UA77641C2 - Insemination of mammals, while controlling the sex, with small amount of spermatozoa - Google Patents

Abstract

The present invention relates to flowing cytometry systems that are designed for emitting required cells, specifically generative cells. The said systems are used in combination with different means that provide a possibility to reduce the loss of sparmatozoa to a minimal level. Such means include 2.9 % sodium citrate solution and medium containing generative cells of a bull.

Description

Description of the invention

This invention generally relates to the field of sexual selection of mammalian offspring. It specifically addresses 2 aspects of low-dose artificial insemination and increasing egg production to produce sexed offspring. In particular, the invention relates to the provision of artificial insemination with sex control at low doses regardless of sorting methods, sperm sorting systems by the flow cytometry method for artificial insemination with sex control at low doses and methods of increasing ovulation results, or similar topics. 70 For centuries it was desirable to choose the sex of a particular offspring. Apart from the obvious psychological aspects, effective control of the sex of mammalian offspring has significant economic implications when applied to food producing animals such as cattle as well as prized prize animals such as horses and the like. This great desire caused various attempts to ensure offspring of the desired sex. Perhaps the attempts that seem closest to achieving the desired results have been attempts 12 to sort and select X and Y sperm prior to insemination.

One of the problems encountered in trying to sort X and Y sperm was the large number of sperm. During natural insemination, billions of sperm are produced in some species; artificial insemination uses a smaller but still significant number of sperm. For example, artificial insemination methods usually use from ten million to five hundred million sperm (depending on the species). Thus, even under the conditions of artificial insemination, a significant number of spermatozoa is required.

Attempts have been made to use many methods to ensure the separation of spermatozoa that carry the X- and Y-chromosome. These techniques have included the use of various separation techniques, from magnetic separation techniques such as those described in U.S. Patent No. 4,276,139 to column separation techniques as described in U.S. Patent No. 5,514,537, gravimetric techniques as described in U.S. Pat. with 4092229, 4067965 and 4155831. Attempts were made to use the electrical properties, as described in the patent Ge)

U.S. Mo. 4,083,957, and combinations of electrical and gravimetric properties as described in U.S. Pat.

MoMo 4225405, 4698142 and 4749458. Attempts have also been made to use mobility as described in US Pat.

MoMo 4009260 and 4339434, Chemical techniques such as those described in US MoMo 4511661 and 4999283 (using monoclonal antibodies) and US MoMo 5021244, 5346990, 5439362 and 5660997 (using membrane proteins), and US MoMo 3687803, 4,191,749, 4,448,767 and 4,680,258 (with "And using antibodies) and the addition of serum components as described in US Pat. No. 4,085,205. Although each of these techniques has been described as highly effective, in fact none of these techniques currently provide the desired level of preliminary sex determination. However, regardless of the /-separation technique ultimately used, the competing combination of a large number of sperm under natural conditions and an approach to separating 3o spermatozoa that carry the X- and Y-chromosome made it desirable to develop methods to ensure insemination with fewer sperm.

Currently, the only quantitative technique used to separate X- and Y-bearing spermatozoa is one that involves individual discrimination and separation of "spermatozoa" by flow cytometry. This technique became possible thanks to the achievements and discoveries related to the differential absorption of the dye by spermatozoa carrying X- and X-chromosomes. It was first described in US patent No. 4,362,246 and has been greatly expanded using techniques described by I am/hepse

I" U9oppsop in the US patent Mo5135759. .doppzop's technique of using flow cytometry to separate X- and Y-chromosome-carrying spermatozoa was an extremely significant advance, making commercial separation of such spermatozoa possible for the first time. While still in the experimental stage, separation has been greatly improved using high-speed flow cytometers, such as the MoRioF flow cytometer manufactured by Suotaiop, Ips., as described in various other patents, including U.S. Patent Nos. 5,150,313, 5,602,039, 5,602,349, and 5,643,796. , as well as in the international patent publication PCT UMO 96/12171.

Although the use of MoBio(F) cytometers from Suiotayop has made it possible to significantly increase the speed, and although this increase in speed is very appropriate in view of the large number of spermatozoa that are often used, certain problems still remain. Despite the almost tenfold increase in speed that is possible with using the current MoBRiobF flow cytometer, even shorter times and sorting are desirable for several reasons. First, it was found that, from a practical point of view, spermatozoa are time-critical cells. The longer they remain unused, the more they lose their efficiency Second, the schedule of collection, sorting and insemination makes speed a factor of high commercial importance 29. Therefore, the dependence of sperm cell properties and process results

GF) from time to time makes speed an essential element in ensuring high efficiency and the rate of successful attempts. yuyu There are also other problems, from practical to theoretical. From a practical point of view, it was desirable to obtain sex-sorted sperm samples using inexpensive consumable components and substances. Also, taking into account the costs, it was desirable to carry out sorting (as well as harvesting and insemination) with 60 as little effort as possible. Therefore, for commercial production and success under practical conditions, any improvements that can lead to increased efficiency can be significant. The practical aspect of costs is connected with the practical aspect of delicacy and sensitivity of the whole process. In this regard, it would be desirable to simplify the process and make it procedurally as reliable as possible to reduce the role of operator error or skill. Together, they also make low-dose insemination even more desirable. for In addition to the delicacy of the process, it has always been known that the spermatozoa themselves are extremely delicate cells. Although at first glance this factor seems to be easily understood, in fact, the sensitivity of cells has not yet been fully investigated. In general, in the context of flow cytometry, most sorted cells or particles are often spherical or capable of other physical characteristics to resist various harmful factors. In the case of sperm, this is not the case. Indeed, as described in the present invention, processing using conventional flow cytometry techniques may not be acceptable in certain cases for cytometric sorting of spermatozoa. Various sensitivities, ranging from sensitivity to dilution and the need to isolate and distinguish each cell individually in flow cytometry, as well as the pressure and other stresses inherent in typical flow cytometry, have, prior to the present invention, greatly affected 7/0 cells or other substances, that were subject to sorting. In the case of spermatozoa, this factor can be of particular importance, because even if it appears that the spermatozoa have been subjected to flow cytometry and sorting without any visually noticeable side effects, in reality the cells themselves may have been so stressed that during the insemination process their performance will be below optimal . So, probably, from the point of view of sperm sorting and their final use for artificial insemination, there is an interaction of factors, which /5 leads to the emergence of unusual problems.

Another problem that remains is despite the great success that has been achieved thanks to the patent

Schoppzop and related technologies - there is the fact that, prior to this invention, it was extremely difficult to ensure insemination with smaller doses of sexed spermatozoa, regardless of the separation technology used. Although there has historically been some success in low-dose insemination, it has been largely theoretical or laboratory-based, not under conditions likely to exist or be applicable to commercial use. In this regard, there is a desire not simply to provide low-dose insemination, but to provide low-dose insemination with fertility rates comparable to existing attempts at high-dose artificial insemination without sex selection.

Thus, the successes achieved by the authors of this invention are significant achievements that can be made for the first time by

Commercial use is possible.

Another problem faced by specialists in this area is also despite the great successes that o); was achieved thanks to the Chdoppzop patent and related technologies - there is the fact that the problem itself, namely artificial insemination with high fertility rates, is statistical in nature and related to the interaction of many factors. Therefore, the proposed solutions may, to a certain extent, include combinations of various factors, the necessity of which, individually or in combination with other factors, will be shown by careful statistical analysis. Such a definition is further complicated by the fact that the results themselves also vary between species and are difficult to estimate because testing and statistical sampling on a large enough Ge database is unlikely to justify the effort in the early stages. For these reasons, the invention may also include a combination of factors that may, individually or in combination, provide a suitable solution for a particular i-

From Tasks. Thus, this description may be considered broad enough to provide for various combinations and combinations of the techniques described. There may be undetected synergies with other factors. Such factors can range from factors related to sorting stages or perhaps flow cytometry to factors related to the harvesting stage as well as insemination. To date, studies have been conducted mainly on bovine animals, but it is believed that these methods are not limited to these species, or, for that matter, only to spermatozoa. It is believed that the used techniques can be used not only for spermatozoa in areas related to the sorting of sensitive objects or simply to minimize the impact of stress during current cytometry on the objects being sorted. )" Interestingly, while this invention is aimed at reducing the effects of sorting or stress on sperm, others have moved away from this direction, increasing the pressure and requirements for speed and other performance indicators. In essence, the pursuit of low-dose insemination and high-rate -I processing may, individually or perhaps in an interrelated manner, pose mutually limiting problems. Therefore, although the need for high-speed insemination with low doses with sex control has been felt for a long time, and although the methods and elements necessary for its implementation have been available for a long time, before this invention, experts did not

Ge" noted achievements or perhaps combinations of achievements. Perhaps, to some extent, they failed to understand that this problem is related to the interplay of factors, as well as the special requirements for the types of cells (sperm or, perhaps, species-specific sperm) used in this field. It is interesting that, as shown by the above

Ck above the list of works, considerable efforts have been made, but they have apparently failed to make clear the problem inherent in such a field as sex-controlled low-dose insemination and perhaps the assumption has been made that since the natural act of insemination involves perhaps billions of sperm, then there may be 5B Physical limitations to achieving artificial insemination using quantities that are four orders of magnitude smaller. Therefore, there is nothing surprising in the fact that there was an opinion that differed from the technical IF, direction of this invention. Perhaps the results may even be considered somewhat surprising, as they showed that sex-controlled low-dose artificial insemination can be achieved with fertilization rates comparable to those for high-dose non-sex-controlled artificial insemination. It may even come as a surprise to some that the techniques and new techniques of this invention actually combine to achieve the significant results described. While each technique alone may be considered insignificant by some, the subtle changes, considered alone or in combination with other subtle changes, lead to significant improvements in the end result.

Thus, prior to this invention, ensuring low-dose artificial insemination with sex-controlled insemination rates was not possible with the required technical and economic characteristics or simplified procedures that may be necessary for commercial implementation. In addition to sex-controlled low-dose insemination, however it may be achieved, the present invention also discloses techniques that provide increased efficiency and thereby contribute to the desired end result, namely low-dose sex-controlled artificial insemination on a commercial basis.

In accordance with the above, the present invention claims to achieve, at a commercial level, insemination with low doses and results related to the preliminary determination of the sex of a mammal. It also offers an advanced sheath and collection system for sorting sperm for sex determination using flow cytometry separation. In this separation technique, the membrane fluid typically used in a flow cytometer is replaced with a fluid that minimizes stress on the sperm during sorting. 7/0 In addition, the collection system has been improved to minimize physical and chemical stress to the spermatozoa. Various techniques and substances are described, but those skilled in the art will readily appreciate that various combinations and modifications may be used to optimize performance depending on the animal species, separation techniques, objectives and other parameters of the particular conditions of use.

Thus, the object of this invention is simply to provide sex-controlled insemination at lower doses in /5 such a way that works under real industrial conditions. The object is also to provide better sorting of substances such as spermatozoa. A related objective is to minimize the effect of the sorting function itself on cells or other sensitive substances that may be subjected to sorting. For flow cytometry sorting, the goal is, in particular, to minimize the effect that the membrane fluid has on the cells and potentially introduce a membrane fluid that has a positive effect by helping the cells to tolerate various stresses. A parallel goal is to develop techniques and substances that are particularly suitable for spermatozoa in general, for bovine spermatozoa, for equine spermatozoa, and for the separation of such spermatozoa into X- and Y-bearing components. Similarly, the aim is to minimize the impact on the cells during the collection stage (eg after sorting) and to minimize the physical and chemical effects on such sex-sorted spermatozoa. Therefore, the goal is to obtain as a result of the sorting material, which is damaged as little as possible.

Another object of the invention is to provide insemination with low doses of sorted material at the level and with o); with fertilization rates comparable to typical high-dose artificial insemination without sex control.

In accordance with this goal, a general artificial insemination system is proposed that can achieve this goal in a commercially practical manner. Therefore, the goals described above to minimize stress or potential sperm damage are important. An important goal is also a sorting method that provides high speed and low stress during sorting and that is particularly adapted for sorting - spermatozoa in a low dose context. Also important are the goals of creating sheath fluids and others that do not adversely affect sperm's ability to fertilize and that are compatible with artificial insemination.

Of course, other objects of the invention are disclosed in other parts of the description and claims. uh-

Figure 1 is a schematic diagram of a sorting system using the flow cytometer separation technique of the present invention.

Figure 2 is a diagram depicting trapped cells in the free-fall region of a typical flow cytometer.

Figure C is a schematic diagram that tentatively depicts the differences resulting from the present invention. "

Figure 4 is a diagram of the flow of sorted cells collected in the receiving area. As will be seen later, the basic principles of this invention can be combined and embodied in different ways.

The present invention includes simple commercially practical low-dose sex-controlled insemination and results. )" In terms of flow cytometry separation techniques, the invention also includes improved flow cytometer systems, as well as systems for creating sex-specific sperm samples that

Can be used for artificial insemination, and animals produced using such techniques. -I The invention includes general processes, thanks to which high speeds are possible even in industrial conditions. In addition, general techniques that can be applied to specific systems and areas of application are described

Sh- when understanding the general principles. When describing device improvements, it should be understood that these improvements are not only

Ge" provide the implementation of certain methods, but can also be changed and combined in various ways.

Importantly, for all of the above, each of these features should be understood as being covered by the invention. shk As mentioned, the main goal is to separate spermatozoa carrying X-chromosomes from spermatozoa carrying Y-chromosomes. This is done in a way that isolates two types of sperm so that each can be packaged and used separately. At present, selection is best done using two flow cytometry. In general, flow cytometry is a technique that is well understood. For example, its main aspects are described and discussed in various Suyuotaiyop, Ips. patents, such as the US patents and other previously mentioned publications. Each of these patents and the references made therein are incorporated herein by reference; therefore, specialists in this field will easily understand the basic principles.

Essentially, flow cytometry involves the sorting of substances, such as cells, that are fed to the flow cytometer instrument using some source of cells. The tool is shown schematically in Figure 1.

The flow cytometer instrument includes a cell source (1) that produces or supplies cells or some other type of material for analysis by the flow cytometer. The cells are introduced into the nozzle (2) in such a way that they are surrounded by the membrane liquid (3). The shell liquid (3) is usually supplied by some source of shell liquid (4) in such a way that when the cells are supplied by the cell source (1), the shell liquid (3) is supplied together with them through the 65 nozzles (2). Thus, it is easy to understand that the membrane fluid forms a liquid membrane environment for the cells.

As the various fluids are supplied to the flow cytometer under pressure, they leave the nozzle (2) and flow out through the orifice of the nozzle (5). By means of some type of oscillator (6), which can be very precisely controlled by the oscillator control device (19), pressure waves can be created in the nozzle (2) which are transmitted to the liquids that exit the nozzle (2) through the orifice of the nozzle (5). Since the oscillator (6) exerts an influence on the liquid of the shell (3), the flow (7) coming out of the opening of the nozzle (5) regularly forms drops (8) as a result. Since the cells are surrounded by a liquid shell medium, the droplets (8) may contain individual isolated cells or other materials.

Since the droplets (8) generally contain isolated cells, the flow cytometer can distinguish and separate the droplets based on whether or not the droplet contains the corresponding cell or cells. This is done using 7/0 bistem cell detection (9). The cell detection system includes at least some type of sensor (10) that responds to the cells inside each droplet (8), as described in detail in the seminal work (no joke) of Iagu doppwop, namely, US Patent No. 5,135,759. As explained in the doppzop sperm patent, the cell detection system (Y) can be triggered by the relative presence or absence of a particular dye, which can be excited by some stimulus such as a laser exciter (11). Although the dye stains each type of sperm, the different lengths of the X chromosome and the Y chromosome cause different levels of staining. Thus, by analyzing the degree of dye presence in spermatozoa, it is possible to distinguish between X-bearing spermatozoa and Y-bearing spermatozoa by their different levels of emission.

To ensure the final separation and isolation of the appropriate cells by flow cytometry separation, the signals received by the sensor (10) are fed to some type of dividing sorting system (12) which makes very fast decisions and can differentially charge each droplet (8) on the basis of its decision about whether or not there is a cell inside this droplet (8). This operation of the dividing sorting system (12) enables the electrostatic deflection plates (13) to reject the droplets (8) based on the presence or absence of a corresponding cell or other material in them. As a result, the current cytometer performs the function of sorting cells, forcing them to fall into one or more sc collectors (14). Thus, by analyzing some property of cells or other materials, a current cytometer can distinguish cells by some specific characteristic and place them in the appropriate collection (14). In o); system currently used to sort sperm, droplets containing X-bearing spermatozoa are positively charged and therefore deflected in one direction, droplets containing Y-bearing spermatozoa are negatively charged and therefore deflected in the other direction, and the idle stream (that is, cells that are not sorted) is uncharged and therefore collected in an undeviated stream to a suction nozzle or similar device. -

With the help of Figure 2, the process can be understood even better. As shown in this figure, the nozzle (2) emits a He flow (7), which thanks to the oscillator (6) (not shown in Figure 2) forms droplets (8). Since the source of cells (1) (not shown in Figure 2) can supply spermatozoa (15) that have been stained by the y-d9oppsop method, the light excitation by the laser exciter (11) is differentially determined by the sensor (10) in such a way that y- the presence or the absence of charge on each droplet (8) when it is separated from the flow (7) can be monitored by a flow cytometer. This control makes it possible to distinguish positively charged, negatively charged and uncharged droplets (8) according to their content. As depicted in Figure 2, certain droplets are shown as deflected droplets (16). These rejected drops (16) are those containing spermatozoa (15) of one or the other "sex." They are then deposited in the appropriate collector (14) for further use. шв с One of the aspects of flow cytometry, which is particularly important for its application in sperm sorting, is the high speed of operation of the flow cytometer. Especially improved are current cytometers manufactured by Suyutaiyop, Ips. under the MoRiof trademark. These flow cytometers have greatly increased the speed of sorting and thereby made flow cytometry a technique that can probably become suitable for

Application in industrial sperm sorting (along with other options for industrial application). They -AND function in such a way as to provide high-speed sorting, that is, at a speed much higher than that which can be achieved in other cases. In particular, current MoBioF cytometers manufactured by Suotaiyop

Sh- work with oscillation frequencies higher than about five kilohertz, more precisely, they can work in the range

Ge" frequencies from 10 to 30, or even up to 50 kilohertz. Thus, droplets are formed at very high frequencies, and 5or Cells that are in the liquid shell environment can very quickly emit from the nozzle (2). As a result, each of the components that make up the flow cytometer system and are part of it, such as the nozzle (2), the oscillator (6), etc., can be arranged or selected in such a way as to create a high-speed sorting device. cells When a high-speed cell sorter was applied to sperm sorting, sorting at rates greater than approximately 500 elementary sorting events per second was achieved. In fact, when using high-speed cell sorting devices, sorting speeds of up to 1,200 events have already been achieved. It is important to understand that the term iF, "high speed" is a relative term, so with new advances in current cytometry and specific applications, what is considered "high" may change or remain absolute. In either definition, the general principle is that sorting can occur at a rate at which the parameters and physical characteristics of the current cytometer are essential for the cells themselves when sorting certain cells, such as spermatozoa.

One aspect of high-speed sorting that plays a role in sorting sperm by the flow cytometer separation method is the pressure and other stresses to which the sperm are subjected in the flow cytometer. For example, when operating at high speed (and as an alternative definition of 65 "high speed") current flow cytometers can operate at pressures of 345kPa (5Opsi) and even 414kPa (bopsi). This pressure can be considered high because it can affect the cells undergoing sorting. A key factor for this aspect of the present invention is the fact that stress thresholds for specific cells are the determining factor. Furthermore, with further knowledge, stress thresholds may be shown to be a function of combined effects such as sperm species or

TYPE of pre- or post-treatment of cells. A key factor in this regard is that the stress that cells experience can actually alter their viability and their ability to achieve a desired outcome.

In the case of pressure, it is possible that simply subjecting the sperm to high pressure as a result of performing an operation on a flow cytometer at that pressure may result in decreased cell counts. In one aspect, the present invention minimizes these stresses and. thus providing increased efficacy as well as lower 7/0 doses as described below.

When considered in terms of cellular stress, the present invention works to minimize stress. These stresses can be reduced at any stage of the entire cycle or process of harvesting, sorting, or even inseminating the animal. Importantly, the stress created by processing cells in a flow cytometer is essential for this application. In one embodiment of the invention, the sheath liquid is specifically chosen so that it can be used in concert with both (or any) of the pre-sorting liquid medium of the cells or the post-sorting liquid medium of the cells. While it is of course possible to modify any of the pre-sorting and post-sorting fluids, one embodiment of the invention modifies the shell fluid (3) so that it causes significantly less cell stress than has previously been achieved. In one respect, the invention is notable in that it shifts the overall focus from the operation of the current cytometer to methods of handling and de-stressing the cells themselves. For example, while it has been known to use fluids with an appropriate pH or osmotic value, the present invention recognizes that there may be certain chemical compositions to which cells may be hypersensitive. These sensitizing chemical compositions may, of course, vary depending on the type of cells or even the pretreatment of the cells. In this case, it is important that for spermatozoa, certain metabolic chemicals, such as citrate, prevent extremely high levels of cell stress. Thus, sensitizing chemical compositions can be defined as those to which the cells are particularly sensitive in the context of their functionality and processing techniques existing at the time. In the case of spermatozoa, it appears that metabolic compositions can be very important, namely citrate composition constancy for bovine spermatozoa and Perez buffer constancy for equine spermatozoa. Thus, the present invention seeks to minimize the changes by selecting the type of operation or substances that can act as a means of minimizing the changes that the cells undergo. "

According to one embodiment of the invention, the substance for the shell liquid is chosen in such a way that it can be chemically compatible with the requirement of minimal changes. Thus, by selecting the appropriate membrane fluid not only in the context of the parameters of the flow cytometry, but also in the context of the parameters of the cells themselves, it is possible to improve the changes that the cells undergo and, above all, the sorting result. This is shown schematically in Fig

Figure 3. Figure C depicts some type of chemical factor (such as citrate or other factors) that may exist at various stages of the process. For example, the four phases depicted may, for a flow cytometry separation technique, denote the following, but are not limited to: phase | may indicate the existence of cells in the source of cells (1), phase I may indicate the existence of cells during their sorting in the liquid medium of the shell, phase III may show cells during their collection after sorting, and phase IM shv s may depict reconstituted cells in the medium for storage after sorting. These four phases, as depicted in the prior art, can be exposed to very different chemical environments. )» However, as shown schematically, the cells of the present invention can undergo very small changes, the most significant being that the immersion or droplet formation that occurs between the phases | and II, may be essentially

Absent. This is a result of selecting the appropriate sheath fluid as mentioned above. Thus, as a result of the fact that according to the present invention, the cells are exposed to the appropriate membrane fluid, they can experience a significantly lower level of stress.

Sh- One of the potential generalizations that can arise in connection with this phenomenon is the fact that certain

Some chemical compositions may be chemical compositions that cause more hypersensitivity than others.

Although, of course, this may vary depending on the type of sperm, processing, or even the type of cells to which it concerns, it turns out that the viability of the cells is necessary for their purpose (in this case - artificial

Kk of insemination) can vary greatly for natural reasons or as a result of sorting, or for both reasons, so that cells will show increased sensitivity to this chemical composition. It turns out that by selecting a specific metabolic chemical composition, preferably citrates or citric acid cycle chemicals, significant improvements can be achieved. Thus, for use with bovine semen, the sheath fluid (3) is selected and formulated to contain approximately 2.9 percent sodium citrate and F. In particular, a solution of sodium citrate with a concentration of 2.9 percent can be obtained as follows: 1. Place 29.0 g of sodium citrate dihydrate (MazSeНО7.2Н2О) in a 1000 ml volumetric flask. Dissolve sodium citrate in 3/4 volume of water, and then add water to the desired amount. in 2. Add deionized water or water obtained at the Maporyge plant to the final volume of 1000 ml. 3. Transfer to beakers and autoclave at 15 psi (1182 (2452E)) for at least 30 minutes a. Autoclave the solution under conditions that ensure reduced evaporation (loosely fitting lid) b. Make sure that the water does not boil. 65 4. Slowly cool to room temperature. 5. Store closed in a cold room at 526.

In the future, the sodium citrate solution can be filtered during the preparation of the shell liquid. 6. Filter on a 0.22 micron filter using the aseptic filtration method.

It is interesting that such a composition is suitable to a lesser extent for horse spermatozoa. Instead, it has been found that a Perez-buffered medium, such as Perez-Buffered Bovine Gamete Medium, particularly NVOMZ, which was developed by .).).Raitisy for working with bovine materials, is preferred for equine spermatozoa. This environment is described in the article "Sarasigayop oi Vomipe Zregt Bu Neragip", 38, Vioirodu oi

Kergodisiiop, 1171 (1988), which is incorporated herein by reference). This is surprising not only because the type of substance is different from that used for bovine semen, but also because this buffer itself was originally 7/0 designed to work with bovine material. Therefore, for equine material, a shell liquid is chosen, which contains the Perez buffer. This solution may have a pH of approximately 7.54 at room temperature (pH at 399

E 7.4), with the following composition:

Reagent Dry weight (g/BOOml) sas 0.145

KSI 0115

Masio-vnoo 0.004

ManROd-nO 0.018 mass 2.525

Pyruvate Ma 0.011

Lactic acid (6095) 1, in 4 ml

NEREV8 4.165 mansoz 0.420

VZA (fraction M) 30 s o

One of the other aspects that may play a role in the present invention is the fact that the cells in question may exhibit unusual sensitivities. In one respect, this may be due to the fact that spermatozoa belong to a class of cells that are non-regenerating cells. That is, they do not have the ability to restore themselves and therefore may require much more careful treatment than usual for current cytometers or other processing equipment. Therefore, it may be found that this improvement is particularly useful in cases where a flow cytometer or other separation device is used as the sperm source. Another potentially related aspect that may be unique to a class of cells such as spermatozoa is the fact that their DNA is incapable of repair, replication, and transcription. h-

Any of these factors may play a role, so that they may have some significance individually or in combination. Therefore, it may appear that the description of this invention applies to all gametic cells, or even viruses - etc., which are cells incapable of repair, translation and transcription.

A separate aspect of processing with a flow cytometer that may also be important is that the processing of cells after sorting must be done properly, both chemically and physically. "

As shown in Figure 4, when the cells within the droplets (8) enter the collector (14), it may be important that the container which is the collector is of an appropriate size so that it provides some means of preventing collision between the cells and the container itself. . Although the technique of placing it on the bottom was known

I" of the container of the initial fluid collector (17) to collect the cells in such a way that they do not hit the bottom of the container, it appears that a simple increase in the width of the container can be used to improve the result to account for variations in the shape of the flow, as well as the inevitable splashing due to the collision of cells with container In one respect, this may act as a cushioning element to provide appropriate treatment for cells that may be mechanically fragile, ie may be destroyed or damaged by impact. Thus, when the cell source of the sorting cytometer is cells that are physically delicate cells, it may be important to use some type of cushioning element, such as a wide

Me. a collection tube, for which the width of the opening (18) allows the walls of the container to be positioned so that the tray 20 avoids contact with the cells. Therefore, the side walls of the tube are not close enough that there is any significant likelihood of contact between the cells being sorted and the walls of the tube. In this method, it may be desirable, in addition to the collection fluid (17), to use a wide tube to collect the material. It may be sufficient to just use the wide opening of the container that is part of the collection (14). For uses that apply high sperm sorting rate, it has been found to be sufficient to use a container having an inside diameter of at least

GF) 15 mm. In particular, it was found that the physical damage to the cells associated with the collector (14) was minimal when a 14 ml Falcon test tube was used for this purpose. It should be noted that even Falcon's 14 ml test tube may not be the best option. In particular, it is believed that the most optimal option may be to use a collector container that corresponds to the flow geometry (ie, a "flow geometry-consistent container"). This flow geometry compliant container may have any or all of the following characteristics: a relatively wide opening, an elliptically shaped opening, a smaller height to width ratio than currently used, an oblique or otherwise defined shape so that it can have side walls parallel to the falling flow, etc. It may also be desirable to provide a mounting element, such as a movable element or means, such as ball bearings, etc., to provide variable orientation of the tube depending on the shape of the incident flow that is desired to be collected. In addition, the physical characteristics of a class of containers such as the existing test tube (which is called a "Falcon-type" test tube) may include not only the width of the tube, but also the material (such as polystyrene, to which cells do not adhere) from which it is made, etc. .p. (These possible materials for Falcon's 14ml test tubes are well known). Thus, the container and its collection fluid can also act as a cushioning element to minimize physical damage to the cells.

Due to its size, it can also contribute to the collection of a sufficient number of spermatozoa without a significant dilution effect.

Another aspect of the collector fluid (17) may be the fact that it can also be used to 7/0 Reduce chemical stress on cells. In one respect, since it may be important to provide nutrients to the cells both before and after sorting, the reservoir fluid (17) can be chosen to provide a consistent level of nutrients so that the levels before and after sorting are balanced.

It has been found that for bull semen, in which citrate with two percent egg yolk is used as the nutrient, good results are obtained using citrate with a level of six percent egg yolk (ie, a citrate solution with a content of six percent egg yolk). This is a consequence of the difference in volumes before and after sorting. The collection liquid can start (before sorting) with a volume of approximately 2 ml. As a result of sorting, approximately double the volume (final volume three times the initial volume) may be added at a very low level of citrate with egg yolk in the solution (due to clumping and taking into account other features of the current cytometer). Thus, the final result based on the indicators of the quantitative level of the available 2o citrate with the egg yolk can be equivalent to the initial data, that is, the content of the egg yolk in the citrate solution is two percent due to the used volumes of liquid. Thus, the reservoir fluid (17) can be selected to create a final reservoir fluid medium that is balanced with the initial nutrient level or other fluid medium. In this way, it can be used to reduce the time of action on cells of a composition with a changed level of components. Of course, these liquid media may already be in use in the current cytometer or may have existed at any other time in the past, and the important thing is simply to minimize the stress that the cells experience at any point in their life cycle. In addition, about); since the initial chemical content can be varied (for example, the percentage of egg yolk in citrate can be changed up or down), the initial composition of the liquid collection medium or the different volumes can also be changed so that the final result was the same. Thus, -" where before the start of the sorting process there is a collection fluid with six percent egg yolk in citrate solution, and after the end of sorting, the collection fluid - with sex-specific sperm - can have a content of - egg yolk in citrate solution, which is equal to two percent, similar to the initial content of nutrients (au substances.

Note that these spermatozoa may be treated with citrated egg yolk fluid up to 2095 in further use for other reasons, but these changes are not thought to stress the M cells as they are only a known part of the overall insemination process . While of course the levels may vary, those skilled in the art will readily appreciate that a citrate buffer of 2095 egg yolk may have the following composition:

Y. Final composition: « 8095 sodium citrate solution (72 mM) sv with 2090 (vol./vol.) egg yolk

II. Recipe for 1 liter: )» A. Sodium citrate solution 1. Place 29.0 g of sodium citrate dihydrate (MazSeНО»7.2Н»20О) in a 1000 ml volumetric flask 2. Add deionized water or water obtained at the Maporyge plant to the final volume to him 1000 ml. -I 3. Transfer to beakers and autoclave at 15 psi (1182 (2452P)) for at least 30 minutes. and. Autoclave the solution under conditions that ensure reduced evaporation (loosely fitting lid)

Ge") b. Make sure that the water does not boil. 4. Slowly cool to room temperature. 5. Store closed in a cold room at 526. - B. Egg preparation 1. Obtain fresh chicken eggs from a reliable commercial source. 2. Wash eggs from dirt (without using too much detergent) and rinse. 3. Immerse the eggs in 7095 ethanol for 2-5 minutes. o 4. Eggs are taken out and left to dry (or wiped dry) and placed on a clean towel.

B. Preparation of diluent (emo) 1. Use sterile, clean glassware. 2. A-fraction (glycerol-free fraction) 60 a. Place 800Oml of 2.995 sodium citrate solution in a 1000ml graduated cylinder. b. Antibiotic levels in the glycerol-free fraction (A-fraction of diluent) can be as follows:

Y. Tylosin - 100mcg/ml

II. Gentamicin - 500 mg/ml

PI. Linco-spectin - 300/60Omkg/ml 65 in. Add 200ml of fresh egg yolk as described below (Section D).

Y. Mix very thoroughly.

g. As a result, the A-fraction of the diluent is obtained based on 2.995 sodium citrate with 2095 egg yolk and a concentration of antibiotics that is probably non-toxic to bovine sperm. d. The diluent can be stored overnight at 59C. e. The next day, decant the supernatant liquid (upper 800 ml). is. The next day, before use, it is heated to 3720.

D. The following procedure works well for adding egg yolk to a buffered solution. 1. Wash and clean the eggs (see B above). 2. Open the egg and separate the yolk from the albumen using a yolk separator. 70 According to another option, pour the yolk back and forth between the two halves of the shell. Do not damage the membrane around the yolk. 3. Place the yolk on a sterile piece of filter paper 15 cm in size. 4. Hold the filter paper over a graduated buffer cylinder and crush the yolk (rupturing the membrane), allowing the yolk to flow from the filter paper into the cylinder. Typically, approximately 12-15 ml of yolk can be obtained from one egg.

Another aspect that may play a role in the various factors of the present invention is that of use for artificial insemination, etc. low doses of sperm. An additional description of the state of the art in artificial insemination with sex control can be found in the book Kyregi R. Attap apa Seogde E. Zeyaei, Og., "Rgozresiv Tog Bogiipd Mattaiyap bZregt", SoIogado Avzosiaiei Opimegeyu Rgezz (1982), which is incorporated herein by link). As mentioned, during natural insemination, a quantity of sperm containing billions of spermatozoa is used. A typical artificial insemination is now carried out using millions of spermatozoa for bovine animals and hundreds of millions of spermatozoa for equine animals. The term "low dose" means that the dose of sperm used for insemination is less than half, or even better, less than about 1095 of the typical number of sperm typically used for artificial insemination. Therefore, the term "low dose" should be considered in the context of typical doses for artificial insemination or as an absolute amount. For bovine semen, where 1 to 10 million spermatozoa are currently used, the process may be considered low-dose at an absolute number of approximately 500,000 spermatozoa, or even 300,000 spermatozoa or less. In fact, by using the methods of the present invention, artificial inseminations with good fertilization rates have been performed at insemination sperm counts of 100,000 and 250,000 sperm (respective fertilization rates of 4190 and 5095), as described in the article " Neiiegv Mp Megu om Mitbegv toi Mop-igogep apa Zehei Zreptaiozoa", published in 48, Tnegiodepoiodu, 1255 (1997), which is incorporated herein by (Se) reference). Because spermatozoa appear to be sensitive to dilution, these results may reflect the particular interdependence of using low-dose sperm samples with different techniques of the present invention. Absolute values may depend on the type of animal. For equines, a low-dose process can be considered a process with less than about twenty-five, ten, five, or even one million spermatozoa.

Another aspect that may be important is the fact that the artificial insemination system uses "sperm separated by the methods of this invention according to sex characteristics, or vice versa." Therefore, when, for the method using a flow cytometer, a collector (14) is used to collect sperm for artificial insemination, the methods of this invention may be particularly appropriate. In addition, it is possible that the combination of the use of artificial insemination and its implementation with a low dose can create synergistic effects that make the various techniques of this invention particularly appropriate. Of course, sexed sperm can be used not only in the mode of artificial insemination, but also in other ways, such as intrauterine insemination, etc. - I The process of collecting, sorting, and subsequently inseminating an animal using flow cytometry or other -1 separation techniques includes various stages. In the context of insemination of cows, first the family of the bull is collected with the help of an artificial vagina. At the same time, the levels are approximately 1.5 billion sperm per ml. This (is) undiluted family can be tested using a spectrophotometer to estimate concentration and can be analyzed microscopically to determine compliance with motility and viability standards. After that, antibiotics can be added. As a result, the original sample may have approximately 60 to 70 percent - M serially moving spermatozoa in the ejaculate. For treatment, dilution with some type of TAGR (thyrode albumin lactate pyruvate) may be used to bring the sperm count down to a suitable (for current analysis) level of approximately 100 million per ml. TAIR not only nourishes spermatozoa, but also increases their activity at the coloration stage. Centrifugation may be performed prior to staining for some animal species, such as horses. Staining is carried out according to the protocol of multiple or single staining, and the latter is the object of a patent of Chdoypvzop and related technologies. Staining can be done with simultaneous modification of the diluent to create a suitable nutrient medium. For bovine material, this may involve adding approximately 2095 egg yolks to the citrate solution immediately after staining. In addition, it has been found that in the staining of spermatozoa, better results can be expected to a certain degree when using larger amounts of dye. These high staining concentrations may involve the use of a dye with a concentration of tens of µM (micromole), as described below in the examples where the dye was used

Noesnzi 33342 with a concentration of ZamkM. 65 After dye addition, an incubation period such as incubation for one hour at 342C can be used to accelerate dye uptake at a concentration of approximately 100 million spermatozoa per ml. Filtration may then be performed to remove sperm clots, and dilution or dilution may then be performed to obtain the desired concentration of approximately 100 million sperm per mL. This can be followed by sorting according to the various techniques described earlier, in which the spermatozoa are collected in the collector phase. As previously mentioned, harvesting may result in material samples with an egg yolk citrate concentration of approximately 295 (for bovine material). The sample can then be concentrated to approximately 3-5 million spermatozoa per ml using centrifugation, after which the coat fluid and preservative fluid can be removed.

This can be followed by a final dilution using 2095 egg yolk citrate or /o Sogpeyi Opimegwa diluent! Ehiepdeg, etc. Diluent Sogpeii OpimegvaI Ehiepdeg can have the following formulation per 100Oml: 14.5g sodium citrate dihydrate 21g Mansoz

Oh, 4g KSI

3.0 g of glucose, 9.37 g of glycine, 0.87 g of citric acid

For a composition of 2095 egg yolk, 800 ml of the above preparation and approximately 200 ml of egg yolk can be used.

After this final dilution, a concentration of 3 to 5 million spermatozoa per ml (for bovine material) can be obtained. This sample can be refrigerated to slow the metabolism of the sperm and allow for longer periods of use. In horses, a sample of the material can then be used using oviduct or other methods of insemination, as is well understood by specialists in this field. For bull semen, the material sample can be diluted again to the desired dosage level. It has been found that dilution can affect sperm viability and therefore it may be convenient to avoid too high a dilution level by using a smaller sample of material. Currently o); time, regardless of the separation technique used, low doses of 300,000 spermatozoa per 0.184ml can be achieved. In addition, it may be desirable to maintain a seminal plasma content of approximately five percent, although the results of compliance with this requirement are currently uncertain. The sperm sample can then be placed in straw intended for use in artificial insemination and delivered to cows or heifers to be inseminated. -

To provide a convenient artificial insemination schedule, estrus in cows or heifers can be synchronized with Ge using known methods, such as the use of prostaglandin B2 in accordance with methods known in the art. The latter substance may be of particular value, as it has been reported to potentially increase the ability of heifers to conceive, as described in the article "Rgoviadiapaiy 2 o - A Repiyu Ogd zr ip Oaigu SashShe?", 18, Tpegiodepoiodu, 245 ( 1982), which is incorporated herein by reference). Although recent results do not support this assumption, it is possible that the present invention demonstrates its special viability in the case of low-dose sex-controlled insemination. After this, artificial insemination can be carried out for bovine animals by using embryo transfer equipment with the introduction of spermatozoa deep into the horns of the uterus. This may not be done at the peak time, which is usually used in artificial insemination, but at a slightly later time point, such as 12 hours after it, since there is some probability that fertilization in artificial insemination with sex control may occur slightly later . and' Embryo transfer equipment may be used because there may be high sensitivity of the uterine wall to such low-dose, sex-controlled insemination.

In addition, these techniques can be combined to achieve higher production efficiency. In particular, any of the invented processes that allow high-speed sorting and insemination with low doses of sex-selected embryos can be used in superovulated animals. Ovulation can be achieved by using a drug to induce (is) superovulation or by any other method. A drug to induce superovulation can act directly or indirectly, for example, through a sequence of reactions, in order to ensure increased compared to normal egg production. The combination with superovulation is unexpected, since it was previously thought that superovulation prevented such a combination. Sperm transport in superovulated animals is risky, so artificial insemination of animals was often carried out several times and/or with several doses of semen. In addition, the known procedures for dividing families by gender were relatively slow; therefore, it was of interest to determine the fertilization rates after a single insemination of cattle that had received a drug to induce superovulation, such as EHN (follicle-stimulating hormone), with a total of only 600,000 unfrozen sex-selected spermatozoa using these new combinations of methods.

For example, twelve crossbred Aberdeen-Angus heifers were induced to superovulate 60 times using standard techniques: 6, 6, 4, 4, 2.2, 2, and 2 mg of EN were administered intramuscularly at half-day intervals starting between days 9 and 12 of the cycle. oestrus; with the second and seventh injections of EN, 25 and 12.5 mg of prostaglandin E-2 alpha were injected intramuscularly. Sperm from bulls of unknown fertility were stained with Noespzi 33342 and then sorted using a flow cytometer/MoRiob cell sorter) yielding 700-800 live spermatozoa of each sex/sec. The average sorting purity was 65,8996 of the desired sex. The sorted sperm was concentrated to 3.36x109 spermatozoa/ml by centrifugation at 650°C for 10 minutes, cooled to 52°C and stored for 4 hours. Then 184 μl was placed in 0.25 ml plastic straws; half of each dose was administered for insemination to each horn of the uterus during the period from 20 to 24 hours. from the beginning of oestrus using automatic embryo transfer capsules with a side opening.

Embryos were collected by standard non-surgical techniques on day 7 or 16 after oestrus. Similar results were obtained for material collected on days 7 and 16 and for sperm sorted by X- and

Y-chromosomes. Embryos were obtained from 9 heifers. There were 52 embryos (average -4.3--5.3/donor) at normal stages of development, 13 with developmental delay and 31 unfertilized eggs. For 46 embryos, the sex was determined by the RSC method using primers for the DNA sequence specific to the U-chromosome; of these, 43 (9390) had a predicted gender. Although these studies involved only a few animals, surprisingly inseminating 70 superovulated heifers with a total of only 600,000 (live) sex-sorted unfrozen spermatozoa produced results similar to conventional procedures. Variations of the above procedure may be performed, including, but not limited to, sorting by means other than current cytometry, other methods of achieving superovulation, other methods of increasing fertility, and the like.

Furthermore, the combination of DNA-based sperm sexing methods, high-speed 75 current cytometers/cell sorters, and heifer insemination procedures using a total of less than 500,000 sperm without compromising fertility rates resulted in the emergence within a few years of an efficient sex-sorted sperm industry 'I. For sperm sorted by gender with an accuracy of 28,595, there are many possible applications. Perhaps the most obvious is the insemination of one herd of cattle (both dairy and beef) to restore the female part of the herd, and the insemination of another herd (dairy or beef) from completely different types of bulls to produce males for meat. A very important variation of the above is the insemination of heifers with X-carrying spermatozoa to produce female calves that have significantly lower rates of dystocia than male calves, primarily due to smaller size. In addition, the evaluation of young breeders of the dairy herd will become much more effective with the predominance of heifers. Gaining more than 8,595 heifers will enable dairy cows to be managed so that they have an average lifetime of fewer than two surviving calves, which is attractive due to reduced problems associated with pregnancy and parturition. Systems of single-sex i9) beef production will also become feasible, in which each female reproduces herself and is slaughtered at the age of 2 to 3 years, thanks to which a much larger percentage of nutrients in the system will be spent on growth, and a smaller percentage - on maintenance. Sex-sorted family will be useful for insemination of ip miigo and insemination of cows subjected to "- superovulation for embryo transfer. Often one sex of the calf is much more valuable than the other, and although there are accurate methods of selecting embryos by sex, they take time, and half of the C embryos produced are of the less valuable sex, so it is assumed that exactly sexed families I will become Ge) will be widely used for artificial insemination of livestock, if the premium for sorting by sex will be small, and the deterioration of fertility rates will be minimal. It is likely that the fate of the meat industry will increase significantly -

35 cattle that are artificially inseminated in gender-sorted families. uh-

It is interesting that better results can be obtained with insemination into the depth of the uterine horn than with insemination into the body of the uterus, as is usually done with insemination. It is also perhaps surprising that the specimens studied so far have not shown any difference between ipsilateral and contralateral insemination when performed deep in the uterine horn. By deep it should be understood that the introduction is carried out far inside the horn of the uterus using « 70 equipment for transferring embryos. The fact that the results of the ipsi- and contralateral sh

The results of insemination are not significantly different, which enabled the authors of this invention to propose the use of both insemination options, so that the process of identifying the appropriate uterine horn may be unnecessary. )» The desired result of insemination is, of course, the production of an animal of the desired sex. This animal can be produced using the systems described above using samples of sex-sorted sperm material. It should also be understood that the techniques of this invention may find application in other methods, such as laparoscopic insemination, IVF, or similar methods.

As examples, the following experiments were conducted. Although not all of them use all aspects of the 7 inventions described here, they show possible improvements in operational performance due to various aspects of the invention.

Gee! indicators In addition, a brief description of some experiments is given in the article "Shegipe Nogp Ipzetipayop oi Neyiegz MUyY Megu Gom/ Mitrege oi Mop-yitogep apa Zehei Zreptaiyo2oa", which was mentioned earlier). This article summarizes some data showing the effectiveness of this invention. As for the experiments, one of them was -Z conducted using sex-sorted unfrozen spermatozoa with high success rates as follows:

Example 1

Heifers of the Aberdeen-Angus breed at the age of 13-14 months, of average condition, were synchronized (synchronized the processes in time) with the help of 25 mg of prostaglandin E-2 alpha at intervals of 12 days and inseminated after 6-26 hours.

F, after the appearance of continuous estrus. A freshly collected family from three bulls aged 14-26 months was incubated with ZOMkM (name) Noesnvi 33342 at 75x105 spermatozoa/ml in TAY R medium for 1 hour at 342C. Spermatozoa were sorted by sex chromosomes based on epifluorescence from laser excitation at 351 and Zb4inm at 60 power of 150 mW using a MoRio F flow cytometer/cell sorter operated at 345 kPa (5 Org) using 2.995 Ma citrate as the sheath fluid. Carrying sperm

The X-chromosome (purity 790906 as verified by re-sorting the sound aliquots of sperm) was collected at a rate of 500 live spermatozoa/sec into 2 ml Eppendorf tubes containing 100 μl of Soptea diluent! Opimegza! Echiepdeg (SOE) from 20965 egg yolk. The collected spermatozoa were centrifuged at 65 rpm for 10 minutes and resuspended in SOE with a concentration of 1.63x109 live spermatozoa/ml.

For control, a rare family was used without sorting by gender; sperm stained with Noesnpvi 33342,

diluted with liquid shell to 9x10? spermatozoa/ml and centrifuged and resuspended in SOE to 1.63x109 persistently motile spermatozoa/ml. Sex-sorted semen and liquid control semen were cooled to 59C for 75 minutes. and loaded into straws at 0.25 ml (0.184 μl/straw). Straws were transported in a temperature-controlled beverage cooler at 3-59 over 240km for insemination 5-9 hours after sorting. Sex-sorted families and rare control families were inseminated using blue side-opening capsules (IMM), half the contents of each straw to each of the uterine horns (3X10? live spermatozoa/heifer). As a standard control, sperm from the same bulls were frozen in 0.5 cm straws according to standard methods (an average of 15.6x105 motile spermatozoa/dose after thawing), then thawed at 352C for Zbsec and insemination was carried out in the uterine body. Procedures were distributed between Z steers and 2 inseminators in a 3:2:2 insemination ratio for a sex-sorted family and two controls.

Pregnancy was determined by ultrasound methods 31-34 days after insemination and confirmed after 64-67 days, when the sex of the fetus was also determined (blindly). The data are given in the table. and 2. Sh , , 2,

The ratio of articles for values with different indices differs (Р «0.02).

Although the pregnancy rate for the sex-matched family was only 8095 from the controls, this difference was not statistically significant (20.1). One pregnancy each was lost by day 64-67 in groups with sex-sorted and frozen control families; 18 of 19 fetuses (9595) in the sex-controlled group were female, whereas 20 of 30 (6795) were female in the control groups. The liquid control sperm gave essentially identical pregnancy rates to the frozen control sperm, which contained more than 50 times more motile (Fo) spermatozoa (more than 120-fold higher total sperm count), demonstrating the efficacy of low-dose insemination at introduction into the horn of the uterus. Using the technology of flow cytometry and artificial insemination, the authors significantly changed the sex ratio in cattle. "-

Similarly, an experiment was conducted with sex-unsorted unfrozen spermatozoa, to which the following report is attached: "And

Example 2 "co

The objective was to determine pregnancy rates when inseminating heifers using extremely low amounts of frozen sperm under ideal field conditions. A family of three Holstein bulls with above-average fertility was bred in homogenized milk, 7906 glycerin thinner (C55) plus 596 homologous seminal plasma to a total amount of 2x10 U, 5x10? or 10x105 (control) spermatozoa on French straw (Egeps wigamus) per 0.25 ml and frozen in a flow of liquid nitrogen vapor. The family was thawed in water at 379 for 20 seconds. Holstein heifers aged 13-15 months weighing 350-450 kg were given two injections of 25 mg of prostaglandin E-2 alpha (and cyuseF) with an interval of 12-70 days and inseminated using a straw syringe for embryo transfer and a capsule with a side hole, sh

Gani by injecting half a seed deep into each horn of the uterus 12 or 24 hours after detection of estrus. The experiment was carried out with five parallel experiments over a period of 5 months, and was distributed between two )» fertilization technicians. The ambient temperature during insemination was often from -10 to -202C, so measures were taken to keep the insemination equipment warm. Pregnancy was determined by detecting a viable fetus using ultrasound on the 40-44th day after estrus and confirmed on 55-62 days after - the 1st estrus; 4 out of 202 conceptions were lost during this time period. Pregnancy rates for days 55-62 were -1 55/103 (5395), 71/101 (7095) and 72/102 (7195) for 2x105, 5x102 and 10x105 total sperm count/insemination (P<0.1). Pregnancy rates were different (P<0.05) between bulls (59, 62 and 7495). but not (o) between techniques (64905 and 6595) or time of insemination after estrus (6595 for 12 h and 6495 for 24 h, M-153 for their 50 each time). For the methods described, pregnancy rates in heifers were the same when used in general

BH105 and 10Xx109 spermatozoa for insemination. An experiment was also conducted with sex-sorted unfrozen spermatozoa, to which the following report is provided:

Example C 29 A family was collected from AMapiis Vgeedegs Sooregaiime bulls, diluted 1:4 with a diluent with NEREZ buffer of 0.196

GF) VZA, and transported 1b0Okm (-2h.) to the city of VeyivmShe, Maguiapa, where it was sorted at ambient temperature by the method of flow cytometry to the thinner for collecting sperm TE5T (2095) using the previously described methods (Vio!. Bergod. , 41:199). Sorting rates of up to 2x10 9 spermatozoa of each sex were achieved in 5-bh. with a purity of -9095. Spermatozoa were concentrated by centrifugation (30,049 rpm for 4 min.) to 2x10 U spermatozoa/ml. Some of the spermatozoa were sorted into a diluent containing homologous seminal plasma (final concentration 595). Sorted spermatozoa were transported by air to Colorado (-2600 km) and stored at ambient temperature or at 52C (during transportation, they were cooled for 24 hours in an Ediiaipeg device or in an isolated device with an ice compartment). Heifers or barren cows in which estrus was detected during the period from 11 to Zbhod. before that, they were inseminated during the period from 9 a.m. to 9 p.m. after sorting the sperm. Sperm (from 1 to 2x10 5 in O,Tml) were injected deep into the uterine horn ipsilateral to the ovary with the largest follicle, which was determined by ultrasound during insemination.

None of the 10 females became pregnant after insemination with spermatozoa that were transported and stored at ambient temperature. Of the 29 females inseminated with sperm cooled to 59 during transport. 14 were pregnant in the 4th week of pregnancy, and 12 (4195) in the 8th week. Eleven out of 22 fertilized within 1 hour. after sorting, were pregnant in the 8th week, but only 1 out of 7 fertilized within 17-24 hours. after sorting, she was pregnant. The addition of seminal plasma did not have a significant effect. One of the 12 fetuses did not belong to the expected sex, one had an unknown sex, and 10 had the expected 70 sex, as determined by ultrasonography on day 60-70 of gestation.

After that, an additional 33 heifers were inseminated with the introduction of 0.05 ml of material (the family was diluted as described above) into each horn of the uterus without the use of ultrasonography; only C were pregnant 4 weeks after insemination and only 1 remained pregnant by week 8. However, different bulls from the previous group were used, and all inseminations were performed during the 18-29h period. after sorting. Similarly, 75 more 38 heifers were fertilized (-22 hours after sorting) 200 km from our laboratory using sorted sperm from another bull; none of them were pregnant 8 weeks after insemination.

In summary, it is possible to induce pregnancy in cattle by artificial insemination using flow cytometry sex-sorted sperm, with fetal sex ratios approaching those predicted by repeated DNA analysis of sorted sperm (9095). However, fertilization rates were highly variable in these earlier experiments, which required sperm to be transported over long distances. Fertilization decreased sharply by 5 p.m. after sorting, but there were some misunderstandings because different bulls were used at different times. Further studies are needed to determine whether the observed variation was due to differences between bulls, insemination techniques, the time period between sorting and insemination, or other factors. with

Finally, an experiment was also carried out with non-sex-sorted, non-frozen spermatozoa, to which the following report is attached: o

Example 4

The aim was to determine the fertility rate when inseminating heifers with a very low number of spermatozoa under ideal conditions for the experiment. A family of three bulls of the Holstein breed was bred with the Sogpeia breeder! MOpimegwa! Ehiepdeg plus 5905 homologous seminal plasma up to 1x10 5 or 2.5x105 spermatozoa per 0.iuml; samples with a total number of spermatozoa of 2.5x105 na were used as a control

Oh, 25 ml. The fully diluted seed was packed into modified plastic French straws (Egepsp (Se) vigamlv) of 0.25 ml for the introduction of fertilization doses of 0.1 or 0.25 ml. The family was cooled for 5 seconds and used after 26-57 hours. after harvesting Holstein heifers aged 13-15 months with weight

From 350-45O0kg, injections of prostaglandin E-2 alpha (I shayuseF) were made at intervals of 12 days and fertilized according to v. using a straw-syringe to transfer embryos and a capsule with a side opening into one corner of the uterus after 24 hours. after detection of estrus. Insemination was performed ipsilaterally to the side where the largest follicle was located, as determined by ultrasound after 12 hours. after the start of estrus; the side of ovulation was checked by detecting the corpus luteum by ultrasound on the 7-9th day after estrus. Pregnancy was determined by ultrasound detection of the fetus on the 42-45th day after estrus. The experiment was carried out with four parallel experiments, and C was distributed among three fertilization technicians. The side of ovulation was determined correctly in 205 out of 225 heifers (9195); unexpectedly, the fertility rates for ipsilateral and contralateral insemination were almost identical.

Fertilization rates were 38/93 (41905), 45/87 (5290) and 25/45 (5695) for 1x10 5, 2.5X109 and 2.5X105 sperm/insemination (P 20.1). There was a significant difference in fertility rates (P<0.05) for different technicians, but not for different bulls. When using the described methods, it may be possible to reduce - the number of spermatozoa per insemination to a sufficient extent so that sperm, sorted by sex using -I current cytometer, find industrial application.

As mentioned and as can be seen from various experiments, this field of technology is statistical in nature,

Therefore, a variety of additional experiments can be conducted to identify strategies for creating appropriate combinations and constraints. In this way, synergistic effects between different factors will be identified, as, for example, dye exposure and the combination of dye exposure and laser excitation can be investigated. The description included in this application should serve as the main description. The reader should understand that the specific description cannot fully cover all possible uses; many alternatives remain speculative.

Nor can it fully disclose the generalizable nature of the invention and cannot exhaustively show how each 22 feature or element may in fact represent a wider function or a greater variety of alternative or

GF) of equivalent elements. They are also definitely included in this description. Where the invention is described using device-oriented terminology, it is understood that each element of the device performs a specific function. Not only the claims in which the apparatus is claimed apply to the described device, but also the method or process claims may be used to describe the functions performed by the invention and each element. 60 Neither description nor terminology should limit the scope of claims that may be submitted for consideration.

It should be understood that various changes may be made without departing from the spirit of the invention. Such changes are also definitely included in the description. All of them are included in the scope of this invention. This description covers a wide range of both the explicitly described embodiment(s) and various implicit alternative embodiments, as well as a broad description of methods and processes, etc. for In addition, each of the different variants of the invention and the claims may also be carried out in different ways. This description should be understood as covering every such variation, whether a variation in any embodiment of the apparatus, in the embodiment of the method or process, or simply in the embodiment of any element thereof. In particular, it should be understood that where the description refers to elements of the invention, the terms used for

Each element can be expressed in equivalent terms relating to the apparatus or method - even when only the function or result remains the same. Such equivalent, broader or even generalizing terms shall be deemed to be covered by the description of each element or function. Such terms may be inserted where it is desirable to express in an explicit manner a generalized broad description to which the present invention claims. As just one example, it is to be understood that all actions can be expressed through a means /0 for performing that action or through an element causing that action. Likewise, each described physical element should be understood as including a description of the action performed by that physical element. Just one example of this aspect is that the description of "gathering" should be understood as including the description of the act of "gathering" - whether it is described in explicit form or not - and, conversely, if only the description of the act of "gathering" is given, this description should be understood as such , which covers the description of the "collection". Such substitutions and alternative terms should be understood as expressly incorporated into the description. Furthermore, it should be understood that in addition to the claims originally presented, the claims may be modified to more broadly encompass at least: i) the devices disclosed and described herein; i) related methods disclosed and described here; ii) similar, equivalent, and even implicitly expressed variations of each of these devices and methods; im) those alternative designs that perform each of the functions disclosed and described here; M) those alternative designs and methods that perform each

Of those functions that are implicitly necessary for the implementation of what is disclosed and described; mi) each feature, component and stage, which are described as separate and independent inventions; and mine) of various combinations and changes of all the above.

Various published works may be useful to assist in understanding the invention. They are listed below and incorporated herein by reference; however, to the extent that this listing may be deemed inconsistent with the patentability of this invention(s), these references shall be expressly deemed not to have been made by the applicant(s). Potentially useful references include: United States patents o);

МоМо 5660997, 5589457, 5514537, 5439362, 5346990, 5135759, 5021244, 4999283, 4749458, 4698142, 4680258, 4511661, 4448767, 4362246, 4339434, 4276139, 4225405, 4191749, 4155831, 4092229, 4085205, 4083957, 4067965, 4009260, 3894529 , 3687806, KE32350. Useful links may also include the following publications: | Ipzetipaiop "- zo oi Noivivip Neiiige MUUyp Megu Gom/ Mitbrege oi Opitolep Zreptafogoa", O.E.bZeide!i, Og., S.N.AiPep, 7.Vhipk,

U.K. Ohayat apa M.V. Satsnei, Soiogado Ziaie Opimegeiu, Bogi SoPShpz, AYapiis Vgeedege Sooregaime, "

I apsazieg, RA, OO Oiagu, Iomeiapa, SO, Oshu 1995; "Agiisiai Ipvzeptipaiop MUyi Kh- apa MU-Breagipd Bomipe Ge zvregt", O.E. Zeideii, g, H. ALoppsop, S.A.AMep, sS.MK.MUeisR, M.O.NoMapa, 2.V'pK apa M.V. Satsei, Apitai

Kergodisiop apa VioiesppoiIodu I arogayugu, SoYogado Biaie Opimegeiu, Rog Soipv, SO; Septriazt apa Sateiye i-

Rpuzioisdu Ga, AKB, OBOA, VeiivmShe, MO; Ayapiis Vgeedege Sooregaiime, I Apsazieg, RA; SHO Oiagu, I omeiapa, i-

SO, OA daptsag 1996; "Ipveptipayop oi Ppeyege m/yyp megu Yum/ pitbrege oi iogep zregtafogoa", O.E. Zeidei, Og.,

S.N.Ashep, 2.Vhipk, M.O.Noyapa apa M.V.Saitsei, Soiogado Z(afie OMPimegeiu, Rog Soiipz, AiYapiis Vgeedege

Sooregaiime, I apsazieg, RA, OO Oiagu, I omeiapa, SO, Oshu 1996; "Rgodisiop oi Iatre ru Iom/ dose ipigashiegipe

Ipzetipayop my Pyom/ suioteigisayu vogpei apa pzopyei zvetep, O.Ob.Stap, MU.A.S.MesKeimeu, M.E.Kipa, "

O.R. YuoYitap, T.. MesEmosu, R.). SoIede, Stairviope, VisKebygp, Aregdeep, AV21 UMA, ShK, TNegiodepoiodu, r.267; "(HZhegipe pogp ipzetipayope oi Neyege m/yy megu m pitbrege oi popitolep apa zehey zreptai(og2oa", O.E.Zeiaei, )" Ug., S.N.Aishep, GO.A.oppsop, M.O.NoyPapa .

Septriazt apa Sateii RPuzioiodu I arogaiogu AK5, OBOA, VeiivmShe, MO 20705, SHO Oiagu, I omeiapa, SO 80538, -I Tpegiodepoiodu, 48: 1255-1264, 1997; "Sarasytsaiop oi Bromipe zregpt Bu Perahip", 9.9U.Raitivy, 9.BizKo-Raitivy,

M.A.M/ipeg apa M..Rigvi, YuOeragitepi oi Meai apa Apita! Zsiepse, Opimegeyu oi MUivsopvip, Madizop, UM! 53706,

Sh-Vioosdu oi Kergodisiop, 38, 1171-1180 (1988); "Rgoviadiapapy B2a - A Tepyyu agod ip aaigu sashShe?",

Ge» K.S. Mastiyap apa A.M. Oau, KiaKiga Apita! Kezeagsp 5iayop, RgiimaSe Vad, Natiyop, Mem/ 7eaiapa, 5o Tpegiodepoiodu, Zerietreg 1982, moY.18, Mo.Z, pp. 245-253; "Rgozresiv og zehipd tattaiap vregt", Soiogado ve Azzvosiagye(y Opimegeyu Rgevzv, Apita! Kergodisiop Iarogaiogu, SoMPede oi Meyegipagu Meadisipe apa Viotedisai shk Zsiepsez, SoYogado biafe Opimegeyu, Rog Soipv, SO 80523, edey ru Kiregi R. E. Attap apa Seogde Zeyaei, g., 1982; "EPChesiv oiedd-usikK-suigabe apa tik ehiepdeg op o sPpgotayip vigysishge apa khmiariyu oi sguorgezegumei By vregt", 9. Oiagu Zsi., 74:3836, O.5. Kagaripiz and O.R. Emepvop apa M.T. Kargoii; "Azzezvtepi dvo oi tat apa oroag zregtailoa digipuo sei-zopipo Bu Yomu sutenu, Kergod. Oot. Apit., 32:251; "Biregoutsiaiop oi doaiv m/y rigyei rEBN zirrietepiei m/y deiipedy atoipiv oi ri N", Tpegio., 43:797,

F) M.A. MomuzNagi, 9.E. VesKegz apa MU. NoiU; "Sepadeg rzheiesiop ip tattaiIv: Ap omegamiem, Oivsp. (egagai|. ka Uuvspg, 103:285, G.A .doppzopi.

Throughout this description, unless the context otherwise requires, the word "include" or its variants, such as "Includes" or "that includes", shall be understood to imply the inclusion of a specified element or unit or group of elements or units, but not to the exclusion of any other element or unit or group of elements or units.

Claims (38)

The formula of the invention b5 1. A flow cytometer system for isolating desired cells, comprising a cell source that supplies cells for analysis by said flow cytometer, a shell fluid source that provides said cells with a shell fluid medium containing about 2.9 95 sodium citrate, a pipe, through through which said Cells pass when fluid envelope medium is supplied to them, an oscillator that acts on said fluid envelope as it passes through said nozzle, a cell analysis system responsive to said cells, a dividing sorting system that sorts cells having the desired characteristics, and a collection into which cells with the desired characteristics are placed. 2. A flow cytometer system for isolating desired cells according to claim 1, characterized in that the source of said cells is bovine sperm cells. 3. A flow cytometer system for isolating desired cells according to claim 2, characterized in that said nozzle, oscillator, cell analysis system and dividing sorting system are part of a flow cytometer system, and said flow cytometer system includes a flow cytometer for sorting said spermatozoa at a speed at least 500 sort events per second. 4. A flow cytometer system for isolating the desired cells according to claim 1, characterized in that the source of said cells are cells that are hypersensitive to the chemical composition in the surrounding liquid medium. 5. A flow cytometer system for isolating the desired cells according to claim 1, which is characterized by the fact that the specified collector is used to provide a low dose of spermatozoa. 6. A flow cytometer system for isolating desired cells according to claim 5, characterized in that said 2o nozzle, oscillator, cell analysis system, and dividing sorting system are part of a flow cytometer system, and said flow cytometer system includes a flow cytometer for sorting said spermatozoa from at a rate of at least 500 sort events per second. 7. A flow cytometer system for isolating desired cells, which includes a cell source that supplies cells for flow cytometer analysis, a sheath fluid source that creates a sheath fluid medium for the cells that contains pPerez-buffered medium, a tube through which the specified cells when a liquid envelope medium is supplied to them, an oscillator that acts on said liquid envelope, (io) as it passes through said nozzle, a sperm analysis system responsive to said cells, a dividing sorting system that sorts cells having the desired characteristics, and a collection into which cells with the desired characteristics are placed. "- zo 8. A flow cytometer system for isolating the desired cells according to claim 7, characterized in that the source of said cells is equine sperm cells. " 9. A flow cytometer system for isolating the desired cells according to claim 7, which is characterized in that the source of He for the specified cells are cells that are hypersensitive to the chemical composition in the surrounding liquid medium. 10. A duct cytometer system for isolating the desired cells according to claim 7, which is characterized by the fact that the indicated collector is used to ensure a low dose of spermatozoa. uh- 11. A flow cytometer system for isolating desired cells according to claim 10, characterized in that said nozzle, oscillator, cell analysis system, and dividing sorting system are part of a flow cytometer system, and said flow cytometer system includes a flow cytometer for sorting said spermatozoa at a speed at least 500 sort events per second. " 12. A flow cytometer system for isolating desired cells, which includes a cell source that supplies cells for analysis by the flow cytometer, a source of sheath fluid that creates a sheath fluid environment for said cells, a tube through which said cells pass when fed to them fluid )" sheath medium, an oscillator that acts on said sheath fluid as it passes through said nozzle, a cell analysis system responsive to said cells, a dividing sorting system that sorts said spermatozoa having desired characteristics, and a collector, to which the cells are placed, -And having the desired characteristics, including a citrate collection fluid containing approximately six percent egg yolk. Sh- 13. The flow cytometer system for isolation of desired cells according to claim 12, characterized in that the He source of the sheath fluid contains a solution of approximately 2.9 95 sodium citrate. 14. A flow cytometer system for isolating desired cells according to any one of claims 12 or 13, characterized in that the source of said cells is bovine sperm cells. - M 15. A flow cytometer system for isolating desired cells according to claim 12, characterized in that the source of said cells are cells that are hypersensitive to the chemical composition in the surrounding liquid medium. 16. A flow cytometer system for isolating the desired cells according to claim 12, which is characterized in that said two Collection is used to ensure a low dose of spermatozoa. 17. A flow cytometer system for isolating desired cells according to claim 16, characterized in that said iF) nozzle, oscillator, cell analysis system, and dividing sorting system are part of a flow cytometer system, and said flow cytometer system includes a flow cytometer for sorting said sperm at a rate of at least 500 sorting events per second. 60 18. A flow cytometer system for cell isolation, comprising a source of cells that supplies cells for analysis by said flow cytometer, a source of chemically modified membrane fluid that creates a membrane fluid environment for said cells that is selected to be compatible with the fluid environment of the cells both before and after sorting, a nozzle through which said cells pass when the shell fluid is supplied to them, an oscillator that acts on said shell fluid as it passes through said nozzle, a cell analysis system responsive to said cells , a dividing sorting system that sorts specified types of cells having the desired characteristics, and a collection into which the cells with the desired characteristics are placed. 19. A flow cytometer system for isolation of desired cells according to claim 18, characterized in that said fluid medium of cells both before and after sorting contains at least one hypersensitive chemical composition to which said cells are particularly sensitive and in which the source of chemically modified fluid shell minimizes changes in the indicated ultra-sensitive chemical composition. 20. The flow cytometer system for isolation of desired cells according to claim 19, characterized in that said ultrasensitive chemical composition contains a metabolic chemical composition. 21. A flow cytometer system for isolating desired cells according to claim 19, characterized in that said ultrasensitive chemical composition contains citrate. 22. A flow cytometer system for isolating desired cells according to claim 21, wherein said source of chemically modified fluid contains a solution of approximately 2.99% sodium citrate. 23. A flow cytometer system for isolating desired cells according to claim 22, characterized in that the source of said cells is bovine sperm cells. 24. A flow cytometer system for isolating the desired cells according to claim 23, which is characterized by the fact that the specified collector is used to ensure artificial insemination with spermatozoa. 25. A flow cytometer system for isolation of desired cells according to claim 23, characterized in that said collector is used to provide a low dose of spermatozoa for artificial insemination. 26. A flow cytometer system for isolating desired cells according to claim 19, characterized in that said 2o source of cells comprises non-renewable cells. 27. A flow cytometer system for isolating desired cells according to claim 26, wherein said source of cells comprises cells with DNA incapable of transcription. 28. A flow cytometer system for isolating desired cells according to claim 26, wherein said source of cells comprises cells with DNA incapable of replication. high school 29. A flow cytometer system for isolating desired cells according to claim 26, characterized in that the source of said cells is sperm cells. io) 30. A flow cytometer system for isolation of desired cells according to claim 29, characterized in that said collector is used to provide a low dose of spermatozoa. C1. A flow cytometer system for the isolation of desired cells according to claim 30 characterized in that the low dose of sperm contains no more than about ten percent of a dosage selected from the group consisting of one million bovine spermatozoa, ten million bovine spermatozoa, and one hundred million horse spermatozoa. Ge 32. A flow cytometer system for isolating desired cells according to claim 3, characterized in that said spermatozoa contain bovine spermatozoa, and said low dose of spermatozoa contains a dose of less than approximately five hundred thousand bovine spermatozoa. uh- 33. The flow cytometer system for isolating desired cells according to claim 3, wherein said spermatozoa comprise bovine spermatozoa, and said low dose spermatozoa comprises a dose of less than about three hundred thousand bovine spermatozoa. 34. The flow cytometer system for isolating desired cells according to claim 3, wherein said spermatozoa comprise equine spermatozoa and said low dose spermatozoa comprises a dose of less than about ten million spermatozoa. 35. A flow cytometer system for isolating the desired cells according to any one of claims 19 or 31, )" characterized in that said source of cells comprises bovine spermatozoa. 36. A flow cytometer system for isolating desired cells according to claim 19, wherein said cell source comprises equine spermatozoa. -and 37. The flow cytometer system for isolating desired cells according to claim 18, wherein said source generates said liquid medium prior to cell sorting and said receiver generates said medium. indicated medium after cell sorting. F 38. A flow cytometer system for isolating desired cells according to claim 18, characterized in that said 5p source of cells comprises cells that are hypersensitive to the chemical composition in the sheath fluid. sh» - F) name) 60 b5