HUE029701T2 - Process for enriching a population of sperm cells - Google Patents

Description

Description

FIELD OF THE INVENTION

[0001] The present invention generally relates to the enrichment of a population of sperm cells. In particular, the present invention generally relates to the enrichment of a population of viable sperm cells without physically sorting the cells.

BACKGROUND

[0002] The fertilization of animals by artificial insemination (AI) and embryo transplant following in vitro fertilization is an established practice. In the livestock production industry, the ability to influence the reproductive outcome toward offspring having one or more desired characteristics has obvious advantages. By way of example, there would be an economic benefit in the dairy industry to preselect offspring in favor of the female sex to ensure the production of dairy cows. The separation of sperm into enriched populations of X and Y chromosome-bearing cells, known as gender enriched semen or gender enriched sperm, is one method of achieving preselected offspring.

[0003] Johnson et al. (U.S. Patent No. 5,135,759) describe the separation of intact X and Y chromosomebearing sperm populations according to DNA content using a flow cytometer/cell sorter into X and Y chromosomebearing sperm enriched populations. As described, the sperm is combined with a DNA selective dye at a temperature of 30°C to 39°C for a period of 1 hour (39°C) to 1.5 hours (30°C). A flow cytometer is then used to measure the amount of fluorescent light given off when the sperm passes through a laser beam. Because the X chromosome-bearing sperm contains more DNA than the Y chromosome-bearing sperm, approximately 3% to 5% depending upon the species, theX chromosome-bearing sperm yields a greater intensity of fluorescent light than the Y chromosome-bearing sperm. Droplets containing single sperm of a predetermined fluorescent intensity are given a charge and electrostatically deflected into collection vessels. The collected, gender enriched sperm population, is then used for microinjection or artificial insemination. Notably, this method requires that the sperm cells be physically sorted to achieve the gender enriched sperm population. Physically sorting according to Johnson requires time and cost.

[0004] Koller et al. (WO 01/6811OA) describes methods and apparatus for selectively identifying, and individually targeting with an energy beam, specific cells within a mixed cell population to induce a response in the targeted cells.

[0005] Didion et al. (WO 02/41906A2) describes methods, compositions of matter, and apparatus for sorting sperm to produce subpopulations enriched in sperm carrying chromosome determinants for male or female offspring and further describes preparing sperm cells by staining them using DNA binding dyes for sperm cells at low temperature in a flow cytometer.

SUMMARY OF THE INVENTION

[0006] The present invention is directed to a process for selectively decreasing the capacity of a subpopulation of non-human sperm cells in a sperm cell dispersion, the process comprises a) labeling a sperm cell sample with a labeling mixture including a chemical agent that induces sperm immotility and DNA selective dye at a temperature between 30°C and 39°C to form a dispersion of labeled sperm cells in a liquid, wherein the amount of the label associated with a sperm cell indicates a genetic, proteomic structural, orfunctional characteristic of a subpopulation of sperm cells in the dispersion, b) optically inspecting the dispersion to identify individual sperm cells as members of the subpopulation, c) determining the position of members of the subpopulation in the dispersion; and d) delivering a dose of energy to different positions within the dispersion to selectively decrease the capacity of members of the subpopulation to fertilize an egg without similarly affecting sperm cells at other positions in the dispersion to produce an enriched sperm cell population.

[0007] Other aspects and features of the invention will be in part apparent and in part pointed out hereinafter.

DETAILED DESCRIPTION OF THE INVENTION

[0008] Advantageously, a population of viable sperm cells may be enriched with respect to a characteristic in accordance with the present invention without physically sorting the cells. This characteristic may be, for exam pie, whether the sperm cells carry an X or a Y chromosome. Alternatively, the characteristic may be another genetic characteristic such as the presence of a single nucleotide polymorphism ("SNP") coding for improved animal productivity (such as, for example, improved milk production) or coding for a lipid to improve cryopreservation of the selected cells. The characteristic may also be a proteomic characteristic such as a protein to improve the performance of sperm, such as, for example, a protein that would improve in utero performance by improving beneficial acrosomal characteristics. The characteristic may also be a structural characteristic, such as, for example, acrosomal integrity, or a functional characteristic, such as, for example, progressive motility.

[0009] Enrichment of a sperm cell population with respect to the genetic, proteomic, structural, or functional characteristic may be achieved, for example, by labeling sperm cells in the population having (or, alternatively, lacking) the characteristic, rendering the sperm cells substantially immotile, and selectively dosing the immotile sperm cells with a dose of energy to decrease the viability of the dosed cells or at least decrease the capacity of the dosed cells to fertilize an egg in vitro or in vivo (i.e., after insemination). Because the sperm cells in the dispersion, sometimes referred to as a suspension, are substantially immotile and selectively labeled, the energy beam may be delivered to a specific position in the dispersion to dose an individual sperm cell; by repeating this process step, i.e., individually dosing immotile sperm cells at discrete positions in the dispersion, a subpopulation of sperm cells having a desired characteristic in the dispersion may be effectively enriched, for example, with respect to the percentage of cells of the subpopulation having the desired characteristic; with respect to the percentage of offspring having a certain genetic or proteomic characteristic as a result of being produced by fertilization with the sperm cells; or with respect to both.

[0010] In any event, the population of sperm cells may be enriched for a particular subpopulation without physically separating cells having the desired characteristic from those lacking the desired characteristic (i.e., without separating the dosed cells from the non-dosed cells). Optionally, further enrichment of the cells may be achieved by additionally purifying the cells by physically separating the dosed and non-dosed cells into separate subpopulations according to methods described below.

Sperm Cell Dispersion

Density of the Sperm Cells [0011] In general, sperm cell dispersions having a population that may be enriched in some characteristic may be prepared with a wide range of sperm cell densities. Typically, however, the sperm cell density will be at least about 1 X 103 sperm/ml, and generally not in excess of about 5 X 1010 sperm/ml, and more preferably not in excess of about 5 X 108 sperm/ml of dispersion. For example, in one example the dispersions may contain spermatozoa in a "relatively low" density, i.e., in a density of less than about 1 X 107 sperm/ml, preferably less than about 1 X 106 sperm/ml, more preferably about 1 X 103 to about 5 X 106 sperm/ml, still more preferably about 1 X 103 to about 1 X 106 sperm/ml, even more preferably about 1 X 104 to about 1 X 105 sperm/ml, and most preferably about 1 X 105 sperm/ml of dispersion. In an alternative example, the dispersions may contain spermatozoa in an "intermediate" density, i.e., in a density of about 1 X 107 to about 1 x 108 sperm/ml of dispersion. In yet another alternative example, the dispersions may contain spermatozoa in a "relatively high" density, i.e., in a density of at least about 1 x 108 sperm/ml, preferably about 1 x 108 to about 5 x 1010 sperm/ml, more preferably about 1.5 x 108 to about 2 x 1010 sperm/ml, even more preferably about 1.5 x 108 to about 2 x 108 sperm/ml, and still more preferably about 1.5 x 108 sperm/ml of dispersion. Thus, for example, the dispersions may contain at least about 0.04 x 106 sperm/ml of dispersion in one example; at least about 1 x 106 in another example; at least about 1.5 x 106 in another example; at least about 2 x 106 in another example; at least about 3 x 106 in another example; at least about 0.5 x 107 in another ex ample; at least about 1 x 107 in another example; at least about 1.25 x 107 in another example; at least about 2 x 107 in another example; at least about 3 x 107 in another example; at least about 4 x 107 in another example; at least about 5 x 107 in another example; at least about 6 x 107 in another example; at least about 7.0 x 107 in another example; at least about 8 x 107 in another example; at least about 9 x 107 in another example; at least about 10 x 107 in another example; at least about 11 x 107 in another example; at least about 12x 107 in another example; at least about 1.0 x 108 in another example; at least about 1.25 x 108 in another example; at least about 1.5 x 108 in another example; at least about 1.75 x 108 in another example; at least about 2.0 x 108 in another example; at least about 2.25 x 108 in another example; at least about 2.5 x 108 in another example; at least about 2.75 x 108 in another example; at least about 3 x 108 in another example; at least about 5 x 108 in another example; at least about 7.0 x 108 in another example; or even at least about 8 x 108 sperm/ml of dispersion. In an alternative example, the dispersion may contain less than about 9 x 105, less than about 7 x 105, less than about 5x 105, less than about 2 x 105, less than about 1 x 105, less than about 1 x 104, or even less than about 1 x 103 sperm/ml of dispersion.

[0012] The density of spermatozoa may vary based upon a number of factors, including, for example, the variations among different species of mammals, variations among the mammals of a single species, and even variations among different ejaculates of a single mammal. For example, bovine spermatozoa may be in a dispersion at a higher density, but typically in a smaller volume, such as for example 0.5 x 106 sperm/ml to about 8 x 107 sperm/ml in a volume of about 0.5ml to about 25ml. Swine spermatozoa, however, may be in a dispersion at a lower density, but typically in a greater volume, such as for example 0.04 x 106 sperm/ml to about 1 x 107 sperm/ml in a volume of about 50ml to about 250ml.

[0013] The density of spermatozoa in the sperm dispersions may also depend upon the method by which the sperm cells may be subsequently enriched or sorted. For example, the sperm cells may be sorted using flow cytometry as described in U.S. Patent Application Publication No. US 2005/0112541. In such an instance, the dispersion may typically be of an "intermediate" or "relatively high" density of spermatozoa. Other sorting or enrichment techniques, as described in greater detail below, may benefit from a lesser density of spermatozoa, such as a "relatively low" density of spermatozoa, labeled with a marker, such as for example the dyes and labels described herein.

[0014] The density of the spermatozoa in the sperm dispersions may also be artificially manipulated to achieve a dispersion of a specific spermatozoa density. Manipulations to the density of spermatozoa in a sperm dispersion, for example, contained in an insemination straw, may be made based upon factors such as the temperature at which the dispersion may be stored, the length of the storage period, whether the spermatozoa in the sperm dispersion are sorted or unsorted, the species of the male mammal from which the spermatozoa were collected, the fertility of the mammal from which the spermatozoa were collected, and the species of the female mammal to be inseminated.

[0015] The density of the spermatozoa in a sperm dispersion may also be afFected by simply concentrating the spermatozoa, such as for example, by centrifugation. In such an instance, the dispersion would substantially separate into what is commonly referred to as a pellet (a mass of cells containing a minimal amount of fluid) and a supernatant (a soluble liquid fraction). The supernatant may then be decanted without disruption of the pellet, thereby resulting in a relatively dense pellet of sperm cells containing a minimal amount of the inhibitor, the effect being to reduce the volume of the dispersion without changing the components of the dispersion. As a result, the sperm cells of the pellet remain in an immotile state.

Immotility of the Sperm Cells [0016] The dispersion of sperm cells contains sperm cells that have a substantially reduced motility. Substantial reduction of the motility of the sperm cells in the sperm cell dispersion may be achieved in a number of ways, including for example, by contacting the sperm cells with a motility inhibitor, by reducing the temperature of the sperm cells or the immediate environment surrounding the sperm cells (i.e., the sperm dispersion), or by a combination of both. Sperm cells in the sperm dispersion may behave, in certain respects, in a manner characteristic of epididymal spermatozoa; for example, the sperm cells in the population are substantially immotile and/or they may have a lesser rate of endogenous respiration as compared to washed or freshly ejaculated spermatozoa. Advantageously, the immotile sperm cells, sometimes referred to as quiescent sperm cells, have the ability, upon separation from the inhibitor (s) or exposure to an increase in temperature, to behave in a manner characteristic of ejaculated spermatozoa (and not characteristic of epididymal spermatozoa) with respect to motility.

[0017] In one example, the inhibitor, the reduction in temperature, or a combination of both reduces path velocity (sometimes referred to as motility or path motility), progressive velocity (sometimes referred to as progressive motility), or both, as measured by HTM-IVOS sperm analysis (Hamilton-Thorne HTM-IVOS computer assisted sperm analysis system Hamilton-Thorne Research, Beverly MA) of at least about 50% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in afresh ejaculate of the same species. Preferably, the motility inhibitor, the reduction in temperature, ora combination of both reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, of at least about 60% of the sperm cells in the dispersion relative to the path ve locity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. More preferably, the motility inhibitor, the reduction in temperature, or a combination of both reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, of at least about 70% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Still more preferably, the motility inhibitor, the reduction in temperature, ora combination of both reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, of at least about 80% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Even more preferably, the motility inhibitor, the reduction in temperature, or a combination of both reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, of at least about 90% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Even more preferably, the motility inhibitor, the reduction in temperature, ora combination of both reduces path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, of at least about 95% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species. Most preferably, the motility inhibitor reduces path velocity, progressive velocity, or both, as measured by an HTM-IVOS sperm analysis, of at least about 99% of the sperm cells in the dispersion relative to the path velocity, progressive velocity, or both of sperm cells in a fresh ejaculate of the same species.

[0018] A motility inhibitor may be used to substantially reduce the motility of the sperm cells in the sperm cell dispersion. The inhibitor may be any of a range of compositions having a depressive effect upon sperm motility. Such compositions include, for example, sodium channel inhibitors, such as, ouabain; compositions comprising potassium ions; and compositions comprising potassium and sodium ions. For example, relatively high concentrations of potassium ions in the dispersion tend to depress sperm motility. In general, therefore, it is preferred that the dispersion contain a source of potassium ions and that the potassium concentration in the dispersion be at least about 0.05 moles/L. More preferably, the potassium concentration may be at least about 0.05 moles/L to about 0.5 moles/L. Still more preferably, the potassium concentration may be at least about 0.1 moles/L to about 0.3 moles/L. Most preferably, the potassium concentration may be at about 0.173 moles/L. Such dispersions will typically, but not necessarily, also contain a source of sodium ions. When sodium is present, the molar ratio of potassium to sodium is generally equal to or greater than 1:1, respectively, but will generally not exceed a molar ratio of 8:1. Preferably, the molar ratio of potassium to sodium is at least about 1.25:1. Still more preferably, the molar ratio of potassium to sodium is at least about 1.5:1. Still more preferably, the molar ratio of potassium to sodium is at least about 1.75:1. Still more preferably, the molar ratio of potassium to sodium is at least about 1.78:1. In one particular example, the molar ratio of potassium to sodium may be at least about 2:1. In yet another example, the molar ratio of potassium to sodium may be at least about 3:1. In still another example, the molar ratio of potassium to sodium may be at least about 4:1. In still another example, the molar ratio of potassium to sodium may be at least about 5:1. In still another example, the molar ratio of potassium to sodium may be at least about 6:1. In still another example, the molar ratio of potassium to sodium may be at least about 7:1. In still another example, the molar ratio of potassium to sodium may be at least about 8:1.

[0019] The sperm dispersion may additionally comprise an ion or source of carbon dioxide capable of enhancing the down-regulation of motility. In one example, the source of carbon dioxide may be, for example, one or more carbonates. In one example, the sperm dispersion comprises NaHC03 and KHC03, thereby providing a source of potassium and sodium ions as well as an increased partial pressure of carbon dioxide (relative to the ambient atmosphere). For example, the dispersion may comprise NaHC03 and KHC03 in an aqueous solution, preferably NaHC03, KHC03, and C6H807-H20 in water; in general, the KHCOs concentration in the dispersion may be at least about 0.05 moles/L. More preferably, the KHC03 concentration may be at least about 0.05 moles/L to about 0.5 moles/L. Still more preferably, the KHC03 concentration may be at least about 0.1 moles/L to about 0.3 moles/L. In one example, the dispersion may be formed using a motility inhibitor comprising 0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0.090 moles/L C6H807-H20 in water as disclosed in Salisbury & Graves, J. Repród. Fertil., 6:351-359 (1963). The sperm cells will generally remain quiescent as long as they are exposed to the motility inhibitor (s).

[0020] When C6H807-H20 is present in the dispersion, the molar ratio of KHC03 to NaHC03 may be as described above. The molar ratio of KHC03 to C6H807-H20 may generally be equal to or greater than 1:1, respectively, but will generally not exceed a molar ratio of 8:1. Preferably, the molar ratio of KHC03 to C6H807-H20 is from atleastabout 1.25:1. Still more preferably, the molar ratio of KHC03 to C6H807-H20 is at least about 1.5:1. Still more preferably, the molar ratio of KHC03 to C6H807-H20 is at least about 1.75:1. In one example, the molar ratio of KHC03 to C6H807-H20 is at least about 1.78:1. In another particular example, the molar ratio of KHC03 to C6H807-H20 may be at least about 2:1. In yet another example, the molar ratio of KHC03 to C6H807-H20 may be at least about 3:1. In still another example, the molar ratio of KHC03 to C6H807-H20 may be at least about 4:1. In still another example, the molar ratio of KHC03 to C6H807-H20 may be at least about 5:1. In still another example, the molar ratio of KHC03 to C6H807-H20 may be at least about 6:1. In still another example, the molar ratio of KHC03 to C5H807-H20 may be at least about 7:1. In still another example, the molar ratio of KHC03 to C6H807-H20 may be at least about 8:1. In one particularly preferred example, the dispersion may be formed using an inhibitory buffer comprising 0.097 moles/L of NaHCOs, 0.173 moles/L of KHCOs, 0.090 moles/L C6H807-H20 in water as disclosed in Salisbury & Graves, J. Repród. Fertil., 6:351-359 (1963). The sperm cells will generally remain quiescent as long as they are exposed to the motility inhibitor (s).

[0021] Experimental evidence to date further suggests that the overall health and other vital characteristics of sperm cells may be improved if the sperm dispersion is maintained under an atmosphere that reduces or prevents the diffusion of oxygen into the dispersion. This can be achieved by replacing the atmosphere of gas above the sperm dispersion with an atmosphere having an enhanced partial pressure of, for example, carbon dioxide, nitrogen, or other inert gases relative to ambient air. In one example, the dispersion may be maintained under an atmosphere having an enhanced partial pressure of carbon dioxide relative to air. I n t another exam pie, the atmosphere over the dispersion has a partial pressure of carbon dioxide of at least about 0.0001 atm, but generally less than about 5 atm at atmospheric pressure. In one example, the partial pressure of carbon dioxide may be about 0.5 atm to about 2 atm at atmospheric pressure; in another example, the partial pressure of carbon dioxide may be about 0.9 atm to about 2 atm at atmospheric pressure; in another example, the partial pressure of carbon dioxide may be about 0.95 atm to about 2 atm at atmospheric pressure. In a particularly preferred example, the atmosphere over the dispersion has a partial pressure of carbon dioxide of at least 0.9 atm; more preferably, at least about 0.95 atm.

[0022] Alternatively, or in addition to the use of a motility inhibitor, the temperature of the sperm cells or the dispersion may be altered in order to induce the sperm cells to become immotile. The temperature induced sperm immotility may be induced, for example, by reducing the temperature of the sperm cells or the dispersion to about 0°C to about 15°C, preferably from about 1°C to about 10°C; more preferably from about 2°C to about 8°C, still more preferably from about 3°C to about 6°C, and even more preferably from about 4°C to about 5°C, and still more preferably about 5°C. Preferably, however, the sperm cells are not exposed to temperatures that substantially detrimentally affect the viability of the cells or significantly affect the ability of the sperm cells to bind or uptake a label.

[0023] In another example, the temperature of the sperm cells or the sperm dispersion may be altered such that the sperm cells or the sperm dispersion may be at a temperature within the range of about 4°C to about 50°C,-preferably from about 7°C to about 43°C; more preferably from about 10°C to about 39°C; still more pref- erably from about 15°C to about 30°C; and most preferably from about 17°C to about 25°C. In a particularly preferred embodiment, the temperature of the sperm cells or the surrounding dispersion may be about 4°C.

[0024] The sperm cells may be exposed to the reduced temperature, and thereby rendered substantially immo-tile, at any time once the cells have been obtained from the source mammal. For example, the temperature of the sperm cells may be reduced, thereby inducing sperm immotility, upon collection of the cells from the source mammal, upon combining the cells with a buffer, upon formation of the labeling mixture, including before, during, or after the labeling process, or upon formation of the dispersion of labeled cells. Generally, however, sperm immotility may be induced by a reduction in temperature prior to the optical inspection of the dispersion.

[0025] For example, the temperature of the sperm cells may be reduced (i.e., sperm immotility may be induced) subsequent to labeling of the cells, thereby allowing for labeling to occur at a more preferred temperature as discussed below. In one example, the temperature of the sperm cells or surrounding dispersion may be reduced (i.e., sperm immotility may be induced) subsequent to labeling and prior to optical inspection of the cells.

[0026] Exposure of the sperm cells to the inhibitor, to the reduced temperature, or to a combination of both induces the sperm cells to become immotile. In one embodiment, for example, the motility inhibitor, the reduction in temperature, ora combination of both reduces the motility, progressive motility, or both of at least 60% of the sperm cells in the dispersion relative to the motility, progressive motility, or both of sperm cells in a fresh ejaculate of the same species. Preferably, the motility inhibitor, the reduction in temperature, or a combination of both reduces the motility, progressive motility, or both of at least 70% of the sperm cells in the dispersion relative to the motility, progressive motility, or both of sperm cells in afresh ejaculate of the same species. More preferably, the motility inhibitor, the reduction in temperature, or a combination of both reduces the motility, progressive motility, or both of at least 80% of the sperm cells in the dispersion relative to the motility, progressive motility, or both of sperm cells in a fresh ejaculate of the same species. Preferably, the motility inhibitor, the reduction in temperature, or a combination of both reduces the motility, progressive motility, or both of at least 90% of the sperm cells in the dispersion relative to the motility, progressive motility, or both of sperm cells in a fresh ejaculate of the same species. Preferably, the motility inhibitor, the reduction in temperature, or a combination of both reduces the motility, progressive motility, or both of at least 99% of the sperm cells in the dispersion relative to the motility, progressive motility, or both of sperm cells in a fresh ejaculate of the same species.

[0027] The cells are preferably rendered immotile, regardless of the method used, fora time sufficient to allow for the optical inspection of the dispersion, the determination of the position of the member cells of the subpop ulation; and the dosing of the member cells of the subpopulation with an energy source. If it is desired to physically separate the dosed from the non-dosed cells, it may also be preferred to maintain the sperm cells in an immotile state through this process step. Similarly, if the sperm cells are to be cryopreserved, they may be maintained in an immotile state through the cryopreservation step (independent of whether the dosed cells are physically separated from the non-dosed cells prior to cryopreservation). In a preferred embodiment, the cells are kept immotile through the step of cryopreservation.

[0028] Immotile cells may be returned to an active state, i.e., behavior characteristic of fresh ejaculate, by separating the cells from the motility inhibitor, exposing them to air, increasing the temperature of the cells or cell dispersion (preferably to the typical temperature of freshly ejaculated spermatozoa), by dilution with physiological saline (Salisbury et al., 1963) or a buffer such as a TCA buffer or PBS, or by any combination of the above, depending upon, for example, the method used to induce immotility. Typically, at least about 20%, preferably at least about 50%, more preferably at least about 60%, still more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 90%, still more preferably at least about 95%, and most preferably at least about 99% of the cells returned to an active state (i.e., reactivated cells) will have a path velocity, progressive velocity, or both, as measured by HTM-IVOS sperm analysis, that is at least about 50%, preferably at least about 60%, more preferably at least about 70%, still more preferably at least about 80%, even more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 99% of the path velocity, progressive velocity, or both of the sperm cells prior to being combined with the motility inhibitor (i.e., of sperm cells of a fresh ejaculate) .

Collection of the Cells from a Mammal [0029] Various methods of collection of viable sperm are known. Such methods include, for example, the gloved-hand method, useofan artificial vagina, and electro-ejaculation.

[0030] At the time of collection, or subsequently, the collected sperm may be combined with any of a number of various buffers that are compatible with sperm, such as TCA, HEPES, PBS, or any of the other buffers disclosed in U.S. Patent Application Publication No. US 2005/0003472. For example, a bovine semen sample typically containing about 0.5 to about 10 billion sperm cells per milliliter may be collected directly from the source mammal into a vessel containing a buffer to form a sperm suspension. Alternatively, the semen sample may be collected into an empty vessel and then subsequently contacted with a buffer within several minutes to hours after collection to form the sperm suspension.

[0031] Alternatively, the sperm cells may be collected and contacted with a motility inhibitor in lieu of or in ad- dition to a buffer, thereby forming a sperm dispersion. The sperm cells may be collected directly from the animal into a vessel containing a motility inhibitor to form the sperm dispersion, or alternatively, may be collected into an empty vessel and then subsequently combined with a motility inhibitor within several minutes (or even hours) of collection to form the sperm dispersion.

[0032] The sperm dispersion may also contain a range of other additives to enhance sperm viability. Exemplary additives include protein sources, antibiotics, growth factors, and compositions that regulate oxidation/reduction reactions intracellularly and/or extracellularly. Examples of each of these additives are well known in the art, as demonstrated in the disclosure of, for example, U.S. Application Serial Nos. 60/557,407 and 11/092,313.

Labeling of the Cells [0033] Sperm cells may be labeled with any of a number of different labels, including labels that bind to the exteriorof the cell (such as, for example, fluorescently labeled antibodies) as well as labels that cross the cell membrane and bind to the internal contents of the cell (such as, for example, fluorescent DNA selective dyes). Generally, the labeling process comprises contacting the sperm cells with a concentration of label (thereby forming a labeling mixture, sometimes referred to as a staining mixture), at a temperature and pH that allow for rapid and efficient binding or uptake of the label, for a time sufficiently long to obtain the desired degree of labeling, without substantially affecting the viability of the cells.

[0034] The sperm may be in the form of neat semen, or alternatively, a sperm-containing semen derivative obtained by centrifugation or the use of other means to separate semen into fractions. The sperm cells are then contacted or otherwise combined with the label to form a labeling mixture; optionally, the label may be in the form of a solid or a solution. Generally, however, the label, the sperm cells, or both are in a medium such as a buffer.

[0035] The sperm cells may be combined with a buffer to form a sperm suspension. Any of a number of various buffers that are compatible with sperm, such as for example, TCA, HEPES, PBS, or the buffers disclosed in U.S. Patent Application Publication No. US 2005/0003472 may be used. Once formed, the sperm suspension may be combined with a source of label to form a labeling mixture; optionally, the label may be in solid or liquid form and, as a further option, may additionally comprise any of the previously mentioned buffers.

[0036] In one example, the label may be combined with a buffer to form a labeling suspension and the labeling suspension combined with a sperm source in the form of neat semen, a sperm-containing semen derivative, or a sperm suspension to form the labeling mixture.

[0037] A buffer comprising a motility inhibitor may be used to form the labeling mixture. For example, the motility inhibitor may be included in the buffer used to form a sperm suspension (which is then combined with the label) or a labeling suspension (which is then combined with a source of sperm) to form the labeling mixture. In either event, the result is a sperm dispersion containing a motility inhibitor and label.

[0038] The labeling mixture may be formed by using any of a number of labels, such as for example, one or more UV or visible light excitable, DNA selective dyes, as previously described in U.S. Patent No. 5,135,759 and WO 02/41906. Exemplary UV light excitable, DNA selective dyes include Hoechst 33342 and Hoechst 33258, each of which is commercially available from Sigma-Aldrich (St. Louis, MO). Exemplary visible light excitable dyes include SYBR-14, commercially available from Molecular Probes, Inc. (Eugene, OR) and bisbenzimide-BODIPY®conjugate6-{[3-( (2Z)-2-{ [1 -(difluoroboryI)-3, 5-dimethyl-1H-pyrrol-2-yl] methylene} -2H-pyrrol-5-yl) propanoyl] amino} -N- [3 -(methyl {3- [ ({4- [6- (4-methyl-pi perazin-1-y I) -1H, 3’H-2,5’ -bibenzimidazol-2’ -yl] phe-noxy(acetyl) amino] propyl} amino) -propyl] hexanamide ("BBC") described in WO 02/41906. Each of these dyes may be used alone or in combination; alternatively, other cell permeant UV and visible light excitable dyes may be used, alone or in combination with the aforementioned dyes, provided the dye does not detrimentally affect the viability of the sperm cells to an unacceptable degree when used in concentrations which enable sorting as described elsewhere.

[0039] Alternatively, the labeling mixture may be formed using fluorescent polyamides, and more specifically polyamides with a fluorescent label or reporter conjugated thereto. Such labels will fluoresce when bound to nucleic acids. Examples of polyamides with a fluorescent label or reporter attached thereto include, for example, those disclosed in Best et al. , Proc. Natl. Acad. Sei. USA, 100(21): 12063-12068(2003); Gygi, étal. , Nucleic Acids Res., 30(13): 2790-2799 (2002); U.S. Patent No. 5,998,140; U.S. Patent No. 6,143,901; and U.S. Patent No. 6,090,947.

[0040] Fluorescent nucleotide sequences may also be used to label the sperm cells. Such nucleotide sequences fluoresce when hybridized to a nucleic acid containing a target or complementary sequence, but are otherwise non-fluorescent when in a non-hybridized state. Such oligonucleotides are disclosed, for example, in U.S. Patent Application Publication No. 2003/0113765.

[0041] Sex specific antibodies may also be used to label the sperm cells in a labeling mixture. For example, a sex specific antibody may be conjugated with a fluorescent moiety (or equivalent reporter molecule). Because the antibody binds to antigens present on only an X chromosome-bearing or, alternatively, a Y chromosomebearing cell, such cells can be selectively identified based upon their fluorescence (versus the non-fluorescence of an unlabeled cell). Moreover, more than one sex specific antibody, each antibody having a different fluorescent moiety attached thereto, may be used simultaneously. This allows for differentiation of X chromosome-bearing and Y chromosome-bearing cells based upon the differ- ing fluorescence of each.

[0042] Luminescent, color-selective nanocrystals may also be used to label sperm cells in a labeling mixture. Also referred to as quantum dots, these particles are well known in the art, as demonstrated by U.S. Patent No. 6,322,901 and U.S. Patent No. 6,576,291. These nanocrystals have been conjugated to a number of biological materials, including for example, peptides, antibodies, nucleic acids, streptavidin, and polysaccharides, (see, for example, U.S. Patent Nos. 6,207,392; 6,423,551; 5,990,479, and 6,326,144), and have been used to detect biological targets (see, for example, U.S. Patent Nos. 6,207,392 and 6,247,323).

[0043] The preferred concentration of the label in the labeling mixture is a function of a range of variables which include, for example, whether the label binds to the exterior of the cell or whether it must cross the cell membrane; if it must cross the cell membrane, the permeability of the cells to the selected label; the temperature of the labeling mixture; the amount of time allowed for labeling to occur; and the degree of selectivity desired. Ingeneral, the concentration of the label is preferably sufficient to achieve the desired degree of labeling of the cells in a reasonably short period of time without substantially detrimentally affecting sperm viability. For example, the concentration ofHoechst 33342, Hoechst 33258, SYBR-14, or BBC in the labeling mixture will generally be between about 0.1 μΜ and about 1.0M, preferably from about 0.1 μΜ to about 700μΜ, and more preferably from about 100μΜ to about 200μΜ. The concentration of Hoechst 33342, Hoechst 33258, SYBR-14, or BBC in the staining mixture may be generally be between about 400μΜ to about 500μΜ, and most preferably about 450μΜ. Accordingly, under one set of labeling conditions, the concentration ofHoechst 33342 is preferably about 100μΜ. Under another set of labeling conditions, the concentration ofHoechst 33342 is about 150μΜ. Under still another set of labeling conditions the concentration is preferably about 200μΜ. Under yet another set of staining conditions the concentration ofHoechst 33342 is most preferably about 450μΜ.

[0044] As another example, the concentration of a fluorescent polyamide, such as for example, those described in U.S. Application Publication No. 2001/0002314, will generally be between about 0.1 μΜ and about 1mM, preferably from about 1μΜ to about 1mM, more preferably about 5μΜ to about 100μΜ, even more preferably about 10μΜ.

[0045] Once formed, the labeling mixture may be maintained at any of a range of temperatures. For example, labeling with Hoechst 33342 or Hoechst 33258 typically will be performed within a range of about 4°C to about 50°C. For example, the labeling mixture may be maintained at a "relatively low" temperature, i.e., a temperature of about 4°C to about 30°C; preferably about 20°C to about 30°C, more preferably from about 25°C to about 30°C, and most preferable at about 28°C. Alternatively, and according to the present invention, the labeling mixture may be maintained within an "intermediate" temperature range, i.e., a temperature of about 30°C to about 39°C. In another example, the labeling mixture may be maintained within a "relatively high" temperature range, i.e., a temperature of about 40°C to about 50°C; preferably from about 40°C to about 45°C, more preferably from about 40°C to about 43°C, and most preferably at about 41 °C. Selection of a preferred temperature generally depends upon a range of variables, including for example, whether the label binds to the exterior of the cell or whether it must cross the cell membrane; if it must cross the cell membrane, the permeability of the cells to the selected label; the concentration of the label (s) in the labeling mixture; the amount of time allowed for labeling to occur; and the degree of selectivity desired.

[0046] The pH of the labeling mixture may be maintained at any of a range of pH’s. For example, labeling with Hoechst 33342 or Hoechst 33258 typically will be performed in a pH range of about 5.0 to about 9.0. For example, the labeling mixture may be maintained at a "slightly acidic" pH, i.e., from about 5.0 to about 7.0. In one example, the pH may preferably be from about 6.0 to about 7.0, more preferably from about 6.0 to about 6.5, and most preferably at about 6.2. In an alternative example, the labeling mixture may be maintained at a "slightly basic" pH, i.e., from about 7.0 to about 9.0. The pH may be preferably from about 7.0 to about 8.0, more preferably from about 7.0 to about 7.5, and most preferably at about 7.3. Generally, however, if labeling is performed at a pH other than about 7.0, once a period of time sufficient to obtain the desired degree of labeling has occurred, the labeling mixture will be adjusted to a pH of about 7.0.

[0047] Optionally, the labeling mixture may also contain additives to enhance sperm viability. Exemplary additives include an antibiotic, a growth factor, or a composition which regulates oxidation/reduction reactions in-tracellularly and/or extracellularly as discussed above with respect to cell sample collection. These additives may be added to the labeling mixture in accordance therewith.

[0048] Uptake of the label by or binding of the label to the sperm cells in the labeling mixture is allowed to continue for a period of time sufficient to obtain a dispersion of sperm cells labeled to the desired degree. That period is typically a period sufficient for the label to bind to the sperm cells or the DNA of the sperm cells such that a member of a subpopulation of cells may be identified and its position in the dispersion determined. Selection of a preferred period generally depends upon a range of variables, including for example, whether the label binds to the exterior of the cell or whether it must cross the cell membrane; if it must cross the cell membrane, the permeability of the cells to the selected label; the concentration of the label (s) in the labeling mixture; the temperature of the labeling mixture; and the degree of selectivity desired. For example, the period may be a period sufficient for a fluorescent DNA selective dye to bind to the DNA of X and Y chromosome-bearing sperm cells such that they may be selected based upon the differing and measurable fluorescence intensity between the two. When, labeling with Hoechst 33342 or Hoechst 33258, for example, typically this period will be no more than about 160 minutes, preferably no more than about 90 minutes, still more preferably no more than about 60 minutes, and most preferably from about 5 minutes to about 40 minutes.

[0049] Certain labels, and in particular certain dyes, are capable of permeating the sperm cells and specifically binding the DNA without further intervention to increase the permeability of the cells. With other labels, however, it may be desirable to treat the sperm cells prior to labeling to increase the rate of permeation without unacceptably reducing viability or motility. Any suitable method known to those skilled in the art may be used. Such methods include electroporation, the use of cell-permeation-enhancing solutions, e.g., mild surfactants, or chemical shock. Where it is desired or advantageous to use other or more stringent techniques, such treatments can include the use of liposomes or many of the techniques which are used by those skilled in the art to introduce stains, dyes, genes, or vectors into living cells. These methods include, but are not limited to microinjection such as used by Gordon et al. (Proc. Natl. Acad. Sei. USA, 77(12): 7380-4 (1980)) and since extended to rabbits, sheep, cattle and pigs; DEAE-dextran-mediated transfer; coprecipitation with calcium phosphate; and other techniques, all of which are well known to one of skill in the art. In yet other instances, it may be desirable to centrifuge the sperm and re-suspend the centrifuged sperm in another medium, albeit based on the same or substantially the same buffer system, to remove certain components (which may have previously been added to the sperm dispersion) that may interfere with later processing steps.

[0050] One particularly preferred method of increasing the permeability of a sperm cell to a label is the well known method of optoinjection as disclosed in U.S. Patent No. 6,753,161. Generally, optoinjection is a method of transiently permeabilizing a cell by contacting the cell with a pulse of radiation. A cell is illuminated, identified and located based upon the detection of the illumination of the cell, and irradiated with a pulse of radiation sufficient to transiently permeabilize the cell. For example, optoinjection may be used to transiently permeabilize sperm cells and thereby allow labels that bind to the internal contents of a cell (such as, for example, labels that bind to DNA or RNA) to more easily and efficiently enter into the cells. Therefore, optoinjection may be used, for example, to decrease the time needed to sufficiently label sperm cells with a fluorescent DNA selective dye, such as Hoechst 33342, Hoechst 33258, or with a fluorescent polyamide.

[0051] Optoinjection may also be used to label cells at reduced temperatures. Previously, sperm cells were generally labeled with, for example, fluorescent DNA selective dyes, at temperatures in excess of 30°C and even 40°C, as the higher temperatures aided in increased dye uptake. While labeling at such temperatures is certainly feasible, it may be beneficial to avoid exposing the sperm cells to higher temperatures, especially for an extended period of time. Therefore, optoinjection may be used to permeabilize sperm cells, thereby allowing for the labeling of the cells at a lower temperature while still maintaining or exceeding the staining efficiency and speed typically associated with labeling at higher temperatures.

[0052] For example, a labeling mixture may be formed comprising sperm cells, a motility inhibitor, and a dye in a concentration from about 100μΜ to about 200μΜ, and the staining mixture held for a period of time at a temperature of about 41 °C. In another example, the motility inhibitor comprises 0.204g NaHC03, 0.433g KHC03, and 0.473g C6H807-H20 per 25mL of purified water (0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0.090 moles/L C6H807-H20 in water).

[0053] In one example, a labeling mixture may be formed comprising sperm cells, a motility inhibitor, and a dye in a concentration of about 400μΜ to about 500μΜ, and the staining mixture held for a period of time at a temperature of about 41 °C. In another example, the dye concentration may be 450μΜ. In another example, the motility inhibitor comprises 0.204g NaHC03, 0.433g KHC03, and 0.473g C6H807-H20 per 25mL of purified water (0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0.090 moles/L C6H807-H20 in water).

[0054] In still another example, a labeling mixture may be formed comprising sperm cells, a motility inhibitor, and a dye in a concentration from about 100μΜ to about 200μΜ and the staining mixture held for a period of time at a temperature of about 28°C. In another example, the motility inhibitor comprises 0.204g NaHC03, 0.433g KHC03, and 0.473g C6H807-H20 per 25mL of purified water (0.097 moles/L of NaHCOs, 0.173 moles/L of KHC03, 0.090 moles/L C6H807-H20 in water).

[0055] In yet another example, a labeling mixture may be formed comprising sperm cells, a motility inhibitor, and a dye in a concentration from about 400μΜ to about 500μΜ, and the staining mixture held fora period of time at a temperature of about 28°C. In another example, the dye concentration may be 450μΜ. In another embodiment, the motility inhibitor comprises 0.204g NaHC03, 0.433g KHC03, and 0.473g C6H807-H20 per 25mL of purified water (0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0.090 moles/L C6H807-H20 in water).

Formation of a Dispersion of Labeled Cells [0056] Once a labeling mixture is formed, the labeling mixture is used to form a dispersion of labeled cells, which is subsequently inspected and dosed. Such a dispersion comprises labeled sperm cells and a chemical agent that induces sperm immotility. Alternatively, or in addition to, the dispersion may comprise a liquid, such as a buffer as described above, in addition to the labeled sperm cells, and wherein the temperature of the cells or the liquid induces sperm immotility.

[0057] The labeled sperm cells may be in any of a number of forms. For example, the labeled cells may still be part of a labeling mixture. As such, the labeled cells may still be in excess or unbound label. Alternatively, the labeled cells may have been separated from any excess or unbound label, such as for example by washing the cells or by spinning down the cells, such as by centrifugation, and then separating the cells from the unbound label. In such an instance, the labeled cells will generally thereafter be combined with a buffer as discussed above with respect to collection of a cell sample. In any event, the sperm cells in the dispersion are labeled such that the absence or amount of label associated with one or more of the sperm cells allows for the identification of a genetic, proteomic, structural, orfunctional characteristic of a subpopulation of sperm cells in the dispersion. The sperm cells may be maintained at a temperature that induces or increases sperm immotility.

[0058] The dispersion of labeled cells may also contain a chemical agent that induces sperm immotility, such as, for example, a motility inhibitor as discussed above. The chemical agent may be added to the labeling mixture or labeled cells at any time before the optical inspection of the dispersion, such as for example, before, during, or after labeling of the sperm cells. The chemical agent may be combined with labeled cells, the labeled cells being in any of the number of forms discussed above (i.e., still in the labeling mixture or removed therefrom). In a particular example, a labeling mixture may be formed comprising sperm cells and a label, and then the labeling mixture combined with the chemical agent that induces sperm immotility. Alternatively, or in addition to the chemical agent, the temperature of the labeling mixture may be reduced as discussed above in order to induce or increase sperm immotility.

Inspection, Determination, and Dosing of the Cells [0059] Once a dispersion of labeled cells has been formed, the dispersion is optically inspected to identify individual sperm cells as members of a subpopulation, the positions of the members of the subpopulation in the dispersion are determined, and an energy beam is delivered to different positions within the dispersion to selectively dose members of the subpopulation with an energy source, thereby decreasing the viability of the dosed cells, or at least their capacity to fertilize an egg, without similarly affecting sperm cells at other positions in the dispersion.

[0060] These steps are typically performed by a device and in a manner commercially referred to as LEAP® (Laser-Enabled Analysis and Processing) Technology Platform (Cyntellect, Inc., San Diego, CA). Generally, this process requires that cells be labeled with a marker to identify and locate individual cells of a subpopulation of cells within a mixture or larger population of cells. The population of cells is then illuminated, allowing for the position of the individuals cells of the subpopulation to be identified. A treatment laser is then positioned in a manner such that it can emit a beam of energy to induce a change in the identified cells of the subpopulation. The induced change is usually cell death. These processes and devices are further described in U.S. Patent Nos. 6,534,308; 6,514,722; 6,753,161 ; and 6,642,018.

[0061] The energy source as used may be any source that, when applied in a certain dose to the sperm cells, decreases the viability of the dosed cells, or at least their capacity to fertilize an egg, with minimal or no similar affect to sperm cells at other positions in the dispersion. Typically, the energy source will be in the form of an energy beam. Examples of suitable energy sources include lasers, collimated or focused non-laser light, RF energy, accelerated particles, focused ultrasonic energy, electron beams, or other radiation beams. Preferably, however, the energy source is a laser, as a laser provides the advantages of high intensity and relatively efficient use of energy in a compact size and with minimal heat generation, thereby allowing dosing of a single cell without significantly adversely affecting surrounding cells.

[0062] The cells may be placed on any surface suitable for optical inspection and dosing of the cells. Generally, such surfaces will have a horizontal surface (either a top, a bottom, or both) that is optically transparent to the energy source used to optically inspect the cells as well as the energy source used to dose members of the subpopulation. Such suitable surfaces include, for example, glass, plastics or other related polymers, and Pyrex®, and may be in the form of a flat slide, a petri dish, a singlewell plate, or a multi-well plate. Examples are discussed in, for example, U.S. Patent Nos. 6,534,308 and 6,514, 722.

[0063] A sample of sperm cells may be divided into several smaller, individual samples, such as for exam pie, by being divided into a number of individual samples for use with a multi-well plate. Each sample (for example, within each well) may be enriched for the same characteristic, thereby producing multiple samples each of which is enriched for a single characteristic. Advantageously, however, each of the samples may be enriched foradifferent characteristic. By way of example, a sample of sperm cells may be divided into smaller, individual samples, and each individual sample placed in one well of a 96 well plate. The individual sample of each well may then be enriched with respect to a single characteristic different from that of the samples in each of the other wells, resulting in 96 individual samples, each enriched with respect to a different characteristic.

[0064] Once the member cells of the subpopulation have been dosed with an energy source, the cell population may be further enriched by purifying the non-dosed cells (i.e., the sperm cells that were not dosed with energy). The purification of the non-dosed cells may occur by removal of either the dosed cells or the non-dosed cells from the dispersion, resulting in a subpopulation comprising non-dosed cells that are enriched for a par- ticular characteristic. For example, if the particular characteristic is Y chromosome-bearing sperm cells, the non-dosed cells may be purified such that they comprise at least about 85% Y chromosome-bearing sperm cells; preferably at least about 90% Y chromosome-bearing sperm cells; more preferably at least about 95% Y chromosome-bearing sperm cells; even more preferably at least about 97% Y chromosome-bearing sperm cells,-and most preferably at least about 99% Y chromosomebearing sperm cells. Alternatively, if the particulardesired characteristic is X chromosome-bearing sperm cells, the non-dosed cells may be purified such that they comprise at least about 85% X chromosome-bearing sperm cells; preferably at least about 90% X chromosome-bearing sperm cells; more preferably at least about 95% X chromosome-bearing sperm cells; even more preferably at least about 97% X Chromosom e-bearing sperm cells; and most preferably at least about 99% X chromosome-bearing sperm cells.

[0065] Removal of either the dosed or non-dosed cells from the dosed dispersion (i.e., from the larger population ofsperm cells comprising both the dosed and non-dosed cells) may be achieved by any of a number of means known to those of skill in the art. Such methods include, for example, spinning down the entire dispersion, such as by centrifugation, and then removing or wicking the supernatant containing the dosed cells. Another method includes the addition of a high-density medium to the dispersion. High-density mediums that may be added to the dispersion include, for example, Percoll® and Isolate®. Generally, in a high-density separation, viable cells (i.e., non-dosed cells with respect to the present application) are able to swim to the top of the high-density medium and may thereafter be skimmed from the top of the medium, whereas damaged ordead cells (i.e., dosed cells) will remain dispersed within the high-density medium, generally within the bulk phase. Methods of using such mediums are well known in the art.

[0066] Advantageously, a dispersion of labeled cells may contain a subpopulation of cells labeled with different labels. Each label may identify a different genetic, proteomic, structural, orfunctional characteristic of a subpopulation of sperm cells in the dispersion. Moreover, each label may be individually detectible when bound to a sperm cell; that is to say, it is possible to separately detect the different labels. For example, the labels may each fluoresce at different wavelengths.

[0067] A different label may be added to the labeling mixture or to the dispersion of labeled cells. Alternatively, a different label may be added subsequent to any of the steps of inspection, determination, or dosing of the cells. Preferably, however, a different label will be added subsequent to the dosing of the dispersion. For example, once the members of a subpopulation ofsperm cells have been dosed, the dosed dispersion (including both the dosed cells and the non-dosed cells) or a purified dosed dispersion (including only the non-dosed cells) may be labeled again, but with a different label, and the process of inspection, determination, and dosing of the cells may be repeated, generally as disclosed above, based upon the absence or amount of the different label associated with a sperm cell.

[0068] Generally, the different label may be used to identify an additional genetic, proteomic, structural, or functional characteristic of the non-dosed cells that may be different from the characteristic used to previously identify members of a subpopulation to which a dose of energy was delivered (i.e., that is different from the characteristic used to previously determine cells to be dosed or not dosed). This provides a manner of further enriching an already enriched population of cells.

[0069] By way of example, adispersion of labeled cells may be formed using a fluorescent DNA selective dye. The dispersion may then be optically inspected to identify individual sperm cells that are X chromosome-bearing. The position of the X chromosome-bearing sperm cells may subsequently be determined, and a dose of energy may then be delivered to one or more of the X chromosome-bearing cells, thereby achieving an enriched Y chromosome-bearing viable cell population. Thereafter, the dosed dispersion (including both the dosed (X chromosome-bearing) and non-dosed (Y chromosome-bearing) cells) or a purified dosed dispersion (including only the non-dosed cells) may be labeled with another label that indicates acrosomal integrity, such as for example, phycoerythrin-conjugated peanut agglutinin (PE-PNA) that induces cell fluorescence, and in particular acrosomal fluorescence, when contacted with a cell having a reacted or damaged acrosome. The steps of optical identification and determination of the position of PE-PNA fluorescing cells may then be performed, and those cells dosed with energy. The result is a subpopulation of non-dosed cells that are Y chromosome-bearing and that have unreacted and undamaged (i.e., intact) acrosomes. See, for example, Nagy et al., Biol Repród, 68:1828-1835 (2003).

Cryoextension of the Cells [0070] Once the member cells of the subpopulation have been dosed with an energy source, the entire sperm cell population (both dosed and non-dosed cells) or a subset of the population (the non-dosed cells only) may be cooled or frozen for use at a later date, for example, in fertilization procedures. In such instances, the non-dosed sperm cells may benefit from the addition of a cry-oextender to minimize the impact upon viability or postthaw motility as a result of cooling and freezing.

[0071] Generally, a cryoextender may comprise a protein source, a cryoprotectant, and a motility inhibitor. If included, a protein source may be added to provide support to the cells. The protein source may be any protein source that does not interfere with the viability of the non-dosed sperm cells and is compatible with the motility inhibitor. Examples of common protein sources include milk (including heat homogenized and skim), milk extract, egg yolk, egg yolk extract, soy protein and soy protein extract. Such proteins may be found in a concentration from about 10% (v/v) to about 30% (v/v), preferably from about 10% (v/v) to about 20% (v/v), and more preferably about 20% (v/v).

[0072] A cryoprotectant may preferably be included in the cryoextender to lessen or prevent cold shock or to maintain fertility of the non-dosed sperm cells. Numerous cryoprotectants are known in the art. Selection of a cryoprotectant suitable for use with a given extender may vary, and depends upon the species from which the sperm to be frozen were obtained. Examples of suitable cryoprocectants include, for example, glycerol, dimethyl sulfoxide, ethylene glycol, propylene glycol, trehalose, Triladyl®, and combinations thereof. If included, generally, these cryoprotectants are present in the cryoextender in an amount of about 1% (v/v) to about 15% (v/v), preferably in an amount of about 5% (v/v) to about 10% (v/v), more preferably in an amount of about 7% (v/v), and most preferably in an amount of about 6% (v/v).

[0073] In addition, the cryoextender may contain a motility inhibitor as discussed above with respect to cell sample collection. The motility inhibitor (s) may be added to the cryoextender in accordance therewith.

[0074] In one example, the cryoextender comprises a motility inhibitor, water, Triladyl®, egg yolk, and pyruvic acid. I n yet another eaxmple, the cryoextender com prises 0.097 moles/L of NaHC03, 0.173 moles/L of KHC03, 0.090 moles/L C6H807-H20 in water, and 25g Triladyl®, 25g egg yolk, and 10mM pyruvic acid per 75mL of water.

[0075] In another particular example, the cryoextender comprises a motility inhibitor, water, Triladyl®, and egg yolk. Inyetanother example, the cryoextender com prises 0.097 moles/L of NaHCOs, 0.173 moles/L of KHC03, 0.090 moles/L C6H807-H20 in water, and 25g Triladyl®, and 25g egg yolk per 75mL of water.

[0076] Optionally, the cryoextender may also contain an antibiotic or a composition which regulates oxida-tion/reduction reactions intracellularly and/or extracellu-larly as discussed above with respect to cell sample collection. Each of these additives may be added to the cryoextender in accordance therewith.

[0077] Cryopreservation of the entire sperm population (i.e., cryopreservation of the dosed dispersion) results in the formation of a frozen dispersion having two subpopulations, each of these subpopulations being substantially different from the other. However, each subpopulation is composed of substantially homogenous cells. That is to say, each subpopulation is comprised of cells, each of the individual cells of a single subpopulation having a characteristic common to each of the other cells in the same subpopulation. In example, the dispersion may be further enriched prior to cryopreservation by purifying the dispersion, based upon the presence or the absence of the common characteristic (s), according to methods described above.

[0078] Therefore, for example, the present process could be used to form a frozen sperm dispersion, the dispersion comprising a dosed subpopulation of cells, wherein all the cells of the dosed subpopulation are X chromosome-bearing cells, and a non-dosed subpopulation of cells, wherein all the cells of the non-dosed subpopulation are Y chromosome-bearing cells. According to this example, the cells not receiving a dose of energy (i.e., the non-dosed Y chromosome-bearing cells) will comprise at least about 85% Y chromosome-bearing sperm cells; preferably at least about 90% Y chromo-some-bearing sperm cells; more preferably at least about 95% Y chromosome-bearing sperm cells; even more preferably at least about 97% Y chromosome-bearing sperm cells; and most preferably at least about 99% Y chromosome-bearing sperm cells.

[0079] Alternatively, the present process could be used to form a frozen sperm dispersion, the dispersion comprising a dosed subpopulation of cells, wherein all the cells of the dosed subpopulation are Y chromosomebearing cells, and a non-dosed subpopulation of cells, wherein all the cells of the non-dosed subpopulation are X chromosome-bearing cells. According to this example, the non-dosed X chromosome-bearing cells will comprise at least about 85% X chromosome-bearing sperm cells; preferably at least about 90% X chromosome-bearing sperm cells; more preferably at least about 95% X chromosome-bearing sperm cells, even more preferably at least about 97% X chromosome-bearing sperm cells; and most preferably at least about 99% X chromosomebearing sperm cells.

Fertilization [0080] A novel process forfertilizing an egg or a female mammal may be used for selectively decreasing the viability of a subpopulation of sperm cells in a cell dispersion as described above.

[0081] Once the dosing of the dispersion of labeled cells has occurred, the dosed dispersion (comprising both the dosed and non-dosed cells) may be used to fertilize a female mammal. Fertilization may be performed according to any of a number of methods well known to those of skill in the art. These methods include, for example, microinjection, artificial insemination, and other methods well known to those of skill in the art. For example, a dosed dispersion comprising both the dosed and non-dosed cells, a purified dispersion comprising only the non-dosed cells, or a derivative of either may be used to inseminate a female mammal, such as for example, by artificial insemination.

[0082] Alternatively, once the dosing of the dispersion of labeled cells has occurred, the dispersion may be used to fertilize an egg, and more particularly, an egg in vitro. The fertilized egg may thereafter be introduced into the uterus of a female mammal by any of a number of means well known to those of skill in the art, such as for example embryo transplant. For example, a dosed dispersion, a purified dispersion, or a derivative of either may be used to fertilize an egg in vitro. Subsequently, the fertilized egg may be introduced into the uterus of a female mammal.

[0083] Fertilization of a female mammal or an egg in vitro using any of the aforementioned dispersions may occur shortly after dosing of the dispersion is complete, such as for example, within about7days, preferably within about 5 days, more preferably within about 3 days, still more preferably within about 2 days, and in one example, within about 1 day after dosing of the dispersion is complete. In such an instance, generally the dispersion may not have been cryopreserved prior to fertilization of a female mammal or an egg in vitro (i.e., the dispersion is fresh or comprises fresh sperm cells); instead it may have been maintained in a motility inhibitor and/or may have been refrigerated at temperatures of about 2°C to about 7°C, more preferably from about 3°C to about 5°C, and most preferably at about 4°C. Alternatively, the dispersion may be cryopreserved and then thawed prior to fertilization of a female mammal or an egg in vitro (i.e., the dispersion is frozen/thawed or comprises frozen/thawed sperm cells). Typically, in such an instance, the cryopreserved dispersion will be thawed immediately before fertilization of a female mammal or an egg in vitro.

Claims 1. A process for selectively decreasing the capacity of a subpopulation of non-human sperm cells in a sperm cell dispersion, the process comprising: a) labeling a sperm cell sample with a labeling mixture including a chemical agent that induces sperm immotility and DNA selective dye at a temperature between 30°C and 39°C to form a dispersion of labeled sperm cells in a liquid, wherein the amount of the label associated with a sperm cell indicates a genetic, proteomic structural, or functional characteristic of a subpopulation of sperm cells in the dispersion; b) optically inspecting the dispersion to identify individual sperm cells as members of the subpopulation; c) determining the position of members of the subpopulation in the dispersion; and d) delivering a dose of energy to different positions within the dispersion to selectively decrease the capacity of members of the subpopulation to fertilize an egg without similarly affecting sperm cells at other positions in the dispersion to produce an enriched sperm cell population. 2. The process as claimed in claim 1, characterised in that the amount of the label associated with the sperm cell indicates that the sperm cell is an X chromosome-bearing sperm cell or a Y chromosomebearing sperm cell. 3. The process as claimed in any one of claims 1 or 2, characterised in that the label is selected from the group consisting of fluorescent dyes, DNA selective dyes, polyamides, oligonucleotides, and a polypeptide that binds to a surface specific characteristic of a sperm cell. 4. The process as claimed in claim 3, characterised in that the label is a DNA selective fluorescent dye. 5. The process as claimed in claim 4, characterised in that the label is Hoechst 33342, Hoechst 33258, or SYBR-14. 6. The process as claimed in claim 1, characterised in that the dose of energy is selected from the group consisting of radiation beams, laser beams, collimated non-laser light, focused non-laser light, and focused ultrasonic energy. 7. The process as claimed in claim 1, characterised in that the process further comprises purifying the sperm cells not receiving a dose of energy, the non-dosed cells. 8. The process as claimed in claim 7, characterised in that purifying the non-dosed cells comprises centrifuging the dispersion and removing the dosed cells. 9. The process as claimed in any one of claims 1 to 4, characterised in that optically inspecting the dispersion to identify individual sperm cells as members of the subpopulation comprises optically inspecting a captured image of the cells. 10. The process as claimed in any one of claims 1 to 4, characterised in that prior to optically inspecting the dispersion, the dispersion is distributed onto a multi-well plate. 11. The process as claimed in any one of claims 1 to 4, characterised in that the dose of energy is sufficient to decrease the viability and/or cause the death of the members of the subpopulation as compared to the viability of sperm cells not receiving a dose of energy. 12. The process as claimed in claim 1, characterised in that the process further com prises cryopreserving the dispersion subsequent to delivering the dose of energy. 13. The process as claimed in any one of the preceding claims characterised in that the female mammal is bovine, equine, porcine, or swine. 14. The process as claimed in any one of claims 2 to 13, characterised in that the dispersion is cryopre-served. 15. The process as claimed in any one of claims 1 to 11, characterised in that the process further comprises: a) labeling the dosed dispersion with an additional label, wherein the presence, absence or amount of the additional label associated with a sperm cell indicates a genetic, proteomic, structural, or functional characteristic of a subpopulation of sperm cells in the dosed dispersion; b) optically inspecting the dosed dispersion to identify individual sperm cells associated with the additional label; c) determining the position of sperm cells associated with the additional label in the dosed dispersion; and d) d) delivering a dose of energy to different positions within the dispersion.

Patentansprüche 1. Prozess zum selektiven Verringern der Kapazität einer untergeordneten Population von nicht menschlichen Spermazellen in einer Suspension von Spermazellen, wobei der Prozess umfasst: a) Markieren einer Spermazellenprobe mit einem Markierungsgemisch umfassend eine chemische Substanz, die eine Unbeweglichkeit von Sperma herbeiführt, und einen DNA-selektiven Farbstoff bei einer Temperatur zwischen 30°C und 39°C, um eine Suspension von markierten Spermazellen in einer Flüssigkeit zu bilden, wobei die Menge der zugehörigen Markierung für eine Spermazelle auf eine genetische, proteo-mische, strukturelle oder funktionelle Eigenschaft einer untergeordneten Population von Spermazellen in der Suspension hinweist; b) optisches Prüfen der Suspension, um einzelne Spermazellen als Teile der untergeordneten Population zu identifizieren; c) Bestimmen der Position von Teilen der untergeordneten Population in der Suspension; und d) Bereitstellen einer Energiedosis an verschiedenen Positionen in der Suspension, um die Kapazität von Teilen der untergeordneten Population selektiv zu verringern, ein Ei zu befruchten, ohne sich gleichermaßen auf Spermazellen an anderen Positionen in der Suspension auszuwirken, eine angereicherte Spermazellenpopulation zu produzieren. 2. Prozess nach Anspruch 1, dadurch gekennzeichnet, dass die Menge der zugehörigen Markierung für die Spermazelle angibt, dass die Spermazelle eine X-Chromosom enthaltende Spermazelle oder eine Y-Chromosom enthaltende Spermazelle ist. 3. Prozess nach einem der Ansprüche 1 oder 2, dadurch gekennzeichnet, dass die Markierung aus der Gruppe bestehend aus fluoreszierenden Farbstoffen, DNA-selektiven Farbstoffen, Polyamiden, Oligonukleotiden und einem Polypeptid, das sich an eine oberflächenspezifische Eigenschaft einer Spermazelle bindet, ausgewählt wird. 4. Prozess nach Anspruch 3, dadurch gekennzeichnet, dass die Markierung ein DNAselektiver fluoreszierender Farbstoff ist. 5. Prozess nach Anspruch 4, dadurch gekennzeichnet, dass die Markierung Hoechst 33342, Hoechst 33258 oder SYBR-14 ist. 6. Prozess nach Anspruch 1, dadurch gekennzeichnet, dass die Energiedosis aus der Gruppe bestehend aus Strahlenbündeln, Laserstrahlen, kollimier-tem Nicht-Laserlicht, fokussiertem Nicht-Laserlicht und fokussierter Ultraschallenergie ausgewählt wird. 7. Prozess nach Anspruch 1, dadurch gekennzeichnet, dass der Prozess ferner das Reinigen der Spermazellen umfasst, die keine Energiedosis erhalten, die nicht dosierten Zellen. 8. Prozess nach Anspruch 7, dadurch gekennzeichnet, dass das Reinigen der nicht dosierten Zellen das Zentrifugieren der Suspension und das Entfernen der dosierten Zellen umfasst. 9. Prozess nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass das optische Prüfen der Suspension zum Identifizieren einzelner Spermazellen als Teile der untergeordneten Population das optische Prüfen eines erfassten Bilds der Zellen umfasst. 10. Prozess nach einem der Ansprüche 1 bis4, dadurch gekennzeichnet, dass die Suspension vor dem optischen Prüfen der Suspension auf eine Mikrotiterplatte verteilt wird. 11. Prozess nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass die Energiedosis ausreicht, um die Lebensfähigkeit zu verringern und/oder um den Tod der Teile der untergeordneten Population zu verursachen, im Vergleich zur Lebensfähigkeit von Spermazellen, die keine Energiedosis erhalten. 12. Prozess nach Anspruch 1, dadurch gekennzeichnet, dass der Prozess ferner das Kryokonservieren der Suspension nachdem Bereitstellen der Energiedosis umfasst. 13. Prozess nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass das weibliche Säugetier ein Rind, Pferd oder Schwein ist. 14. Prozess nach einem der Ansprüche 2 bis 13, dadurch gekennzeichnet, dass die Suspension kry-okonserviert wird. 15. Prozess nach einem der Ansprüche 1 bis 11, dadurch gekennzeichnet, dass der Prozess ferner umfasst: a) Markieren der dosierten Suspension mit einer zusätzlichen Markierung, wobei das Vorhandensein, Fehlen oder die Menge der zugehörigen zusätzlichen Markierung für eine Spermazelle auf eine genetische, proteomische, strukturelleoderfunktionelle Eigenschaft einer untergeordneten Population von Spermazellen in der dosierten Suspension hinweist; b) optisches Prüfen der dosierten Suspension, um einzelne Spermazellen zu identifizieren, die zu der zusätzlichen Markierung gehören; c) Bestimmen der Position von zugehörigen Spermazellen für die zusätzliche Markierung in der dosierten Suspension; und d) Bereitstellen einer Energiedosis an verschiedenen Positionen in der Suspension.

Revendications 1. Procédé de diminution sélective de la capacité d’une sous-population de spermatozoïdes non humains dans une dispersion de spermatozoïdes, le procédé comprenant : a) le marquage d’un échantillon de spermatozoïdes avec un mélange de marquage incluant un agent chimique qui induit une immotilité de sperme et un colorant sélectif de l’ADN à une température entre 30 °C et 39 °C pour former une dispersion de spermatozoïdes marqués dans un liquide, dans lequel la quantité du marqueur associé à un spermatozoïde indique une caractéristique génétique, protéomique, structurelle ou fonctionnelle d’une sous-population de spermatozoïdes dans la dispersion ; b) l’examen optique de la dispersion pour iden-tifierdes spermatozoïdes individuels en tant que membres de la sous-population ; c) la détermination de la position de membres de la sous-population dans la dispersion ; et d) l’administration d’une dose d’énergie à des positions différentes dans la dispersion pour di minuer sélectivement la capacité des membres de la sous-population à fertiliser un oeuf sans affecter de façon similaire les spermatozoïdes à d’autres positions dans la dispersion pour produire une population de spermatozoïdes enrichie. 2. Procédé selon la revendication 1, caractérisé en ce que la quantité du marqueur associé au spermatozoïde indique que le spermatozoïde est un spermatozoïde porteur du chromosome X ou un spermatozoïde porteur du chromosome Y. 3. Procédé selon l’une quelconque des revendications 1 ou 2, caractérisé en ce que le marqueur est choisi dans le groupe constitué des colorants fluorescents, des colorants sélectifs de l’ADN, des polyamides, des oligonucléotides, et d’un polypeptide qui se lie à une caractéristique spécifique de surface d’un spermatozoïde. 4. Procédé selon la revendication 3, caractérisé en ce que le marqueur est un colorant fluorescent sélectif de l’ADN. 5. Procédé selon la revendication 4, caractérisé en ce que le marqueur est Hoechst 33342, Hoechst 33258, ou SYBR-14. 6. Procédé selon la revendication 1, caractérisé en ce que la dose d’énergie est choisie dans le groupe constitué de faisceaux de rayonnement, de faisceaux laser, d’une lumière non-laser collimatée, d’une lumière non-laser focalisée, et d’une énergie ultrasonore focalisée. 7. Procédé selon la revendication 1, caractérisé en ce que le procédé comprend en outre la purification des spermatozoïdes ne recevant pas de dose d’énergie, les cellules non dosées. 8. Procédé selon la revendication 7, caractérisé en ce que la purification des spermatozoïdes non dosés comprend la centrifugation de la dispersion et l’élimination des cellules dosées. 9. Procédé selon l’une quelconque des revendications 1 à 4, caractérisé en ce que l’examen optique de la dispersion pour identifier des spermatozoïdes individuels en tant que membres de la sous-population comprend l’examen optique d’une image capturée des cellules. 10. Procédé selon l’une quelconque des revendications 1 à 4, caractérisé en ce que préalablement à l’examen optique de la dispersion, la dispersion est répartie sur une plaque multipuits. 11. Procédé selon l’une quelconque des revendications 1 à 4, caractérisé en ce que la dose d’énergie est suffisante pour diminuer la viabilité et/ou provoquer la mort des membres de la sous-population en comparaison à la viabilité des spermatozoïdes ne recevant pas de dose d’énergie. 12. Procédé selon la revendication 1, caractérisé en ce que le procédé comprend en outre la cryoconservation de la dispersion suite à l’administration de la dose d’énergie. 13. Procédé selon l’une quelconque des revendications précédentes, caractérisé en ce que le mammifère femelle est bovin, équin, porcin, ou suidé. 14. Procédé selon l’une quelconque des revendications 2 à 13, caractérisé en ce que la dispersion est cryo-conservée. 15. Procédé selon l’une quelconque des revendications 1 à 11, caractérisé en ce que le procédé comprend en outre : a) le marquage de la dispersion dosée avec un marqueur supplémentaire, dans lequel la présence, l’absence ou la quantité du marqueur supplémentaire associé à un spermatozoïde indique une caractéristique génétique, protéomique, structurelle, ou fonctionnelle d’une sous-population de spermatozoïdes dans la dispersion dosée ; b) l’examen optique de la dispersion dosée pour identifier des spermatozoïdes individuels associés au marqueur supplémentaire ; c) la détermination de la position de spermatozoïdes associés au marqueur supplémentaire dans la dispersion dosée ; et d) l’administration d’une dose d’énergie à des positions différentes dans la dispersion.

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description • US 5135759 A [0003] [0038] · US 6322901 B [0042] • WO 0168110 A, Koller [0004] · US 6576291 B [0042] • WO 0241906 A2, Didion [0005] · US 6207392 B [0042] • US 20050112541 A [0013] · US 6423551 B [0042] • US 20050003472 A [0030] [0035] · US 5990479 A [0042] • US 557407 P [0032] · US 6326144 B [0042] • US 11092313 B [0032] · US 6247323 B [0042] • WO 0241906 A [0038] · US 20010002314 A [0044] • US 5998140 A [0039] · US 6753161 B [0050] [0060] • US 6143901 A [0039] · US 6534308 B [0060] [0062] • US 6090947 A [0039] · US 6514722 B [0060] [0062] • US 20030113765 A [0040] · US 6642018 B [0060]

Non-patent literature cited in the description • SALISBURY ; GRAVES. J. Repród. Fertil., 1963, vol. · GORDON et al. Proc. Natl. Acad. Sei. USA, 1980, 6, 351-359 [0019] [0020] vol. 77 (12), 7380-4 [0049] • BEST et al. Proc. Natl. Acad. Sei. USA, 2003, vol. · NAGY et al. Biol Repród, 2003, vol. 68, 1828-1835 100 (21), 12063-12068 [0039] [0069] • GYGI et al. Nucleic Acids Res., 2002, vol. 30 (13), 2790-2799 [0039]

Claims (2)

to exacerbate the population of junction sxmnium to S1b. A method for reducing the ability of non-human sperm to diminish the ability of the sphygmomania of the spermatozoa, which method comprises: the designation of a sperm-mmta label. which provides the yparndum motion disruption to the Tefakhafo Patterns of carbon and DNA-selective dyes to give a dispersion of the parent dispensing at 3 ° C and 39 ° C of coughing. where spermatozoa: the amount of labeled 6d6 is indicated by the subspecies of the sub-population of sperm in the spermic-dispersed sperm, sphyphoid, structural ft onί ket élΐϊ ioy io Í ΐϊΐϊ ΐΐ ΐϊäs äs äs bj Visual inspection of 8 dispersions to identify individual members of iSpemilton: AAS; ej determining the position of members of the α1 population in the dispersion; and dj transmission of dj energy dose to different dispersions of dispersion, so that you have the chance to do 11¾: sdekdé for a long time: the ability of your sponges to find out what you are looking for. it is like influencing the dispatcher: in another position, he is a lover. 2, The method of claim 1, wherein the labeled strain attached to the spernnunmph is indicative of the presence of sperm or sperm with Y-chromium. A method according to claim 1 or 2, characterized by a signal; that g marking is selected from the group of stone masons, lysine dyes, DNA selector dyes, polyamides, pligon clips, and polytepltd associated with spenoiapr Speeifikio surface characteristics. 4, A method according to claim 3, wherein the label is a γhis-refractory ilescent dye; 5 <A 4> jgéUypöht number process, to denote ifoeéhst 33342, Iteeohst 3323: 8 or SYBRS4 oh <The first Jgabypod Sboti Szzdi Jehilian ytm that the interdependence of the group formed by the stone hands is wild: radiation, laser beam, dirty laser light, focused non-laser creature and focus? ultrasound energy. % yas. characterized in that the method further comprises purifying non-dosirozoites, which are not dosed with m energy doses. 8. A ?. Claim 7, characterized by the fact that the purification of the non-dosed sperm includes the dispensing of the dispersion and the dose! cells obtained. fe. A method according to any one of claims 1 to 3, characterized in that it comprises visual inspection of the image of the individual sperm of the individual spermatozoa of the spermatozoa. Hl, 1-4. A method according to any one of claims 1 to 4, characterized in that the wind deflection of the dispersion is effected by the dispersion of the dispersion onto a plurality of discs having a thickness of 1Ϊ, 1 to 4. Method according to any one of claims 1 to 3, characterized in that the energy dose is sufficient? To reduce the viability of the ai-populápó, the desire to thwart them, the total viability of the spermatozoa of the Etergládosi: <zkä? i. claim discard, inks, »tz * 8t jetlcroeave ,. abstinence $$$: »SágábSft #glW | § ·. - a dispersion of krsôpesærvik'ôj for the transmission of energwsdose according to any one of claims 1 to 4. bass jefjeípssive, with the nóstósiy otnlős SMfvâsniarhafôis, Sóleie, áfflfltifélé Desire pig, 14 A 2'L>. Claims PpMs * according to claim 1, wherein the dispersion is in the form of a dispersion. W> A Ml. That is, in addition, the following: (a) the addition of the dispersed dispersion to a further marking,. wherein the presence, absence, or amount of additional labeling of spornvíürnboK: capSoSoSo indicates the sperm 8d: $ pófc: .- gl-ppfüiMee of the dispersed dispersion: genetic, prototype, structural or functional property; b) visualization of the dispersed dispersion of the doppler is further ai-popnIa. has been identified to identify the members of its unique spinine; y) the definitions of positionalJia # t «of the labeled chapel members of dioiKbti: di ^ PiSi ^ Sii.ii ^ bbi; and d) the positioning of the self-energizing ZSS mediation dispersion in the interdependent positions.