BR132015024816E2 - selected sex insemination dose and sex-selected sperm production method - Google Patents

Abstract

"selected sex insemination dose and sex-selected sperm production method". Embodiments of the claimed invention relate to sex-selected sperm that have improved dose response and can be produced by the steps of extending a sperm sample with buffering and adjusting the sperm sample concentration extended to a target range of concentration. Extended sperm can then be marked with a dna-selective ink and sex-selected with a flow cytometer in a seizure fluid. During sperm sample extension and selection, the ph can be kept in a targeted ph. In addition, at least one element of the buffering medium, DNA selective ink and seizure fluid may be supplemented with at least one antioxidant.

Description

DESCRIPTIVE REPORT

Patent Application for “SELECTED SEX INSEMINATION DOSE AND METHOD OF PRODUCTION OF SEX SELECTED SPERMATOZOES” - Certificate of Addition of Invention Patent Application No. BR11 2015 007479 0, filed 02/04/2015 .

Technical Field In general, inventive technology refers to marking and selection methods, such as those employed for sperm selection, and more particularly refers to sperm selection methods and flow cytometry methods that improve the efficiency and recovery associated with sexual sperm selection as well as improved dose response in sexually selected sperm.

Fundamentals [002 | The most widely used sperm selection methods are based on the detection of quantifiable differences in the content of X-chromosome-bearing and Y-chromosome-bearing spermatozoa in DNA. Various modifications to flow cytometers for this purpose are described in US 5135759, 6263745, 7371517 and 7758811. In many species, the difference in DNA content may be small. In cattle, for example, Holstein bulls have a difference of about 3.8% in DNA content, while Jersey bulls have a difference of about 4.1%. The inaccurate nature of stoichiometric DNA labeling makes these small variations difficult to verify, and require strict labeling protocols.

Although the Hoechst 33342 fluorescent dye is suitable for distinguishing such variations at non-toxic concentrations, sperm must be incubated at elevated temperatures and at a high pH for the penetration of Hoechst 33342 to provide a uniform marking. Sperm are relatively fragile cells that are unable to replicate and usually have a short lifespan. Thus, damage caused by both sperm temperature rise and sperm pll change can result in significant damage to sperm health. In addition, pressure and shear forces applied to sperm within a flow cytometer may also compromise sperm membranes. The selection process further includes exposure of the squamous cells to an UV laser at an interrogation stage, and to an electrical charge to divert droplets containing sperm cells to be collected. Finally, since the selected sperm are ejected from the flow cytometer, they are impacted with a collection medium at speeds of approximately 80 - 90 km / h. Damage imposed at each of these steps in the flow selection process adversely affects sperm health and can accelerate sperm membrane deterioration, further reducing the already limited life span of viable sperm for use in artificial insemination or other breeding procedures. assisted.

A major disadvantage of using sexually selected sperm in assisted reproduction technologies is the reduced fertility associated with sexually selected sperm. Studies suggest that fertility of sexually selected sperm by flow cytometry is approximately 75 - 80% of conventional unselected semen fertility. In addition, increasing the amount of sexually selected sperm used in AI does not fully compensate for subfertility. DeJarmette et al., “Effects of sex-sorting and sperm dosage on conception rates of Holestien Heifers: Is it comparable to fertility of sex-sorted and conventional semen plausible?” Journal of Dairy Science. Vol. 94, pgs. 3477-3483 (2011).

Brief Description of the Invention | 005 | Certain embodiments of the claimed invention are summarized below. Such embodiments are not intended to limit the scope of the claimed invention, but rather serve as brief descriptions of possible forms of the invention. The invention may encompass a variety of forms that differ from these summary descriptions. | 006 | Certain embodiments refer to a sexually selected insemination dosage, such as a population of at least 4 million labeled and sexually selected sperm capable of artificial insemination fertilization at at least the same fertility level as a conventional dose of 15 million sperm. In certain aspects of such embodiments, the insemination dosage may include a residual amount of antioxidant from a buffering support medium in which the sperm sample has been processed, a residual amount of antioxidant from a paint in which the sperm sample has been processed. has been processed, and / or a residual amount of antioxidant from a seizure fluid in which sperm have been processed.

[007] Certain embodiments refer to a sexually selected sperm production method that has an enhanced dose reaction, including the steps of extending a sperm sample with a buffering buffer and adjusting the sperm sample concentration. extended within a target concentration range. Extended sperm can then be labeled with a DNA sorting ink and sexually selected with a flow cytometer in a seizure fluid. During sperm sample extension and selection, the pH can be maintained at a target pH. In addition, at least one component of the buffering support medium, DNA sorting ink and seizure fluid may be supplemented with at least one antioxidant.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates a schematic diagram of a flow cytometer for sperm selection according to various embodiments described herein.

[009] Figure 2 illustrates a graphical representation of selection parameters acquired on a flow cytometer during sperm selection according to various embodiments described herein. 1010] Figure 3 illustrates a graphical representation of selection parameters acquired on a flow cytometer during sperm selection according to various embodiments described herein.

Figure 4 illustrates a graphical representation of selection parameters acquired on a flow cytometer during sperm selection according to various embodiments described herein.

[012 | While the present invention may be embodied with various modifications and alternative forms, specific embodiments are illustrated in the figures and described herein as illustrative examples. It is to be understood that the figures and detailed description are not intended to limit the scope of the invention to the particular form disclosed, and that all modifications, alternatives and equivalents within the spirit and scope of the claims should be considered to be encompassed.

Embodiments of Invention | 013 | The term "sperm sample" should be understood to refer broadly to sperm cells in any medium, including natural fluids such as seminal plasma. For example, the term "sperm sample" may include crude ejaculatory materials, unchanged ejaculatory materials, or semen, as well as processed or partially processed sperm suspended in any combination of buffers, extenders, markers, other media, or the like. 10141 As used herein, the term "instrumental parameters" shall be understood to include settings related to analysis to selection conditions on or from an instrument or related to an instrument, and such settings may be modified by manual or automatic adjustments to the instrument. In the case of a flow cytometer or other similar instruments, instrumental parameters may include sample pressure, sample flow rate, circulating flow rate, circulating fluid pressure, drip control frequency, drip control amplitude, abortive match logic, collection regions, selection logic, and other similar settings. 1015] The expression “selection parameters” may include conditions related to selection made on a particle selection instrument. Selection parameters may include selection parameters measured in addition to parameters that are determined offline, estimated by an operator, and conditions related to a selected population of particles or cells. 1016] “Measured selection parameters” may include conditions related to selections measured directly, calculated or determined on a particle selection instrument during analysis and / or selection of a particle or cell population. In the case of a flow cytometer or other similar instruments, the measured selection parameters may include: event rate, selection rate, selective efficiency, abortive rate, percentage of dead collections, percentage of live oriented collections, valley over peak ratio , or the percentage of events in other selective collections, such as an X chromosome selection collection or a Y chromosome selection collection.

[017] As used herein, the term "coincident event" can be understood as a single event in a particle selection instrument where one or more particles or cells are too close to be separated for individual collection, and only One of two cells or particles is desirable for collection. In the case of a droplet selector air jet cytometer, a coincident event may occur when two sperm are close enough to be contained in the same droplet, but only one of these two cells is desired for collection.

[018] The term "selective efficiency" may be understood to refer to the recovery of particles or cells in terms of the percentage of particles or cells selected or collected from a group of cells or particles that are analyzed. The analyzed group of cells may be the total number of cells analyzed or may be a subset of the total number of cells analyzed, such as the analyzed cells determined to be viable or desirable for another reason for analysis and potential collection. | 019 | With respect to selection, the term "purity" may refer to an actual or estimated percentage of cells or particles in the collected or selected population of particles or cells having the characteristic by which the particles have been selected. In the case of sperm, purity may refer to the percentage of Y chromosome-bearing sperm in a selected population for Y-chromosome-bearing sperm regardless of the viability of the selected sperm. (020] The term "antioxidant" refers to any molecule among a wide variety of molecules that inhibit the oxidation of another molecule, or become oxidized in place of the target molecule, or that bind to harmful free radical intermediates and disrupt oxidative chain reactions within a cell Most antioxidants have a dual function, some are enzymes, some are non-enzymatic, some are vitamins and some are cofactors, but this diversity corresponds to the diversity of antioxidants, but because of their known ability In order to minimize cell damage, they are often grouped as a single class of compounds that have a single function, to bind free radicals.

Certain aspects disclosed herein relate to a method of selecting a sperm sample in a particle selection instrument; however, the methods described are not limited to any specific instruments. Particle selection instruments may include water jet flow cytometers, such as the Legacy MoFlo® SX flow cytometer and the MoFlo® XDP flow cytometer (Beckmcm Coulter, Miami FL, USA); however, other commercially available flow cytometers may be modified for sperm selection as well. Water jet flow cytometers can be equipped with guidance features such as orientation nozzles to guide sperm, optics for uniform cell illumination and / or radially uniform optics for collecting fluorescent emissions from all cells. regardless of your orientation. Cytometers with different flow chambers may also be used, such as closed chamber flow cytometers, or cuvettes. In addition, devices such as microfluidic chip with selection functions, such as acoustic or fluidic means for particle deflection at divergent outputs, may be used in accordance with certain embodiments described herein.

Pre-selected sperm processing [022J] Certain aspects described here relate to sexually selected sperm fertility enhancement methods, such as methods that result in sexually selected sperm that have a dose response, or that are extended into a sperm extender. suitable for transportation to a selection facility. Once obtained, sperm can be kept at room temperature, cooled, or even frozen in an extender suitable for later use. Labeling and selection sperm may be purchased from a mammal, or may be storage-acquired sperm, such as a frozen or chilled rod obtained from storage. Alternatively, frozen or extended sperm may be grouped. | 023 | The sperm population may originate from mammals, such as the mammals listed in the documents whose full contents are incorporated herein by reference. l «24 | At the time of collection, or melting, or even clustering, sperm can be checked for concentration, pH, motility and / or morphology. In addition, antibiotics may be added prior to further processing steps. In one embodiment, once obtained, sperm may optionally be standardized to a target concentration range and / or to a target pH range. As used herein, the term "standardization" can be understood as driving characteristics of an ejaculatory material into or near a targeted range. Although bovine ejaculatory materials, for example, may vary widely in pH and sperm concentration, the step of standardizing sperm concentration or pH may include the addition of a buffering support that serves to both standardize pH and sperm. to protect against the tendency of ejaculatory materials to become more acidic over time. As a non-limiting example, the pH and concentration of a sperm sample may be standardized before selection with a buffering support solution having a target pH. The sperm sample can be diluted in buffer support medium at ratios of 1: 1 to 1:10, or perhaps 1: 3. The buffered sperm sample can then be centrifuged and the supernatant removed to achieve a target concentration range. Alternatively, buffered sperm sample may then be centrifuged as a pellet and resuspended in the buffer support medium, or other similar extender.

[026 | Both the predetermined concentration and pH may be specific to different species, or even different animal breeds within a species. In one embodiment, a sperm sample may be combined with an initial extender, such as a buffering support medium in the form of a high capacity buffer or an extender having a high pH buffering capacity. Buffer support media, and other media described below, may include trisodium citrate, sodium citrate, sodium bicarbonate, HEPES, TRIS, TEST, MOPS, KMT, TALP, and combinations thereof. Other extenders having a buffer with a high pH buffering capacity may also be employed, and may be used in combination with additional components that promote sperm viability. As an example of an additive, proteins may be incorporated in the form of egg yolk, milk, lipoproteins, lecithin, casein or albumin, or other protein sources. An energy source may also be incorporated in the form of a monosaccharide such as fructose, glucose or mannose, or even a disaccharide or trisaccharide. In addition, antioxidants and antibiotics may be employed in buffering support media to promote sperm viability. Additional additives such as bovine serum albumin (BSA) may also be supplemented in the initial extender or buffering support medium.

The buffering support medium may be adjusted to a predetermined pH target to standardize the pH of all sperm samples obtained, such as a pH between approximately 6.8 and 7.4. In one embodiment, the buffering support medium is adjusted to a pH of 7.2. In addition, semen can become increasingly acidic over time, possibly because of proteins in the seminal fluid, or because of acid metabolism products or dead or dying cell byproducts. Buffer support means introduces free proton binding sites (e.g., H +) to maintain the pH near a target pll. Even in the face of the natural tendency of sperm to become more acidic over time, the buffering support medium provides a uniform starting point for pH stabilization of multiple sperm samples throughout further processing steps. Buffer support medium may include antibiotics to prevent the proliferation of bacteria. As non-limiting examples, tylosin, gentamicin, lincomycin, lincoespectin, spectinomycin, penicillin, streptomycin, and combinations thereof may be incorporated into the buffering support medium. (0291 Antioxidants may also be incorporated into the buffering support medium to reduce free radicals and oxidative stress. Although the present discussion involves the use of antioxidants in a buffering support medium, antioxidants may be incorporated at multiple stages of the selection process. of sperm, independently or in combination, as described in international publication WO2012167151, the full contents of which are incorporated herein by reference A non-limiting list of antioxidants that may be incorporated includes: catalase, SOD, an SOD mimic, glutathione, glutathione reductase, glutathione peroxidase, pyruvate, caproic acid, mercaptoethanol, BHT, lipoic acid, flavins, quinines, vitamin K (and related vitamers), vitamin BI2, vitamin B2, vitamin E (and related vitamers), tocopherols, tocotrienols, α-tocopheryl , alpha-ketoglutarate (AKG), malondialdehyde (MDA), asymmetric dimethylarginine (ADMA), and biologically active derivatives thereof, and combinations thereof.

[030 | The concentration of antioxidants may be in the range of 0.01 mg / ml to 0.5 mg / ml, and as non-limiting examples the antioxidants listed above may be provided in the concentration of 0.01 mg / ml to 5.0 mg / ml. ml; from 0.01 mg / ml to 0.25 mg / ml; from 0.01 mg / ml to 0.5 mg / ml; from 0.01 mg / ml to 1 mg / ml; from 0.01 mg / ml to 2.5 mg / ml; from 0.01 mg / ml to 5 mg / ml; from 0.05 mg / ml to 0.1 mg / ml; from 0.05 mg / ml to 1.0 mg / ml; from 0.05 mg / ml to 2.5 mg / ml; from 0.1 mg / ml to 0.25 mg / ml; from 0.1 mg / ml to 0.5 mg / ml; from 0.1 mg / ml to 1 mg / ml; from 0.1 mg / ml to 2.5 mg / ml; from 0.1 mg / ml to 5 mg / ml; from 0.15 mg / ml to 0.45 mg / ml; from 0.15 mg / ml to 0.5 mg / ml; from 0.25 mg / ml to 0.35 mg / ml; from 0.25 mg / ml to 0.5 mg / ml; from 0.25 mg / ml to 1 mg / ml; from 0.25 mg / ml to 2.5 mg / ml; from 0.25 mg / ml to 5 mg / ml; from 0.35 mg / ml to 0.5 mg / ml; from 0.35 mg / ml to 1 mg / ml; from 0.35 mg / ml to 2.5 mg / ml; from 0.35 mg / ml to 5 mg / ml; from 0.5 mg / ml to 1 mg / ml; from 0.5 mg / ml to 2.5 mg / ml; from 0.5 mg / ml to 5 mg / ml; from 1 mg / ml to 2.5 mg / ml; and from 1 mg / ml to 5 mg / ml.

[031 | The sperm sample can be diluted in the buffer support medium in ratios from approximately 1: 1 to approximately 1:10. For example, an unchanged ejaculatory material may be diluted in a sperm buffering medium to buffer ratios between 1: 2 and 1: 5. In one embodiment, the sperm sample may be diluted in the buffer support medium at a ratio of 1: 3. Dilution may reduce sperm exposure to some harmful factors present in seminal plasma. The diluted sperm sample can then be reconcentrated by centrifugation, removal of the supernatant and resuspension. The centrifuged sperm sample may be resuspended in the buffering medium, or other similar buffering medium in a volume that brings the sperm concentration to or near the target concentration. The target concentration can be selected based on additional sperm processing steps. For example, in the case of sexual selection of cattle, sperm can be reconcentrated between approximately 2,400 million sperm per ml and approximately 500 million sperm per ml to simulate a natural range of concentrations. Other concentrations, such as between approximately 1,400 million sperm per ml and approximately 2,100 million sperm per ml, or between approximately 1,700 million sperm per ml and approximately 2,100 million sperm per ml, may also be used for further processing. Thereby seminal plasma that originates with sperm can be diluted and replaced with one or more buffers, such as a buffering support medium. 1032] A sperm population will include both X-chromosome-bearing sperm and Y-chromosome-bearing sperm. In addition, each of the X-chromosome-bearing and Y-chromosome-bearing sperm groups will include viable sperm and non-viable sperm. The distinction between viable sperm and unviable sperm in conventional sperm selection is determined by the inclusion of a deactivating ink that permeates sperm with compromised membranes. Dead or dying sperm absorb the deactivating ink and produce fluorescence signals distinct from the remaining sperm population, while sperm with intact membranes tend to be viable and will prevent the deactivating ink from catching. Viable sperm, at the appropriate dosage, will usually be able to achieve fertilization in artificial insemination, while non-viable sperm, or sperm with compromised membranes, may be unable to achieve fertilization in artificial insemination or have a greatly reduced capacity. to do so. However, some fertilizing sperm may have compromised membranes, and some intact membrane sperm may be unable to fertilize.

Whether or not extended in a buffering support medium, a sperm sample can be labeled with a labeling solution that includes a DNA selector ink. In the labeling step, at least a portion of the sperm population is incubated with a labeling solution and a fluorescent DNA sorting ink to stoichiometrically label the DNA content of each cell in the sperm sample. Hoechst 33342 tends to be less toxic than other DNA selector inks. The vehicle for dispensing this ink may be in the form of a modified TALP buffer adjusted to a pl of 7.4. Hoechst 33342 is described in US Patent 5135759 and is commonly used for this purpose. However, other UV excitable inks as well as visible light excitable inks, fluorescent polyamides, fluorescent nucleotide sequences and sexually specific antibodies may also be used. 1034] Natural sperm are often not permeable to such inks. In order to produce uniform labeling, the first labeling step may include incubating at least a portion of the sperm population at an elevated temperature in a labeling solution (sometimes referred to herein as a labeling buffer) at a high pH. , besides the ink. Examples of suitable labeling solutions may be TALP, TES-TRIS, TRIS citrate, sodium citrate, or a HEPES based medium, each of which is described in W02005 / 095960, which is incorporated herein by reference. An exemplary modified TALP described in WO2001 / 37655 is illustrated in Table 1.

TzXBELA 1 - Modified TALP Buffer Ingredient 95.0 mM NaCl Concentration

3.0 mM KC1

0.3 mM NaHPO4

10.0 mM NaHCCb

0.4mM MgCl26H2O

Na Pyruvate 2.0 mM

5.0 mM glucose

Na 25.0 mM Lactate

HEPES 40.0 mM BSA 3.0 mg / ml [035] As an example, the sperm population, or a portion of the sperm population, could be diluted with the labeling solution to between 640 x 106 and 40 x 106 sperm. / ml, between approximately 320 x 106 and 80 x 106 sperm / ml, or to approximately 160 x 106 sperm / ml in the buffer. Fluorescent DNA sorting ink can be added to sperm suspended in the buffer at concentrations between approximately 10 μΜ and 200μΜ, between approximately 20 μΜ and 100μΜ, or between approximately 30 μΜ and 70μΜ. The pH of the buffer may be from approximately 6.8 to 7.9, from approximately 7.1 to 7.6, or approximately 7.4, to help ensure uniform labeling of nuclear DNA. Those skilled in the art will appreciate that the pH may be increased with the addition of NaOH and decreased with the addition of HCl. Optionally, the antioxidants and concentrations described above may be incorporated into the labeling solution.

[036 | The sperm sample may be incubated at 30-39 ° C, approximately 32-37 ° C, or approximately 34-36 ° C. The incubation period may range from about 20 minutes to about 3 hours, about 30 minutes to about 90 minutes, or about 45 minutes to about 60 minutes. For example, the sperm population can be incubated for approximately 45 minutes at 34 ° C. Even within a single species, sperm concentration, pH, and other factors affecting markability may vary from animal to animal. Minor variations occur for incubation of sperm across species or even between races or animals of the same race to achieve uniform marking without over-marking a sperm population. 10371 In addition to DNA selector dye, a deactivating dye may be applied for the purpose of permeating sperm with compromised membranes and deactivating the signals they produce. A deactivating ink can be understood to include inks that differentially associate with compromised membrane sperm. These inks may more easily enter sperm with compromised membranes because the membranes are breaking or becoming increasingly porous. It may also be that deactivating inks readily enter all sperm membranes, and healthy sperm actively pump out deactivating inks faster than sperm with compromised membranes. Whatever the case, the sperm that deactivating inks associate with includes a large portion of dead or dying sperm, although not necessarily all of them are dead or dying. Deactivated signals produced from compromised membrane sperm in association with deactivating ink are sufficiently distinct from healthy sperm signals that they can be removed from further analysis and selections applied to viable sperm.

[038 | In one embodiment, the deactivating ink and DNA selector ink are applied together in a single dilution. In this embodiment, the deactivating ink is incubated along with the DNA selector ink at a high temperature in the labeling solution. As an example, the labeling solution may be a modified TALP with a pH of 7.4. However, other markings may be employed, including TES-TRIS, TRIS citrate, sodium citrate or a HEPES-based medium with the DNA sorting and deactivating ink, and the pH may range from 7.0 to 7, 8 In one embodiment, a synergy may exist when sperm are standardized at a high pH of approximately 7.2 prior to labeling at a pH of 7.4. Thus, the pH to which sperm are exposed remains within a constant range with minimal variations. Since both the labeling solution and buffering medium have high buffering capacities, it is believed that the natural tendency of sperm to become more acidic over time can be avoided. In addition, by minimizing sperm-supported pH changes, sperm are believed to be in a healthier condition to better cope with various sperm-borne pressures and stresses in the process of sexual selection, including but not limited to additional and induced shear forces in flow cytometers operated above 40 psi. Marking sperm in a single dilution can help sperm better survive selection at high shear fluid pressures such as fluid shear pressures greater than 40 psi, fluid shear pressures between 40 and 65 psi, between 50 and 60 psi , or at about 60 psi.

[039] In another embodiment, the deactivating ink is added in a second labeling step that further reduces sperm concentration in the sperm sample. The pH of the second labeling solution can be directed to reach a target pH in the final sperm sample. Non-limiting examples of two-step marking processes are described in international publication WO 2011/123166 and international application PCT / US12 / 58008. | 040 | The labeling solution may be supplemented with additives such as an antioxidant in the concentration ranges described above. In some embodiments, elevated temperatures may increase free radicals and oxidative stresses borne by sperm during labeling. Thus, antioxidants can serve to neutralize free radicals and reduce oxidative stresses borne by sperm being labeled. A non-exclusive list of antioxidants that can be incorporated into the labeling process includes: catalase, SOD, SOD mimic, glutathione, glutathione reductase, glutathione peroxidase, pyruvate, caproic acid, mercaptoethanol, BHT, lipoic acid, flavins, quinines, vitamin K (and related vitamers), vitamin B 2, vitamin B 2, vitamin E (and related vitamers), tocopherols, tocotrienols, α-tocopheryl, alpha ketoglutarate (AKG), malondialdehyde (MDA), asymmetric dimethylarginine (ADMA), and biologically derived assets, and combinations thereof. Any of the concentrations described above may be used.

[041] Certain aspects of the preselective methodologies described above may be combined in synergistic ways. For example, sperm may be standardized at target pHs and concentrations, kept within a relatively narrow range of pHs throughout the preselection process, and exposed to uniform levels of antioxidants throughout the preselection process. Alone or in combination, these selection process modifications can help sperm to better survive flow cytometer selection. Sperm can then be selected at higher concentrations and at higher pressures, perhaps even at pressures previously considered detrimental to sperm health, for improved efficiency and yield. Finally, the improved sperm health obtained in the examples below may also provide sexually selected sperm, which demonstrates a dose response in artificial insemination. Sexually selected sperm capable of demonstrating a dose reaction can then be used in artificial insemination at dosages with fertility comparable to unselected conventional sperm.

Sperm Selection [042] Whether standardized at a predetermined or undetermined pH and concentration, and marked at a single dilution or two dilutions, the sperm sample can be selected by a particle selection instrument such as a flow cytometer. . Referring to Fig. 1, an air jet flow cytometer (10) is illustrated, although selection may be performed with microfluidic chips or other types of flow cytometers, including flow cytometers having closed chambers and cytometers incorporating lasers. ablative. The flow cytometer (10) includes a cell source (12) for producing a sperm sample flow, such as a selectable sperm sample flow. The rate at which the sperm sample is distributed to the nozzle (14) can be considered the sample flow rate, and can be determined by the sample pressure. The flow of labeled sperm sample is deposited into a nozzle (14) and introduced or drained into a fluid stream (16) of circulating fluid (18). Circulating fluid (18) may be supplied by a circulating fluid source, so that while cell source (12) supplies sperm to circulating fluid (18), they are concurrently supplied through the nozzle (14). Circulating fluid 18 may be supplied at a circulating flow rate determined by the circulating fluid pressure. Thereby, the circulating fluid (18) forms a fluid stream that coaxially surrounds the sample with labeled sperm exiting the nozzle (14) through the nozzle outlet port (22). By providing an oscillator (24) that can be precisely controlled with an oscillator control (26), pressure waves can be established within the nozzle (14) and transmitted to the fluids leaving the nozzle (14) through the outlet port ( 22) of the nozzle. In reaction to pressure waves, the fluid stream (16) exiting through the nozzle outlet port (22) eventually forms regular droplets (28) at precise intervals. The frequency, and to some extent the shape of the formed droplets, can be controlled by a drip control frequency and a drip control amplitude provided to the oscillator (24) or the oscillator controller (26).

[043 | Each droplet formed retains circulating fluid and the sperm sample that previously formed a portion of the fluid stream (16). Since labeled sperm are surrounded by the fluid stream (16) or circulating fluid environment, the droplets (28) ideally contain individually isolated sperm. However, sample concentration, sample pressure, nozzle outlet port diameter 22, and other instrumental parameters determine the frequency at which multiple cells will regularly occupy a single droplet, as well as the percentage of sperm-containing droplets. | 044] The flow cytometer (10) acts to select droplets based on the sperm characteristics expected to be contained in the droplets. This can be achieved through a cellular detection system (30) in communication with an analyzer (36). The cellular detection system (30) includes at least one sensor (32) responsive to the cells contained in the fluid stream (16). To cite an example, two orthogonal PMTs may be incorporated into a sperm selection flow cytometer for fluorescence detection at 0 ° and 90 °, although other sensor configurations may be readily employed, such as those described in WO2010. / 021627.

[045] The cellular detection system (30) provides data to the analyzer (36), which may cause an action depending on the relative presence or relative absence of a cellular characteristic in the fluid stream (16). Certain characteristics, such as the relative DNA content of sperm cells, can be detected by excitation with an electromagnetic radiation source (34), such as a laser that generates an irradiating beam to which sperm react. The electromagnetic radiation source 34 may be a laser operated at UV wavelength, such as approximately 355 nm. An example of such a laser may be a Vanguard 50 (available from Spectra-Physics), which operates at 350 mW. Various optical features can be employed to shape the laser beam profile, split the beam into more than one current, or reduce the beam power in one current. Nonlimiting examples of such optical resources can be found in WO / 2004/104178 and WO / 2001/85913, each of which is incorporated herein by reference.

[0461] The characteristics of individual sperm, particularly the presence of an X chromosome or a Y chromosome, can be determined from the detected fluorescence produced in response to the source of electromagnetic radiation (34). In particular, the configurations of the cellular detection system (30) may be in communication with an analyzer (36) to provide a variety of fluorescence in formation, such as forward fluorescence of an event, lateral fluorescence of an event, or amount of scatter associated with an event. The analyzer (36) may include written instructions for analyzing the signals produced by one or more sensors (32) in the cellular detection system (30). Fluorescent DNA sorting ink stoichiometrically binds to sperm DNA. Because X-chromosome-bearing sperm contain more DNA than Y-chromosome-bearing sperm, X-chromosome-carrying sperm can bind more DNA-selective fluorescent ink than Y-chromosome-sized sperm. fluorescence emitted by the excitation-linked ink, it is possible to differentiate between X-chromosome-bearing sperm and Y-chromosome-bearing sperm. Distinctions such as viability or non-viability of sperm can be distinguished by the analyzer-oriented or non-oriented sperm (36). ) according to the selection logic incorporated in the collecting regions.

[047 | In order to achieve separation and isolation based on tagged sperm characteristics, the emitted light can be detected by sensor (32) and information sent to an analyzer (36) coupled to a droplet carrier that differentially charges each droplet (28). based on the characteristics of the labeled sperm contained in that droplet (28). In this way the analyzer (36) acts to allow the electrostatic deflection plates (38) to deflect the droplets (28) depending on whether or not they contain the appropriate particle or cell. | 048 | Consequently, the flow cytometer (10) acts to separate labeled sperm by causing droplets (28) containing sperm to be directed to one or more collection vessels (40). For example, when the analyzer differentiates sperm based on a sperm characteristic, the X-chromosome-bearing sperm droplets may be positively charged and thus drift in one direction, whereas the Y-chromosome-bearing sperm droplets may be positively charged. negatively charged and thus deviate in another direction, and the wasted current (ie droplets that do not carry a particle or cell or that carry undesirable or unselectable cells) can be left unloaded and thus collected in an undistorted current. for a suction tube or similar. Alternatively, one group from X-chromosome-bearing sperm or Y-chromosome-bearing sperm can be collected, while the other group is discarded with the remainder. Once diverted, the droplets can then be collected in collection vessels that include a seizure fluid. The seizure fluid may comprise an extender, similar to the buffering medium, or similar to a subsequent extender that may be used for cooling or freezing selected sperm. By way of example, the seizure fluid may comprise a buffer component such as TRIS citrate, sodium citrate, sodium bicarbonate, HEPES, TRIS, TEST, MOPS, KMT, TALP, or combinations thereof. Other buffers that have a high pH buffering capacity may be employed, any of which may be used in conjunction with additional components that promote sperm viability. As an example of additive, proteins may be incorporated in the form of egg yolk, milk, lipoproteins, lecithin, casein, albumin, or other protein sources. An energy source may also be incorporated in the form of a monosaccharide, such as fructose, glucose or mannose, or even a disaccharide or trisaccharide. Additionally, antioxidants and antibiotics may be employed in the initial extender to promote sperm viability. J049 | A non-limiting list of antioxidants that may be incorporated into the seizure fluid include: catalase, SOD, SOD mimic, glutathione, glutathione reductase, glutathione peroxidase, pyruvate, caproic acid, mercaptoethanol, BHT, lipoic acid, flavins, quinines, vitamin K (and related vitamers), vitamin B 2, vitamin B 2, vitamin E (and related vitamers), tocopherols, tocotrienols, α-tocopheryl, alpha ketoglutarate (AKG), malondialdehyde (MDA), asymmetric dimethylarginine (ADMA), and derivatives biologically active compounds thereof, and combinations thereof.

[050] Antioxidant concentrations may be in the range 0.01 mg / ml to 0.5 mg / ml, and as non-limiting examples the antioxidants listed above may be provided in the concentration of 0.1 mg / ml to 5 mg / ml. 0.0 mg / ml; from 0.01 mg / ml to 0.25 mg / ml; from 0.01 mg / ml to 0.5 mg / ml; from 0.01 mg / ml to 1 mg / ml; from 0.01 mg / ml to 2.5 mg / ml; from 0.01 mg / ml to 5 mg / ml; from 0.05 mg / ml to 0.1 mg / ml; from 0.05 mg / ml to 1.0 mg / ml; from 0.05 mg / ml to 2.5 mg / ml; from 0.1 mg / ml to 0.25 mg / ml; from 0.1 mg / ml to 0.5 mg / ml; from 0.1 mg / ml to 1 mg / ml; from 0.1 mg / ml to 2.5 mg / ml; from 0.1 mg / ml to 5 mg / ml; from 0.15 mg / ml to 0.45 mg / ml; from 0.15 mg / ml to 0.5 mg / ml; from 0.25 mg / ml to 0.35 mg / ml; from 0.25 mg / ml to 0.5 mg / ml; from 0.25 mg / ml to 1 mg / ml; from 0.25 mg / ml to 2.5 mg / ml; from 0.25 mg / ml to 5 mg / ml; from 0.35 mg / ml to 0.5 mg / ml; from 0.35 mg / ml to 1 mg / ml; from 0.35 mg / ml to 2.5 mg / ml; from 0.35 mg / ml to 5 mg / ml; from 0.5 mg / ml to 1 mg / ml; from 0.5 mg / ml to 2.5 mg / ml; from 0.5 mg / ml to 5 mg / ml; from 1 mg / ml to 2.5 mg / ml; and from 1 mg / ml to 5 mg / ml. 1051] In one embodiment, antioxidants are supplemented in each buffering medium, labeling solution and seizure fluid. The antioxidants may comprise the same additives or a combination of additives may be present at similar concentrations in each buffering medium, labeling solution and seizure fluid. Thus oxidative stress can be mitigated in sperm throughout the selection process. Thus, aspects of the selection methodologies described above can be combined synergistically throughout the selection process. For example, sperm can be standardized at target pHs and concentrations and kept within a relatively narrow range of pHs throughout the selection process. Sperm can also be exposed to uniform levels of antioxidants throughout the selection process. Alone or in combination, these modifications can help sperm to better survive flow cytometer selection, allowing sperm to be selected at higher pressures, perhaps even at pressures previously thought to be harmful to sperm health, or to achieve a dose response with sexually selected sperm in artificial insemination. 1052] A controller (42) may form a portion of the analyzer (36) or may be a component external to the analyzer (36). The illustrated controller 42 may also represent a collection of individual controllers. Controller (42) may receive signals or instructions from analyzer (36), and in response may modify one or more instrumental parameters such as sample flow rate, sample pressure, circulating flow rate, circulating fluid, drip control frequency, drip control amplitude, among others. Controller 42 may also provide an operator input interface for manual adjustment of sample flow rate, sample pressure, circulating flow rate, circulating fluid pressure, drip control frequency, drip control amplitude. , among others. The analyzer 36 may include written instructions for modifying instrumental parameters in response to measured selection parameters, or instrument parameter modifications may be performed manually by an operator adjusting various settings. Modifications of the instrumental parameters can be performed on the analyzer (36), for example to change the selection logic, interrupt logic, selection regions or collection regions, and other parameters specific to the selection on the analyzer. Additional modifications of the instrumental parameters may be made by a controller (42) to control various external components to the analyzer, such as to control the sample flow rate, sample pressure, circulating flow rate, pressure of circulating fluid, drip control frequency and drip control amplitude.

[053 | Fig. 2 illustrates a bivariate histogram plot representative of lateral fluorescence and forward fluorescence from a labeled sperm jet stream flow cytometer that can be generated by an analyzer (36). Visual representation of the data can be used by an operator to receive information related to the sample being selected, and to graphically demonstrate certain aspects of logic if present selection. R1, for example, can be viewed as a collection region that can be applied to the flow cytometer selection logic. Additional numerical results can be provided on an analyzer screen (36). Such numerical results may be in the form of measured selection parameters such as event rate, abortion rate, selection rate, selective efficiency, or the percentage of particles in any region or input. IR is illustrated as a region that can be considered the living oriented region, as the IR boundaries include two dense cell populations that reflect an X-chromosome-bearing sperm population and a closely related Y-chromosome-bearing sperm population. R2 is a collection region positioned around unviable sperm, or compromised membrane sperm whose fluorescence is deactivated by a deactivating ink. Although several selection logics may be employed, two strategies related to RI and R2 can be a first step in a selection logic, where all events in RI are accepted for further processing or collection. Alternatively, all events outside of R2 are accepted for further processing or collection.

Fig. 3 illustrates a univariate graph in the form of a histogram that can be produced by the analyzer 36 and generated as a graphical presentation for an operator. The data illustrated in Fig. 3 can represent the number of occurrences of peak signal intensities from lateral or forward fluorescence within a certain period. In the case of sperm, X-chromosome-bearing sperm and Y-chromosome-bearing sperm tend to have peak intensities ranging from 2 to 5%, depending on the species, and this difference is reflected in the bimodal distribution of peak intensities seen in Fig. 2. Because X-chromosome-bearing sperm and Y-chromosome-bearing sperm tend to have different fluorescence values, each of the peaks represents either X-chromosome-bearing sperm or Y-chromosome-based sperm. Based on the selection logic applied within From the analyzer (36), the cell population in the histogram may be only cells that have been determined to be viable oriented cells, such as those found in R1 in Fig. 2, or may represent cells that have not been determined to be dead or undesirable, such as any event other than those in R2. Various selection parameters can be derived from the information contained in this histogram. For example, the level of distinction between the two peaks may provide an indication of how selective purity can look. Fig. 3 further illustrates relative intensity measurements at the lowest point between the two groups, which can be considered a V value and a second relative intensity at the peak or peaks of the P histogram. Visual inspection of a histogram can provide the An operator has an idea of the performance of the flow cytometer, but computer-executed instructions for determining a P value, a V value, and a V over P ratio have not been implemented in commercial sperm selectors. The valley-to-peak ratio can be determined as a selection parameter measured periodically during the course of selection. The valley-to-peak ratio, while not necessarily completely determining selective purity, can provide a means of quickly estimating purity values, either automatically by executing instructions written to the analyzer (36) or manually by visual inspection of an operator. Alternatively, the inverse relationship, that is, a peak to valley ratio, provides information similar to that of the inverse value.

Referring to Fig. 4, a second bimodal graph may be generated by the analyzer (36) in response to signals acquired by the cellular detection system (30). The bimodal plot may represent a first axis illustrating the peak intensity value of a forward fluorescence signal or the peak intensity of the lateral fluorescence signal. As in Fig. 3, the data illustrated in Fig. 4 can be collected so that only events that fall within IR in Fig. 2 are included. Alternatively, in the case of sperm, all events that are not in the dead collection R2 can also be displayed.

[056] R3 may represent an X-selection port for the collection of X-chromosome-bearing sperm. The expression "X-selection port" may be used here synonymously with the X-expression. Referring to Fig. 4, it can be demonstrated how changing size of collection regions can affect efficiency, purity and productivity. If the R3 region were expanded, it would be possible to see that every second more sperm would be selected as X-chromosome-bearing sperm, resulting in higher selective efficiency and higher productivity. However, expansion of the R3 gate or region would begin to include events with an increasing likelihood of being Y chromosome-bearing sperm. In order to increase selective sperm purity, the R3 region may be made smaller and / or away from the chromosome region. Y. Because fewer events are within selection port X, fewer sperm are selected from the population of X-chromosome-bearing sperm, and those that are will be more likely to actually be X-chromosome-bearing sperm, which means that Collected purity can be increased. However, both efficiency in terms of collected cells and productivity in terms of selections per second will decrease as fewer events are within the R3 region and more coincident events are aborted. In addition, as other instrumental parameters are modified, the illustrated graphs of Figs. 2, 3 and 4 may change shape and nature. For example, increasing sample pressure or sample flow rate may result in a reduction in the valley-to-peak ratio, or decrease the bimodal distinction between X-chromosome-bearing sperm and Y-chromosome-bearing sperm.

Dose Response and Increased Sexualized Doses 1057] Conventional semen can be understood as semen collected according to standard industry practices. As a non-limiting example of a standard industrial practice for conventional semen collection, the methodology described as conventional by DeJamette et al. of Dairy Science, Vol. 93, pgs. 4079-7085 (2010) provided a conception rate of 62% with 15 million conventional (non-sexualized) sperm. As illustrated in Example 7, certain selection methods provided herein generate sexually selected sperm with fertility of 59.9%, or up to 66.7%. Compared to a control of 15 million conventional sperm (66.5%), 3 million sexually selected sperm (60.0%) and 4 million sexually selected sperm (66.7%) provided a relative fertility of 90%. and 100%, respectively. 4 million sexually selected sperm did not produce a statistically significant loss of fertility (P <0.05) compared to a dose of 15 million conventional sperm. It should be noted that a large number of factors may influence fertility, such as time or year, room temperature, heifers vs. cows, the accuracy of heat detection, the skills of AI technicians, as well as the particular characteristics of specific breeds, or even different herds or specific animals. Thus, fertility can be understood as variable for both sexually selected sperm and conventional sperm for a number of reasons. Thus, in one embodiment, sexualized sperm should be considered to have a fertility that is equal to 90% of the fertility of a conventional non-sexualized control. In another embodiment, the fertility of sexually selected bovine sperm according to certain methods described herein may be at least about 50%, at least about 55%, at least about 60%, at least about 65%, or even at least about 67% or more. | 058 | Sperm selected according to certain embodiments described herein may possess fertility characteristics including a fertility of at least 90% of the fertility of conventional semen or even as high as conventional semen. As shown in Example 7, doses of 3 million sexually selected sperm were able to achieve 90% fertility of conventional semen and doses of 4 million sexually selected sperm were able to achieve the same fertility as doses of 15 million sperm (untreated). sexualized) conventional.

[059 | As a non-limiting example, a rod can be filled with between approximately 3 million sperm and approximately 4 million sperm, between approximately 4 million sperm and approximately 5 million sperm, between approximately 5 million sperm and approximately 6 million sperm. , between approximately 6 million sperm and approximately 7 million sperm, between approximately 7 million sperm and approximately 8 million sperm, between approximately 8 million sperm and approximately 9 million sperm, between approximately 9 million sperm and approximately 10 million between approximately 10 million sperm and approximately 11 million sperm, between approximately 11 million sperm and approximately 12 million sperm, between approximately 12 million sperm and approximately 13 million sperm, between approximately 13 million sperm and approximately 14 million sperm, between approximately 14 million sperm and approximately 15 million sperm, between approximately 15 million sperm and approximately 16 million sperm. between approximately 16 million sperm and approximately 17 million sperm, between approximately 17 million sperm and approximately 18 million sperm, between approximately 18 million sperm and approximately 19 million sperm, and between approximately 19 million sperm and approximately 20 million sperm. 10601 Although the selected sperm reconcentration step can largely replace extenders and markings used throughout the selection process, residual amounts of these extenders and their various components may remain. For example, in an embodiment in which an antioxidant is added with an initial extender (buffering support medium), that antioxidant may be present in a residual amount in the final sperm dosage. In addition, an antioxidant provided in the labeling step may also be present in a residual amount in the final sperm dosage. Thus, a dosage of sperm, such as between 3 million and 20 million sexually selected sperm, may contain a residual amount of a first antioxidant, a residual amount of a second antioxidant, and a residual amount of a third antioxidant. In one embodiment, the same antioxidant, or combination of antioxidants, may be added to the initial extender, labeling and seizing fluid, and may further be considered as a first, a second and a third antioxidant in the final form.

[061] Once selected, individual sperm dosages can be prepared for artificial insemination (AI), in vitro fertilization (IVF), or intracytoplasmic sperm injection (ICSI). Sperm for use in assisted reproduction technologies can typically be stored on sticks. Previously, because sexually selected sperm did not provide a dose response, sexually selected sperm were often loaded on 0.25 ml rods at concentrations of approximately 10x106 sperm per ml. Because of the small volume occupied on each rod by sealing caps inside, approximately 2.1 million sperm cells are in each selected sperm rod. Modalities of the present invention include between 3 million and 20 million sperm in a dosage for IA or IVF. Certain embodiments described herein incorporate an antioxidant at an early stage in a buffering support medium. It should be noted that a residual amount of antioxidant can be retained with the sperm sample through subsequent steps of labeling, selection, and in some cases freezing. In this context, the antioxidant presented in the buffering medium may be found in residual amounts on the final dipstick, and may be referred to as a first residual amount of antioxidant. Similarly, a second residual amount of antioxidants present in the labeling solution can be retained in the sperm sample through the selection and perhaps freezing steps. For this reason, a dipstick may include a second residual amount of antioxidants introduced in the labeling step. In addition, a residual amount of antioxidants present in the seizure fluid may be retained in the sperm sample and end up in a dipstick as a third residual amount of antioxidant. Finally, a fourth amount of antioxidant may be present before or after reconcentration of a sperm sample for loading into rods with a freezing medium. For this reason, the fourth amount of antioxidant may be present in a residual amount or may be present in a concentration between approximately 0.1 mg / ml and approximately 5 mg / ml.

Example 1 [063 | Collection - Sperm were collected from five different bulls in a routine collection scheme using an artificial vagina. Each bull was collected two or three times in one day. Of the five bulls, two were Jersey bulls and three were Holstein bulls. All ejaculatory materials contained progressive motility greater than 60% and sperm concentration ranged from 857 million sperm per ml to 2,480 million sperm per ml. The ejaculatory materials collected from the same bull were grouped and then divided into nine sperm samples for collection and marking treatments.

[064] Sperm Processing and Marking - Portions of each bull's ejaculatory material were processed and labeled by nine different methods, each described below: [065 | (A) Control (no standardization, two-step labeling) - A control was established that did not include the standardization stage of collected ejaculatory materials and in which sperm were labeled in two steps. Prior to labeling, sperm samples were concentrated between 1,700 million sperm per ml and 1,800 million sperm per ml by centrifugation or by the addition of an egg yolk extender with a pH of 6.8, depending on the initial concentration. of the samples. The sperm in the control group were diluted to 160 x 106 sperm per ml in a modified TALP buffer as described in Table 1 at a pH of 7.4. Each sperm sample in the control group was then incubated with 16-17pL Hoechst 33342 per ml (64-68 μΜ) sample for 45 minutes at 34 ° C. After incubation, an equal volume of a second modified TALP was added, reducing the concentration to 80x106 sperm per ml. The second modified TALP includes the components described in Table 1 with the addition of 4% egg yolk, 50 μΜ No. 6 yellow food ink (20 g / 1) and the pH was lowered to 5.5 with the addition of HCl. | 067 | (B) Extended (no standardization, two-step labeling) - In the second group, sperm were not standardized, but were extended with an extender containing 20% egg yolk. Sperm were then concentrated between 1,700 million sperm per ml and 1,800 million sperm per ml in the same manner as described for group (A). The sperm were then diluted to 160x10 ° sperm per ml in a modified TALP buffer, and similarly labeled in two steps as described for group (A).

[068] (C) Single Step (Nonstandard, Single Step Marking with 1% Egg Yolk) - In a third group, sperm were collected and the concentration adjusted in the same way as the control group (A). Each sperm sample was then diluted to 160x10 ° sperm per ml in a modified TALP buffer at a pH of 7.4. The modified TALP buffer was substantially identical to the buffer described in Table 1, except that it additionally included 1% egg yolk and No. 6 yellow food ink at a concentration of 25 μΜ. Each sperm sample in this group was then incubated with 14-15pL Hoechst 33342 per ml (56-60 μΜ) of sample for 45 minutes at 34 ° C. After incubation, sperm remained at a concentration of 160x106 sperm per ml. [069] (D) Standardized I (standardization with 3% egg yolk extender, two-step labeling) - In this group, sperm were standardized by adjusting both pH and sperm concentration prior to labeling and selection. After collection, sperm were diluted 1: 3 in an initial extender with a pH of 7.2 and a high pH buffering capacity. The high capacity buffer was supplemented with 3% egg yolk. All samples were then centrifuged to reduce sperm concentration to between 1,700 million sperm per ml and 1,800 million sperm per ml. Standard sperm were then labeled according to the two-step method described in (A). (070] (E) Standardized II (standardization with 10% egg yolk extender, two-step labeling) - In this group, sperm were standardized by adjusting both pH and sperm concentration prior to labeling in the same manner as described for the group. (D), except that the initial extender was 10% egg yolk. | 0711 (F) Single step and standardized I (standardization with 3% egg yolk extender, single step marking with 7 (% egg yolk) - In this group, sperm were standardized by adjusting both pH and sperm concentration prior to labeling in the same manner as described for group (D) .The standardized sample was then marked by a single step labeling process. as described for group (C).

[072] (G) Single step and standardized II (10% egg yolk extender standardization, 1% egg yolk single step marking) - In this group, sperm were standardized by adjusting both pH and sperm concentration before marking in the same manner as described for group (E). The standardized sample was then labeled by a single step labeling process as described for group (C).

[073] (H) Single step and standardized III (standardization with 3% egg yolk extender, single-step marking without egg yolk) -In this group, sperm were standardized by adjusting both pH and sperm concentration before labeling. in the same way as described for group (D). The standardized sample was then labeled by a single step labeling process as described for group (C), except that no egg yolk was added to the single step labeling TALP.

[074 | (I) Single step and standardized III (standardization with 10% egg yolk extender, single step marking without egg yolk) -In this group, sperm were standardized by adjusting both pH and sperm concentration prior to labeling in the same manner. described for group (E). The standardized sample was then labeled by a single step labeling process as described for group (C), except that no egg yolk was added to the single step labeling TALP.

[075 | Data Selection and Acquisition - Each labeled sample was selected on a Legacy MoFlo® SX flow cytometer (Beckman Coulter, USA) with a Genesis digital upgrade (Cytonome / ST, Boston MA, USA). Those samples that had been labeled in a two step process were selected at the concentration of 80x106 sperm per ml, and the samples that were labeled by the single step process were selected at the concentration of 160 x106 sperm per ml. Data recorded by the flow cytometer were recorded, including information related to selection rates and sperm subpopulation collection. For example, the percentage of sperm collected as dead as well as the percentages of sperm collected as live-oriented and above ranges were recorded and an average was taken for the five bulls. Results - A comparison of the percentage of oriented, unoriented, and dead sperm as determined by the flow cytometer selection parameters is summarized in Table 2 below.

Compared to control (A), the Single Step I (C), Standardized I (D), and Standardized II (E) groups had significantly lower dead populations with reductions of 4.58%, 7,18 % and 10.85% respectively. Based on these improvements, the steps of standardizing sperm samples prior to labeling and modifying the single-step labeling process independently improve the ability of sperm to survive the labeling process. In addition, the Single and Standardized Stage I (F), Single and Standardized Stage II (G), Single and Standardized Stage III (H), and Single and Standardized Stage IV (I) groups demonstrate a synergy in which the combined effect of standardization of an ejaculatory material and the one-step ejaculatory material marking is superior to any of the individual enhancements.

[078 | Referring to Table 2, it can be seen that the Standardized I (D), Standardized II (E), Single Step, and Standardized I (F) / and Single Step and Standardized II (G) groups appear to provide significant benefits in terms of reduction in the number of dead sperm, but the percentage of targeted sperm did not improve. This could be related to the column indicated as overband. Although more sperm have been collected as live for selection, there appears to be an increase in scattered signals above the selection collection ranges. These signals may represent sperm that stick together, or they may represent sperm that bind to egg yolk lipids. In either case, the general pattern arises that larger amounts of egg yolk reduce the numbers of dead sperm, but may introduce a new problem and a balance may therefore be required.

[079 | In addition, tests incorporating the single-step labeling methodology provided a more efficient means of associating Iloechst 33342 DNA sorting ink with sperm cell nuclear DNA. Labeling quality was maintained under all conditions tested, but tests that included the single labeling step used 2μ1 minus Hoechst per ml sample. Less Hoechst marking ability can contribute to improved overall sperm health.

Example 2 Collection - Sperm were collected from six different Jersey bulls in a routine collection scheme using an artificial vagina. All ejaculatory materials contained progressive motility greater than 65% and sperm concentration ranged from 765 million sperm per ml to 1,710 million sperm per ml. Each sperm sample was divided into two parts in 15 ml tubes for two collection and labeling treatments. PH measurements were taken at collection and at each subsequent processing step. 81θ81 | Sperm Processing and Marking - Portions of each bull's ejaculatory material were processed and labeled by two comparison methods.

[082] Control (no standardization, two-step labeling) - A control was established that did not include the standardization stage of collected ejaculatory materials and in which sperm were labeled in two stages. Prior to labeling, sperm samples were concentrated between 1,700 million sperm per ml and 1,800 million sperm per ml by centrifugation or by the addition of an egg yolk extender with a pH of 6.8, depending on the initial concentration. of the samples.

[083] The sperm in the control group were diluted to 160 x 106 sperm per ml in a modified TALP buffer, as described in Table 1, at a pH of 7.4. Each sperm sample in the control group was then incubated with 16-17pL Hoechst 33342 per ml (64-68 μΜ) sample for 45 minutes at 34 ° C. After incubation, an equal volume of a second modified TALP was added, reducing the concentration to 80x106 sperm per ml. The second modified TALP includes the components described in Table 1 with the addition of 4% egg yolk, 50 μΜ No. 6 yellow food ink (20 g / 1) and the pH was lowered to 5.5 with the addition of HCl.

[084] Single step and standardized (10% egg yolk standardization, single step marking with 1% egg yolk) - Sperm were standardized by adjusting both pH and sperm concentration before labeling. After collection, sperm were diluted 1: 3 in an initial extender with a pH of 7.2 and a high pH buffering capacity. The high capacity buffer was supplemented with 1% egg yolk. All samples were then centrifuged to reduce sperm concentration to between 1,700 million sperm per ml and 1,800 million sperm per ml. (085 (The sperm samples were then diluted to 160 x 106 sperm per ml in a TALP buffer at a pH of 7.4. The modified TALP buffer was substantially identical to the buffer described in Table 1, except that it additionally included 1% egg yolk and No. 6 yellow food ink at a concentration of 25 μΜ Each sperm sample in this group was then incubated with 16 - 17 pL Hoechst 33342 per ml (64 - 68 μΜ) for 45 minutes at 34 °. C. Following incubation, sperm remained at a concentration of 160 x 106 sperm per ml. (086] Data selection and acquisition - Each sample was selected on an MoFlo® SX flow cytometer (Beckman Coulter, USA) with an upgrade. Genesis (Cytonome / ST, Boston MA, USA) Control was selected at a concentration of 80x106 sperm per ml, whereas standardized sperm were selected at 160x106 sperm per ml. by the flow cytometer and then averaged for the six bulls. (087] Results - Table 3 illustrates the pH recorded for both control (A) and standardized ejaculatory material (B). These values are reflected in Table 3 below. Although standardized ejaculatory material is subject to an initial increase, a subsequent increase is avoided during labeling and the next drop is also avoided.Furthermore, Table 4 illustrates similar benefits in reducing dead sperm as seen in Example 1. Specifically, the standardized sample that was marked in one step had 5.67% less dead sperm.

Table 3 Example 3 [088 | Collection - Sperm were collected from three different Jersey bulls in a routine collection regime for a total of 17 collections. Each ejaculatory material was divided into two treatments. | 089 | Sperm Processing and Marking - Portions of each bull's ejaculatory material were processed and labeled by two comparison methods. | 090 | Control (no standardization, two-step labeling) - A control was established that did not include the standardization stage of collected ejaculatory materials and in which sperm were labeled in two steps. The sperm in the control group were diluted to 160 x 106 sperm per ml in a modified TALP buffer, as described in Table 1, at a pH of 7.4. Each sperm sample in the control group was then incubated with 16 - 17 pL Hoechst 33342 per ml (64 - 68 μΜ) for 45 minutes at 34 ° C. After incubation, an equal volume of a second modified TALP was added, reducing the concentration to 80 x 106 sperm per ml. The second modified TALP includes the components described in Table 1 with the addition of 4% egg yolk, 50 μΜ red food ink No. 40 (20 g / 1) and the pH was lowered to 5.5 with the addition of HCl. r [091] Standardized III and Single Step (standardization with 3% egg yolk, single step marking) - Remaining sperm were standardized by adjusting both pLI and sperm concentration prior to marking and selection. After collection, sperm were diluted 1: 3 in an initial extender with a pH of 7.2 and a high pH buffering capacity. The high capacity buffer was supplemented with 3% egg yolk. The sperm sample was then diluted to 160x106 sperm per ml in a modified TALP buffer at a pH of 7.4. The modified TALP buffer was substantially identical to the buffer described in Table 1, except that it additionally included 1% egg yolk and No. 6 yellow food ink at a concentration of 25 μΜ. Each sperm sample in this group was then incubated with 14-15μΕ Hoechst 33342 per ml (56-60 μΜ) sample for 45 minutes at 34 ° C. After incubation, sperm remained at a concentration of 160x106 sperm per ml.

[0921 Marking and Results - The control group was processed using a Legacy MoFlo® SX flow cytometer (Beckman Coulter, Maimi FL, USA) with a Genesis digital upgrade (Cytonome / ST, Boston MA, USA) at a concentration of 80 x 106 sperm per ml, while the Standardized 111 and Step One group was selected at a concentration of 160 x 106 sperm per ml. Table 5 illustrates the percentage of cells in the dead collection of each ejaculatory material and the mean. After selection, percentages of sperm occurring in the dead collections (R2 seen in Fig. 3) were indicated for both samples. It can be seen that the average for 17 bulls was 17% of sperm collected as dead in control and only 10% of sperm collected as dead for treated sperm, meaning that the treatment offered a significant benefit to sperm health.

Table 4 Example 4 [093] Collection and Selection - Sperm were collected from a Holstein bull and labeled according to the Standardized t-group III and Single Step protocol described in Examples 1 and 3. The sample was placed on the Legacy MoFlo flow cytometer. ® SX (Beckman Coulter, Maimi FL, USA) with a Genesis digital upgrade (Cytonome / ST, Boston MA, USA). During selection, the circulating fluid pressure was set at 40 psi and the drip control frequency was set at 64.9 KHz. The sample pressure was adjusted to target event rates of approximately 1500, 3500, 7500, 8500, 10,000 15000, 20000, 25000, and 30000.

[094] Results - Selection parameters measured from each target event rate were recorded in Table 6. The ejaculatory material in this example demonstrated approximately 3% - 5% dead collection, allowing large portions of sperm to be included in the sample. oriented living collection, between 79.1% and 85.4%. The selective logic used in this selection collected in a region of living oriented sperm (R1). IR was established by an operator to retain a large portion of sperm. X-selection collection was similarly established by an operator with a target purity of 90%. Data were periodically digitally recorded for several samples at each event rate. Data were averaged at each event rate to provide productivity averages (selective rate), selective efficiency (selective rate / event rate), valley-to-peak ratio, abortion rate, as well as the percentage of population in the dead collection. (R2), the percentage of the population in the living oriented collection (Rl), the percentage of the sperm population in the X-selection collection (R3), and the percentage of viable (living) sperm in the X-selection collection. In addition, the purities were determined offline for each sperm selected in each event rate setting. Purity was determined by sonication of the 1 million sperm tails and collected at each event rate group, and measured on an offline purity analyzer. This measurement was taken twice for each group and an average was calculated.

Table 6 1095] It can be seen that low event rates reduce abortion rates and improve selective efficiency. In particular, the abortion rate is equal to 7% of the selective rate when the event rate is 1,722. | 096 | In addition, the synergistic effect of reducing dead sperm is illustrated by the fact that over 50% of the sperm sample was collected at the X-selection port for event rates below 10,000 events per second. The low percentage of dead sperm in combination with the high percentage of live oriented sperm allows the collection of an R3 region to be adjusted so that R3 invades the region of Fig. 4 where sperm are more likely to be chromosome-bearing sperm. Y than X-chromosome-bearing sperm. Even when this region is slightly invaded, post-selection purity remained at 96%, although 54% of all sperm were included in the X-selection collection and 57% of all. live sperm have been included in the X-selection collection.

[097 | The synergistic combination of improved marking techniques with efficiency-enhancing selection methods demonstrates to provide reliable selection methods that can provide approximately 25% to approximately 40% yield over the total sperm population, and maintain purity greater than 90%. | 098 | One aspect of this disclosure designs spatially more efficient flow cytometers, which may allow more selection heads in available space. In such an arrangement, more flow cytometer selective heads can be dedicated to a single sperm sample, and each can be operated with improved efficiency, thereby combining the benefits of efficient selection methods with high productivity.

Example 5 | 099 | Sperm Manipulation - A sperm sample was collected from five bulls, including two Holstein bulls, two Jersey bulls and a Simmental bull. At the time of collection, volume, concentration, motility, morphology and pEI were verified, and then antibiotics were added according to industrial practices. Each bull used in the example had motility of 70% or greater. The sperm sample was then standardized by placement in a pH buffered extender and centrifuged for reconcentration between 1,700 - 1,800 sperm per ml. 3 ml of each bull was labeled with Hochest 33342 TALP and a deactivating yellow food ink, and concentrations after labeling were 160 million sperm per ml according to the single-step labeling described in previous examples. ΐοιοθι The sperm of each bull were divided into four treatments. Two treatments were performed at 40 psi and two treatments were performed at 65 psi. In each of these 40 and 65 psi groups, one treatment was established as the high throughput treatment and one treatment was established as the high efficiency treatment. | 01011 Treatment 1 - In the first treatment, the selector circulating fluid pressure was set to 40 psi. An event rate of approximately 35,000 events per second was then established by adjusting the sample pressure. The drip control frequency and other droplet generation signals were adjusted until a side calibration current was established without spraying. A drip delay calibration was then performed to determine a load delay.

Treatment 2 - In the second treatment, the selector was maintained at a pressure of 40 psi and the event rate was decreased to 20,000 events per second with sample pressure adjustments. The collection was then readjusted on the flow cytometer.

Treatment 3 - In the third treatment, the circulating fluid pressure was set to 65 psi and an event rate of approximately 35,000 events per second was set at the same pressure as the sample. The drip control frequency and other droplet generation signals until a side calibration current was established without spraying. A drip delay calibration was then performed to determine a load delay.

[0104 | Treatment 4 - In the fourth treatment, circulating fluid pressure was maintained at 65 psi and an event rate of 20,000 events per second was established by adjusting the sample pressure. The collection was then readjusted on the flow cytometer.

Sperm Selection and Freezing - The sperm from each of the five bulls were selected with each calibration described. During each selection, data were recorded from the flow cytometer and are shown in Table 7. A total of 10 million X-chromosome-bearing sperm were collected in a seizure tube with an extender A fraction including approximately 20% of egg yolk for each. The collected sperm were refrigerated for 90 minutes at approximately 5 ° C. An extender fraction B including 12% glycerol was added in two equal portions. After fraction B was added, the sample was centrifuged and resuspended in an equal extender in fraction A and fraction B having approximately 20% egg yolk and approximately 6% glycerol. Multiple 0.25 ml rods were filled for each bull and each treatment and then frozen in liquid nitrogen.

Post Melting - Frozen rods for each bull and treatment were selected to pass quality control tests. Motility was checked at 0 hours and then again after 3 hours. In addition, viability was determined by flow cytometric analysis of a portion of the molten sperm which were then labeled with Sybr Green and propidium iodide. The acrosome health of another portion of the molten sperm was analyzed by flow cytometry marked PI / PNA. In addition, the sperm from each rod were sonicated and the sperm nuclei were analyzed for purity. The results are seen in Table 8.

Results - The data recorded in Table 7 illustrate several trends, with a significant trend being that slower event rates from Treatments 2 and 4 slightly increased the percentage of sperm in collection X and moderately improved the valley-to-valley ratio. (RVP) and percentage of sperm in the guided collection, compared to Treatments 1 and 3, respectively. In addition, increased pressure from Treatments 3 and 4 independently decreased abortion rates and further increased sperm percentage in collection X compared to Treatments 1 and 2, respectively.

Example 6 Sperm Manipulation - A sperm sample was collected from seven bulls, including four Holstein bulls and three Jersey bulls. At the time of collection, volume, concentration, motility, morphology and pH were checked, and then antibiotics were added. Each bull used in the example had 60% motility or greater. The sperm sample was then standardized by placement in a pH buffered extender and centrifuged for reconcentration between 1,700 -1,800 sperm per ml. The sperm were labeled according to the single step procedures outlined in Example 1, without the addition of egg yolk at the time of labeling. | 0109 | Selector Calibration - A first dataset was produced with a Legacy MoFlo® SX flow cytometer featuring a Genesis digital upgrade available from Cytonome / ST (Boston, MA, USA), configured for 40 psi operating circulating fluid pressure. . The drip control frequency has been set to the highest frequency, providing good quality side current within a recommended existing range. The drip delay was then determined with a selection test on a microscope slide. The initial seizure fluid level of a collection tube was positioned 11.43 cm below the flow cytometer deflector plates, or in the standard position of a 50 ml seizure tube on a MoFlo® flow cytometer. (0110] A second dataset was produced with the same Legacy MoFlo® SX flow cytometer operating at a circulating fluid pressure of 60 psi. The drip control frequency was set to the highest frequency, providing a side current of good quality within a recommended existing range Drip retardation was then determined with a selection test on a microscope slide The initial seizure fluid level of a collection tube was positioned 11.43 cm below the cytometer deflector plates or at the standard position of a 50 ml seizure tube on a MoFlo® flow cytometer.

10111] A third data set was produced in the same way as the second data set, except that the collection tube was moved down 3,175 cm through a workbench cut on which the stream was located. The initial seizure fluid level was 14.605 cm below the flow cytometer deflector plates. | 0112 | Sperm Selection and Freezing - Sperm from each of the seven bulls were selected with each calibration described. A total of 15 million X-chromosome-bearing sperm were collected in a seizure tube having an extender fraction A including approximately 20% egg yolk for each.

The collected sperm were refrigerated for 90 minutes at approximately 5 ° C. An extender fraction B including 12% glycerol was added in two equal portions. After fraction B was added, the sample was centrifuged and resuspended in an equal extender in fraction A and fraction B having approximately 20% egg yolk and approximately 6% glycerol. Multiple 0.25 ml rods were filled for each bull and each treatment and then frozen in liquid nitrogen. (0114] Post-melting - Frozen rods for each bull and treatment were selected to pass quality control testing, rods were melted and motility was checked at 0 hours and then again after 3 hours, plus sperm viability. was determined by flow cytometry after Syòr / PI labeling. Five of the seven bulls were selected for IVF. In addition, the sperm from each rod were sonicated and the sperm nuclei were analyzed for purity.] 0115] Results - Means The measured selection parameters determined by data logging software were compiled for all selections made at 40 psi and for all selections made at 60 psi. In addition, the average time for selection of 15 million X-chromosome-bearing sperm at 40 psi was 48:17, and the average time to select 15 million chromosome-bearing sperm X at 60 psi was 34:23.

Table 9 [0116] The benefits of 60 psi selection over 40 psi can be readily seen in terms of productivity as well as efficiency in the average measured selection parameters recorded in Table 9. With respect to productivity, an average of 7175 selections per second allowed 15 million sperm to be selected 13:54 faster. In addition, 60 psi offered higher event rates, improved sperm orientation, and a better distinction between X-chromosome-bearing sperm and Y-chromosome-bearing sperm.

Each material of the seven bulls was frozen, one rod for each bull was melted and evaluated for motility, compromised sperm membranes (Sybr / PI) and purity. Five of the seven bulls, including three Holstein bulls and two Jersey bulls, were selected for IVF. The conversion of oocytes to embryos for each treatment is shown in Table 11.

[0118] In addition, a benefit was realized by changing the distance of the seizure fluid, particularly for 60 psi selection. Table 10 illustrates the average post-melt motility, viability and purity for each treatment in the five bulls selected for the IVF tests. | 0119 | Especially for the five bulls rated at 60 psi, the standard location of the seizing fluid provided slightly lower post-melt motility and slightly lower viability compared to selection at 40 psi at the same site. However, moving the seizure tube down an additional 3.18 cm downwards provided a 10% improvement in 0 hour motility at 60 psi and an 11% improvement in 3 hour motility. For the five bulls used for IVF, sperm selected at 60 psi and collected in the second position of the seizure tube showed slightly better motility and viability than selection at 40 psi.

Table 11 Example 7 [0120] A sperm sample was collected from five bulls. At the time of collection, volume, concentration, motility, morphology and pH were checked, and then antibiotics were added. Each bull used in the example had 60% motility or greater. The sperm sample was then divided into two groups.

Treatment I - The sperm sample in Treatment I was labeled in two steps, in substantially the same manner as described above. In particular, Treatment I was maintained as unchanged semen until it was labeled at a first dilution to 160 x 106 sperm per ml in a modified TALP buffer as described in Table 1 at a pH of 7.4. Each sperm sample in the second group was then incubated with Hoechst 33342. After incubation, an equal volume of a second modified TALP was added, reducing the concentration to 80 x 106 sperm per ml. The second modified TALP includes the components described in Table 1 with the addition of red food ink No. 40 and 4% egg yolk, and the pH was lowered to 5.5 with the addition of HCl.

Treatment II - The sperm sample in Treatment II was then standardized with a 3 to 1 dilution in an initial extender, or a buffering support extender. The buffering extender had a high buffering capacity and a pH of about 7.2. In addition, antioxidants were added to the initial extender to reduce oxidative stress on sperm. The extended suspension was centrifuged and reconcentrated to between 1,600 ± 400 sperm per ml. Sperm were labeled in a single dilution to a concentration of 160 x 106 in a modified ASB-containing TALP supplemented with an antioxidant component. | 0123] Sperm Selection - A Genesis II flow cytometer provided by Cytonome / ST (Boston, MA, USA) was used for the selection of both labeled samples. Sperm were selected for X-chromosome (female) in both treatments at event rates between 25,000 and approximately 40,000 events per second, depending on projected purity. | 0124 | Sperm prepared according to Treatment I were selected from a TRIS citrate extender seizure containing 20% egg yolk (sometimes referred to as TRIS A).

Sperms prepared according to Treatment II were selected on a TRIS citrate extender seizure containing 20% egg yolk, with the addition of an antioxidant treatment.

[0126] Post-selection / counseling - Treatment I sperm were collected and refrigerated for 90 minutes at approximately 5 ° C. An extender fraction B including 12% glycerol was added in two equal portions. After fraction B was added, the sample was centrifuged and resuspended in an equal extender in fraction A and fraction B having approximately 20% egg yolk and approximately 6% glycerol. Multiple 0.25 ml rods were filled for each bull and each treatment at a dosage of approximately 2.1 million sperm, and then frozen in liquid nitrogen.

Treatment II sperm were collected and refrigerated for 90 minutes at approximately 5 ° C. An extender fraction B including 12% glycerol and a combination of antioxidants was added in two equal portions to reduce oxidative stress. After fraction B was added, the sample was centrifuged and resuspended in an equal extender in fraction A and fraction B having approximately 20% egg yolk, approximately 6% glycerol and approximately 25 mg / μΐ antioxidants. Treatment II sperm were divided into three groups. A first group filled 0.25 ml rods at a dosage of 2.1 million sperm per rod, a second group filled 0.25 ml rods at a dosage of 3 million sperm per rod, and a third group filled rods 0.25 ml at a dosage of 4 million sperm per stick, and were then frozen in liquid nitrogen.

[0128] Insemination was performed with each of the treatments and dosages plus a control of unselected sperm at a conventional dosage (15 million sperm per stick). (0129J Results - For the first time the detrimental effects of sperm selection by flow cytometry were sufficiently compensated for a dose response effect with sexually selected sperm to be seen. In addition, for the first time, a dosage of sexually selected sperm showed performance as good as conventional dosages of non-sexualized sperm. In Table 12, a non-return rate (TNR) of 56 days was compared for each treatment and dosage. In addition, relative fertility was compared as a percentage with conventional insemination.

Table 12 Data from cows and heifers. TNR results with different envelopes are significantly different P <0.05.

[0130 | Having established a sperm selection methodology that provides sperm capable of demonstrating dose response, it is expected that further increases in dosage may begin to outweigh the fertility of conventional semen. Although sexual selection is a sperm-damaging procedure, the selection step actively removes dead or compromised membrane sperm cells from the selected sperm sample. It may be that in the improved selection method the detrimental steps of the sexual selection process irreparably damage the less likely sperm cells to fertilize an egg, thereby removing the most subfertile sperm from the insemination sample. Thus, once overall process damage has been reduced sufficiently to provide a dose response, further increases in sperm dosages may begin to outperform conventional unselected semen due to the removal of more subfertile sperm from the selected population.

Those skilled in the art will recognize that the invention described above includes many inventive aspects, which may be provided in any combination and include at least the following: | 0132 | Al - A sexually selected sperm production method having an improved dose response, comprising: a) extending a sperm sample with a buffering medium; b) adjusting the concentration of the extended sperm sample to a target concentration range; c) marking the sperm sample with a DNA selective dye; d) sexual selection of the sperm sample marked with a flow cytometer in a seizure fluid; e) preserving the pH of the sperm sample through steps (a) to (c) within a target pH range; and f) supplementing at least one element from the buffering medium, DNA selective dye and seizure fluid with at least one antioxidant. A method according to claim A1, characterized in that the steps of extending a sperm sample with buffering means and adjusting the concentration of the sperm sample extended to a target concentration range further comprise: (a) the extent of the sperm sample in the buffering medium in a target pH range between 7.0 and 7.4; and (b) adjusting the concentration of the extended sperm sample to a target concentration range between approximately 800 x 106 sperm / ml and approximately 2,100 x 106 sperm / ml. A method according to claim A1 or A2, further comprising maintaining a sperm sample exposure to antioxidants from steps (a) to (ç). A method according to any one of the preceding claims, characterized in that a dose of at least 4 million sexually selected sperm processed by steps (a) to (e) has at least the same fertility as a conventional dose. than 15 million unselected sperm by sex. | 0136 | A method according to any one of the preceding claims, characterized in that the buffering support means comprises one or more components selected from the group consisting of: TRIS citrate, sodium citrate, sodium bicarbonate, HEPES, TRIS, TEST, MOPS, KMT, TALP, and combinations thereof. A method according to any one of the preceding claims, characterized in that the buffering support means further comprises egg yolk.

The method according to claim A6, characterized in that the buffering support means further comprises from about 1% to about 10% egg yolk. | 0139 | A method according to any one of the preceding claims, characterized in that the buffering means further comprises citric acid or citrates. | 0140 | A method according to any one of the preceding claims, characterized in that the buffering support means further comprises one or more antioxidants. (0141 (Α10 - Method) according to any one of the preceding claims, characterized in that the pH of the sperm sample is stabilized by steps (a) to (e). I0142J AI1 - Method according to claim A10, characterized by the pH of the sample. maintained within a difference of 0.5 from the target pH by steps (a) to (e). A12 - A method according to claim A10, characterized in that the pH of the sperm sample is maintained at approximately 6, 65 and approximately 7.35 through steps (a) to (e).

Method according to any one of the preceding claims, characterized in that the antioxidants are selected from the group consisting of: catalase, superoxide dismutase (SOD), an SOD mimic, glutathione, glutathione reductase, glutathione peroxidase, pyruvate , caproic acid, mercaptoethanol, BHT, lipoic acid, flavins, quinines, vitamin K (and related vitamers), vitamin BI2, vitamin B 2, vitamin E (and related vitamers), tocopherols, tocotrienols, α-tocopheryl, alpha- ketoglutarate (AKG), malondialdehyde (MDA), asymmetric dimethylarginine (ADMA), and biologically active derivatives thereof, and combinations thereof.

[0145 | Method according to any one of the preceding claims, characterized in that the antioxidants are selected from the group consisting of: vitamin B 2, vitamin B 2, vitamin E, vitamin E, tocopherols, tocotrienols, α-tocopheryl, alpha -ketoglutarate, derivatives thereof, and combinations thereof. A method according to any one of the preceding claims, characterized in that the antioxidants are present in concentrations of approximately 0.01 and approximately 50 mg / ml. A method according to any one of claims Al - A14, characterized in that the antioxidants are present in concentrations selected from the group consisting of: from 0.01 mg / ml to 5.0 mg / ml; 01 mg / ml to 0.25 mg / ml, from 0.01 mg / ml to 0.5 mg / ml, from 0.01 mg / ml to 1 mg / ml, from 0.01 mg / ml to 2.5 mg / ml; 0.01 mg / ml to 5 mg / ml; 0.05 mg / ml to 0.1 mg / ml; 0.05 mg / ml to 1.0 mg / ml; mg / ml to 2.5 mg / ml, from 0.1 mg / ml to 0.25 mg / ml, from 0.1 mg / ml to 0.5 mg / ml, from 0.1 mg / ml to 1 mg / ml, from 0.1 mg / ml to 2.5 mg / ml, from 0.1 mg / ml to 5 mg / ml, from 0.15 mg / ml to 0.45 mg / ml, from 0, 15 mg / ml to 0.5 mg / ml, 0.25 mg / ml to 0.35 mg / ml, 0.25 mg / ml to 0.5 mg / ml, 0.25 mg / ml to 1 mg / ml 0.25 mg / ml to 2.5 mg / ml 0.25 mg / ml to 5 mg / ml 0.35 mg / ml to 0.5 mg / ml 0 to 35 mg / ml to 1 mg / ml 0.35 mg / ml to 2.5 mg / ml 0.35 mg / ml to 5 mg / ml 0.5 mg / ml to 1 mg / ml 0.5 mg / ml to 2.5 mg / ml, 0.5 mg / ml to 5 mg / ml, 1 mg / ml to 2.5 mg / ml and 1 mg / ml to 5 mg / ml.

A method according to any one of claims Al-A14, characterized in that the concentration of antioxidants is selected from the group consisting of: approximately 0.05 mg / ml; approximately 0.1 mg / ml; approximately 0.15 mg / ml; approximately 0.25 mg / ml; approximately 0.35 mg / ml; approximately 0.45 mg / ml; and approximately 0.5 mg / ml.

A method according to any one of the preceding claims, characterized in that the target concentration comprises a concentration between approximately 800 x 106 sperm per ml and approximately 2,100 x 106 sperm per ml.

A method according to any one of the preceding claims, characterized in that the marking step is performed in a single dilution with a DNA sorting dye and a dead deactivating dye. Method according to any one of the preceding claims, characterized in that it further comprises the step of freezing the selected sperm sample. | 0152 | A21 - A method according to claim A20, characterized in that one or more antioxidants are added to the medium in which the selected sperm sample is frozen.

A method according to claim A21, characterized in that one or more antioxidants are present in concentrations selected from the group consisting of: from 0.01 mg / ml to 5.0 mg / ml; from 0.01 mg / ml to 0.25 mg / ml; from 0.01 mg / ml to 0.5 mg / ml; from 0.01 mg / ml to 1 mg / ml; from 0.01 mg / ml to 2.5 mg / ml; from 0.01 mg / ml to 5 mg / ml; from 0.05 mg / ml to 0.1 mg / ml; from 0.05 mg / ml to 1.0 mg / ml; from 0.05 mg / ml to 2.5 mg / ml; from 0.1 mg / ml to 0.25 mg / ml; from 0.1 mg / ml to 0.5 mg / ml; from 0.1 mg / ml to 1 mg / ml; from 0.1 mg / ml to 2.5 mg / ml; from 0.1 mg / ml to 5 mg / ml; from 0.15 mg / ml to 0.45 mg / ml; from 0.15 mg / ml to 0.5 mg / ml; from 0.25 mg / ml to 0.35 mg / ml; from 0.25 mg / ml to 0.5 mg / ml; from 0.25 mg / ml to 1 mg / ml; from 0.25 mg / ml to 2.5 mg / ml; from 0.25 mg / ml to 5 mg / ml; from 0.35 mg / ml to 0.5 mg / ml; from 0.35 mg / ml to 1 mg / ml; from 0.35 mg / ml to 2.5 mg / ml; from 0.35 mg / ml to 5 mg / ml; from 0.5 mg / ml to 1 mg / ml; from 0.5 mg / ml to 2.5 mg / ml; from 0.5 mg / ml to 5 mg / ml; from 1 mg / ml to 2.5 mg / ml; and from 1 mg / ml to 5 mg / ml. | 0154 | A23 - A method according to claim A21, characterized in that the concentration of antioxidants is selected from the group consisting of: approximately 0.05 mg / ml; approximately 0.1 mg / ml; approximately 0.15 mg / ml; approximately 0.25 mg / ml; approximately 0.35 mg / ml; approximately 0.45 mg / ml; and approximately 0.5 mg / ml. A method according to any one of the preceding claims, characterized in that the step of extending a sperm sample with a buffering support means further comprises extending the sperm sample in the buffering support medium in a ratio of: approximately 1 :1; approximately 1: 2; approximately 1: 3; approximately 1: 4; approximately 1: 5; approximately 1: 6; approximately 1: 7; approximately 1: 8; approximately 1: 9; or about 1:10.

BI - Sexually selected insemination dosage produced by the method defined in claim 1, characterized in that it has a dose response at dosages greater than 3 million. | 0157] B2 - Sexually selected insemination dosage characterized by comprising: a population of at least 4 million marked and sexually selected sperm capable of fertilization in artificial insemination at at least the same fertility level as a conventional dose of 15 million sperm. | 0158 | B3 - Sexually selected insemination dosage comprising: at least 3 million labeled and sexually selected sperm capable of achieving 90% of fertility from a conventional dose of 15 million sperm.

Sexually selected insemination dosage according to any one of claims BI - B3, characterized in that sexually selected sperm comprises sperm selected for X chromosome by flow cytometry.

[0160] B5 - Sexually selected insemination dosage according to any one of claims BI - B3, characterized in that sexually selected sperm comprises sperm selected for Y chromosome by flow cytometry.

Sexually selected insemination dosage according to any one of claims BI - B5, further comprising a first residual amount of antioxidant from a buffering support medium in which the sperm sample has been processed.

A sexually selected insemination dosage according to any one of claims BI - B6, further comprising a second residual amount of antioxidant from a label in which the sperm sample has been processed.

A sexually selected insemination dosage according to any one of claims B-B7, further comprising a third residual amount of antioxidant from a seizing fluid in which the sperm sample has been processed.

Sexually selected insemination dosage according to any one of claims B-B8, characterized in that the sperm dosage comprises between approximately 4 million and approximately 20 million selected sperm with fertility characteristics comparable to those of a conventional semen dose. 15 million sperm.

[0165] B10 - Sexually selected insemination dosage according to any one of Claims B - B9, characterized in that the sperm dosage comprises a range selected from: between approximately 3 million sperm and approximately 4 million sperm, between approximately 4 million sperm. and approximately 5 million sperm, between approximately 5 million sperm and approximately 6 million sperm, between approximately 6 million sperm and approximately 7 million sperm, between approximately 7 million sperm and approximately 8 million sperm, between approximately 8 million of sperm and approximately 9 million sperm, between approximately 9 million sperm and approximately 10 million sperm, between approximately 10 million sperm and approximately 11 million between approximately 11 million sperm and approximately 12 million sperm, between approximately 12 million sperm and approximately 13 million sperm, between approximately 13 million sperm and approximately 14 million sperm, between approximately 14 million sperm and approximately 15 million sperm, between approximately 15 million sperm and approximately 16 million sperm, between approximately 16 million sperm and approximately 17 million sperm, between approximately 17 million sperm and approximately 18 million sperm, approximately 18 million sperm sperm and approximately 19 million sperm, and between approximately 19 million sperm and approximately 20 million sperm.

[0166 | Sexually selected insemination dosage according to any one of claims BI - B10, characterized in that the sperm comprise frozen sperm.

Sexually selected insemination dosage according to any one of claims BI - B11, characterized in that it comprises a freezing extender, and that the freezing extender further comprises an antioxidant present at a concentration between 0.01 mg / ml and 0.5. mg / ml.

As can be readily understood from the foregoing, the basic concepts of the present invention may be embodied in a variety of ways. The invention involves numerous and varied embodiments of sexual sperm selection, including, but not limited to, the best embodiment of the invention. | 0169 | Thus, the particular embodiments or elements of the invention disclosed by the description or shown in the accompanying figures or tables are not intended to be limiting, but, on the contrary, non-limiting examples of the numerous and varied embodiments generally encompassed by the invention or equivalents encompassed with respect to the invention. any specific element of it. Further, the specific description of a single embodiment or element of the invention may not explicitly describe all possible embodiments or elements; Many alternatives are implicitly revealed by the description and the figures. In addition, for each term used herein, it should be understood that, unless their use in this application is inconsistent with such an interpretation, common dictionary definitions should be taken as included in the description for each term according to Random. House Webster's Uriabridged Dictionary, second edition, each definition hereby incorporated by reference.

Further, for purposes of the present invention, the term "one" or "one" entity refers to one or more of this entity. Thus, the terms "one" or "one", "one or more" or "at least one" may be used synonymously here. | 0172 | All numeric values disclosed herein are taken to be modified by the term "approximately", whether or not explicitly indicated. For purposes of the present invention, ranges may be expressed as from "approximately" a specific value to "approximately" another specific value. When such a range is expressed, another embodiment will include from that specific value to the other specific value. Quotation of numeric ranges at the extremes includes all numerical values covered by the ranges. A numerical range from 1 to 5 includes, for example, the numerical values 1.5; 2; 2.75; 3; 3.80; 4, 5, and so on. It should be further understood that the extremes of each range are significant both in relation to the other end and independently of the other end. When a value is expressed as an approximation by the use of the "approximately" antecedent, it must be understood that the specific value forms another modality.

[0173] The introductory section of this patent application provides a statement of the search field to which the invention belongs. This section may incorporate or contain citations of certain patents, patent applications, publications, or materials of the claimed invention that are useful for reporting information, problems, or questions about the state of the art to which the invention is directed. Any patents, patent applications, publications, declarations, or other information cited or incorporated herein are not intended to be construed, read, or considered to be prior art admission to the invention. | 0174 | The claims contained in this report, as the case may be, are hereby incorporated by reference as part of the description of the invention, and the Depositor expressly reserves its right to use all or a portion of such incorporated content of such claims as further description to substantiate any one or all of the claims or any elements or components thereof, and the Depositor also expressly reserves its right to move any portion or all embedded content of such claims or any element or component thereof from the description to the claims or vice versa, as necessary to define the matter for which protection is sought through this application or any subsequent application or continuation, division or certificate of addition, or to obtain any benefit, reduction of fees, or in compliance with patent laws, rules or regulations of any country or treaty o, and such content incorporated by reference shall remain for the entire duration of this application, including any subsequent continuation, division or certificate of addition, or any new publication or extension thereof.

Claims (15)

1. Selected sex insemination dose comprising: a population of at least 4 million sex-labeled and selected sperm capable of artificial insemination fertilization at at least the same fertility level as a conventional dose of 15 million sperm. 2. Selected sex insemination dose comprising: at least 3 million sex-labeled and selected sperm capable of achieving 90% fertility from a conventional dose of 15 million sperm. A sex-selected insemination dose according to claim 1 or 2, characterized in that the sex-selected sperm comprises X-chromosome-selected sperm by flow cytometry. Sex insemination dose selected according to any one of claims 1 to 3, characterized in that the sex-selected sperm comprises Y-chromosome-selected sperm by flow cytometry. Sex insemination dose selected according to any one of claims 1 to 4, characterized in that it further comprises a first residual amount of antioxidant from a buffering medium in which the sperm sample has been processed. Sex insemination dose selected according to any one of claims 1 to 4, characterized in that it further comprises a second residual amount of antioxidant from a label in which the sperm sample has been processed. Sex insemination dose selected according to any one of claims 1 to 4, characterized in that it further comprises a third residual amount of antioxidant from a seizure fluid in which the sperm sample has been processed. A selected sex insemination dose according to any one of claims 1 to 7, characterized in that the sperm dose comprises from approximately 4 million to approximately 20 million selected sperm having fertility characteristics comparable to those of a 15 million sperm dose. of conventional semen. A sex-selected insemination dose according to any one of claims 1 to 7, characterized in that the sex-selected sperm dose comprises a range selected from approximately 3 million to approximately 4 million sperm, from approximately 4 million and approximately 5 million sperm, between approximately 5 million and approximately 6 million sperm, between approximately 6 million and approximately 7 million sperm, between approximately 7 million and approximately 8 million sperm, between approximately 8 million and approximately 9 million between 9 million and approximately 10 million sperm, between approximately 10 million and approximately 11 million sperm, between approximately 11 million and approximately 12 million sperm, between approximately approximately 12 million and approximately 13 million sperm, between approximately 13 million and approximately 14 million sperm, between approximately 14 million and approximately 15 million sperm, between approximately 15 million and approximately 16 million sperm, between approximately 16 million and approximately 17 million sperm, between approximately 17 million and approximately 18 million sperm, between approximately 18 million and approximately 19 million sperm, and between approximately 19 million and approximately 20 million sperm. Sex insemination dose selected according to any one of claims 1 to 9, characterized in that the sperm comprise frozen sperm. Sex insemination dose selected according to any one of claims 1 to 10, characterized in that it comprises a freezing extender and the freezing extender further comprises an antioxidant present in a concentration between 0.01 mg / ml and 0.5 mg / ml. A method of producing sex-selected sperm having an improved dose response, comprising: a) extending a sperm sample with a buffering medium; b) adjusting the concentration of the extended sperm sample to a target concentration range; c) marking the sperm sample with a DNA selective dye; <7 / sexual selection of sperm sample marked with a flow cytometer in a seizure fluid; e) preserving the pH of the sperm sample through steps (a) to (c) within a target pH range; and supplementing at least one element from the buffering medium, the DNA-selective dye and the seizure fluid with at least one antioxidant. A method according to claim 12, characterized in that the steps of extending a sperm sample with buffering means and adjusting the concentration of the sperm sample extended to a target concentration range further comprise: (a) extending the sperm sample buffer sperm sample in a target pH range between 7.0 and 7.4; and (b) adjusting the concentration of the extended sperm sample to a target concentration range between approximately 800 x 10 6 sperm / ml and approximately 2100 x 106 sperm / ml. Method according to claim 12 or 13, characterized in that a dose of at least 4 million sex-selected sperm processed by steps (a) to (e) has at least the same fertility as a conventional dose of sperm. 15 million unselected sperm by sex. A selected sex insemination dose characterized by being produced by the method defined in any one of claims 12 to 14 with a dose response in dosages greater than 3 million.