US20150112125A1 - Methods for sorting sperm and producing sexed embryos - Google Patents

Methods for sorting sperm and producing sexed embryos Download PDF

Info

Publication number
US20150112125A1
US20150112125A1 US14/581,142 US201414581142A US2015112125A1 US 20150112125 A1 US20150112125 A1 US 20150112125A1 US 201414581142 A US201414581142 A US 201414581142A US 2015112125 A1 US2015112125 A1 US 2015112125A1
Authority
US
United States
Prior art keywords
sperm cells
sperm
method
cells
sheath fluid
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.)
Abandoned
Application number
US14/581,142
Inventor
George E. Seidel
Lisa A. Herickhoff
John L. Schenk
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
XY Inc
Original Assignee
XY Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Family has litigation
Priority to US09/001,394 priority Critical patent/US6149867A/en
Priority to US09/511,959 priority patent/US6524860B1/en
Priority to US10/378,109 priority patent/US7195920B2/en
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=21695819&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20150112125(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to US11/536,492 priority patent/US20070026379A1/en
Priority to US13/764,408 priority patent/US9365822B2/en
Application filed by XY Inc filed Critical XY Inc
Priority to US14/581,142 priority patent/US20150112125A1/en
Assigned to COLORADO STATE UNIVERSITY RESEARCH FOUNDATION reassignment COLORADO STATE UNIVERSITY RESEARCH FOUNDATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SEIDEL, GEORGE E.
Assigned to XY, INC. reassignment XY, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HERICKHOFF, LISA, SCHENK, JOHN
Assigned to XY, INC. reassignment XY, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: COLORADO STATE UNIVERSITY RESEARCH FOUNDATION
Assigned to XY, LLC reassignment XY, LLC CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: XY, INC.
Assigned to COMPASS BANK reassignment COMPASS BANK SECURITY INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: XY, LLC
Publication of US20150112125A1 publication Critical patent/US20150112125A1/en
Application status is Abandoned legal-status Critical

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues ; Not used, see subgroups
    • C12N5/0602Vertebrate cells
    • C12N5/0608Germ cells ; Not used, see subgroups
    • C12N5/0612Germ cells ; Not used, see subgroups sorting of gametes, e.g. according to sex or motility
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027New breeds of vertebrates
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES, AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02Preservation of living parts
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES, AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02Preservation of living parts
    • A01N1/0205Chemical aspects
    • A01N1/021Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61DVETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
    • A61D19/00Instruments or methods for reproduction or fertilisation
    • A61D19/02Instruments or methods for reproduction or fertilisation for artificial insemination
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61DVETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
    • A61D19/00Instruments or methods for reproduction or fertilisation
    • A61D19/04Instruments or methods for reproduction or fertilisation for embryo transplantation
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30Staining; Impregnating Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N2015/149Sorting the particles
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/10Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/101666Particle count or volume standard or control [e.g., platelet count standards, etc.]
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/10Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/108331Preservative, buffer, anticoagulant or diluent

Abstract

A method of sorting sperm cells and a method of producing at least one sexed embryo. The method of sorting sperm cells includes the steps of establishing a sheath fluid environment including a citrate for stained sperm cells, establishing a stream including stained sperm cells in said sheath fluid environment, sensing a property of the stained sperm cells, and discriminating between stained sperm cells for a desired sex characteristic. The method of producing at least one sexed embryo includes the steps for sorting sperm cells in addition to fertilizing at least one egg with the sexed sperm to form at least one sexed embryo.

Description

  • This application is a continuation of U.S. patent application Ser. No. 13/764,408, filed Feb. 11, 2013, which is continuation of U.S. patent application Ser. No. 11/536,492, filed on Sep. 28, 2006, which is a continuation of U.S. patent application Ser. No. 10/378,109, filed Feb. 25, 2003, now U.S. Pat. No. 7,195,920 which is a divisional of U.S. patent application Ser. No. 09/511,959, filed on Feb. 23, 2000, now U.S. Pat. No. 6,524,860, which is a divisional of U.S. patent application Ser. No. 09/001,394, filed on Dec. 31, 1997, now U.S. Pat. No. 6,149,867, each of which are incorporated herein by reference.
  • I. BACKGROUND OF THE INVENTION
  • This invention relates generally to the field of sex selection in mammalian offspring. It is especially relevant to the aspect of low dose artificial insemination for creating the desired sex of offspring. Particularly, the invention relates to systems for sorting sperm via flow cytometry for sex-specific and low dose efforts at artificial insemination or the like.
  • For ages it has been desired to select the sex of specific offspring. Beyond obvious psychological aspects, the actual sex selection of mammalian offspring has significant economic consequences when one considers its application to food producing animals such as cattle as well as celebrated trophy animals such as horses and the like. This great desire has resulted in a significant variety of efforts to achieve sex-selected offspring. Probably the effort which has appeared most likely to achieve the desired results has been efforts at sorting and selecting between X and Y sperm prior to insemination.
  • One of the challenges the sperm sorting effort faces is the large numbers of sorted sperm required. In natural insemination sperm are produced in some species by the billions; in artificial insemination less, but still significantly large numbers of sperm are used. For instance, artificial insemination techniques commonly use ten million to five hundred million sperm (depending on species). Thus a significant number of sperm are necessary even in an artificial insemination environment.
  • Many methods have been attempted to achieve the separation of X- and Y-chromosome bearing sperm. These methods have ranged from magnetic techniques such as appears disclosed in U.S. Pat. No. 4,276,139 to columnar techniques as appears disclosed in U.S. Pat. No. 5,514,537 to gravimetric techniques as discussed in U.S. Pat. No. 3,894,529, reissue Pat. No. 32,350, U.S. Pat. Nos. 4,092,229, 4,067,965, and 4,155,831. Electrical properties have also been attempted as shown in U.S. Pat. No. 4,083,957 as well as a combination of electrical and gravimetric properties as discussed in U.S. Pat. Nos. 4,225,405, 4,698,142, and 4,749,458. Motility efforts have also been attempted as shown in U.S. Pat. Nos. 4,009,260 and 4,339,434. Chemical techniques such as those shown in U.S. Pat. Nos. 4,511,661 and 4,999,283 (involving monoclonal antibodies) and U.S. Pat. Nos. 5,021,244, 5,346,990, 5,439,362, and 5,660,997 (involving membrane proteins), and U.S. Pat. Nos. 3,687,803, 4,191,749, 4,448,767, and 4,680,258 (involving antibodies) as well as the addition of serum components as shown in U.S. Pat. No. 4,085,205. While each of these techniques has been presented as if to be highly efficient, in fact at present none of those techniques yield the desired level of sex preselection.
  • At present, the only quantitative technique used to achieve the separation of X- and Y-chromosome bearing sperm has been that involving individual discrimination and separation of the sperm through the techniques of flow cytometry. This technique appeared possible as a result of advances and discoveries involving the differential dye absorption of X- and Y-chromosome bearing sperm. This was discussed early in U.S. Pat. No. 4,362,246 and significantly expanded upon through the techniques disclosed by Lawrence Johnson in U.S. Pat. No. 5,135,759. The Johnson technique of utilizing flow cytometry to separate X- and Y-chromosome bearing sperm has been so significant an advancement that it has for the first time made the commercial separation of such sperm feasible. While still experimental, separation has been significantly enhanced through the utilization of high speed flow cytometers such as the MoFlo® flow cytometer produced by Cytomation, Inc. and discussed in a variety of other patents including U.S. Pat. Nos. 5,150,313, 5,602,039, 5,602,349, and 5,643,796 as well as international PCT patent publication WO 96/12171. While the utilization of Cytomation's MoFlo® cytometers has permitted great increases in speed and while these speed increases are particularly relevant given the high number of sperm often used, certain problems have still remained. In spite of the almost ten-fold advances in speed possible by the MoFlo® flow cytometer, shorter and shorter sorting times have been desired for several reasons. First, it has been discovered that as a practical matter, the sperm are time-critical cells. They lose their effectiveness the longer they remain unused. Second, the collection, sorting, and insemination timings has made speed an item of high commercial importance. Thus, the time critical nature of the sperm cells and the process has made speed an essential element in achieving high efficacy and success rates.
  • Other problems also exist ranging from the practical to the theoretical. On the practical side, it has been desired to achieve sex-sorted sperm samples using inexpensive disposable components and substances. Also on the expense side, it has been desired to be able to achieve sorting (as well as collection and insemination) in as efficient a labor event as possible. Thus, for commercial production and success in this field, improvements which might only represent an increase in efficiency may still be significant. Related to the practical aspect of expense, is the practical aspect of the delicateness and sensitivity of the entire process. In this regard, it has been desired to simplify the process and make it as procedurally robust as possible so that operator error or skill can play an ever decreasing role.
  • In addition to the delicateness of the process, it has always been known that the sperm themselves are extremely delicate cells. While this factor at first glance seems like it might be considered easily understood, in fact, the full extent of the cells' sensitivities have not yet been fully explored. In the context of flow cytometry in general, most sorted cells or particles have often been spherical or otherwise physically able to withstand a variety of abuses. This is not the case for sperm cells. In fact, as the present invention discloses, the processing through normal flow cytometer techniques may, in fact, be unacceptable for cytometric sorting of sperm cells in certain applications. The sensitivities range from dilution problems and the flow cytometer's inherent need to isolate and distinguish each cell individually as well as the pressure and other stresses which typical flow cytometry has, prior to the present invention, imposed upon the cells or other substances that it was sorting. This may also represent a unique factor for sperm cells because it appears that even though the sperm cell may appear to pass through the flow cytometer and be sorted with no visually discernable side-effects, in fact, the cells themselves may have been stressed to the point that they perform less than optimally in the insemination process. Thus, an interplay of factors seems involved and has raised unusual problems from the perspective of sperm cell sorting and ultimate use for artificial insemination.
  • Another problem which has remained—in spite of the great advances achieved through the Johnson patent and related technology—is the fact that prior to the present invention it has been extremely difficult to achieve lower dosage insemination with sexed sperm. While historically, some achievement of low dose insemination has occurred, it has appeared to be more on a theoretical or laboratory environment rather than from environments which are likely to be experienced in or applicable to a commercial application. In this regard, the desire has not been merely to achieve low dose insemination but rather to achieve low dose insemination with pregnancy success rates which are comparable to existing unsexed, high dosage artificial insemination efforts. Thus, the advances achieved by the present inventors in both sexed and low dose artificial insemination represent significant advances which may, for the first time, make commercial applications feasible.
  • Another problem which has been faced by those in the industry—again, in spite of the great advances by the Johnson patent and related technology—is the fact that the problem itself, namely, artificial insemination with a high success rate is one of a statistical nature in which a multitude of factors seem to interplay. Thus, the solutions proposed may to some degree involve a combination of factors which, when thoroughly statistically studied, will be shown to be necessary either in isolation or in combination with other factors. Such a determination is further compounded by the fact that the results themselves vary by species and may be difficult to ascertain due to the fact that testing and statistical sampling on a large enough data base is not likely to be worth the effort at the initial stages. For these reasons the invention can also involve a combination of factors which may, individually or in combination, represent the appropriate solutions for a given application. This disclosure is thus to be considered broad enough so that the various combinations and permeations of the techniques disclosed may be achieved. Undiscovered synergies may exist with other factors. Such factors may range from factors within the sorting or flow cytometer steps to those in the collection as well as insemination steps. At present, studies have been primarily achieved on bovine species, however, it is not believed that these techniques will be limited to such species or, for that matter to only sperm cells. It appears that the techniques used may have application beyond just sperm cells into areas which involve either sensitive items to be sorted or merely minimization of the impacts of the stresses of flow cytometry upon the item sorted.
  • Interestingly, while the present invention takes an approach to minimize the impacts and stresses upon the sperm cells, others appear to have actually taken steps away from this direction by increasing pressures and demands for speed and other such performance. Essentially, the drive for low dose insemination and high speed processing may, in an individual or perhaps interrelated fashion have posed problems which limited one another. Thus, while there has been a long felt but unsatisfied need for high speed, low dose sexed insemination, and while the implementing arts and elements have long been available, prior to the present invention the advances or perhaps combinations of advances had apparently been overlooked by those skilled in the art. Perhaps to some degree they failed to appreciate that the problem involved an interplay of factors as well as peculiar necessities for the types of cells (sperm cells or perhaps species-specific sperm cells) involved in this field. Interestingly, as the listing of efforts earlier in this discussion shows, substantial attempts had been made but they apparently failed to understand the problem inherent in such an area as low dose, sexed insemination and had perhaps assumed that because the natural service event involves perhaps billions of sperm, there may have been physical limitations to the achievement of artificial insemination with numbers which are as many as four orders of magnitude less in number. Thus, it may not be surprising that there was to some extent an actual teaching away from the technical direction in which the present inventors went. Perhaps the results may even be considered unexpected to a degree because they have shown that sexed, low dose artificial insemination can be achieved with success rates comparable to those of unsexed, high dose artificial insemination. It might even be surprising to some that the techniques and advances of the present invention in fact combine to achieve the great results shown. While each technique could, in isolation, be viewed by some as unremarkable, in fact, the subtle changes appear to afford significant advances in the end result whether considered alone or in combination with other subtle changes.
  • Thus, until the present invention the achievement of success rates for low dose, sexed artificial insemination has not been possible with levels of performance necessary or simplified procedures likely to be necessary to achieve commercial implementation. The present invention discloses techniques which permit the achievement of improved performances and thus facilitate the end result desired, namely, low dose, sexed artificial insemination on a commercial basis.
  • II. SUMMARY OF THE INVENTION
  • Accordingly, the present invention provides improved sheath and collector systems for sorting of sperm cells to determine their sex through a flow cytometer. The sheath fluid as typically used in a flow cytometer is replaced with a fluid which minimizes the stress on the sperm cells as they are sorted. Furthermore, the collection system is improved to minimize both the physical and chemical stress to which the sperm cells are subjected. Various techniques and substances are represented but as those skilled in the art will readily understand, various combinations and permutations can be used in the manner which may be optimized for performance based in the species, goals and other parameters involved in a specific processing application.
  • An object of the invention is thus to achieve better sorting for substances such as sperm cells. A goal is to minimize the impact the sorting function itself has on the cells or other sensitive items which may be sorted. A particular goal is to minimize the impact the sheath fluid imposes upon the cells and to potentially provide a sheath fluid which affirmatively acts to assist the cells in handling the various stresses involved. A parallel goal is to provide substances and techniques which are especially suited for sperm cells in general, for bovine sperm cells, for equine sperm cells, and for the separation of such sperm cells into X- and Y-chromosome bearing components. Similarly a goal is to minimize the impacts that the collection phase (e.g., after sorting) has upon the cells and to minimize the physical impact as well as chemical impacts on such sex sorted sperm cells. Thus a goal is to achieve as unaffected a sorted result as possible.
  • Another object of the invention is to achieve low dose, sorted insemination on levels and with success rates which are comparable to those of the typical unsexed, high dose artificial insemination. Thus the prior goals of minimizing the stress or potential damage upon the sperm cells is important. Sorting in a manner which affords both high speed and low stress sorting, and which is especially adapted for sperm cell sorting in a low dose context is an important goal as well. The goals of providing sheath and other fluids which do not negatively affect the fertility of the sperm and which are compatible with artificial insemination are also important.
  • Naturally further objects of the invention are disclosed throughout other areas of the specification and claims.
  • III. BRIEF DESCRIPTION OF DRAWINGS
  • FIG. 1 is a schematic diagram of a sorter system according to the present invention.
  • FIG. 2 is a diagram of the entrained cells in the free fall area of a typical flow cytometer.
  • FIG. 3 is a conceptual diagram showing differences as they roughly appear as a result of the present invention.
  • FIG. 4 is a diagram of the sorted cell stream as they are collected in the landing zone area.
  • IV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
  • As will be seen, the basic concepts of the present invention can be combined and embodied in a variety of ways. The invention involves both improved flow cytometer systems as well a systems for the creation of sex-specific sperm samples which may be used in artificial insemination and the animals produced by such techniques. Furthermore, the techniques are disclosed in a general fashion so that they may be applied to specific systems and applications once the general principals are understood. While device enhancements are disclosed it should be understood that these enhancements not only accomplish certain methods but also can be varied and combined in a number of ways. Importantly, as to all of the foregoing, each of these facets should be understood to be encompassed by this disclosure.
  • As mentioned, the basic goal is that of separating the X-bearing sperm from the Y-bearing sperm. This is done in a manner which isolates the two types of sperm so that each can be separately packaged and dealt with. The isolation is preferably done through the use of flow cytometry. Flow cytometry in general is a technique which is well understood. For instance, the basic aspects of it are shown and discussed in a variety of patents to Cytomation, Inc. such as the U.S. Patents and other publications listed earlier. Each of these patents and the references cited therein, are incorporated by reference, thus those skilled in the art can easily understand the basic principles involved.
  • Essentially, flow cytometry involves sorting items, such as cells, which are provided to the flow cytometer instrument through some type of cell source. A conceptual instrument is shown in FIG. 1. The flow cytometer instrument includes a cell source (1) which acts to establish or supply cells or some other type of item to be analyzed by the flow cytometer. The cells are deposited within a nozzle (2) in a manner such that the cells are surrounded by a sheath fluid (3). The sheath fluid (3) is usually supplied by some sheath fluid source (4) so that as the cell source (1) supplies its cells, the sheath fluid (3) is concurrently fed through the nozzle (2). In this manner it can be easily understood how the sheath fluid (3) forms a sheath fluid environment for the cells. Since the various fluids are provided to the flow cytometer at some pressure, they flow out of nozzle (2) and exit at the nozzle orifice (5). By providing some type of oscillator (6) which may be very precisely controlled through an oscillator control (19), pressure waves may be established within the nozzle (2) and transmitted to the fluids exiting the nozzle (2) at nozzle orifice (5). Since the oscillator (6) thus acts upon the sheath fluid (3), the stream (7) exiting the nozzle orifice (5) eventually and regularly forms drops (8). Because the cells are surrounded by a sheath fluid environment, the drops (8) may contain within them individually isolated (generally) cells or other items.
  • Since the drops (8) generally contain isolated cells, the flow cytometer can distinguish and separate droplets based upon whether or not the appropriate cell or cells is/are contained within the drop. This is accomplished through a cell sensing system (9). The cell sensing system involves at least some type of sensor (10) which responds to the cells contained within each drop (8) as discussed at length in the seminal work (no pun intended) by Larry Johnson, namely, U.S. Pat. No. 5,135,759. As the Johnson patent explains for sperm cells, the cell sensing system (9) may cause an action depending upon the relative presence or relative absence of a particular dye which may be excited by some stimulant such as the laser exciter (11). While each type of sperm cell is stained by the dye, the differing length of the X-chromosome and the Y-chromosome causes different levels of staining Thus, by sensing the degree of dye present in the sperm cells it is possible to discriminate between X-bearing sperm and Y-bearing sperm by their differing emission levels.
  • In order to achieve the ultimate separation and isolation of the appropriate cells, the signals received by sensor (10) are fed to some type of sorter discrimination system (12) which very rapidly makes the decision and can differentially charge each drop (8) based upon whether it has decided that the desired cell does or does not exist within that drop (8). In this manner the sorter discrimination system (12) acts to permit the electrostatic deflection plates (13) to deflect drops (8) based on whether or not they contain the appropriate cell or other item. As a result, the flow cytometer acts to sort the cells by causing them to land in one or more collectors (14). Thus by sensing some property of the cells or other items the flow cytometer can discriminate between cells based on a particular characteristic and place them in the appropriate collector (14). In the system presently used to sort sperm, the X-bearing sperm droplets are charged positively and thus deflect in one direction, the Y-bearing sperm droplets are charged negatively and thus deflect the other way, and the wasted stream (that is unsortable cells) is uncharged and thus is collected in an undeflected stream into a suction tube or the like.
  • Referring to FIG. 2, the process can be even further understood. As shown in that figure, the nozzle (2) emits a stream (7) which because of the oscillator (6) (not shown in FIG. 2) forms drops (8). Since the cell source (1) (not shown in FIG. 2) may supply sperm cells (15) which have been stained according to the Johnson technique, the light stimulation by laser exciter (11) is differentially determined by sensor (10) so that the existence or nonexistence of a charge on each drop (8) as it separates from stream (7) can be controlled by the flow cytometer. This control results in positively charged, negatively charged, and uncharged drops (8) based upon their content. As shown in FIG. 2, certain drops are shown as deflected drops (16). These deflected drops (16) are those containing sperm cells (15) of the one or the other sex. They are then deposited in the appropriate collector (14) for later use.
  • One of the aspects of flow cytometry which is particularly important to its application for sperm sorting is the high speed operation of a flow cytometer. Advances have been particularly made by the flow cytometers available through Cytomation, Inc. under the MoFlo® trademark. These flow cytometers have increased sorting speeds extraordinarily and have thus made flow cytometry a technique which is likely to make feasible the commercial application of sperm sorting (among other commercial applications). They act to achieve high speed sorting, that is at a speed which is notably higher than those otherwise utilized. Specifically, Cytomation's MoFlo® flow cytometers act with oscillator frequencies of greater than about five kilohertz and more specifically can be operated in the 10 to 30 or even the 50 kilohertz ranges. Thus, droplets are formed at very high frequencies and the cells contained within the sheath fluid environment can be emitted very rapidly from the nozzle (2). As a result, each of the components such as the nozzle (2) oscillator (6), and the like which make up and are part of a flow cytometer system result in a high speed cell sorter.
  • In the application of a high speed cell sorter to the sorting of sperm cells, sorting at rates of greater than about 500 sorts per second is achieved. In fact, rates of sorting in the thousand and twelve hundred ranges have already been achieved through a high speed cell sorter. Importantly, it should be understood that the term “high speed” is a relative term such that as other advances in flow cytometry and specific applications are achieved, the aspect which is considered “high” may be varied or may remain absolute. In either definition, the general principle is that the sorting may occur at rates at which the parameters and physical characteristics of the flow cytometer are significant to the cells themselves when sorting particular cells such as sperm cells.
  • One aspect of high speed sorting which appears to come into play when sorting sperm cells is that of the pressures and other stresses to which the sperm cells are subjected within the flow cytometer. For instance, when operating at high speeds (and an alternative definition of “high speed”), flow cytometers can be operated at a pressure of 50 pounds per square inch and even 60 and higher pounds per square inch. These pressures may be considered high because they may result in effects upon the cells being sorted. The key as disclosed in the present invention for this facet is the fact that the stress thresholds of the particular cells are the determining factor. Additionally as further knowledge is gained it may be shown that the stress thresholds are a function of combined effects such as the particular species or the particular prior or subsequent handling of the cells. The key in this regard is that the stress imposed upon the cells can, in fact, alter their viability and their ability to achieve the desired result. In the pressure case, it may be that merely subjecting the sperm cells to a higher pressure as a result of the operation of the flow cytometer at that pressure may result in decreased performance of the cells. The present invention in one regard acts to minimize these stresses and thus results in greater efficacies as well as lower dosages as discussed later.
  • In considering the stress aspect of the cells, the present invention acts in a fashion which minimizes the stresses. These stresses can be minimized at any point in the overall cycle or process of collecting, sorting or even inseminating the animal. Importantly, the stress imposed by the handling of the cells within the flow cytometer appears significant for this application. In one embodiment of the invention, the sheath fluid is specifically selected so that it can serve in a coordinated fashion with both (or either) the pre-sort cell fluid environment or the post-sort cell fluid environment. While naturally it is possible to adjust either the pre- or post-sort fluids, in one embodiment the invention adjusts the sheath fluid (3) so that it imposes significantly less stress upon the cells than was previously accomplished. In one regard the invention is remarkable in that it removes the total focus from that of operation of the flow cytometer to a focus on handling and removing stress from the cells themselves. For instance, while it has been known to utilize fluids having a proper pH factor or osmolality, the present invention recognizes that there may be certain chemical compositions to which the cells may be hyper-responsive. These hyper-responsive chemical compositions may naturally vary based upon the cells or even the prior handling of the cells. Importantly at present it appears that for sperm cells certain metabolic chemical compositions such as citrate seem to prevent unusually high stresses upon the cells. Thus, the hyper-responsive chemical compositions can be defined as those to which the cells are particularly responsive in the context of their functionality and the then-existing handling techniques. As to sperm cells it appears that metabolic compositions, specifically citrate constancy for bovine sperm cells and hepes buffer constancy for equine sperm cells may be very important. Thus the present invention acts to minimize the changes through the type of operation or the selection of substances which may act as a means for minimizing the changes which the cells experience.
  • For the sheath fluid, a substance is selected according to one embodiment of the invention so that it may be chemically coordinated to prevent minimal changes. Thus, by selecting the appropriate sheath fluid not only in context of flow cytometry parameters, but rather also in context of the cell parameters themselves, the changes experienced by the cells and the overall result of the sorting can be enhanced. This is shown conceptually in FIG. 3. FIG. 3 shows some type of chemical factor (such as citrate or other factors) as it may exist throughout the various phases of the process. For instance, the four phases shown might represent the following: phase I may represent the existence of the cells within the cell source (1), phase II might show the existence of the cells as they are sorted in the sheath fluid environment, phase III might show the cells as they are collected after sorting and phase IV might show the reconstituted cells in a storage medium after sorting. These four phases as shown for the prior art may experience vastly different chemical factor environments. As shown conceptually, however, in the present invention the cells may experience very little change, most notably the dip or drop experienced between phases I and II may be virtually absent. This is as a result of the selection of the appropriate sheath fluid as mentioned above. Thus, as a result of being subjected to an appropriate sheath fluid, the cells in the present invention may experience a much lower level of stress.
  • One of the potential generalities that may exist with respect to this phenomenon is the fact that certain chemical compositions may represent more hyper-responsive chemical compositions than others. While naturally this may vary based upon the species of sperm, the handling, or even the type of cell involved, it appears that the viability of the cells for their intended purpose (here, artificial insemination) varies greatly, naturally or because of sorting or both, and so the cells exhibit a hyper-responsive character with respect to that chemical composition. By selecting certain metabolic chemical compositions, most notably citrates or chemicals which are within the citric acid cycle, great advances appear possible. Thus for the bovine sperm application, the sheath fluid (3) is selected and coordinated so that it presents about a 2.9 percent sodium citrate composition. Specifically, the 2.9 percent sodium citrate solution may be created as follows:
      • 1. Place 29.0 grams of sodium citrate dihydrate (Na3C6H5O7.2H20) in a 1,000 ml volumetric flask
        • a. Dissolve sodium citrate in ¾ of water batch, then add water to volume.
      • 2. Add deionized or Nanopure water to make 1,000 ml final volume.
      • 3. Transfer to bottles and autoclave at 15 lbs pressure (245° F.) for at least 30 minutes
        • a. Autoclave solution using conditions to minimize evaporation (loose cover)
        • b. Be careful that water does not boil away.
      • 4. Cool slowly at room temperature.
      • 5. Store sealed in a 5° C. cold room.
  • Further, for a sheath fluid, the sodium citrate solution may be filtered.
      • 6. Filter with a 0.22 micron filter using aseptic techniques.
  • Interestingly, for equine sperm cells such a composition does not perform as well. Rather, it has been discovered that for equine sperm cells, a hepes buffered medium such as a hepes bovine gamete medium—particularly HBGM3 as previously created by J. J. Parrish for a bovine application—works well. This medium is discussed in the article “Capacitation of Bovine Sperm by Heparin”, 38 Biology of Reproduction 1171 (1988) hereby incorporated by reference. Not only is this surprising because it is not the same type of substance as is utilized for bovine sperm, but the actual buffer, originally was developed for a bovine application. Thus in the equine application the sheath fluid is selected which contains the hepes buffer. This solution may have a pH at room temperature of about 7.54 (pH at 39° C.=7.4) with the following composition:
  • Chemical Dry weight (g/500 ml) CaCl2 0.145 KCl2 0.115 MgCl2•6H20 0.004 NaH2PO4•H2O 0.018 NaCl 2.525 NaPyruvate 0.011 Lactic Acid (60%) 1.84 ml HEPES 4.765 NaHCO3 0.420 BSA (fraction V) 3.0
  • One other aspect which may interplay in the present invention is the fact that the cells involved may experience unusual sensitivities. In one regard this may be due to the fact that sperm cells are in a class of cells which are non-repairing cells. That is, they do not have the ability to repair themselves and hence, they may need to be treated much more sensitively than is typical for flow cytometers or other handling equipment. Thus, it may be appropriate that the enhancement is particularly applicable when the flow cytometer acts to establish a source of sperm cells. Another potentially related aspect which may be unique to a class of cells such as sperm cells is the fact that their DNA is non-repairing, non-replicating, and non-transcribing. Either of these factors may come into play and so they may be relevant either individually or together. Thus, it may be that the teachings of the present invention apply to all gamete cells or even to viruses and the like which are non-repairing, non-translating, non-transcribing cells.
  • A separate aspect of the flow cytometer processing which may also be important is the fact of properly treating the cells both chemically and physically after they are sorted. As shown in FIG. 4, as the cells within drops (8) land in collector (14), it may be important that the container which makes up the collector be properly sized so that it acts as some means of avoiding an impact between the cells and the container itself. While it has been known to place an initial collector fluid (17) in the bottom of the container to collect the cells so that they do not hit the bottom of the container, it appears that a simple widening of the container to address variations in stream presentation as well as the inevitable splashing due to the impact of the cells into the container can be used to enhance the result. In one regard this can act as a cushioning element so that cells which may be mechanically delicate, that is, they may break or be damaged by an impact can be treated appropriately. Thus when the cytometer source establishes cells which are physically delicate cells as the cells to be sorted, it may be important to provide some type of cushioning element such as a wide collection tube for which the opening width (18) serves to position the walls of the container in a manner which avoids contact with the cells. Thus the tube does not present side walls so close that there is any significant probability of contact between those cells being sorted and the walls of the tube. In this manner, in addition to the collector fluid (17), it may be desirable to include a wide collection tube as well. Perhaps merely providing a wide opening to the container which serves as part of the collector (14) may be sufficient. For applications utilizing high speed sorting of sperm cells, it has been found that providing a container having an inner diameter opening of at least 15 millimeters is believed to be sufficient. Specifically when utilizing a 14 ml Falcon test tube in such an application, minimal physical damage to the cells as a result of the collector (14) has been discovered.
  • It should be noted that even the 14 ml Falcon test tube may not be optimum. Specifically, it is believed that designing a collection container which matches the geometry of the stream (that is, a “stream-matched container”) may be most optimal. This stream-matched container may have any or all of the following characteristics: a relatively wide orifice, an elliptically shaped orifice, a lesser height to width ratio than currently involved, an angled or otherwise coordinated presentation such as may present side walls which are parallel to the falling streams, and the like. It may also be desirable to provide a mounting element such as a movable element or medium like ball bearings or the like to permit variable orientation of the tube to match the falling stream desired to be collected. In addition, the physical characteristics for the class of containers such as the existing tube (described as a “Falcon-type” test tube) may include not only the width of the tube but also the material (such polystyrene to which the cells do not stick) out of which it is made and the like. (These material options are well known for the 14 ml Falcon tube.) Thus the container and it collection fluid may also serve as a cushioning element to minimize physical damage to the cells. It also can serve, by its size, to facilitate collection of adequate numbers of sperm without a significant dilution effect.
  • Another aspect of the collector fluid (17) can be the fact that it, too, may serve to minimize chemical stresses upon the cells. In one regard, since it may be important to provide a nutrient to the cells both before and after sorting, the collector fluid (17) may be selected so as to provide a coordinated level of nutrient so that the levels are balanced both before and after sorting. For bovine sperm in which a nutrient of egg yolk citrate is utilized at a two percent egg yolk level, it has been discovered that utilizing a six percent egg yolk citrate level (that is six percent egg yolk content in a citrate solution) provides good results. This is as result of the volumes existing before and after the sorting event. The collector fluid (17) may start (before sorting) with about 2 ml of volume. The sorting event may add about double this volume (ending at three times the initial starting volume) with very little egg yolk citrate in solution (due to clogging and other flow cytometer considerations). Thus, the end result in terms of the level of the amount of egg yolk citrate present may be equivalent to the starting result, namely, two percent egg yolk content in a citrate solution due to the volumes involved. Thus the collector fluid (17) may be selected so as to create an ending collector fluid environment which is balanced with the initial nutrient or other fluid environment. In this manner, it may serve to minimize the time and changed level of composition to which the cells are subjected. Naturally, these fluid environments may be presented within the flow cytometer or may exist at some other prior time, the important point being merely minimizing the stress to which the cells are subjected at any time in their life cycle. Furthermore, since the initial chemical substance content can be varied (for instance the percent egg yolk content in the citrate may be varied up or down), likewise the starting collection fluid environment or various volumes may also be varied so that the ending result is the same. Thus, prior to commencing the sorting process, the collector fluid exists with a six percent egg yolk content in the citrate solution and after completion of the sorting event the collector fluid with the sex-specific sperm may result in a two percent egg yolk content in the citrate solution similar to the initial nutrient content.
  • Note that in later use these sperm cells may be treated to a 20% egg yolk content in the citrate fluid for other reasons, however these changes are not deemed to provide stress to the cells as they are merely a known part of the total insemination process. While naturally the levels may be varied as those skilled in the art readily understand, a 20% egg yolk citrate buffer may be constituted as follows:
  • I. Final Composition:
  • 80% sodium citrate solution (72 mM)
  • 20% (vol/vol) egg-yolk
  • II. Preparation for 1 Liter:
  • A. Sodium citrate solution
      • 1. Place 29.0 grams of sodium citrate dihydrate (Na3C6H5O7.2H2O) in a 1,000 ml volumetric flask
      • 2. Add deionized or Nanopure water to make 1,000 ml final volume.
      • 3. Transfer to bottles and autoclave at 15 lbs pressure (245° F.) for at least 30 minutes.
        • a. Autoclave solution using conditions to minimize evaporation (loose cover)
        • b. Be careful that water does not boil away.
      • 4. Cool slowly at room temperature.
      • 5. Store sealed in a 5° C. cold room.
  • B. Egg preparation
      • 1. Obtain fresh hen's eggs from a good commercial source.
      • 2. Wash the eggs free of dirt (do not use too much detergent) and rinse.
      • 3. Immerse eggs in 70% ethanol for 2-5 minutes.
      • 4. Remove eggs and allow to dry (or wipe dry) and store on a clean towel.
  • C. Preparation of extender
      • 1. Use sterile, clean glassware
      • 2. A-fraction (non-glycerol fraction)
        • a. Place 800 ml of 2.9% sodium citrate solution in a 1,000 ml graduated cylinder.
        • b. Antibiotic levels for the non-glycerol containing fraction (A-fraction) of the extender may be as follows:
          • i. Tylosin=100 μg/ml
          • ii. Gentamicin=500 μg/ml
          • iii. Linco-spectin=300/600 μg/ml
        • c. Add 200 ml of fresh egg-yolk as outlined below (Section D)
          • i. Mix very thoroughly.
        • d. This provides A-fraction extender based on 2.9% sodium citrate, with 20% egg-yolk and antibiotics at concentrations known to be non-toxic to bull sperm.
        • e. Extender can be stored overnight at 5° C.
        • f Decant supernatant (upper 800 ml) the next day.
        • g. Warm to 37° C. prior to use the next day.
  • D. To add egg-yolk to a buffered solution, the following procedure works well.
      • 1. Wash egg and clean the eggs (see B above)
      • 2. Open egg and separate yolk from albumin using a yolk separator. Alternatively, pour yolk back and forth 2-3 times between the two half shells. Do not rupture the membrane around the yolk.
      • 3. Place the yolk onto a sterile piece of 15 cm filter paper.
      • 4. Hold the filter paper over the graduated cylinder containing buffer and squeeze the yolk (rupturing the membrane) and allow the yolk to run out of the folded filter paper into the cylinder. Typically about 12-15 ml of the yolk can be obtained from one egg.
  • Another aspect which may interplay in the various factors of the present invention is that of utilizing low dose amounts of sperm for artificial insemination or the like. Additional background on the aspect of sexed, artificial insemination may be found in “Prospects for Sorting Mammalian Sperm” by Rupert P. Amman and George E. Seidel, Jr., Colorado Associated University Press (1982) hereby incorporated by reference. As mentioned, natural insemination involves numbers of sperm on the order of billions of sperm. Typical artificial insemination is presently conducted with millions of sperm for bovine species and hundreds of millions of sperm for equine species. By the term “low dose” it is meant that the dosage of sperm utilized in the insemination event are less than one-half or preferably even less than about 10% of the typical number of sperm provided in a typical artificial insemination event. Thus, the term “low dose” is to be viewed in the context of the typical artificial insemination dosage or also as an absolute number. For bovine sperm where currently 1 to 10 million sperm are provided, a low dose process may be considered an absolute number of about 500,000 sperm or perhaps as low as 300,000 sperm or lower. In fact, through utilization of the techniques of the present invention, artificial insemination with good percentages of success has been shown with levels of insemination of sperm at 100,000 and 250,000 sperm (41% and 50%, respectively pregnancy rates). As shown in the article “Uterine Horn Insemination of Heifers With Very Low Numbers of Non-frozen and Sexed Spermatozoa” as published in 48 Theriogenology 1255 (1997) hereby incorporated by reference. Since sperm cells appear to display a sensitivity to dilution, these results may display particular interdependence on the utilization of low dose sperm samples with regards to various techniques of the present invention. The absolute numbers may be species dependent, for equine species, merely less than about ten, five, or even one million sperm may be considered a low dose process.
  • Another aspect which may be important is the fact that the sperm sexed through the present invention techniques is utilized in an artificial insemination system. Thus, when the collector (14) is used to provide sperm for artificial insemination the techniques of the present invention may be particularly relevant. Further, it is possible that the combination of both artificial insemination use and the use in a low dose environment may together create synergies which makes the various techniques of the present invention particularly appropriate. Naturally, the sexed sperm can be utilized not just in an artificial insemination mode, but in other techniques such as in vitro fertilization and the like.
  • The process of collecting, sorting, and eventually inseminating an animal through the use of flow cytometry involves a variety of steps. In the context of bovine insemination, first the semen is collected from the bull through the use of an artificial vagina. This occurs at rates of approximately 1.5 billion sperm per ml. This neat semen may be checked through the use of a spectrophotometer to assess concentration and may be microscopically evaluated to assure that it meets appropriate motility and viability standards. Antibiotics may then added. As a result the initial sample may have approximately 60 to 70 percent of the progressively motile sperm per ejaculate. For processing, a dilution through of some type TALP (tyrode albumin lactate pyruvate) may be used to get the numbers of sperm at a manageable level (for flow analysis) of approximately 100 million per ml. The TALP not only nurtures the sperm cells, but it may make them hyper-activated for the staining step. Prior to staining, in some species such as the equine species, centrifugation may be accomplished. Staining may be accomplished according to a multi-stained or single-stained protocol, the latter, the subject of the Johnson Patent and related technology. The staining may be accomplished while also adjusting the extender to create the appropriate nutrient environment. In bovine applications this may involve adding approximately 20% egg yolk content in a citrate solution immediately after staining. Further, in staining the sperm cells, it has been discovered that by using higher amounts of stain than might to some extent be expected better results may be achieved. This high concentration staining may involve using amounts of stain in the tens of micro-molar content such as discussed in the examples below where 38 micro-molar content of Hoechst 33342 stain was used.
  • After adding the stain, an incubation period may be used such as incubating at one hour at 34° C. to hasten the dye uptake with concentrations at about 100 million sperm cells per ml. Filtration may then be accomplished to remove clumps of sperm cells and then dilution or extending may or may not be accomplished to the desired sort concentration of approximately 100 million sperm cells per ml may be accomplished. Sorting according to the various techniques discussed earlier may then be accomplished from which sperm cells may be recovered in the collection phase. As mentioned earlier, the collection may result in samples with approximately 2% egg yolk citrate concentrate content (for bovine species). This sample may then be concentrated to about 3-5 million sperm cells per ml through the use of centrifugation after which the sheath fluid and preserving fluid may be removed. A final extension may then be accomplished with either 20% egg yolk citrate or a Cornell Universal Extender or the like. The Cornell Universal Extender may have the following composition for 1000 ml:
      • 14.5 g sodium citrate dihydrate
      • 2.1 g NaHCO3
      • 0.4 g KCl
      • 3.0 g glucose
      • 9.37 g glycine
      • 0.87 g citric acid
      • For 20% egg-yolk
        using 800 ml of above preparation and may include about 200 ml of egg-yolk composition.
  • After this last extending, 3 to 5 million sperm per ml (for bovine species) may result. This sample may then be cooled to slow the sperm's metabolism and to permit use over longer periods of time. In the equine species the sample may then be used in oviductal or other insemination processes as those skilled in the art well understand. In bovine sperm, the sample may be diluted yet one more time to the desired dosage level. It has been discovered that dilution may create an effect upon the sperm cell's viability and so it may be appropriate to avoid too large a level of dilution by providing a smaller sample. At present, low dosages of approximately 300,000 sperm per 0.184 ml may be achieved. Furthermore, it may be desirable to maintain a level of seminal plasma at approximately a five percent level, although the results of this requirement are, at present, mixed. The sperm cell specimen may then be placed in a straw for use in artificial insemination and may be transported to the cows or heifers to be inseminated.
  • In order to achieve conveniently timed artificial insemination, heifer or cow estrus may be synchronized using known techniques such as the utilization of prostaglandin F2α, according to techniques well known in the art. This latter substance may be particularly valuable in that it has been reported to potentially achieve enhanced fertility in heifers as discussed in the article “Prostoglandin F2α, —A Fertility Drug in Dairy Cattle?”, 18 Theriogenology 245 (1982) hereby incorporated by reference. While recent results have not maintained this premise, it may be that the present invention demonstrates its particular viability in situations of sexed, low dose insemination. For bovine species, artificial insemination may then be accomplished through the use of embryo transfer equipment with placement of the sperm cells deep within the uterine horns. This may be accomplished not at the peak moment as typically used in artificial insemination, but rather at a somewhat later moment such as 12 hours after that time since there is some possibility that fertility for sexed artificial insemination may occur slightly later. The utilization of embryo transfer equipment may be used because there may be high sensitivity of the uterine wall for such low dose, sexed inseminations.
  • Interestingly, rather than inseminating within the uterine body where such insemination are usually placed, by insemination deep within the uterine horn, better results may be achieved. Perhaps it is also surprising that the samples thus far studied have shown no difference between ipsi- and contra-lateral inseminations when accomplished deep within the uterine horn. By deep, it should be understood that the insertion is placed well into the uterine horn using the embryo transfer equipment. The fact that results do not appear significantly different using ipsi- and contra-lateral inseminations has led the present inventors to propose the use of insemination in both so that the process of identifying the appropriate uterine horn may no longer be needed.
  • As a result of the insemination, it is of course desired that an animal of the desired sex be produced. This animal may be produced according to the systems discussed earlier through the use of the sexed sperm specimen. It should also be understood that the techniques of the present invention may find application in other techniques such as laproscopic insemination, oviductal insemination, or the like.
  • As examples, the following experiments have been conducted. While not all use every aspect of the inventions described here, they do show the performance enhancements possible through differing aspects of the invention. Further, a summary of some experiments is contained in the article “Uterine Horn Insemination of Heifers With Very Low Numbers of Non-frozen and Sexed Spermatozoa” as referenced earlier. This article summarizes some of the data showing the efficacy of the present invention. As to the experiments, one has been conducted with sexed, unfrozen sperm cells with high success as follows:
  • Example 1
  • Angus heifers, 13-14 mo of age and in moderate body condition, were synchronized with 25 mg of prostaglandin F-2 alpha at 12-day intervals and inseminated 6-26 h after observed standing estrus. Freshly collected semen from three 14-26 mo old bulls was incubated in 38 μM Hoechst 33342 at 75×106 sperm/ml in a TALP medium for 1 h at 34° C. Sperm were sorted by sex chromosomes on the basis of epiflourescence from laser excitation at 351 and 364 nm at 150 mW using a MoFlo® flow cytometer/cell sorter operating at 50 psi and using 2.9% Na citrate as sheath fluid. X chromosome-bearing sperm (˜90% purity as verified by resorting sonicated sperm aliquots) were collected at ˜500 live sperm/sec into 2-ml Eppendorf tubes containing 100 μl Cornell Universal Extender (CUE) with 20% egg yolk. Collected sperm were centrifuged at 600×g for 10 min and resuspended to 1.63×106 live sperm/ml in CUE. For a liquid semen unsexed control; Hoechst 33342-stained sperm were diluted with sheath fluid to 9×105 sperm/ml and centrifuged and resuspended to 1.63×106 progressively motile sperm/ml in CUE. Sexed semen and liquid control semen were cooled to 5° C. over 75 min and loaded into 0.25-ml straws (184 μl/straw). Straws were transported at 3 to 5° C. in a temperature-controlled beverage cooler 240 km for insemination 5 to 9 h after sorting. Sexed semen and liquid control semen were inseminated using side-opening blue sheaths (IMV), one half of each straw into each uterine horn (3×105 live sperm/heifer). As a standard control, semen from the same bulls had been frozen in 0.5-cc straws by standard procedures (mean 15.6×106 motile sperm/dose post-thaw), thawed at 35° C. for 30 sec, and inseminated into the uterine body. Treatments were balanced over the 3 bulls and 2 inseminators in a ratio of 3:2:2 inseminations for the sexed semen and two controls. Pregnancy was determined ultrasonically 31-34 days after insemination and confirmed 64-67 days later when fetuses also were sexed (blindly). Data are presented in the table.
  • No. Heifers No. Pregnant No. Pregnant No female Treatment bred d31-34 d64-67 fetuses Sexed semen 45 20 (44%) 19 (42%) 18 (95%)a Liquid control 28 15 (54%) 15 (54%)  8 (53%)b Frozen control 29 16 (55%) 15 (52%) 12 (80%)c a,bSex ratios of values with different superscripts differ (P < 0.02).
  • Although the pregnancy rate with sexed semen was only 80% of controls, this difference was not statistically significant (>0.1). One pregnancy was lost by 64-67 d in each of the sexed and frozen control groups; 18 of 19 fetuses (95%) were female in the sexed group, and 20 of 30 (67%) were female in the control groups. The liquid semen control yielded a virtually identical pregnancy rate to the frozen semen control containing over 50 times more motile sperm (over 120 times more total sperm), demonstrating the efficacy of low-dose insemination into the uterine horns. We have altered the sex ratio in cattle significantly using flow cytometer technology and artificial insemination.
  • Similarly, an experiment was conducted with unsexed, unfrozen sperm cells and may be reported as follows:
  • Example 2
  • The objective was to determine pregnancy rates when heifers are inseminated with extremely low numbers of frozen sperm under ideal field conditions. Semen from three Holstein bulls of above average fertility was extended in homogenized milk, 7% glycerol (CSS) extender plus 5% homologous seminal plasma to 2×105, 5×105 or 10×106 (control) total sperm per 0.25 ml French straw and frozen in moving liquid nitrogen vapor. Semen was thawed in 37° C. water for 20 sec. Holstein heifers 13-15 mo of age weighing 350-450 kg were injected with 25 mg prostaglandin F-2-alpha (Lutalyse®) twice at a 12-day interval and inseminated with an embryo transfer straw gun and side-opening sheath, half of the semen deep into each uterine horn 12 or 24 h after detection of estrus. The experiment was done in five replicates over 5 months, and balanced over two insemination technicians. Ambient temperature at breeding was frequently −10 to −20° C., so care was taken to keep insemination equipment warm. Pregnancy was determined by detection of a viable fetus using ultrasound 40-44 days post-estrus and confirmed 55-62 days post-estrus; 4 of 202 conceptuses were lost between these times. Day 55-62 pregnancy rates were 55/103 (53%), 71/101, (70%), and 72/102 (71%) for 2×105, 5×105 and 10×106 total sperm/inseminate (P<0.1). Pregnancy rates were different (P<0.05) among bulls (59, 62, and 74%), but not between technicians (64 and 65%) or inseminations times post-estrus (65% for 12 h and 64% for 24 h, N=153 at each time). With the methods described, pregnancy rates in heifers were similar with 5×105 and 10×106 total sperm per inseminate.
  • Prior experiment has also been conducted on sexed, unfrozen sperm cells and may be reported as follows:
  • Example 3
  • Semen was collected from bulls at Atlantic Breeders Cooperative, diluted 1:4 with a HEPES-buffered extender+0.1% BSA, and transported 160 km (˜2 HR) to Beltsville, Md. where it was sorted at ambient temperature by flow cytometry into a TEST yield (20%) extender using methods described previously (Biol Reprod 41:199). Sorting rates of up to 2×106 sperm of each sex per 5-6 h at ˜90% purity were achieved. Sperm were concentrated by centrifugation (300 g for 4 min) to 2×106 sperm/ml. Some sperm were sorted into extender containing homologous seminal plasma (final concentration, 5%). Sorted sperm were shipped by air to Colorado (˜2,600 km) and stored at either ambient temperature or 5° C. (cooled during shipping over 6 hr in an Equitainer, an insulated device with an ice-containing compartment). Heifers or dry cows detected in estrus 11 to 36 h earlier were inseminated within 9 to 29 h of the end of the sperm sorting session. Sperm (1 to 2×105 in 0.1 ml) were deposited deep in the uterine horn ipsilateral to the ovary with the largest follicle as determined by ultrasound at the time of insemination.
  • None of 10 females became pregnant when inseminated with sperm shipped and stored at ambient temperature. Of 29 females inseminated with sperm cooled to 5° C. during shipping, 14 were pregnant at 4 weeks of gestation, and 12 (41%) at 8 weeks. Eleven of the 22 inseminated within 10 h of the end of sorting were pregnant at 8 weeks, but only 1 of 7 inseminated 17-24 h after sorting was pregnant. There was no significant effect of adding seminal plasma. One of the 12 fetuses was not of the predicted sex, one was unclear, and 10 were of the predicted sex, as determined by ultrasonography at 60-70 days of gestation.
  • Subsequently, 33 additional heifers were inseminated with 0.05 ml (semen extended as described above) into each uterine horn without using ultrasonography; only 3 were pregnant 4 weeks after insemination, and only 1 remained pregnant at 8 weeks.
  • However, different bulls were used from the previous group, and all inseminations were done 18-29 h post-sorting. An additional 38 heifers were inseminated similarly (˜22 h post-sorting) 200 km from our laboratory with sorted sperm from another bull; none of these was pregnant 8 weeks after insemination.
  • To summarize, it is possible to achieve pregnancies in cattle via artificial insemination of sperm sorted for sex chromosomes by flow cytometry, and the sex ratio of fetuses approximates that predicted by reanalysis of sorted sperm for DNA content (90%). However, pregnancy rates varied greatly in these preliminary experiments which required shipping sperm long distances. Fertility decreased drastically by 17 h post-sorting, but there was some confounding because different bulls were used at the different times. Further studies are needed to determine whether variation observed in pregnancy rates was due to bull differences, insemination techniques, interval between sorting and insemination, or other factors.
  • Finally, an experiment also has been conducted with unsexed, unfrozen sperm cells and may be reported as follows:
  • Example 4
  • The objective was to determine pregnancy rates when heifers were inseminated with very low numbers of sperm under ideal experimental conditions. Semen from three Holstein bulls was extended in Cornell Universal Extender plus 5% homologous seminal plasma to 1×105 or 2.5×105 sperm per 0.1 ml; 2.5×106 total sperm per 0.25 ml was used as a control. Fully extended semen was packaged in modified 0.25 ml plastic French straws to deliver the 0.1 or 0.25 ml inseminate doses. Semen was cooled to 5° C. and used 26-57 h after collection. Holstein heifers 13-15 mo of age weighing 350-450 kg were injected with 25 mg prostaglandin F-2 alpha (Lutalyse®) at 12-day intervals and inseminated with an embryo transfer straw gun and side-opening sheath into one uterine horn 24 h after detection of estrus. Insemination was ipsilateral to the side with the largest follicle determined by ultrasound 12 h after estrus; side of ovulation was verified by detection of a corpus luteum by ultrasound 7-9 days post-estrus. Pregnancy was determined by detection of a fetus by ultrasound 42-45 days post estrus. The experiment was done in four replicates and balanced over three insemination technicians. Side of ovulation was determined correctly in 205 of 225 heifers (91%); surprisingly, pregnancy rates were nearly identical for ipsilateral and contralateral inseminates. Pregnancy rates were 38/93 (41%), 45/87 (52%), and 25/45 (56%) for 1×105, 2.5×105 and 2.5×106 sperm/inseminate (P>0.1). There was a significant difference in pregnancy rate (P<0.05) among technician, but not among bulls. With the methods described, it may be possible to reduce sperm numbers per inseminate sufficiently that sperm sorted by sex with a flow cytometer would have commercial application.
  • As mentioned and as can be seen from the various experiments, the field is statistically base and thus a variety of additional experiments may be conducted to show the appropriate combination and limitation strategies. Thus synergies among various affects will further be identified, such as instances in which the dye effects and combined dye effects with laser excitation may be studied.
  • The discussion included in this application is intended to serve as a basic description. The reader should be aware that the specific discussion may not explicitly describe all embodiments possible; many alternatives are implicit. It also may not fully explain the generic nature of the invention and may not explicitly show how each feature or element can actually be representative of a broader function or of a great variety of alternative or equivalent elements. Again, these are implicitly included in this disclosure. Where the invention is described in device-oriented terminology, each element of the device implicitly performs a function. Apparatus claims may not only be included for the device described, but also method or process claims may be included to address the functions the invention and each element performs. Neither the description nor the terminology is intended to limit the scope of the claims which may be submitted. It should be understood that a variety of changes may be made without departing from the essence of the invention. Such changes are also implicitly included in the description. They still fall within the scope of this invention. A broad disclosure encompassing both the explicit embodiment(s) shown, the great variety of implicit alternative embodiments, and the broad methods or processes and the like are encompassed by this disclosure.
  • In addition, each of the various elements of the invention and claims may also be achieved in a variety of manners. This disclosure should be understood to encompass each such variation, be it a variation of an embodiment of any apparatus embodiment, a method or process embodiment, or even merely a variation of any element of these. Particularly, it should be understood that as the disclosure relates to elements of the invention, the words for each element may be expressed by equivalent apparatus terms or method terms—even if only the function or result is the same. Such equivalent, broader, or even more generic terms should be considered to be encompassed in the description of each element or action. Such terms can be substituted where desired to make explicit the implicitly broad coverage to which this invention is entitled. As but one example, it should be understood that all actions may be expressed as a means for taking that action or as an element which causes that action. Similarly, each physical element disclosed should be understood to encompass a disclosure of the action which that physical element facilitates. As but one example of this aspect, the disclosure of a “collector” should be understood to encompass disclosure of the act of “collecting”—whether explicitly discussed or not—and, conversely, were there only disclosure of the act of “collecting”, such a disclosure should be understood to encompass disclosure of a “collector.” Such changes and alternative terms are to be understood to be explicitly included in the description.
  • Any references mentioned in the application for this patent as well as all references listed in any information disclosure filed with the application are hereby incorporated by reference. In addition, the table of references as presented below are hereby incorporated by reference. However, to the extent statements might be considered inconsistent with the patenting of this/these invention(s) such statements are expressly not to be considered as made by the applicant(s).

Claims (20)

We claim:
1. A method of sorting sperm cells comprising:
establishing a sheath fluid environment for stained sperm cells, the sheath fluid environment including a citrate;
establishing a stream comprising said stained sperm cells in said sheath fluid environment;
sensing a property of the stained sperm cells; and
discriminating between stained sperm cells for a desired sex characteristic.
2. The method of claim 1, wherein the step of discriminating between stained sperm cells for a desired sex characteristic comprises the step of differentiating X chromosome bearing sperm cells from Y chromosome bearing sperm cells.
3. The method of claim 1, wherein changes in the amount of citrate are minimized between the sheath fluid environment and a pre-sort and/or a post-sort fluid environments.
4. The method of claim 1, further comprising:
forming drops from the stream containing the sperm cells;
charging drops having sperm cells with the desired sex characteristic; and
collecting drops having sperm cells with the desired sex characteristic.
5. The method of claim 4, wherein the step of collecting drops having sperm cells with the desired characteristic further comprises: collecting drops having sperm cells with the desired characteristic in a wide collection tube.
6. The method of claim 4, wherein the step of collecting drops having sperm cells with the desired characteristic further comprises: collecting drops having sperm cells with the desired characteristic in a stream matched collection tube.
7. A method of producing a sexed sperm specimen according to the process of claim 1.
8. The method of claim 7, further comprising preparing an artificial insemination dosage having less than one half of the typical number of sperm provided in a typical artificial insemination dosage.
9. A method of sorting sperm cells comprising:
chemically coordinating a sheath fluid environment for stained sperm cells with a pre-sort fluid environment or post-sort fluid environment;
supplying stained sperm cells in a stream comprising said chemically coordinated;
sensing a property of said stained sperm cells; and
discriminating between stained sperm cells having a desired sex characteristic.
10. The method of claim 9, wherein the step of chemically coordinating a sheath fluid environment with a pre-sort environment or a post-sort fluid environment further comprises incorporating a citrate into the sheath fluid.
11. The method of claim 10, wherein changes in the amount of citrate are minimized between the sheath fluid environment and a pre-sort and/or a post-sort fluid environments.
12. The method of claim 10, wherein the step of chemically coordinating a sheath fluid environment with a pre-sort environment or a post-sort fluid environment further comprises incorporating comprises a chemical in the citric acid cycle into the sheath fluid.
13. The method of claim 9, further comprising:
forming drops from the stream containing the sperm cells;
charging drops having sperm cells with the desired sex characteristic; and
collecting drops having sperm cells with the desired sex characteristic.
14. The method of claim 13, wherein the step of collecting drops having sperm cells with the desired characteristic further comprises: collecting drops having sperm cells with the desired characteristic in a wide collection tube.
15. The method of claim 13, wherein the step of collecting drops having sperm cells with the desired characteristic further comprises: collecting drops having sperm cells with the desired characteristic in a stream matched collection tube.
16. A method of producing a sexed sperm specimen according to the process of claim 1.
17. The method of claim 16, further comprising preparing an artificial insemination dosage having less than one half of the typical number of sperm provided in a typical artificial insemination dosage.
18. A method of producing at least one sexed embryo comprising:
establishing a sheath fluid environment for stained sperm cells, the sheath fluid environment including a citrate;
establishing a stream comprising said stained sperm cells in said sheath fluid environment;
sensing a property of the stained sperm cells;
discriminating between stained sperm cells having a desired sex characteristic;
and fertilizing at least one egg with the sexed sperm to form at least one sexed embryo.
19. The method of claim 18, wherein the at least one egg is fertilized in vitro with the sex selected sperm.
20. The method of claim 18, wherein the step of fertilized in vivo further comprises the step of delivering sperm cells into both uterine horns of a uterus.
US14/581,142 1997-12-31 2014-12-23 Methods for sorting sperm and producing sexed embryos Abandoned US20150112125A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US09/001,394 US6149867A (en) 1997-12-31 1997-12-31 Sheath fluids and collection systems for sex-specific cytometer sorting of sperm
US09/511,959 US6524860B1 (en) 1997-12-31 2000-02-23 Methods for improving sheath fluids and collection systems for sex-specific cytometer sorting of sperm
US10/378,109 US7195920B2 (en) 1997-12-31 2003-02-25 Collection systems for cytometer sorting of sperm
US11/536,492 US20070026379A1 (en) 1997-12-31 2006-09-28 Collection Systems for Cytometer Sorting of Sperm
US13/764,408 US9365822B2 (en) 1997-12-31 2013-02-11 System and method for sorting cells
US14/581,142 US20150112125A1 (en) 1997-12-31 2014-12-23 Methods for sorting sperm and producing sexed embryos

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US14/581,142 US20150112125A1 (en) 1997-12-31 2014-12-23 Methods for sorting sperm and producing sexed embryos

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US13/764,408 Continuation US9365822B2 (en) 1997-12-31 2013-02-11 System and method for sorting cells

Publications (1)

Publication Number Publication Date
US20150112125A1 true US20150112125A1 (en) 2015-04-23

Family

ID=21695819

Family Applications (8)

Application Number Title Priority Date Filing Date
US09/001,394 Expired - Lifetime US6149867A (en) 1997-12-31 1997-12-31 Sheath fluids and collection systems for sex-specific cytometer sorting of sperm
US09/511,959 Expired - Lifetime US6524860B1 (en) 1997-12-31 2000-02-23 Methods for improving sheath fluids and collection systems for sex-specific cytometer sorting of sperm
US10/378,109 Expired - Fee Related US7195920B2 (en) 1997-12-31 2003-02-25 Collection systems for cytometer sorting of sperm
US11/536,492 Abandoned US20070026379A1 (en) 1997-12-31 2006-09-28 Collection Systems for Cytometer Sorting of Sperm
US11/613,605 Abandoned US20070099260A1 (en) 1997-12-31 2006-12-20 Use of a Composition which Regulates Oxidation/Reduction Reactions Intracellularly and/or Extracellularly in a Staining or Sorting Process
US13/764,408 Expired - Lifetime US9365822B2 (en) 1997-12-31 2013-02-11 System and method for sorting cells
US13/764,390 Expired - Lifetime US9422523B2 (en) 1997-12-31 2013-02-11 System and method for sorting cells
US14/581,142 Abandoned US20150112125A1 (en) 1997-12-31 2014-12-23 Methods for sorting sperm and producing sexed embryos

Family Applications Before (7)

Application Number Title Priority Date Filing Date
US09/001,394 Expired - Lifetime US6149867A (en) 1997-12-31 1997-12-31 Sheath fluids and collection systems for sex-specific cytometer sorting of sperm
US09/511,959 Expired - Lifetime US6524860B1 (en) 1997-12-31 2000-02-23 Methods for improving sheath fluids and collection systems for sex-specific cytometer sorting of sperm
US10/378,109 Expired - Fee Related US7195920B2 (en) 1997-12-31 2003-02-25 Collection systems for cytometer sorting of sperm
US11/536,492 Abandoned US20070026379A1 (en) 1997-12-31 2006-09-28 Collection Systems for Cytometer Sorting of Sperm
US11/613,605 Abandoned US20070099260A1 (en) 1997-12-31 2006-12-20 Use of a Composition which Regulates Oxidation/Reduction Reactions Intracellularly and/or Extracellularly in a Staining or Sorting Process
US13/764,408 Expired - Lifetime US9365822B2 (en) 1997-12-31 2013-02-11 System and method for sorting cells
US13/764,390 Expired - Lifetime US9422523B2 (en) 1997-12-31 2013-02-11 System and method for sorting cells

Country Status (3)

Country Link
US (8) US6149867A (en)
CN (7) CN101504405B (en)
AR (2) AR016442A1 (en)

Families Citing this family (80)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998034094A1 (en) * 1997-01-31 1998-08-06 The Horticulture & Food Research Institute Of New Zealand Ltd. Optical apparatus
US6149867A (en) * 1997-12-31 2000-11-21 Xy, Inc. Sheath fluids and collection systems for sex-specific cytometer sorting of sperm
US6071689A (en) * 1997-12-31 2000-06-06 Xy, Inc. System for improving yield of sexed embryos in mammals
WO1999060381A1 (en) 1998-05-16 1999-11-25 The Perkin-Elmer Corporation Instrument for monitoring polymerase chain reaction of dna
US6818437B1 (en) 1998-05-16 2004-11-16 Applera Corporation Instrument for monitoring polymerase chain reaction of DNA
US7498164B2 (en) * 1998-05-16 2009-03-03 Applied Biosystems, Llc Instrument for monitoring nucleic acid sequence amplification reaction
AT383869T (en) * 1998-07-30 2008-02-15 Xy Inc System for the artificial non-surgical occupation of horses
US7208265B1 (en) * 1999-11-24 2007-04-24 Xy, Inc. Method of cryopreserving selected sperm cells
US6263745B1 (en) 1999-12-03 2001-07-24 Xy, Inc. Flow cytometer nozzle and flow cytometer sample handling methods
EP2258168B1 (en) 2000-05-09 2019-07-10 Xy, Llc Apparatus to isolate X-chromosome bearing and Y-chromosome bearing populations of spermatozoa
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 herd management system that utilizes an isolated population of X-chromosome bearing sperm and the y-chromosome bearing sperm
US20040031071A1 (en) * 2000-10-05 2004-02-12 Xy, Inc. System of hysteroscopic insemination of mares
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
CA2468772C (en) 2000-11-29 2013-10-29 George E. Seidel System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations
WO2002074078A2 (en) * 2001-01-30 2002-09-26 Rebholtz, Amy, K. Method for embryo after impregnation of the recipient female
US7280207B2 (en) * 2001-07-25 2007-10-09 Applera Corporation Time-delay integration in a flow cytometry system
US7265833B2 (en) * 2001-07-25 2007-09-04 Applera Corporation Electrophoretic system with multi-notch filter and laser excitation source
EP1468266A4 (en) * 2002-01-22 2009-03-11 Beckman Coulter Inc Environmental containment system for a flow cytometer
CN1787739B (en) * 2002-07-22 2011-03-30 Xy有限责任公司 Sperm cell process system
EP2889879B1 (en) * 2002-07-31 2017-09-06 Premium Genetics (UK) Limited System and method of sorting materials using holographic laser steering
US7699767B2 (en) 2002-07-31 2010-04-20 Arryx, Inc. Multiple laminar flow-based particle and cellular separation with laser steering
EP2275533B9 (en) 2002-08-01 2016-10-19 Xy, Llc Method of assessing sperm cells
US8486618B2 (en) 2002-08-01 2013-07-16 Xy, Llc Heterogeneous inseminate system
US20050229046A1 (en) * 2002-08-02 2005-10-13 Matthias Marke Evaluation of received useful information by the detection of error concealment
US6880414B2 (en) * 2002-08-08 2005-04-19 Becton Dickinson And Company Sort block and liquid collection device for sorting flow cytometer
CA2534394C (en) 2002-08-15 2013-01-08 Xy, Inc. High resolution flow cytometer
US7169548B2 (en) * 2002-09-13 2007-01-30 Xy, Inc. Sperm cell processing and preservation systems
JP3891925B2 (en) * 2002-12-03 2007-03-14 ベイバイオサイエンス株式会社 Device for obtaining information on biological particles
NZ577678A (en) 2003-03-28 2010-10-29 Inguran Llc Apparatus and methods for providing sex-sorted animal sperm
CA2752247C (en) * 2003-03-28 2014-06-17 Inguran, Llc Apparatus, methods and processes for sorting particles and for providing sex-sorted animal sperm
DE602004008241T2 (en) * 2003-03-28 2008-05-08 Monsanto Technology Llc. Process for coloring sperm
DK1625203T3 (en) 2003-05-15 2015-07-06 Xy Llc EFFECTIVE SEPARATION OF haploid cells FOR FLOWCYTOMETRISYSTEMER
US20050011582A1 (en) * 2003-06-06 2005-01-20 Haug Jeffrey S. Fluid delivery system for a flow cytometer
US7118676B2 (en) * 2003-09-04 2006-10-10 Arryx, Inc. Multiple laminar flow-based particle and cellular separation with laser steering
PL2269617T3 (en) * 2004-07-22 2016-12-30 Process for enriching a population of sperm cells
NZ550197A (en) * 2004-03-29 2009-10-30 Inguran Llc Use of a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly in a staining or sorting process of spermatozoa
AU2005266930B2 (en) * 2004-07-22 2010-09-16 Inguran, Llc Process for enriching a population of sperm cells
MXPA06011344A (en) 2004-03-29 2006-12-15 Monsanto Technology Llc Sperm suspensions for sorting into x or y chromosome-bearing enriched populations.
US7355696B2 (en) * 2005-02-01 2008-04-08 Arryx, Inc Method and apparatus for sorting cells
US20070025879A1 (en) * 2005-07-27 2007-02-01 Dakocytomation Denmark A/S Method and apparatus for syringe-based sample introduction within a flow cytometer
US7618770B2 (en) * 2005-07-29 2009-11-17 Xy, Inc. Methods and apparatus for reducing protein content in sperm cell extenders
AU2007343701B2 (en) * 2007-01-16 2013-01-31 Texas Tech University System Method and apparatus for gender selection based on pH
US8101426B2 (en) * 2007-03-02 2012-01-24 Icyt Mission Technology, Inc. System and method for the measurement of multiple fluorescence emissions in a flow cytometry system
US20080269549A1 (en) * 2007-04-27 2008-10-30 The Jackson Laboratory Method, article, and apparatus for cryopreservation of biological samples
CA2701170A1 (en) 2007-09-29 2009-08-27 Timothy James Williams Composition and method to modify sperm function and increase male gender ratio in mammals
EP2229457A4 (en) 2007-12-14 2011-01-26 Minitube America Inc Gender-specific separation of sperm cells and embryos
US20090171138A1 (en) * 2007-12-26 2009-07-02 Uri Eli Ultrasonic device for fertility control and management and navigation
KR100938926B1 (en) 2008-03-05 2010-01-27 재단법인서울대학교산학협력재단 Cell Sorting Apparatus using Ultrasonic Wave
WO2009149733A1 (en) * 2008-06-13 2009-12-17 Embl Heidelberg Next generation flow cytometer sorter
JP4572973B2 (en) * 2008-06-16 2010-11-04 ソニー株式会社 Microchip and flow-feeding method in microchip
US8251887B2 (en) * 2009-01-24 2012-08-28 Xihe Li Reproductive technology of low dose semen production and in vitro/in vitro fertilization in domestic animals
US8512224B2 (en) * 2009-01-24 2013-08-20 Xy, Llc Method of producing an inseminate
US20110001963A1 (en) * 2009-07-02 2011-01-06 Durack Gary P System and method for the measurement of multiple emissions from multiple parallel flow channels in a flow cytometry system
EP2494036A4 (en) * 2009-10-30 2013-08-28 Inguran Llc Methods and systems for reducing dna fragmentation in a population of sperm cells
US20110223587A1 (en) * 2010-03-11 2011-09-15 Schulman Joseph D Optical particle characterization system
US20110223586A1 (en) * 2010-03-11 2011-09-15 David Karabinus Optical particle characterization system
US20110236923A1 (en) * 2010-03-23 2011-09-29 Genetics & Ivf Institute Method for staining and sorting of a small volume of sperm
US9539652B2 (en) 2010-04-30 2017-01-10 Kennametal Inc. Rotary cutting tool having PCD cutting tip
NZ603807A (en) 2010-06-09 2014-09-26 Xy Llc A heterogeneous inseminate system
DE102011006081A1 (en) 2011-03-24 2012-09-27 Masterrind Gmbh Nozzle for flow cytometer for producing fraction of particles from mixture of particles, comprises inner flow channel, which connects inlet cross-section and opposite outlet section with outlet cross-section at its inlet section
DE102011006080B4 (en) 2011-03-24 2015-06-18 Masterrind Gmbh Apparatus and method for fractionating mammalian spermatozoa
DE102011075711A1 (en) 2011-05-12 2012-11-15 Masterrind Gmbh Nozzle for particle orientation in the liquid stream
SG194699A1 (en) 2011-05-12 2013-12-30 Xy Llc Uv diode laser excitation in flow cytometry
US9781919B2 (en) 2011-06-01 2017-10-10 Inguran, Llc Compositions and methods for improving the quality of processed sperm
AU2012261941B2 (en) 2011-06-01 2015-10-22 Inguran, Llc Compositions and methods for improving the quality of processed sperm
CN103013811A (en) * 2011-09-20 2013-04-03 北京富通华投资有限公司 Sperm sorter
EP2761275B1 (en) 2011-09-30 2017-06-21 Inguran, LLC Sperm staining and sorting methods
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
WO2014032017A1 (en) 2012-08-23 2014-02-27 Carrell Douglas T Sperm separation devices and associated methods
US20150219546A1 (en) 2012-09-19 2015-08-06 Inguran, Llc Nozzle assembly for a flow cytometry system and methods of manufacture
AU2013318001B2 (en) 2012-09-19 2016-06-16 Inguran, Llc Flow cytometer nozzle tip
AU2013325223A1 (en) 2012-10-05 2015-04-23 Inguran, Llc High efficiency methods of sex sorting sperm
DE102013208584A1 (en) 2013-05-08 2014-11-13 Masterrind Gmbh Nozzle and method for flow cytometry
CN103344543B (en) * 2013-06-28 2016-07-06 浙江星博生物科技股份有限公司 A kind of sperm mitochondrial membrane potential detection reagent based on flow cytometry
EP3063529A2 (en) 2013-10-30 2016-09-07 Premium Genetics (UK) Ltd. Microfluidic system and method with focused energy apparatus
FR3018679A1 (en) * 2014-03-18 2015-09-25 Imv Technologies Animal semen collection system and animal semen collection method using such a system
US20190060904A1 (en) 2017-08-22 2019-02-28 10X Genomics, Inc. Devices having a plurality of droplet formation regions
USD864415S1 (en) 2018-01-30 2019-10-22 Becton, Dickinson And Company Particle sorting system
RU184197U1 (en) * 2018-06-13 2018-10-18 Федеральное государственное бюджетное образовательное учреждение высшего образования "Кубанский государственный аграрный университет им. И.Т. Трубилина" Device for insemination of rabbits

Family Cites Families (648)

<
* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US32350A (en) * 1861-05-21 Improvement in machines for turning and spreading hay
US34782A (en) * 1862-03-25 Improvement in lamps
US3005756A (en) 1958-11-14 1961-10-24 Noland L Van Demark Diluter containing carbon dioxide for preserving semen
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
US4474875A (en) 1969-04-10 1984-10-02 Wallace Shrimpton Method and means for controlling the sex of mammalian offspring and product therefor
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
DE1919628C3 (en) 1969-04-18 1975-04-10 Wolfgang Prof. Dr. Dittrich
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
US3687803A (en) 1970-11-09 1972-08-29 American Cyanamid Co Acid chloride activators for hydrogen peroxide bleaching
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 An apparatus for analyzing and sorting particles quickly such as biological cells
US3826364B1 (en) 1972-05-22 1984-09-25
US3973196A (en) 1974-05-24 1976-08-03 Coulter Electronics, Inc. Method and apparatus for ejecting a metered amount of particulate sample
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
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
US3893766A (en) 1973-06-14 1975-07-08 Coulter Electronics Apparatus for orienting generally flat particles for slit-scan photometry
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
US3854470A (en) 1973-11-23 1974-12-17 L Augspurger Reproduction processes for cellular bodies
FR2254639B1 (en) 1973-12-18 1978-06-02 Agronomique Inst Nat Rech
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
USRE32350E (en) 1974-11-22 1987-02-10 Bhairab C. Bhattacharya 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
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
US4302166A (en) 1976-04-22 1981-11-24 Coulter Electronics, Inc. Droplet forming apparatus for use in producing uniform particles
US4162282A (en) 1976-04-22 1979-07-24 Coulter Electronics, Inc. Method for 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
DE2716095C2 (en) 1977-04-12 1987-02-19 Becton, Dickinson And Co., Paramus, N.J., Us
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
US4191749A (en) * 1977-10-11 1980-03-04 Bryant Bernard J Method and material 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
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
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 performing 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
NO144002C (en) 1979-04-10 1981-05-27 Norsk Hydro S Inst For Kreftfo Apparatus for use in vaeskestroemsfotometri
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
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
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
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
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
DE2943116C2 (en) 1979-10-25 1986-06-19 Gesellschaft Fuer Strahlen- Und Umweltforschung Mbh, 8000 Muenchen, De
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
US4511166A (en) * 1980-08-29 1985-04-16 The Eastern Company Handle-operated door lock with latch-operator override
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
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
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
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
US4673288A (en) 1981-05-15 1987-06-16 Ratcom, Inc. Flow cytometry
US4818103A (en) 1981-05-15 1989-04-04 Ratcom Flow cytometry
EP0068404B1 (en) 1981-06-24 1985-10-02 Becton, Dickinson and Company Analyzer for simultaneously determining volume and light emission characteristics of particles
FR2510393B1 (en) 1981-07-31 1983-12-30 Cassou Bertrand
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
SU1056008A1 (en) 1982-01-25 1983-11-23 Предприятие П/Я Р-6681 Flow-type cytofluorimeter
JPS59500340A (en) 1983-01-31 1984-03-01
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
DE3315194A1 (en) 1982-04-29 1983-11-03 Int Remote Imaging Systems Inc A method for separating a fluid sample flowing into particles
DE3315195A1 (en) 1982-04-29 1983-11-03 Int Remote Imaging Systems Inc A method for aligning particles 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
EP0120854A4 (en) 1982-10-05 1985-06-10 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
JPH043528B2 (en) 1983-02-07 1992-01-23
IT1197570B (en) 1983-02-11 1988-12-06 Serono Ist Farm Mixtures of fsh and lh from porcine pituitary defined in relationship
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
CH651930A5 (en) 1983-03-24 1985-10-15 Coulter Corp Apparatus for analysis and sorting of particles
JPH0145018B2 (en) 1983-03-25 1989-10-02 Kogyo Gijutsu Incho
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
AT58017T (en) 1983-12-24 1990-11-15 Inotech Ag Device for guiding and collecting light in the fotometrie od. Like.
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 Laser optical arrangement for measuring the degree of dispersion in the pouring 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 Apparatus for artificial insemination, particularly carnivores
FR2566543B1 (en) 1984-06-20 1988-02-26 Commissariat Energie Atomique An optical device has high performance and cytofluorimeter collection by applying
EP0171676B1 (en) * 1984-07-31 1990-11-07 Hitachi, Ltd. Free-flow electrophoretic separation method and apparatus therefor
AT48477T (en) 1984-09-11 1989-12-15 Partec Ag Method and apparatus for sorting of 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
FR2574656B1 (en) 1984-12-13 1988-08-05 Cassou Robert Gynecological probe including injector of sperm or embryos in the cavity of the animals, such as mares
FR2575063B1 (en) 1984-12-21 1988-07-01 Cassou Robert gynecological probe for artificial insemination, particularly for pigs
JPS61159135A (en) 1984-12-31 1986-07-18 Canon Inc Particle analyzing device
SU1260778A1 (en) 1985-01-31 1986-09-30 Центральный научно-исследовательский рентгено-радиологический институт Device for fluorescent analysis of individual microparticles in flow
US4702598A (en) 1985-02-25 1987-10-27 Research Corporation Flow cytometer
US4683202B1 (en) 1985-03-28 1990-11-27 Cetus Corp
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
CN85104679A (en) 1985-06-13 1987-02-04 王公佐 Process for controlling the sex of embryo of calves
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 Stroemningskammer for vaeskestroemsfotometer.
NO156917C (en) 1985-07-16 1987-12-16 Harald B Steen A device for measuring biological cell light scattering in vaeskestroemsfotometere.
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
US5756696A (en) 1986-01-16 1998-05-26 Regents Of The University Of California Compositions for chromosome-specific staining
US5447841A (en) 1986-01-16 1995-09-05 The Regents Of The Univ. Of California Methods for chromosome-specific staining
US4683195B1 (en) 1986-01-30 1990-11-27 Cetus Corp
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.
US4790653A (en) 1986-05-22 1988-12-13 Becton Dickinson And Company Housing for a flow cytometry apparatus with particle unclogging feature
JPS62274238A (en) 1986-05-22 1987-11-28 Becton Dickinson Co Gel for optical bonding used for flow sight metry device
US4786165A (en) 1986-07-10 1988-11-22 Toa Medical Electronics Co., Ltd. Flow cytometry and apparatus therefor
US4867908A (en) 1986-08-29 1989-09-19 Becton, Dickinson And Company Method and materials for calibrating flow cytometers and other analysis instruments
US4704891A (en) 1986-08-29 1987-11-10 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, embryo transfer or sampling follicular liquids in mammals
US4780451B1 (en) 1987-01-23 1995-04-04 Asua International Inc Composition and method for producing superovulation in cattle
US5162306A (en) 1987-01-23 1992-11-10 Donaldson Lloyd E Composition and method for producing superovulation in mammals
AT91789T (en) 1987-02-17 1993-08-15 Ratcom Inc Flow cytometry.
KR970007077B1 (en) 1987-03-13 1997-05-02 월레이스 엣취 코울터 Multi-part diefferential analyzing apparatus using light scatter techniques
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
EP0286088B1 (en) * 1987-04-08 1994-09-14 Hitachi, Ltd. A 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
JPH0575352B2 (en) 1987-04-20 1993-10-20 Hitachi Ltd
DE3851176D1 (en) 1987-04-27 1994-09-29 Preikschat F K Device and method for the examination of particles.
EP0289677A3 (en) 1987-04-27 1989-05-10 Ekhard Preikschat Apparatus and method for particle analysis
FR2614626B1 (en) * 1987-04-30 1989-07-21 Ranoux Claude Container for fertilization oocytes and embryos replacement in humans and animals
JP2642632B2 (en) 1987-07-03 1997-08-20 株式会社日立製作所 Particle measuring apparatus and particulate 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
DE3832901C2 (en) 1987-10-02 1992-09-17 Hitachi, Ltd., Tokio/Tokyo, Jp
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
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
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
US4845025A (en) 1987-11-10 1989-07-04 Coulter Corporation Biological sample mixing apparatus and method
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
US4986659A (en) 1988-02-29 1991-01-22 Aerometrics, Inc. Method for measuring the size and velocity of spherical particles using the phase and intensity of scattered light
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 The flow cell apparatus
JP2635126B2 (en) 1988-09-30 1997-07-30 東亜医用電子株式会社 Particle analyzing apparatus and method for determining the lobulated index of nuclei
JP2635125B2 (en) 1988-09-30 1997-07-30 東亜医用電子株式会社 Particle analyzing apparatus and method for determining the lobulated index of nuclei
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
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
WO1990013303A1 (en) * 1989-05-10 1990-11-15 The United States Of America, Represented By The Secretary, United States Department Of Commerce Method to preselect the sex of offspring
US4942305A (en) 1989-05-12 1990-07-17 Pacific Scientific Company Integrating sphere aerosol particle detector
EP0475936B1 (en) 1989-05-12 1995-09-13 Cytogam, Inc. Sex-associated membrane proteins and methods for increasing the probability that offspring will be of a desired sex
FR2647668A1 (en) 1989-06-06 1990-12-07 Medizin Labortechnik Veb K Transfer pipette for embryos
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
US5005981A (en) * 1989-09-08 1991-04-09 Becton, Dickinson And Company Apparatus for method for causing vortices in a test tube
US5215376A (en) 1989-09-08 1993-06-01 Becton, Dickinson And Company Method for causing vortices in a test tube
DE69014507T2 (en) 1989-09-13 1995-04-13 Tiyoda Seisakusho Koushoku Kk Device for pretreatment cells for flow cytometry.
JP2808321B2 (en) * 1989-09-19 1998-10-08 東亜医用電子株式会社 Cell analysis methods and apparatus
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 An apparatus for counting and determination of at least one leukocyte 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
EP0448931B1 (en) * 1990-01-26 1996-04-03 Canon Kabushiki Kaisha Method for measuring a specimen by the use of fluorescence light
JP3049254B2 (en) 1990-02-08 2000-06-05 シスメックス株式会社 Optical particle analyzer having two kinds of light sources
IL93634D0 (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
US5076472A (en) 1990-06-13 1991-12-31 Becton, Dickinson And Company Cleaning cycle for flow cytometers
US5087295A (en) * 1990-06-13 1992-02-11 Becton Dickinson And Company Cleaning cycle for flow cytometers
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
US5366888A (en) 1990-07-09 1994-11-22 Amrad Corporation Limited Enhanced maintenance of pregnancy using leukaemia inhibitory factor in embryo culturing
JP2939647B2 (en) 1990-07-24 1999-08-25 シスメックス株式会社 Automatic focus adjustment method in 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
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
US5116125A (en) 1990-10-31 1992-05-26 Biophos Medical Ab Fertility analyzer
DE69013366D1 (en) 1990-10-31 1994-11-17 Biophos Medical Ab Fertility analyzer.
JP2874746B2 (en) 1990-11-22 1999-03-24 シスメックス株式会社 The flow cell mechanism in the flow imaging cytometer
US5991028A (en) 1991-02-22 1999-11-23 Applied Spectral Imaging Ltd. Spectral bio-imaging methods for cell classification
JP3121849B2 (en) 1991-02-27 2001-01-09 シスメックス株式会社 Flow image cytometer
JPH0734012B2 (en) 1991-02-27 1995-04-12 東亜医用電子株式会社 Flow image cytometer
US5204884A (en) 1991-03-18 1993-04-20 University Of Rochester System for high-speed measurement and sorting of particles
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 Process for fertilization in spontaneous cycle.
US20020186874A1 (en) 1994-09-07 2002-12-12 Jeffrey H. Price Method and means for image segmentation in fluorescence scanning cytometry
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
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
US5488469A (en) * 1991-08-30 1996-01-30 Omron Corporation Cell analyzing apparatus
EP0529666B1 (en) 1991-08-30 2000-04-12 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 the extraction of liquids of different nature in animals, particularly for gynecological treatments in cattle, horses and the like
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
GB9125327D0 (en) 1991-11-28 1992-01-29 Nat Res Dev Bovine semen sexing
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 the sex of bovine embryos
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
EP0556748B1 (en) 1992-02-20 1998-10-28 Canon Kabushiki Kaisha Method and apparatus for particle manipulation, and measuring apparatus utilizing the same
GB2278679B (en) 1992-02-21 1995-09-06 Secr Defence Analysis of particle characteristics
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
US5273527A (en) 1992-05-12 1993-12-28 Ovamed Corporation Delivery catheter
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
CH685922A5 (en) 1992-07-13 1995-11-15 Pall Corp Automates device for processing a biological fluid and its use.
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
US5466572A (en) 1992-09-03 1995-11-14 Systemix, Inc. High speed flow cytometric separation of viable cells
PT662124E (en) 1992-09-03 2002-10-31 Univ California High-speed citometric flux separation of mamifero 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
FR2699678B1 (en) 1992-12-23 1997-02-14
JPH06197665A (en) 1993-01-07 1994-07-19 Meiji Seika Kaisha Ltd Medium for ectosomatic fertilization and ectosomatic fertilization method
AT178439T (en) 1993-01-16 1999-04-15 James Andrew Bangham Signal processing system
JP2525713B2 (en) * 1993-01-19 1996-08-21 社団法人 家畜改良事業団理事長 Low molecular thio - culture and transport method of Ushihai using Le 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 The flow cell apparatus
CA2113957A1 (en) 1993-01-29 1994-07-30 University Of Guelph Nucleotide sequences for bovine sex determination
US5453575A (en) 1993-02-01 1995-09-26 Endosonics Corporation Apparatus and method for detecting blood flow in intravascular ultrasonic imaging
AU673245B2 (en) 1993-02-01 1996-10-31 Seq, Ltd. Methods and apparatus for DNA sequencing
US5367474A (en) 1993-02-08 1994-11-22 Coulter Corporation Flow cytometer
US5556764A (en) 1993-02-17 1996-09-17 Biometric Imaging, Inc. Method and apparatus for cell counting and cell classification
US5547849A (en) 1993-02-17 1996-08-20 Biometric Imaging, Inc. Apparatus and method for volumetric capillary cytometry
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
WO1994020883A1 (en) 1993-03-01 1994-09-15 General Signal Corporation Variable annular illuminator for photolithographic projection imager
NO930980L (en) 1993-03-18 1994-09-19 Flowtech As Optical configuration for væskeströmcytofotometer
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
DE69418248D1 (en) 1993-06-03 1999-06-10 Hamamatsu Photonics Kk A laser optical sensing system with axicon
JPH08501718A (en) * 1993-06-04 1996-02-27 クワハック・インターナショナル・カンパニー・リミテッド Artificial insemination and embryo transfer device
NO932088L (en) 1993-06-08 1995-01-05 Oddbjoern Gjelsnes A device for use in flow cytometry
US5483469A (en) 1993-08-02 1996-01-09 The Regents Of The University Of California Multiple sort flow cytometer
US5464581A (en) 1993-08-02 1995-11-07 The Regents Of The University Of California Flow cytometer
US6328071B1 (en) 1993-08-06 2001-12-11 Cary Austin Well pressure tank
DE69434551T2 (en) * 1993-09-16 2006-06-14 Sysmex Corp Particle analyzer
US5602533A (en) * 1993-10-04 1997-02-11 Boverio; Antonello Device for sensing a state change in a mechanical system, method for monitoring the state of a mechanical system and use of said 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.