out in Example 8 wherein percent progressive motility of sperm is measured for sperm stained with 600~M Hoechst 33342 dye at 28°C in either a TCA buffer or a TCA buffer containing lOmM pyruvate.
[0033] FIGURE 15 graphically depicts the results of the study carried out in Example 9 wherein percent progressive motility of sperm is measured for sperm stained with 600uM Hoechst 33342 dye at 28°C in either a TCA buffer or a TCA buffer containing 100~.M vitamin K.
[0034] FIGURE 16 graphically depicts the results of the study carried out in Example 10 wherein percent progressive motility of sperm is measured for sperm stained with 600~CM Hoechst 33342 dye at 28°C in either a TCA buffer or a TCA buffer containing 1mM lipoic acid.
[0035] FIGURE 17 graphically depicts the results of the study carried out in Example 11 wherein percent progressive motility of sperm is measured for sperm stained with 600~,M Hoechst 33342 dye at 28°C in a TCA buffer, a TCA
buffer containing 2.5mM pyruvate, a TCA buffer containing lOmM pyruvate, a TCA buffer containing 25mM pyruvate, and a TCA buffer containing 50mM pyruvate.
[0036] FIGURE 18 graphically depicts the results of the study carried out in Example 12 wherein percent progressive motility of sperm is measured for sperm stained with 20~,M SYBR-14 dye at 28°C in either a TCA buffer or a TCA buffer containing lOmM pyruvate.
[0037] FIGURE 19 graphically depicts the results of the study carried out in Example 13 wherein percent progressive motility of sperm is measured for sperm stained with 100~,M BBC dye at 28°C in either a TCA buffer or a TCA
buffer containing lOmM pyruvate.
[0038] FIGURE 20 graphically depicts the results of the study carried out in Example 14 wherein percent progressive motility of sperm is measured for sperm stained with 200~CM BBC dye at 28°C in either a TCA buffer or a TCA
buffer containing lOmM pyruvate.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Surprisingly, it has been determined that spermatozoa having reduced motility relative to endogenous ejaculated spermatozoa (of the same species) tend to have a greater capacity for enduring the various process steps typically associated with the sorting of sperm cells into an enriched population of X or Y chromosome-bearing spermatozoa. In a preferred embodiment, therefore, gender enriched populations of spermatozoa may be prepared for artificial insemination which have an increased number of viable cells or an increased number of motile sperm, particularly progressively motile sperm, in a post-stain or post-sort composition.
[0040] In accordance with the process of the present invention, a suspension, sometimes referred to as a dispersion, is formed containing spermatozoa and one or more compositions which inhibit the motility of the spermatozoa; such a state of inhibited motility sometimes being referred to as immotility or sperm quiescence. In general, the suspensions will contain spermatozoa in a density of about 1 X 103 sperm/ml to about 5 X 101° sperm/ml of suspension. For example, in one embodiment the suspensions may contain spermatozoa in a "relatively low"
density, i.e., in a density of less than about 1 X 10' sperm/ml, preferably less than about 1 X 106 sperm/ml, more preferably about 1 X 103 to about 5 X 106 sperm/ml, still more preferably about 1 X 103 to about 1 X 106 sperm/ml, even more preferably about 1 X 104 to about 1 X 105 sperm/ml, and most preferably about 1 X 105 sperm/ml of suspension. In an alternative embodiment, the suspensions may contain spermatozoa in an "intermediate" density, i.e., in a density of about 1 X 10' to about 1 X 108 sperm/ml of suspension. In yet another alternative embodiment, the suspensions may contain spermatozoa in a "relatively high"
density, i.e., in a density of at least about 1 x 108 sperm/ml, preferably about 1 X 108 to about 5 X 1010 sperm/ml, more preferably about 1.5 X 10$ to about 2 X 1010 sperm/ml, even more preferably about 1.5 X 108 to about 2 X 108 sperm/ml, and still more preferably about 1.5 X 108 sperm/ml of suspension. Thus, for example, in one embodiment the suspension may contain at least about 1.25 x 108, at least about 1.5 x 108, at least about 1.75 x 108, at least about 2 x 108, at least about 2.25 x 108, at least about 2.5 x 108, at least about 2.75 x 108, or even at least about 3 x 108 sperm/ml of suspension. In an alternative embodiment, the suspension may contain less than about 9 X 105, less than about 7 X 105, less than about 5 X 105, less than about 2 X 105, less than about 1 X 105, less than about 1 X 104, or even less than about 1 X 103 sperm/ml of suspension.
[0041] The density of spermatozoa in the sperm suspensions depends upon several considerations, including the method by which the sperm cells may be subsequently enriched or sorted. For example, the sperm cells may be sorted using flow cytometry as described in greater detail below. In such an instance, the buffered sperm suspension may typically be of an "intermediate" or "relatively high"
density of spermatozoa. Other sorting or enrichment techniques may benefit from a lesser density of spermatozoa, such as a "relatively low" density of spermatozoa, labeled with a marker, such as for example the dyes and labels described herein.
[0042] In a preferred embodiment, spermatozoa in suspensions of the present invention behave, in certain respects, in a manner characteristic of epididymal spermatozoa; for example, the spermatozoa may be immotile 5 and/or they may have a lesser rate of endogenous respiration and a greater rate of aerobic glycolysis as compared to washed or freshly ejaculated spermatozoa.
Advantageously, the inhibited spermatozoa have the ability, upon separation from the inhibitor(s), to behave in a 10 manner characteristic of ejaculated spermatozoa (and not characteristic of epididymal spermatozoa) with respect to mots.lity and, in one embodiment, with respect to motility and respiration.
[0043] In one embodiment, for example, the motility inhibitor reduces path velocity, progressive velocity, or bot h, as measured by HTM-IVOS sperm analysis (Hamilton-Tho rne HTM-IVOS computer assisted sperm analysis system Hamilton-Thorne Research, Beverly MA) of at least about 50%
of t he sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Preferably, the mot ility inhibitor reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, of at least about 60% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or bot h of sperm cells in a fresh ejaculate of the same spe ties. More preferably, the motility inhibitor reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, of at least about 70% of the sperm cells in the dispersion relative to the path veto city, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Still more preferably, the motility inhibitor reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS
sperm analysis, of at least about 800 of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Even more preferably, the motility inhibitor reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, of at least about 90% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Even more preferably, the motility inhibitor reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS
sperm analysis, of at least about 95% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Most preferably, the motility inhibitor reduces path velocity, progressive velocity, or both, as measured by an HTM-IVOS sperm analysis, of at least about 99% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species.
(0044] In addition to or in lieu of an inhibitory buffer, the temperature of the sperm cells or the immediate environment surrounding the sperm cells (i.e., a sperm dispersion) may solely be reduced to affect the motility of the cells. Such a reduction in temperature will generally increase immotility. Moreover, for example, the reduction of temperature of the sperm cells or the sperm dispersion may permit a reduction in the concentration of inhibitor used to induce immotility. Accordingly, the sperm dispersion may be at a temperature not in excess of 5°C;
preferably between about D°C and about 5°C; more preferably between about 3°C and about 5°C; and most preferably about 5°C. Alternatively, the sperm dispersion may be at a temperature wit hin the range of about 4°C to about 50°C;
preferably from about 7°C to about 43°C; more preferably from about 10°C to about 39°C; still more preferably from about 15°C to about 30°C; even more preferably from about 17°C to about 2 5°C; and most preferably at about 18°C.
Preferably, hovaever, the sperm cells are not exposed to temperatures that substantially detrimentally affect the viability of the cells.
[0045] The inhibitor may be any of a range of compositions having a depressive effect upon sperm motility. Such compositions include, for example, sodium/potassium ATPase inhibitors, such as, ouabain;
compositions comprising potassium ions; and compositions comprising potassium and sodium ions. For example, relatively high concentrations of potassium ions in the suspension tend to depress sperm motility. In general, therefore, it i s preferred that the suspension contain a source of potassium ions and that the potassium concentration i n the suspension be at least about 0.05 moles/L. More preferably, the potassium concentration is at least about 0.05 moles/L to about 0.5 moles/L. Still more preferably, the potassium concentration is at least about 0.1 moles /L to about 0.3 moles/L. Most preferably, the potassium concentration is at about 0.173 moles/L.
Such suspension s will typically, but not necessarily, also contain a sours a of sodium ions. When sodium is present, the molar ratio of potassium to sodium is generally equal to or greater than 1:1, respectively. Preferably, the molar ratio of potassium to sodium is at least about 1.25:1. Still more preferably, the molar ratio of potassium to sodium is at least about 1.5:1. Still more preferably, the molar ratio of potassium to sodium is at least about 1.75:1. Still more preferably, the molar ratio of potassium to sodium is at least about 1.78:1. In one particular embodiment, the molar ration of potassium to sodium is at least about 2:1. In yet another embodiment, the molar ratio of potassium to sodium is at least about 3:1. In still another embodiment, the molar ratio of potassium to sodium is at least about 4:1. In still another embodiment, the molar ratio of potassium to sodium is at least about 5:1. In still another embodiment, the molar ratio of potassium to sodium is at least about 6:1.
In still another embodiment, the molar ratio of potassium to sodium is at least about 7:1. In still another embodiment, the molar ratio of potassium to sodium is at least about 8:1.
[0046] The sperm suspension may additionally comprise an ion or source of carbon dioxide capable of down-regulating uptake of carbohydrate. In this embodiment, the source of carbon dioxide may be, for example, one or more carbonates. In one presently preferred embodiment, the sperm suspension comprises NaHC03 and KHCO3, thereby providing a source potassium and sodium ions as well as a partial pressure of carbon dioxide. For example, in one presently preferred embodiment, the suspension comprises NaHC03 and KHC03 in an aqueous solution, preferably NaHC03, KHC03 , and CgH80~ ~ H20 in water; In general , the KFiC03 concentration in the dispersion may be at least about 0.05 moles/L. More preferably, the KHC03 concentration is at least about 0.05 moles/L to about 0.5 moles/L. Still more preferably, the KHCO3 concentration is at least about 0.1 moles/L to about 0.3 moles/L. In one particularly preferred embodiment, the suspension is formed using an inhibitory buffer comprising 0.097 moles/L of NaHCO3, 0.173 moles/L of KHC03, 0.090 moles/L C6H80~~H20 in water as disclosed in Salisbury & Graves, J. Reprod. Fertil., 6:351-359 (1963). The sperm cells will generally remain quiescent as long as they are exposed to the motility inhibitor(s).
[0047] When C6Ha0~~HZO is present in the dispersion, the molar ratio of KHC03 to NaHC03 may be as described above . The molar ratio of KHC03 to C6H80~ ~ HZO may generally be equal to or greater than 1:1, respectively, but will generally not exceed a molar ratio of 8:1. Preferably, the molar ratio of KHC03 to C6H807~H~O is from at least about 1.25:1. Still more preferably, the molar ratio of KHC03 to C6H80~~H20 is at least about 1.5:1. Still more preferably, the molar ratio of KHC03 to C6Ha0~~H20 is at least about 1.75:1. In one particular embodiment, the molar ratio of KHC03 to C6H80~~H20 is at least about 1.78:1. In another particular embodiment, the molar ratio of KHC03 to C6H80~-H20 is at least about 2:1. In yet another embodiment, the molar ratio of KHC03 to C6Ha0~~HZO is at least about 3:1. In still another embodiment, the molar ratio of KHC03 to C6H$OwH~O is at least about 4:1. In still another embodiment, the molar ratio of KHC03 to C6Hs0~~H~O is at least about 5:1. In still another embodiment, the molar ratio of KHC03 to C6H80~ ~ H~0 i s at least about 6 : 1 . In still another embodiment, the molar ratio of KHC03 to C6H80~-H20 is at least about 7:1. In still another embodiment, the molar ratio of KHC03 to C6H80~ ~ H20 i s at least about 8 : 1 . In one particularly preferred embodiment, the dispersion is formed using an inhibitory buffer comprising 0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0.090 moles/L C6H80~~H20 in water as disclosed in Salisbury & Graves, J. Reprod.
Fertil., 6:351-359 (1963). The sperm cells will generally remain quiescent as long as they are exposed to the motility inhibitor(s).
[0048] Experimental evidence to date further suggests that the overall health and other vital characteristics of sperm cells may be improved if the cell suspension is maintained under an atmosphere having an enhanced partial 5 pressure of carbon dioxide relative to air. In a preferred embodiment, the atmosphere over the suspension has a partial pressure of carbon dioxide of at least 0.9; more preferably, at least about 0.95.
[0049] Quiescent cells may be returned to an active 10 state by separating the cells from the motility inhibitor and exposing them to air. In addition, the initiation of an active state may be further induced by the dilution of the cells in a physiological saline (Salisbury et al., 1963) or a buffer such as TCA buffer or PBS. Typically, at 15 least about 20%, preferably at lea st about 50%, more preferably at least about 60%, still more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 90%, still more preferably at least about 95%, and most preferably at least about 99% of the cells returned to an active state (i.e., reactivated cells) will have a path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, that is at least about 50%, preferably at least about 60%, more preferably at least about 70%, still more preferably at least about 80%, even more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 99% of the path velocity, progressive velocity, or both of the sperm cells prior to being combined with the motility inhibitor (i.e., of sperm cells of a fresh ejaculate).
[0050] In general, the cell sorting process comprises a series of discrete steps, i.e., collection of a cell sample, staining of the cells, sorting of the cells, collection of the sorted cells, and optionally, cryoextension of the sorted cells. Advantageously, the motility inhibitor may be included in sperm suspensions formed or employed in one or more of then a steps.
Collection of the cell sample [0051] Intact viable bovine, porcine, equine, or other mammalian sperm cells, may be collected and contacted with the motility inhibitor. Various methods of collection of viable sperm are known and include, for example, the gloved-hand method, use of an artificial vagina, and electro-ejaculation. As an example, a bovine semen sample, typically containing about 0.5 to about 10 billion sperm cells per milliliter, may be collected directly from the source mammal into a vessel containing a motility inhibitor to form a sperm suspension. Alternatively, the semen sample may be collected into an empty vessel and then subsequently contacted with the motility inhibitor within several minutes to hours after collection to form the sperm suspension.
[0052] In addition to a buffer, the sperm suspension may also contain a range of additives to enhance sperm viability. Exemplary additives include protein sources, antibiotics, and compositions which regul ate oxidation/reduction reactions intracellul arty and/or extracellularly.
[0053] Exemplary protein sources include egg yolk, egg yolk extract, milk (including heat homogenised and skim), milk extract, soy protein, soy protein extract, serum albumin, bovine serum albumin, human serum substitute supplement, and combinations thereof. Albumin, and more particularly bovine serum albumin (BSA), is a preferred protein source. For example, if included, BSA may be present in the sperm suspension in an amount of less than about 5.Oa (w/v), preferably less than about 2% (w/v), more preferably less than about to (w/v), and most preferably in an amount of about O.lo (w/v).
[0054] The use of a protein source, such BSA, alone may initiate the process of oapaoitation in a percentage of the sperm cells in the suspension. It is preferred that this process take place in the female reproductive tract.
Therefore, in order to inhibit the initiation of capacitation during dilution, as well as during the subsequent staining and sorting, an alternative protein source or a protein substitute may be included in the sperm suspension. The alternative protein source or protein substitute possess the advantageous effects of a typical protein source, such as BSA, in addition to the ability to inhibit the initiation of capacitation in a larger percentage of the cells in the sperm suspension. Examples of a alternative protein sources includes human se rum substitute supplement (SSS) (Irvine Scientific, Santa Ana, CA) and cholesterol enhanced BSA, while an example of a protein substitute includes a polyvinyl alcohol, such as for example, a low to medium viscosity polyvinyl alcohol generally of a molecular weight of about 30,000 to about 60,000. Generally, if included, these compositions will be present in the same amounts as disclosed above with respect to BSA, with the total albumin content of the buffer or buffered solution generally not exceeding about 5_0% (w/v).
[0055] Exemplary compositions which regulate s oxidation/reduction reactions intracellularly andO or extracellularly include for example pyruvate, vitamin K, lipoic acid, glutathione, flavins, quinones, superoxide dismutase (SOD), and SOD mimics. If included in the sperm suspension, such a composition may be present in a concentration sufficient to effect the protective effect without detrimentally affecting sperm health. Exemplary concentration ranges include from about 10~M to about 20 mM
depending upon such factors as the particular composition being used or the concentration of sperm in the suspensi on.
For example, pyruvate may be present in the sperm suspension in a concentration from about 1mM to about 20 mM, preferably from about 5mM to about l5mM, and more preferably about lOmM. Vitamin K may be present in the sperm suspension in a concentration from about 1~M to ab out 100~M, preferably from about 10~M to about 100~M, and mo re preferably about 100~.M. Lipoic acid may be present in t he sperm suspension in a concentration from about 0.lmM to about lmM, preferably from about 0.5mM to about lmM, and more preferably about lmM.
[0056] An antibiotic may be included in the sperm suspension in order to inhibit bacterial growth. Exemplary antibiotics include, for example, tylosin, gentamicin, lincomycin, spectinomycin, Linco-Spectin° (lincomycin hydrochloride-spectinomycin), penicillin, streptomycin, ticarcillin, or any combination thereof. If included, t he antibiotics may be present in a concentration of about 50~.g to about 800~g per ml of semen, regardless of whether the semen is neat, buffered, or contains additional substances, such as for example, any of the additives mentioned here in.
The Certified Semen Services (CSS) and National Associat ion of Animal Breeders (NAAB) have promulgated guidelines regarding the use of antibiotics with respect to sperm collection and use.
[0057] A growth factor may be added to the sperm dispersion in order to help maintain the viability of the sperm cells. Exemplary growth factors include, for example, transforming growth factors ( "TGF) , such as, for example, TGF~i-1 and TGF,~-2, and insulin-like growth factors ("IGF"), such as for example, IGF-1. Generally, TGF may be present in the sperm dispersion in the form of TGF(3-1 in a concentration of about 0.lng/L t o about 10~.g/L or as TGF,~-2 in a concentration of about O.lrig/L to about 200ng/L, and IGF may be present in the sperm dispersion in the form of IGF-1 in a concentration of about O.lng/L to about 50~,g/L.
The use of such growth factors i_s well known in the art and is disclosed, for example, in U. S. Patent Application Publication No. 2003/0157473, the content of which is hereby incorporated herein by reference.
[0058] Once collected, the cells may be stored in a quiescent state for several hour s at room temperature, for several weeks at a reduced temps nature, such as for example at 5°C, or stored for several months in a cryoextender as discussed below. Preferably, the atmosphere above the cells has a high partial pressure of COZ as discussed above.
Alternatively, the collected cells may be used within several hours, such as for example in a fertilization process, a staining process, or a sorting process.
Staining of the cells [0059] A motility inhibitor may be used to render cells immotile during staining of the cells. A process of staining sperm cells typically comprises the formation of a staining mixture, sometimes refs rred to as a labeling mixture, containing intact viabL a sperm cells, a motility inhibitor, and a dye, sometimes referred to as a label. In this aspect of the invention, the motility inhibitor may be contacted with the sperm cells t o form a sperm suspension, and then the suspension contacts d with a DNA selective dye.
In this embodiment, the sperm source may be neat semen, or alternatively, a sperm-containing semen derivative obtained by centrifugation or the use of other- means to separate semen into fractions.
[0060] In an alternative embodiment, the dye may be 5 combined with a motility inhibitor, t hereby forming a dye solution. Thus, for example, dye in the form of a neat solid, including a free-flowing powder r, or a liquid composition may be combined with the inhibitor to form a dye solution, which may then be combined with neat semen, a 10 sperm suspension, or a sperm-containing semen derivative.
[0061] In any event, the sperm cells will generally remain quiescent as long as they are maintained in the inhibitor. (Salisbury et al., 1963) Preferably, however, the staining mixture is maintained under an atmosphere 15 having an enriched partial pressure o f carbon dioxide relative to air; for example, providing an atmosphere over the staining mixture which is 99%+ C02 is generally preferred.
[0062] The pH of the staining mixture may be 20 maintained at any of a range of pH's; typically this will be in the range of about 5.0 to about 9Ø For example, the staining mixture may be maintaine d at a "slightly acid"
pH, i.e., from about 5.0 to about 7.0 . In this embodiment, the pH is preferably from about 6.0 t o about 7.0, more preferably from about 6.0 to about 6. 5, and most preferably at about 6.2. Alternatively, the sta fining mixture may be maintained at a "slightly basic" pH, i.e., from about 7.0 to about 9Ø In this embodiment, th a pH is preferably from about 7.0 to about 8.0, more pre ferably from about 7.0 to about 7.5, and most preferably at about 7.3.
[0063] The staining mixture may be formed by using one or more UV or visible light excitable , DNA selective dyes as previously described in U.S. Paten t No. 5,135,759 and W002/41906. Exemplary UV light excitable, selective dyes include Hoechst 33342 and Hoechst 33258, each of which is commercially available from Sigma-Aldrich (St. Louis, MO).
Exemplary visible light excitable dyes include SYBR-14, commercially available from Molecular Probes, Inc. (Eugene, OR) and bisbenzimide-BODIPY conjugate 6- f [3- ( (2Z) -2-~ [1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2-yl]methylene~-2H-pyrrol-5-yl)propanoyl] amino}-N- [3- (methyl (3- [ (~4- [6- (4-methylpiperazin-1-yl)-1H,3'H-2,5'-biben ~ imidazol-2'-yl]phenoxy~acetyl)amino]propyl~amino)propyl]hexanamide ("BBC") described in WO 02/41906. Each of these dyes may be used alone or in combination; alternatively, other cell permeant UV and visible light excitable dyes may be used, alone or in combination with the aforementioned dyes, provided the dye does not detrimentally affect the viability of the sperm cells to an unacceptable degree when used in concentrations which enable sort ing as described elsewhere.
[0064] Alternatively, the staining mixture may be formed using fluorescent polyamides, and more specifically polyamides with a fluorescent label or reporter conjugated thereto. Such labels will fluoresce when bound to nucleic acids. Examples of polyamides with a fluorescent label or reporter attached thereto include, for example, those disclosed in Best et al., Proc. Natl. A cad. Sci. USA, 100 (21) : 12063-12068 (2003) ; Gygi, et a~ . , Nucleic Acids Res., 30(13): 2790-2799 (2002); U.S. Patent No. 5,998,140;
U.S. Patent No. 6,143,901; and U.S. Patent No. 6,090,947, the content of each of which is hereby incorporated herein by reference.
[0065] Fluorescent nucleotide sequences may also be used to label the sperm cells. Such nucleotide sequences fluoresce when hybridized to a nucleic acid containing a target or complementary sequence, but are otherwise non-fluorescent when in a non-hybridized state. Such oligonucleotides are disclosed, for example, in U.S. Patent Application Publication No. 2003/0113765 (hereby incorporated herein by reference).
[0066] Sex specific antibodies may also be used to label the sperm cells in a staining mixture. In this embodiment, for example, a sex specific antibody may be conjugated with a fluorescent moiety (or equivalent reporter molecule). Because the antibody binds to antigens present on only an X chromosome-bearing or, alternatively, a Y chromosome-bearing cell, such cells can be selectively identified based upon their fluorescence (versus the rion-fluorescence of an unlabeled cell). Moreover, more tL-pan one sex specific antibody, each antibody having a different fluorescent moiety attached thereto, may be used simultaneously. This allows for differentiation of X chromosome-bearing and Y chromosome-bearing cells based upon the differing fluorescence of each.
[0067] Luminescent, color-selective nanocrystals may also be used to label sperm cells in a staining mixture.
Also referred to as quantum dots, these particles are well known in the art, as demonstrated by U.S. Patent No.
6,322,901 and U.S. Patent No. 6,576,291, each of which is hereby incorporated herein by reference. These nanocrystals have been conjugated to a number of biological materials, including for example, peptides, antibodies, nucleic acids, streptavidin, and polysaccharides, (see, for example, U.S. Patent Nos. 6,207,392; 6,423,551; 5,990, 479, and 6,326,144, each of which is hereby incorporated herein by reference), and have been used to detect biological targets (see, for example, U.S. Patent Nos. 6,207,392 and 6,247,323, each of which is hereby incorporated herein by ref erence ) .
[0068] The preferred concentration of the DTTA
selective or of any other type of dye in the sta i ning mixture is a function of a range of variables which include the permeability of the cells to the selected dye, the temperature of the staining mixture, the amount of time allowed for staining to occur, and the degree of enrichment desired in the subsequent sorting step. In general, the dye concentration is preferably sufficient to achieve the desired degree of staining in a reasonably short period of time without substantially detrimentally affecting sperm viability. For example, the concentration of Hoe chst 33342, Hoechst 33258, SYBR-14, or BBC in the staining mixture will generally be between about 0.1~.M ancZ about 1. OM, preferably from about 0.1~CM to about 700~CM~ and more preferably from about 100~,M to about 200~CM. In a.
particularly preferred embodiment, the concentration of Hoechst 33342, Hoechst 33258, SYBR-14, or BBC in the staining mixture will generally be between about 400~cM to about 500~,M, and most preferably about 450~.M. Accordingly, under one set of staining conditions, the concentrration of Hoechst 33342 is preferably about 100~,M. Under another set of staining conditions, the concentration of Hoechst 33342 is about 150~.M. Under still another set of staining conditions the concentration is preferably about 200~,M.
Under yet another set of staining conditions the concentration of Hoechst 33342 is most preferably about 450~.M.
[0069] As another example, the concentration of a fluorescent polyamide, such as for example, those described in U.S. Application Publication No. 2001/0002314, will generally be between about O.l~cM and about lmM, preferably from about 1~,M to about lmM, more preferably about 5~,M to about 100~.M, even more preferably about 10~.M.
[0070] Optionally, the staining mixture may also contain additives to enhance sperm viability. Exemplary additives include an antibiotic, a growth factor or a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly as discussed above with respect to cell sample collection. These additives may be added to the collection fluid in accordance therewith.
[0071] Once formed, the staining mixture may be maintained at any of a range of temperatures; typically, this will be within a range of about 4°C to about 50°C.
For example, the staining mixture may be maintained at a "relatively low" temperature, i.e., a temperature of about 4°C to about 30°C; in this embodiment, the temperature is preferably from about 20°C to about 30°C, more preferably from about 25°C to about 30°C, and most preferable at about 28°C. Alternatively, the staining mixture may be maintained within an "intermediate" temperature range, i.e., a temperature of about 30°C to about 39°C; in this embodiment, the temperature is preferably at about 34°C to about 39°C, and more preferably about 37°C. In addition, the staining mixture may be maintained within a "relatively high" temperature range, i.e., a temperature of about 40°C
to about 50°C; in this embodiment, the temperature is preferably from about 40°C to about 45°C, more preferably from about 40°C to about 43°C, and most preferably at about 41°C. Selection of a preferred temperature generally depends upon a range of variables, including for example, the permeability of the cells to the dyes) being used, the concentration of the dyes) in the staining mixture, the amount of time the cells will be maintained in the staining mixture, and the degree of enrichment desired in the sorting step.
[0072] Uptake of dye by the sperm cells in the staining mixture is allowed to continue for a period of 5 time sufficient to obtain the desired degree of DNA
staining. That period is typically a period sufficient for the dye to bind to the DNA of the sperm cells such that X
and Y chromosome-bearing sperm cells may be sorted based upon the differing and measurable fluorescence intensity 10 between the two. Generally, this will be no more than about 160 minutes, preferably no more than about 90 minutes, still more preferably no more than about 60 minutes, and most preferably from about 5 minutes to about 40 minutes.
15 [0073] Accordingly, in one embodiment, a staining mixture is formed comprising sperm cells, a motility inhibitor, and a dye in a concentration from about 100~cM to about 200~.M, and the staining mixture is held for a period of time at a temperature of about 41°C. In another 20 embodiment, the motility inhibitor comprises 0.2048 NaHC03, 0.4338 KHC03, and 0.4738 C6HeO~~H20 per 25mL of purified water (0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0 . 090 moles/L C6H80~ ~H20 in water) .
[0074] In another embodiment, a staining mixture is 25 formed comprising sperm cells, a motility inhibitor, and a dye in a concentration of about 400~.M to about 500~,M, and the staining mixture is held for a period of time at a temperature of about 41°C. In another embodiment, the dye concentration is 450~M. In another embodiment, the motility inhibitor comprises 0.2048 NaHC03, 0.4338 KHC03, and 0.4738 C6HsO~~H~O per 25mL of purified water (0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0.090 moles/L
C6H80~ ~ H20 in water) .
L0025] FIGURE 7 graphically depicts the results of the study carried out in Example 3 wherein percent progressive motility of sperm cells is measured for sperm cells stained with 300~,M Hoechst 33342 dye at 41°C in TCA containing lOmM
pyruvate and then diluted 1 to 3 with either TCA containing lOmM pyruvate or a carbonate-based inhibitor at pH 6.2.
L0026] FIGURE 8 graphically depicts the results of the study carried out in Example 3 wherein percent progressive motility of sperm cells is measured for sperm cells stained with 300~,M Hoechst 33342 dye at 41°C in (1) TCA containing lOmM pyruvate and diluted 1 to 3 with the same or (2) a carbonate-based buffer at pH 7.3 and diluted 1 to 3 with carbonate-based inhibitor at pH 6.2.
[0027] FIGURE 9 graphically depicts the results of the study carried out in Example 3 wherein percent progressive motility of sperm cells is measured for sperm cells stained with 300~,M Hoechst 33342 dye at 41°C in TCA containing lOmM
pyruvate or a carbonate-based inhibitor at pH 6.2.
[0028] FIGURE 10 graphically depicts the results of the study carried out in Example 4 wherein percent progressive motility of sperm is measured for sperm stained with 400~tM Hoechst 33342 dye at 41°C in either a TCA buffer or a TCA buffer containing lOmM pyruvate.
[0029] FIGURE 11 graphically depicts the results of the study carried out in Example 5 wherein percent progressive motility of sperm is measured for sperm stained with 400~,M Hoechst 33342 dye at 41°C in either a TCA buffer or a TCA buffer containing 10~,M vitamin K.
[0030] FIGURE 12 graphically depicts the results of the study carried out in Example 6 wherein percent progressive motility of sperm is measured for sperm stained with 400~,M Hoechst 33342 dye at 41°C in either a TCA buffer or a TCA buffer containing 100~M vitamin K.
[0031] FIGURE 13 graphically depicts the results of the study carried out in Example 7 wherein percent progressive motility of sperm is measured for sperm stained with 400~CM Hoechst 33342 dye at 41°C in either a TCA buffer or a TCA buffer containing 1mM lipoic acid.
[0032] FIGURE 14 graphically depicts the results of the study carried out in Example 8 wherein percent progressive motility of sperm is measured for sperm stained with 600~M Hoechst 33342 dye at 28°C in either a TCA buffer or a TCA buffer containing lOmM pyruvate.
[0033] FIGURE 15 graphically depicts the results of the study carried out in Example 9 wherein percent progressive motility of sperm is measured for sperm stained with 600uM Hoechst 33342 dye at 28°C in either a TCA buffer or a TCA buffer containing 100~.M vitamin K.
[0034] FIGURE 16 graphically depicts the results of the study carried out in Example 10 wherein percent progressive motility of sperm is measured for sperm stained with 600~CM Hoechst 33342 dye at 28°C in either a TCA buffer or a TCA buffer containing 1mM lipoic acid.
[0035] FIGURE 17 graphically depicts the results of the study carried out in Example 11 wherein percent progressive motility of sperm is measured for sperm stained with 600~,M Hoechst 33342 dye at 28°C in a TCA buffer, a TCA
buffer containing 2.5mM pyruvate, a TCA buffer containing lOmM pyruvate, a TCA buffer containing 25mM pyruvate, and a TCA buffer containing 50mM pyruvate.
[0036] FIGURE 18 graphically depicts the results of the study carried out in Example 12 wherein percent progressive motility of sperm is measured for sperm stained with 20~,M SYBR-14 dye at 28°C in either a TCA buffer or a TCA buffer containing lOmM pyruvate.
[0037] FIGURE 19 graphically depicts the results of the study carried out in Example 13 wherein percent progressive motility of sperm is measured for sperm stained with 100~,M BBC dye at 28°C in either a TCA buffer or a TCA
buffer containing lOmM pyruvate.
[0038] FIGURE 20 graphically depicts the results of the study carried out in Example 14 wherein percent progressive motility of sperm is measured for sperm stained with 200~CM BBC dye at 28°C in either a TCA buffer or a TCA
buffer containing lOmM pyruvate.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Surprisingly, it has been determined that spermatozoa having reduced motility relative to endogenous ejaculated spermatozoa (of the same species) tend to have a greater capacity for enduring the various process steps typically associated with the sorting of sperm cells into an enriched population of X or Y chromosome-bearing spermatozoa. In a preferred embodiment, therefore, gender enriched populations of spermatozoa may be prepared for artificial insemination which have an increased number of viable cells or an increased number of motile sperm, particularly progressively motile sperm, in a post-stain or post-sort composition.
[0040] In accordance with the process of the present invention, a suspension, sometimes referred to as a dispersion, is formed containing spermatozoa and one or more compositions which inhibit the motility of the spermatozoa; such a state of inhibited motility sometimes being referred to as immotility or sperm quiescence. In general, the suspensions will contain spermatozoa in a density of about 1 X 103 sperm/ml to about 5 X 101° sperm/ml of suspension. For example, in one embodiment the suspensions may contain spermatozoa in a "relatively low"
density, i.e., in a density of less than about 1 X 10' sperm/ml, preferably less than about 1 X 106 sperm/ml, more preferably about 1 X 103 to about 5 X 106 sperm/ml, still more preferably about 1 X 103 to about 1 X 106 sperm/ml, even more preferably about 1 X 104 to about 1 X 105 sperm/ml, and most preferably about 1 X 105 sperm/ml of suspension. In an alternative embodiment, the suspensions may contain spermatozoa in an "intermediate" density, i.e., in a density of about 1 X 10' to about 1 X 108 sperm/ml of suspension. In yet another alternative embodiment, the suspensions may contain spermatozoa in a "relatively high"
density, i.e., in a density of at least about 1 x 108 sperm/ml, preferably about 1 X 108 to about 5 X 1010 sperm/ml, more preferably about 1.5 X 10$ to about 2 X 1010 sperm/ml, even more preferably about 1.5 X 108 to about 2 X 108 sperm/ml, and still more preferably about 1.5 X 108 sperm/ml of suspension. Thus, for example, in one embodiment the suspension may contain at least about 1.25 x 108, at least about 1.5 x 108, at least about 1.75 x 108, at least about 2 x 108, at least about 2.25 x 108, at least about 2.5 x 108, at least about 2.75 x 108, or even at least about 3 x 108 sperm/ml of suspension. In an alternative embodiment, the suspension may contain less than about 9 X 105, less than about 7 X 105, less than about 5 X 105, less than about 2 X 105, less than about 1 X 105, less than about 1 X 104, or even less than about 1 X 103 sperm/ml of suspension.
[0041] The density of spermatozoa in the sperm suspensions depends upon several considerations, including the method by which the sperm cells may be subsequently enriched or sorted. For example, the sperm cells may be sorted using flow cytometry as described in greater detail below. In such an instance, the buffered sperm suspension may typically be of an "intermediate" or "relatively high"
density of spermatozoa. Other sorting or enrichment techniques may benefit from a lesser density of spermatozoa, such as a "relatively low" density of spermatozoa, labeled with a marker, such as for example the dyes and labels described herein.
[0042] In a preferred embodiment, spermatozoa in suspensions of the present invention behave, in certain respects, in a manner characteristic of epididymal spermatozoa; for example, the spermatozoa may be immotile 5 and/or they may have a lesser rate of endogenous respiration and a greater rate of aerobic glycolysis as compared to washed or freshly ejaculated spermatozoa.
Advantageously, the inhibited spermatozoa have the ability, upon separation from the inhibitor(s), to behave in a 10 manner characteristic of ejaculated spermatozoa (and not characteristic of epididymal spermatozoa) with respect to mots.lity and, in one embodiment, with respect to motility and respiration.
[0043] In one embodiment, for example, the motility inhibitor reduces path velocity, progressive velocity, or bot h, as measured by HTM-IVOS sperm analysis (Hamilton-Tho rne HTM-IVOS computer assisted sperm analysis system Hamilton-Thorne Research, Beverly MA) of at least about 50%
of t he sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Preferably, the mot ility inhibitor reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, of at least about 60% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or bot h of sperm cells in a fresh ejaculate of the same spe ties. More preferably, the motility inhibitor reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, of at least about 70% of the sperm cells in the dispersion relative to the path veto city, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Still more preferably, the motility inhibitor reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS
sperm analysis, of at least about 800 of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Even more preferably, the motility inhibitor reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, of at least about 90% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Even more preferably, the motility inhibitor reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS
sperm analysis, of at least about 95% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Most preferably, the motility inhibitor reduces path velocity, progressive velocity, or both, as measured by an HTM-IVOS sperm analysis, of at least about 99% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species.
(0044] In addition to or in lieu of an inhibitory buffer, the temperature of the sperm cells or the immediate environment surrounding the sperm cells (i.e., a sperm dispersion) may solely be reduced to affect the motility of the cells. Such a reduction in temperature will generally increase immotility. Moreover, for example, the reduction of temperature of the sperm cells or the sperm dispersion may permit a reduction in the concentration of inhibitor used to induce immotility. Accordingly, the sperm dispersion may be at a temperature not in excess of 5°C;
preferably between about D°C and about 5°C; more preferably between about 3°C and about 5°C; and most preferably about 5°C. Alternatively, the sperm dispersion may be at a temperature wit hin the range of about 4°C to about 50°C;
preferably from about 7°C to about 43°C; more preferably from about 10°C to about 39°C; still more preferably from about 15°C to about 30°C; even more preferably from about 17°C to about 2 5°C; and most preferably at about 18°C.
Preferably, hovaever, the sperm cells are not exposed to temperatures that substantially detrimentally affect the viability of the cells.
[0045] The inhibitor may be any of a range of compositions having a depressive effect upon sperm motility. Such compositions include, for example, sodium/potassium ATPase inhibitors, such as, ouabain;
compositions comprising potassium ions; and compositions comprising potassium and sodium ions. For example, relatively high concentrations of potassium ions in the suspension tend to depress sperm motility. In general, therefore, it i s preferred that the suspension contain a source of potassium ions and that the potassium concentration i n the suspension be at least about 0.05 moles/L. More preferably, the potassium concentration is at least about 0.05 moles/L to about 0.5 moles/L. Still more preferably, the potassium concentration is at least about 0.1 moles /L to about 0.3 moles/L. Most preferably, the potassium concentration is at about 0.173 moles/L.
Such suspension s will typically, but not necessarily, also contain a sours a of sodium ions. When sodium is present, the molar ratio of potassium to sodium is generally equal to or greater than 1:1, respectively. Preferably, the molar ratio of potassium to sodium is at least about 1.25:1. Still more preferably, the molar ratio of potassium to sodium is at least about 1.5:1. Still more preferably, the molar ratio of potassium to sodium is at least about 1.75:1. Still more preferably, the molar ratio of potassium to sodium is at least about 1.78:1. In one particular embodiment, the molar ration of potassium to sodium is at least about 2:1. In yet another embodiment, the molar ratio of potassium to sodium is at least about 3:1. In still another embodiment, the molar ratio of potassium to sodium is at least about 4:1. In still another embodiment, the molar ratio of potassium to sodium is at least about 5:1. In still another embodiment, the molar ratio of potassium to sodium is at least about 6:1.
In still another embodiment, the molar ratio of potassium to sodium is at least about 7:1. In still another embodiment, the molar ratio of potassium to sodium is at least about 8:1.
[0046] The sperm suspension may additionally comprise an ion or source of carbon dioxide capable of down-regulating uptake of carbohydrate. In this embodiment, the source of carbon dioxide may be, for example, one or more carbonates. In one presently preferred embodiment, the sperm suspension comprises NaHC03 and KHCO3, thereby providing a source potassium and sodium ions as well as a partial pressure of carbon dioxide. For example, in one presently preferred embodiment, the suspension comprises NaHC03 and KHC03 in an aqueous solution, preferably NaHC03, KHC03 , and CgH80~ ~ H20 in water; In general , the KFiC03 concentration in the dispersion may be at least about 0.05 moles/L. More preferably, the KHC03 concentration is at least about 0.05 moles/L to about 0.5 moles/L. Still more preferably, the KHCO3 concentration is at least about 0.1 moles/L to about 0.3 moles/L. In one particularly preferred embodiment, the suspension is formed using an inhibitory buffer comprising 0.097 moles/L of NaHCO3, 0.173 moles/L of KHC03, 0.090 moles/L C6H80~~H20 in water as disclosed in Salisbury & Graves, J. Reprod. Fertil., 6:351-359 (1963). The sperm cells will generally remain quiescent as long as they are exposed to the motility inhibitor(s).
[0047] When C6Ha0~~HZO is present in the dispersion, the molar ratio of KHC03 to NaHC03 may be as described above . The molar ratio of KHC03 to C6H80~ ~ HZO may generally be equal to or greater than 1:1, respectively, but will generally not exceed a molar ratio of 8:1. Preferably, the molar ratio of KHC03 to C6H807~H~O is from at least about 1.25:1. Still more preferably, the molar ratio of KHC03 to C6H80~~H20 is at least about 1.5:1. Still more preferably, the molar ratio of KHC03 to C6Ha0~~H20 is at least about 1.75:1. In one particular embodiment, the molar ratio of KHC03 to C6H80~~H20 is at least about 1.78:1. In another particular embodiment, the molar ratio of KHC03 to C6H80~-H20 is at least about 2:1. In yet another embodiment, the molar ratio of KHC03 to C6Ha0~~HZO is at least about 3:1. In still another embodiment, the molar ratio of KHC03 to C6H$OwH~O is at least about 4:1. In still another embodiment, the molar ratio of KHC03 to C6Hs0~~H~O is at least about 5:1. In still another embodiment, the molar ratio of KHC03 to C6H80~ ~ H~0 i s at least about 6 : 1 . In still another embodiment, the molar ratio of KHC03 to C6H80~-H20 is at least about 7:1. In still another embodiment, the molar ratio of KHC03 to C6H80~ ~ H20 i s at least about 8 : 1 . In one particularly preferred embodiment, the dispersion is formed using an inhibitory buffer comprising 0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0.090 moles/L C6H80~~H20 in water as disclosed in Salisbury & Graves, J. Reprod.
Fertil., 6:351-359 (1963). The sperm cells will generally remain quiescent as long as they are exposed to the motility inhibitor(s).
[0048] Experimental evidence to date further suggests that the overall health and other vital characteristics of sperm cells may be improved if the cell suspension is maintained under an atmosphere having an enhanced partial 5 pressure of carbon dioxide relative to air. In a preferred embodiment, the atmosphere over the suspension has a partial pressure of carbon dioxide of at least 0.9; more preferably, at least about 0.95.
[0049] Quiescent cells may be returned to an active 10 state by separating the cells from the motility inhibitor and exposing them to air. In addition, the initiation of an active state may be further induced by the dilution of the cells in a physiological saline (Salisbury et al., 1963) or a buffer such as TCA buffer or PBS. Typically, at 15 least about 20%, preferably at lea st about 50%, more preferably at least about 60%, still more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 90%, still more preferably at least about 95%, and most preferably at least about 99% of the cells returned to an active state (i.e., reactivated cells) will have a path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, that is at least about 50%, preferably at least about 60%, more preferably at least about 70%, still more preferably at least about 80%, even more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 99% of the path velocity, progressive velocity, or both of the sperm cells prior to being combined with the motility inhibitor (i.e., of sperm cells of a fresh ejaculate).
[0050] In general, the cell sorting process comprises a series of discrete steps, i.e., collection of a cell sample, staining of the cells, sorting of the cells, collection of the sorted cells, and optionally, cryoextension of the sorted cells. Advantageously, the motility inhibitor may be included in sperm suspensions formed or employed in one or more of then a steps.
Collection of the cell sample [0051] Intact viable bovine, porcine, equine, or other mammalian sperm cells, may be collected and contacted with the motility inhibitor. Various methods of collection of viable sperm are known and include, for example, the gloved-hand method, use of an artificial vagina, and electro-ejaculation. As an example, a bovine semen sample, typically containing about 0.5 to about 10 billion sperm cells per milliliter, may be collected directly from the source mammal into a vessel containing a motility inhibitor to form a sperm suspension. Alternatively, the semen sample may be collected into an empty vessel and then subsequently contacted with the motility inhibitor within several minutes to hours after collection to form the sperm suspension.
[0052] In addition to a buffer, the sperm suspension may also contain a range of additives to enhance sperm viability. Exemplary additives include protein sources, antibiotics, and compositions which regul ate oxidation/reduction reactions intracellul arty and/or extracellularly.
[0053] Exemplary protein sources include egg yolk, egg yolk extract, milk (including heat homogenised and skim), milk extract, soy protein, soy protein extract, serum albumin, bovine serum albumin, human serum substitute supplement, and combinations thereof. Albumin, and more particularly bovine serum albumin (BSA), is a preferred protein source. For example, if included, BSA may be present in the sperm suspension in an amount of less than about 5.Oa (w/v), preferably less than about 2% (w/v), more preferably less than about to (w/v), and most preferably in an amount of about O.lo (w/v).
[0054] The use of a protein source, such BSA, alone may initiate the process of oapaoitation in a percentage of the sperm cells in the suspension. It is preferred that this process take place in the female reproductive tract.
Therefore, in order to inhibit the initiation of capacitation during dilution, as well as during the subsequent staining and sorting, an alternative protein source or a protein substitute may be included in the sperm suspension. The alternative protein source or protein substitute possess the advantageous effects of a typical protein source, such as BSA, in addition to the ability to inhibit the initiation of capacitation in a larger percentage of the cells in the sperm suspension. Examples of a alternative protein sources includes human se rum substitute supplement (SSS) (Irvine Scientific, Santa Ana, CA) and cholesterol enhanced BSA, while an example of a protein substitute includes a polyvinyl alcohol, such as for example, a low to medium viscosity polyvinyl alcohol generally of a molecular weight of about 30,000 to about 60,000. Generally, if included, these compositions will be present in the same amounts as disclosed above with respect to BSA, with the total albumin content of the buffer or buffered solution generally not exceeding about 5_0% (w/v).
[0055] Exemplary compositions which regulate s oxidation/reduction reactions intracellularly andO or extracellularly include for example pyruvate, vitamin K, lipoic acid, glutathione, flavins, quinones, superoxide dismutase (SOD), and SOD mimics. If included in the sperm suspension, such a composition may be present in a concentration sufficient to effect the protective effect without detrimentally affecting sperm health. Exemplary concentration ranges include from about 10~M to about 20 mM
depending upon such factors as the particular composition being used or the concentration of sperm in the suspensi on.
For example, pyruvate may be present in the sperm suspension in a concentration from about 1mM to about 20 mM, preferably from about 5mM to about l5mM, and more preferably about lOmM. Vitamin K may be present in the sperm suspension in a concentration from about 1~M to ab out 100~M, preferably from about 10~M to about 100~M, and mo re preferably about 100~.M. Lipoic acid may be present in t he sperm suspension in a concentration from about 0.lmM to about lmM, preferably from about 0.5mM to about lmM, and more preferably about lmM.
[0056] An antibiotic may be included in the sperm suspension in order to inhibit bacterial growth. Exemplary antibiotics include, for example, tylosin, gentamicin, lincomycin, spectinomycin, Linco-Spectin° (lincomycin hydrochloride-spectinomycin), penicillin, streptomycin, ticarcillin, or any combination thereof. If included, t he antibiotics may be present in a concentration of about 50~.g to about 800~g per ml of semen, regardless of whether the semen is neat, buffered, or contains additional substances, such as for example, any of the additives mentioned here in.
The Certified Semen Services (CSS) and National Associat ion of Animal Breeders (NAAB) have promulgated guidelines regarding the use of antibiotics with respect to sperm collection and use.
[0057] A growth factor may be added to the sperm dispersion in order to help maintain the viability of the sperm cells. Exemplary growth factors include, for example, transforming growth factors ( "TGF) , such as, for example, TGF~i-1 and TGF,~-2, and insulin-like growth factors ("IGF"), such as for example, IGF-1. Generally, TGF may be present in the sperm dispersion in the form of TGF(3-1 in a concentration of about 0.lng/L t o about 10~.g/L or as TGF,~-2 in a concentration of about O.lrig/L to about 200ng/L, and IGF may be present in the sperm dispersion in the form of IGF-1 in a concentration of about O.lng/L to about 50~,g/L.
The use of such growth factors i_s well known in the art and is disclosed, for example, in U. S. Patent Application Publication No. 2003/0157473, the content of which is hereby incorporated herein by reference.
[0058] Once collected, the cells may be stored in a quiescent state for several hour s at room temperature, for several weeks at a reduced temps nature, such as for example at 5°C, or stored for several months in a cryoextender as discussed below. Preferably, the atmosphere above the cells has a high partial pressure of COZ as discussed above.
Alternatively, the collected cells may be used within several hours, such as for example in a fertilization process, a staining process, or a sorting process.
Staining of the cells [0059] A motility inhibitor may be used to render cells immotile during staining of the cells. A process of staining sperm cells typically comprises the formation of a staining mixture, sometimes refs rred to as a labeling mixture, containing intact viabL a sperm cells, a motility inhibitor, and a dye, sometimes referred to as a label. In this aspect of the invention, the motility inhibitor may be contacted with the sperm cells t o form a sperm suspension, and then the suspension contacts d with a DNA selective dye.
In this embodiment, the sperm source may be neat semen, or alternatively, a sperm-containing semen derivative obtained by centrifugation or the use of other- means to separate semen into fractions.
[0060] In an alternative embodiment, the dye may be 5 combined with a motility inhibitor, t hereby forming a dye solution. Thus, for example, dye in the form of a neat solid, including a free-flowing powder r, or a liquid composition may be combined with the inhibitor to form a dye solution, which may then be combined with neat semen, a 10 sperm suspension, or a sperm-containing semen derivative.
[0061] In any event, the sperm cells will generally remain quiescent as long as they are maintained in the inhibitor. (Salisbury et al., 1963) Preferably, however, the staining mixture is maintained under an atmosphere 15 having an enriched partial pressure o f carbon dioxide relative to air; for example, providing an atmosphere over the staining mixture which is 99%+ C02 is generally preferred.
[0062] The pH of the staining mixture may be 20 maintained at any of a range of pH's; typically this will be in the range of about 5.0 to about 9Ø For example, the staining mixture may be maintaine d at a "slightly acid"
pH, i.e., from about 5.0 to about 7.0 . In this embodiment, the pH is preferably from about 6.0 t o about 7.0, more preferably from about 6.0 to about 6. 5, and most preferably at about 6.2. Alternatively, the sta fining mixture may be maintained at a "slightly basic" pH, i.e., from about 7.0 to about 9Ø In this embodiment, th a pH is preferably from about 7.0 to about 8.0, more pre ferably from about 7.0 to about 7.5, and most preferably at about 7.3.
[0063] The staining mixture may be formed by using one or more UV or visible light excitable , DNA selective dyes as previously described in U.S. Paten t No. 5,135,759 and W002/41906. Exemplary UV light excitable, selective dyes include Hoechst 33342 and Hoechst 33258, each of which is commercially available from Sigma-Aldrich (St. Louis, MO).
Exemplary visible light excitable dyes include SYBR-14, commercially available from Molecular Probes, Inc. (Eugene, OR) and bisbenzimide-BODIPY conjugate 6- f [3- ( (2Z) -2-~ [1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2-yl]methylene~-2H-pyrrol-5-yl)propanoyl] amino}-N- [3- (methyl (3- [ (~4- [6- (4-methylpiperazin-1-yl)-1H,3'H-2,5'-biben ~ imidazol-2'-yl]phenoxy~acetyl)amino]propyl~amino)propyl]hexanamide ("BBC") described in WO 02/41906. Each of these dyes may be used alone or in combination; alternatively, other cell permeant UV and visible light excitable dyes may be used, alone or in combination with the aforementioned dyes, provided the dye does not detrimentally affect the viability of the sperm cells to an unacceptable degree when used in concentrations which enable sort ing as described elsewhere.
[0064] Alternatively, the staining mixture may be formed using fluorescent polyamides, and more specifically polyamides with a fluorescent label or reporter conjugated thereto. Such labels will fluoresce when bound to nucleic acids. Examples of polyamides with a fluorescent label or reporter attached thereto include, for example, those disclosed in Best et al., Proc. Natl. A cad. Sci. USA, 100 (21) : 12063-12068 (2003) ; Gygi, et a~ . , Nucleic Acids Res., 30(13): 2790-2799 (2002); U.S. Patent No. 5,998,140;
U.S. Patent No. 6,143,901; and U.S. Patent No. 6,090,947, the content of each of which is hereby incorporated herein by reference.
[0065] Fluorescent nucleotide sequences may also be used to label the sperm cells. Such nucleotide sequences fluoresce when hybridized to a nucleic acid containing a target or complementary sequence, but are otherwise non-fluorescent when in a non-hybridized state. Such oligonucleotides are disclosed, for example, in U.S. Patent Application Publication No. 2003/0113765 (hereby incorporated herein by reference).
[0066] Sex specific antibodies may also be used to label the sperm cells in a staining mixture. In this embodiment, for example, a sex specific antibody may be conjugated with a fluorescent moiety (or equivalent reporter molecule). Because the antibody binds to antigens present on only an X chromosome-bearing or, alternatively, a Y chromosome-bearing cell, such cells can be selectively identified based upon their fluorescence (versus the rion-fluorescence of an unlabeled cell). Moreover, more tL-pan one sex specific antibody, each antibody having a different fluorescent moiety attached thereto, may be used simultaneously. This allows for differentiation of X chromosome-bearing and Y chromosome-bearing cells based upon the differing fluorescence of each.
[0067] Luminescent, color-selective nanocrystals may also be used to label sperm cells in a staining mixture.
Also referred to as quantum dots, these particles are well known in the art, as demonstrated by U.S. Patent No.
6,322,901 and U.S. Patent No. 6,576,291, each of which is hereby incorporated herein by reference. These nanocrystals have been conjugated to a number of biological materials, including for example, peptides, antibodies, nucleic acids, streptavidin, and polysaccharides, (see, for example, U.S. Patent Nos. 6,207,392; 6,423,551; 5,990, 479, and 6,326,144, each of which is hereby incorporated herein by reference), and have been used to detect biological targets (see, for example, U.S. Patent Nos. 6,207,392 and 6,247,323, each of which is hereby incorporated herein by ref erence ) .
[0068] The preferred concentration of the DTTA
selective or of any other type of dye in the sta i ning mixture is a function of a range of variables which include the permeability of the cells to the selected dye, the temperature of the staining mixture, the amount of time allowed for staining to occur, and the degree of enrichment desired in the subsequent sorting step. In general, the dye concentration is preferably sufficient to achieve the desired degree of staining in a reasonably short period of time without substantially detrimentally affecting sperm viability. For example, the concentration of Hoe chst 33342, Hoechst 33258, SYBR-14, or BBC in the staining mixture will generally be between about 0.1~.M ancZ about 1. OM, preferably from about 0.1~CM to about 700~CM~ and more preferably from about 100~,M to about 200~CM. In a.
particularly preferred embodiment, the concentration of Hoechst 33342, Hoechst 33258, SYBR-14, or BBC in the staining mixture will generally be between about 400~cM to about 500~,M, and most preferably about 450~.M. Accordingly, under one set of staining conditions, the concentrration of Hoechst 33342 is preferably about 100~,M. Under another set of staining conditions, the concentration of Hoechst 33342 is about 150~.M. Under still another set of staining conditions the concentration is preferably about 200~,M.
Under yet another set of staining conditions the concentration of Hoechst 33342 is most preferably about 450~.M.
[0069] As another example, the concentration of a fluorescent polyamide, such as for example, those described in U.S. Application Publication No. 2001/0002314, will generally be between about O.l~cM and about lmM, preferably from about 1~,M to about lmM, more preferably about 5~,M to about 100~.M, even more preferably about 10~.M.
[0070] Optionally, the staining mixture may also contain additives to enhance sperm viability. Exemplary additives include an antibiotic, a growth factor or a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly as discussed above with respect to cell sample collection. These additives may be added to the collection fluid in accordance therewith.
[0071] Once formed, the staining mixture may be maintained at any of a range of temperatures; typically, this will be within a range of about 4°C to about 50°C.
For example, the staining mixture may be maintained at a "relatively low" temperature, i.e., a temperature of about 4°C to about 30°C; in this embodiment, the temperature is preferably from about 20°C to about 30°C, more preferably from about 25°C to about 30°C, and most preferable at about 28°C. Alternatively, the staining mixture may be maintained within an "intermediate" temperature range, i.e., a temperature of about 30°C to about 39°C; in this embodiment, the temperature is preferably at about 34°C to about 39°C, and more preferably about 37°C. In addition, the staining mixture may be maintained within a "relatively high" temperature range, i.e., a temperature of about 40°C
to about 50°C; in this embodiment, the temperature is preferably from about 40°C to about 45°C, more preferably from about 40°C to about 43°C, and most preferably at about 41°C. Selection of a preferred temperature generally depends upon a range of variables, including for example, the permeability of the cells to the dyes) being used, the concentration of the dyes) in the staining mixture, the amount of time the cells will be maintained in the staining mixture, and the degree of enrichment desired in the sorting step.
[0072] Uptake of dye by the sperm cells in the staining mixture is allowed to continue for a period of 5 time sufficient to obtain the desired degree of DNA
staining. That period is typically a period sufficient for the dye to bind to the DNA of the sperm cells such that X
and Y chromosome-bearing sperm cells may be sorted based upon the differing and measurable fluorescence intensity 10 between the two. Generally, this will be no more than about 160 minutes, preferably no more than about 90 minutes, still more preferably no more than about 60 minutes, and most preferably from about 5 minutes to about 40 minutes.
15 [0073] Accordingly, in one embodiment, a staining mixture is formed comprising sperm cells, a motility inhibitor, and a dye in a concentration from about 100~cM to about 200~.M, and the staining mixture is held for a period of time at a temperature of about 41°C. In another 20 embodiment, the motility inhibitor comprises 0.2048 NaHC03, 0.4338 KHC03, and 0.4738 C6HeO~~H20 per 25mL of purified water (0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0 . 090 moles/L C6H80~ ~H20 in water) .
[0074] In another embodiment, a staining mixture is 25 formed comprising sperm cells, a motility inhibitor, and a dye in a concentration of about 400~.M to about 500~,M, and the staining mixture is held for a period of time at a temperature of about 41°C. In another embodiment, the dye concentration is 450~M. In another embodiment, the motility inhibitor comprises 0.2048 NaHC03, 0.4338 KHC03, and 0.4738 C6HsO~~H~O per 25mL of purified water (0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0.090 moles/L
C6H80~ ~ H20 in water) .
[0075] In still another embodiment, a staining mixture is formed comprising sperm cells, a motility inhibitor, and a dye in a concentration from about 100~,M to about 200~.M, and the staining mixture is held for a period of time at a temperature of about 28°C. In another embodiment, the motility inhibitor comprises 0.2048 NaHC03, 0.4338 KHC03, and 0.4738 C6H80~~H20 per 25mL of purified water (0.097 moles/L of NaHCO3, 0.173 moles/L of KHC03, 0.090 moles/L
C6H80~-H20 in water) .
[0076] In yet another embodiment, a staining mixture is formed comprising sperm cells, a motility inhibitor, and a dye in a concentration from about 400~,M to about 500~,M, and the staining mixture is held for a period of time at a temperature of about 28°C. In another embodiment, the dye concentration is 450~.M. In another embodiment, the motility inhibitor comprises 0.2048 NaHC03, 0.4338 KHC03, and 0.4738 C6H80~~H20 per 25mL of purified water (0.097 moles/L of NaHC03, 0.173 moles/L of KHCO3, 0.090 moles/L
C6H80~ ~ H20 in water) .
Sorting [0077] A motility inhibitor may also be used to render the sperm cells immotile during sorting of the sperm cells.
Generally, once the sperm are stained according to the present invention, they may be sorted according to any known means that allows for separation based upon fluorescence. Commonly used and well known methods include flow cytometry systems, as exemplified by and described in U.S. Patent Nos. 5,135,759, 5,985,216, 6,071,689, 6,149,867, and 6,263,745, International Patent Publications WO 99/33956 and WO 01/37655, and U.S. Patent Application Serial No. 10/812,351, the content of which is hereby incorporated herein by reference, and corresponding International Patent Publication WO 2004/088283. When sorting according to such methods, the sperm are introduced into the nozzle of a flow cytometer in a sample fluid. In one embodiment, therefore, the sample fluid may comprise the stained sperm cells and a motility inhibitor.
[0078] Likewise, the sheath fluid used to surround the stream of sample fluid as it travels through the cytometer may also comprise a motility inhibitor. Generally, the sheath fluid may be introduced into a nozzle of the cytometer using pressurized gas or by a syringe pump.
Preferably, the pressurized gas is carbon dioxide or nitrogen, more preferably nitrogen. Alternatively, the pressurized gas may be carbon dioxide, although under such.
circumstances, care may be taken to minimize effervescence.
[0079] Optionally, the sample fluid or sheath fluid may also contain additive, such as, an antibiotic, a composition which regulates oxidation/reduction reactions intracellularly or extracellularly, or a growth factor as discussed above with respect to cell sample collection Each of these additives may be added to either fluid in accordance therewith.
Collection of the sorted cells [0080] Once sorted, the sorted cells are collected in a vessel that contains a collection fluid. Generally, the purpose of the collection fluid includes cushioning the impact of the sperm cells with the collection vessel or providing a fluid support for the cells.
[0081] In one embodiment, the collection fluid comprises a motility inhibitor and a protein source. If included, the protein source may be any protein source that does not interfere with the viability of the sperm cells and is compatible with the motility inhibitor. Examples of common protein sources include milk (including heat homogenized and skim), milk extract, egg yolk, egg yolk extract, soy protein and soy protein extract. Such proteins may be used in a concentration from about 1% (v/v) to about 30% (v/v), preferably from about 10o (v/v) to about 200 (v/v), and more preferably about l00 (v/v).
[0082] Optionally, the collection fluid may also contain additives such as, an antibiotic, a growth factor or a composition which regulates oxidation/reduction reactions intracellularly or extracellularly as discussed above with respect to cell sample collection. Each of these additives may be added to the collection fluid in accordance therewith.
[0083] Accordingly, in a certain embodiment, the collection fluid comprises 0.097 moles/L of NaHC03, 0.173 moles/L of I~HC03, 0.090 moles/L C6H80wHZ0 and 10% (v/v) egg yolk in water, at a pH of about 6.2, more preferably of about 7.0, and even more preferably of about 6.5. Preferably, the collection fluid is maintained under an atmosphere having an enriched partial pressure of carbon dioxide relative to air; for example, the atmosphere may have a partial pressure of carbon dioxide in excess of 0.9, more preferably 0.95 and still more preferably 0.99.
[0084] In lieu of the use of a more traditional collection fluid, the sorted cells may be collected into a vessel containing or coated with a cryoextender.
Accordingly, in one particular embodiment, the sorted cells are collected into a cryoextender comprising a motility inhibitor. In another embodiment, the sorted cells are collected into a cryoextender comprising a motility inhibitor, water, Triladyl~ (Minitube, Verona, WI, comprising glycerol, tris, citric acid, fructose, 5mg/100m1 tylosin, 25mg/100m1 gentamycin, 30mg/100m1 Spectinomycin, and l5mg/100m1 Lincomycin), egg yolk, and pyruvic acid. In yet another embodiment, the collection fluid is the cryoextender comprising 0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0.090 moles/L C6HeO~~HaO in water, and 25g Triladyl~, 25g egg yolk, and lOmM pyruvic acid per 75mL of water.
Cryoextension of the sorted cells [0085] Once the sperm have been sorted and collected into collection vessels, they may be used for inseminating female mammals. This can occur almost immediately, requiring little additional treatment of the sperm.
Likewise, the sperm may also be cooled or frozen for use at a later date. In such instances, the sperm may benefit from the addition of a cryoextender to minimize the impact upon viability or post-thaw motility as a result of cooling and freezing.
[0086] A motility inhibitor may be used to render cells in the cryoextender immotile. Generally, a cryoextender may comprise a motility inhibitor, a protein source, and a cryoprotectant. If included, a protein source may be added to provide support to the cells and to cushion the contact of the cells with the collection vessel. The protein source may be any protein source that does not interfere with the viability of the sperm cells and is compatible with the motility inhibitor. Examples of common protein sources include milk (including heat homogenized and skim), milk extract, egg yolk, egg yolk extract, soy protein and soy protein extract. Such proteins may be found in a concentration from about 10a (v/v) to about 300 (v/v), preferably from about 100 (v/v) to about 200 (v/v), and more preferably about 20% (v/v) .
[0087] A cryoprotectant is preferably included in the cryoextender to lessen or prevent cold shock or to maintain fertility of the sperm. Numerous cryoprotectants are known in the art. Selection of a cryoprotectant suitable for use 5 with a given extender may vary, and depends upon the species from which the sperm to be frozen were obtained.
Examples of suitable cryoprotectants include, for example, glycerol, dimethyl sulfoxide, ethylene glycol, propylene glycol, trehalose, Triladyl~, and combinations thereof. If 10 included, generally, these cryoprotectants are present in the cryoextender in an amount of about 1% (v/v) to about 15-°s (v/v), preferably in an amount of about 5% (v/v) to about 10% (v/v), more preferably in an amount of about 7% (v/v), and most preferably in an amount of about 15 6 0 (v/v) .
[0088] In one particular embodiment, the cryoextender comprises a motility inhibitor, water, Triladyl~, egg yolk, and pyruvic acid. In yet another embodiment, the cryoextender comprises 0.097 moles/L of NaHC03, 20 0.173 moles/L of KHC03, 0.090 moles/L C6HgOwH20 in water, and 25g Triladylo, 25g egg yolk, and lOmM pyruvic acid per 75mL of water.
[0089] In another particular embodiment, the cryoextender comprises a motility inhibitor, water, 25 Triladyl~, and egg yolk. In yet another embodiment, the cryoextender comprises 0.097 moles/L of NaHC03, 0.173 moles/L of KHCO3, 0.090 moles/L C6H80wH20 in water, and 25g Triladyl~, and 25g egg yolk per 75mL of water.
[0090] Optionally, the cryoextender may also contain 30 an antibiotic, a growth factor or a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly as discussed above with respect to cell sample collection Each of these additives may be added to the collection fluid in accordance therewith.
[0091] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing the scope of the invention defined in the appended claims.
EXAMPIiES
[0092] The following non-limiting examples are provided to further illustrate the present invention.
Example 1 [0093] Bull semen was collected from a sexually mature bull using an artificial vagina and transported at 25°C in a temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al . Theriogenology, 49 (4) 871- 9 (Mar 1998)). Based on the semen concentration, several tubes of 150 X 106 sperm/ml suspensions were prep ared by suspending semen in a TCA buffer or a carbonate-based inhibitor. Table I. below illustrates the comp ositions and staining conditions used.
C6H80~-H20 in water) .
[0076] In yet another embodiment, a staining mixture is formed comprising sperm cells, a motility inhibitor, and a dye in a concentration from about 400~,M to about 500~,M, and the staining mixture is held for a period of time at a temperature of about 28°C. In another embodiment, the dye concentration is 450~.M. In another embodiment, the motility inhibitor comprises 0.2048 NaHC03, 0.4338 KHC03, and 0.4738 C6H80~~H20 per 25mL of purified water (0.097 moles/L of NaHC03, 0.173 moles/L of KHCO3, 0.090 moles/L
C6H80~ ~ H20 in water) .
Sorting [0077] A motility inhibitor may also be used to render the sperm cells immotile during sorting of the sperm cells.
Generally, once the sperm are stained according to the present invention, they may be sorted according to any known means that allows for separation based upon fluorescence. Commonly used and well known methods include flow cytometry systems, as exemplified by and described in U.S. Patent Nos. 5,135,759, 5,985,216, 6,071,689, 6,149,867, and 6,263,745, International Patent Publications WO 99/33956 and WO 01/37655, and U.S. Patent Application Serial No. 10/812,351, the content of which is hereby incorporated herein by reference, and corresponding International Patent Publication WO 2004/088283. When sorting according to such methods, the sperm are introduced into the nozzle of a flow cytometer in a sample fluid. In one embodiment, therefore, the sample fluid may comprise the stained sperm cells and a motility inhibitor.
[0078] Likewise, the sheath fluid used to surround the stream of sample fluid as it travels through the cytometer may also comprise a motility inhibitor. Generally, the sheath fluid may be introduced into a nozzle of the cytometer using pressurized gas or by a syringe pump.
Preferably, the pressurized gas is carbon dioxide or nitrogen, more preferably nitrogen. Alternatively, the pressurized gas may be carbon dioxide, although under such.
circumstances, care may be taken to minimize effervescence.
[0079] Optionally, the sample fluid or sheath fluid may also contain additive, such as, an antibiotic, a composition which regulates oxidation/reduction reactions intracellularly or extracellularly, or a growth factor as discussed above with respect to cell sample collection Each of these additives may be added to either fluid in accordance therewith.
Collection of the sorted cells [0080] Once sorted, the sorted cells are collected in a vessel that contains a collection fluid. Generally, the purpose of the collection fluid includes cushioning the impact of the sperm cells with the collection vessel or providing a fluid support for the cells.
[0081] In one embodiment, the collection fluid comprises a motility inhibitor and a protein source. If included, the protein source may be any protein source that does not interfere with the viability of the sperm cells and is compatible with the motility inhibitor. Examples of common protein sources include milk (including heat homogenized and skim), milk extract, egg yolk, egg yolk extract, soy protein and soy protein extract. Such proteins may be used in a concentration from about 1% (v/v) to about 30% (v/v), preferably from about 10o (v/v) to about 200 (v/v), and more preferably about l00 (v/v).
[0082] Optionally, the collection fluid may also contain additives such as, an antibiotic, a growth factor or a composition which regulates oxidation/reduction reactions intracellularly or extracellularly as discussed above with respect to cell sample collection. Each of these additives may be added to the collection fluid in accordance therewith.
[0083] Accordingly, in a certain embodiment, the collection fluid comprises 0.097 moles/L of NaHC03, 0.173 moles/L of I~HC03, 0.090 moles/L C6H80wHZ0 and 10% (v/v) egg yolk in water, at a pH of about 6.2, more preferably of about 7.0, and even more preferably of about 6.5. Preferably, the collection fluid is maintained under an atmosphere having an enriched partial pressure of carbon dioxide relative to air; for example, the atmosphere may have a partial pressure of carbon dioxide in excess of 0.9, more preferably 0.95 and still more preferably 0.99.
[0084] In lieu of the use of a more traditional collection fluid, the sorted cells may be collected into a vessel containing or coated with a cryoextender.
Accordingly, in one particular embodiment, the sorted cells are collected into a cryoextender comprising a motility inhibitor. In another embodiment, the sorted cells are collected into a cryoextender comprising a motility inhibitor, water, Triladyl~ (Minitube, Verona, WI, comprising glycerol, tris, citric acid, fructose, 5mg/100m1 tylosin, 25mg/100m1 gentamycin, 30mg/100m1 Spectinomycin, and l5mg/100m1 Lincomycin), egg yolk, and pyruvic acid. In yet another embodiment, the collection fluid is the cryoextender comprising 0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0.090 moles/L C6HeO~~HaO in water, and 25g Triladyl~, 25g egg yolk, and lOmM pyruvic acid per 75mL of water.
Cryoextension of the sorted cells [0085] Once the sperm have been sorted and collected into collection vessels, they may be used for inseminating female mammals. This can occur almost immediately, requiring little additional treatment of the sperm.
Likewise, the sperm may also be cooled or frozen for use at a later date. In such instances, the sperm may benefit from the addition of a cryoextender to minimize the impact upon viability or post-thaw motility as a result of cooling and freezing.
[0086] A motility inhibitor may be used to render cells in the cryoextender immotile. Generally, a cryoextender may comprise a motility inhibitor, a protein source, and a cryoprotectant. If included, a protein source may be added to provide support to the cells and to cushion the contact of the cells with the collection vessel. The protein source may be any protein source that does not interfere with the viability of the sperm cells and is compatible with the motility inhibitor. Examples of common protein sources include milk (including heat homogenized and skim), milk extract, egg yolk, egg yolk extract, soy protein and soy protein extract. Such proteins may be found in a concentration from about 10a (v/v) to about 300 (v/v), preferably from about 100 (v/v) to about 200 (v/v), and more preferably about 20% (v/v) .
[0087] A cryoprotectant is preferably included in the cryoextender to lessen or prevent cold shock or to maintain fertility of the sperm. Numerous cryoprotectants are known in the art. Selection of a cryoprotectant suitable for use 5 with a given extender may vary, and depends upon the species from which the sperm to be frozen were obtained.
Examples of suitable cryoprotectants include, for example, glycerol, dimethyl sulfoxide, ethylene glycol, propylene glycol, trehalose, Triladyl~, and combinations thereof. If 10 included, generally, these cryoprotectants are present in the cryoextender in an amount of about 1% (v/v) to about 15-°s (v/v), preferably in an amount of about 5% (v/v) to about 10% (v/v), more preferably in an amount of about 7% (v/v), and most preferably in an amount of about 15 6 0 (v/v) .
[0088] In one particular embodiment, the cryoextender comprises a motility inhibitor, water, Triladyl~, egg yolk, and pyruvic acid. In yet another embodiment, the cryoextender comprises 0.097 moles/L of NaHC03, 20 0.173 moles/L of KHC03, 0.090 moles/L C6HgOwH20 in water, and 25g Triladylo, 25g egg yolk, and lOmM pyruvic acid per 75mL of water.
[0089] In another particular embodiment, the cryoextender comprises a motility inhibitor, water, 25 Triladyl~, and egg yolk. In yet another embodiment, the cryoextender comprises 0.097 moles/L of NaHC03, 0.173 moles/L of KHCO3, 0.090 moles/L C6H80wH20 in water, and 25g Triladyl~, and 25g egg yolk per 75mL of water.
[0090] Optionally, the cryoextender may also contain 30 an antibiotic, a growth factor or a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly as discussed above with respect to cell sample collection Each of these additives may be added to the collection fluid in accordance therewith.
[0091] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing the scope of the invention defined in the appended claims.
EXAMPIiES
[0092] The following non-limiting examples are provided to further illustrate the present invention.
Example 1 [0093] Bull semen was collected from a sexually mature bull using an artificial vagina and transported at 25°C in a temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al . Theriogenology, 49 (4) 871- 9 (Mar 1998)). Based on the semen concentration, several tubes of 150 X 106 sperm/ml suspensions were prep ared by suspending semen in a TCA buffer or a carbonate-based inhibitor. Table I. below illustrates the comp ositions and staining conditions used.
Table I.
S ample Conc.(uM) Temperature Composition pH
Name Hoechst (C) mM pyr lomM pyruvate in 7.3 600,uM 28C
TcA
TCA
1 lOmM pyruvate in TCA
0 mM pyr blanket with C02 7 . 6 0 O,uM 2 8 C
balloon Carbonate Carbonate based 6.2 600,uM 28C
6.2 inhibitor, pH 6.2 Carbonate Carbonate based 7.3 600,uM 28C
'7,3 inhibitor, pH 7.3 [0094] To the sperm suspensions, aliquots of a lOmM
5 Hoechst solution in water were added to yield a concentration of 600~.M Hoechst. The sperm suspensions were maintained in a 28°C water bath for the duration of the staining period (approximately 1 hour). Sperm suspensions were analyzed by removing a 50~.L aliquot from the stained 10 sperm suspension, adding 200~L of 25°C lOmM pyruvate in TCA
at pH 7.3 to initiate the reversal of the quiescence, allowing at least a five minute equilibration period, and analyzing by IVOS to measure percent progressive motility (% Prog. Mot.). Comparisons of the IVOS percent progressive motilities are seen in Figures 1-3.
S ample Conc.(uM) Temperature Composition pH
Name Hoechst (C) mM pyr lomM pyruvate in 7.3 600,uM 28C
TcA
TCA
1 lOmM pyruvate in TCA
0 mM pyr blanket with C02 7 . 6 0 O,uM 2 8 C
balloon Carbonate Carbonate based 6.2 600,uM 28C
6.2 inhibitor, pH 6.2 Carbonate Carbonate based 7.3 600,uM 28C
'7,3 inhibitor, pH 7.3 [0094] To the sperm suspensions, aliquots of a lOmM
5 Hoechst solution in water were added to yield a concentration of 600~.M Hoechst. The sperm suspensions were maintained in a 28°C water bath for the duration of the staining period (approximately 1 hour). Sperm suspensions were analyzed by removing a 50~.L aliquot from the stained 10 sperm suspension, adding 200~L of 25°C lOmM pyruvate in TCA
at pH 7.3 to initiate the reversal of the quiescence, allowing at least a five minute equilibration period, and analyzing by IVOS to measure percent progressive motility (% Prog. Mot.). Comparisons of the IVOS percent progressive motilities are seen in Figures 1-3.
Example 2 [0095] Bull semen was collected from a sexually mature bull using an artificial vagina and transported at 25°C in a temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al. Theriogenology, 49(4):
871-9 (Mar 1998)). Based on the semen concentration, several tubes of 450 X 106 sperm/ml suspensions were prepared by su spending semen in either a TCA buffer or a carbonate base d inhibitor. Table II. below illustrates the compositions and staining conditions used.
Table II.
Sample Conc. (uM) Temperature Composition pH
Name Hoechst (C) lOmM pyr lOmM pyruvate in 7 . 10 0 O,uM 2 $ C
TCA
Carbonate Carbonate based 6.2 1000,uM 28C
6.2 inhibitor, pH 6.2 Carbonate Carbonate based 7.3 1000,uM 28C
7.3 inhibitor, pH 7.3 [0096] To the sperm suspensions, aliquots of a lOmM
Hoechst solution in water were added to yield a concentration of 1000~M Hoechst. The sperm suspensions were maintaine d in a 28°C water bath for 1 hour, and were then diluted t o 150 X 106 sperm/ml with lOmM pyruvate in TCA
or a carbonate -based inhibitor at a pH~6.2 as specifically indicated in a ach figure to dilute to a concentration typical for sorting. Sperm suspensions were analyzed by removing a 50~.L aliquot from the stained and diluted sperm suspension at the time period designated within each figure and adding 200~L of 25°C lOmM pyruvate in TCA at pH 7.3 to initiate the reversal of the quiescence, allowing at least a five minute equilibration period, and analyzing the aliquot by IVOS to measure the percent progressive motility. Compari sons of the IVOS percent progressive motilities are seen in Figures 4-6.
Example 3 [0097] Bull semen was collected from a sexually mature bull using an artificial vagina and transported at 25°C in a temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al. Theriogenology, 49(4):
871-9 (Mar 1998)). Based on the semen concentration, several tubes of 450 X. 106 sperm/ml suspensions were prepared by suspending semen in either a TCA buffer or a carbonate based inhibitor. Table II. below illustrates the compositions and staining conditions used.
Table III
Sample Cone (uM) Temperature Buffer pH
Name Hoechst (C) lOmM pyr lOmM pyruvate in 7 . 3 0 O,uM 41 C
TCA
CarbOriate Carbonate based 6.2 300,uM 41C
6.2 inhibitor, pH 6.2 Carbonate Carbonate based 7.3 300,uM 41C
7.3 inhibitor, pH 7.3 [0098] To the sperm suspensions, aliquots of a IOmM
Hoechst solution in water were added to yield a concentration of 300~,M Hoechst. The sperm suspensions were maintained in a 41°C water bath for 30 minutes, and then 5 diluted to 150 X 106 sperm/ml with 10 mM pyruvate in TCA or a carbonate-based inhibitor at pH 6.2 as specifically indicated in each figure to dilute to a concentration typical for sorting. Sperm suspensions were analyzed by removing a 50~cL aliquot from the stained and diluted sperm 10 suspension at the time period designated within each figure and adding 200~,L of 25°C lOmM pyruvate in TCA at pH 7.3 to initiate the reversal of the quiescence, allowing at least a five minute equilibration period, and analyzing by IVOS
to measure the percent progressive motility. Comparisons 15 of the IVOS percent progressive motilities are seen in Figures 7-9.
Example 4 [0099] Bull semen was collected from a sexually mature 20 bull using an artificial vagina and the sample diluted in 2 parts carbonate buffer for transportation at 25°C in a temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn 25 Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al. Theriogenology, 49(4):
871-9 (Mar 1998)). Based on the semen concentration, 1mL
of 150 X 106 sperm/ml suspension was prepared by removing an aliquot of the carbonate sperm suspension centrifuging the 30 sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-suspending the pellet in 41°C TCA buffer pH 7.3. An additional 1mL of 150 X 106 sperm/ml was prepared by suspending an aliquot of semen in 41°C TCA
buffer containing lOmM pyruvate at pH 7.3. To the sperm suspensions, aliquots of a lOmM Hoechst solution in water were added to yield the dye cons entration of 400~M Hoechst.
The sperm suspensions were maim ained in a 41°C water bath for the duration of the staining period. Sperm suspensions were analyzed by removing a 50~L aliquot from the staining sperm suspension, adding 200~,L of the same buffer at the same temperature and analyzing by IVOS to measure o progressive motility (% Prog Mot). Results of the IVOS
analysis are summarized in Figure 10.
Example 5 [00100] Sperm samples were obtained and prepared in the 1 5 same manner as in Example 4 with the following exception.
The buffer used to suspend the sperm for staining and IVOS
analysis were TCA and TCA com a i ning lOuM Vitamin K.
Results of the IVOS analysis are summarized in Figure 11.
Example 6 [00101] Sperm samples were obtained and prepared in the same manner as in Example 4 with the following exception.
The buffer used to suspend the sperm for staining and IVOS
analysis were TCA and TCA comaining 100uM Vitamin IC.
Results of the IVOS analysis are summarized in Figure 12.
Example 7 [00102] Sperm samples were obtained and prepared in the same manner as in Example 4 with the following exception.
The buffers used to suspend the sperm for staining and IVOS
analysis were TCA and TCA com a i ning 1mM Lipoic Acid.
Results of the IVOS analysis ara summarized in Figure 13.
Example 8 [00103) Bull semen was collect ed from a sexually mature bull using an artificial vagina and the sample diluted in 2 parts carbonate buffer for transportation at 25°C in a temperature-controlled container t o the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al. Theriogenology, 49(4):
871-9 (Mar 1998)). Based on the semen concentration, 1mL
of 150 X 106 sperm/ml suspension was prepared by centrifuging the sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-su spending the pellet in 28°C TCA buffer pH 7.3. An additional 1mL of 150 X 106 sperm/ml was prepared by suspending an aliquot of semen in 28°C TCA buffer containing lOmM pyruvate at pH 7.3. To the sperm suspensions, aliquots of a 10 mM Hoechst solution in water were added to yield the dye concentration of 600~uM
Hoechst. The sperm suspensions we re maintained in 28°C
water bath for the duration of the staining period. Sperm suspensions were analyzed by removing a 50~L aliquot from the staining sperm suspension, add ing 200~,L of the same buffer at the same temperature and analyzing by IVOS to measure percent progressive motility (% Prog Mot). Results of the IVOS analysis are summarize d in Figure 14.
Example 9 [00104) Sperm samples were obtained and prepared in the same manner as in Example 8 with t he following exception.
The buffer used to suspend the sperm for staining and IVOS
analysis were TCA and TCA containing 100uM Vitamin K.
Results of the IVOS analysis are summarized in Figure 15.
Example 10 [00105] Sperm samples were obtained and prepared in the same manner as in Example 8 with the following exception.
The buffer used to suspend the sperm for staining and IVOS
analysis were TCA and TCA containing 1mM Lipoic Acid.
Results of the IVOS analysis are summarized in Figure 16.
Example 11 [00106] Bull semen was collected from a sexually mature bull using an artificial vagina and the sample diluted in 2 parts carbonate buffer for transport ation at 25°C in a temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al. Thariogenology, 49 (4) 871-9 (Mar 1998)). Based on the semen concentration, 1mL
of 150 X 106 sperm/ml suspensions we re prepared by removing an aliquots of the carbonate sperm suspension, centrifuging the sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-suspending the pallet in 1 ml TCA buffer or in 1 ml TCA buffer with 2.5 mM, 10 mM, 25 mM, or 50 mM
pyruvate. To the samples was added MON33342 solution to yield the final dye concentrations of 600~M. The suspensions were incubated in a 28°C water bath. Stained sperm suspensions were analyzed by removing a 50~L aliquot from the staining sperm suspension, adding 200~,L of the same buffer at the same temperature and analyzing by IVOS
to measure percent progressive motility (% Prog Mot). IVOS
results for % Prog Mot are shown in Figures 17.
Example 12 [001071 Bull semen was collected from a sexually mature bull using an artificial vagina and the sample diluted in 2 parts carbonate buffer for transportation at 25°C in a temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), ace ording to standard and well known procedures (Farrell et al. Theriogenology, 49(4):
871-9 (Mar 1998)). Based on the semen concentration, 1mL
of 150 X 106 sperm/ml suspension in TCA buffer was prepared by removing an aliquot of the carbonate sperm suspension, centrifuging the sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-suspending the pellet in 1mL TCA buffer. 1ml of 150 X 106 sperm/ml suspension in lOmM pyruvate in TCA was prepared by removing an aliquot of the carbonate sperm suspension, centrifuging the sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-suspending the pellet in 1mL of 10 mM
pyruvate TCA buffer. To samples was added SYBR 14 dye solution to yield the final dye concentrations of 20~M The suspensions were incubated in a 28°C water bath. Sperm suspensions were analyzed by removing a 50~.~,L aliquot from the staining sperm suspension, adding 200~L of the same buffer at the same temperature and analyzing by IVOS to measure percent progressive motility (% Prog Mot). IVOS
results for % Prog Mot are shown in Figures 18.
Example 13 [00108] Bull semen was collected from a sexually mature bull using an artificial vagina and the sample diluted in 2 parts carbonate buffer for transportation at 25°C in a 5 temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al. Theriogenology, 49 (4) 10 871-9 (Mar 1998)). Based on the semen concentration, 1mL
of 150 X 106 sperm/ml suspension in TCA buffer was prepared by removing an aliquot of the carbonate sperm suspension, centrifuging the sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-suspending the pellet in 15 1mL TCA buffer. 1m1 of 150 X 106 sperm/ml suspension in lOmM pyruvate in TCA was prepared by removing an aliquot of the carbonate sperm suspension, centrifuging the sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-suspending the pellet in lml of 10 mM
20 pyruvate TCA buffer. To t he samples was added BBC solution to yield the final dye concentrations of 100~M. The suspensions were incubated in a 28°C water bath. Stained sperm suspensions were analyzed by removing a 50~.L aliquot from the staining sperm suspension, adding 200~L of the 25 same buffer at the same temperature and analyzing by IVOS
to measure percent progres sine motility (% Prog Mot). IVOS
results for o Prog Mot are shown in Figures 19.
Example 14 (00109] Sperm samples were obtained and prepared in the same manner as in Examp 1e 4 with the following exception.
The staining concentrat ion was 200uM BBC. Results of the IVOS analysis are summarized in Figure 20.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al. Theriogenology, 49(4):
871-9 (Mar 1998)). Based on the semen concentration, several tubes of 450 X 106 sperm/ml suspensions were prepared by su spending semen in either a TCA buffer or a carbonate base d inhibitor. Table II. below illustrates the compositions and staining conditions used.
Table II.
Sample Conc. (uM) Temperature Composition pH
Name Hoechst (C) lOmM pyr lOmM pyruvate in 7 . 10 0 O,uM 2 $ C
TCA
Carbonate Carbonate based 6.2 1000,uM 28C
6.2 inhibitor, pH 6.2 Carbonate Carbonate based 7.3 1000,uM 28C
7.3 inhibitor, pH 7.3 [0096] To the sperm suspensions, aliquots of a lOmM
Hoechst solution in water were added to yield a concentration of 1000~M Hoechst. The sperm suspensions were maintaine d in a 28°C water bath for 1 hour, and were then diluted t o 150 X 106 sperm/ml with lOmM pyruvate in TCA
or a carbonate -based inhibitor at a pH~6.2 as specifically indicated in a ach figure to dilute to a concentration typical for sorting. Sperm suspensions were analyzed by removing a 50~.L aliquot from the stained and diluted sperm suspension at the time period designated within each figure and adding 200~L of 25°C lOmM pyruvate in TCA at pH 7.3 to initiate the reversal of the quiescence, allowing at least a five minute equilibration period, and analyzing the aliquot by IVOS to measure the percent progressive motility. Compari sons of the IVOS percent progressive motilities are seen in Figures 4-6.
Example 3 [0097] Bull semen was collected from a sexually mature bull using an artificial vagina and transported at 25°C in a temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al. Theriogenology, 49(4):
871-9 (Mar 1998)). Based on the semen concentration, several tubes of 450 X. 106 sperm/ml suspensions were prepared by suspending semen in either a TCA buffer or a carbonate based inhibitor. Table II. below illustrates the compositions and staining conditions used.
Table III
Sample Cone (uM) Temperature Buffer pH
Name Hoechst (C) lOmM pyr lOmM pyruvate in 7 . 3 0 O,uM 41 C
TCA
CarbOriate Carbonate based 6.2 300,uM 41C
6.2 inhibitor, pH 6.2 Carbonate Carbonate based 7.3 300,uM 41C
7.3 inhibitor, pH 7.3 [0098] To the sperm suspensions, aliquots of a IOmM
Hoechst solution in water were added to yield a concentration of 300~,M Hoechst. The sperm suspensions were maintained in a 41°C water bath for 30 minutes, and then 5 diluted to 150 X 106 sperm/ml with 10 mM pyruvate in TCA or a carbonate-based inhibitor at pH 6.2 as specifically indicated in each figure to dilute to a concentration typical for sorting. Sperm suspensions were analyzed by removing a 50~cL aliquot from the stained and diluted sperm 10 suspension at the time period designated within each figure and adding 200~,L of 25°C lOmM pyruvate in TCA at pH 7.3 to initiate the reversal of the quiescence, allowing at least a five minute equilibration period, and analyzing by IVOS
to measure the percent progressive motility. Comparisons 15 of the IVOS percent progressive motilities are seen in Figures 7-9.
Example 4 [0099] Bull semen was collected from a sexually mature 20 bull using an artificial vagina and the sample diluted in 2 parts carbonate buffer for transportation at 25°C in a temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn 25 Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al. Theriogenology, 49(4):
871-9 (Mar 1998)). Based on the semen concentration, 1mL
of 150 X 106 sperm/ml suspension was prepared by removing an aliquot of the carbonate sperm suspension centrifuging the 30 sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-suspending the pellet in 41°C TCA buffer pH 7.3. An additional 1mL of 150 X 106 sperm/ml was prepared by suspending an aliquot of semen in 41°C TCA
buffer containing lOmM pyruvate at pH 7.3. To the sperm suspensions, aliquots of a lOmM Hoechst solution in water were added to yield the dye cons entration of 400~M Hoechst.
The sperm suspensions were maim ained in a 41°C water bath for the duration of the staining period. Sperm suspensions were analyzed by removing a 50~L aliquot from the staining sperm suspension, adding 200~,L of the same buffer at the same temperature and analyzing by IVOS to measure o progressive motility (% Prog Mot). Results of the IVOS
analysis are summarized in Figure 10.
Example 5 [00100] Sperm samples were obtained and prepared in the 1 5 same manner as in Example 4 with the following exception.
The buffer used to suspend the sperm for staining and IVOS
analysis were TCA and TCA com a i ning lOuM Vitamin K.
Results of the IVOS analysis are summarized in Figure 11.
Example 6 [00101] Sperm samples were obtained and prepared in the same manner as in Example 4 with the following exception.
The buffer used to suspend the sperm for staining and IVOS
analysis were TCA and TCA comaining 100uM Vitamin IC.
Results of the IVOS analysis are summarized in Figure 12.
Example 7 [00102] Sperm samples were obtained and prepared in the same manner as in Example 4 with the following exception.
The buffers used to suspend the sperm for staining and IVOS
analysis were TCA and TCA com a i ning 1mM Lipoic Acid.
Results of the IVOS analysis ara summarized in Figure 13.
Example 8 [00103) Bull semen was collect ed from a sexually mature bull using an artificial vagina and the sample diluted in 2 parts carbonate buffer for transportation at 25°C in a temperature-controlled container t o the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al. Theriogenology, 49(4):
871-9 (Mar 1998)). Based on the semen concentration, 1mL
of 150 X 106 sperm/ml suspension was prepared by centrifuging the sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-su spending the pellet in 28°C TCA buffer pH 7.3. An additional 1mL of 150 X 106 sperm/ml was prepared by suspending an aliquot of semen in 28°C TCA buffer containing lOmM pyruvate at pH 7.3. To the sperm suspensions, aliquots of a 10 mM Hoechst solution in water were added to yield the dye concentration of 600~uM
Hoechst. The sperm suspensions we re maintained in 28°C
water bath for the duration of the staining period. Sperm suspensions were analyzed by removing a 50~L aliquot from the staining sperm suspension, add ing 200~,L of the same buffer at the same temperature and analyzing by IVOS to measure percent progressive motility (% Prog Mot). Results of the IVOS analysis are summarize d in Figure 14.
Example 9 [00104) Sperm samples were obtained and prepared in the same manner as in Example 8 with t he following exception.
The buffer used to suspend the sperm for staining and IVOS
analysis were TCA and TCA containing 100uM Vitamin K.
Results of the IVOS analysis are summarized in Figure 15.
Example 10 [00105] Sperm samples were obtained and prepared in the same manner as in Example 8 with the following exception.
The buffer used to suspend the sperm for staining and IVOS
analysis were TCA and TCA containing 1mM Lipoic Acid.
Results of the IVOS analysis are summarized in Figure 16.
Example 11 [00106] Bull semen was collected from a sexually mature bull using an artificial vagina and the sample diluted in 2 parts carbonate buffer for transport ation at 25°C in a temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al. Thariogenology, 49 (4) 871-9 (Mar 1998)). Based on the semen concentration, 1mL
of 150 X 106 sperm/ml suspensions we re prepared by removing an aliquots of the carbonate sperm suspension, centrifuging the sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-suspending the pallet in 1 ml TCA buffer or in 1 ml TCA buffer with 2.5 mM, 10 mM, 25 mM, or 50 mM
pyruvate. To the samples was added MON33342 solution to yield the final dye concentrations of 600~M. The suspensions were incubated in a 28°C water bath. Stained sperm suspensions were analyzed by removing a 50~L aliquot from the staining sperm suspension, adding 200~,L of the same buffer at the same temperature and analyzing by IVOS
to measure percent progressive motility (% Prog Mot). IVOS
results for % Prog Mot are shown in Figures 17.
Example 12 [001071 Bull semen was collected from a sexually mature bull using an artificial vagina and the sample diluted in 2 parts carbonate buffer for transportation at 25°C in a temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), ace ording to standard and well known procedures (Farrell et al. Theriogenology, 49(4):
871-9 (Mar 1998)). Based on the semen concentration, 1mL
of 150 X 106 sperm/ml suspension in TCA buffer was prepared by removing an aliquot of the carbonate sperm suspension, centrifuging the sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-suspending the pellet in 1mL TCA buffer. 1ml of 150 X 106 sperm/ml suspension in lOmM pyruvate in TCA was prepared by removing an aliquot of the carbonate sperm suspension, centrifuging the sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-suspending the pellet in 1mL of 10 mM
pyruvate TCA buffer. To samples was added SYBR 14 dye solution to yield the final dye concentrations of 20~M The suspensions were incubated in a 28°C water bath. Sperm suspensions were analyzed by removing a 50~.~,L aliquot from the staining sperm suspension, adding 200~L of the same buffer at the same temperature and analyzing by IVOS to measure percent progressive motility (% Prog Mot). IVOS
results for % Prog Mot are shown in Figures 18.
Example 13 [00108] Bull semen was collected from a sexually mature bull using an artificial vagina and the sample diluted in 2 parts carbonate buffer for transportation at 25°C in a 5 temperature-controlled container to the staining facility.
Upon receipt, the semen was analyzed for concentration, motility and progressive motility by the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (Farrell et al. Theriogenology, 49 (4) 10 871-9 (Mar 1998)). Based on the semen concentration, 1mL
of 150 X 106 sperm/ml suspension in TCA buffer was prepared by removing an aliquot of the carbonate sperm suspension, centrifuging the sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-suspending the pellet in 15 1mL TCA buffer. 1m1 of 150 X 106 sperm/ml suspension in lOmM pyruvate in TCA was prepared by removing an aliquot of the carbonate sperm suspension, centrifuging the sperm suspension at 500 X g for 5 minutes, removing the supernatant and re-suspending the pellet in lml of 10 mM
20 pyruvate TCA buffer. To t he samples was added BBC solution to yield the final dye concentrations of 100~M. The suspensions were incubated in a 28°C water bath. Stained sperm suspensions were analyzed by removing a 50~.L aliquot from the staining sperm suspension, adding 200~L of the 25 same buffer at the same temperature and analyzing by IVOS
to measure percent progres sine motility (% Prog Mot). IVOS
results for o Prog Mot are shown in Figures 19.
Example 14 (00109] Sperm samples were obtained and prepared in the same manner as in Examp 1e 4 with the following exception.
The staining concentrat ion was 200uM BBC. Results of the IVOS analysis are summarized in Figure 20.
Claims
WHAT IS CLAIMED:
1.~A sperm cell suspension comprising viable, immotile sperm, the concentration of spermatozoa in the suspension being less than about, 1 × 10 6 or at least about 1 × 10 8 spermatozoa per ml.
2.~A sperm cell suspension comprising viable spermatozoa, the spermatozoa having a motility more characteristic of epididymal spermatozoa than endogenous ejaculated spermatozoa of the same species, the concentration of spermatozoa in the suspension being less than about 1 × 10 6 or at least about 1 × 10 8 spermatozoa per ml.
3.~A sperm cell suspension comprising viable sperm, potassium, and sodium, the concentration of spermatozoa in the suspension being less than about 1 × 10 6 or at least about 1 × 10 8 spermatozoa per ml and the molar ratio of potassium to sodium being greater than 1:1, respectively.
4. The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 1.25:1, respectively.
5. The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 1.5:1, respectively.
6.~The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 1.75:1, respectively.
7.~The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 2:1, respectively.
8.~A sperm cell suspension comprising viable, immotile sperm and a DNA-selective dye.
9.~A sperm cell suspension comprising viable spermatozoa and a DNA-selective dye, the spermatozoa having a metabolic rate and motility more characteristic of epididymal spermatozoa than endogenous ejaculated spermatozoa of the same species.
10. A sperm cell suspension comprising viable, immotile spermatozoa, the spermatozoa having a DNA-selective dye associated with their DNA.
11. A sperm cell suspension comprising viable spermatozoa, the spermatozoa having a metabolic rate and motility more characteristic of epididymal spermatozoa than endogenous ejaculated spermatozoa of the same species, the spermatozoa also having a DNA-selective dye associated, with their DNA.
12.~The suspension of claim 8, wherein the dye is a DNA
selective fluorescent dye.
13.~The suspension of claim 8, wherein the dye is a UV
excitable or a visible light excitable dye.
14.~The suspension of claim 8, wherein the dye is selected from the group consisting of Hoechst 33342, Hoechst 33258, SYBR-14, and bisbenzimide-BODIPY~ conjugate 6-([3-((2Z)-2-([1-(difluoroboryl)-3, 5-dimethyl-1H-pyrrol-2-yl]methylene}-2H-pyrrol-5-yl)propanoyl]amino}-N-[3-(methyl(3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3'H-2,5'-bibenzimidazol-2'-yl]phenoxy}acetyl)amino]propyl}amino)propyl]hexanamide.
15. The sperm suspension of claim 1, wherein the suspension comprises a source of carbonate.
16.~The sperm suspension of claim 1, wherein the suspension comprises NaHCO3, KHCO3, and C6H8O7.H2O.
17.~The sperm suspension of claim 1, wherein the suspension is formed from a buffer comprising 0.097 moles/L
of NaHCO3, 0.173 moles/L of KHCO3, 0.090 moles/L C6H8O7.H2O in water.
18.~The sperm suspension of claim 1, wherein the concentration of spermatozoa in the suspension is at least 1.25 × 10 8 spermatozoa per ml 19.~The sperm suspension of claim 1, wherein the concentration of spermatozoa in the suspension is at least 1.5 × 10 8 spermatozoa per ml.
20. The sperm suspension of claim 1, wherein the concentration of spermatozoa in the suspension is at least 1.75 × 10 8 spermatozoa per ml.
21. The suspension of claim 1, wherein the concentration of spermatozoa in the suspension is less than about 9.0 × 10 5 spermatozoa per ml.
22.~The suspension of claim 1, wherein the concentration of spermatozoa in the suspension is less than about 7 × 10 5 epermatozoa per ml.
22.~The suspension of claim 1, wherein the concentration of spermatozoa in the suspension is less than about 5 × 10 5 spermatozoa per ml.
24. The suspension of claim 1, wherein the concentration of spermatozoa in the suspension is less than about 2 × 10 5 spermatozoa per ml.
25.~The suspension of claim 1, wherein the concentration of spermatozoa in the suspension is less than about 1 × 10 5 sgermatozoa per ml.
26. A process for staining sperm cells, the process comprising forming a staining mixture containing intact viable sperm cells, a motility inhibiting amount of potassium, and sodium and a DNA selective dye.
27. The process of claim 26 wherein the staining mixture is under an atmosphere having an enriched partial pressure of CO2 relative to air.
28.~A process of forming a sperm cell suspension for use in cell sorting, the process comprising combining a sperm cell source with a composition which inhibits the motility of sperm cells to form a sperm cell suspension, the concentration of sperm cells in the suspension being less than about 1 × 10 6 or at least 1 × 10 8 sperm cells per milliliter.
29.~The process of claim 28, wherein the cell suspension comprises a DNA-selective dye.
30. The suspension o~ claim 16, further comprising a DNA-selective dye.
1.~A sperm cell suspension comprising viable, immotile sperm, the concentration of spermatozoa in the suspension being less than about, 1 × 10 6 or at least about 1 × 10 8 spermatozoa per ml.
2.~A sperm cell suspension comprising viable spermatozoa, the spermatozoa having a motility more characteristic of epididymal spermatozoa than endogenous ejaculated spermatozoa of the same species, the concentration of spermatozoa in the suspension being less than about 1 × 10 6 or at least about 1 × 10 8 spermatozoa per ml.
3.~A sperm cell suspension comprising viable sperm, potassium, and sodium, the concentration of spermatozoa in the suspension being less than about 1 × 10 6 or at least about 1 × 10 8 spermatozoa per ml and the molar ratio of potassium to sodium being greater than 1:1, respectively.
4. The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 1.25:1, respectively.
5. The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 1.5:1, respectively.
6.~The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 1.75:1, respectively.
7.~The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 2:1, respectively.
8.~A sperm cell suspension comprising viable, immotile sperm and a DNA-selective dye.
9.~A sperm cell suspension comprising viable spermatozoa and a DNA-selective dye, the spermatozoa having a metabolic rate and motility more characteristic of epididymal spermatozoa than endogenous ejaculated spermatozoa of the same species.
10. A sperm cell suspension comprising viable, immotile spermatozoa, the spermatozoa having a DNA-selective dye associated with their DNA.
11. A sperm cell suspension comprising viable spermatozoa, the spermatozoa having a metabolic rate and motility more characteristic of epididymal spermatozoa than endogenous ejaculated spermatozoa of the same species, the spermatozoa also having a DNA-selective dye associated, with their DNA.
12.~The suspension of claim 8, wherein the dye is a DNA
selective fluorescent dye.
13.~The suspension of claim 8, wherein the dye is a UV
excitable or a visible light excitable dye.
14.~The suspension of claim 8, wherein the dye is selected from the group consisting of Hoechst 33342, Hoechst 33258, SYBR-14, and bisbenzimide-BODIPY~ conjugate 6-([3-((2Z)-2-([1-(difluoroboryl)-3, 5-dimethyl-1H-pyrrol-2-yl]methylene}-2H-pyrrol-5-yl)propanoyl]amino}-N-[3-(methyl(3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3'H-2,5'-bibenzimidazol-2'-yl]phenoxy}acetyl)amino]propyl}amino)propyl]hexanamide.
15. The sperm suspension of claim 1, wherein the suspension comprises a source of carbonate.
16.~The sperm suspension of claim 1, wherein the suspension comprises NaHCO3, KHCO3, and C6H8O7.H2O.
17.~The sperm suspension of claim 1, wherein the suspension is formed from a buffer comprising 0.097 moles/L
of NaHCO3, 0.173 moles/L of KHCO3, 0.090 moles/L C6H8O7.H2O in water.
18.~The sperm suspension of claim 1, wherein the concentration of spermatozoa in the suspension is at least 1.25 × 10 8 spermatozoa per ml 19.~The sperm suspension of claim 1, wherein the concentration of spermatozoa in the suspension is at least 1.5 × 10 8 spermatozoa per ml.
20. The sperm suspension of claim 1, wherein the concentration of spermatozoa in the suspension is at least 1.75 × 10 8 spermatozoa per ml.
21. The suspension of claim 1, wherein the concentration of spermatozoa in the suspension is less than about 9.0 × 10 5 spermatozoa per ml.
22.~The suspension of claim 1, wherein the concentration of spermatozoa in the suspension is less than about 7 × 10 5 epermatozoa per ml.
22.~The suspension of claim 1, wherein the concentration of spermatozoa in the suspension is less than about 5 × 10 5 spermatozoa per ml.
24. The suspension of claim 1, wherein the concentration of spermatozoa in the suspension is less than about 2 × 10 5 spermatozoa per ml.
25.~The suspension of claim 1, wherein the concentration of spermatozoa in the suspension is less than about 1 × 10 5 sgermatozoa per ml.
26. A process for staining sperm cells, the process comprising forming a staining mixture containing intact viable sperm cells, a motility inhibiting amount of potassium, and sodium and a DNA selective dye.
27. The process of claim 26 wherein the staining mixture is under an atmosphere having an enriched partial pressure of CO2 relative to air.
28.~A process of forming a sperm cell suspension for use in cell sorting, the process comprising combining a sperm cell source with a composition which inhibits the motility of sperm cells to form a sperm cell suspension, the concentration of sperm cells in the suspension being less than about 1 × 10 6 or at least 1 × 10 8 sperm cells per milliliter.
29.~The process of claim 28, wherein the cell suspension comprises a DNA-selective dye.
30. The suspension o~ claim 16, further comprising a DNA-selective dye.
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Families Citing this family (47)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2264428B1 (en) | 1997-01-31 | 2017-05-03 | Xy, Llc | Optical apparatus with focussing reflector for converging radiation onto a flow of particles |
US6149867A (en) | 1997-12-31 | 2000-11-21 | Xy, Inc. | Sheath fluids and collection systems for sex-specific cytometer sorting of sperm |
NZ527659A (en) | 1998-07-30 | 2006-02-24 | Colorado State University Thro | Equine artificial insemination when there has been sex selection of the sperm to produce an equine of the desired sex |
US7208265B1 (en) * | 1999-11-24 | 2007-04-24 | Xy, Inc. | Method of cryopreserving selected sperm cells |
AR034121A1 (en) | 2000-05-09 | 2004-02-04 | Xy Inc | METHOD FOR INSULATING Sperm Cells Containing X Chromosome from Sperm Cells Containing Chromosome and |
AU2002220018A1 (en) | 2000-11-29 | 2002-06-11 | Colorado State University | System for in-vitro fertilization with spermatozoa separated into x-chromosome and y-chromosome bearing populations |
US7713687B2 (en) | 2000-11-29 | 2010-05-11 | Xy, Inc. | System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations |
AU2003265362B2 (en) | 2002-08-01 | 2009-11-05 | Xy, Llc. | Low pressure sperm cell separation system |
US8486618B2 (en) | 2002-08-01 | 2013-07-16 | Xy, Llc | Heterogeneous inseminate system |
BRPI0313476B1 (en) | 2002-08-15 | 2015-06-23 | Xy Llc | High resolution flow cytometer |
US7169548B2 (en) | 2002-09-13 | 2007-01-30 | Xy, Inc. | Sperm cell processing and preservation systems |
DE602004024874D1 (en) | 2003-03-28 | 2010-02-11 | Inguran Llc | EASTER-ASSORTED TIERSPERMIES |
AU2004242121B2 (en) * | 2003-05-15 | 2010-06-24 | Xy, Llc. | Efficient haploid cell sorting for flow cytometer systems |
ES2397678T3 (en) | 2004-03-29 | 2013-03-08 | Inguran, Llc | Sperm suspensions for classification in enriched populations carrying the X or Y chromosome |
MX2007000888A (en) | 2004-07-22 | 2007-04-02 | Monsanto Technology Llc | Process for enriching a population of sperm cells. |
DE102005044530B4 (en) * | 2004-09-16 | 2010-02-18 | Masterrind Gmbh | Method for the sex-specific selection of mammalian spermatozoa |
US9433484B2 (en) * | 2007-07-27 | 2016-09-06 | Brad K. Stroud | Artificial breeding techniques for bovines including semen diluents and AI apparatus |
US8415094B2 (en) * | 2007-12-21 | 2013-04-09 | Jaffar Ali bin M. Abdullah | Protein-free gamete and embryo handling and culture media products |
CN101554152B (en) * | 2009-05-21 | 2012-05-09 | 西北农林科技大学 | Frozen semen diluent and preparation method thereof |
NZ600737A (en) * | 2009-11-24 | 2013-05-31 | Biassex Pty Ltd | Method for influencing sex selection in artificial insemination and apparatus for same |
US10610343B2 (en) | 2013-07-03 | 2020-04-07 | Brad K. Stroud | Method, apparatus and kit for artificial insemination of bovine |
AU2010359044B2 (en) | 2010-08-10 | 2015-04-30 | Brad K. Stroud | Method and apparatus to reduce the number of sperm used in artificial insemination of cattle |
US11622844B2 (en) | 2010-08-10 | 2023-04-11 | Maximate, Llc | Method, apparatus and kit for artificial insemination of bovine |
US9340762B2 (en) | 2010-08-20 | 2016-05-17 | Yu Sun | Method for automated sperm manipulation and device for holding sperm and oocytes |
US9347038B2 (en) | 2011-06-01 | 2016-05-24 | Inguran, Llc | Compositions and methods for processing sperm |
US9781919B2 (en) | 2011-06-01 | 2017-10-10 | Inguran, Llc | Compositions and methods for improving the quality of processed sperm |
EP3964285A1 (en) | 2011-09-26 | 2022-03-09 | Thermo Fisher Scientific Geneart GmbH | High efficiency, small volume nucleic acid synthesis |
WO2014153188A2 (en) | 2013-03-14 | 2014-09-25 | Life Technologies Corporation | High efficiency, small volume nucleic acid synthesis |
BR112014022817A8 (en) | 2012-03-14 | 2021-07-13 | Membrane Protective Tech Inc | process of increasing cryopreservation of biological cells and cryopreservation composition |
EP2834357B1 (en) | 2012-04-04 | 2017-12-27 | Life Technologies Corporation | Tal-effector assembly platform, customized services, kits and assays |
US9888990B2 (en) | 2012-06-06 | 2018-02-13 | Inguran, Llc | Methods for use of sex sorted semen to improve genetic management in swine |
US9433195B2 (en) | 2012-06-06 | 2016-09-06 | Inguran, Llc | Methods for increasing genetic progress in a line or breed of swine using sex-selected sperm cells |
US9034161B2 (en) | 2012-08-23 | 2015-05-19 | Douglas T. Carrell | Sperm separation devices and associated methods |
BR112015007480B1 (en) * | 2012-10-05 | 2020-04-22 | Inguran Llc | high efficiency sex based sperm separation methods. |
US10620213B2 (en) | 2012-10-05 | 2020-04-14 | Inguran, Llc | High pressure sperm sorting and flow cytometer methods |
EP3169781B1 (en) | 2014-07-15 | 2020-04-08 | Life Technologies Corporation | Compositions and methods for nucleic acid assembly |
EP3209766A4 (en) * | 2014-10-20 | 2018-08-15 | University of Utah Research Foundation | Tissue sample processing system and associated methods |
US11326146B2 (en) | 2014-11-20 | 2022-05-10 | Inguran, Llc | Low sugar sperm media and compositions |
CA3147259A1 (en) | 2014-12-09 | 2016-06-16 | Life Technologies Corporation | High efficiency, small volume nucleic acid synthesis |
WO2017007925A1 (en) | 2015-07-07 | 2017-01-12 | Thermo Fisher Scientific Geneart Gmbh | Enzymatic synthesis of nucleic acid sequences |
WO2017062343A1 (en) | 2015-10-06 | 2017-04-13 | Pierce Biotechnology, Inc. | Devices and methods for producing nucleic acids and proteins |
PT110231B (en) * | 2017-08-02 | 2021-02-22 | Universidade Do Porto | MTOR POTENTIATORS AND THEIR USES TO IMPROVE THE QUALITY AND FUNCTION OF SPERM DURING STORAGE |
EP3814494B1 (en) | 2018-06-29 | 2023-11-01 | Thermo Fisher Scientific GENEART GmbH | High throughput assembly of nucleic acid molecules |
US10603075B1 (en) | 2018-11-30 | 2020-03-31 | Ohana Biosciences, Inc. | Compositions and methods for enhancing sperm function |
GB201905303D0 (en) | 2019-04-15 | 2019-05-29 | Thermo Fisher Scient Geneart Gmbh | Multiplex assembly of nucleic acid molecules |
WO2021178809A1 (en) | 2020-03-06 | 2021-09-10 | Life Technologies Corporation | High sequence fidelity nucleic acid synthesis and assembly |
DE102021002257A1 (en) | 2021-04-29 | 2022-11-03 | Forschungsverbund Berlin E.V. | Composition for preserving mammalian sperm and use of the composition |
Family Cites Families (635)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3005756A (en) * | 1958-11-14 | 1961-10-24 | Noland L Van Demark | Diluter containing carbon dioxide for preserving semen |
US3005759A (en) * | 1959-04-24 | 1961-10-24 | American Zinc Inst | Zinc electroplating |
US3299354A (en) | 1962-07-05 | 1967-01-17 | Coulter Electronics | Aperture tube structure for particle study apparatus |
US3499435A (en) | 1967-06-02 | 1970-03-10 | Paul E Rockwell | Esophageal probe for use in monitoring |
US3547526A (en) | 1967-10-26 | 1970-12-15 | Kollsman Instr Corp | Optical beam cross-section converter |
US3810010A (en) | 1968-11-02 | 1974-05-07 | Telefunken Patent | Particle analysis method and apparatus wherein liquid containing particles is sucked into a constricted flow path |
US3829216A (en) | 1968-11-26 | 1974-08-13 | M Persidsky | Optical system and method for counting sperm cells |
DE1815352C3 (en) | 1968-12-18 | 1975-03-20 | Wolfgang Prof. Dr. Dittrich | Automatic measuring and counting device for the particles of a dispersion |
US3894529A (en) | 1969-04-10 | 1975-07-15 | Bio Controls Inc | Method and means for controlling the sex of mammalian offspring and product therefor |
US4327177A (en) | 1969-04-10 | 1982-04-27 | Wallace Shrimpton | Method and means for controlling the sex of mammalian offspring and product therefor |
US4474875A (en) * | 1969-04-10 | 1984-10-02 | Wallace Shrimpton | Method and means for controlling the sex of mammalian offspring and product therefor |
DE1919628C3 (en) | 1969-04-18 | 1975-04-10 | Wolfgang Prof. Dr. Dittrich | Arrangement for the automatic counting and / or classification of particles dispersed in a flowable medium |
US3687806A (en) | 1969-11-04 | 1972-08-29 | Bio Controls Inc | Method for controlling sex of mammalian offspring |
US3788744A (en) | 1970-01-14 | 1974-01-29 | Bio Physics Systems Inc | Method and apparatus for photoanalysis |
US3661460A (en) | 1970-08-28 | 1972-05-09 | Technicon Instr | Method and apparatus for optical analysis of the contents of a sheathed stream |
US3816249A (en) | 1970-11-23 | 1974-06-11 | B Bhattacharya | Universal medium and method for extending the useful life of semen in vitro |
US3644128A (en) | 1970-12-28 | 1972-02-22 | Stuart Lipner | Method of preparing comminuted meat products |
US3756459A (en) | 1971-01-12 | 1973-09-04 | Damon Corp | Method and apparatus for metering fluid utilizing pressure differentials |
US3791384A (en) * | 1971-07-15 | 1974-02-12 | Schaumann H | Artificial insemination of sows |
US3833796A (en) | 1971-10-13 | 1974-09-03 | Georgia Tech Res Inst | Method and apparatus for chromosome digitizing |
BE793185A (en) | 1971-12-23 | 1973-04-16 | Atomic Energy Commission | APPARATUS FOR QUICKLY ANALYZING AND SORTING PARTICLES SUCH AS BIOLOGICAL CELLS |
US3826364A (en) | 1972-05-22 | 1974-07-30 | Univ Leland Stanford Junior | Particle sorting method and apparatus |
US3761941A (en) | 1972-10-13 | 1973-09-25 | Mead Corp | Phase control for a drop generating and charging system |
CA1029833A (en) | 1973-02-23 | 1978-04-18 | Hildegarde Goehde | Apparatus for the automatic counting and measuring of suspended particles |
US3791517A (en) | 1973-03-05 | 1974-02-12 | Bio Physics Systems Inc | Digital fluidic amplifier particle sorter |
US4009260A (en) | 1973-04-19 | 1977-02-22 | Schering Aktiengesellschaft | Fractionation of sperm |
US3893766A (en) | 1973-06-14 | 1975-07-08 | Coulter Electronics | Apparatus for orienting generally flat particles for slit-scan photometry |
USRE29141E (en) | 1973-06-14 | 1977-02-22 | Coulter Electronics, Inc. | Apparatus for orienting generally flat particles for sensing |
US3947093A (en) | 1973-06-28 | 1976-03-30 | Canon Kabushiki Kaisha | Optical device for producing a minute light beam |
US3944917A (en) | 1973-08-13 | 1976-03-16 | Coulter Electronics, Inc. | Electrical sensing circuitry for particle analyzing device |
US3909744A (en) | 1973-09-24 | 1975-09-30 | United Technologies Corp | Unstable resonator system producing a high irradiance beam in the far field |
US3906929A (en) | 1973-11-23 | 1975-09-23 | Lynn Lawrence Augspurger | Processes for reproduction of cellular bodies |
FR2254639B1 (en) | 1973-12-18 | 1978-06-02 | Agronomique Inst Nat Rech | |
US4070617A (en) | 1974-05-08 | 1978-01-24 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | Device for controlling the particle flow in an apparatus for measuring the properties of particles suspended in liquid |
US3973196A (en) | 1974-05-24 | 1976-08-03 | Coulter Electronics, Inc. | Method and apparatus for ejecting a metered amount of particulate sample |
US3963606A (en) | 1974-06-03 | 1976-06-15 | Coulter Electronics, Inc. | Semi-automatic adjusting delay for an electronic particle separator |
US3877430A (en) | 1974-07-17 | 1975-04-15 | Horst K Wieder | Artificial insemination apparatus |
US4083957A (en) | 1974-07-26 | 1978-04-11 | Lang John L | Process for the alteration of the sex-ratio of mammals |
US4006360A (en) | 1974-08-21 | 1977-02-01 | Block Engineering, Inc. | Method of discriminating between dyed particles and background fluorescence of the dye |
US3976197A (en) | 1974-11-22 | 1976-08-24 | Bhattacharya Bhairab C | Thermal convection counter streaming sedimentation method and apparatus for controlling the sex of mammalian offspring |
US4092229A (en) | 1975-12-17 | 1978-05-30 | Bhattacharya Bhairab C | Thermal convection counter streaming sedimentation and forced convection galvanization method for controlling the sex of mammalian offspring |
US4014611A (en) | 1975-04-30 | 1977-03-29 | Coulter Electronics, Inc. | Aperture module for use in particle testing apparatus |
DE2521236C3 (en) | 1975-05-10 | 1978-12-14 | Hildegard Dr. 4400 Muenster Goehde Geb. Kuhl | Device for counting and measuring particles suspended in a liquid |
US3960449A (en) | 1975-06-05 | 1976-06-01 | The Board Of Trustees Of Leland Stanford Junior University | Measurement of angular dependence of scattered light in a flowing stream |
US4058732A (en) | 1975-06-30 | 1977-11-15 | Analytical Radiation Corporation | Method and apparatus for improved analytical fluorescent spectroscopy |
US4007087A (en) | 1975-10-17 | 1977-02-08 | Gametrics Limited | Sperm fractionation and storage |
AU2154077A (en) | 1976-01-27 | 1978-07-27 | Univ Edinburgh | Control of sex ratio in mammalian offspring |
US4162282A (en) | 1976-04-22 | 1979-07-24 | Coulter Electronics, Inc. | Method for producing uniform particles |
US4302166A (en) | 1976-04-22 | 1981-11-24 | Coulter Electronics, Inc. | Droplet forming apparatus for use in producing uniform particles |
GB1563856A (en) | 1976-06-10 | 1980-04-02 | Coulter Electronics | Methods and apparatus for delectively separating small particles suspended in a liquid |
GB1583150A (en) | 1976-08-02 | 1981-01-21 | Milk Marketing Board | Apparatus for collecting eggs |
US4110604A (en) | 1976-11-04 | 1978-08-29 | Becton, Dickinson And Company | Particle density measuring system |
DE2709399C3 (en) | 1977-03-04 | 1980-07-24 | Goehde, Wolfgang, Dr., 4400 Muenster | Device for measuring cell properties |
DE2716095A1 (en) | 1977-04-12 | 1978-10-19 | Zoeld Tibor Dr Phys | GAS CONTROLLED PROCESS FOR SORTING PARTICLES SUSPENDED IN AN ELECTROLYTE AND DEVICE FOR CARRYING OUT THE PROCESS |
US4191749A (en) | 1977-10-11 | 1980-03-04 | Bryant Bernard J | Method and material for increasing the percentage of mammalian offspring of either sex |
US4448767A (en) | 1977-10-11 | 1984-05-15 | Sumar Corporation | Preparation of monospecific male-specific antibody and the use thereof for increasing the percentage of mammalian offspring of either sex |
US4189236A (en) | 1978-03-20 | 1980-02-19 | Coulter Electronics, Inc. | Ellipsoid-conic radiation collector and method |
US4179218A (en) | 1978-05-15 | 1979-12-18 | The Boeing Company | Particle size analyzer |
US4251733A (en) * | 1978-06-29 | 1981-02-17 | Hirleman Jr Edwin D | Technique for simultaneous particle size and velocity measurement |
DE2832091A1 (en) | 1978-07-21 | 1980-01-31 | Eidenschink Henning | OPTICAL METHOD FOR DETERMINING THE PARTICLE SIZE OF COLLOIDAL SOLUTIONS AND MEASURING DEVICE FOR IMPLEMENTING THE METHOD |
US4276139A (en) | 1978-08-16 | 1981-06-30 | Lawson Rommon L | Process for magnetic separation and collection of viable female and male spermatozoa |
US4225405A (en) | 1978-08-16 | 1980-09-30 | Lawson Rommom L | Process for separation and collection of viable female and male spermatozoa |
US4230558A (en) | 1978-10-02 | 1980-10-28 | Coulter Electronics, Inc. | Single drop separator |
US4341471A (en) | 1979-01-02 | 1982-07-27 | Coulter Electronics, Inc. | Apparatus and method for measuring the distribution of radiant energy produced in particle investigating systems |
US4267268A (en) * | 1979-03-12 | 1981-05-12 | Nelson Jr Robert A | Spermatozoa extenders |
US4274408A (en) | 1979-03-26 | 1981-06-23 | Beatrice Nimrod | Method for guide-wire placement and novel syringe therefor |
US4200802A (en) | 1979-03-28 | 1980-04-29 | The United States Of America As Represented By The United States Department Of Energy | Parabolic cell analyzer |
US4408877A (en) | 1979-04-10 | 1983-10-11 | Ernst Leitz Wetzlar Gmbh | Device for hydrodynamic focussing of a particle-suspension in a liquid flow cytophotometer |
NO144002C (en) | 1979-04-10 | 1981-05-27 | Norsk Hydro S Inst For Kreftfo | DEVICE FOR USE IN LIQUID PHOTOMETRY |
US4263508A (en) | 1979-04-20 | 1981-04-21 | Research Corporation | Pulse edge measurement for determining particle dimensional characteristics |
US4255021A (en) | 1979-04-20 | 1981-03-10 | The United States Of America As Represented By The United States Department Of Energy | Optical device with conical input and output prism faces |
US4318480A (en) | 1979-08-20 | 1982-03-09 | Ortho Diagnostics, Inc. | Method and apparatus for positioning the point of droplet formation in the jetting fluid of an electrostatic sorting device |
US4318481A (en) | 1979-08-20 | 1982-03-09 | Ortho Diagnostics, Inc. | Method for automatically setting the correct phase of the charge pulses in an electrostatic flow sorter |
US4325483A (en) | 1979-08-20 | 1982-04-20 | Ortho Diagnostics, Inc. | Method for detecting and controlling flow rates of the droplet forming stream of an electrostatic particle sorting apparatus |
US4317520A (en) | 1979-08-20 | 1982-03-02 | Ortho Diagnostics, Inc. | Servo system to control the spatial position of droplet formation of a fluid jet in a cell sorting apparatus |
US4318482A (en) | 1979-08-20 | 1982-03-09 | Ortho Diagnostics, Inc. | Method for measuring the velocity of a perturbed jetting fluid in an electrostatic particle sorting system |
DE2943116C2 (en) | 1979-10-25 | 1986-06-19 | Gesellschaft für Strahlen- und Umweltforschung mbH, 8000 München | Device for flow cytometric reaction and / or diffusion measurement |
US4284355A (en) | 1979-10-29 | 1981-08-18 | Ortho Diagnostics, Inc. | Automated method for cell volume determination |
US4400764A (en) | 1980-02-05 | 1983-08-23 | The Boeing Company | Low backscatter illumination system |
US4367043A (en) | 1980-05-05 | 1983-01-04 | Leland Stanford Junior University | Method and means for delivering liquid samples to a sample scanning device |
US4362246A (en) | 1980-07-14 | 1982-12-07 | Adair Edwin Lloyd | Method of treating collected mammal semen and separating sperm into X Y components |
US4348107A (en) | 1980-07-18 | 1982-09-07 | Coulter Electronics, Inc. | Orifice inside optical element |
EP0046345A3 (en) | 1980-08-15 | 1982-03-03 | Ortho Diagnostic Systems Inc. | Controlled hydrodynamic flow in flow cytometry systems |
US4350410A (en) | 1980-10-08 | 1982-09-21 | The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration | Multiprism collimator |
US4487320A (en) | 1980-11-03 | 1984-12-11 | Coulter Corporation | Method of and apparatus for detecting change in the breakoff point in a droplet generation system |
US4691829A (en) | 1980-11-03 | 1987-09-08 | Coulter Corporation | Method of and apparatus for detecting change in the breakoff point in a droplet generation system |
US4395676A (en) | 1980-11-24 | 1983-07-26 | Coulter Electronics, Inc. | Focused aperture module |
US4361400A (en) | 1980-11-26 | 1982-11-30 | The United States Of America As Represented By The United States Department Of Energy | Fluidic assembly for an ultra-high-speed chromosome flow sorter |
US4680258A (en) | 1981-03-24 | 1987-07-14 | Sloan-Kettering Institute For Cancer Research | Process for sex determination in man by use of monoclonal antibodies to the H-Y antigen |
US4818103A (en) | 1981-05-15 | 1989-04-04 | Ratcom | Flow cytometry |
US4673288A (en) | 1981-05-15 | 1987-06-16 | Ratcom, Inc. | Flow cytometry |
DE3266669D1 (en) | 1981-06-24 | 1985-11-07 | Becton Dickinson Co | Analyzer for simultaneously determining volume and light emission characteristics of particles |
US4339434A (en) | 1981-08-17 | 1982-07-13 | Gametrics Limited | Method of increasing the incidence of female offspring |
US4395397A (en) | 1981-09-17 | 1983-07-26 | Sidney Farber Cancer Institute, Inc. | Apparatus and method for killing unwanted cells |
US4422761A (en) | 1981-09-28 | 1983-12-27 | Frommer Joseph C | Photo-electric particle sensing system |
US4515274A (en) | 1981-12-02 | 1985-05-07 | Coulter Corporation | Particle analyzing and sorting apparatus |
JPS59500340A (en) | 1982-03-08 | 1984-03-01 | モトロ−ラ・インコ−ポレ−テツド | integrated circuit lead frame |
US4511661A (en) | 1982-03-19 | 1985-04-16 | University Patents, Inc. | ATCC HB8116 And its monoclonal anti-H-Y antibody, Hyclonalan |
US4498766A (en) | 1982-03-25 | 1985-02-12 | Becton, Dickinson And Company | Light beam focal spot elongation in flow cytometry devices |
DE3315195A1 (en) | 1982-04-29 | 1983-11-03 | International Remote Imaging Systems Inc., 91311 Chatsworth, Calif. | METHOD FOR ALIGNING PARTICLES IN A FLUID SAMPLE |
DE3315194A1 (en) | 1982-04-29 | 1983-11-03 | International Remote Imaging Systems Inc., 91311 Chatsworth, Calif. | METHOD FOR SEPARATING PARTICLES FLOWING IN A FLUID SAMPLE |
US4629687A (en) | 1982-07-29 | 1986-12-16 | Board Of Trustees Of Michigan State University | Positive selection sorting of cells |
GB2125181B (en) | 1982-08-11 | 1986-01-29 | Coulter Electronics | Flow cells for particle study |
US4559309A (en) | 1982-09-01 | 1985-12-17 | Memorial Sloan Kettering Cancer Center | Flow cytometry-fluorescence measurements for characterizing sperm |
WO1984001265A1 (en) | 1982-10-05 | 1984-04-12 | Genetic Engineering Inc | Method of treating collected mammal semen and separating sperm into x and y components |
US4501366A (en) | 1982-12-14 | 1985-02-26 | Adolph Coors Company | Photomultiplier tube assembly |
DE3372137D1 (en) | 1982-12-21 | 1987-07-23 | Crosfield Electronics Ltd | Light beam-splitter |
US4492436A (en) | 1983-01-03 | 1985-01-08 | At&T Bell Laboratories | Polarization independent beam splitter |
JPS59143146A (en) | 1983-02-07 | 1984-08-16 | Nippon Kogaku Kk <Nikon> | Mirror condenser type illuminating optical system |
CA1206559A (en) | 1983-03-04 | 1986-06-24 | Robert E. Auer | Method of and apparatus for detecting change in the breakoff point of a droplet generation system |
IT1197570B (en) | 1983-02-11 | 1988-12-06 | Serono Ist Farm | MIXTURES OF FSH AND LH FROM PORCINE HYPOPHYSIS IN DEFINED RATIO |
CH651930A5 (en) | 1983-03-24 | 1985-10-15 | Coulter Corp | Apparatus for analysis and sorting of particles |
JPS59174742A (en) | 1983-03-25 | 1984-10-03 | Agency Of Ind Science & Technol | Method and apparatus for dividing and sorting fine particle |
GB2145112B (en) | 1983-04-27 | 1987-02-18 | Milk Marketing Board | Sorting living spermatozoa |
US4523809A (en) | 1983-08-04 | 1985-06-18 | The United States Of America As Represented By The Secretary Of The Air Force | Method and apparatus for generating a structured light beam array |
NZ207393A (en) | 1983-08-05 | 1987-03-31 | Neal Lloyd First | Staining dna in living cells |
US4538733A (en) | 1983-10-14 | 1985-09-03 | Becton, Dickinson And Company | Particle sorter with neutralized collection wells and method of using same |
US4780406A (en) | 1983-10-18 | 1988-10-25 | The Regents Of The University Of California | Flow cytometric measurement of total DNA and incorporated halodeoxyuridine |
US4585736A (en) | 1983-10-18 | 1986-04-29 | The United States Of America As Represented By The United States Department Of Energy | Flow cytometric measurement of total DNA and incorporated halodeoxyuridine |
US4735504A (en) | 1983-10-31 | 1988-04-05 | Technicon Instruments Corporation | Method and apparatus for determining the volume & index of refraction of particles |
ATE58017T1 (en) | 1983-12-24 | 1990-11-15 | Inotech Ag | DEVICE FOR GUIDING AND COLLECTING LIGHT IN PHOTOMETRY OD. DGL. |
US4573796A (en) | 1984-01-06 | 1986-03-04 | The United States Of America As Represented By The United States Department Of Energy | Apparatus for eliminating background interference in fluorescence measurements |
US4631483A (en) | 1984-02-01 | 1986-12-23 | Coulter Electronics, Inc. | Particle analyzing apparatus and method of moving particles in suspension through such apparatus |
US4965204A (en) | 1984-02-06 | 1990-10-23 | The Johns Hopkins University | Human stem cells and monoclonal antibodies |
US4714680B1 (en) | 1984-02-06 | 1995-06-27 | Univ Johns Hopkins | Human stem cells |
EP0172242A1 (en) | 1984-02-29 | 1986-02-26 | Research Corporation | Flow cytometers |
US4545677A (en) | 1984-03-05 | 1985-10-08 | Becton, Dickinson And Company | Prismatic beam expander for light beam shaping in a flow cytometry apparatus |
US4600302A (en) | 1984-03-26 | 1986-07-15 | Becton, Dickinson And Company | Flow cytometry apparatus with uniform incoherent light excitation |
DE3412620A1 (en) | 1984-04-04 | 1985-10-17 | Basf Ag, 6700 Ludwigshafen | LASER OPTICAL ARRANGEMENT FOR MEASURING THE DEGREE OF DISPERSION IN FLOWING SYSTEMS |
US4609286A (en) | 1984-04-16 | 1986-09-02 | Becton, Dickinson And Company | Dispersion prism for separation of wavelengths of spectrally rich light in a flow cytometry apparatus |
US4605558A (en) | 1984-04-20 | 1986-08-12 | Wallace Shrimpton | Process for cell separation |
US4660971A (en) | 1984-05-03 | 1987-04-28 | Becton, Dickinson And Company | Optical features of flow cytometry apparatus |
FR2563726B1 (en) | 1984-05-04 | 1986-10-10 | Robert Cassou | ARTIFICIAL INSEMINATION APPARATUS, PARTICULARLY CARNIVORES |
FR2566543B1 (en) | 1984-06-20 | 1988-02-26 | Commissariat Energie Atomique | HIGH COLLECTION AND CYTOFLUORIMETER OPTICAL DEVICE USING THE SAME |
EP0171676B1 (en) | 1984-07-31 | 1990-11-07 | Hitachi, Ltd. | Free-flow electrophoretic separation method and apparatus therefor |
ATE48477T1 (en) | 1984-09-11 | 1989-12-15 | Partec Ag | METHOD AND DEVICE FOR SORTING MICROSCOPIC PARTICLES. |
US4661913A (en) | 1984-09-11 | 1987-04-28 | Becton, Dickinson And Company | Apparatus and method for the detection and classification of articles using flow cytometry techniques |
US4598408A (en) | 1984-10-22 | 1986-07-01 | Trw Inc. | High extraction efficiency cylindrical ring resonator |
JPS61139747A (en) | 1984-12-12 | 1986-06-27 | Canon Inc | Particle analyser |
JPS61159135A (en) | 1984-12-31 | 1986-07-18 | Canon Inc | Particle analyzing device |
US4702598A (en) | 1985-02-25 | 1987-10-27 | Research Corporation | Flow cytometer |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
US4683195A (en) | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
CA1250808A (en) | 1985-04-29 | 1989-03-07 | David W. Dresser | Semen sexing |
US4662742A (en) | 1985-05-10 | 1987-05-05 | Becton, Dickinson And Company | Scatter/fluorescene beam splitter in a flow cytometry apparatus |
USRE34782E (en) | 1985-07-01 | 1994-11-08 | Diatron Corporation | Fluorometer |
US4877965A (en) | 1985-07-01 | 1989-10-31 | Diatron Corporation | Fluorometer |
US4744090A (en) | 1985-07-08 | 1988-05-10 | Trw Inc. | High-extraction efficiency annular resonator |
NO156916C (en) | 1985-07-10 | 1987-12-16 | Harald B Steen | FLOW CLEANING FOR FLUID FLOW PHOTOMETER. |
NO156917C (en) | 1985-07-16 | 1987-12-16 | Harald B Steen | DEVICE FOR MEASUREMENT OF BIOLOGICAL CELLS LIGHT DISTRIBUTION IN LIQUID CURRENT PHOTOMETERS. |
US4989977A (en) | 1985-07-29 | 1991-02-05 | Becton, Dickinson And Company | Flow cytometry apparatus with improved light beam adjustment |
US4794086A (en) | 1985-11-25 | 1988-12-27 | Liquid Air Corporation | Method for measurement of impurities in liquids |
US4770992A (en) | 1985-11-27 | 1988-09-13 | Den Engh Gerrit J Van | Detection of specific DNA sequences by flow cytometry |
US4999283A (en) | 1986-01-10 | 1991-03-12 | University Of Kentucky Research Foundation | Method for x and y spermatozoa separation |
US5447841A (en) | 1986-01-16 | 1995-09-05 | The Regents Of The Univ. Of California | Methods for chromosome-specific staining |
US5756696A (en) | 1986-01-16 | 1998-05-26 | Regents Of The University Of California | Compositions for chromosome-specific staining |
US4710635A (en) | 1986-04-14 | 1987-12-01 | Becton, Dickinson And Company | Dual laser excitation from single laser source |
NL8601000A (en) | 1986-04-21 | 1987-11-16 | Jan Greve T H Twente Afdeling | THE USE OF POLARIZED LIGHT IN FLOW CYTOMETRY. |
JPS62274238A (en) | 1986-05-22 | 1987-11-28 | ベクトン・デイツキンソン・アンド・カンパニ− | Gel for optical bonding used for flow sight metry device |
US4790653A (en) | 1986-05-22 | 1988-12-13 | Becton Dickinson And Company | Housing for a flow cytometry apparatus with particle unclogging feature |
US4786165A (en) | 1986-07-10 | 1988-11-22 | Toa Medical Electronics Co., Ltd. | Flow cytometry and apparatus therefor |
US4704891A (en) | 1986-08-29 | 1987-11-10 | Becton, Dickinson And Company | Method and materials for calibrating flow cytometers and other analysis instruments |
US4867908A (en) | 1986-08-29 | 1989-09-19 | Becton, Dickinson And Company | Method and materials for calibrating flow cytometers and other analysis instruments |
FR2609885B1 (en) | 1987-01-22 | 1989-04-14 | Cassou Robert | INSTRUMENT FOR ARTIFICIAL INSEMINATION, TRANSFER OF EMBRYOS OR COLLECTION OF FOLLICULAR LIQUIDS FROM MAMMALS |
US5162306A (en) | 1987-01-23 | 1992-11-10 | Donaldson Lloyd E | Composition and method for producing superovulation in mammals |
US4780451B1 (en) | 1987-01-23 | 1995-04-04 | Asua International Inc | Composition and method for producing superovulation in cattle |
ATE91789T1 (en) | 1987-02-17 | 1993-08-15 | Ratcom Inc | FLOW CYTOMETRY. |
US4764013A (en) | 1987-03-23 | 1988-08-16 | The United States Of America As Represented By The United States Department Of Energy | Interferometric apparatus and method for detection and characterization of particles using light scattered therefrom |
US4765737A (en) | 1987-03-30 | 1988-08-23 | Cornell Research Foundation | Cell size measurements using light in flow cytometry and cell sorting |
US5021244A (en) | 1988-12-06 | 1991-06-04 | Cytogam, Inc. | Sex-associated membrane antibodies and their use for increasing the probability that offspring will be of a desired sex |
US4983038A (en) | 1987-04-08 | 1991-01-08 | Hitachi, Ltd. | Sheath flow type flow-cell device |
US5346990A (en) | 1987-04-08 | 1994-09-13 | Cytogam, Inc. | Sex-associated membrane proteins and methods for increasing the probability that offspring will be of a desired sex |
JPS63262565A (en) | 1987-04-20 | 1988-10-28 | Hitachi Ltd | Flow cell |
DE3851176T3 (en) | 1987-04-27 | 1999-02-11 | Laser Sensor Technology Inc | Device and method for examining particles. |
EP0289677A3 (en) | 1987-04-27 | 1989-05-10 | Fritz K. Preikschat | Apparatus and method for particle analysis |
FR2614626B1 (en) | 1987-04-30 | 1989-07-21 | Ranoux Claude | CONTAINER FOR FERTILIZATION OF OVOCYTES AND REPLACEMENT OF EMBRYOS IN HUMANS AND ANIMALS |
JP2642632B2 (en) | 1987-07-03 | 1997-08-20 | 株式会社日立製作所 | Particle measuring device and particle measuring method |
GB8716285D0 (en) | 1987-07-10 | 1987-08-19 | Medical Res Council | Light collecting device |
US4979093A (en) | 1987-07-16 | 1990-12-18 | Cavro Scientific Instruments | XYZ positioner |
US4987539A (en) | 1987-08-05 | 1991-01-22 | Stanford University | Apparatus and method for multidimensional characterization of objects in real time |
US4796788A (en) | 1987-08-26 | 1989-01-10 | Liqui-Box Corporation | Bag-in-box packaging and dispensing of substances which will not readily flow by gravity |
US4758729A (en) | 1987-08-28 | 1988-07-19 | Spectra-Physics, Inc. | Apparatus and method for measuring the included angle of a reflective cone |
DE3832901A1 (en) | 1987-10-02 | 1989-04-20 | Hitachi Ltd | PARTICLE MEASURING DEVICE |
US4793705A (en) | 1987-10-07 | 1988-12-27 | The United States Of America As Represented By The United States Department Of Energy | Single molecule tracking |
US4831385A (en) | 1987-10-14 | 1989-05-16 | Burlington Industries, Inc. | Vacuum tray fluid-jet start-up system |
US4980277A (en) | 1987-10-16 | 1990-12-25 | Cultor Ltd. | Cryoprotectant solution and method |
US5712807A (en) | 1987-10-21 | 1998-01-27 | Bangham; James Andrew | Pulse analyzing method and apparatus |
US5789155A (en) * | 1987-10-30 | 1998-08-04 | California Institute Of Technology | Process for identifying nucleic acids and triple helices formed thereby |
US4845025A (en) | 1987-11-10 | 1989-07-04 | Coulter Corporation | Biological sample mixing apparatus and method |
EP0386061A1 (en) | 1987-11-10 | 1990-09-12 | Secretary Of State For Defence In Her Britannic Majesty's Gov. Of The United Kingdom Of Great Britain And Northern Ireland | Particle monitoring system |
GB8726305D0 (en) | 1987-11-10 | 1987-12-16 | Secr Defence | Portable particle analysers |
GB8726304D0 (en) | 1987-11-10 | 1987-12-16 | Secr Defence | Particle asymmetry analyser |
US5040890A (en) | 1987-11-25 | 1991-08-20 | Becton, Dickinson And Company | Sheathed particle flow controlled by differential pressure |
US4887721A (en) | 1987-11-30 | 1989-12-19 | The United States Of America As Represented By The United States Department Of Energy | Laser particle sorter |
US4988619A (en) | 1987-11-30 | 1991-01-29 | United States Department Of Energy | Flow cytometry apparatus |
US4936465A (en) | 1987-12-07 | 1990-06-26 | Zoeld Tibor | Method and apparatus for fast, reliable, and environmentally safe dispensing of fluids, gases and individual particles of a suspension through pressure control at well defined parts of a closed flow-through system |
US5219729A (en) | 1988-02-25 | 1993-06-15 | Serono Laboratories, Inc. | Fertility assay |
US5622820A (en) | 1988-03-10 | 1997-04-22 | City Of Hope | Method for amplification and detection of RNA and DNA sequences |
US4836038A (en) | 1988-03-18 | 1989-06-06 | Aim Instruments Ltd. | Automated sampler-injector apparatus and method for sampling a quantity of sample and testing portions of said quantity |
US5057413A (en) | 1988-06-13 | 1991-10-15 | Becton, Dickinson And Company | Method for discriminating between intact and damaged cells in a sample |
US5070080A (en) | 1988-08-10 | 1991-12-03 | Fahim Mostafa S | Method of inhibiting generation, maturation, motility and viability of sperm with minerals in bioavailable form |
JPH0718785B2 (en) | 1988-09-19 | 1995-03-06 | 株式会社日立製作所 | Flow cell device |
JP2635125B2 (en) | 1988-09-30 | 1997-07-30 | 東亜医用電子株式会社 | Particle analysis apparatus and method for determining nuclear leaf index |
JP2635126B2 (en) | 1988-09-30 | 1997-07-30 | 東亜医用電子株式会社 | Particle analysis apparatus and method for determining nuclear leaf index |
JPH02168160A (en) | 1988-12-22 | 1990-06-28 | Omron Tateisi Electron Co | Cell selecting apparatus |
US5726009A (en) | 1989-03-20 | 1998-03-10 | Anticancer, Inc. | Native-state method and system for determining viability and proliferative capacity of tissues in vitro |
JPH02289808A (en) | 1989-04-28 | 1990-11-29 | Olympus Optical Co Ltd | Lighting optical system |
US4981580A (en) | 1989-05-01 | 1991-01-01 | Coulter Corporation | Coincidence arbitration in a flow cytomery sorting system |
ES2091823T3 (en) * | 1989-05-10 | 1996-11-16 | Us Agriculture | METHOD TO PRESELECT THE SEX OF THE PROGENIE. |
US4942305A (en) | 1989-05-12 | 1990-07-17 | Pacific Scientific Company | Integrating sphere aerosol particle detector |
DE68924275T2 (en) | 1989-05-12 | 1996-05-02 | Cytogam Inc | MEMBRANE PROTEINS ASSOCIATED WITH GENDER AND METHODS FOR INCREASING THE LIKELIHOOD THAT THE DEScendants HAVE THE DESIRED GENDER. |
US5055393A (en) | 1989-06-13 | 1991-10-08 | Salk Institute Biotechnology/Industrial Associates, Inc. | Prenatal sex determination of bovine cells using male-specific oligonucleotides |
US4954715A (en) | 1989-06-26 | 1990-09-04 | Zoeld Tibor | Method and apparatus for an optimized multiparameter flow-through particle and cell analyzer |
JPH0353164A (en) | 1989-07-20 | 1991-03-07 | Canon Inc | Sample supply device and sample measuring instrument using the same |
US5098657A (en) | 1989-08-07 | 1992-03-24 | Tsi Incorporated | Apparatus for measuring impurity concentrations in a liquid |
US5030002A (en) | 1989-08-11 | 1991-07-09 | Becton, Dickinson And Company | Method and apparatus for sorting particles with a moving catcher tube |
US5215376A (en) | 1989-09-08 | 1993-06-01 | Becton, Dickinson And Company | Method for causing vortices in a test tube |
US5005981A (en) | 1989-09-08 | 1991-04-09 | Becton, Dickinson And Company | Apparatus for method for causing vortices in a test tube |
US5167926A (en) | 1989-09-13 | 1992-12-01 | Kabushiki Kaisha Tiyoda Seisakusho | Apparatus for pretreating cells for flow cytometry |
JP2808321B2 (en) | 1989-09-19 | 1998-10-08 | 東亜医用電子株式会社 | Cell analysis method and device |
US5072382A (en) | 1989-10-02 | 1991-12-10 | Kamentsky Louis A | Methods and apparatus for measuring multiple optical properties of biological specimens |
US5275787A (en) | 1989-10-04 | 1994-01-04 | Canon Kabushiki Kaisha | Apparatus for separating or measuring particles to be examined in a sample fluid |
EP0422616B1 (en) | 1989-10-11 | 1996-02-07 | Canon Kabushiki Kaisha | Apparatus for and method of fractionating particle in particle-suspended liquid in conformity with the properties thereof |
JPH03140840A (en) | 1989-10-26 | 1991-06-14 | Hitachi Ltd | Flow cytoanalyser |
FR2653885B1 (en) | 1989-10-27 | 1994-01-14 | Abx | APPARATUS FOR COUNTING AND DETERMINING AT LEAST ONE LEUKOCYTAIC SUB-POPULATION. |
US5034613A (en) | 1989-11-14 | 1991-07-23 | Cornell Research Foundation, Inc. | Two-photon laser microscopy |
US5101978A (en) | 1989-11-27 | 1992-04-07 | The United States Of America As Represented By The Secretary Of The Army | Fluidic sorting device for two or more materials suspended in a fluid |
AU647741B2 (en) | 1989-12-01 | 1994-03-31 | Regents Of The University Of California, The | Methods and compositions for chromosome-specific staining |
US5274240A (en) | 1990-01-12 | 1993-12-28 | The Regents Of The University Of California | Capillary array confocal fluorescence scanner and method |
DE69118429T2 (en) | 1990-01-26 | 1996-09-12 | Canon Kk | Method of measuring a species using fluorescent light |
JP3049254B2 (en) | 1990-02-08 | 2000-06-05 | シスメックス株式会社 | Optical particle analyzer with two types of light sources |
IL93634A0 (en) | 1990-03-05 | 1990-12-23 | Galai Lab Ltd | Particle size analyzer |
US5153117A (en) | 1990-03-27 | 1992-10-06 | Genetype A.G. | Fetal cell recovery method |
US5492534A (en) | 1990-04-02 | 1996-02-20 | Pharmetrix Corporation | Controlled release portable pump |
US5150313A (en) | 1990-04-12 | 1992-09-22 | Regents Of The University Of California | Parallel pulse processing and data acquisition for high speed, low error flow cytometry |
US5087295A (en) | 1990-06-13 | 1992-02-11 | Becton Dickinson And Company | Cleaning cycle for flow cytometers |
US5076472A (en) | 1990-06-13 | 1991-12-31 | Becton, Dickinson And Company | Cleaning cycle for flow cytometers |
JPH0710226B2 (en) | 1990-06-20 | 1995-02-08 | 株式会社ピーシーシーテクノロジー | Protoplast stirring device, cell fusion device and flow cytometer device including the same |
JPH0716392B2 (en) | 1990-06-20 | 1995-03-01 | 株式会社ピーシーシーテクノロジー | Cell fusion and fused cell sorting system |
JPH04126065A (en) | 1990-06-25 | 1992-04-27 | Saburo Okonogi | Device for molding food raw material into bar-like shape |
US5160974A (en) | 1990-06-25 | 1992-11-03 | Flow Science, Inc. | Closed sample cell for use in flow cytometry |
US5559032A (en) | 1990-06-29 | 1996-09-24 | Pomeroy; Patrick C. | Method and apparatus for post-transfer assaying of material on solid support |
WO1992001040A1 (en) | 1990-07-09 | 1992-01-23 | Amrad Corporation Limited | Enhanced implantation, development and maintenance of embryos using leukaemia inhibitory factor |
JP2939647B2 (en) | 1990-07-24 | 1999-08-25 | シスメックス株式会社 | Automatic focus adjustment method for flow imaging cytometer |
IE76732B1 (en) | 1990-08-07 | 1997-11-05 | Becton Dickinson Co | One step test for absolute counts |
US5259593A (en) | 1990-08-30 | 1993-11-09 | University Of Southern California | Apparatus for droplet stream manufacturing |
JPH04115136A (en) | 1990-09-05 | 1992-04-16 | Hitachi Ltd | Particle measuring apparatus |
US5132548A (en) | 1990-09-14 | 1992-07-21 | High Yield Technology | High sensitivity, large detection area particle sensor for vacuum applications |
JPH04126064A (en) | 1990-09-14 | 1992-04-27 | Nitto Shokai:Kk | Method for preparing doubly covered bean jam food |
JP2957246B2 (en) | 1990-09-14 | 1999-10-04 | 大和化成株式会社 | Microbial carboxypeptidase B-like enzyme |
JPH04126080A (en) | 1990-09-14 | 1992-04-27 | Juzo Udaka | Production of heat-resistant xylose isomerase |
US5204884A (en) | 1991-03-18 | 1993-04-20 | University Of Rochester | System for high-speed measurement and sorting of particles |
US5273527A (en) | 1992-05-12 | 1993-12-28 | Ovamed Corporation | Delivery catheter |
US5663048A (en) | 1990-10-04 | 1997-09-02 | University Of Calgary | Y-chromosome specific polynucleotide probes for prenatal sexing |
US5840482A (en) | 1990-10-10 | 1998-11-24 | The Regents Of The University Of California | Y chromosome specific nucleic acid probe and method for determining the Y chromosome in situ |
WO1992008120A1 (en) | 1990-10-29 | 1992-05-14 | Macquarie University | Pulsed laser flow cytometry |
EP0555212B1 (en) | 1990-10-31 | 1994-10-12 | Biophos Medical Ab | Fertility analyzer |
US5116125A (en) | 1990-10-31 | 1992-05-26 | Biophos Medical Ab | Fertility analyzer |
JP2874746B2 (en) | 1990-11-22 | 1999-03-24 | シスメックス株式会社 | Flow cell mechanism in flow imaging cytometer |
US5991028A (en) | 1991-02-22 | 1999-11-23 | Applied Spectral Imaging Ltd. | Spectral bio-imaging methods for cell classification |
JPH0734012B2 (en) | 1991-02-27 | 1995-04-12 | 東亜医用電子株式会社 | Flow image cytometer |
JP3121849B2 (en) | 1991-02-27 | 2001-01-09 | シスメックス株式会社 | Flow image cytometer |
US5144224A (en) | 1991-04-01 | 1992-09-01 | Larsen Lawrence E | Millimeter wave flow cytometer |
US5199576A (en) | 1991-04-05 | 1993-04-06 | University Of Rochester | System for flexibly sorting particles |
EP0515211A3 (en) | 1991-05-23 | 1993-04-07 | Becton Dickinson And Company | Apparatus and method for phase resolved fluorescence lifetimes of independent and varying amplitude pulses |
DE9107792U1 (en) | 1991-06-25 | 1991-09-12 | Labotect-Labor-Technik, Goettingen, Gmbh, 3406 Bovenden, De | |
FR2678506B1 (en) | 1991-07-01 | 2000-03-10 | Claude Ranoux | SPONTANEOUS FERTILIZATION PROCESS. |
US5548661A (en) | 1991-07-12 | 1996-08-20 | Price; Jeffrey H. | Operator independent image cytometer |
US5412466A (en) | 1991-07-26 | 1995-05-02 | Toa Medical Electronics Co., Ltd. | Apparatus for forming flattened sample flow for analyzing particles |
EP0529666B1 (en) | 1991-08-30 | 2000-04-12 | Omron Corporation | Cell analyzing apparatus |
US5488469A (en) | 1991-08-30 | 1996-01-30 | Omron Corporation | Cell analyzing apparatus |
US5548395A (en) | 1991-09-20 | 1996-08-20 | Toa Medical Electronics Co., Ltd. | Particle analyzer |
US5578449A (en) | 1991-10-03 | 1996-11-26 | Hilding Ohlsson, S.A. | Procedure for the sex determination of embryos in mammals especially applied to bovine embryos |
IT1253226B (en) | 1991-10-24 | 1995-07-11 | Piero Serra | CATHETER FOR THE INTRODUCTION OR ASPIRATION OF LIQUIDS OF DIFFERENT NATURES IN ANIMALS PARTICULARLY FOR GYNECOLOGICAL TREATMENTS IN CATTLE, EQUINE AND SIMILAR |
JP3212647B2 (en) | 1991-10-24 | 2001-09-25 | シスメックス株式会社 | Imaging flow cytometer |
US5919621A (en) | 1991-10-24 | 1999-07-06 | Brown; David B. | Methods for diagnosing human male infertility |
US5866344A (en) | 1991-11-15 | 1999-02-02 | Board Of Regents, The University Of Texas System | Antibody selection methods using cell surface expressed libraries |
US5370842A (en) | 1991-11-29 | 1994-12-06 | Canon Kabushiki Kaisha | Sample measuring device and sample measuring system |
JP2593021B2 (en) | 1991-12-13 | 1997-03-19 | 伊藤ハム株式会社 | How to identify bovine embryo sex |
JP3130628B2 (en) | 1992-01-30 | 2001-01-31 | シスメックス株式会社 | Particle determination device |
US5400179A (en) | 1992-02-18 | 1995-03-21 | Asahi Kogaku Kogyo Kabushiki Kaisha | Optical multilayer thin film and beam splitter |
DE69321748T2 (en) | 1992-02-20 | 1999-06-17 | Canon Kk | Method and measuring apparatus for handling particles |
JP3248910B2 (en) | 1992-02-21 | 2002-01-21 | イギリス国 | Analysis of particle properties |
US5558998A (en) | 1992-02-25 | 1996-09-24 | The Regents Of The Univ. Of California | DNA fragment sizing and sorting by laser-induced fluorescence |
US6120735A (en) | 1992-02-26 | 2000-09-19 | The Ohio States University | Fractional cell sorter |
JPH084601B2 (en) * | 1992-04-10 | 1996-01-24 | 岡山県 | Sperm freezing method and sperm freezing device |
US5298967A (en) | 1992-06-02 | 1994-03-29 | Pacific Scientific Company | Measurement of concentrations of dissolved solvent |
JP3145486B2 (en) | 1992-06-12 | 2001-03-12 | シスメックス株式会社 | Imaging flow cytometer |
FR2696551B1 (en) | 1992-07-13 | 1995-06-23 | Pall Corp | AUTOMATIC SYSTEM AND METHOD FOR THE TREATMENT OF A BIOLOGICAL FLUID. |
JP3215175B2 (en) | 1992-08-10 | 2001-10-02 | シスメックス株式会社 | Particle analyzer |
US5315122A (en) | 1992-08-25 | 1994-05-24 | Becton, Dickinson And Company | Apparatus and method for fluorescent lifetime measurement |
WO1994005775A1 (en) | 1992-09-03 | 1994-03-17 | Systemix, Inc. | High speed flow cytometric separation of viable mammalian cells |
US5466572A (en) | 1992-09-03 | 1995-11-14 | Systemix, Inc. | High speed flow cytometric separation of viable cells |
US5736410A (en) | 1992-09-14 | 1998-04-07 | Sri International | Up-converting reporters for biological and other assays using laser excitation techniques |
US5275933A (en) | 1992-09-25 | 1994-01-04 | The Board Of Trustees Of The Leland Stanford Junior University | Triple gradient process for recovering nucleated fetal cells from maternal blood |
US5371585A (en) | 1992-11-10 | 1994-12-06 | Pacific Scientific Company | Particle detecting instrument with sapphire detecting cell defining a rectangular flow path |
US5359907A (en) | 1992-11-12 | 1994-11-01 | Horiba Instruments, Inc. | Method and apparatus for dry particle analysis |
US5395588A (en) | 1992-12-14 | 1995-03-07 | Becton Dickinson And Company | Control of flow cytometer having vacuum fluidics |
AU5839694A (en) | 1993-01-16 | 1994-08-15 | James Andrew Bangham | Signal processing system |
JP2525713B2 (en) | 1993-01-19 | 1996-08-21 | 農林水産省畜産試験場長 | Culture and transport method of bovine embryo using low molecular weight thiol compound |
US5467189A (en) | 1993-01-22 | 1995-11-14 | Venturedyne, Ltd. | Improved particle sensor and method for assaying a particle |
JP3052665B2 (en) | 1993-01-26 | 2000-06-19 | 株式会社日立製作所 | Flow cell device |
CA2113957A1 (en) | 1993-01-29 | 1994-07-30 | University Of Guelph | Nucleotide sequences for bovine sex determination |
JPH08506664A (en) | 1993-02-01 | 1996-07-16 | セック,リミテッド | Method and apparatus for DNA sequencing |
US5453575A (en) | 1993-02-01 | 1995-09-26 | Endosonics Corporation | Apparatus and method for detecting blood flow in intravascular ultrasonic imaging |
US5367474A (en) | 1993-02-08 | 1994-11-22 | Coulter Corporation | Flow cytometer |
US5547849A (en) | 1993-02-17 | 1996-08-20 | Biometric Imaging, Inc. | Apparatus and method for volumetric capillary cytometry |
US5556764A (en) | 1993-02-17 | 1996-09-17 | Biometric Imaging, Inc. | Method and apparatus for cell counting and cell classification |
US5563059A (en) | 1993-02-23 | 1996-10-08 | Genentech, Inc. | Use of human inhibin and human activin to increase the number of mature primate oocytes |
DE69418131D1 (en) | 1993-03-01 | 1999-06-02 | Gen Signal Corp | DEVICE FOR GENERATING AN ADJUSTABLE RING-SHAPED LIGHTING FOR A PHOTOLITHOGRAPHIC PROJECTION APPARATUS |
NO930980L (en) | 1993-03-18 | 1994-09-19 | Flowtech As | Optical configuration for fluid flow cytophotometer |
US5658751A (en) | 1993-04-13 | 1997-08-19 | Molecular Probes, Inc. | Substituted unsymmetrical cyanine dyes with selected permeability |
US5494795A (en) | 1993-05-05 | 1996-02-27 | The United States Of America As Represented By The Secretary Of The Navy | Specific oligonucleotide primers for detection of pathogenic campylobacter bacteria by polymerase chain reaction |
US5439578A (en) | 1993-06-03 | 1995-08-08 | The Governors Of The University Of Alberta | Multiple capillary biochemical analyzer |
EP0627643B1 (en) | 1993-06-03 | 1999-05-06 | Hamamatsu Photonics K.K. | Laser scanning optical system using axicon |
PL307305A1 (en) | 1993-06-04 | 1995-05-15 | Kwahak International Co Ltd | Apparatus for arificial fertilisation and transfer of embryos |
NO932088L (en) | 1993-06-08 | 1995-01-05 | Oddbjoern Gjelsnes | Device for use in liquid flow cytometry |
US5464581A (en) | 1993-08-02 | 1995-11-07 | The Regents Of The University Of California | Flow cytometer |
US5483469A (en) | 1993-08-02 | 1996-01-09 | The Regents Of The University Of California | Multiple sort flow cytometer |
US6328071B1 (en) | 1993-08-06 | 2001-12-11 | Cary Austin | Well pressure tank |
US5596401A (en) | 1993-09-16 | 1997-01-21 | Toa Medical Electronics Co., Ltd. | Particle analyzing apparatus using a coherence lowering device |
US5503994A (en) | 1993-10-08 | 1996-04-02 | The Board Of Trustees Of The Leland Stanford Junior University | System for sample detection with compensation for difference in sensitivity to detection of components moving at different velocities |
US5480774A (en) | 1993-10-14 | 1996-01-02 | A/F Protein, Inc. | Determination of genomic sex in salmonids |
JP3290786B2 (en) | 1993-11-26 | 2002-06-10 | シスメックス株式会社 | Particle analyzer |
GB9324938D0 (en) | 1993-12-04 | 1994-01-26 | Atomic Energy Authority Uk | Aerosol generator |
FI96452C (en) | 1994-01-26 | 1996-06-25 | Pekka Haenninen | Method for excitation of dyes |
AU693107B2 (en) | 1994-02-28 | 1998-06-25 | Sunkyong Industries Co., Ltd. | Pyrimidine acyclonucleoside derivatives |
DE69534484T2 (en) | 1994-03-25 | 2006-07-06 | Marukin Chuyu Co. Ltd. | epimerase |
US5475487A (en) | 1994-04-20 | 1995-12-12 | The Regents Of The University Of California | Aqueous carrier waveguide in a flow cytometer |
DE4414940C2 (en) | 1994-04-28 | 1998-07-02 | Pekka Haenninen | Luminescence scanning microscope with two photons excitation |
US5595866A (en) | 1994-05-27 | 1997-01-21 | Methodist Hospital Of Indiana, Inc. | Step-wise method to remove cryoprotectant from sperm |
DE4419894A1 (en) | 1994-06-07 | 1995-12-14 | Gip Medizin Technik Gmbh | Endoscopic puncture needle with elastic catheter |
EP0687956B2 (en) | 1994-06-17 | 2005-11-23 | Carl Zeiss SMT AG | Illumination device |
US5601234A (en) | 1994-08-01 | 1997-02-11 | Abbott Laboratories | Fluid nozzle and method of introducing a fluid |
US5891734A (en) | 1994-08-01 | 1999-04-06 | Abbott Laboratories | Method for performing automated analysis |
JP3375203B2 (en) | 1994-08-08 | 2003-02-10 | シスメックス株式会社 | Cell analyzer |
FR2723735B1 (en) | 1994-08-18 | 1996-10-31 | Abx Sa | AUTOMATIC CONNECTION BOX FOR DISPENSING REAGENTS IN AN APPARATUS IN PARTICULAR A HEMATOLOGICAL ANALYZER. |
US5790692A (en) | 1994-09-07 | 1998-08-04 | Jeffrey H. Price | Method and means of least squares designed filters for image segmentation in scanning cytometry |
US20020186874A1 (en) | 1994-09-07 | 2002-12-12 | Jeffrey H. Price | Method and means for image segmentation in fluorescence scanning cytometry |
US5700692A (en) | 1994-09-27 | 1997-12-23 | Becton Dickinson And Company | Flow sorter with video-regulated droplet spacing |
US5934885A (en) | 1994-10-07 | 1999-08-10 | Bayer Corporation | Reagent pump assembly |
IL115327A (en) | 1994-10-07 | 2000-08-13 | Bayer Ag | Diaphragm pump |
US5602349A (en) | 1994-10-14 | 1997-02-11 | The University Of Washington | Sample introduction system for a flow cytometer |
US5643796A (en) | 1994-10-14 | 1997-07-01 | University Of Washington | System for sensing droplet formation time delay in a flow cytometer |
US5602039A (en) | 1994-10-14 | 1997-02-11 | The University Of Washington | Flow cytometer jet monitor system |
AU698929B2 (en) | 1994-10-14 | 1998-11-12 | University Of Washington | High speed flow cytometer droplet formation system |
US6861265B1 (en) | 1994-10-14 | 2005-03-01 | University Of Washington | Flow cytometer droplet formation system |
US5495719A (en) | 1994-11-14 | 1996-03-05 | Gray, Jr.; Carl O. | Method of preserving spermatozoa |
JP3347495B2 (en) | 1994-11-14 | 2002-11-20 | シスメックス株式会社 | Particle analyzer |
US5514537A (en) | 1994-11-28 | 1996-05-07 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Process and apparatus for sorting spermatozoa |
EP0746865B1 (en) | 1994-12-08 | 2003-03-26 | Molecular Dynamics, Inc. | Fluorescence imaging system employing a macro scanning objective |
US5632754A (en) | 1994-12-23 | 1997-05-27 | Devices For Vascular Intervention | Universal catheter with interchangeable work element |
DE69533469T2 (en) | 1994-12-26 | 2005-09-22 | Sysmex Corp. | flow cytometer |
US5835262A (en) | 1994-12-28 | 1998-11-10 | Research Development Corporation Of Japan | Multi-wavelength optical microscope |
FI98765C (en) | 1995-01-16 | 1997-08-11 | Erkki Soini | Flow cytometric method and apparatus |
US5793485A (en) | 1995-03-20 | 1998-08-11 | Sandia Corporation | Resonant-cavity apparatus for cytometry or particle analysis |
US5608519A (en) | 1995-03-20 | 1997-03-04 | Gourley; Paul L. | Laser apparatus and method for microscopic and spectroscopic analysis and processing of biological cells |
US5620842A (en) | 1995-03-29 | 1997-04-15 | Becton Dickinson And Company | Determination of the number of fluorescent molecules on calibration beads for flow cytometry |
US5786560A (en) | 1995-03-31 | 1998-07-28 | Panasonic Technologies, Inc. | 3-dimensional micromachining with femtosecond laser pulses |
US5699152A (en) * | 1995-04-03 | 1997-12-16 | Alltrista Corporation | Electro-optical inspection system and method |
US5528045A (en) | 1995-04-06 | 1996-06-18 | Becton Dickinson And Company | Particle analyzer with spatially split wavelength filter |
US5682038A (en) | 1995-04-06 | 1997-10-28 | Becton Dickinson And Company | Fluorescent-particle analyzer with timing alignment for analog pulse subtraction of fluorescent pulses arising from different excitation locations |
US5641457A (en) | 1995-04-25 | 1997-06-24 | Systemix | Sterile flow cytometer and sorter with mechanical isolation between flow chamber and sterile enclosure |
US5687727A (en) | 1995-05-01 | 1997-11-18 | Danforth Biomedical Incorporated | Catheter adaptor with slitting blade and improved manual control and method of use |
EP0957797A4 (en) | 1995-05-09 | 2001-05-16 | Univ Missouri | A system for introducing a fluid into the uterus of an animal |
FR2734637B1 (en) | 1995-05-24 | 1997-08-14 | Abx Sa | DEVICE FOR OPTICAL INSPECTION OF A FLUID, PARTICULARLY FOR HEMATOLOGICAL ANALYSIS |
US5798276A (en) | 1995-06-07 | 1998-08-25 | Molecular Probes, Inc. | Reactive derivatives of sulforhodamine 101 with enhanced hydrolytic stability |
US5650847A (en) | 1995-06-14 | 1997-07-22 | Erkki Soini | Method and device for determination of parameters of individual microparticles |
US5716852A (en) | 1996-03-29 | 1998-02-10 | University Of Washington | Microfabricated diffusion-based chemical sensor |
US6454945B1 (en) | 1995-06-16 | 2002-09-24 | University Of Washington | Microfabricated devices and methods |
US5589457A (en) | 1995-07-03 | 1996-12-31 | Ausa International, Inc. | Process for the synchronization of ovulation |
US6411835B1 (en) | 1997-01-13 | 2002-06-25 | Medispectra, Inc. | Spectral volume microprobe arrays |
JPH11511252A (en) | 1995-08-11 | 1999-09-28 | ユニバーシティ・オブ・グエルフ | Methods for identifying sex-specific and species-specific molecules, molecules identified using the methods, and uses of the molecules |
WO1997009043A1 (en) | 1995-09-06 | 1997-03-13 | The Research Foundation Of State University Of New York | Two-photon upconverting dyes and applications |
DE19533092A1 (en) | 1995-09-07 | 1997-03-13 | Basf Ag | Device for parallelized two-photon fluorescence correlation spectroscopy (TPA-FCS) and its use for drug screening |
US6329158B1 (en) | 1995-09-15 | 2001-12-11 | Becton Dickinson And Company | Use of dimly fluorescing nucleic acid dyes in the identification of nucleated cells |
US6040139A (en) * | 1995-09-19 | 2000-03-21 | Bova; G. Steven | Laser cell purification system |
US5726751A (en) | 1995-09-27 | 1998-03-10 | University Of Washington | Silicon microchannel optical flow cytometer |
US5780230A (en) | 1995-10-06 | 1998-07-14 | Coriell Institute For Medical Research | Compositions and methods for human sperm activation and quantitative assessment of human sperm genome replication |
US5736330A (en) | 1995-10-11 | 1998-04-07 | Luminex Corporation | Method and compositions for flow cytometric determination of DNA sequences |
JP4421681B2 (en) | 1995-10-19 | 2010-02-24 | バイオ‐オリジン エルエルシー | Methods and compositions for improving survival and function of germ cells and embryos |
US6117068A (en) | 1995-10-19 | 2000-09-12 | Elite Genetics, Inc | Artificial insemination system |
DE69529538T2 (en) | 1995-11-09 | 2003-11-06 | Medical Res Council London | USE OF CYB MEDIUM FOR TRANSPORTING AND STORING SPERM |
DE19549015C1 (en) | 1995-12-28 | 1997-04-03 | Siemens Ag | Method of monitoring precise location at which fluid stream breaks up into droplets |
JP3584108B2 (en) | 1996-01-08 | 2004-11-04 | キヤノン株式会社 | Lens barrel |
US6641708B1 (en) | 1996-01-31 | 2003-11-04 | Board Of Regents, The University Of Texas System | Method and apparatus for fractionation using conventional dielectrophoresis and field flow fractionation |
WO1997029354A1 (en) | 1996-02-05 | 1997-08-14 | Bayer Aktiengesellschaft | Process and device for sorting and for extraction of biological objects arranged on planar means, such as biological cells or cell organelles, histological sections, chromosome particles etc. using laser beams |
BR9600722A (en) | 1996-02-14 | 1997-12-30 | Jorge Antonio Rodrigues Claro | Infusion pump for contained in flexible plastic bags |
AU1852297A (en) | 1996-02-16 | 1997-09-02 | Inphocyte, Inc. | System and method for rapid analysis of cells using spectral cytometry |
JP3127111B2 (en) | 1996-02-22 | 2001-01-22 | 株式会社日立製作所 | Flow type particle image analysis method and apparatus |
US6143901A (en) | 1996-07-31 | 2000-11-07 | Genesoft, Inc. | Complex formation between dsDNA and pyrrole imidazole polyamides |
US6090947A (en) | 1996-02-26 | 2000-07-18 | California Institute Of Technology | Method for the synthesis of pyrrole and imidazole carboxamides on a solid support |
US5895922A (en) | 1996-03-19 | 1999-04-20 | Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of National Defence | Fluorescent biological particle detection system |
US5701012A (en) | 1996-03-19 | 1997-12-23 | Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of National Defence | Fluorescent biological particle detection system |
US5747349A (en) | 1996-03-20 | 1998-05-05 | University Of Washington | Fluorescent reporter beads for fluid analysis |
JP3590470B2 (en) * | 1996-03-27 | 2004-11-17 | アルプス電気株式会社 | Cleaning water generation method and cleaning method, and cleaning water generation device and cleaning device |
JP3640461B2 (en) | 1996-04-03 | 2005-04-20 | シスメックス株式会社 | Particle analyzer |
US5707808A (en) | 1996-04-15 | 1998-01-13 | The Regents Of The University Of California | Optical selection and collection of DNA fragments |
ATE357663T1 (en) | 1996-04-25 | 2007-04-15 | Genicon Sciences Corp | ANALYTICAL TASSAY USING PARTICLE MARKER |
WO1997043620A1 (en) | 1996-05-15 | 1997-11-20 | International Remote Imaging Systems, Inc. | Selectively emphasizing particles of interest from a fluid sample for analysis |
JP2963393B2 (en) | 1996-06-14 | 1999-10-18 | 浜松ホトニクス株式会社 | Voltage divider circuit for photomultiplier tube |
US5846737A (en) | 1996-07-26 | 1998-12-08 | Molecular Probes, Inc. | Conjugates of sulforhodamine fluorophores with enhanced fluorescence |
US5909278A (en) | 1996-07-29 | 1999-06-01 | The Regents Of The University Of California | Time-resolved fluorescence decay measurements for flowing particles |
US5745308A (en) | 1996-07-30 | 1998-04-28 | Bayer Corporation | Methods and apparatus for an optical illuminator assembly and its alignment |
US6042249A (en) | 1996-07-30 | 2000-03-28 | Bayer Corporation | Illuminator optical assembly for an analytical instrument and methods of alignment and manufacture |
EP0822404B1 (en) | 1996-07-30 | 2009-06-17 | Siemens Healthcare Diagnostics Inc. | Optical system for a hematology analytical instrument |
EP0822401A3 (en) | 1996-07-30 | 1999-05-06 | Bayer Corporation | Hydraulic system for a hematology analytical instrument |
US5883378A (en) | 1996-07-30 | 1999-03-16 | Bayer Corporation | Apparatus and methods for transmitting electrical signals indicative of optical interactions between a light beam and a flowing suspension of particles |
US5872627A (en) | 1996-07-30 | 1999-02-16 | Bayer Corporation | Method and apparatus for detecting scattered light in an analytical instrument |
US5719667A (en) | 1996-07-30 | 1998-02-17 | Bayer Corporation | Apparatus for filtering a laser beam in an analytical instrument |
US6074827A (en) | 1996-07-30 | 2000-06-13 | Aclara Biosciences, Inc. | Microfluidic method for nucleic acid purification and processing |
US5844685A (en) | 1996-07-30 | 1998-12-01 | Bayer Corporation | Reference laser beam sampling apparatus |
US5998140A (en) * | 1996-07-31 | 1999-12-07 | The Scripps Research Institute | Complex formation between dsDNA and oligomer of cyclic heterocycles |
US5804436A (en) | 1996-08-02 | 1998-09-08 | Axiom Biotechnologies, Inc. | Apparatus and method for real-time measurement of cellular response |
ATE280317T1 (en) | 1996-08-07 | 2004-11-15 | Cuno Inc | DISPENSING DEVICE FOR ADDITIVES |
DK0925494T3 (en) | 1996-09-04 | 2002-07-01 | Scandinavian Micro Biodevices | Microfluidic system for particle separation and analysis |
US5873254A (en) | 1996-09-06 | 1999-02-23 | Interface Multigrad Technology | Device and methods for multigradient directional cooling and warming of biological samples |
JP3574665B2 (en) | 1996-09-06 | 2004-10-06 | メダレツクス・インコーポレーテツド | Cyanidin compositions and their therapeutic and diagnostic uses |
US6221654B1 (en) | 1996-09-25 | 2001-04-24 | California Institute Of Technology | Method and apparatus for analysis and sorting of polynucleotides based on size |
US5759767A (en) | 1996-10-11 | 1998-06-02 | Joseph R. Lakowicz | Two-photon and multi-photon measurement of analytes in animal and human tissues and fluids |
US6002471A (en) | 1996-11-04 | 1999-12-14 | California Institute Of Technology | High resolution scanning raman microscope |
US5799830A (en) | 1996-11-08 | 1998-09-01 | Carroll; David C. | Pressure vessel access port |
US5696157A (en) | 1996-11-15 | 1997-12-09 | Molecular Probes, Inc. | Sulfonated derivatives of 7-aminocoumarin |
EP2264428B1 (en) | 1997-01-31 | 2017-05-03 | Xy, Llc | Optical apparatus with focussing reflector for converging radiation onto a flow of particles |
US6534308B1 (en) | 1997-03-27 | 2003-03-18 | Oncosis, Llc | Method and apparatus for selectively targeting specific cells within a mixed cell population |
US5874266A (en) | 1997-03-27 | 1999-02-23 | Palsson; Bernhard O. | Targeted system for removing tumor cells from cell populations |
US6753161B2 (en) | 1997-03-27 | 2004-06-22 | Oncosis Llc | Optoinjection methods |
GB9707096D0 (en) | 1997-04-08 | 1997-05-28 | Smithkline Beecham Plc | Novel device |
JP2968231B2 (en) | 1997-04-11 | 1999-10-25 | 株式会社荏原製作所 | Air conditioning system |
US6050935A (en) | 1997-05-09 | 2000-04-18 | Biofertec | Container assembly for intravaginal fertilization and culture and embryo transfer and method of intravaginal fertilization and culture employing such a container |
US6133995A (en) | 1997-05-09 | 2000-10-17 | Sysmex Corporation | Particle measuring apparatus |
EP2302368B1 (en) | 1997-06-09 | 2017-04-05 | EMD Millipore Corporation | Method and apparatus for detecting microparticles in fluid samples |
US6139800A (en) | 1997-06-23 | 2000-10-31 | Luminex Corporation | Interlaced lasers for multiple fluorescence measurement |
EP0894498B1 (en) | 1997-07-01 | 2006-05-17 | Vicam, L.P. | Method for sex determination of mammalian offspring |
US6111398A (en) | 1997-07-03 | 2000-08-29 | Coulter International Corp. | Method and apparatus for sensing and characterizing particles |
BR9704313A (en) | 1997-07-08 | 1999-04-06 | Alves Elias Walter | Use of chicken egg ig-y associated with monoclonal antibodies against sex specific antigens in bovine sperm immunosexation |
US5899848A (en) | 1997-07-14 | 1999-05-04 | Haubrich; Mark A. | Device and process for artificial insemination of animals |
US5876942A (en) | 1997-07-24 | 1999-03-02 | National Science Council Of Republic Of China | Process for sexing cow embryos |
US5985216A (en) * | 1997-07-24 | 1999-11-16 | The United States Of America, As Represented By The Secretary Of Agriculture | Flow cytometry nozzle for high efficiency cell sorting |
US5985538A (en) | 1997-08-01 | 1999-11-16 | Saint Barnabas Medical Center | Cryopreservation and cell culture medium comprising less than 50 mM sodium ions and greater than 100 mM choline salt |
US6003678A (en) | 1997-08-21 | 1999-12-21 | University Of Washington | Particle separating apparatus and method |
US5819948A (en) | 1997-08-21 | 1998-10-13 | Van Den Engh; Gerrit J. | Particle separating apparatus and method |
US5880474A (en) | 1997-08-29 | 1999-03-09 | Becton Dickinson And Company | Multi-illumination-source flow particle analyzer with inter-location emissions crosstalk cancelation |
EP1028622A1 (en) | 1997-09-22 | 2000-08-23 | University Of Guelph | Reduction of sperm sensitivity to chilling |
US6540895B1 (en) | 1997-09-23 | 2003-04-01 | California Institute Of Technology | Microfabricated cell sorter for chemical and biological materials |
US6322901B1 (en) * | 1997-11-13 | 2001-11-27 | Massachusetts Institute Of Technology | Highly luminescent color-selective nano-crystalline materials |
AU754775B2 (en) | 1997-11-19 | 2002-11-21 | University Of Washington | High throughput optical scanner |
US6086574A (en) | 1997-11-21 | 2000-07-11 | Hyclone Laboratories, Inc. | Fluid delivery systems with diptube connector |
US5895764A (en) | 1997-11-24 | 1999-04-20 | University Of New Mexico | Controlled sheath flow injection cytometry |
US5990479A (en) | 1997-11-25 | 1999-11-23 | Regents Of The University Of California | Organo Luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes |
US6207392B1 (en) | 1997-11-25 | 2001-03-27 | The Regents Of The University Of California | Semiconductor nanocrystal probes for biological applications and process for making and using such probes |
US6221671B1 (en) | 1997-12-12 | 2001-04-24 | Chemunex S.A. | Digital flow cytometer and method |
US6071689A (en) | 1997-12-31 | 2000-06-06 | Xy, Inc. | System for improving yield of sexed embryos in mammals |
US6149867A (en) | 1997-12-31 | 2000-11-21 | Xy, Inc. | Sheath fluids and collection systems for sex-specific cytometer sorting of sperm |
EP1058688A4 (en) | 1998-02-03 | 2004-10-06 | Xy Inc | Specific oligonucleotide primers for detection of bovine male chromosome presence by polymerase chain reaction and method |
KR20010041113A (en) | 1998-02-20 | 2001-05-15 | 헤릭호프, 리사 | A vibratory system for a sorting flow cytometer |
US6746873B1 (en) | 1998-02-20 | 2004-06-08 | Xy, Inc. | Vibratory system for a sorting flow cytometer |
US6154276A (en) | 1998-02-23 | 2000-11-28 | The Regents Of The University Of California | Waveguide detection of right-angle-scattered light in flow cytometry |
US6211477B1 (en) | 1998-02-26 | 2001-04-03 | Becton Dickinson And Company | Electrostatic deceleration system for flow cytometer |
US6248590B1 (en) | 1998-02-27 | 2001-06-19 | Cytomation, Inc. | Method and apparatus for flow cytometry |
US6042025A (en) * | 1998-03-13 | 2000-03-28 | Smith Et Al. | Two hole dispenser with baffles |
ATE254762T1 (en) | 1998-03-16 | 2003-12-15 | Partec Partikelzaehlgeraete Gm | ELECTRONIC DEVICE FOR PRECISE DISPENSING SMALL QUANTITIES OF LIQUID |
JP3594794B2 (en) | 1998-03-24 | 2004-12-02 | 独立行政法人 科学技術振興機構 | Nanosecond time-gated spectroscopic diagnostic equipment |
US6642018B1 (en) * | 1998-03-27 | 2003-11-04 | Oncosis Llc | Method for inducing a response in one or more targeted cells |
JP2002510045A (en) | 1998-03-30 | 2002-04-02 | バイオシャフ リミテッド | Flow-based cell count calculator for analyzing common diagnostic factors in cells and body fluids |
US6175409B1 (en) | 1999-04-02 | 2001-01-16 | Symyx Technologies, Inc. | Flow-injection analysis and variable-flow light-scattering methods and apparatus for characterizing polymers |
JP3350442B2 (en) | 1998-04-09 | 2002-11-25 | 科学技術振興事業団 | Microscope system |
JP3946444B2 (en) | 1998-05-14 | 2007-07-18 | ルミネックス コーポレイション | Configuration and method for zero dead time of flow cytometer |
EP1046032A4 (en) | 1998-05-18 | 2002-05-29 | Univ Washington | Liquid analysis cartridge |
ATE530891T1 (en) | 1998-05-22 | 2011-11-15 | California Inst Of Techn | MINIATURIZED CELL SORTER |
US6079836A (en) | 1998-07-20 | 2000-06-27 | Coulter International Corp. | Flow cytometer droplet break-off location adjustment mechanism |
NZ527659A (en) * | 1998-07-30 | 2006-02-24 | Colorado State University Thro | Equine artificial insemination when there has been sex selection of the sperm to produce an equine of the desired sex |
US20040107150A1 (en) | 1998-07-31 | 2004-06-03 | Chata Biosystems, Inc. Of Colorado | Apparatus and method with vessel for containing/transporting a fluent substance |
US6729369B2 (en) | 1998-07-31 | 2004-05-04 | Chata Biosystems, Inc. | Vessel for containing/transporting a fluent substance |
DE29813921U1 (en) | 1998-08-04 | 1998-10-08 | Kisfeld Alfons | Device for inseminating animals, especially sows |
US6309815B1 (en) * | 1998-08-07 | 2001-10-30 | University Of Kansas Medical Center | Composition and method for preparation, storage and activation of large populations of immotile sperm |
WO2000009113A1 (en) | 1998-08-17 | 2000-02-24 | Trout William E | Use of zeranol to modulate reproductive cycles |
AU754644B2 (en) | 1998-08-21 | 2002-11-21 | Union Biometrica, Inc. | Instrument for selecting and depositing multicellular organisms and other large objects |
US6326144B1 (en) * | 1998-09-18 | 2001-12-04 | Massachusetts Institute Of Technology | Biological applications of quantum dots |
US6495333B1 (en) | 1998-09-22 | 2002-12-17 | Becton Dickinson And Company | Flow cytometric, whole blood dendritic cell immune function assay |
US6208411B1 (en) | 1998-09-28 | 2001-03-27 | Kla-Tencor Corporation | Massively parallel inspection and imaging system |
US6707555B1 (en) | 1998-10-15 | 2004-03-16 | Sysmex Corporation | Optical information measuring apparatus |
US6423505B1 (en) | 1998-12-03 | 2002-07-23 | Becton Dickinson And Company | Methods and reagents for quantitation of HLA-DR and CD11b expression on peripheral blood cells |
US7116407B2 (en) | 1998-12-15 | 2006-10-03 | Union Biometrica, Inc. | System for axial pattern analysis of multicellular organisms |
US6128133A (en) | 1998-12-22 | 2000-10-03 | Lucent Technologies Inc. | Optical beamsplitter |
EP1018644A2 (en) | 1999-01-06 | 2000-07-12 | Bayer Corporation | Variable rate particle counter and method of use |
US20010006416A1 (en) | 1999-01-11 | 2001-07-05 | Johnson Paul E. | Ribbon flow cytometry apparatus and methods |
US6256096B1 (en) | 1999-01-11 | 2001-07-03 | Softray | Flow cytometry apparatus and method |
US6150119A (en) | 1999-01-19 | 2000-11-21 | Caliper Technologies Corp. | Optimized high-throughput analytical system |
US6580504B1 (en) | 1999-01-25 | 2003-06-17 | Amnis Corporation | Multipass cavity for illumination and excitation of moving objects |
US6671044B2 (en) | 1999-01-25 | 2003-12-30 | Amnis Corporation | Imaging and analyzing parameters of small moving objects such as cells in broad flat flow |
US6473176B2 (en) | 1999-01-25 | 2002-10-29 | Amnis Corporation | Imaging and analyzing parameters of small moving objects such as cells |
US6119465A (en) | 1999-02-10 | 2000-09-19 | Mullens; Patrick L. | Shipping container for storing materials at cryogenic temperatures |
FR2789778B1 (en) | 1999-02-12 | 2001-09-14 | France Telecom | METHOD FOR ASSOCIATING ROUTING REFERENCES WITH DATA PACKETS BY MEANS OF A SORTED MEMORY, AND PACKET ROUTER APPLYING THIS METHOD |
US6323632B1 (en) | 1999-08-13 | 2001-11-27 | Coulter International Corp. | Solid state RF oscillator-detector for flow cytometer |
JP2000239101A (en) * | 1999-02-18 | 2000-09-05 | Japan Science & Technology Corp | Refrigeration of rat sperm |
CH690557A5 (en) | 1999-03-01 | 2000-10-13 | Arson Ag | Illuminator. |
US6097485A (en) | 1999-03-08 | 2000-08-01 | Integrated Waveguides, Inc. | Microchip optical transport technology for use in a personal flow cytometer |
FR2791645B1 (en) | 1999-04-02 | 2001-06-15 | Valois Sa | FLUID PRODUCT SAMPLE FOR PRESS |
US6193647B1 (en) | 1999-04-08 | 2001-02-27 | The Board Of Trustees Of The University Of Illinois | Microfluidic embryo and/or oocyte handling device and method |
US6793387B1 (en) | 1999-05-08 | 2004-09-21 | Chata Biosystems, Inc. | Apparatus for automatic preparation of a mixture and method |
FR2793495B1 (en) | 1999-05-14 | 2001-08-10 | Imv Technologies | THINNER FOR CRYOPRESERVATION OF CATTLE SPERMATOZOIDS |
FR2793708B1 (en) | 1999-05-21 | 2001-08-03 | Valois Sa | FLUID PRODUCT DISPENSING DEVICE |
US6372506B1 (en) | 1999-07-02 | 2002-04-16 | Becton, Dickinson And Company | Apparatus and method for verifying drop delay in a flow cytometer |
JP3361778B2 (en) * | 1999-07-09 | 2003-01-07 | 社団法人家畜改良事業団 | Injector for sperm for artificial insemination or fertilized egg transplantation of livestock and method of operating the same |
IT1307787B1 (en) | 1999-07-26 | 2001-11-19 | Univ Firenze | PROCESS TO INCREASE THE MOTILITY OF SPERMATOZOI AND SPERMATOZOIA SUPERIOR MOTILITY SO OBTAINED. |
DE19935766A1 (en) | 1999-07-29 | 2001-02-01 | Friedrich Schiller Uni Jena Bu | Process for the optical excitation of fluorophore-labeled DNA and RNA |
US6664550B2 (en) | 1999-08-30 | 2003-12-16 | Sandia National Laboratories | Apparatus to collect, classify, concentrate, and characterize gas-borne particles |
US6495366B1 (en) | 1999-09-03 | 2002-12-17 | Therakos, Inc. | Uninterrupted flow pump apparatus and method |
US6813017B1 (en) | 1999-10-20 | 2004-11-02 | Becton, Dickinson And Company | Apparatus and method employing incoherent light emitting semiconductor devices as particle detection light sources in a flow cytometer |
JP2003512605A (en) | 1999-10-21 | 2003-04-02 | サイトメーション, インコーポレイテッド | Temporary dynamic flow cytometer analysis system |
US6472153B1 (en) | 1999-10-26 | 2002-10-29 | Epoch Biosciences, Inc. | Hybridization-triggered fluorescent detection of nucleic acids |
US7208265B1 (en) | 1999-11-24 | 2007-04-24 | Xy, Inc. | Method of cryopreserving selected sperm cells |
WO2001040454A1 (en) | 1999-11-30 | 2001-06-07 | Oncosis | Method and apparatus for selectively targeting specific cells within a cell population |
US6263745B1 (en) * | 1999-12-03 | 2001-07-24 | Xy, Inc. | Flow cytometer nozzle and flow cytometer sample handling methods |
US6558911B1 (en) | 1999-12-10 | 2003-05-06 | Oregon Health Sciences University | Sperm quality assay |
EP1250897B1 (en) | 2000-01-03 | 2004-09-29 | Iberica de Reproduccion Asistida, S.L. | Artificial insemination device for pigs |
ATE216182T1 (en) | 2000-01-14 | 2002-05-15 | Artemis Pharmaceuticals Gmbh | METHOD FOR CRYOPRESERVING ZEBRAFISH SEEDS |
US6465169B2 (en) | 2000-01-14 | 2002-10-15 | Artemis Pharmaceuticals Gmbh | Method for cryoconservation of Zebrafish sperm |
IT1317724B1 (en) * | 2000-01-14 | 2003-07-15 | Istituto Sperimentale Italiano | PROCEDURE FOR THE PRODUCTION OF NON-HUMAN SEX EMBRYOS WITH A HIGH GENETIC VALUE. |
CA2398107C (en) | 2000-01-28 | 2013-11-19 | Althea Technologies, Inc. | Methods for analysis of gene expression |
US6587203B2 (en) | 2000-02-17 | 2003-07-01 | Cornell Research Foundation, Inc. | Sort stream stabilizer for flow cytometer |
US6618143B2 (en) | 2000-02-18 | 2003-09-09 | Idexx Laboratories, Inc. | High numerical aperture flow cytometer and method of using same |
US6646742B1 (en) | 2000-02-19 | 2003-11-11 | Mwi, Inc. | Optical device and method for multi-angle laser light scatter |
JP3587755B2 (en) | 2000-03-14 | 2004-11-10 | シスメックス株式会社 | Particle measuring apparatus and method |
GB2360360B (en) | 2000-03-14 | 2002-03-20 | Univ Bristol | A method of sorting cells |
US6482652B2 (en) | 2000-03-23 | 2002-11-19 | The Board Of Trustees Of The Leland Stanford Junior University | Biological particle sorter |
CA2403992A1 (en) | 2000-03-30 | 2001-10-11 | Max F. Rothschild | Genetic markers for improved meat characteristics in animals |
JP2003530568A (en) | 2000-04-11 | 2003-10-14 | シェモメテック・アクティーゼルスカブ | Method and apparatus for detecting fluorescence of a sample |
EP1147774A1 (en) | 2000-04-20 | 2001-10-24 | Stichting Dienst Landbouwkundig Onderzoek | Method for improving the quality of sperm for artificial insemination of animals |
AR034121A1 (en) | 2000-05-09 | 2004-02-04 | Xy Inc | METHOD FOR INSULATING Sperm Cells Containing X Chromosome from Sperm Cells Containing Chromosome and |
JP2001328901A (en) * | 2000-05-19 | 2001-11-27 | Seatsuku Yoshitomi Kk | Kit for cryopreservation of mouse sperm |
EP1287111A4 (en) | 2000-05-19 | 2005-12-28 | Dakocytomation Colorado Inc | A rapid multi-material sample input system |
US6590911B1 (en) | 2000-06-02 | 2003-07-08 | Coherent, Inc. | Passively modelocked harmonic-generating laser |
US6700130B2 (en) | 2001-06-29 | 2004-03-02 | Honeywell International Inc. | Optical detection system for flow cytometry |
US6784981B1 (en) * | 2000-06-02 | 2004-08-31 | Idexx Laboratories, Inc. | Flow cytometry-based hematology system |
US7351376B1 (en) | 2000-06-05 | 2008-04-01 | California Institute Of Technology | Integrated active flux microfluidic devices and methods |
US6503698B1 (en) | 2000-06-16 | 2003-01-07 | The United States Of America As Represented By The Secretary Of Agriculture | Cryopreservation of swine embryos |
JP2004503231A (en) | 2000-06-12 | 2004-02-05 | エックスワイ,インコーポレイテッド | Integrated swarm management system using isolated populations of X chromosome-bearing sperm and Y chromosome-bearing sperm |
DE10031028B4 (en) | 2000-06-26 | 2008-09-04 | Gnothis Holding Sa | Method for the selection of particles |
GB0016920D0 (en) | 2000-07-10 | 2000-08-30 | Univ Cambridge Tech | Decondensation of DNA |
US20040005582A1 (en) | 2000-08-10 | 2004-01-08 | Nanobiodynamics, Incorporated | Biospecific desorption microflow systems and methods for studying biospecific interactions and their modulators |
KR20030018069A (en) | 2000-08-10 | 2003-03-04 | 지티씨바이오쎄라퓨틱스,인크. | Cryopreservation of sperm |
FR2813283B1 (en) | 2000-08-25 | 2003-02-14 | Valois Sa | INTEGRATED PUMP DISPENSER |
EP1190684A1 (en) | 2000-09-05 | 2002-03-27 | Universiteit Gent | Device and method for artificial insemination of bovines and other animals |
US20020028434A1 (en) | 2000-09-06 | 2002-03-07 | Guava Technologies, Inc. | Particle or cell analyzer and method |
CA2421754C (en) | 2000-09-08 | 2012-04-10 | Iowa State University Research Foundation, Inc. | Novel prkag3 alleles and use of the same as genetic markers for reproductive and meat quality traits |
WO2002023163A1 (en) | 2000-09-15 | 2002-03-21 | California Institute Of Technology | Microfabricated crossflow devices and methods |
GB0023041D0 (en) | 2000-09-20 | 2000-11-01 | Univ Manchester | Identification apparatus |
WO2002026114A2 (en) | 2000-09-27 | 2002-04-04 | Bitensky Mark W | Cellular diagnostic arrays, methods of using and processes for producing same |
CN1325909C (en) | 2000-09-27 | 2007-07-11 | 清华大学 | Apparatus for particle operation and guide and use method thereof |
BR0005045A (en) | 2000-09-28 | 2002-05-14 | Marcos Fernando De Resende Mat | Sperm sexing method using the classic complement system pathway, eliminating the alternative pathway by inactivating protein "b" |
WO2002029106A2 (en) | 2000-10-03 | 2002-04-11 | California Institute Of Technology | Microfluidic devices and methods of use |
US20040031071A1 (en) | 2000-10-05 | 2004-02-12 | Xy, Inc. | System of hysteroscopic insemination of mares |
BR0114442A (en) * | 2000-10-05 | 2004-02-17 | Xy Inc | Hysteroscopic Mares Insemination System |
US6563583B2 (en) | 2000-10-12 | 2003-05-13 | Amnis Corporation | Multipass cavity for illumination and excitation of moving objects |
JP2004537712A (en) | 2000-10-18 | 2004-12-16 | バーチャル・アレイズ・インコーポレーテッド | Multiple cell analysis system |
MXPA03003556A (en) | 2000-10-23 | 2005-04-11 | Medical Instill Tech Inc | Fluid dispenser having a rigid vial and flexible inner bladder. |
US20040034879A1 (en) | 2000-11-09 | 2004-02-19 | Steve Rothstein | Mammalian sex selection using genetic modification |
SE0004777D0 (en) | 2000-12-22 | 2000-12-22 | Amersham Pharm Biotech Ab | Separation of X and Y sperm cells |
AU1980202A (en) * | 2000-11-22 | 2002-06-03 | Pharmacia Corporation | Methods and apparatus for producing gender enriched sperm |
US6849423B2 (en) | 2000-11-29 | 2005-02-01 | Picoliter Inc | Focused acoustics for detection and sorting of fluid volumes |
US7713687B2 (en) * | 2000-11-29 | 2010-05-11 | Xy, Inc. | System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations |
AU2002220018A1 (en) | 2000-11-29 | 2002-06-11 | Colorado State University | System for in-vitro fertilization with spermatozoa separated into x-chromosome and y-chromosome bearing populations |
US20020064808A1 (en) | 2000-11-29 | 2002-05-30 | Mutz Mitchell W. | Focused acoustic energy for ejecting cells from a fluid |
US20020064809A1 (en) | 2000-11-29 | 2002-05-30 | Mutz Mitchell W. | Focused acoustic ejection cell sorting system and method |
WO2002044720A2 (en) | 2000-11-30 | 2002-06-06 | The Netherlands Cancer Institute | Membrane molecule indicator compositions and methods |
US6576291B2 (en) | 2000-12-08 | 2003-06-10 | Massachusetts Institute Of Technology | Preparation of nanocrystallites |
EP1352237A4 (en) | 2000-12-15 | 2009-03-04 | Beckman Coulter Inc | Electrical conductive containment system |
US6577387B2 (en) | 2000-12-29 | 2003-06-10 | Johnson & Johnson Vision Care, Inc. | Inspection of ophthalmic lenses using absorption |
CN1231472C (en) | 2001-01-29 | 2005-12-14 | 日内瓦大学 | Pyrimidine acyclonucleoside derivatives, preparation method and use thereof |
US6673095B2 (en) | 2001-02-12 | 2004-01-06 | Wound Healing Of Oklahoma, Inc. | Apparatus and method for delivery of laser light |
US7029916B2 (en) | 2001-02-21 | 2006-04-18 | Maxcyte, Inc. | Apparatus and method for flow electroporation of biological samples |
WO2002077011A2 (en) | 2001-03-12 | 2002-10-03 | The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon | Oxidation-reduction sensitive green fluorescent protein variants |
US6706163B2 (en) | 2001-03-21 | 2004-03-16 | Michael Seul | On-chip analysis of particles and fractionation of particle mixtures using light-controlled electrokinetic assembly of particles near surfaces |
US20050081256A1 (en) | 2001-03-22 | 2005-04-14 | Forsberg Erik J. | Sex- specific selection of sperm from transgenic animals |
DE60218074T2 (en) | 2001-03-29 | 2008-02-07 | Sysmex Corp. | flow cytometer |
CA2442282A1 (en) | 2001-03-29 | 2002-10-10 | Cellect Technologies Corp. | Methods devices and systems for sorting and separating particles |
JP2002308897A (en) * | 2001-03-30 | 2002-10-23 | Council Scient Ind Res | Protein from coelomic fluid of indian earthworm causing immotility of sperm |
WO2002081934A2 (en) | 2001-04-03 | 2002-10-17 | Micronics, Inc. | Pneumatic valve interface for use in microfluidic structures |
JP2002311027A (en) | 2001-04-09 | 2002-10-23 | Hitachi Software Eng Co Ltd | Beads, manufacturing method of beads, flow cytometer, and program |
US6416190B1 (en) * | 2001-04-27 | 2002-07-09 | University Of Chicago | Apparatus for using optical tweezers to manipulate materials |
US20020186375A1 (en) | 2001-05-01 | 2002-12-12 | Asbury Charles L. | Device and methods for detecting samples in a flow cytometer independent of variations in fluorescence polarization |
WO2002092161A1 (en) | 2001-05-10 | 2002-11-21 | Biophan, Llc | Miniaturized particle analyzer |
US7345758B2 (en) | 2001-05-17 | 2008-03-18 | Cytopeia | Apparatus for analyzing and sorting biological particles |
US7012689B2 (en) | 2001-05-17 | 2006-03-14 | Dako Colorado, Inc. | Flow cytometer with active automated optical alignment system |
US20020182590A1 (en) | 2001-05-25 | 2002-12-05 | Vanderbilt University | Determining protein function in cell culture using RNA interference |
US20030048433A1 (en) | 2001-06-01 | 2003-03-13 | Jean-Marie Desjonqueres | Cytometer signal processing system and method |
FR2825987B1 (en) | 2001-06-19 | 2003-12-12 | Valois Sa | FLUID PRODUCT DISPENSER |
AU2002318269A1 (en) | 2001-07-18 | 2003-03-03 | The Regents Of The University Of Michigan | Gas-focusing flow cytometer cell and flow cytometer detection system with waveguide optics |
US7704728B2 (en) | 2001-07-18 | 2010-04-27 | The University Of Michigan | Microfluidic gravity pump with constant flow rate |
FR2828281B1 (en) | 2001-08-02 | 2004-12-31 | Biocytex | DEVICE FOR ANALYZING A SAMPLE IN PARTICULAR BY FLOW CYTOMETRY |
AU2002320399A1 (en) | 2001-09-01 | 2003-03-18 | Pharmacia Corporation | Staining and sorting genomes and chromosomes using sequence-specific polyamides |
DE10151216A1 (en) | 2001-10-16 | 2003-04-24 | Zeiss Carl Jena Gmbh | Method for the optical detection of characteristic quantities of an illuminated sample |
US20030078703A1 (en) | 2001-10-19 | 2003-04-24 | Surromed, Inc. | Cytometry analysis system and method using database-driven network of cytometers |
US6838289B2 (en) | 2001-11-14 | 2005-01-04 | Beckman Coulter, Inc. | Analyte detection system |
US6831279B2 (en) | 2001-11-27 | 2004-12-14 | Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of National Defence | Laser diode-excited biological particle detection system |
WO2003053055A1 (en) | 2001-12-18 | 2003-06-26 | Thomson Licensing S.A. | Internally generated close captioning/tele-texting for set-up menus of network-capable signal processing apparatus |
AU2002363895A1 (en) | 2001-12-31 | 2003-07-15 | Institut Fur Physikalische Hochtechnologie E.V. | Cell sorting system for the size-based sorting or separation of cells suspended in a flowing fluid |
WO2003056335A2 (en) | 2001-12-31 | 2003-07-10 | Institut für Physikalische Hochtechnologie e.V. | Micro-recess array for size-dependent sorting or separation of cells suspended in a flowing medium and method for analysing the functional activity of individual cells |
WO2003062796A1 (en) | 2002-01-22 | 2003-07-31 | Dakocytomation Denmark A/S | Environmental containment system for a flow cytometer |
US6698627B2 (en) | 2002-02-19 | 2004-03-02 | Valois S.A.S. | Fluid dispenser |
US6849394B2 (en) | 2002-02-21 | 2005-02-01 | Minitube Of America | Compositions comprising reproductive cell media and methods for using such compositions |
EP1478739A1 (en) | 2002-02-27 | 2004-11-24 | The Regents of the University of Michigan | Process for sorting motile particles from lesser-motile particles and apparatus suitable therefor |
US20030175980A1 (en) | 2002-03-14 | 2003-09-18 | Hayenga Jon W. | Ribbon flow cytometry and cell sorting |
US7223371B2 (en) | 2002-03-14 | 2007-05-29 | Micronics, Inc. | Microfluidic channel network device |
JP2004000144A (en) | 2002-03-29 | 2004-01-08 | Aisin Seiki Co Ltd | Cell separating and sorting apparatus and substrate for cell alignment |
US7312085B2 (en) * | 2002-04-01 | 2007-12-25 | Fluidigm Corporation | Microfluidic particle-analysis systems |
US20050251476A1 (en) | 2002-06-28 | 2005-11-10 | Monsanto Technology Llc | Swine genetics business system |
EP1531813A1 (en) | 2002-07-10 | 2005-05-25 | Applied Research Systems ARS Holding N.V. | Use of compounds for increasing spermatozoa motility |
EP1542529A4 (en) | 2002-07-22 | 2006-08-09 | Xy Inc | Sperm cell process system |
CN1774623B (en) | 2002-07-31 | 2012-02-01 | 阿尔利克斯公司 | System and method of sorting materials using holographic laser steering |
AU2003265362B2 (en) | 2002-08-01 | 2009-11-05 | Xy, Llc. | Low pressure sperm cell separation system |
BRPI0313476B1 (en) | 2002-08-15 | 2015-06-23 | Xy Llc | High resolution flow cytometer |
US7169548B2 (en) | 2002-09-13 | 2007-01-30 | Xy, Inc. | Sperm cell processing and preservation systems |
JP3983151B2 (en) | 2002-09-25 | 2007-09-26 | ダイワ精工株式会社 | Fishing spinning reel |
US7201875B2 (en) | 2002-09-27 | 2007-04-10 | Becton Dickinson And Company | Fixed mounted sorting cuvette with user replaceable nozzle |
US20040061853A1 (en) | 2002-09-27 | 2004-04-01 | Blasenheim Barry J. | Prism-based flow cytometry excitation optics |
US6941005B2 (en) | 2002-11-01 | 2005-09-06 | Coulter International Corp. | Monitoring and control of droplet sorting |
CA2506935A1 (en) | 2002-11-20 | 2004-06-03 | University Of Virginia Patent Foundation | Isolation of sperm cells from other biological materials using microfabricated devices and related methods thereof |
WO2004059282A2 (en) | 2002-12-19 | 2004-07-15 | Monsanto Technology Llc | Method and means for early detection of pregnancy in animals by combination testing |
DE602004024874D1 (en) | 2003-03-28 | 2010-02-11 | Inguran Llc | EASTER-ASSORTED TIERSPERMIES |
WO2004087177A1 (en) * | 2003-03-28 | 2004-10-14 | Monsanto Technology Llc | Process for the staining of sperm |
AU2004242121B2 (en) | 2003-05-15 | 2010-06-24 | Xy, Llc. | Efficient haploid cell sorting for flow cytometer systems |
US20050011582A1 (en) | 2003-06-06 | 2005-01-20 | Haug Jeffrey S. | Fluid delivery system for a flow cytometer |
US7460223B2 (en) * | 2003-09-19 | 2008-12-02 | Applied Biosystems Inc. | Inverted orientation for a microplate |
EP1730523B1 (en) | 2004-03-29 | 2010-01-13 | Inguran, LLC | Use of a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly in a staining or sorting process of spermatozoa |
ES2397678T3 (en) | 2004-03-29 | 2013-03-08 | Inguran, Llc | Sperm suspensions for classification in enriched populations carrying the X or Y chromosome |
MX2007000888A (en) | 2004-07-22 | 2007-04-02 | Monsanto Technology Llc | Process for enriching a population of sperm cells. |
DK2884258T3 (en) | 2004-07-27 | 2017-01-02 | Beckman Coulter Inc | IMPROVING FLOW CYTOMETRIC DISCRIMINATION WITH COMPUTER IMPLEMENTED GEOMETRIC TRANSFORMATION |
US20060118167A1 (en) | 2004-12-03 | 2006-06-08 | Xy, Inc. | Pressure regulated continuously variable volume container for fluid delivery |
US20060147894A1 (en) | 2004-12-30 | 2006-07-06 | Vicam, L.P. | Jacketed vessel for holding semen for sex biasing mammals through artificial insemination and systems and methods for enhancing the probability of sex biasing using the same |
US7591064B2 (en) | 2005-07-20 | 2009-09-22 | Hitachi Global Storage Technologies Netherlands B.V. | Method of fabrication for tape medium read head with unitary formation of multiple elements |
US7618770B2 (en) | 2005-07-29 | 2009-11-17 | Xy, Inc. | Methods and apparatus for reducing protein content in sperm cell extenders |
US7732408B2 (en) * | 2005-10-19 | 2010-06-08 | Iversync Ii Llc | Reproductive management |
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