RU2775530C2 - Method for sperm treatment, device and related medium compositions - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to a method for sperm treatment. The method for sperm treatment includes: coloring of a sperm sample containing viable sperm carrying X-chromosome and viable sperm carrying Y-chromosome by a coloring medium; bringing of the colored sperm sample into contact with flowing liquid in a flow; and manipulation of a ratio of viable sperm carrying X-chromosome to viable sperm carrying Y-chromosome to form at least one sperm population subjected to manipulation. In this case, one or more of flowing liquid and coloring medium contains cryoprotector in a concentration from 0.1% to 6% vol./vol. or wt./vol.

EFFECT: above-described method allows for an increase in sperm motility after thawing and improvement of its sorting, namely improvement of the efficiency, throughput and rate.

16 cl, 37 dwg, 18 tbl, 17 ex

Description

Technical field

In general, the present disclosure relates to the processing of sperm, and more specifically, to methods, systems, and compositions suitable for manipulating the ratio of viable X-bearing to viable Y-bearing sperm in at least one sperm population. , and suitable for conservation of the resulting manipulated sperm population.

State of the art

The chromosome set of male haploid gametes determines the sex of mammalian offspring. More specifically, fertilization of an oocyte with sperm carrying a Y chromosome results in the birth of male offspring, and fertilization with sperm carrying an X chromosome results in the birth of a female offspring. Although a number of technologies have been explored for the preliminary sex determination of mammalian offspring, only sorting by flow cytometry has gained wide acceptance in the market.

Flow cytometers modified for semen sorting detect relative differences in DNA content between X chromosome-bearing and Y-bearing sperm. To determine the DNA content of semen, the semen population is usually stained stoichiometrically with a DNA-selective fluorescent dye that binds to nuclear DNA. One such DNA selective fluorescent dye, Hoechst 33342, sometimes referred to as Hoechst Bisbenzimide 33342, can be used in sufficient amounts to differentiate small variations in nuclear DNA without exhibiting the toxicity associated with other dyes.

These relative differences between sperm carrying an X chromosome and sperm carrying a Y chromosome are usually small variations. In cattle, for example Holstein bulls, the difference in DNA content is about 3.8%, and in Jersey bulls it is about 4.1%. Due to the unspecified nature of stoichiometric DNA staining, these small differences are difficult to ascertain. Semen samples, even samples within the same breed, can vary greatly in concentration, pH, motility and morphology. Thus, staining conditions that work well in one circumstance may stain other semen samples weaker or stronger, even semen from the same breed or even from the same animal. The shape of the sperm creates additional difficulties in separating X chromosome-bearing cells from Y-bearing cells. In particular, the sperm heads, which contain nuclear DNA, are paddle-shaped in most species. This geometry provides another distorting effect by generating different levels of fluorescence at different angles, and these differences outweigh the detectable differences in X and Y chromosome-bearing sperm. providing a more uniform orientation of the sperm.

Despite these difficulties, Hoechst 33342 can be used at non-toxic concentrations. Unfortunately, the staining method is detrimental to non-regenerating, time-sensitive cells. In particular, uniform staining with Hoechst 33342 requires incubation at elevated temperatures and elevated pH values, and both an increase in the temperature of the semen and an increase in the pH of the semen contribute to damage to the sperm. After staining, the pressure experienced by the sperm in the flow cytometer can cause additional damage to the sperm, and the associated shear forces can cause more damage.

The limited lifespan of sperm often requires freezing for storage and transport. Intracellular fluids, including water, are removed from the cell during this method to reduce the volume of intracellular fluids that freeze. Otherwise, intracellular fluids will crystallize and increase in volume compared to the liquid. This expansion causes intracellular stress and mechanical damage to the sperm and reduces sperm fertility. A number of cryoprotectants may be suitable for this purpose, the most common of which is glycerol. However, even glycerin has a number of disadvantages. For example, glycerol has at least a certain degree of toxicity to sperm, the effect of which may become more pronounced as the amount of glycerol is increased. In addition, glycerol can be hyperosmotic to semen, which can result in some degree of shock to sperm to which glycerol has been added. Such hyperosmotic properties can lead to rapid contraction or expansion of sperm that comes into contact with glycerol as a result of the difference in solute concentration on the other side of the sperm cell membrane. This rapid contraction and expansion can damage the sperm. Such toxicity and damage can have a combined effect, especially for sorted semen that has been damaged by the destructive staining and sorting steps.

However, most often in commercial settings, the benefits of freezing sorted semen generally outweigh the negative effects of the damage caused, and certain techniques have been developed to minimize the adverse effects of glycerol on sperm. In one example, glycerol may be combined with semen at low temperatures to mitigate the toxic effects of glycerol on sperm. For this purpose, fillers or other media containing glycerol can be prepared using a multi-stage process involving two or more filler fractions. In particular, semen fillers may contain an "A" fraction without glycerol and a "B" fraction with glycerol. Fractions "A" and "B" allow the introduction of the semen filler in two or more stages, for example, the first stage, in which fraction A is added to the semen, for example, sex-selected semen, which may be at room temperature, is followed by a second stage, in which semen and fraction A are cooled to a lower temperature, and fraction B containing glycerol is added at this lower temperature. In addition, to reduce the hyperosmotic effect of glycerol on sperm cells, Fraction B can be added in several steps, possibly to reduce shock to sperm cells by exposing the sperm cells to reduced amounts of glycerol at each step of adding glycerol. As described in WO/200137655, for example, filler-added semen can be reconcentrated by centrifugation and suspension in a freezing vehicle, sometimes referred to as "AB" vehicle, which has a glycerol content that is half that of the "B" fraction.

Brief description of the invention

Certain embodiments of the claimed invention are summarized below. These embodiments are not intended to limit the scope of the claimed invention, but instead serve as brief descriptions of possible forms of the invention. The present invention may cover a variety of forms other than these brief descriptions.

One broad embodiment described herein relates to a sperm stain medium composition that includes a buffer, a DNA selective dye; and cryoprotectant. The cryoprotectant may be a sugar alcohol such as ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite or any combination of sugar alcohols. Alternatively, the cryoprotectant may be a glycol such as propylene glycol, butanetriol, or combinations thereof. The cryoprotectant in the staining medium may have a volume/volume (v/v) concentration of from about 0.1% to about 1%; from about 1% to about 2%; from about 2% to about 3%; from about 3% to about 4%; from about 2% to about 4%; or from about 1.5% to about 3%. The buffer can be any of HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)), sodium bicarbonate, MOPS ((3-(N-morpholino)propanesulfonic acid)), TRIS (tris(hydroxymethyl)aminomethane) , TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), TALP (Tyrode solution, albumin, lactate, pyruvate), TCA (trichloroacetic acid), PBS ( phosphate buffered saline) milk, its derivatives or combinations thereof. The DNA selective dye may be a DNA selective fluorescent dye such as Hoechst 33342, which can be supplied at a concentration of about 10 µM to about 200 µM, about 20 µM to about 100 µM, about 30 µM to about 70 µM. The sperm staining medium may comprise an incubated sperm medium composition incubated at a temperature of from about 30°C to about 39°C, from about 32°C to about 37°C, or at a temperature of about 34°C.

Another broad embodiment of the present invention described herein relates to a semen treatment method in which a cryoprotectant is administered at earlier stages than previously thought. A sperm sample containing viable X-bearing sperm and viable Y-bearing sperm is stained with staining medium and brought into contact with sheath fluid in a stream. The staining medium and/or sheath contains a cryoprotectant. The method can be continued by manipulating the ratio of viable sperm carrying a Y chromosome to viable sperm carrying a Y chromosome in a sperm sample to form at least one manipulated sperm population. Some embodiments may further include the step of collecting the manipulated sperm population into a collection medium, sometimes referred to as capture fluid or collection fluid. In some embodiments, each of the staining medium, the sheath liquid, and the collection medium includes an amount of a cryoprotectant. Some embodiments may further include the step of cryopreserving/freezing the manipulated sperm sample. The cryoprotectant may be a polyhydric alcohol, a low molecular weight amide, or methylamide; in one embodiment, the polyhydric alcohol is a sugar alcohol such as ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite; or any combination of sugar alcohols. In another embodiment, the polyhydric alcohol may be a glycol such as propylene glycol, butanetriol, or combinations thereof.

If present in the medium for staining, the cryoprotectant may have about./about. or a weight/volume (w/v) concentration of from about 0.1% to about 1%; from about 1% to about 2%; from about 2% to about 3%; from about 4% to about 5%; from about 2% to about 4%; about 1%, 2%, 3%, 4%, 5%; or from about 1.5% to about 3%.

If present in the sheath fluid, the cryoprotectant may have about./about. or wt./about. a concentration of from about 0.1% to about 6%; from about 0.1% to about 2%; from about 2% to about 4%; from about 4% to about 6%; from about 1% to about 2%; from about 2% to about 3%; about 3%; from about 3% to about 4%; from about 4% to about 5%; from about 5% to about 6%; from about 2% to about 6%; about 1%, 2%, 3%, 4%, 5%; or from about 3% to about 5%. If present in the collection medium, the cryoprotectant may have about./about. or wt./about. a concentration of from about 1% to about 2% cryoprotectant by volume; about 2% to about 4% cryoprotectant by volume; about 4% to about 6% cryoprotectant by volume; about 3% to about 5% cryoprotectant by volume; about 1%, 2%, 3%, 4%, or 5% cryoprotectant by volume; from about 3.5% to about 5.5% cryoprotectant by volume, or a concentration of about 4.5% by volume.

In particular embodiments of the broad method, the cryoprotectant may be added to more than one of staining medium, sheath fluid, collection medium. The cryoprotectant may be present in varying amounts in each of the staining medium, sheath fluid, collection medium. In one embodiment, the concentration of the cryoprotectant is increased, if present, at each successive step in which the cryoprotectant is present. Similarly, in embodiments in which a manipulated semen sample is diluted in a freeze vehicle to effect a freeze, and where a cryoprotectant is added to more than one of stain medium, sheath fluid, collection medium, the cryoprotectant may be present in the freeze vehicle in vol./rev. or wt./about. concentrations less than 6%. In such an embodiment, the implementation of the cryoprotectant may be present in the filler for freezing in v/v. or wt./about. about 1% to about 6%, about 3% to about 5%, about 3.5% to about 5.5%, about 3.5%, about 4% to about 5%, or about 4.5%.

One broad embodiment described herein relates to a semen sample manipulation system in the presence of a cryoprotectant. The system includes a sample source containing a semen sample and a sheath fluid source containing a sheath fluid containing a cryoprotective additive. The fluid delivery structure forms a coaxial flow of the sperm sample from the sample source surrounded by sheath fluid from the sheath fluid source and directs the coaxial flow through the measurement site. An electromagnetic radiation source illuminates the semen at the measurement site, and at least one detector generates signals in response to such illumination. The analyzer determines the characteristics of the sperm based on the signals generated by at least one detector.

In some embodiments of the semen sample handling system, the fluid delivery structure includes a nozzle, such as an orienting nozzle. While in other embodiments, the fluid delivery structure comprises a flow channel such as a cuvette or a microfluidic chip. Some system implementations include the necessary components to form, load, and deflect droplets from the coaxial flow, while in other implementations, a photodestructive (ie, ablative) laser damages selected sperm in the coaxial flow.

The cryoprotectant is present in the sheath fluid if such a system can be a sugar alcohol such as ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite or any combination of sugar alcohols. Alternatively, the cryoprotectant may be a glycol such as propylene glycol, butanetriol, or combinations thereof. The cryoprotectant may be present in the sheath fluid in about./about. or wt./about. concentrations from about 0.1% to about 6%; from about 0.1% to about 2%; from about 2% to about 4%; from about 4% to about 6%; from about 1% to about 2%; from about 2% to about 3%; from about 3% to about 4%; from about 4% to about 5%; from about 5% to about 6%; from about 2% to about 6%; or from about 3% to about 5%.

Yet another broad embodiment described herein relates to a semen processing method in which the cryoprotectant is administered at earlier stages than previously thought. A sperm sample containing viable X-chromosome-bearing sperm and a Y-chromosome-bearing viable sperm is stained with a staining medium containing a DNA selective dye and injected into a sheath. The stained sperm in the sperm sample is then exposed to an electromagnetic radiation source that causes a detectable DNA selective dye response. Then, a detectable DNA selective dye response is detected and the ratio of viable X-chromosome-bearing sperm to Y-chromosome-bearing viable sperm is manipulated to form at least one manipulated sperm population. At least one manipulated semen population is collected in one or more collection vessels containing collection media. In this broad method, one or more of the staining medium, the sheath fluid, and the collection medium comprise a cryoprotectant.

The cryoprotectant may be a sugar alcohol such as ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite or any combination of sugar alcohols. Alternatively, the cryoprotectant may be a glycol such as propylene glycol, butane triol, or combinations thereof.

If present in the medium for staining, the cryoprotectant may have about./about. or wt./about. a concentration of from about 0.1% to about 1%; from about 1% to about 2%; from about 2% to about 3%; from about 4% to about 5%; from about 2% to about 4%; or from about 1.5% to about 3%.

If present in the sheath fluid, the cryoprotectant may have about./about. or wt./about. concentration less than 6%; less than 5%; less than 4%; less than 3%; less than 2%; less than 1%; from about 0.1% to about 6%; from about 0.1% to about 2%; from about 2% to about 4%; from about 4% to about 6%; from about 1% to about 2%; from about 2% to about 3%; from about 3% to about 4%; from about 4% to about 5%; from about 5% to about 6%; from about 2% to about 6%; or from about 3% to about 5%.

Similarly, if present in the collection medium, the cryoprotectant may have about./about. or wt./about. a concentration of from about 1% to about 2%; from about 2% to about 4%; from about 4% to about 6%; from about 3% to about 5%; from about 3.5% to about 5.5%; or a concentration of about 4.5.

The cryoprotectant may be present in varying amounts in each of the staining medium, sheath fluid, collection medium. In one embodiment, the concentration of the cryoprotectant is increased, if present, at each successive step in which the cryoprotectant is present.

In embodiments in which a manipulated sperm sample is diluted in a freeze vehicle to effect a freeze, and where a cryoprotectant is added to more than one of stain medium, sheath fluid, collection medium, the cryoprotectant may be present in the freeze vehicle in vol./ about. or wt./about. concentrations less than 6%. In such an embodiment, the cryoprotectant may be present at a concentration of 1% to 6%, 3.5% to 5.5%, 4% to 5%, or about 4.5%.

Another broad embodiment described herein relates to a method for freezing/cryopreserving a manipulated sperm population harvested in a mixture that includes a cryoprotectant. One such implementation includes bringing the stained sperm sample into contact with sheath fluid in a stream; manipulating the ratio of viable X-bearing sperm to viable Y-bearing sperm to form at least one manipulated sperm population; collecting a manipulated semen sample in the presence of a cryoprotectant; concentrating the collected semen sample; resuspension of the concentrated semen sample in the freezer; and freezing the resuspended sperm sample. The method may further include the step of cooling the sperm for a period of less than 90 minutes. In addition, the step of collecting the mixture further comprises: collecting the mixture into a container that contains from about 5 ml to about 50 ml of collection medium. In some embodiments, the step of collecting the mixture further comprises collecting a sorted sperm sample from the flow cytometer until the container is filled to about 60% to about 90% of the container capacity. In further embodiments, the step of concentrating the collected mixture is carried out in a vessel. In yet another further embodiment, the step of concentrating the collected semen sample directly follows the step of collecting the manipulated semen sample, in the presence of a cryoprotectant, without any further dilution occurring between said steps. The freeze filler in this embodiment may be less than 6% v/v. or wt./about. cryoprotectant; from 3.5 to 5.5% v/v or wt./about. cryoprotectant; or from 4 to 5% vol./about. or wt./about. cryoprotectant. In a further embodiment, the step of manipulating the ratio of viable X-bearing sperm to viable Y-bearing sperm to form at least one manipulated sperm population may include either forming droplets that are expected to contain the selected sperm, either diverting these droplets to collect or photodamaging isolated semen in the stream.

An additional aspect of the present invention includes the use of semen processed using any of the methods described herein to fertilize an oocyte. Such fertilization can be achieved by bringing the treated sperm into contact with the oocyte. This embodiment of the present invention encompasses the use of any in vitro fertilization (IVF) methods known in the art, or any artificial insemination methods known in the art, to achieve fertilization.

With respect to any and all embodiments of the present invention disclosed herein, including the aforementioned embodiments, sheath liquid, staining medium, or collection medium may contain any of the aforementioned cryoprotectants in v/v. or wt./about. concentrations of less than 8%; less than 7%; 6%; less than 6%; less than 5%; 4.5%; less than 4%; less than 3%; less than 2%; or less than 1%.

In addition, with respect to any and all embodiments of the present invention disclosed herein, including the aforementioned embodiments, the sheath fluid, stain medium, or collection medium may contain any of the aforementioned cryoprotectants at any of the following concentrations: 10 mM to 1000 mM ; from 10 mM to 500 mM; from 10 mM to 300 mM; from 10 mM to 200 mM; from 10 mM to 150 mM; less than 1000 mm; less than 900 mm; less than 800 mm; less than 700 mm; less than 600 mm; less than 500 mm; less than 400 mm; less than 300 mm; less than 200 mm; less than 150 mm; less than 100 mm; less than 50 mm; or less than 25 mM.

In a further embodiment of the present invention, the cryoprotectant for use in any of the above embodiments contained in the stain medium, sheath liquid, capture liquid, or freezing medium may contain erythritol. In a further embodiment, the erythritol is at a concentration of from about 10 to 300 mM, from about 15 to 250 mM, from about 25 to 125 mM, from about 40 to 80 mM, from about 0.01 to 1000 mM, from about 0.01 up to 400 mM, about 35 mM, about 65 mM, about 135 mM, about 270 mM, about 400 mM, 400 to 1000 mM, 400 to 500 mM, or 400 to 600 mM.

Brief description of the drawings

Fig. 1 illustrates a schematic of a flow cytometer for manipulating a sperm sample, in accordance with some of the embodiments described herein.

Fig. 2 illustrates a graphical representation of the parameters obtained in a flow cytometer while manipulating a sperm sample in accordance with the various embodiments described herein.

Fig. 3 illustrates a graphical representation of the parameters obtained in a flow cytometer when manipulating a sperm sample in accordance with the various embodiments described herein.

Fig. 4 illustrates a graphical representation of sorting parameters obtained in a flow cytometer when semen is sorted according to the various embodiments described herein.

Fig. 5-7 illustrate alternative applications of the cryoprotectant at various stages of semen sample processing.

Fig. 8 illustrates an implementation involving freezing of a sperm sample.

In FIG. 9 shows the ratios of the maximum signal to the minimum obtained using sheath fluid containing various amounts of glycerol.

In FIG. 10 shows the motility of semen after thawing, sorted using different amounts of glycerol in the sheath.

In FIG. 11 shows viability after thawing and percentage of intact acrosomes (PIA) of semen sorted using various amounts of glycerol in the sheath.

In FIG. 12 shows maximum to minimum signal ratios (PVR) obtained using sheath fluid containing various amounts of glycerol.

In FIG. 13 shows thaw motility, viability and PIA of semen sorted using various amounts of glycerol in the sheath.

In FIG. 14 shows the convergence of post-thaw motility, viability, and PIA of semen sorted using different amounts of glycerol in the sheath.

In FIG. 15 shows thaw motility, viability and PIA of semen sorted using various amounts of glycerol in the sheath.

In FIG. 16 shows the convergence of post-thaw motility, viability and PIA of semen sorted using different amounts of glycerol in the sheath.

In FIG. 17 shows post-thaw motility, viability and PIA of semen sorted using various glycols in sheath fluid.

In FIG. 18 shows the convergence of post-thaw motility, viability and PIA of semen sorted using different glycols in sheath fluid.

In FIG. 19 shows the motility after thawing of semen sorted using different amounts of glycerol in the sheath.

In FIG. 20 shows viability after thawing and PIA of semen sorted using various amounts of glycerol in the sheath.

In FIG. 21 shows the motility after thawing of semen sorted using various glycols in sheath fluid.

In FIG. 22 shows viability after thawing and PIA of semen sorted using various glycols in sheath fluid.

In FIG. 23 shows motility at 0 hours after thawing for semen sorted using 3% glycerol in sheath fluid and cryopreserved in media having various concentrations of glycerol.

In FIG. 24 shows viability and PIA at 0 hours after thawing for semen sorted using 3% glycerol in sheath fluid and cryopreserved in media having various concentrations of glycerol.

In FIG. 25 shows motility 3 hours after thawing for semen sorted using 3% glycerol in sheath fluid and cryopreserved in media having various concentrations of glycerol.

In FIG. 26 shows viability and PIA 3 hours after thawing for semen sorted using 3% glycerol in sheath fluid and cryopreserved in media having various concentrations of glycerol.

In FIG. 27 shows the convergence of motility after thawing for semen sorted using 3% glycerol in sheath fluid and cryopreserved in media having various concentrations of glycerol.

In FIG. 28 shows the convergence of thaw viability and PIA for semen sorted using 3% glycerol in sheath fluid and cryopreserved in media having various concentrations of glycerol.

In FIG. 29 shows 0 hour post-thaw motility, viability, and PIA for semen sorted using various amounts of glycerol in sheath fluid and cryopreserved in media having various concentrations of glycerol.

In FIG. 30 shows motility 3 hours after thawing, viability and PIA for semen sorted using various amounts of glycerol in sheath fluid and cryopreserved in media having various concentrations of glycerol.

In FIG. 31 shows the convergence of post-thaw motility, viability, and PIA for semen sorted using different amounts of glycerol in sheath fluid and cryopreserved in media having different concentrations of glycerol.

In FIG. 32 shows the results of IVF using semen sorted with either glycerol in the sheath fluid or no sheath fluid.

In FIG. 33 shows post-thaw motility, PIA, convergence, and viability of semen sorted with either glycerol in the sheath fluid or no sheath fluid.

In FIG. 34 shows conception rates for semen sorted with either glycerol in the sheath fluid or none in the sheath fluid.

In FIG. 35 shows post-thaw motility, viability and PIA for sorted semen frozen with cryopreserved AB or 100% v/v AB. glycerin.

In FIG. 36 shows post-thaw motility, viability, and PIA for semen sorted with or without glycerol in the capture fluid and sheath fluid or none in the fluid and sheath fluid.

In FIG. 37 shows the motility of sperm in Caprogen with glycerol and in Caprogen with various concentrations of erythritol.

Methods for implementing the invention

Sperm can be obtained or donated by obtaining a semen sample or semen solution that contains sperm. As is commonly used, the term "sperm" refers to the singular or plural of a male reproductive cell, while "sperm sample" refers to a carrier fluid in addition to the sperm it contains. Examples of semen samples include pure seminal fluid or semen diluted in another solution such as a diluent or buffer, and include frozen thawed semen. The semen sample may be obtained at the same location as the rest of the steps, or may be diluted in a suitable semen diluent for transport to the sorting facility. Once obtained, semen can be kept at room temperature, refrigerated or even frozen in a suitable diluent for later use. Sperm for staining and sorting may be obtained from a mammal or may be stored semen, such as a frozen or chilled storage tube. Alternatively, frozen or diluted semen may be pooled.

The sperm sample may be from a mammal, such as the non-human mammals listed by Wilson, D.E. and Reeder, D.M., Mammal Species of the World. Smithsonian Institution Press, (1993), the contents of which are incorporated herein by reference in their entirety. During collection, thawing or even pooling, the concentration, pH, motility and/or morphology of the semen can be checked. Additionally, antibiotics may be added prior to further processing steps.

Upon receipt, the sperm may optionally be standardized to a predetermined concentration and/or to a predetermined pH. As used herein, the term "standardization" can be understood as the action performed in order to bring various characteristics of the ejaculate into a predetermined range or approach a specified predetermined range. Although bovine ejaculate, for example, can vary greatly in pH and semen concentration, the step of standardizing semen concentration or pH may include the addition of a high capacity buffer that serves to standardize the pH and a buffer against the tendency of ejaculates to acidify over time.

Each of the predetermined concentration and pH may be specific to a different species, or even to a different breed of animal within a species. In one embodiment, the sperm can be combined with the original diluent in the form of a high capacity buffer or with a diluent having a high buffering pH. Suitable diluents may include TRIS citrate, sodium citrate, sodium bicarbonate, HEPES, TRIS, TEST, MOPS, KMT, TALP, derivatives thereof, and combinations thereof. Any diluent containing a high pH buffering capacity can also be used and can be used in combination with additional components that promote sperm viability. As an example of a supplement, the protein may be included in the form of egg yolk, milk, lipoproteins, lecithin, casein or albumin, or other protein sources. The energy source may also be included in the form of a monosaccharide such as fructose, glucose or mannose, or even a disaccharide or trisaccharide. In addition, antioxidants and antibiotics can be used in the original extender to improve the viability of the sperm.

The initial diluent may be set to a predetermined pH to standardize the pH of all semen samples obtained, for example a pH of about 6.8 to 7.4. In one embodiment, the diluent is adjusted to pH 7.2. In addition, seminal fluid can become progressively more acidic over time, possibly due to the presence of proteins in the seminal fluid or due to the presence of acidic metabolic products or by-products of dead or dying cells. The initial diluent introduces enough free proton binding sites (ie, H ⁺ ) to keep the pH close to the target value. Even in light of the natural tendency for semen to become more acidic over time, the original diluent provides a means of stabilizing the pH in all additional processing steps.

The initial filler may contain additives for the purpose of maintaining healthy sperm. The initial fill may include antibiotics to prevent bacterial growth. As non-limiting examples, tylosin, gentamicin, lincomycin, lincospectin, spectinomycin, penicillin, streptomycin, and combinations thereof may be included in the original vehicle.

Antioxidants may also be included in the original filler to reduce free radicals and oxidative stress. Although the present discussion relates to the use of antioxidants in the initial diluent, it should be understood that antioxidants can be included in several steps of the semen sorting process, independently or in combination, as described in international patent application WO2012167151, the entire content of which is incorporated herein by reference. A non-limiting list of antioxidants that may be included include: catalase, SOD, SOD mimic, glutathione, glutathione reductase, glutathione peroxidase, pyruvate, caproic acid, mercaptoethanol, BHT, lipoic acid, flavins, quinines, vitamin K (and other vitamers), vitamin B12 , vitamin B12 vitamers, vitamin E (and related vitamers), tocopherols, tocotrienols, a-tocopheryl, alpha-ketoglutarate (AKG), malondialdehyde (MDA), asymmetric dimethylarginine (ADMA) and their biologically active derivatives and combinations thereof.

The concentration of antioxidants can range from 0.01 mg/ml to 0.5 mg/ml, and as non-limiting examples, the above listed antioxidants can be offered at a concentration of 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; 1 mg/ml to 2.5 mg/ml; and from 1 mg/ml to 5 mg/ml.

As one example, a semen sample can be diluted in high capacity buffer in ratios of 1:1 to 1:10. The resulting mixture will have a sperm concentration many times lower than natural sperm concentrations for a given species. The diluted semen can be centrifuged to re-concentrate the semen. Centrifuging the semen and removing the supernatant allows the semen to be re-concentrated to a predetermined concentration. The predetermined concentration may be selected based on additional processing steps for the semen. For example, in the case of sex sorting, bovine semen can be reconcentrated from about 2400 million semen per ml to about 500 million semen per ml to mimic the natural range of concentrations when the seminal plasma components are replaced with diluent. Other concentrations, such as from about 1400 million sperm per ml to about 2100 million sperm per ml, or from about 1700 million sperm per ml to about 2100 million sperm per ml, can also be achieved for further processing.

In one embodiment, the semen concentration and pH may provide a single starting point for further processing. For example, relatively constant pH and concentration can provide greater predictability when staining sperm, for example with a DNA selective stain. If each sample is adjusted to the same predetermined pH and concentration, each new selection may require fewer tests to ensure proper staining for sex sorting.

The sperm population will include both sperm carrying the X chromosome and sperm carrying the Y chromosome. In addition, X-chromosome-carrying sperm and Y-chromosome-carrying sperm will each include viable and non-viable sperm. Viable sperm can be considered sperm with intact membranes, while non-viable sperm can be considered sperm with damaged membranes. The difference between viable and non-viable sperm in conventional semen sorting is determined by the inclusion of a quenching dye that penetrates the membrane-damaged semen. Sperm that tend to die or die will take up the quenching dye and generate different fluorescent signals from the rest of the sperm population, while sperm that have intact membranes tend to be viable and prevent uptake of the quenching dye. Viable sperm at the appropriate dosage will generally be able to achieve insemination by artificial insemination, while non-viable sperm or sperm with a damaged membrane may be unable to achieve insemination by artificial insemination or will have a significantly reduced ability to do so. However, some sperm that are capable of fertilization may have damaged membranes, and some sperm with intact membranes may be unable to fertilize.

Whether standardized or not, sperm can be stained with staining medium to introduce a DNA selective dye. In the staining step, at least a portion of the sperm population is incubated with staining medium and a DNA selective fluorescent dye to stoichiometrically stain the DNA content of each cell in the sperm population. Hoechst 33342 tends to be less toxic than other DNA selective stains. The dye delivery vehicle may be in the form of a modified TALP buffer adjusted to a pH of about 7.4. Hoechst 33342 is described in US Pat. No. 5,135,759 and is commonly used for this purpose. However, other UV exciter dyes as well as visible light exciters, fluorescent polyamides, fluorescent nucleotide sequences and sex-specific antibodies can also be used.

Sperm in its natural state is often not easily permeable to such dyes. To obtain uniform staining, the first staining step may include incubating at least a portion of the sperm population at elevated temperature in a staining medium (sometimes referred to herein as "staining buffer") at elevated pH in addition to the stain. Examples of suitable staining solutions may be TALP, TES-TRIS, TRIS citrate, sodium citrate, or HEPES-based media, each of which is described in WO 2005/095960, which is incorporated herein by reference. A non-limiting example of the modified TALP described in WO 2001/37655, which is incorporated herein by reference, is illustrated in Table 1.

Table 1. Ingredient Concentration of Modified TALP Buffer

Ingredient Concentration NaCl KC1 NaHPO ₄ NaHCO ₃ MgCL ₂ 6H ₂ O 0.4mm Pyruvate Na Glucose Na lactate HEPES Bovine serum albumin 3.0 mg/ml

As one example, a sperm population or a portion of a sperm population can be diluted with staining medium from 640 x 10 ⁶ to 40 x 10 ⁶ sperm/ml to about 320 x 10 ⁶ to 80 x 10 ⁶ sperm/ml, or up to about 160 x 10 ⁶ sperm/ml in buffer. The DNA selective fluorescent dye can be added to buffered semen at a concentration of about 10 μM to 200 μM; from about 20 μM to 100 μM, or from about 30 μM to 70 μM. The pH of the buffer may be from about 6.8 to 7.9; from about 7.1 to 7.6; or about 7.4 to help ensure uniform nuclear DNA staining. Those skilled in the art will appreciate that the pH can be raised with the addition of NaOH and lowered with the addition of HCl. Optionally, previously colored antioxidants and concentrations may be included in the staining solution.

The sperm population can be incubated at 30 to 39°C, about 32 to 37°C, or about 34°C. The incubation period may vary from about 20 minutes to about three hours, from about 30 minutes to about 90 minutes, or from about 45 minutes to about 60 minutes. As one example, the sperm population can be incubated for about 45 minutes at 34°C. Even within the same species, sperm concentration and pH and other factors that affect staining can vary from animal to animal. Those skilled in the art can appreciate subtle variations in sperm incubation between species and even between breeds or animals of the same breed to achieve uniform staining without over-staining the sperm population.

In addition to the DNA selective fluorescent dye, a quenching dye can be used to penetrate membrane-damaged sperm and quench the signals it generates. It can be understood that the quenching dye includes dyes that otherwise bind to membrane-damaged sperm. It is possible that these dyes can more easily penetrate semen with damaged membranes, as the membranes break down or become more and more porous. It is also possible that quenching dyes easily penetrate all sperm membranes, and that healthy sperm actively expels quenching dyes faster than membrane-damaged sperm. In any case, the semen to which the quenching dyes bind includes most of the dead and dying sperm, although not necessarily all of the dead and dying sperm. The quenching signals produced by membrane-damaged sperm associated with the quenching dye are sufficiently different from those of healthy sperm that they can be removed from further analysis and sorting applied to viable sperm.

In one embodiment, a second staining step is preliminarily performed, in which the sperm concentration is further reduced and a quenching dye is added. The pH of the second staining medium can be targeted to achieve the target pH in the final semen sample. Non-limiting examples of two-step staining methods are described in PCT Published International Application WO 2011/123166 and International Application PCT/US12/58008, both of which are hereby incorporated by reference in their entirety.

In another embodiment, the quenching dye and the DNA selective dye are applied together in the same dilution. In this embodiment, the quenching dye is incubated with the DNA selective dye at elevated temperature in the staining solution. As an example, the staining medium may be modified TALP pH 7.4. However, other dyes can be used, including TES-TRIS, TRIS citrate, sodium citrate, or a HEPES-based medium containing a DNA selective dye and a quenching dye, and the pH can vary from about 7.0 to 7.8. In one embodiment, synergy may exist when semen is standardized at an elevated pH of about 7.2 prior to staining at pH 7.4. In this way, the pH that affects the sperm remains in a constant range with minimal changes. Since the staining medium and initial diluent have a high buffering capacity, it is believed that the natural tendency of sperm to become acidic over time can be avoided.

In one embodiment, whether using a one-step or two-step staining protocol, the cryoprotectant may be included in the staining step or steps. As used herein, the term "cryoprotectant" should be understood to refer to a substance that protects cells or biological tissue from freezing damage. Cryoprotectants may include those substances that act to remove intracellular water to prevent damage associated with the formation and expansion of intracellular ice. This action can be caused by an increase in the concentration of the solute in the cells. Substances that protect cells and tissues from other types of damage, such as osmotic shock and cooling, but not freezing, are not considered cryoprotectants. As a few examples, sources of lipoproteins, phospholipids, lecithin, and the like, such as egg yolk, egg yolk extract, milk, milk extract, casein, albumin, lecithin, and cholesterol, are not cryoprotectants as the term is used herein. In addition, energy sources such as monosaccharides, disaccharides and trisaccharides are not cryoprotectants used herein.

Cryoprotectants that may be included in the staining step include a range of sugar alcohols and glycols. As non-limiting examples, sugar alcohols such as ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite, and combinations thereof, may be included as a cryoprotectant introduced during staining. US Pat. No. 3,185,623, the entire contents of which are incorporated herein by reference, describes a number of carbohydrate alcohols (ie, sugar alcohols or alditols) that may be suitable cryoprotectants useful in the present invention. In addition, glycols such as propylene glycol, butanetriol may be included as a cryoprotectant. Many such cryoprotectants are toxic to sperm at certain concentrations and temperatures. Glycerin, for example, is commonly added to semen after the semen has been cooled to 5°C, especially because of this toxicity. Surprisingly, as described in more detail in the examples, it has been shown that the presence of a cryoprotectant during the staining step improves the motility after thawing of subsequently frozen semen and improves semen sorting. This is a particularly surprising and unexpected result, given the fact that semen staining for sex sorting is usually carried out at temperatures above 34°C.

The cryoprotectant can be provided during the staining step, for example, in a staining medium, v/v. or wt./about. concentrations from about 0.1% to about 5%. For example, a stained semen sample for further processing, such as sorting in a flow cytometer, may contain a v/v cryoprotectant. or wt./about. concentrations from about 0.1% to about 1%; from about 1% to about 2%; from about 2% to about 3%; from about 3% to about 4%; from about 2% to about 4%; or from about 1.5% to about 3%.

The dye may be supplemented with an antioxidant in the previously described concentration ranges. In some embodiments, increased blood pressure may increase the amount of free radicals and oxidative stress experienced when semen is stained. Accordingly, antioxidants can serve to neutralize free radicals and reduce oxidative stress experienced by stained sperm. A non-limiting list of antioxidants that can be included in the staining method include: catalase, SOD, SOD mimic, glutathione, glutathione reductase, glutathione peroxidase, pyruvate, caproic acid, mercaptoethanol, BHT, lipoic acid, flavins, quinines, vitamin K (and other vitamers) , vitamin B12, vitamin B12 vitamers, vitamin E (and related vitamers), tocopherols, tocotrienols, a-tocopheryl, alpha-ketoglutarate (AKG), malondialdehyde (MDA), asymmetric dimethylarginine (ADMA) and their biologically active derivatives and combinations thereof . Any of the previously described concentrations may be used.

The staining step or steps may optionally include components to reduce sperm motility or respiration during staining. As a few examples, the staining step or steps can reduce sperm respiration by the inclusion of additives or by reducing and/or reducing the impact of certain substances on the sperm. In particular, a low sugar color can be used in which only a residual level of fructose, sucrose, glucose or other sugar is present. Similarly, sperm respiration can be reduced by staining with partial pressure of nitrogen or carbon dioxide to displace the oxygen in the dye. As an alternative, substances can be added to the dye that have specific ratios of potassium to sodium that tend to reduce sperm respiration. As another alternative, fluoride or sodium fluoride may be added during the staining step or steps to reduce respiration of sperm.

Whether standardized or not, and stained at one stage or two stages, the sperm population can be sorted using a particle sorting instrument such as a flow cytometer, including, but not limited to, an air jet cytometer, a flow cytometer with cuvette or microfluidic chip. With reference to FIG. 1, a jet-in-air flow cytometer (10) is illustrated, although sorting can be done with microfluidic chips or other types of flow cytometers, including closed chamber flow cytometers and cytometers containing ablative lasers. The flow cytometer (10) includes a cell source (12) to create a sperm sample stream, such as a stained sperm sample stream, for sorting. The rate at which the semen sample enters the nozzle (14) can be taken from the sample flow rate and can be determined by the sample pressure. The flow of the colored sperm sample is deposited in the nozzle (14) and introduced or poured into the liquid flow (16) of the sheath fluid (18). The sheath fluid (18) can be supplied by a sheath fluid source (20), so the cell source (12) supplies sperm to the sheath fluid (18), they simultaneously enter through the nozzle (14). The sheath fluid (18) can be supplied at a flow rate that is determined by the pressure of the sheath fluid.

Whereas prior art sheath fluid used in flow cytometry typically contained phosphate buffered saline (PBS), possibly supplemented with bovine serum albumin (BSA), semen sorting used sodium citrate, TRIS citrate, or citric acid as sheath fluid to maintaining the health of the sperm during the sorting process. As described in International Patent Application WO 99/33956, the entire content of which is incorporated herein by reference, suitable buffers may be included as sheath fluids. In certain embodiments of the present invention, the prior art sheath fluid is supplemented with a cryoprotectant. As described above, cryoprotectants refer to substances that protect cells or biological tissue from freezing, and may include those substances that act to remove intracellular water to prevent damage associated with the formation and expansion of intracellular ice. This action can be caused by an increase in the concentration of the solute in the cells. Substances that protect cells and tissues from other types of damage, such as osmotic shock and cooling, but not freezing, are not considered cryoprotectants.

Cryoprotectants include the range of sugar alcohols and glycols described above. As non-limiting examples, sugar alcohols such as ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite and combinations thereof may be included in the sheath fluid as a cryoprotectant. In addition, glycols such as propylene glycol, butanetriol can be included in the sheath fluid as a cryoprotectant. Surprisingly, as described in more detail in the examples, the presence of a cryoprotectant in the sheath fluid has been shown to improve motility after thawing of subsequently frozen semen and improve semen sorting. Surprisingly, as seen in Example 1, modifying the sorting tool by incorporating a cryoprotectant into the sheath fluid, in accordance with some embodiments of the present invention, improved sorting resolution, meaning that throughput, throughput, and purity can all be improved to varying degrees at the same time.

In some embodiments, the same cryoprotectant can be used both in the staining step and in the sheath. As a non-limiting example of such an implementation option, glycerol can be offered in the first v/v. or wt./about. concentration at the stage of staining and in the second vol./about. or wt./about. concentration in the flowing fluid. In one embodiment, the implementation of the first about./about. or wt./about. the concentration provided during staining may be lower than the second vol./about. or wt./about. concentration provided in the sheath fluid. Thus, the sperm cells undergoing the semen sorting method can be subjected to a gradual increase in the relative concentration of the sheath fluid. Alternatively, the amount of cryoprotectant present in the staining medium and in the sheath can be coordinated such that during any method of handling the semen sample, a predetermined concentration of cryoprotectant is reached at the end of the manipulation. In alternative embodiments, the cryoprotectant may be included in only one of the sheath fluid and staining medium.

In some embodiments, the cryoprotectant in the sheath fluid may be provided at concentrations from about 0.1% v/v. or wt./about. up to about 6% vol./about. or wt./about. For example, a stained semen solution for further processing, such as sorting in a flow cytometer, may contain a v/v cryoprotectant. or wt./about. concentrations from about 0.1% to about 2%; from about 2% to about 4%; from about 4% to about 6%; from about 1% to about 2%; from about 2% to about 3%; from about 3% to about 4%; from about 4% to about 5%; from about 5% to about 6%; from about 2% to about 6%; or from about 3% to about 5%.

Those skilled in the art may appreciate that the described embodiment relates to a jet-in-air droplet-forming flow cytometer, but that a flow fluid containing a cryoprotectant additive provides the same benefits in other sorting systems, such as systems that employ shifting liquids, systems using photodamaging lasers, and in microfluidic chips.

During the semen sorting operation, the sheath fluid (18) forms a fluid stream coaxially surrounding the semen sample containing colored semen that exits the nozzle (14) into the orifice of the nozzle (22). Due to the presence of an oscillator (24), which can be precisely controlled from the oscillator controller (26), it is possible to create pressure waves inside the nozzle (14) and transmit them to the liquids exiting the nozzle (14) through the nozzle opening (22). In response to the pressure waves, the fluid flow (16) exiting the orifice of the nozzle (22) eventually forms even droplets (28) at precise intervals. The frequency and to some extent the shape of the droplets formed can be controlled by the droplet movement frequency and the droplet movement amplitude applied to the oscillator (24) or the oscillator controller (26).

Each drop thus formed retains the sheath fluid and the semen sample that previously formed part of the fluid stream (16). Since the colored sperm is surrounded by a fluid stream (16) or a flowing liquid medium, the droplets (28) ideally contain individually isolated sperm. However, sample concentration, sample pressure, and other instrument parameters determine the frequency with which multiple cells are regularly found in a single drop, as well as the percentage of droplets containing sperm.

The flow cytometer (10) sorts the droplets based on the characteristics of the semen predicted to be in the droplets. This can be achieved with a cell sensing system (30) connected to an analyzer (36). Cell sensing system (30) includes at least one sensor (32) responsive to cells contained in fluid flow (16). As one example, two orthogonal photomultiplier tubes (PMT) can be built into a sperm sorting flow cytometer to detect fluorescence at 0 and 90 degrees, although other sensor configurations such as those described in WO2010/021627, which is incorporated herein, can easily be used. application by reference.

The cell sensing system (30) transmits data to the analyzer (36) which can trigger an action depending on the relative presence or relative absence of cell characteristics in the fluid stream (16). Certain characteristics, such as the relative DNA content of the semen, can be detected by excitation with an electromagnetic radiation source (34), such as a laser, which generates a beam of radiation to which the colored semen responds. The source (34) of electromagnetic radiation may be a laser operating at a wavelength of UV radiation, for example at a wavelength of about 355 nm. An example of such a laser would be the Vanguard 350 (available from Spectra-Physics) which operates at 350 mW. Various optics can be used to profile the laser beam, split the beam into more than one stream, or reduce the beam power in a stream. Non-limiting examples of such optics can be found in WO/2004/104178 and WO/2001/85913, each of which is incorporated herein by reference.

Characteristics of an individual sperm, in particular the presence of an X chromosome or a Y chromosome, can be determined by detectable fluorescence generated in response to an electromagnetic radiation source (34). In particular, configurations of the cell sensing system (30) may be coupled to the analyzer (36) to provide a variety of fluorescence in the formation, such as forward event fluorescence, event side fluorescence, or event-related scatter. The analyzer (36) may include written instructions for analyzing the signals generated by one or more sensors (32) in the cell sensing system (30). The DNA-selective fluorescent dye binds stoichiometrically to sperm DNA. Because X-bearing sperm contains more DNA than Y-bearing sperm, X-bearing sperm can bind more DNA-selective fluorescent dye than Y-bearing sperm. Thus, by measuring the fluorescence emitted by the associated dye upon excitation, it is possible to distinguish between X-bearing and Y-bearing spermatozoa. sperm according to sorting logic including gated areas.

Once the analyzer differentiates the sperm based on the characteristics of the sperm, the droplets capturing sperm carrying the X chromosome can be positively charged and thus deflected in one direction, while the droplets capturing sperm carrying the Y chromosome can be negatively charged. and thus be deflected in the other direction, and the empty stream (i.e., droplets that do not entrain a particle or cell or entrain unwanted or unsortable cells) can be left uncharged and thus collected from the undiverted stream into a suction tube or the like. . Alternatively, one of the X-chromosome-carrying sperm or the Y-chromosome-carrying sperm can be collected and the other discarded with an empty stream.

As a result, the flow cytometer (10) acts to separate the stained semen by causing the drops (28) containing the semen to be directed to one or more containers (40) for collection. The collection containers (40) may be collection tubes such as centrifuge tubes or Falcon tubes. In one embodiment, one or more collection vials contain a 50 ml centrifuge tube. The one or more collection vessels may include a collection medium that simultaneously cushions cells in deflected droplets from hitting the bottom of the vessel and includes components to maintain the health of sorted sperm cells. As an example, a capture fluid containing TRIS-citrate and egg yolk can be used with some embodiments of the present invention. Other capture fluids that may be used in certain embodiments include TRIS citrate, sodium citrate, sodium bicarbonate, HEPES, TRIS, TEST, MOPS, KMT, TALP, derivatives thereof, and combinations thereof. However, other diluents containing pH buffering buffer can also be used and can be used in combination with additional components that promote sperm viability after the sorter. As an example of an additive, the protein is usually included in the capture liquid in the form of an egg yolk. However, other forms of protein such as milk, lipoproteins, lecithin, casein or albumin or other protein sources can be used as an alternative or even used in combination with egg yolk. In one embodiment, an egg yolk substitute such as a phospholipid available from soy lecithin can be used in place of egg yolk. The energy source may also be included in the form of a monosaccharide such as fructose, glucose or mannose, or even a disaccharide or trisaccharide. In addition, antioxidants and antibiotics can be used in the original extender to improve the viability of the sperm.

In one embodiment, the collection medium includes a cryoprotectant. Cryoprotectants suitable for addition to the collection medium include the range of sugar alcohols and glycols described above. As non-limiting examples, sugar alcohols such as ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite and combinations thereof may be included in the capture fluid as a cryoprotectant. In addition, glycols such as propylene glycol, butanetriol can be included in the capture fluid as a cryoprotectant.

In some embodiments, the collection of the manipulated semen sample in the collection medium may be the first time the sperm is brought into contact with the cryoprotectant. In some embodiments, the staining medium and/or the sheath fluid includes a cryoprotectant in addition to the collection medium. In other embodiments, the staining medium and/or sheath contains a cryoprotectant while the collection medium does not. In each of these embodiments, semen collected in the collection medium is collected in a mixture that includes both semen and a cryoprotectant, due to the presence of the cryoprotectant in the collection medium, or alternatively due to any amounts of cryoprotectant present in the collection medium. stain or sheath that is collected from manipulated or harvested semen.

In embodiments where the cryoprotectant is present in the collection medium and at least one other fluid, the same cryoprotectant may be used in the collection medium as in the staining medium and/or sheath fluid. As a non-limiting example of such an implementation option, glycerol can be offered in the first v/v. or wt./about. concentration at the stage of staining and in the second vol./about. or wt./about. concentration in the flowing fluid. In one embodiment, the implementation of the first about./about. or wt./about. the concentration provided during staining may be lower than the second vol./about. or wt./about. concentration provided in the sheath fluid. Thus, sperm cells subjected to the semen sorting method may be subject to a gradual increase in the relative concentration of the sheath fluid. Alternatively, the amount of cryoprotectant present in the collection medium can be coordinated with the amount of cryoprotectant present in the staining medium and/or in the sheath so that during the method of manipulating the semen sample, a predetermined concentration of cryoprotectant is reached at the end of this manipulation. In alternative embodiments, the cryoprotectant may be included in only one of the sheath fluid, stain medium, and collection medium.

In some embodiments, the cryoprotectant may be provided in a v/v collection medium. or wt./about. concentrations from about 1% to about 2%; from about 2% to about 4%; from about 4% to about 6%; from about 3% to about 5%; from about 3.5% to about 5.5%; or about 4.5%.

In one embodiment, the cryoprotectant is added to the capture fluid, but not to the sheath fluid or staining step(s). In another embodiment, the cryoprotectant is added to any two of the capture fluid, sheath fluid, and staining step(s). In yet another embodiment, a cryoprotectant is added to each of the capture fluid, sheath fluid, and dye. As a non-limiting example of an implementation where the cryoprotectant is added in more than one processing step, glycerin can be added in the first v/v. or wt./about. concentration at the stage of staining and in the second vol./about. or wt./about. concentration in the flowing fluid. In one embodiment, the implementation of the first about./about. or wt./about. the concentration provided during staining may be lower than the second vol./about. or wt./about. concentration provided in the sheath fluid. Thus, the sperm cells undergoing the semen sorting method can be subjected to a gradual increase in the relative concentration of the sheath fluid. As a non-limiting example of an embodiment in which a cryoprotectant is added to each of the capture fluid, sheath fluid, and staining step(s), glycerol can be added in the first v/v. or wt./about. concentration at the staining stage, the second vol./about. or wt./about. concentrations in the sheath fluid and in the third vol./about. or wt./about. concentration in the capture liquid. In one embodiment, about./about. or wt./about. the concentration of the cryoprotectant may be increased at each successive stage at which it is administered. For example, the first concentration of cryoprotectant in the staining step may be lower than the second concentration of cryoprotectant in the sheath fluid and the third concentration of cryoprotectant in the capture fluid. The gradual introduction of a cryoprotectant in particular may have a positive effect on the ability of sperm to ultimately survive possible freezing and thawing, or may improve the health of viable sperm that is ultimately frozen and thawed.

In an alternative implementation, the deflection plates (38) and other components required for droplet formation, such as the oscillator (24) or oscillator controller (26), can be replaced with a photodamage laser, sometimes also referred to as laser ablation. In this embodiment, the analyzer (36) and the controller (42) can interact when starting a photodestructive laser adjusted to hit cells in the fluid stream (16) in the second section after the source (34) of electromagnetic radiation based on cell classification. Such a laser may operate at a different wavelength than the electromagnetic radiation source (34) or at a higher power to ensure that the sperm cells are removed, deactivated and rendered infertile. Embodiments using such a photodestructive laser still use the staining medium and sheath and may additionally use a collection medium. In accordance with some embodiments of the present invention, any of the staining medium, sheath liquid, and collection medium, if present, may include a cryoprotectant.

In a further alternative, specific aspects of the present invention can be equally applied to microfluidic chips. As one example, for microfluidic chips such as those described in WO 2011/097032 and WO 2011/097032, each of which is incorporated herein by reference, the inclusion of a sheath fluid containing a cryoprotectant may also be advantageous. Similarly, in embodiments that include the use of flow channels on microfluidic chips, staining medium is still used and sheath liquid and collection medium can be additionally used. In accordance with some embodiments of the present invention, any of the staining medium, sheath liquid, and collection medium, if present, may include a cryoprotectant.

Fig. 2 illustrates a representative two-dimensional plot of lateral fluorescence and forward fluorescence from an air jet flow cytometer of stained semen that the analyzer can generate (36). The visual representation of the data may be used by the operator to provide feedback regarding the sample being sorted and to graphically demonstrate certain aspects of the sorting logic. For example, R1 can be thought of as a gate area that can be applied to the sorting logic of a flow cytometer. An additional numeric output can be shown on the analyzer display (36). Such numerical output can be in the form of measured sorting parameters such as event rate, failure rate, sorting speed, sorting efficiency, or percentage of particles in any area or gate. R1 is illustrated as a region that can be considered a living oriented region, since the boundaries of R1 include two dense populations of cells that reflect a closely related population of sperm bearing the X chromosome and a population of sperm bearing the Y chromosome. R2 is a gating region located around non-viable or membrane-damaged sperm whose fluorescence is quenched with a quenching dye. Although different sorting logic can be applied, the two strategies related to R1 and R2 can represent the first stage in the sorting logic whereby all events falling into R1 are accepted for further processing or gating. Alternatively, all events outside R2 are accepted for further processing or gating.

Fig. 3 illustrates a one-dimensional bar graph that can be created by the analyzer (36) and generated in a graphical representation for the operator. The data illustrated in FIG. 3 may represent the number of occurrences of peak signal intensities from side or forward fluorescence over a specified period. In the case of sperm, X-bearing sperm and Y-bearing sperm tend to have peak intensities that vary from 2 to 5%, depending on the species, and this difference is reflected in the bimodal distribution of peak intensities shown in FIG. 2. Because X-bearing sperm and Y-bearing sperm generally have different fluorescence values, each of the peaks represents either an X-bearing sperm or a Y-bearing sperm. Based on the sorting logic used in the analyzer (36), the cell population in the histogram may represent only those cells that have been determined to be viable oriented cells, such as cells falling into R1 in FIG. 2, or they may represent cells that were not determined to be dead or unwanted, such as every event other than those that fall in R2. A variety of sort options can be obtained from the information contained in this histogram. For example, the level of discrimination between two peaks may indicate what sorted purity might look like. Fig. 3 further illustrates measurements of the relative intensity at the lowest point between the two groups, which can be thought of as a V value and a second relative intensity at the histogram peak or peaks at P. Visual inspection of the histogram can give the operator an idea of how the flow cytometer works, but is performed by the computer instructions for determining P value, V value, and V to P ratio have not been implemented in commercial semen sorting devices. The ratio of the minimum signal to the maximum can be periodically determined as the sorting parameter is measured periodically during sorting. The ratio of the minimum signal to the maximum, while not necessarily fully determining the purity of the sort, can provide a means of quickly estimating purity values, either automatically by following a written instruction in the analyzer (36) or manually by visual inspection by the operator. Alternatively, the inverse ratio, namely the ratio of the maximum signal to the minimum, provides information similar to the reciprocal.

Referring to FIG. 4, the analyzer (36) may generate a second bimodal plot in response to signals received by the cell sensing system (30). The bimodal graph may represent a first axis illustrating the peak intensity of the direct fluorescence signal or the peak intensity of the side fluorescence signal. Similarly to FIG. 3, the data illustrated in FIG. 4 can be restricted so that only events falling in R1 in FIG. 2. Alternatively, in the case of sperm, all events that do not fall within the R2 dead cell gate can also be displayed.

R3 may represent an X-sorting gate for collecting X-bearing sperm. The term "X-sort gate" in this document can be used interchangeably with the term "X-gate". With reference to FIG. 4, this can demonstrate how resizing the gated areas can affect efficiency, purity, and throughput. If the R3 area is expanded, it can be seen that every second more sperm will be sorted as X-bearing sperm, resulting in higher sorting efficiency and higher throughput. However, an expansion of the gate or R3 region will include events that have an increased likelihood of having Y chromosome-bearing sperm. To increase the purity of sorted sperm, the R3 region can be reduced and/or moved away from the Y chromosome region. The fewer events that enter the X-sort gate, the fewer sperm will be sorted into the population of X-carrying sperm that are more likely to actually be X-carrying sperm, meaning that the purity of the collected sperm can be increased. However, both efficiency in terms of collected cells and throughput in terms of sort per second will decrease as fewer events enter the R3 region and more matching events are rejected. In addition, as other instrument parameters change, the illustrated diagrams in FIG. 2, fig. 3 and FIG. 4 may change in form and character. For example, increasing the sample pressure or sample flow rate may result in a decrease in the minimum to maximum signal ratio, or may otherwise reduce the bimodal difference between X chromosome-bearing and Y-bearing sperm.

The processed semen can then be pooled and frozen according to known methods for normal or sex-selected semen. As one example, for the freezing methods described in international patent application WO/200137655, incorporated herein by reference in its entirety, specific embodiments of the described invention in which a cryoprotectant is introduced into semen sorting methods at earlier stages, for example, during or stains, in sheath fluid or even in capture fluid, can be particularly advantageous. Alternatively, the inclusion of a cryoprotectant in one or more of the earlier steps may be included in the new freezing process.

In one embodiment, the collected sorted sperm can be cooled to a temperature of about 5° C. or another suitable cooling temperature based on the specific sperm species. Alternatively, the semen can be collected in a collection container that is already chilled. Depending on the size of the collection container, the collected semen can be combined with additional collection containers. The pooled collection vessels can be centrifuged and the supernatant discarded. In accordance with some of the previous methods, the isolated semen is resuspended in Fraction A (or the cryoprotectant-free fraction) of diluent, and Fraction B (or the fraction containing cryoprotectant) having twice the desired concentration of cryoprotectant is added to Fraction A in an equal volume. In some previous methods fraction B was added in two equal volumes about 15 minutes apart, while in other methods fraction B was introduced in the form of a constant droplet flow.

In some embodiments of the present invention, cryoprotectants are included in various media during the staining, sorting, and collection steps, which can reduce or even eliminate the need for chilling a manipulated semen sample before reconcentration. In addition, the inclusion of a cryoprotectant in the various staining and sorting steps can eliminate the need for an "A" and "B" fraction model.

Referring to FIG. 5, the illustrated embodiment of the present invention relates to a method in which cryoprotectants are added to a plurality of media during semen processing, such as staining media, sheath fluid, and collection media. In step (510), the method starts with the step of staining the sperm sample in staining medium. The semen sample may be pure seminal fluid or semen diluted in another solution such as described previously. As an example, a semen sample can be standardized by diluting and re-concentrating in the manner described previously. Alternatively, the semen sample may be in the form of pure seminal fluid or pure seminal fluid diluted with a buffer and/or supplemented with antibiotics.

Whether standardized or not, the DNA selective fluorescent dye can be Hoechst 33342 supplied in a modified TALP buffer (Table 1). The staining step itself can be performed in a single dilution that further includes a quenching dye. Alternatively, step 510 may be performed in two dilutions, such as a first dilution in modified TALP containing a DNA selective fluorescent dye, followed by a second dilution in modified TALP containing a quenching dye. It should be understood that other DNA selective fluorescent dyes and other quenching dyes may be used according to this example. For purposes of this embodiment, each will be referred to as a staining medium.

Whether achieved by one or two dilutions, a semen sample can be diluted to between 640xl0 ⁶ and 40xl0 ⁶ semen/ml, between about 320xl0 ⁶ and 80xl0 ⁶ semen/ml, up to about 160x10 ⁶ semen/ml, or up to about 120x10 ⁶ semen /ml in medium for staining. The DNA selective fluorescent dye can be added to sperm suspended in staining medium at a concentration of from about 10 μM to 200 μM; from about 20 μM to 100 μM, or from about 30 μM to 70 μM. The pH of the staining medium may be from about 6.8 to 7.9; from about 7.1 and 7.6; or about 7.4. If two dilutions are used, the second dilution may be performed at or near the same pH as the first dilution, or at a low pH such as 5.0 to 6.0 or about 5.5. Optionally, the previously described antioxidants and concentrations may be included in the staining medium. The semen sample can be incubated at 30-39°C, about 32-37°C or about 34°C. The incubation period may vary from about 20 minutes to about three hours, from about 30 minutes to about 90 minutes, or from about 45 minutes to about 60 minutes.

At step 512, it can be seen that the staining medium used at step 510 includes a first amount of cryoprotectant. The cryoprotectant may be a suitable sugar alcohol such as ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite or combinations thereof. The cryoprotectant may also be a suitable glycol such as propylene glycol, butanetriol, or combinations thereof. It should be understood that the selected cryoprotectant may be species-specific or possibly even breed-specific. As an example, glycerol can be selected in the case of bovine semen and by following the steps shown in FIG. 5, the toxicity of glycerin can be reduced. It should be understood that other suitable cryoprotectants may be used as long as they are suitable for the preservation of the semen sample, and that the steps in FIG. 5 can similarly mitigate the toxic effects of these cryoprotectants.

The first amount of cryoprotectant may be about./about. or wt./about. a cryoprotectant concentration in the staining medium of from about 0.1% to about 5%; from about 0.1% to about 1%; from about 1% to about 2%; from about 2% to about 3%; from about 3% to about 4%; from about 2% to about 4%; or from about 1.5% to about 3%.

After staining, the sperm sample may be brought into contact with the sheath fluid in step 520. The step of bringing the sperm sample into contact may be associated with processing the sperm sample, for example by flow cytometry using an air jet flow cytometer or similarly using a microfluidic chip. In one embodiment, the step of bringing the sperm sample into contact with the sheath fluid occurs in an air jet flow cytometer, as described with reference to FIG. 1. However, it should be understood that step 520 is not limited to air jet flow cytometer processing, but rather encompasses any semen processing method in which a semen sample containing semen comes into contact with a sheath fluid. As a non-limiting example, microfluidic chip sorting typically requires establishing a coaxial flow of sample and sheath fluid through a flow channel. In the case of sperm, the sperm sample must pass through one or more flow channels in laminar flow while surrounded by sheath fluid to focus the location of the sperm cells and prevent them from touching the inside of the flow channel. The laminar flow of the semen sample and sheath fluid in each channel will prevent or at least minimize any mixing of the semen sample and sheath fluid while maintaining fluid contact.

At step 522, it can be seen that the sheath fluid used at step 520 contains a second amount of cryoprotectant. The cryoprotectant may be the same as the cryoprotectant in the staining medium. Alternatively, the cryoprotectant may be another cryoprotectant such as a suitable sugar alcohol including ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite, or combinations thereof, or a suitable glycol such as propylene glycol, butanetriol, or combinations thereof. The sheath fluid may contain a second amount of cryoprotectant from about 0.1% to about 2% cryoprotectant by volume./about. or wt./about.; from about 2% to about 4% cryoprotectant by vol./about. or wt./about.; from about 4% to about 6% cryoprotectant by vol./about. or wt./about.; from about 1% to about 2% cryoprotectant by vol./about. or wt./about.; from about 2% to about 3% cryoprotectant by vol./about. or wt./about.; from about 3% to about 4% cryoprotectant by vol./about. or wt./about.; from about 4% to about 5% cryoprotectant by vol./about. or wt./about.; from about 5% to about 6% cryoprotectant by vol./about. or wt./about.; from about 2% to about 6% cryoprotectant by vol./about. or wt./about.; or from about 3% to about 5% cryoprotectant by vol./about. or wt./about. In one embodiment, the second amount of cryoprotectant in the sheath fluid is about the same as the first amount of cryoprotectant found in the staining medium. In an alternative implementation, the second amount of cryoprotectant in the sheath fluid is greater than v/v. or wt./about. concentration compared to the first amount of cryoprotectant in the staining medium. In another embodiment, about./about. or wt./about. the cryoprotectant concentration in both the staining medium and the sheath can be coordinated to achieve the desired cryoprotectant concentration.

In step 530, the semen sample is manipulated. In one embodiment, the sperm sample is manipulated to obtain a manipulated sperm sample having a manipulated ratio of viable X-chromosome-bearing to viable Y-chromosome-bearing sperm. The manipulation step may be performed by physically separating a subset of cells, such as in the droplet embodiment described above with reference to FIG. 1. But the manipulation step is not so limited and includes changing the fluid or diverting the cells into the flow channels of the microfluidic chips. Alternatively, a photodestructive laser can be used in conjunction with a jet-in-air flow cytometer or microfluidic chip to disrupt selected sperm cells in a sperm sample to produce a manipulated sperm sample with a manipulated ratio of viable X-bearing sperm to viable sperm, carrying a Y chromosome. Alternatively, the semen sample can be manipulated based on other characteristics. As one example, a sperm sample can be passed through a flow cytometer to separate presumably viable sperm from sperm with damaged membranes.

One embodiment of the method shown in FIG. 5 ends with obtaining the manipulated sperm sample at step 530, while other embodiments of the method continue at the step of collecting the manipulated sperm sample 540 and/or at the step of freezing the manipulated sperm sample 550. In one embodiment, the method of FIG. 5 optionally continues to step 540 where the manipulated sperm sample is collected in the collection medium. In the case of flow cytometry, the collection medium can be described as a capture fluid located in a collection vessel or tube into which the selected sperm is withdrawn. As can be understood from step 542, the collection medium in this embodiment also optionally contains an amount of cryoprotectant, which can be considered a third amount of cryoprotectant. Again, the cryoprotectant may be the same as the cryoprotectant in the stain medium and/or sheath. Alternatively, the cryoprotectant may be another cryoprotectant such as a suitable sugar alcohol including ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite, or combinations thereof, or a suitable glycol such as propylene glycol, butanetriol, or combinations thereof. The collection medium may contain a third amount of cryoprotectant from about 1% to about 2% cryoprotectant by volume./about. or wt./about.; from about 2% to about 4% cryoprotectant by vol./about. or wt./about.; from about 4% to about 6% cryoprotectant by vol./about. or wt./about.; from about 6% to about 8% cryoprotectant by vol./about. or wt./about.; from about 3% to about 7% cryoprotectant by vol./about. or wt./about.; or from about 3.5% to about 5.5% cryoprotectant by vol./about. or wt./about.

In specific embodiments of the present invention, the cryoprotectant is only included in the sheath fluid and is not present in the staining medium or collection medium. In another embodiment of the present invention, the cryoprotectant is included in the sheath fluid, in the staining medium, and in the freezing medium, but not included in the collection medium.

In one embodiment, the method proceeds from the step 530 of manipulating the sperm sample directly to the step 550 of freezing the manipulated sperm sample. For example, in the case of manipulation of a semen sample using a microfluidic chip, the use of collection medium may not be required because no drops are formed that require softening of the collection medium. However, embodiments are contemplated in which the collection medium is used in connection with manipulation of a semen sample using a microfluidic chip.

For the method embodied in FIG. 5, freezing at step 550 can be achieved using a conventional method such as that described in WO/200137655. By way of example only, one such method may begin by diluting a manipulated sperm sample to be frozen in Fraction A. Alternatively, the manipulated sperm sample may be collected in a collection medium including Fraction A diluent at collection step 540. By way of example only, fraction A may contain diluent TRIS citrate with 20% egg yolk. Other suitable diluents may include sodium citrate, tris[hydroxymethyl]ammomethane and TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid) and monosodium glutamate buffers; milk; HEPES - buffered medium; and any combination of them.

The freezing step 550 may be continued by cooling the manipulated semen sample. The manipulated semen sample may be cooled to a temperature of about 8°C to 4°C. In one specific example, the manipulated semen sample may be cooled to about 5°C. After cooling, fraction B can be added in one or more steps and the manipulated semen sample can then be reconcentrated. Reconcentration may include centrifuging the manipulated semen sample to pellet and resuspending the semen in a final diluent, sometimes referred to as AB diluent. As one example, diluent AB may have a concentration of cryoprotectant that is half the concentration of cryoprotectant in fraction B. As a non-limiting example, this concentration may be about 6% in the case of glycerol. Other freezing methods are proposed for use in the embodiment depicted in FIG. 5. In particular, some of the freezing methods described below may provide synergy with the processing method depicted in FIG. 5.

The method shown in Fig. 6 largely reflects the method depicted in FIG. 5, except that a cryoprotectant was added to the staining medium. In step (610), the method starts with the step of staining the sperm sample in staining medium. The semen sample may be pure seminal fluid or semen diluted in another solution such as described previously. As an example, a semen sample can be standardized by diluting and re-concentrating in the manner described previously. Alternatively, the semen sample may be in the form of pure seminal fluid or pure seminal fluid diluted with a buffer and/or supplemented with antibiotics.

Whether standardized or not, the DNA selective fluorescent dye can be Hoechst 33342 supplied in a modified TALP buffer (Table 1). The staining step itself can be performed in a single dilution that further includes a quenching dye. Alternatively, step 510 may be performed in two dilutions, such as a first dilution in modified TALP containing a DNA selective fluorescent dye, followed by a second dilution in modified TALP containing a quenching dye. It should be understood that other DNA selective fluorescent dyes and other quenching dyes may be used according to this example. For purposes of this embodiment, each will be referred to as a staining medium.

Whether achieved by one or two dilutions, a semen sample can be diluted to between 640xl0 ⁶ and 40xl0 ⁶ semen/ml, from about 320xl0 ⁶ to 80xl0 ⁶ semen/ml, up to about 160x10 ⁶ semen/ml, or up to about 120x10 ⁶ semen/ml in staining medium. The DNA selective fluorescent dye can be added to sperm suspended in staining medium at a concentration of from about 10 μM to 200 μM; from about 20 μM to 100 μM, or from about 30 μM to 70 μM. The pH of the staining medium may be from about 6.8 to 7.9; from about 7.1 and 7.6; or about 7.4. If two dilutions are used, the second dilution may be performed at or near the same pH as the first dilution, or at a low pH such as 5.0 to 6.0 or about 5.5. Optionally, the previously described antioxidants and concentrations may be included in the staining medium. The semen sample can be incubated at 30-39°C, about 32-37°C or about 34°C. The incubation period may vary from about 20 minutes to about three hours, from about 30 minutes to about 90 minutes, or from about 45 minutes to about 60 minutes.

After staining, the sperm sample may be brought into contact with the sheath fluid in step 620. The step of bringing the sperm sample into contact may be associated with processing the sperm sample, for example by flow cytometry using an air jet flow cytometer or similarly using a microfluidic chip. In one embodiment, the step of bringing the sperm sample into contact with the sheath fluid occurs in an air jet flow cytometer, as described with reference to FIG. 1. However, it should be understood that step 620 is not limited to air jet flow cytometer processing, but rather encompasses any semen processing method in which a semen sample containing semen comes into contact with a sheath fluid. As a non-limiting example, microfluidic chip sorting typically requires establishing a coaxial flow of sample and sheath fluid through a flow channel. In the case of sperm, the sperm sample must pass through one or more flow channels in laminar flow while surrounded by sheath fluid to focus the location of the sperm cells and prevent them from touching the inside of the flow channel. The laminar flow of the semen sample and sheath fluid in each channel will prevent or at least minimize any mixing of the semen sample and sheath fluid while maintaining fluid contact.

At step 622, it can be seen that the sheath fluid used at step 620 includes a first amount of a cryoprotectant, which may be a suitable sugar alcohol, including ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite, or combinations thereof, or a suitable glycol such as propylene glycol, butanetriol, or combinations thereof. The first amount of cryoprotectant in the sheath fluid may be from about 0.1% to about 2% cryoprotectant v/v. or wt./about.; from about 2% to about 4% cryoprotectant by vol./about. or wt./about.; from about 4% to about 6% cryoprotectant by vol./about. or wt./about.; from about 1% to about 2% cryoprotectant by vol./about. or wt./about.; from about 2% to about 3% cryoprotectant by vol./about. or wt./about.; from about 3% to about 4% cryoprotectant by vol./about. or wt./about.; from about 4% to about 5% cryoprotectant by vol./about. or wt./about.; from about 5% to about 6% cryoprotectant by vol./about. or wt./about.; from about 2% to about 6% cryoprotectant by vol./about. or wt./about.; or from about 3% to about 5% cryoprotectant by vol./about. or wt./about. In one embodiment, the second amount of cryoprotectant in the sheath fluid in the sheath fluid is about the same as the first amount of cryoprotectant found in the staining medium. In an alternative implementation, the second amount of cryoprotectant in the sheath fluid is greater than v/v. or wt./about. concentration compared to the first amount of cryoprotectant in the staining medium.

In step 630, the semen sample is manipulated. In one embodiment, the sperm sample is manipulated to obtain a manipulated sperm sample having a manipulated ratio of viable X-chromosome-bearing to viable Y-chromosome-bearing sperm. The manipulation step may be performed by physically separating a subset of cells, such as in the droplet embodiment described above with reference to FIG. 1. But the manipulation step is not so limited and includes changing the fluid or diverting the cells into the flow channels of the microfluidic chips. Alternatively, a photodestructive laser can be used in conjunction with a jet-in-air flow cytometer or microfluidic chip to disrupt selected sperm cells in a sperm sample to produce a manipulated sperm sample with a manipulated ratio of viable X-bearing sperm to viable sperm, carrying a Y chromosome. Alternatively, the semen sample can be manipulated based on other characteristics. As one example, a sperm sample can be passed through a flow cytometer to separate presumably viable sperm from sperm with damaged membranes.

One embodiment of the method shown in FIG. 6 ends with obtaining the manipulated sperm sample at step 630, while other embodiments of the method continue at step 640 for collecting the manipulated sperm sample and/or at step 650 for freezing the manipulated sperm sample. In one embodiment, the method of FIG. 6 optionally continues to step 640 where the manipulated semen sample is collected in the collection medium. In the case of flow cytometry, the collection medium can be described as a capture fluid located in a collection vessel or tube into which the selected sperm is withdrawn. As can be understood from step 642, the collection medium also optionally contains an amount of cryoprotectant, which can be considered a second amount of cryoprotectant. The cryoprotectant may be the same as the cryoprotectant in the sheath fluid. Alternatively, the cryoprotectant may be another cryoprotectant such as a suitable sugar alcohol including ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite, or combinations thereof, or a suitable glycol such as propylene glycol, butanetriol, or combinations thereof. The collection medium may contain a second amount of cryoprotectant from about 1% to about 2% cryoprotectant v/v. or wt./about.; from about 2% to about 4% cryoprotectant by vol./about. or wt./about.; from about 4% to about 6% cryoprotectant by vol./about. or wt./about.; from about 6% to about 8% cryoprotectant by vol./about. or wt./about.; from about 3% to about 7% cryoprotectant by vol./about. or wt./about.; or from about 3.5% to about 5.5% cryoprotectant by vol./about. or wt./about.

In one embodiment, the method proceeds from the step 630 of manipulating the sperm sample directly to the step 650 of freezing the manipulated sperm sample. For example, in the case of manipulation of a semen sample using a microfluidic chip, the use of collection medium may not be required because no drops are formed that require softening of the collection medium. However, embodiments are contemplated in which the collection medium is used in connection with manipulation of a semen sample using a microfluidic chip.

For the method embodied in FIG. 6, freezing at step 650 can be achieved using a conventional method such as that described in WO/200137655. By way of example only, one such method may begin by diluting a manipulated sperm sample to be frozen in Fraction A. Alternatively, the manipulated sperm sample may be collected in a collection medium including Fraction A diluent at collection step 540. By way of example only, fraction A may contain diluent TRIS citrate with 20% egg yolk. Other suitable diluents may include sodium citrate, tris[hydroxymethyl]ammomethane and TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid) and monosodium glutamate buffers; milk; HEPES - buffered medium; and any combination of them.

The freezing step 650 may be continued by cooling the manipulated semen sample. The manipulated semen sample may be cooled to a temperature of about 8°C to 4°C. In one specific example, the manipulated semen sample may be cooled to about 5°C. After cooling, fraction B can be added in one or more steps and the manipulated semen sample can then be reconcentrated. Reconcentration may include centrifuging the manipulated semen sample to pellet and resuspending the semen in a final diluent, sometimes referred to as AB diluent. As one example, diluent AB may have a concentration of cryoprotectant that is half the concentration of cryoprotectant in Fraction B. As a non-limiting example, this concentration may be about 6% v/v. or wt./about. in the case of glycerin. Other freezing methods are proposed for use in the embodiment depicted in FIG. 6. In particular, some of the freezing methods described below may provide synergy with the processing method depicted in FIG. 6.

The method shown in Fig. 7 differs from the method shown in FIG. 5 by the fact that the flowing fluid is not supplemented with a cryoprotectant. In step (710), the method starts with the step of staining the sperm sample in staining medium. The semen sample may be pure seminal fluid or semen diluted in another solution such as described previously. As an example, a semen sample can be standardized by diluting and re-concentrating in the manner described previously. Alternatively, the semen sample may be in the form of pure seminal fluid or pure seminal fluid diluted with a buffer and/or supplemented with antibiotics.

Whether standardized or not, the DNA selective fluorescent dye can be Hoechst 33342 supplied in a modified TALP buffer (Table 1). The staining step itself can be performed in a single dilution that further includes a quenching dye. Alternatively, step 510 may be performed in two dilutions, such as a first dilution in modified TALP containing a DNA selective fluorescent dye, followed by a second dilution in modified TALP containing a quenching dye. It should be understood that other DNA selective fluorescent dyes and other quenching dyes may be used according to this example. For purposes of this embodiment, each will be referred to as a staining medium.

Whether achieved by one or two dilutions, a semen sample can be diluted to between 640×l0 ⁶ and 40×l0 ⁶ semen/ml, from about 320×l0 ⁶ to 80×l0 ⁶ semen/ml, up to about 160×10 ⁶ sperm/ml or up to about 120×10 ⁶ sperm/ml in staining medium. The DNA selective fluorescent dye can be added to the sperm suspended in the staining medium at a concentration of from about 10 μM to 200 μM; from about 20 μM to 100 μM, or from about 30 μM to 70 μM. The pH of the staining medium may be from about 6.8 to 7.9; from about 7.1 and 7.6; or about 7.4. If two dilutions are used, the second dilution may be performed at or near the same pH as the first dilution, or at a low pH such as 5.0 to 6.0 or about 5.5. Optionally, the previously described antioxidants and concentrations may be included in the staining medium. The semen sample can be incubated at 30-39°C, about 32-37°C or about 34°C. The incubation period may vary from about 20 minutes to about three hours, from about 30 minutes to about 90 minutes, or from about 45 minutes to about 60 minutes.

At step 712, it can be seen that the staining medium used at step 710 includes a first amount of cryoprotectant. The cryoprotectant may be a suitable sugar alcohol such as ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite or combinations thereof. The cryoprotectant may also be a suitable glycol such as propylene glycol, butanetriol, or combinations thereof. It should be understood that the selected cryoprotectant may be species-specific or possibly even breed-specific. As an example, glycerol can be selected in the case of bovine semen and by following the steps shown in FIG. 7, the toxicity of glycerol can be reduced. It should be understood that other suitable cryoprotectants may be used as long as they are suitable for the preservation of the semen sample, and that the steps in FIG. 7 can similarly mitigate the toxic effects of these cryoprotectants.

The first amount of cryoprotectant may be about./about. or wt./about. a cryoprotectant concentration in the staining medium of from about 0.1% to about 5%; from about 0.1% to about 1%; from about 1% to about 2%; from about 2% to about 3%; from about 3% to about 4%; from about 2% to about 4%; or from about 1.5% to about 3%.

After staining, the sperm sample may be brought into contact with the sheath fluid in step 720. The step of bringing the sperm sample into contact may be associated with processing the sperm sample, for example by flow cytometry using an air jet flow cytometer or similarly using a microfluidic chip. In one embodiment, the step of bringing the sperm sample into contact with the sheath fluid occurs in an air jet flow cytometer, as described with reference to FIG. 1. However, it should be understood that step 720 is not limited to air jet flow cytometer processing, but rather encompasses any semen processing method in which a semen sample containing semen comes into contact with a sheath fluid. As a non-limiting example, microfluidic chip sorting typically requires establishing a coaxial flow of sample and sheath fluid through a flow channel. In the case of sperm, the sperm sample must pass through one or more flow channels in laminar flow while surrounded by sheath fluid to focus the location of the sperm cells and prevent them from touching the inside of the flow channel. The laminar flow of the semen sample and sheath fluid in each channel will prevent or at least minimize any mixing of the semen sample and sheath fluid while maintaining fluid contact.

In step 730, the semen sample is manipulated. In one embodiment, the sperm sample is manipulated to obtain a manipulated sperm sample having a manipulated ratio of viable X-chromosome-bearing to viable Y-chromosome-bearing sperm. The manipulation step may be performed by physically separating a subset of cells, such as in the droplet embodiment described above with reference to FIG. 1. But the manipulation step is not so limited and includes changing the fluid or diverting the cells into the flow channels of the microfluidic chips. Alternatively, a photodestructive laser can be used in conjunction with a jet-in-air flow cytometer or microfluidic chip to disrupt selected sperm cells in a sperm sample to produce a manipulated sperm sample with a manipulated ratio of viable X-bearing sperm to viable sperm, carrying a Y chromosome. Alternatively, the semen sample can be manipulated based on other characteristics. As one example, a sperm sample can be passed through a flow cytometer to separate presumably viable sperm from sperm with damaged membranes.

One embodiment of the method shown in FIG. 7 ends with obtaining the manipulated sperm sample at step 730, while other embodiments of the method continue at step 740 for collecting the manipulated sperm sample and/or at step 750 for freezing the manipulated sperm sample. In one embodiment, the method of FIG. 7 optionally continues to step 740 where the manipulated semen sample is collected in the collection medium. In the case of flow cytometry, the collection medium can be described as a capture fluid located in a collection vessel or tube into which the selected sperm is withdrawn. As can be understood from step 742, the collection medium in this embodiment also optionally contains an amount of cryoprotectant, which can be considered a third amount of cryoprotectant. Again, the cryoprotectant may be the same as the cryoprotectant in the stain medium and/or sheath. Alternatively, the cryoprotectant may be another cryoprotectant such as a suitable sugar alcohol including ethylene glycol; glycerol; erythritol; trait; Arabite; ribit; xylitol; sorbitol; galactite; goes; volemite; fucite; inositol; glycylglycite, or combinations thereof, or a suitable glycol such as propylene glycol, butanetriol, or combinations thereof. The collection medium may contain a third amount of cryoprotectant from about 1% to about 2% cryoprotectant by volume./about. or wt./about.; from about 2% to about 4% cryoprotectant by vol./about. or wt./about.; from about 4% to about 6% cryoprotectant by vol./about. or wt./about.; from about 3% to about 7% cryoprotectant by vol./about. or wt./about.; or from about 3.5% to about 5.5% cryoprotectant by vol./about. or wt./about; or about 4.5% cryoprotectant vol./about. or wt./about.

In one embodiment, the method proceeds from the step 530 of manipulating the sperm sample directly to the step 750 of freezing the manipulated sperm sample. For example, in the case of manipulation of a semen sample using a microfluidic chip, the use of collection medium may not be required because no drops are formed that require softening of the collection medium. However, embodiments are contemplated in which the collection medium is used in connection with manipulation of a semen sample using a microfluidic chip.

For the method embodied in FIG. 7, freezing at step 750 can be achieved using a conventional method such as that described in WO/200137655. By way of example only, one such method may begin by diluting a manipulated sperm sample to be frozen in Fraction A. Alternatively, the manipulated sperm sample may be collected in a collection medium including Fraction A diluent at collection step 540. By way of example only, fraction A may contain diluent TRIS citrate with 20% egg yolk. Other suitable diluents may include sodium citrate, tris[hydroxymethyl]aminomethane and TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid) and monosodium glutamate buffers; milk; HEPES - buffered medium; and any combination of them.

The freezing step 750 may be continued by chilling the manipulated semen sample. The manipulated semen sample may be cooled to a temperature of about 8°C to 4°C. In one specific example, the manipulated semen sample may be cooled to about 5°C. After cooling, fraction B can be added in one or more steps and the manipulated semen sample can then be reconcentrated. Reconcentration may include centrifuging the manipulated semen sample to pellet and resuspending the semen in a final diluent, sometimes referred to as AB diluent. As one example, diluent AB may have a concentration of cryoprotectant that is half the concentration of cryoprotectant in Fraction B. As a non-limiting example, this concentration may be about 6% v/v. or wt./about. in the case of glycerin. Other freezing methods are proposed for use in the embodiment depicted in FIG. 7. In particular, some of the freezing methods described below may provide synergy with the processing method depicted in FIG. 7.

Fig. 8 illustrates a new semen freezing method that can be used in combination with any of the sorting methods described above or systems in which a cryoprotectant is incorporated into the semen population manipulation method. As non-limiting examples, a sperm sample can be manipulated to change the ratio of viable X-bearing sperm to viable Y-bearing sperm using an air jet flow cytometer or using a microfluidic chip. The mechanism for manipulating the semen sample may be droplet redirection, fluid reversal, photodestruction by a laser, or other means.

Regardless of the means used to manipulate the sperm sample, step 810 begins by collecting the manipulated sperm sample in a mixture that includes a cryoprotectant. The cryoprotectant may be present in the container prior to obtaining the manipulated semen sample, the cryoprotectant may be present in the semen sample prior to manipulation.

As previously described, in one embodiment, the sperm is stained in a staining medium that includes a cryoprotectant. Similarly, during manipulation of the semen sample, the semen may be brought into contact with a sheath fluid containing a cryoprotectant. Whether they are stained in a staining medium that includes a cryoprotectant, or sorted in a system that includes a sheath fluid with a cryoprotectant, or both, the cryoprotectant is left in the semen sample and collected at step 810 along with the manipulated sperm cells.

In embodiments in which semen is collected at a sufficient concentration of cryoprotectant, the freezing process can be simplified to skip or change the steps that were previously necessary to effect the gradual introduction of the cryoprotectant into the semen. For example, in one embodiment, manipulated semen in contact with a staining medium containing glycerol, sheath fluid, or capture fluid may be taken directly from a sorter, concentrated by centrifugation, resuspended in a cryoprotectant, and then cryopreserved. In a further embodiment, after resuspension in the cryoprotectant, the sperm is held for a holding time of less than 24 hours, less than 8 hours, less than 4 hours, 2 hours, less than 2 hours, or less than 1 hour prior to cryopreservation. In yet another further embodiment, the semen is chilled during the holding time. In a specific embodiment of the present invention, the sperm is brought into contact with a sheath fluid containing 3% glycerol (v/v or w/v), concentrated by centrifugation, resuspended in a cryoprotectant containing 4.5% glycerol (v/v or w/v) and then cryopreserved. Thus, the above embodiments do not include the steps of chilling the semen and adding a cryoprotectant to the chilled semen prior to the step of concentrating the semen.

Settings and conditions for examples 1-10

Sorter Settings - All sorting analysis and sorting was performed on a modified MoFlo-SX semen sorter (Beckman Coulter, Brea California) using Genesis digital processing hardware upgrades (available from CytonomeST, Boston, Massachusetts) and dedicated semen sorting software . The Genesis sorting software includes a parameter logging feature that records averages for various parameters such as Event Rate (in kHz), Sorting Rate (in kHz), Bounce Rate (in kHz), Dead Sperm Count (Dead Sperm Event Rate, divided by event rate in %), number of survival cells (event rate of survival sperm divided by event rate in %), X-gated count (as percentage of survival sperm selected for collection) and ratio of maximum signal to minimum (PVR). Mass sorting refers to the use of an X-gated quantity that includes both X-chromosome-bearing sperm and Y-chromosome-bearing sperm to obtain sperm that is sorted but not selected by sex, based on experience that semen quality after sorting and freezing does not depend on sex, and mass sorting occurs about twice as fast as sorting by sex. Pressurized sheath fluid is delivered either from a sterile sheath fluid bag inside a pressure reservoir with an air cap, or from a sheath fluid bag or open beaker feeding a SheathMaster™ Precision Fluid Delivery System (CytonomeST) where pressure is applied to a small volume pressure chamber . In both cases, sheath fluid velocity is precisely controlled by air pressure controlled by the MoFlo sampling station. TRIS-based sheath fluid for the commercial production of sex-specific seminal fluid was used for all sorting.

Batch Sheath Replacement Method - Multiple volumes of TRIS based sheath fluid, each containing a certain amount of glycerol, were placed in sterile bags or open beakers and applied to the sorter from a pressurized reservoir or SheathMaster™ fluid delivery system, respectively. The concentration of glycerol for each sheath fluid is known from its composition.

Sperm Staining - 160 million per ml of freshly ejaculated bovine semen are stained with 65 mM Hoechst 33342 in modified TALP for 60 minutes at 34°C, cooled to room temperature and diluted with 1/3 volume of the same modified TALP with 8% vol. /about. clarified egg yolk to create a final semen concentration of 120 million semen per milliliter and an egg yolk concentration of 2% v/v, then filtered through a 50 micron Partec filter.

Sperm Centering for Stabilization/PVR Measurement - PVR (maximum signal to minimum signal ratio) is an objective measurement of a one-dimensional graph graphically displayed on the graphical user interface of the Genesis computer that controls the sorter. The one-dimensional plot in Fig. 3 illustrates the frequency of events having different forward fluorescence intensities. The representation in the form of a properly aligned stained sperm sample corresponds to two overlapping curves with the same number of events at two different peak intensities (two maxima) and also where the two curves begin to overlap and the number of samples from each of the two curves are approximately the same (one local minimum roughly equally spaced between the two maxima). A local minimum can be called a valley (V), while two local maxima can be called peaks (P), the P value used in the example is the average of the intensity values of the two peaks. The ordinal values for P and V were determined in the software from which the PVR was calculated using the following equation: ((P-V)/P)* 100. Prior to analysis, a trained operator performed centering on the sorter to correct the position of the optics, semen sorting nozzle and tip, as well as a direct fluorescence detector (FAF) and a side fluorescence detector (SAF). In addition, appropriate signal amplification should be applied to the FAF and SAF.

Recorded Data Analysis for PVR - Genesis sorter software provides real-time PVR calculation. For every 25,000 events, PVR was calculated and buffered as a raw value, while 100 consecutive raw measurements were averaged. The average value of PVR can be recorded (registered) at the request of the operator. Typically, running a flow cytometer at a lower event rate provides improved PVR ratios due to the increased accuracy of individual event (sperm) measured values caused by the narrower sample flow cross section. Centering was first optimized for sorting at a rate of 40,000 events per second, a sorting gate (examples of which are denoted as R1, R3 and R4 in Fig. 4) was used to simultaneously collect live X- and live Y-carrier populations (mass sort), and the device for sorting worked without any further modification for a period of time corresponding to 500,000 to 700,000 sorted cells before asking for the average, ensuring that the parameters recorded are representative of the average. After requesting an average at 40,000 events per second, the operator depressurized the stained semen sample to set a new pressure that determined an event rate of approximately 30,000, and a sorting gate applied simultaneously to live X- and Y-containing populations (mass sort), and the sorter was operated without any further modification for a period of time corresponding to 500,000 to 700,000 sorted cells before requesting an average. The same was repeated for a sample processed at a rate of 20,000 events per second. The sample pressure was then increased to establish an average event rate of 40,000 events per second, followed by centering (if necessary) and three consecutive measurements as above. This was done sequentially over time, and the recorded data was then analyzed.

Example 1

Table 2 shows the processing options for example 1.

table 2

Standard. Color-
ing capture The sort volume
rovki Sort-
rovka Cooling Zamora-
living Cont-
role Support-
environment (1:3) TALP 2% EY (120 million/ml) TRIS A (3.5ml) 20 ml 0% Glycerin v/v SF Freezing Medium (12% Glycerol v/v) (1:1) AB (6% Glycerol v/v) (4 million/vial) T1 TRIS A (7.0ml) 40 ml 0% Glycerin v/v SF - T2 TRIS A (7.0ml) 40 ml 1% Glycerin v/v SF - T3 TRIS A (7.0ml) 40 ml 2% Glycerin v/v SF - T4 TRIS A (7.0ml) 40 ml 4% Glycerin v/v SF - T5 TRIS A (7.0ml) 40 ml 8% Glycerin v/v SF -

1. One ejaculate was obtained from a bull.

2. Checked the quality of the ejaculate (QC) for mobility and evaluated the morphology and concentration of sperm cells.

3. The ejaculate was standardized by combining with maintenance medium (physiological saline and buffer with egg yolk and nutrients) in a ratio of 1:3, respectively. and then stained according to the staining procedure described above.

4. Control sheath fluid (SF) was connected to a flow cytometer, and the waste fluid was collected in a pre-weighed tube for 3 minutes.

5. The fluid in the tube was weighed to determine the flow rate of each particular sheath fluid.

6. The stained sample was placed in a sorter and checked for droplet retention.

7. Recorded flow cytometer data were collected for 1 million sex-disaggregated sperm at 30,000, 20,000, and 40,000 eps (events per second).

8. After collecting the recorded data, mass sorting of 1 collection tube with a volume of no more than 20 ml was carried out.

9. For treatment groups, steps 7 and 8 were repeated using each treatment sheath fluid described in Table 2 above.

10. After collecting the recorded data, mass sorting was carried out for 1 collection tube to be processed, with a volume of no more than 40 ml.

11. All collection tubes were placed in a cold room for 90 minutes.

12. 20 ml of freezing medium (12% v/v glycerol) was added to the control (two-step addition).

13. All collection tubes were centrifuged and the supernatant decanted.

14. An appropriate volume of AB (6% v/v glycerol) was added to each collection tube to bring the concentration of sperm in the 1/4 cm ³ artificial insemination tube (AI straw) to 4 million cells/tube.

15. The diluted semen was kept in a cold room overnight.

16. Sperm was placed in test tubes and cryopreserved.

17. Steps 1-16 were repeated two more times using ejaculates from the same bull.

18. PVR was assessed for the control and each treatment. The results are shown in FIG. 9. Sperm motility (IVOS II), viability (PI) and PIA (PNA) were also assessed at 0 and 3 hours after thawing. The results are shown in figures 10 and 11, respectively.

Example 2

Instead of the periodic sheath fluid replacement method described above, in Example 2, the following sheath fluid gradient method was used to replace the sheath fluid.

Sheath Fluid Gradient Method for Sheath Replacement - Beaker 1 providing sheath fluid to the SheathMaster™ fluid delivery system containing bovine sheath fluid containing 0% glycerol is agitated with a magnetic stirrer. Bovine sheath fluid containing 6 about./about. glycerol (820 mm) in beaker 2 was slowly pumped into beaker 1 using a peristaltic pump, providing a continuously increasing concentration of glycerol in beaker 1 for about 3 hours, where the final concentration of glycerol was about 5.5% v/v. (750mm). The concentration of glycerol at intervals was determined by measuring the osmolarity of the sheath liquid exiting the nozzle and comparing the measurement with the corresponding standard curve. A typical curve for glycerol in a sheath fluid is the percentage of glycerol (G) = (measured mOsm (X) - 300) / 161, or in other words, each increase of 161 mOsm corresponds to an increase in the concentration of glycerol by 1% (v/v .).

1. Fresh ejaculates were obtained from three bulls.

2. Checked the quality of the ejaculate, standardized and stained in accordance with the methods described in example 1.

3. A beaker containing 0% glycerol sheath fluid was connected to the fluid delivery system of the flow cytometer. The liquid was constantly stirred with a magnetic stirrer.

4. The stained semen sample was placed in a flow cytometer and the droplet delay was checked.

5. 1 million sperm were sorted by sex at 20,000 eps, then 30,000 eps, and finally 40,000 eps.

6. Flowing fluid containing 6% vol./about. glycerin was pumped with a peristaltic pump into a sheath liquid containing 0% glycerol at an average flow rate of 204 g/h.

7. Recorded flow cytometer data was continuously collected at various event rates until the flow fluid containing 6.0% v/v. glycerin was available.

8. PVR was calculated for each control and treatment sample. The results are shown in FIG. 12.

Example 3

Table 3 shows the processing options for Example 3.

Table 3

Standard. Color-
ing Collection (Volume) ABOUT. sorting-
ki Sorting I cool-
environment Cooling time Freeze-
ing Control Storage medium (1:3) TALP 2% EY (120 million/ml) TRIS A (3.5ml) 20 ml 0% Glycerin v/v SF Medium for freezing
(12% Glycerin v/v) (1:1) 90 minutes COLD AB T1 TRIS A (7.0ml) 40 ml 3.5% Glycerin v/v SF - 90 minutes T2 TRIS A (7.0ml) 40 ml 3.5% Glycerin v/v SF - 30 minutes T3 TRIS A (7.0ml) 40 ml 3.5% Glycerin v/v SF - 0 minutes T4 TRIS A (7.0ml) 40 ml 3.5% Glycerin v/v SF - 0 minutes RT AB

1. Ejaculates were obtained from 10 bulls.

2. Checked the quality of the ejaculate, standardized and stained in accordance with the methods described in example 1.

3. 1 collection tube with a volume of not more than 20 ml was subjected to mass sorting.

Control - The tubes were placed in a cold room for 90 minutes.

20 L of freezing medium (12% v/v glycerol) was added to the control (2 step addition).

4. Step 3 was repeated. 4 collection tubes with a volume of not more than 40 ml were mass sorted using sheath fluid containing 3.5% glycerol v/v. (SF).

Treatment 1 - The tubes were placed in a cold room for 90 minutes.

Treatment 2 - The tubes were placed in a cold room for 30 minutes.

Treatment 3 - Stored at room temperature until ready for centrifugation.

Treatment 4 - Stored at room temperature until ready for centrifugation.

5. All collection tubes were centrifuged and the supernatant decanted.

6. An appropriate volume of cold AV (6% v/v glycerol) was added to each collection tube to bring the concentration of sperm in the 1/4 cm ³ IV tube to 4 million cells/tube, except for the addition of room temperature AB for processing 4.

7. Diluted semen was stored in a cold room overnight.

8. Sperm cells were placed in tubes and cryopreserved (3 tubes per treatment with 4 million cells/tube).

9. Mobility at 0 and 3 hours (IVOS), viability (PI) and PIA (PNA) were assessed after thawing. The results are shown in FIG. 13. Convergence (3h/0h) for motility, viability and PIA are shown in FIG. fourteen.

Example 4

Table 4 shows the processing options for example 4.

Table 4

Standard. Color-
ing Collection (Volume) ABOUT. sorting-
ki Sorting I cool-
environment Cooling time Freeze-
nie Control Storage medium (1:3) TALP 2% EY (120 million/ml) TRIS A (3.5ml) 20 ml 0% Glycerin v/v SF Medium for freezing
(12% Glycerin v/v) (1:1) 90 minutes AB (6% Glycerin v/v) T1 TRIS A (7.0ml) 40 ml 3.0% Glycerin v/v SF - 90 minutes AB (5% Glycerin v/v) T2 TRIS A (7.0ml) 40 ml 3.0% Glycerin v/v SF - 0 minutes AB (5% Glycerin v/v) T3 TRIS A (7.0ml) 40 ml 5.0% Glycerin v/v SF - 90 minutes AB (5% Glycerin v/v) T4 TRIS A (7.0ml) 40 ml 5.0% Glycerin v/v SF - 0 minutes AB (5% Glycerin v/v)

1. Ejaculates were obtained from five bulls.

2. Checked the quality of the ejaculate, standardized and stained in accordance with the methods described in example 1.

3. 1 collection tube with a volume of not more than 20 ml was subjected to mass sorting.

Control - collection tubes were placed in a cold room for 90 minutes.

20 ml of freezing medium (12% v/v glycerol) was added to the control (2 step addition).

All collection tubes were centrifuged and the supernatant decanted.

An appropriate volume of AB (6% v/v glycerol) was added to each collection tube to bring the sperm concentration in the 1/4 cm ³ IV tube to 4 million cells/tube.

4. Connected sheath fluid containing 3.0% glycerol (SF) and pressurized the reservoir.

5. 2 collection tubes with a volume of not more than 40 ml were mass sorted:

Treatment 1 - collection tubes were placed in a cold room for 90 minutes.

Treatment 2 - collection tubes were placed in a cold room for 0 minutes.

6. Connected sheath fluid containing 5.0% glycerol and pressurized the reservoir.

7. 4 collection tubes with a volume of not more than 40 ml were mass sorted:

Treatment 3 - collection tubes were placed in a cold room for 90 minutes.

Treatment 4 - collection tubes were placed in a cold room for 0 minutes.

All tubes were centrifuged and the supernatant was decanted.

AB (5% v/v glycerol) was added to each tube to a final concentration of 4 million sperm per tube.

8. Diluted semen was stored in a cold room overnight.

9. Sperm cells were placed in test tubes and cryopreserved.

10. Mobility at 0 and 3 hours (IVOS), viability (PI) and PIA (PNA) were assessed after thawing. The results are shown in FIG. 15. Convergence (3h/0h) for motility, viability and PIA are shown in FIG. 16.

Example 5

Table 5 shows the processing options for example 5.

Table 5

Standard.; staining Collection volume Sorting volume Concentrated
value in SF (normalized to glycerol) Cooling time Cryoprotectant for freezing Glycerin (control) Storage medium (1:3); TALP 2% EY (120 million/ml) TRIS A (7.0 ml) 40 ml 3.5% Glycerin v/v SF 0 minutes 5% Glycerin v/v AB ethylene glycol TRIS A (7.0ml) 40 ml 3.5% EG v/v SF 0 minutes 5% Glycerin v/v AB propylene glycol TRIS A (7.0ml) 40 ml 3.5% PG v/v SF 0 minutes 5% Glycerin v/v AB erythritol TRIS A (7.0ml) 40 ml 3.5% Erythritol v/v SF 0 minutes 5% Glycerin v/v AB erythritol TRIS A (7.0ml) 40 ml 5.0% Erythritol v/v SF 0 minutes 5% Glycerin v/v AB

1. Ejaculates were obtained from four bulls.

2. Checked the quality of the ejaculate, standardized and stained in accordance with the methods described in example 1.

3. Connect the reference sheath fluid and pressurize the sheath fluid reservoir.

4. The stained sample was placed in the sorter and the droplet retention was checked.

5. 1 collection tube with a volume of not more than 40 ml was subjected to mass sorting using sheath liquid (SF) containing 3.5% vol./about. glycerin.

6. 1 collection tube with a volume of not more than 40 ml was subjected to mass sorting using SF containing 3.5% vol./about. ethylene glycol.

7. 1 collection tube with a volume of not more than 40 ml was mass sorted using SF containing 3.5% v/v. propylene glycol

8. 1 collection tube with a volume of not more than 40 ml was mass sorted using SF containing 3.5% v/v. erythritis.

9. 1 collection tube with a volume of not more than 40 ml was mass sorted using SF containing 5.0% v/v. erythritis.

10. All tubes were centrifuged and decanted.

11. AB containing 5% vol./about. glycerol was added to each tube in the amount necessary to obtain 4 million sperm per tube.

12. Diluted semen was stored in a cold room overnight.

13. Sperm cells were placed in test tubes and cryopreserved.

14. Motility (IVOS), viability (PI) and PIA (PNA) were assessed at 0 and 3 hours after thawing. The results are shown in FIG. 17. Convergence (3h/0h) for motility, viability and PIA are presented in FIG. eighteen.

Example 6

Table 6 shows the processing options for example 6.

Table 6

Standard. Color-
ing Collection volume Sorting volume Glycerin in sheath fluid Cooling time Cryoprotectant for freezing Control storage medium (1:3) TALP 2% EY (120 million/ml) TRIS A (3.5ml) 20 ml 0.0% Glycerin v/v SF 90 minutes + Freeze Medium (12% Glycerin v/v) 6.0% Glycerin v/v AB Control 2 TRIS A (7.0ml) 40 ml 3.0% Glycerin v/v SF 0 minutes 5.0% Glycerin v/v AB T1 TRIS A (7.0ml) 40 ml 1.0% Glycerin v/v SF 0 minutes 1.0% Glycerin v/v AB T2 TRIS A (7.0ml) 40 ml 3.0% Glycerin v/v SF 0 minutes 3.0% Glycerin v/v AB T3 TRIS A (7.0ml) 40 ml 5.0% Glycerin v/v SF 0 minutes 5.0% Glycerin v/v AB T4 TRIS A (7.0ml) 40 ml 7.0% Glycerin v/v SF 0 minutes 7.0% Glycerin v/v AB

1. Fresh ejaculates were obtained from four bulls.

2. Checked the quality of the ejaculate, standardized and stained in accordance with the methods described in example 1.

3. Connect the reference sheath fluid and pressurize the sheath fluid reservoir.

4. 1 collection tube with a volume of not more than 40 ml was mass sorted using sheath fluid (SF) containing 0.0% v/v. glycerin (Control).

10. The control tubes were placed in a cold room for 90 minutes and 20 ml of freezing medium (12% v/v glycerol) was added (2 step addition).

11. All tubes were centrifuged and decanted.

12. Enough AB was added to each tube to produce 4 million sperm per tube:

AB 6.0% v/v glycerin (Control)

AB 1.0% v/v glycerol (T1)

AB 3.0% v/v glycerol (T2)

AB 5.0% v/v glycerol (T3 and Control 2)

AB 7.0% v/v glycerol (T4)

13. Diluted semen was stored in a cold room overnight.

14. Sperm cells were placed in test tubes and cryopreserved.

15. Mobility at 0 and 3 hours after thawing (IVOS II) is shown in FIG. 19. Pot life (PI) and PIA (PNA) at 0 and 3 hours after thawing are shown in FIG. twenty.

Example 7

Table 7 shows the processing options for example 7.

Table 7

Standard. Coloring Collection volume Sorting volume Polyol in Sheath Fluid Cooling time Cryoprote-
freezer-
vaniya Control storage medium (1:3) TALP 2% EY (120 million/ml) TRIS A (3.5ml) 20 ml 0.0% Glycerin v/v SF 90 minutes + freezing medium (12% Gly) 6.0% Glycerin v/v AB Control 2 TRIS A (7.0ml) 40 ml 3.0% Glycerin v/v SF 0 minutes 5.0% Glycerin v/v AB T1 TRIS A (7.0ml) 40 ml 1.0% propylene glycol v/v SF 0 minutes 1.0% propylene glycol v/v AB T2 TRIS A (7.0ml) 40 ml 3.0% propylene glycol v/v SF 0 minutes 3.0% propylene glycol v/v AB T3 TRIS A (7.0ml) 40 ml 5.0% propylene glycol v/v SF 0 minutes 5.0% Glycerin v/v AB T4 TRIS A (7.0ml) 40 ml 7.0% propylene glycol v/v SF 0 minutes 7.0% Glycerin v/v AB

1. Fresh ejaculates were obtained from four bulls.

2. Checked the quality of the ejaculate, standardized and stained in accordance with the methods described in example 1.

3. Connect the reference sheath fluid and pressurize the sheath fluid reservoir.

4. 1 collection tube with a volume of not more than 20 ml was subjected to mass sorting using sheath fluid (SF) containing 0.0% v/v. glycerin (Control)

5. 1 collection tube with a volume of not more than 40 ml was mass sorted using SF containing 3.0% v/v. glycerin (Control 2)

6. 1 collection tube with a volume of not more than 40 ml was mass sorted using SF containing 1.0% v/v. propylene glycol (T1)

7. 1 collection tube with a volume of not more than 40 ml was mass sorted using SF containing 3.0% v/v. propylene glycol (T2)

8. 1 collection tube with a volume of not more than 40 ml was mass sorted using SF containing 5.0% v/v. propylene glycol (T3)

9. 1 collection tube with a volume of not more than 40 ml was mass sorted using SF containing 7.0% v/v. propylene glycol (T4)

10. Control tubes were placed in a cold room for 90 minutes and 20 ml of freezing medium (12% v/v glycerol) was added (2 step addition).

11. All tubes were centrifuged and decanted.

12. Enough AB was added to each tube to obtain 4 million sperm per tube:

AB containing 6.0% vol./about. glycerin (Control)

AB containing 5.0% vol./about. glycerin (Control 2)

AB containing 1.0% vol./about. propylene glycol (T1)

AB containing 3.0% vol./about. propylene glycol (T2)

AB containing 5.0% vol./about. propylene glycol (T3)

AB containing 7.0% vol./about. propylene glycol (T4)

13. Diluted semen was stored in a cold room overnight.

14. Sperm cells were placed in test tubes and cryopreserved.

15. Mobility at 0 and 3 hours after thawing (IVOS II) is shown in FIG. 21. Pot life (PI) and PIA (PNA) at 0 and 3 hours after thawing are shown in FIG. 22.

Example 8

Table 8 shows the processing options for example 8.

Table 8

Standard. Coloring Collection volume Sorting volume Glycerin in sheath fluid Cooling time Cryoprotectant for freezing T1 Storage medium (1:3) TALP 2% EY (120 million/ml) TRIS A (7.0ml) 40 ml 3.0% Glycerin v/v SF 0 minutes 3.0% Glycerin v/v AB T2 3.5% Glycerin v/v AB T3 4.0% Glycerin v/v AB T4 4.5% Glycerin v/v AB T5 5.0% Glycerin v/v AB T6 5.5% Glycerin v/v AB T7 6.0% Glycerin v/v AB

1. Fresh ejaculates were obtained from four bulls.

2. Checked the quality of the ejaculate, standardized and stained in accordance with the methods described in example 1.

3. Connect the reference sheath fluid and pressurize the sheath fluid reservoir.

4. The stained sample was placed in a flow cytometer and the droplet delay was checked.

5. 7 collection tubes with a volume of not more than 40 ml were subjected to mass sorting using a sheath liquid containing 3.0% glycerol (SF).

6. All tubes were centrifuged and decanted.

7. Enough AB was added to each tube to obtain 4 million sperm per tube:

AB containing 3.0% vol./about. glycerol (T1)

AB containing 3.5% vol./about. glycerol (T2)

AB containing 4.0% vol./about. glycerol (T3)

AB containing 4.5% vol./about. glycerol (T4)

AB containing 5.0% vol./about. glycerol (T5)

AB containing 5.5% vol./about. glycerol (T6)

AB containing 6.0% vol./about. glycerol (T7)

8. Diluted semen was stored in a cold room overnight.

9. Sperm cells were placed in test tubes and cryopreserved.

10. Mobility at 0 and 3 hours (visual and IVOS II) is shown in FIG. 23 and 25. Viability (PI) and PIA (PNA) at 0 and 3 hours are shown in FIG. 24 and 26. The convergence (3h/0h) for mobility is shown in FIG. 27. Convergences for viability and PIA are presented in FIG. 28.

Example 9

Table 9 shows the processing options for example 9.

Table 9

Collection volume Sorting volume sheath fluid Cooling time Freezing environment Cryoprotectant for freezing Storage time before freezing
nia Control 1 TRIS A (3.5ml) 20 ml (15 million) 0% Glycerin v/v SF 90 minutes Yes (12% v/v glycerol) 6.0% Glycerin v/v AB 2 hours Control 2 During the night T1 Not 2 hours T2 TRIS A (7.0ml) 40 ml (30 million) 3.0% Glycerin v/v SF + AKG (0.0875mg/ml) 0 minutes 4.5% Glycerin v/v AB During the night

1. Fresh ejaculates were obtained from 10 bulls.

2. The quality of the ejaculates was checked, standardized and stained in accordance with the methods described in example 1.

3. Connect the reference sheath fluid and pressurize the sheath fluid reservoir.

4. 2 collection tubes with a volume of not more than 20 ml (or 15 million semen) were mass sorted using sheath fluid containing 0.0% v/v. glycerin (SF).

5. The tubes were placed in a cold room for 90 minutes and 20 ml of freezing medium (12% v/v glycerol) was added (2 step addition).

6. Connect sheath fluid for treatment, and pressurize reservoir with sheath fluid

7. The stained sample was placed in a flow cytometer and the droplet delay was checked.

8. 1 collection tube with a volume of not more than 40 ml (or 30 million semen) was mass sorted using SF containing 3.0% v/v. glycerol + alpha-ketoglutarate (AKG).

9. All tubes were centrifuged and decanted.

10. AB was added to each tube:

1200 μl of AB containing 6.0% v/v. glycerin (Control 1 and Control 2)

1200 μl of AB containing 4.5% v/v. glycerol (T1 and T2)

11. Control 1 and T1 sperm were kept in AB for 2 hours and then cryopreserved in IVF tubes (2 tubes per treatment with 4 million sperm/tube).

12. Control 2 and T2 were kept in AB overnight and then cryopreserved in IVF tubes (2 tubes per treatment with 4 million sperm/tube).

13. Mobility at 0 and 3 hours (visual and IVOS), viability (PI), PIA (PNA) and convergence (0h/3h) were assessed after thawing. Mobility at 0 h (visual and IVOS), viability and PIA are shown in FIG. 29. Motility at 3 hours (visual and IVOS), viability and PIA are shown in FIG. 30. Convergence (3h/0h) for motility, viability and PIA are shown in FIG. 31.

Example 10

Table 10 shows the processing options for example 10.

Table 10

Mill-
dart. Coloring-
nie Collection volume Sorting volume Glitse-
rin in SF ACG Cooling time
denia Freezing environment Cryopro-
tektor for freezing -
vaniya Storage time before freezing
animating T1- Control role storage medium (1:3) TALP 2% EY (120m/
ml) TRISA (3.5ml) 20 ml (15 million, semen) 0% v/v Not 90 minutes Yes (12% v/v glyce-
rina) 6.0% Glyce-
Rin v/v AB 2 hours T2-3% Glycerol SF TRISA (7.0ml) 40 ml (30 million, semen) 3% v/v Yes
(0.0875 mg/ml) 0 minutes Not 4.5% Glyce-
Rin v/v AB 2 hours

1. Fresh ejaculates were obtained from three bulls.

2. Checked the quality of the ejaculate, standardized and stained in accordance with the methods described in example 1.

3. Treatment sheath fluid 1 (Control) (without glycerin) was connected, and the sheath fluid reservoir was pressurized.

4. 4 control collection tubes with a volume of not more than 20 ml (or 15 million semen) were mass sorted.

5. The tubes were placed in a cold room for 90 minutes and 20 ml of freezing medium (12% v/v glycerol) was added (2 step addition).

6. Treatment sheath fluid 2 (3% v/v glycerol with ACG) was connected and the reservoir was pressurized.

7. 2 collection tubes with a volume of not more than 40 ml (or 30 million semen) were mass sorted.

8. Then all tubes were centrifuged and decanted. The pellet for T1 (4 tubes) was pooled. The pellet for T2 (2 tubes) was pooled.

9. AB was added to each tube:

AB containing 6.0% vol./about. glycerin, for processing 1

AB containing 4.5% vol./about. glycerin, for processing 2

10. Diluted sperm were kept in AB for 2 hours and then cryopreserved in IVF tubes (4 million sperm/tube).

11. 200 oocytes per treatment were then fertilized by IVF. IVF results are shown in Fig. 32.

Example 11

Example 11 consists of a field test to test the degree of conception achieved with sexed seminal fluid sorted using sheath fluid containing 3% v/v. glycerol and 0.0875 mg/ml alpha-ketoglutarate. 5 sires and more than 3000 heifers (up to the 3rd lactation) were used, which received from 1 to 3 artificial inseminations with 90% female seminal fluid.

The results of Example 11 are shown in FIG. 33 and 34. In FIG. 33 shows post-thaw motility, PIA, convergence and viability of semen used in field trials. In FIG. 34 shows the achieved conception rates.

Example 12

Example 12 tested the effectiveness of a dilute glycerol solution (“cryopreservative”) that, when mixed with a calculated amount of TRIS A (TRIS + 20% egg yolk), converted to AB, eliminating the need for pure glycerol in semen sorting laboratories.

Table 11 shows the processing options for example 12.

Table 11

Glycerin (v/v) Grams TRIS A Grams of glycerin stock solution AB total EY% mOsm twenty% 32.761 9.959 42.720 13.9 1077 mOsm 45% 40.183 4.937 45.120 17.5 1084 mOsm 65% 43.325 3.715 47.040 18.3 1085 mOsm 100% 47.628 2.772 50,400 19.1 1090 mOsm

1. Five ejaculates were obtained from a bull.

2. Checked the quality of the ejaculate, standardized and stained in accordance with the methods described in example 1.

3. 4 collection tubes were mass sorted using sheath fluid containing 3.0% v/v. glycerin + alpha-ketoglutarate (AKG).

4. The tubes were placed in a cold room for 15 minutes.

5. All tubes were centrifuged and decanted and the pellets were pooled.

6. The precipitates were divided into 5 tubes containing AB with 4.5% glycerol, obtaining the following:

Control - prepared using pure glycerol (100% v/v)

Treatment 1 - prepared with 20% cryo-preservative

Treatment 2 - prepared using 45% cryopreservative

Treatment 3 - prepared using 65% cryopreservative

7. Sperm was kept overnight and frozen.

8. Motility (visual and IVOS), viability (PI) and PIA (PNA) were determined at 0 and 3 hours.

The results of Example 12 are shown in FIG. 35, which show a beneficial effect when using AB in a cryo-preservative (diluted glycerol) instead of 100% v/v. glycerin.

Example 13

Table 12 shows the processing options for example 13.

Table 12

Glycerin in the liquid to collect vol./about. Sorting volume Glycerin in SF v/v Cooling time Freezing environment Cryoprotector Control 0% 20 ml
(15 million) 0% 90 minutes Yes (12% v/v glycerol) 6.0% Glycerin AB Glycerin in collection liquid 2.4% 40 ml
(30 million) 3% 15 minutes Not 4.5% Glycerin AB

1. Ejaculates were obtained from 8 bulls.

2. Checked the quality of the ejaculate, standardized and stained in accordance with the methods described in example 1.

3. Control - Sheath (0.0% glycerol) was used on sorter A for bulls 1-4, and grader B for bulls 5-8.

1 collection tube with a volume of not more than 20 ml (or 15 million) per bull was mass sorted in Tris A (0.0% glycerol, no ACG).

The tubes were placed in a cold room for 90 minutes. Freezing medium (12% v/v glycerol) was added after cooling.

4. Treatment - A sheath containing 3.0% glycerol and 0.0875 mg/mL AKG was used on grader B for bulls 1-4 and grader A for bulls 5-8.

1 collection tube with a volume of not more than 40 ml (or 30 million) for one bull was subjected to mass sorting in a capture fluid containing 2.4% vol./about. glycerin.

The tube was placed in a cold room for 15 minutes.

5. All tubes were centrifuged and decanted.

6. To the control tubes and processing tubes, respectively, was added:

AB containing 6.0% glycerol into control tubes

AB containing 4.5% glycerol in processing tubes

7. Sperm was kept in AB for 12 hours (overnight) and then frozen.

8. Motility, visual and IVOS, viability (PI) and PIA (PNA) at 0 and 3 hours were determined. Convergence was calculated for each parameter.

The results for Example 13 are shown in FIG. 36.

Example 14

Example 14 demonstrates that up to 400 mM erythritol can be used in fresh bovine extender with good motility support in bovine semen over a 4 day period. This is feasible with "regular" (unsorted) sperm stained with Hoechst 33342 (to facilitate CASA using fluorescence).

The fresh diluent was a caprogen-like medium containing 5% (v/v) egg yolk, which was sparged with nitrogen gas prior to semen dilution.

Caprogen with glycerin is one of the media compared in this example and consists of the following components:

Tribasic sodium citrate dihydrate, 20.0 grams, citric acid monohydrate, 0.25 grams, glycine, 10.0 grams, D-(+)-glucose, 3.0 grams, dibasic potassium phosphate anhydrous, 0.609 grams, n-hexanoic acid (caproic acid), 0.231 ml, cyanocobalamin, 0.250 grams, disodium alphaketoglutarate dihydrate, 0.35 grams, trehalose dihydrate, 0.750 grams, glycerin, 10.0 ml, streptomycin sulfate, 0.150 grams, gentamicin, 0.500 grams, tylosin, 0.100 grams, lincomycin, 0.300 grams, spectinomycin, 0.500 grams, chicken egg yolk, 5.0 ml.

Erythritol-supplemented caprogen is another medium compared in this example and consists of the following components:

Tribasic sodium citrate dihydrate, 20.0 grams, citric acid monohydrate, 0.225 grams, glycine, 8.653 grams, D-(+)-glucose, 2.50 grams, dibasic potassium phosphate anhydrous, 0.609 grams, n-hexanoic acid sodium salt ( caproic acid) 0.276 grams, cyanocobalamin, 0.250 grams, disodium alphaketoglutarate dihydrate, trehalose dihydrate, 0.750 grams, 0.35 grams, erythritol, 14.64 grams, streptomycin sulfate, 0.150 grams, gentamicin, 0.500 grams, tylosin, 0.100 grams, lincomycin , 0.300 grams, spectinomycin, 0.500 grams, chicken egg yolk, 5.0 ml.

In glycerol-added caprogen, the glycine concentration is 135 mmol and the glycerol concentration is 135 mmol. Control medium 1 was prepared with the same concentrations, while the balance of the test medium (media 2-7) was performed with the following concentrations, as shown in Table 13:

Table 13

Concentration of Added Media Components Glycerol Glycine erythritol Wednesday mmol mmol mmol one 135 135 0 2 0 135 0 3 0 135 35 four 0 135 65 5 0 135 135 6 0 135 270 7 0 135 400

Fresh bovine ejaculates from 5 bulls were diluted to 160 million semen per ml in TALP stain combined with Hoechst 33342 DNA stain, incubated for 60 minutes at 34°C (as standard stain for XY semen sorting). After staining, sperm in TALP were diluted in the appropriate medium to a new concentration of 15 million sperm per ml and placed in 0.25 cm ³ tubes, sealed and kept in a horizontal position at 18°C for 4 days. Each day, one vial of semen was heated for 15 minutes at 34° C. and the contents of about 200 microliters from the vial were combined with 100 microliters of fresh medium, identical to the specified test medium used for storing sperm. Semen was analyzed on fluorescent CASA (Hamilton Thome IVOS II) using UV light. Average total and progressive mobility for 5 bulls are summarized in Table 14 and in FIG. 37.

Table 14

Wednesday number Storage days one 2 3 four General
mobile
ness Progress
sivnaya
mobile
ness General
mobile
ness Progress
sivnaya
mobile
ness General
mobile
ness Progress
sivnaya
mobile
ness General
mobile
ness Progress
sivnaya
mobile
ness one 89% 75% 66% 47% 62% 47% 37% 25% 2 74% 68% 68% 53% 56% 45% 46% 37% 3 73% 65% 65% 57% 59% 48% 42% 31% four 73% 65% 69% 55% 52% 41% 33% 26% 5 68% 61% 73% 56% 45% 38% 38% 27% 6 78% 70% 63% 52% fifty% 42% 43% 29% 7 69% 57% 65% 44% 44% 29% 53% 34%

This test shows that 135 mM glycerol has a beneficial effect on maintenance of sperm motility for up to 4 days, and that replacing glycerol with erythritol in fresh medium with caprogen provides similar support for sperm motility, with the beneficial effects of erythritol occurring beyond 4 days. .

Example 15

Example 15 demonstrates that in order to increase the shelf life of graded bovine semen to nine days at 17° C., glycerol in the standard caprogen formulation can be completely replaced with erythritol to allow longer semen storage time when comparing visual and CASA motility. The standard caprogen (with glycerol) is called "CaproGLY" and the caprogen containing erythritol is called "CaproERY".

Preparation of the medium in a volume of 100 ml: 95 ml of the appropriate working medium solution was mixed with 5 ml of egg yolk, stirred for 15 minutes, kept overnight at 4°C, centrifuged for 60 minutes at 1200 G in 50 ml Falcon tubes (clarified ). On the day of application, nitrogen gas was bubbled through the clarified medium for 15 minutes. Table 15 shows the properties of 2 media.

Table 15

pH mOsm mM glycine mM glycerol mM erythritol CaproGLY 7.01 512 133 133 CaproERY 7.03 482 120 120

Semen sorting should be carried out in two fresh diluents: fresh ejaculates from 3 Jersey bulls were mass sorted according to standard proprietary sex sorting methods disclosed in other patents. Briefly, semen was washed, concentrated, and resuspended in Hepes, a citrate-buffered TRIS diluent, to normalize the concentration to about 1200 sperm per ml, and a pH of about 7.15. Each 1 ml of stain contains: 0.150 ml of sperm solution (approximately 160 million sperm), 64 nM Hoechst 33342 in 8 microliters, 592 ml of TALP stain and 100 nM of FD&C food coloring. The sample was incubated in a 4 ml sample tube at 34°C for 60 minutes, after which 0.25 ml of 8% (v/v) TALP with egg yolk was added, which diluted the semen to about 120 million per ml and provided a final egg yolk concentration of 2% for the sample to be sorted.

Freshly stained sperm are subjected to mass sorting at a rate of approximately 40,000 events per second, where mass sorting means collecting prone to survival sperm containing both X and Y chromosomes, it is clear that the sorted sperm is equivalent in quality to standard sorted sperm, but not sex-enriched . 80 million semen from each of 3 Jersey bulls were sorted in TRIS A capture fluid, chilled for a minimum of 90 minutes in a cold cabinet (typical temperature 4-7°C), mixed in equal volumes with yolk-free TRIS B solution , concentrated by centrifugation and extraction of the semen sediment and determining the final concentration (concentrated semen) in the range of 100-115 million semen per ml. Then 3 volumes of CaproGLY and CaproERY were combined with 1 volume of concentrated semen to a final concentration obtained of about 20-24 million per ml (about 5.0-5.5 million sperm per 0.25 cm ³ tube).

The prepared sperm was filled into test tubes with a volume of 0.25 cm ³ , hermetically sealed and stored in a horizontal position at 17°C for no more than nine days. On the indicated test days, the tube is opened and visually inspected for motility (calculated with the eyes of a specialist using microscopic analysis during the heating stage), total CASA motility, and progressive CASA motility using the Hamilton Thome IVOS system. In addition to the tube, 0.5 ml of each of the six samples were stored in a 4 ml sample tube at 17° C. for comparison on the eighth day.

Table 16 shows the average movements for three Jersey bulls on different days.

Table 16

Treatment Day Storage visual mobility General mobility CASA Prog. CASA mobility CASA prog/total CaproERY one test tube 68% 76% 55% 72% CaproGLY one test tube 68% 73% 53% 73% CaproERY 2 test tube 61% 66% 51% 77% CaproGLY 2 test tube 58% 61% 44% 72% CaproERY 3 test tube 71% 73% 48% 65% CaproGLY 3 test tube 68% 64% 45% 69% CaproERY four test tube 67% 68% 48% 71% CaproGLY four test tube 51% 51% 35% 68% CaproERY 7 test tube 63% 59% 45% 76% CaproGLY 7 test tube 33% 39% 29% 74% CaproERY eight test tube 55% 52% 35% 68% CaproGLY eight test tube 23% 16% four% 28% CaproERY eight test tube 65% 61% 55% 89% CaproGLY eight test tube 31% thirty% eleven% 38% CaproERY 9 test tube 47% 42% 34% 81% CaproGLY 9 test tube twenty% 9% 2% 23%

The above results show that when the principle of equal molarity of glycine and accompanying polyol (glycerol or erythritol) is followed, complete replacement of glycerol with erythritol provides a fresh semen diluent comparable to caprogen that is as good or better than that prepared with glycerol. If the storage time exceeds 4 days, erythritol retains its quality better, which is manifested in mobility. It also turned out that storage in test tubes may be better than in IVF tubes.

Example 16

Example 16 demonstrates that sex-sorted semen stored in caprogen medium using erythritol instead of glycerol is able to provide a number of pregnancies similar to standard caprogen.

In order to pre-evaluate the possible fertility outcomes with CaproERY as a replacement under conditions in which CaproGLY performed well, a small number (approximately 145) sex-sorted semen stored in CaproGLY. As a rule, heifers were fertilized with fresh diluted seminal fluid within 1-2 days from the date of sorting and receiving the composition.

Table 17 below summarizes the results for the CaproERY field study.

Total number of fertilized heifers Total pregnant women Percentage of pregnancy CaproGLY 277 159 57.4% CaproERY 145 77 53.1%

In this field study, no separate treatment was applied, there was not an evenly distributed number of tubes for each treatment, and it was not evenly distributed between bulls and farms. Accordingly, the relative nominal values (57.4% versus 53.1%) are not statistically different. Both values are within the standard range observed for CaproGLY over a 40-month time period and, accordingly, CaproERY provides the same fertility.

Example 17

Example 17 demonstrates that erythritol can be used (with or without glycine) in semen sorting sheath fluid or freezing diluents at concentrations from 15 to 110 mM and is similar or better than standard erythritol-free control media.

Standard bovine semen sorting used a sheath containing 68 mM fructose, 80 mM citrate and 243 mM TRIS at pH 6.80 and osmolarity 290-300 mOsm. (Control sheath fluid). Combining 80 volumes of this control sheath with 20 volumes of fresh egg yolk, followed by clarification, results in a CONTROL cooling solution called TRIS A (“Fraction A”). Combining 88 volumes of TRIS A with 12 volumes of pure glycerol results in a solution called TRIS B ("Fraction B"). In the case of standard semen sorting, "Fraction B" may not necessarily contain egg yolk, and may be referred to as "Fraction B without eggs", still containing 12 volumes of pure glycerol.

Table 18 shows the millimolar concentrations of the components used in this test, where the control medium does not contain glycine or erythritol, and the test samples contain varying amounts of erythritol and in some cases glycine. The pH of all media was 6.70-6.75 and the osmolarity was 290-305 mOsm.

Table 18

visual mobility Freezing concentrations in mM 0 h 3 h erythritol Glycine Fructose Citrate TRIS Control 36% 27% 0 0 54 64 194 A 42% 34% 51 fifty thirty 35 106 AT 40% 24% 108 0 thirty 36 109 FROM 42% 31% 53 46 thirty 35 105 D 42% 36% 35 34 thirty 43 130 E 54% 35% 25 0 51 45 138 F 37% 31% fourteen fourteen thirty 63 190 G 41% 40% 25 0 thirty 61 186 visual mobility Concentration (mM) in sheath fluid 0 h 3 h erythritol Glycine Fructose Citrate TRIS Control 35% 23% 0 0 68 80 243 H 35% 28% 62 0 61 33 102 I 47% 28% 35 35 eight 75 235 J 47% 37% 65 0 eight 72 223

The same two bulls were used for each of the two tests, and the quality after thawing was determined at the same time. The first test was performed with unstained unsorted semen in a manner similar to the standard "normal freezing". The second test was performed using different semen sorting sheaths.

The first test compared a control (standard medium) with 7 alternative media (AG) with formulations containing varying amounts of erythritol and in some cases glycine. 10 milliliters of semen from each bull at a concentration of 100 million semen per milliliter was cooled in standard TRIS A (control medium) for 90 minutes from room temperature to about 4-6°C. The samples were then divided into 1 ml aliquots and 0.5 ml of cold TEST-B medium was added, held for 15 minutes before an additional 0.5 ml of TEST-B medium was added. This resulted in a final semen concentration of 50 million per ml. These samples were loaded into 0.25 ^cc cryopreservation tubes and frozen using the controlled frosting method commonly used to freeze normal seminal fluid.

Glycerol concentrations in TEST-B medium were always 6.0% (v/v) resulting in final glycerol concentrations of 3.0% (v/v). All TEST-B media contained 0% egg yolk resulting in a final egg yolk concentration of 10% (v/v). The concentrations of the chemical components at the time of freezing are shown in the table (CONTROL vs. A-G). Under these conditions, the semen was cooled in a standard amount of egg yolk (20%), cryopreserving them in 1/2 of the standard amount of egg yolk and glycerol. The results of visual mobility assessment show that under these control conditions, all 7 media containing erythritol maintained cryopreservation as well as or better than the control without any amount of erythritol. The results show that no more than about 110 mM erythritol can be used in freezing bovine semen, with optimal freezing results achieved with 15-50 mM erythritol.

The second test compared a control (standard) sheath fluid with three alternative sheath fluids (HJ). The compositions of all flowing fluids are given in the table. After sorting, all samples were processed identically to standard semen sorting. A volume of 3.5 ml of capture fluid (TRIS A) was placed in each collection tube and sorted semen was sorted into a 20 ml collection tube. This volume of liquid was then cooled over 90 minutes to about 4-6° C., then an equal volume of cold egg-free fraction B was added. Sperm cells were concentrated by centrifugation and removal of the supernatant and resuspended in the TRIS AB fraction containing egg yolk, prepared by mixing equal volumes of fraction A and fraction B. Cells were prepared at 18 ppm and frozen in 0.25 cm ³ tubes by controlled frosting , which is usually used to freeze ordinary seminal fluid (same as above).

The results indicate that up to 60 mM erythritol may be present in the sheath fluid, which may be beneficial for sperm quality in a standard semen processing method after sorting.

As can be easily understood from the foregoing, the basic concepts of the present invention can be implemented in various ways. The present invention includes numerous and varied embodiments of semen processing, including, but not limited to, the best embodiment of the invention.

Thus, the specific embodiments or elements of the invention disclosed in the description or depicted in the figures or tables accompanying this application are not intended to be limiting, but are to be regarded rather as illustrative examples of the many and varied embodiments generally included within the scope of the invention, or equivalents included in the scope in relation to any particular element of the invention. In addition, a specific description of one embodiment or element of the invention may not explicitly describe all possible implementations or elements; many alternatives are implicitly disclosed in the description and in the figures.

It should be understood that each element of the apparatus or each step of the method may be described by the term "apparatus" or the term "method". Such terms may be substituted as desired to specify the broad, implied scope to which this invention is entitled. As one example, it should be understood that all steps of a method can be described as an act, a means for performing that act, or as an element that performs that act. Likewise, each element of the apparatus can be described as a physical element or an action facilitated by that physical element. As one example, it is to be understood that a description of a "sorting device" includes a description of a "sorting" action—either explicitly or implicitly—and conversely, if there is an actual description of a "sorting" action, such a description should be understood to include description of "sorting device" and even "sorting means". It should be understood that such alternative terms for each element or step are expressly included in the description.

In addition, with respect to the use of each term, it is to be understood that, unless its use in this application conflicts with such interpretation, the generally accepted dictionary definitions are to be understood as included in the description for each term, in the meaning indicated in the second edition of Random House Webster's Dictionary. Unabridged Dictionary", and each definition is hereby incorporated into this application by reference.

Moreover, for the purposes of the present invention, indefinite articles before an object refer to the singular or plural of that object. Thus, the indefinite articles, the phrases "one or more" and "at least one" can be used interchangeably herein.

All numerical values are intended to be modified by the term "about", whether or not it is explicitly stated. For purposes of the present invention, ranges may be expressed as being from "about" one specific value to "about" another specific value. When such a range is expressed, another embodiment includes from one specific value to another specific value. The enumeration of numerical ranges by finite values includes all numerical values that are within this range. The numerical range from one to five includes, for example, the numerical values 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, and so on. It should also be understood that the end values of each range are significant both with respect to the other end value and independently of the other end value. When a value is expressed as an approximation by using the preceding word "about", it is to be understood that the specific value constitutes another embodiment.

The Background section of this patent application provides a summary of the technical field to which the invention pertains. This section may also include or contain summaries of certain US patents, patent applications, publications, or subject matter of the claimed invention that are useful in connection with information, problems, or issues relating to the state of the technology to which the invention pertains. It is not intended that any US patent, patent application, publication, assertion, or other information cited or contained herein be construed, construed, or taken as prior art with respect to the invention.

The claims set forth in this application are hereby incorporated herein by reference, and the Applicant expressly reserves the right to use, in whole or in part, this incorporated content of such claims as an additional description to support any claim or all claims. or any of its elements or components, and the applicant also expressly reserves the right to move any part of the content or all included content of such claims, or any element or component thereof, from the description to the claims or vice versa, as necessary to determine the subject matter, for which protection is sought by this application or by any subsequent application or continuation application, divisional application or continuation application in part, or to obtain any advantage, reduction of payments thereunder, or to comply with patent laws, regulations, or and the regulations of any country or any treaty, and such content, incorporated by reference, shall survive the entire consideration of this application, including any subsequent continuation application, divisional application or continuation in part, or any application for a patent reissue or extension of the scope of claims.

Claims (16)

1. A method for processing sperm, including: staining a sperm sample containing viable sperm bearing the X chromosome and viable sperm bearing the Y chromosome with a staining medium; bringing the stained sperm sample into contact with a sheath fluid in the stream; and manipulating the ratio of viable X-chromosome-bearing sperm to Y-chromosome-bearing viable sperm to form at least one manipulated sperm population, characterized in that one or more of the sheath fluid and staining medium contains a cryoprotectant at a concentration of 0.1% to 6% v/v or w/v, where the cryoprotectant contains a polyhydric alcohol selected from the group consisting of: propylene glycol; butanetriol; ethylene glycol; glycerin; erythritis; treita; Arabita; ribite; xylitol; sorbitol; galactite; go; volemite; fucita; inositol; glycylglycite and combinations thereof. 2. The method of claim 1, further comprising the step of collecting the manipulated sperm population in the collection medium. 3. The method of claim 2 wherein the collection medium contains a cryoprotectant. 4. The method of claim 2, wherein each of the staining medium, the sheath liquid and the collection medium contains a cryoprotectant. 5. The method according to claim 1, wherein the step of manipulating the ratio of viable X-chromosome-bearing sperm to Y-chromosome-bearing viable sperm to obtain at least one manipulated sperm population further comprises selecting sperm for separation and collection. or photodamage and collection. 6. The method of claim 1, further comprising the step of freezing the manipulated sperm sample. 7. The method according to p. 1, further comprising one or more of the following processing steps: keeping; transportation; buffering; cooling; the heating; dilution; concentration; laser excitation; loading; abduction; ablation; collection; shaking; oscillation; magnetic separation; oxygen saturation; marking; precipitation; centrifugation; resuspension; mixing; dialysis; cryostabilization; microchip processing; and flow cytometry. 8. The method according to p. 1, characterized in that the stage of processing and the stage of bringing the colored sperm sample into contact with the sheath fluid further includes: injecting the colored sperm sample into the flow of the sheath fluid; exposing the stained sperm sample in the sheath fluid to an electromagnetic radiation source that elicits a detectable DNA selective dye response; detecting the response of the DNA-selective dye to the effect of electromagnetic radiation; analysis of the detected response; semen classification in the semen sample based on the analysis of the detected response; and collecting one or more viable semen subpopulations from the semen sample into one or more collection media based on semen classification. 9. The method of claim 8, further comprising the step of electromagnetically changing the direction of the sperm-containing droplets based on the classification. 10. The method of claim. 1, characterized in that at least one of the sheath fluid and medium for staining additionally contains an antioxidant. 11. The method according to p. 10, characterized in that the antioxidant includes an antioxidant selected from the group consisting of: pyruvate, vitamin B12; vitamers of vitamin B12; vitamin E; vitamin E vitamers; tocopherol; tocotrienol; α-tocopheryl; alpha-ketoglutarate; their derivatives and their combinations. 12. The method according to p. 1, characterized in that the flowing fluid contains a cryoprotectant in about./about. or wt./about. concentrations from 0.1% to 2%; from 2% to 4%; from 4% to 6%; from 1% to 2%; from 2% to 3%; from 3% to 4%; from 4% to 5%; from 5% to 6%; 2% to 6% or 3% to 5%. 13. The method of claim 1 wherein the sheath fluid contains phosphate buffered saline, TRIS citrate, or HEPES. 14. The method according to claim 1, characterized in that the cryoprotectant is added to the staining medium in the first amount and added to the flowing liquid in the second amount, the second amount being greater than the first amount. 15. The method according to p. 1, characterized in that the staining medium contains a cryoprotectant at a concentration of from 0.1% to 5% v/v. or wt./about. 16. The method according to p. 1, characterized in that the staining medium contains a cryoprotectant in about./about. or wt./about. concentrations from 0.1% to 1%; from 1% to 2%; from 2% to 3%; from 3% to 4%; 2% to 4% or 1.5% to 3%.

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